G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
neuroblastoma RAS viral (v-ras) oncogene homolog
G00000226 (Mus musculus)

Databases (7)

Curated Gene
OTTHUMG00000012059 (Vega human gene)
4893 (Entrez Gene)
540 (G2Cdb plasticity & disease)
NRAS (GeneCards)
164790 (OMIM)
Marker Symbol
HGNC:7989 (HGNC)
Protein Sequence
P01111 (UniProt)

Synonyms (1)

  • N-ras

Literature (137)

Pubmed - other

  • Multiple genetic alterations in papillary thyroid cancer are associated with younger age at presentation.

    Moses W, Weng J, Khanafshar E, Duh QY, Clark OH and Kebebew E

    Department of Surgery, University of California, San Francisco, California 94143, USA.

    Background: There is a significant gender and age disparity in thyroid cancer incidence and outcome. The molecular basis for these divergent clinical presentations and outcome are essentially unknown.

    Methods: The primary tumor genotype in 217 patients with papillary thyroid cancer was determined for six common somatic genetic alterations (RET/PTC1, RET/PTC3, and NTRK1 rearrangements, and BRAF V600E, KRAS, and NRAS hotspot mutations) by PCR and direct sequencing, and nested PCR. Univariate and multivariate analyses were performed to determine the association of genetic changes and age, gender, and other clinicopathologic factors.

    Results: One hundred twenty-one of the 190 conventional papillary thyroid carcinoma samples (63.7%) had at least one genetic alteration, and 27 of the samples (14.2%) had more than one alteration. In the follicular variant of papillary thyroid carcinomas, 13 of the 27 samples (48.1%) had at least one genetic alteration and three of the 27 samples (11.1%) had more than one. The presence of multiple genetic alterations was associated with younger age at diagnosis (P=0.034), mean difference of 8 y earlier. We found no significant association with the number or type of genetic alterations present by gender, tumor size, extent of tumor differentiation, multicentricity, lymph node metastasis, distant metastases, TNM stage, and the AMES risk group. The association of multiple genetic alterations and younger age were independent of tumor size, lymph node or distant metastasis, TNM stage, or AMES risk group.

    Conclusions: Multiple genetic alterations are more common in younger patients with papillary thyroid cancer, but there is no difference in the type or number of genetic alterations by gender. Our findings suggest that multiple genetic alterations in thyroid cancer may be associated with earlier disease initiation and or progression.

    The Journal of surgical research 2010;160;2;179-83

  • Coexisting NRAS and BRAF mutations in primary familial melanomas with specific CDKN2A germline alterations.

    Jovanovic B, Egyhazi S, Eskandarpour M, Ghiorzo P, Palmer JM, Bianchi Scarrà G, Hayward NK and Hansson J

    Funded by: NCI NIH HHS: R01 CA088363

    The Journal of investigative dermatology 2010;130;2;618-20

  • Mutual exclusion of ASXL1 and NPM1 mutations in a series of acute myeloid leukemias.

    Carbuccia N, Trouplin V, Gelsi-Boyer V, Murati A, Rocquain J, Adélaïde J, Olschwang S, Xerri L, Vey N, Chaffanet M, Birnbaum D and Mozziconacci MJ

    Leukemia 2010;24;2;469-73

  • c-Kit mutants require hypoxia-inducible factor 1alpha to transform melanocytes.

    Monsel G, Ortonne N, Bagot M, Bensussan A and Dumaz N

    INSERM U976, Hôpital Saint Louis, Paris, France.

    Many studies have highlighted the critical role of c-Kit in normal melanocyte development but its role in melanoma development remains unclear. Although c-Kit expression is often lost during melanoma progression, a subset of melanoma has been found to overexpress c-Kit and mutations activating c-Kit have recently been identified in some acral and mucosal melanoma. To address the role of these c-Kit mutants in the transformation of melanocytes, we characterized the physiological responses of melanocytes expressing the most frequent c-Kit mutants found in melanoma (K642E and L576P) and a novel mutant we identified in an acral melanoma. We analysed signaling pathways activated downstream of c-Kit and showed that all three mutants led to a strong activation of the phosphatidyl-inositol-3 kinase (PI3K) pathway but only weak activation of the Ras/Raf/Mek/Erk pathway, which was not sufficient to promote uncontrolled melanocyte proliferation and transformation. However, in hypoxic conditions or coexpressed with a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha), c-Kit mutants activate the Ras/Raf/Mek/Erk pathway, stimulate proliferation and transform melanocytes. Proliferation of melanocytes transformed by these mutants was specifically inhibited by imatinib. These results show for the first time that melanocytes require a specific epigenetic environment to be transformed by c-Kit mutants and highlight a distinct molecular mechanism of melanocyte transformation.

    Oncogene 2010;29;2;227-36

  • Oncogenic NRAS cooperates with p53 loss to generate melanoma in zebrafish.

    Dovey M, White RM and Zon LI

    Stem Cell Program and Hematology/Oncology, Children's Hospital, Boston, Massachusetts, USA.

    NRAS mutations are a common oncogenic event in skin cancer, occurring frequently in congenital nevi and malignant melanoma. To study the role of NRAS in zebrafish, a transgenic approach was applied to generate fish that express human oncogenic NRAS(Q61K) under the control of the melanocyte-restricted mitfa promoter. By screening the progeny of the injected animals, two strains stably expressing the NRAS transgene were identified: Tg(mitfa:EGFP:NRAS(Q61K))(1) and Tg(mitfa:EGFP:NRAS(Q61K))(2). Stable expression of this transgene results in hyperpigmented fish displaying a complete ablation of the normal pigment pattern. Although oncogenic NRAS expression alone was found to be insufficient to promote tumor formation, loss of functional p53 was found to collaborate with NRAS expression in the genesis of melanoma. The tumors derived from these animals are variably pigmented and closely resemble human melanoma. Underscoring the pathological similarities between these tumors and human disease and suggesting that common pathways are similar in these models and human disease, gene set enrichment analysis performed on microarray data found that the upregulated genes from zebrafish melanomas are highly enriched in human tumor samples. This work characterizes two zebrafish melanoma models that will be useful tools for the study of melanoma pathogenesis.

    Funded by: NIDDK NIH HHS: R01 DK053298, R01 DK053298-08, R01-DK53298-08.

    Zebrafish 2009;6;4;397-404

  • Effect on the Ras/Raf signaling pathway of post-translational modifications of neurofibromin: in silico study of protein modification responsible for regulatory pathways.

    Nasir-ud-Din, Kaleem A, Ahmad I, Walker-Nasir E, Hoessli DC and Shakoori AR

    Institute of Molecular Sciences and Bioinformatics, Lahore, Pakistan. professor_nasir@yahoo.com

    Mapping and chemical characterization of post-translational modifications (PTMs) in proteins are critical to understand the regulatory mechanisms involving modified proteins and their role in disease. Neurofibromatosis type 1 (NF-1) is an autosomal dominantly inherited disorder, where NF1 mutations usually result in a reduced level of the tumor suppressor protein, neurofibromin (NF). NF is a multifunctional cytoplasmic protein that regulates microtubule dynamics and participates in several signaling pathways, particularly the RAS signaling pathway. NF is a Ras GTPase-activating protein (GAP) that prevents oncogenesis by converting GTP-Ras to GDP-Ras. This function of NF is regulated by phosphorylation. Interplay of phosphorylation with O-GlcNAc modification on the same or vicinal Ser/Thr residues, the Yin Yang sites, is well known in cytoplasmic and nuclear proteins. The dynamic aspects of PTMs and their interplay being difficult to follow in vivo, we undertook this in silico work to predict and define the possible role of Yin Yang sites in NF-1. Interplay of phosphorylation and O-GlcNAc modification is proposed as a mechanism controlling the Ras signaling pathway.

    Journal of cellular biochemistry 2009;108;4;816-24

  • RAS and CSF3R mutations in severe congenital neutropenia.

    Germeshausen M, Kratz CP, Ballmaier M and Welte K

    Funded by: NIAID NIH HHS: R24 AI049393

    Blood 2009;114;16;3504-5

  • RAS mutations affect tumor necrosis factor-induced apoptosis in colon carcinoma cells via ERK-modulatory negative and positive feedback circuits along with non-ERK pathway effects.

    Kreeger PK, Mandhana R, Alford SK, Haigis KM and Lauffenburger DA

    Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts02139, USA.

    More than 40% of colon cancers have a mutation in K-RAS or N-RAS, GTPases that operate as central hubs for multiple key signaling pathways within the cell. Utilizing an isogenic panel of colon carcinoma cells with K-RAS or N-RAS variations, we observed differences in tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. When the dynamics of phosphorylated ERK response to TNFalpha were examined, K-RAS mutant cells showed lower activation whereas N-RAS mutant cells exhibited prolonged duration. These divergent trends were partially explained by differential induction of two ERK-modulatory circuits: negative feedback mediated by dual-specificity phosphatase 5 and positive feedback by autocrine transforming growth factor-alpha. Moreover, in the various RAS mutant colon carcinoma lines, the transforming growth factor-alpha autocrine loop differentially elicited a further downstream chemokine (CXCL1/CXCL8) autocrine loop, with the two loops having opposite effects on apoptosis. Although the apoptotic responses of the RAS mutant panel to TNFalpha treatment showed significant dependence on the respective phosphorylated ERK dynamics, successful prediction across the various cell lines required contextual information concerning additional pathways including IKK and p38. A quantitative computational model based on weighted linear combinations of these pathway activities successfully predicted not only the spectrum of cell death responses but also the corresponding chemokine production responses. Our findings indicate that diverse RAS mutations yield differential cell behavioral responses to inflammatory cytokine exposure by means of (a) differential effects on ERK activity via multiple feedback circuit mechanisms, and (b) differential effects on other key signaling pathways contextually modulating ERK-related dependence.

    Funded by: NCI NIH HHS: U54 CA112967, U54 CA112967-05, U54-CA112967; NIGMS NIH HHS: P50 GM068762, P50-GM68762

    Cancer research 2009;69;20;8191-9

  • Characterization of candidate gene copy number alterations in the 11q13 region along with BRAF and NRAS mutations in human melanoma.

    Lázár V, Ecsedi S, Szöllosi AG, Tóth R, Vízkeleti L, Rákosy Z, Bégány A, Adány R and Balázs M

    Department of Preventive Medicine, Faculty of Public Health, Medical and Health Science Center, University of Debrecen, Kassai str. 26, Debrecen, Hungary.

    Amplification of the 11q13 chromosomal region is a common event in primary melanomas. Several candidate genes are localized at this sequence; however, their role in melanoma has not been clearly defined. The aim of this study was to develop an accurate method for determining the amplification pattern of six candidate genes that map to this amplicon core and to elucidate the possible relationship between BRAF, NRAS mutations and CCND1 copy number alterations, all of which are key components of the MAP kinase pathway. Characterization of gene copy numbers was performed by quantitative PCR and, as an alternative method, fluorescence in situ hybridization was used to define the CCND1 amplification pattern at the single cell level. Samples with amplified CCND1 (32%) were further analyzed for copy number alterations for the TAOS1, FGF3, FGF19, FGF4 and EMS1 genes. Co-amplification of the CCND1 and TAOS1 was present in 15% of tumors and was more frequent in ulcerated lesions (P=0.017). Furthermore, 56% of primary melanomas had either BRAF or NRAS mutations, but these two mutations were not present in any of the lesions analyzed. Of these cases, 34% also had CCND1 amplification. There was a significant relationship between NRAS activating mutations and UV exposure (P=0.005). We did not find correlations between CCND1 gene amplification status and any of the patients' clinicopathological parameters. However, CCND1 amplification simultaneously with either BRAF or NRAS activation mutations was observed mainly in primary tumors with ulcerated surfaces (P=0.028). We assume that co-amplification of these candidate genes in the 11q13 region or CCND1 gene alterations along with either BRAF or NRAS mutations might be more important for prognosis than the presence of these alterations alone.

    Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2009;22;10;1367-78

  • Comprehensive analysis of cooperative gene mutations between class I and class II in de novo acute myeloid leukemia.

    Ishikawa Y, Kiyoi H, Tsujimura A, Miyawaki S, Miyazaki Y, Kuriyama K, Tomonaga M and Naoe T

    Department of Infectious Diseases, Nagoya University Graduate School of Medicine, Nagoya, Japan.

    Acute myeloid leukemia (AML) has been thought to be the consequence of two broad complementation classes of mutations: class I and class II. However, overlap-mutations between them or within the same class and the position of TP53 mutation are not fully analyzed. We comprehensively analyzed the FLT3, cKIT, N-RAS, C/EBPA, AML1, MLL, NPM1, and TP53 mutations in 144 newly diagnosed de novo AML. We found 103 of 165 identified mutations were overlapped with other mutations, and most overlap-mutations consisted of class I and class II mutations. Although overlap-mutations within the same class were found in seven patients, five of them additionally had the other class mutation. These results suggest that most overlap-mutations within the same class might be the consequence of acquiring an additional mutation after the completion both of class I and class II mutations. However, mutated genes overlapped with the same class were limited in N-RAS, TP53, MLL-PTD, and NPM1, suggesting the possibility that these irregular overlap-mutations might cooperatively participate in the development of AML. Notably, TP53 mutation was overlapped with both class I and class II mutations, and associated with morphologic multilineage dysplasia and complex karyotype. The genotype consisting of complex karyotype and TP53 mutation was an unfavorable prognostic factor in entire AML patients, indicating this genotype generates a disease entity in de novo AML. These results collectively suggest that TP53 mutation might be a functionally distinguishable class of mutation.

    European journal of haematology 2009;83;2;90-8

  • [Analysis of changes induced by oncogene N-RAS expression in pattern and distribution of pseudopodial activity of fibroblasts].

    Lomakina ME and Aleksandrova AIu

    It is not known which morphological properties of fibroblasts induced by malignant transformation modulate their migration pattern. We studied the changes in the distribution and dynamics of the leading edge of 10(3) mouse fibroblasts after their transformation by oncogene N-RAS(asp13) and analyzed the changes in the pattern of cell migration. Transformation proved to increase the leading edge proportion and to considerably redistribute pseudopodial activity along the cell edge. As the result of transformation, small pseudopodia are formed in the stable lateral regions of the cell edge typical of normal fibroblasts, i.e., the lateral edge is no more truly stable. In addition, pseudopodial activity of the leading edge in transformed fibroblasts proved higher compared to normal ones. It is necessary to notice, the leading edge activity is equally high immediately after induction in both normal and transformed fibroblasts; although, it is suppressed with time in normal cells but not in transformed ones where it remains steadily high. These properties promote the random component of malignant cell motility and modify the cell migration pattern after transformation.

    Ontogenez 2009;40;4;282-93

  • Mutational profile of advanced primary and metastatic radioactive iodine-refractory thyroid cancers reveals distinct pathogenetic roles for BRAF, PIK3CA, and AKT1.

    Ricarte-Filho JC, Ryder M, Chitale DA, Rivera M, Heguy A, Ladanyi M, Janakiraman M, Solit D, Knauf JA, Tuttle RM, Ghossein RA and Fagin JA

    Human Oncology and Pathogenesis Program and Departments of Medicine and Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

    Patients with poorly differentiated thyroid cancers (PDTC), anaplastic thyroid cancers (ATC), and radioactive iodine-refractory (RAIR) differentiated thyroid cancers have a high mortality, particularly if positive on [(18)F]fluorodeoxyglucose (FDG)-positron emission tomography (PET). To obtain comprehensive genetic information on advanced thyroid cancers, we designed an assay panel for mass spectrometry genotyping encompassing the most significant oncogenes in this disease: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes were surveyed in 31 cell lines, 52 primary tumors (34 PDTC and 18 ATC), and 55 RAIR, FDG-PET-positive recurrences and metastases (nodal and distant) from 42 patients. RAS mutations were more prevalent than BRAF (44 versus 12%; P = 0.002) in primary PDTC, whereas BRAF was more common than RAS (39 versus 13%; P = 0.04) in PET-positive metastatic PDTC. BRAF mutations were highly prevalent in ATC (44%) and in metastatic tumors from RAIR PTC patients (95%). Among patients with multiple metastases, 9 of 10 showed between-sample concordance for BRAF or RAS mutations. By contrast, 5 of 6 patients were discordant for mutations of PIK3CA or AKT1. AKT1_G49A was found in 9 specimens, exclusively in metastases. This is the first documentation of AKT1 mutation in thyroid cancer. Thus, RAIR, FDG-PET-positive metastases are enriched for BRAF mutations. If BRAF is mutated in the primary, it is likely that the metastases will harbor the defect. By contrast, absence of PIK3CA/AKT1 mutations in one specimen may not reflect the status at other sites because these mutations arise during progression, an important consideration for therapies directed at phosphoinositide 3-kinase effectors.

    Funded by: NCI NIH HHS: CA50706, P30 CA008748, R01 CA050706, R01 CA050706-20, R01 CA072597

    Cancer research 2009;69;11;4885-93

  • Frequencies of NRAS and BRAF mutations increase from the radial to the vertical growth phase in cutaneous melanoma.

    Greene VR, Johnson MM, Grimm EA and Ellerhorst JA

    The Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.

    A lack of consensus exists with regards to the relative rates of NRAS and BRAF mutations in the radial (RGP) and vertical (VGP) growth phases of individual melanoma tumors. This study was conducted to test the hypothesis that mutations are acquired with progression from RGP to VGP. Using laser capture microdissection, pure tumor DNA was obtained from 15 in situ melanomas, and from the RGP and VGP of 29 invasive tumors. NRAS exon 2 and BRAF exon 15 DNA were amplified by PCR and sequenced. Mutations were present in 6 of 15 in situ melanomas (40%). Of 29 invasive tumors, 16 exhibited RGP mutations (55.2%); 22 showed VGP mutations (75.9%). Paired RGP/VGP mutation analysis revealed a trend toward discordance in the distribution of mutations, favoring VGP localization (P=0.07). Of 15 samples, 12 with mutations in both phases had an increased proportion of mutated DNA in the VGP, measured on DNA chromatograms (P=0.08). Limitations of this study include a relatively small sample cohort selected for technical reasons from a larger population, presenting the risk of selection bias. These concerns notwithstanding our findings support the hypothesis that NRAS and BRAF mutations increase with tumor progression from superficial to invasive disease. JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://network.nature.com/group/jidclub.

    Funded by: NCI NIH HHS: CA16672, P30 CA016672, P50 CA093459, P50 CA093459-05

    The Journal of investigative dermatology 2009;129;6;1483-8

  • Mutational analysis of the BRAF, RAS and EGFR genes in human adrenocortical carcinomas.

    Kotoula V, Sozopoulos E, Litsiou H, Fanourakis G, Koletsa T, Voutsinas G, Tseleni-Balafouta S, Mitsiades CS, Wellmann A and Mitsiades N

    Department of Pathology, School of Medicine, Aristotle University of Thessaloniki, University Campus, Thessaloniki 54006, Greece. vkotoula@auth.gr

    The serine/threonine kinase B-Raf plays a key role in the Ras/Raf/MEK/ERK pathway that relays extracellular signals for cell proliferation and survival. Several types of human malignancies harbor activating BRAF mutations, most frequently a V600E substitution. The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase (TK) receptor that mediates proliferation and survival signaling, is expressed in a wide variety of normal and neoplastic tissues. EGFR inhibitors have produced objective responses in patients with non-small cell lung carcinomas harboring activating EGFR TK domain somatic mutations. We evaluated the presence of mutations in BRAF (exons 11 and 15), KRAS (exons 1 and 2), NRAS (exons 1 and 2), and EGFR (exons 18-21) in adrenal carcinomas (35 tumor specimens and two cell lines) by DNA sequencing. BRAF mutations were found in two carcinomas (5.7%). Four carcinomas (11.4%) carried EGFR TK domain mutations. One specimen carried a KRAS mutation, and another carried two NRAS mutations. No mutations were found in the two adrenocortical cell lines. BRAF- and EGFR-mutant tumor specimens exhibited stronger immunostaining for the phosphorylated forms of the MEK and ERK kinases than their wild-type counterparts. EGFR-mutant carcinomas exhibited increased phosphorylation of EGFR (Tyr 992) compared with wild-type carcinomas. We conclude that BRAF, RAS, and EGFR mutations occur in a subset of human adrenocortical carcinomas. Inhibitors of the Ras/Raf/MEK/ERK and EGFR pathways represent candidate targeted therapies for future clinical trials in carefully selected patients with adrenocortical carcinomas harboring respective activating mutations.

    Endocrine-related cancer 2009;16;2;565-72

  • Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.

    Jaiswal BS, Janakiraman V, Kljavin NM, Eastham-Anderson J, Cupp JE, Liang Y, Davis DP, Hoeflich KP and Seshagiri S

    Department of Molecular Biology, Genentech Inc., South San Francisco, California, United States of America.

    Background: Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors.

    In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRAS(G13D) mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRAS(Q61L) or NRAS(Q61K) mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy.

    Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells.

    PloS one 2009;4;5;e5717

  • Activation of RAS family genes in urothelial carcinoma.

    Boulalas I, Zaravinos A, Karyotis I, Delakas D and Spandidos DA

    Department of Urology, Asklipieio General Hospital, Voula, Athens, Greece.

    Purpose: Bladder cancer is the fifth most common malignancy in men in Western society. We determined RAS codon 12 and 13 point mutations and evaluated mRNA expression levels in transitional cell carcinoma cases.

    Samples from 30 human bladder cancers and 30 normal tissues were analyzed by polymerase chain reaction/restriction fragment length polymorphism and direct sequencing to determine the occurrence of mutations in codons 12 and 13 of RAS family genes. Moreover, we used real-time reverse transcriptase-polymerase chain reaction to evaluate the expression profile of RAS genes in bladder cancer specimens compared to that in adjacent normal tissues.

    Results: Overall H-RAS mutations in codon 12 were observed in 9 tumor samples (30%). Two of the 9 patients (22%) had invasive bladder cancer and 7 (77%) had noninvasive bladder cancer. One H-RAS mutation (11%) was homozygous and the remaining 89% were heterozygous. All samples were WT for K and N-RAS oncogenes. Moreover, 23 of 30 samples (77%) showed over expression in at least 1 RAS family gene compared to adjacent normal tissue. K and N-RAS had the highest levels of over expression in bladder cancer specimens (50%), whereas 27% of transitional cell carcinomas demonstrated H-RAS over expression relative to paired normal tissues.

    Conclusions: Our results underline the importance of H-RAS activation in human bladder cancer by codon 12 mutations. Moreover, they provide evidence that increased expression of all 3 RAS genes is a common event in bladder cancer that is associated with disease development.

    The Journal of urology 2009;181;5;2312-9

  • Esophageal melanomas harbor frequent NRAS mutations unlike melanomas of other mucosal sites.

    Sekine S, Nakanishi Y, Ogawa R, Kouda S and Kanai Y

    Pathology Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo, Japan.

    Mucosal melanomas have genetic alterations distinct from those in cutaneous melanomas. For example, NRAS- and BRAF-activating mutations occur frequently in cutaneous melanomas, but not in mucosal melanomas. We examined 16 esophageal melanomas for genetic alterations in NRAS, BRAF, and KIT to determine whether they exhibit genetic features common to melanomas arising from other mucosal sites. A sequencing analysis identified NRAS mutations in six cases; notably, four of these mutations were located in exon 1, an uncommon mutation site in cutaneous and other mucosal melanomas. BRAF and KIT mutations were found in one case each. Immunohistochemistry showed KIT expression in four cases, including the tumor with a KIT mutation and two other intramucosal tumors. The low frequency of BRAF mutations and the presence of a KIT mutation-positive case are findings similar to those of mucosal melanomas of other sites, but the prevalence of NRAS mutations was even higher than that of cutaneous melanomas. The present study implies that esophageal melanomas have genetic alterations unique from those observed in other mucosal melanomas.

    Virchows Archiv : an international journal of pathology 2009;454;5;513-7

  • Genetic mutations associated with cigarette smoking in pancreatic cancer.

    Blackford A, Parmigiani G, Kensler TW, Wolfgang C, Jones S, Zhang X, Parsons DW, Lin JC, Leary RJ, Eshleman JR, Goggins M, Jaffee EM, Iacobuzio-Donahue CA, Maitra A, Klein A, Cameron JL, Olino K, Schulick R, Winter J, Vogelstein B, Velculescu VE, Kinzler KW and Hruban RH

    Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21231, USA.

    Cigarette smoking doubles the risk of pancreatic cancer, and smoking accounts for 20% to 25% of pancreatic cancers. The recent sequencing of the pancreatic cancer genome provides an unprecedented opportunity to identify mutational patterns associated with smoking. We previously sequenced >750 million bp DNA from 23,219 transcripts in 24 adenocarcinomas of the pancreas (discovery screen). In this previous study, the 39 genes that were mutated more than once in the discovery screen were sequenced in an additional 90 adenocarcinomas of the pancreas (validation screen). Here, we compared the somatic mutations in the cancers obtained from individuals who ever smoked cigarettes (n = 64) to the somatic mutations in the cancers obtained from individuals who never smoked cigarettes (n = 50). When adjusted for age and gender, analyses of the discovery screen revealed significantly more nonsynonymous mutations in the carcinomas obtained from ever smokers (mean, 53.1 mutations per tumor; SD, 27.9) than in the carcinomas obtained from never smokers (mean, 38.5; SD, 11.1; P = 0.04). The difference between smokers and nonsmokers was not driven by mutations in known driver genes in pancreatic cancer (KRAS, TP53, CDKN2A/p16, and SMAD4), but instead was predominantly observed in genes mutated at lower frequency. No differences were observed in mutations in carcinomas from the head versus tail of the gland. Pancreatic carcinomas from cigarette smokers harbor more mutations than do carcinomas from never smokers. The types and patterns of these mutations provide insight into the mechanisms by which cigarette smoking causes pancreatic cancer.

    Funded by: NCI NIH HHS: CA62924, P50 CA062924, P50 CA062924-08S30011, P50 CA062924-090011, P50 CA062924-160011, R01 CA039416, R37 CA043460

    Cancer research 2009;69;8;3681-8

  • Correlation of clinical features with the mutational status of GM-CSF signaling pathway-related genes in juvenile myelomonocytic leukemia.

    Yoshida N, Yagasaki H, Xu Y, Matsuda K, Yoshimi A, Takahashi Y, Hama A, Nishio N, Muramatsu H, Watanabe N, Matsumoto K, Kato K, Ueyama J, Inada H, Goto H, Yabe M, Kudo K, Mimaya J, Kikuchi A, Manabe A, Koike K and Kojima S

    Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.

    Mutations in RAS, neurofibromatosis type 1 (NF1), and PTPN11, constituents of the granulocyte-macrophage colony-stimulating factor signaling pathway, have been recognized in patients with juvenile myelomonocytic leukemia (JMML). We assessed 71 children with JMML for NRAS, KRAS, and PTPN11 mutations and evaluated their clinical significance. Of the 71 patients, three had been clinically diagnosed with neurofibromatosis type 1, and PTPN11 and NRAS/KRAS mutations were found in 32 (45%) and 13 (18%) patients, respectively. No simultaneous aberrations were found. Compared with patients with RAS mutation or without any aberrations, patients with PTPN11 mutation were significantly older at diagnosis and had higher fetal Hb levels, both of which have been recognized as poor prognostic factors. As was expected, overall survival was lower for patients with the PTPN11 mutation than for those without (25 versus 64%; p = 0.0029). In an analysis of 48 patients who received hematopoietic stem cell transplantation, PTPN11 mutations were also associated with poor prognosis for survival. Mutation in PTPN11 was the only unfavorable factor for relapse after hematopoietic stem cell transplantation (p = 0.001). All patients who died after relapse had PTPN11 mutation. These results suggest that JMML with PTPN11 mutation might be a distinct subgroup with specific clinical characteristics and poor outcome.

    Pediatric research 2009;65;3;334-40

  • Active N-Ras and B-Raf inhibit anoikis by downregulating Bim expression in melanocytic cells.

    Goldstein NB, Johannes WU, Gadeliya AV, Green MR, Fujita M, Norris DA and Shellman YG

    Department of Dermatology, School of Medicine, University of Colorado Denver, Aurora, Colorado 80010, USA.

    B-Raf and N-Ras proteins are often activated in melanoma, yet their roles in producing inherent survival signals are not fully understood. In this study, we investigated how N-RAS(Q61K) and B-RAF(V600E) contribute to melanoma's resistance to apoptosis induced by detachment from the extracellular matrix (anoikis). We found that expression of constitutively active N-RAS(Q61K) and B-RAF(V600E) downregulated the proapoptotic Bim protein in an immortalized melanocyte cell line. Bim is one of the main proapoptotic mediators of anoikis. Western blot analysis showed that detachment increased Bim expression in melanocytes, and Annexin V staining indicated that detachment induced cell death significantly in melanocytes. Blocking Bim expression by using RNAi vectors or by expressing N-RAS(Q61K) significantly inhibited anoikis in melanocytes. In summary, this report indicates that N-RAS(Q61K) and B-RAF(V600E) contribute to melanoma's resistance to apoptosis in part by downregulating Bim expression, suggesting that Bim is a possible treatment target for overriding melanoma's inherent defenses against cell death.

    Funded by: NIAMS NIH HHS: R01AR26427-18

    The Journal of investigative dermatology 2009;129;2;432-7

  • RAS and RAF mutations in banal melanocytic aggregates contiguous with primary cutaneous melanoma: clues to melanomagenesis.

    Dadzie OE, Yang S, Emley A, Keady M, Bhawan J and Mahalingam M

    Dermatopathology Section, Department of Dermatology, Boston University School of Medicine, 609 Albany Street, J-301, Boston, MA 02118, USA.

    Background: Distinguishing banal melanocytic aggregates contiguous with malignant melanoma can be a histological challenge but is essential because of the potential for a spurious Breslow measurement.

    Objectives: Our aim was to ascertain whether the histological distinction between the two relates to differences in the prevalence of mutations in genes significant in melanomagenesis.

    Methods: Mutations in BRAF codon 600, NRAS1 codons 12/13, NRAS2 codons 60/61 and KRAS codons 12/13 were ascertained in 18 cases of primary cutaneous malignant melanoma contiguous with banal melanocytic aggregates using laser capture microdissection.

    Results: Overall, 12 of 18 cases (67%) exhibited a mutation in at least one gene. BRAF V600E appeared to be the most commonly mutated gene in both the melanocytic aggregate (seven of 18, 39%) and the melanoma (four of 18, 22%). Both populations demonstrated a similar BRAF genomic profile in 11 of 18 cases (61%) (two BRAF V600E, nine BRAF-WT), a similar KRAS genomic profile in 14 of 18 cases (78%) (one KRAS G12V, 13 KRAS-WT) and a similar NRAS2 genomic profile in 14 of 18 cases (all WT). Of interest, we noted a relatively high prevalence of KRAS mutations (five of 18, 28%). The frequency of KRAS mutations in the melanocytic aggregate (five of 18, 28%) was second to BRAF V600E, while in melanoma, the frequency was also second to BRAF V600E but equalled that of NRAS2 (1 of 18, 6%). No NRAS1 mutations were observed. BRAF and RAS mutations appeared to be mutually exclusive with only three of 18 cases (17%) demonstrating a mutation in both genes (melanocytic aggregate only).

    Conclusions: Our findings hint towards the interpretation of banal melanocytic aggregates serving as precursor lesions.

    The British journal of dermatology 2009;160;2;368-75

  • Oncogenic NRAS has multiple effects on the malignant phenotype of human melanoma cells cultured in vitro.

    Eskandarpour M, Huang F, Reeves KA, Clark E and Hansson J

    Department of Oncology-Pathology, Karolinska Institutet, Cancer Center Karolinska, Karolinska University Hospital Solna, Stockholm, Sweden. malihe.eskandarpour@ki.se

    Activating mutations in the NRAS gene, which occur predominantly in codon 61 (Q61R, Q61K) are among the most common genetic events in malignant melanoma. NRAS protein with oncogenic codon 61 mutations may therefore be good therapeutic targets. In the present study, we used gene expression profiling as a method for global characterization of gene expression alterations that resulted from treatment of melanoma cells with siRNA specifically targeting NRAS(Q61R). Sixteen probe sets representing 15 unique genes were identified whose expression was significantly altered by siRNA against NRAS(Q61R) in 2 melanoma cell lines. The genes with altered expression are involved in several functions, including modulation of cell growth, invasion and migration. The results suggest that downregulation of cyclin E2 and cyclin D1 and also upregulation of the negative cell-cycle regulator HBP1 in NRAS(Q61R) knockdown cells contribute to the inhibition of cell proliferation. Furthermore, suppression of oncogenic NRAS results in reduced migration and invasion, which is accompanied by downregulation of EphA2 (a receptor tyrosine kinase), uPAR (urokinase receptor) and cytoskeleton proteins such as leupaxin, paxillin and vinculin. These studies support the concept that suppression of oncogenic NRAS by siRNA can induce growth arrest and inhibit invasion of human melanoma cells by modulating the levels of these gene products.

    International journal of cancer 2009;124;1;16-26

  • Stimulation of human mast cells by activated T cells leads to N-Ras activation through Ras guanine nucleotide releasing protein 1.

    Shefler I, Mekori YA and Mor A

    The Journal of allergy and clinical immunology 2008;122;6;1222-5

  • KIT gene mutations and copy number in melanoma subtypes.

    Beadling C, Jacobson-Dunlop E, Hodi FS, Le C, Warrick A, Patterson J, Town A, Harlow A, Cruz F, Azar S, Rubin BP, Muller S, West R, Heinrich MC and Corless CL

    Oregon Cancer Institute, Oregon Health & Science University, Portland, Oregon 97239, USA.

    Purpose: We recently identified a KIT exon 11 mutation in an anorectal melanoma of a patient who had an excellent response to treatment with imatinib. To determine the frequency of KIT mutations across melanoma subtypes, we surveyed a large series of tumors.

    One hundred eighty-nine melanomas were screened for mutations in KIT exons 11, 13, and 17. KIT copy number was assessed by quantitative PCR. A subset of cases was evaluated for BRAF and NRAS mutations. Immunohistochemistry was done to assess KIT (CD117) expression.

    Results: KIT mutations were detected in 23% (3 of 13) of acral melanomas, 15.6% (7 of 45) of mucosal melanomas, 7.7% (1 of 13) of conjunctival melanomas, 1.7% (1 of 58) of cutaneous melanomas, and 0% (0 of 60) of choroidal melanomas. Almost all the KIT mutations were of the type predicted to be imatinib sensitive. There was no overlap with NRAS mutations (11.1% of acral and 24.3% of mucosal tumors) or with BRAF mutations (absent in mucosal tumors). Increased KIT copy number was detected in 27.3% (3 of 11) of acral and 26.3% (10 of 38) of mucosal melanomas, but was less common among cutaneous (6.7%; 3 of 45), conjunctival (7.1%; 1 of 14), and choroidal melanomas (0 of 28). CD117 expression, present in 39% of 105 tumors representing all melanoma types, did not correlate with either KIT mutation status or KIT copy number.

    Conclusions: Our findings confirm that KIT mutations are most common in acral and mucosal melanomas but do not necessarily correlate with KIT copy number or CD117 expression. Screening for KIT mutations may open up new treatment options for melanoma patients.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2008;14;21;6821-8

  • Patients with acute myeloid leukemia and RAS mutations benefit most from postremission high-dose cytarabine: a Cancer and Leukemia Group B study.

    Neubauer A, Maharry K, Mrózek K, Thiede C, Marcucci G, Paschka P, Mayer RJ, Larson RA, Liu ET and Bloomfield CD

    Department for Hematology, Oncology and Immunology, Philipps University, Marburg, Germany.

    Purpose: RAS mutations occur in 12% to 27% of patients with acute myeloid leukemia (AML) and enhance sensitivity to cytarabine in vitro. We examined whether RAS mutations impact response to cytarabine in vivo.

    One hundred eighty-five patients with AML achieving complete remission on Cancer and Leukemia Group B study 8525 and randomly assigned to one of three doses of cytarabine postremission were screened for RAS mutations. We assessed the impact of cytarabine dose on cumulative incidence of relapse (CIR) of patients with (mutRAS) and without (wild-type; wtRAS) RAS mutations.

    Results: Thirty-four patients (18%) had RAS mutations. With 12.9 years median follow-up, the 10-year CIR was similar for mutRAS and wtRAS patients (65% v 73%; P = .31). However, mutRAS patients receiving high-dose cytarabine consolidation (HDAC; 3 g/m(2) every 12 hours on days 1, 3, and 5 or 400 mg/m(2)/d x 5 days) had the lowest 10-year CIR, 45%, compared with 68% for wtRAS patients receiving HDAC and 80% and 100%, respectively, for wtRAS and mutRAS patients receiving low-dose cytarabine (LDAC; 100 mg/m(2)/d x 5 days; overall comparison, P < .001). Multivariable analysis revealed an interaction of cytarabine dose and RAS status (P = .06). After adjusting for this interaction and cytogenetics (core binding factor [CBF] AML v non-CBF AML), wtRAS patients receiving HDAC had lower relapse risk than wtRAS patients receiving LDAC (hazard ratio [HR] = 0.67; P = .04); however, mutRAS patients receiving HDAC had greater reduction in relapse risk (HR = 0.28; P = .002) compared with mutRAS patients treated with LDAC.

    Conclusion: AML patients carrying mutRAS benefit from higher cytarabine doses more than wtRAS patients. This seems to be the first example of an activating oncogene mutation favorably modifying response to higher drug doses in AML.

    Funded by: NCI NIH HHS: CA101140, CA114725, CA16058, CA31946, CA77658

    Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2008;26;28;4603-9

  • Novel V600E BRAF mutations in imatinib-naive and imatinib-resistant gastrointestinal stromal tumors.

    Agaram NP, Wong GC, Guo T, Maki RG, Singer S, Dematteo RP, Besmer P and Antonescu CR

    Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.

    BRAF and NRAS are commonly mutated in cancer and represent the most frequent genetic events in malignant melanoma. More recently, a subset of melanomas was shown to overexpress KIT and harbor KIT mutations. Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, about 10% of the cases lack mutations in these genes. It is our hypothesis following the melanoma model that mutations in BRAF or NRAS may play a role in wild-type GIST pathogenesis. Alterations in RAS/MEK/ERK pathway may also be involved in development of imatinib resistance in GIST, particularly in tumors lacking secondary KIT or PDGFRA mutations. Imatinib-naive wild-type GISTs from 61 patients, including 15 children and 28 imatinib-resistant tumors without secondary KIT mutations were analyzed. Screening for hot spots mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3) was performed. A BRAF exon 15 V600E was identified in 3 of 61 GIST patients, who shared similar clinical features, being 49- to 55-years-old females and having their tumors located in the small bowel. The tumors were strongly KIT immunoreactive and had a high risk of malignancy. An identical V600E BRAF mutation was also identified in one of 28 imatinib resistant GIST lacking a defined mechanism of drug resistance. In conclusion, we identified a primary BRAF V600E mutations in 7% of adult GIST patients, lacking KIT/PDGFRA mutations. The BRAF-mutated GISTs show predilection for small bowel location and high risk of malignancy. A secondary V600E BRAF mutation could represent an alternative mechanism of imatinib resistance. Kinase inhibitors targeting BRAF may be effective therapeutic options in this molecular GIST subset.

    Funded by: NCI NIH HHS: CA102613, CA102774, P01 CA047179, P01 CA047179-15A29002, P01CA47179, R01 CA102613, R01 CA102774; NIDDK NIH HHS: HL/DK55748, R01 DK055748

    Genes, chromosomes & cancer 2008;47;10;853-9

  • Silencing of N-Ras gene expression using shRNA decreases transformation efficiency and tumor growth in transformed cells induced by anti-BPDE.

    Zhou L, Jiang Y, Tan A, Greenlee AR, Shen Y, Liu L and Yang Q

    Institute for Chemical Carcinogenesis, State Key Laboratory of Respiratory Diseases, Guangzhou Medical College, Guangzhou 510182, China.

    Anti-benzo[a]pyrene-trans-7,8-diol-9,10-epoxide (anti-BPDE) is the most important metabolite of benzo[a]pyrene which is a ubiquitous environmental pollutant, and may cause human cancer, especially of the lung. Ras genes (H, K, and N) are activated in 40% of human tumors and may contribute to carcinogenesis. Here, we used malignant human bronchial epithelial cells transformed by anti-BPDE (16HBE-T) to help characterize possible molecular mechanisms of carcinogenesis. We compared H-, K-, and N-Ras mRNA and protein expression levels in 16HBE-T cells and untransformed control 16HBE cells (16HBE-N), using reverse transcription-PCR (RT-PCR) and Western blotting. We further used short hairpin RNA to silence N-Ras gene expression in 16HBE-T cells to determine the effects of silencing on the cell cycle, transformation efficiency and tumor growth. We observed overexpression of H-, K-, and N-Ras genes at both mRNA and protein levels in 16HBE-T cells, compared with 16HBE-N cells. Silencing of N-Ras in 16HBE-T cells using stable RNA interference increased the proportion of cells in G(0)/G(1) phase, decreased the proportion in S-phase, decreased transformation efficiency, and inhibited tumor growth. Our findings suggest that overexpression of N-Ras gene plays an important role in malignant transformation of 16HBE cells by anti-BPDE. N-Ras gene may be a useful target for gene therapy.

    Toxicological sciences : an official journal of the Society of Toxicology 2008;105;2;286-94

  • Mutation of genes affecting the RAS pathway is common in childhood acute lymphoblastic leukemia.

    Case M, Matheson E, Minto L, Hassan R, Harrison CJ, Bown N, Bailey S, Vormoor J, Hall AG and Irving JA

    Northern Institute for Cancer Research, Newcastle upon Tyne, Tyne and Wear, United Kingdom.

    Deregulation of the RAS-RAF-mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK signaling cascade is often caused by somatic mutations in genes encoding proteins which influence the activity of this pathway and include NRAS, KRAS2, FLT3, PTPN11, and BRAF. We report the first comprehensive mutational screen of key exons of these genes in a large cohort of unselected acute lymphoblastic leukemia (ALL) cases at diagnosis (n = 86) and in a more selected cohort at disease recurrence (n = 47) using the sensitive method of denaturing high-performance liquid chromatography. We show that somatic mutations that deregulate the pathway constitute one of the most common genetic aberrations in childhood ALL (cALL), being found in 35% of diagnostic and 25% of relapse samples. In matched presentation/relapse pairs, mutations predominating at relapse could be shown to be present at very low levels at diagnosis using allele-specific PCR, thus implicating the mutated clone in disease progression. Importantly, in primary samples, we show that mutations are associated with activated ERK and differential cytotoxicity to MEK-ERK inhibitors was shown for some patients. Inhibitors of the pathway, which are currently undergoing clinical trial, may be a novel therapeutic option for cALL, particularly at relapse.

    Cancer research 2008;68;16;6803-9

  • Phospho-ERK staining is a poor indicator of the mutational status of BRAF and NRAS in human melanoma.

    Houben R, Vetter-Kauczok CS, Ortmann S, Rapp UR, Broecker EB and Becker JC

    Klinik für Dermatologie, Venerologie und Allergologie, Julius-Maximilians-Universität, Würzburg, Germany.

    Mutated BRAF and NRAS are suspected to contribute to melanomagenesis by activation of extracellular signal-regulated kinase (ERK). To test this notion, we analyzed the presence of phosphorylated ERK1/2 in 170 melanomas with established NRAS/BRAF mutational status and well-documented clinical follow-up by immunohistochemistry. Several notable observations were obtained: (i) phospho-ERK staining was very heterogeneous within the tumor; (ii) in most cases, ERK was phosphorylated in only a minority of tumor cells; (iii) the percentage of phospho-ERK-positive cells was not correlated with the mutational status of NRAS and/or BRAF; (iv) the Raf kinase inhibitor protein (RKIP) was expressed homogeneously in virtually all melanoma samples not reflecting the inhomogeneity of phospho-ERK; and, finally, (v) neither the portion of phospho-ERK-positive tumor cells nor the RKIP staining intensity showed any correlation to the clinical course of the patients. Furthermore, the ability of BRAF mutant melanoma cells to downregulate mitogen-activated protein kinase activation was shown in melanoma cell lines cultured at high densities or under nonadherent conditions. Our findings suggest that mitogen-activated protein kinase (MAPK) activity is subject to regulation even in BRAF/NRAS mutant melanoma cells and that high MAPK pathway signaling may be important only in distinct subsets of tumor cells.

    The Journal of investigative dermatology 2008;128;8;2003-12

  • Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells.

    Ibiza S, Pérez-Rodríguez A, Ortega A, Martínez-Ruiz A, Barreiro O, García-Domínguez CA, Víctor VM, Esplugues JV, Rojas JM, Sánchez-Madrid F and Serrador JM

    Departamento de Biología Vascular e Inflamación, Centro Nacional de Investigaciones Cardiovasculares, E-28029 Madrid, Spain.

    Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys(118), suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys(118) contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.

    Proceedings of the National Academy of Sciences of the United States of America 2008;105;30;10507-12

  • Identification of a novel subgroup of melanomas with KIT/cyclin-dependent kinase-4 overexpression.

    Smalley KS, Contractor R, Nguyen TK, Xiao M, Edwards R, Muthusamy V, King AJ, Flaherty KT, Bosenberg M, Herlyn M and Nathanson KL

    The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA. K.smalley@mac.com

    Although many melanomas harbor either activating mutations in BRAF or NRAS, there remains a substantial, yet little known, group of tumors without either mutation. Here, we used a genomic strategy to define a novel group of melanoma cell lines with co-overexpression of cyclin-dependent kinase 4 (CDK4) and KIT. Although this subgroup lacked any known KIT mutations, they had high phospho-KIT receptor expression, indicating receptor activity. Quantitative PCR confirmed the existence of a similar KIT/CDK4 subgroup in human melanoma samples. Pharmacologic studies showed the KIT/CDK4-overexpressing subgroup to be resistant to BRAF inhibitors but sensitive to imatinib in both in vitro and in vivo melanoma models. Mechanistically, imatinib treatment led to increased apoptosis and G(1) phase cell cycle arrest associated with the inhibition of phospho-ERK and increased expression of p27(KIP). Other melanoma cell lines, which retained some KIT expression but lacked phospho-KIT, were not sensitive to imatinib, suggesting that KIT expression alone is not predictive of response. We suggest that co-overexpression of KIT/CDK4 is a potential mechanism of oncogenic transformation in some BRAF/NRAS wild-type melanomas. This group of melanomas may be a subpopulation for which imatinib or other KIT inhibitors may constitute optimal therapy.

    Funded by: NCI NIH HHS: CA098101, CA10815, CA117881, CA25874, CA47159, CA76674, CA80999, CA93372, P01 CA025874, P01 CA025874-180004, P01 CA098101, P01 CA098101-01A1, P01 CA114046, P01 CA114046-01A2, P30 CA010815, P30 CA010815-309017, P30 CA016520, P50 CA093372, P50 CA093372-01, R01 CA047159, R01 CA047159-15, R01 CA076674, R01 CA076674-02, R01 CA080999, R01 CA080999-01, R01 CA118871, R01 CA118871-01A1; NIGMS NIH HHS: GM071695, R01 GM071695, R01 GM071695-01A1

    Cancer research 2008;68;14;5743-52

  • Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia.

    Schlenk RF, Döhner K, Krauter J, Fröhling S, Corbacioglu A, Bullinger L, Habdank M, Späth D, Morgan M, Benner A, Schlegelberger B, Heil G, Ganser A, Döhner H and German-Austrian Acute Myeloid Leukemia Study Group

    University Hospital of Ulm, Ulm, Germany.

    Background: Mutations occur in several genes in cytogenetically normal acute myeloid leukemia (AML) cells: the nucleophosmin gene (NPM1), the fms-related tyrosine kinase 3 gene (FLT3), the CCAAT/enhancer binding protein alpha gene (CEPBA), the myeloid-lymphoid or mixed-lineage leukemia gene (MLL), and the neuroblastoma RAS viral oncogene homolog (NRAS). We evaluated the associations of these mutations with clinical outcomes in patients.

    Methods: We compared the mutational status of the NPM1, FLT3, CEBPA, MLL, and NRAS genes in leukemia cells with the clinical outcome in 872 adults younger than 60 years of age with cytogenetically normal AML. Patients had been entered into one of four trials of therapy for AML. In each study, patients with an HLA-matched related donor were assigned to undergo stem-cell transplantation.

    Results: A total of 53% of patients had NPM1 mutations, 31% had FLT3 internal tandem duplications (ITDs), 11% had FLT3 tyrosine kinase-domain mutations, 13% had CEBPA mutations, 7% had MLL partial tandem duplications (PTDs), and 13% had NRAS mutations. The overall complete-remission rate was 77%. The genotype of mutant NPM1 without FLT3-ITD, the mutant CEBPA genotype, and younger age were each significantly associated with complete remission. Of the 663 patients who received postremission therapy, 150 underwent hematopoietic stem-cell transplantation from an HLA-matched related donor. Significant associations were found between the risk of relapse or the risk of death during complete remission and the leukemia genotype of mutant NPM1 without FLT3-ITD (hazard ratio, 0.44; 95% confidence interval [CI], 0.32 to 0.61), the mutant CEBPA genotype (hazard ratio, 0.48; 95% CI, 0.30 to 0.75), and the MLL-PTD genotype (hazard ratio, 1.56; 95% CI, 1.00 to 2.43), as well as receipt of a transplant from an HLA-matched related donor (hazard ratio, 0.60; 95% CI, 0.44 to 0.82). The benefit of the transplant was limited to the subgroup of patients with the prognostically adverse genotype FLT3-ITD or the genotype consisting of wild-type NPM1 and CEBPA without FLT3-ITD.

    Conclusions: Genotypes defined by the mutational status of NPM1, FLT3, CEBPA, and MLL are associated with the outcome of treatment for patients with cytogenetically normal AML.

    The New England journal of medicine 2008;358;18;1909-18

  • BRAF and NRAS mutations in melanoma: potential relationships to clinical response to HSP90 inhibitors.

    Banerji U, Affolter A, Judson I, Marais R and Workman P

    Cancer Research UK Centre for Cancer Therapeutics, Haddow Laboratories, The Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom.

    Oncogenic BRAF and NRAS mutations are frequent in malignant melanoma. BRAF that is activated by the common V600E and other mutations, as well as by upstream NRAS mutations, has been shown to require the molecular chaperone heat shock protein 90 (HSP90) for stabilization and is depleted by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)]. Here, we explore the possible relationship between tumor BRAF and NRAS mutations and clinical response to 17-AAG in six patients with metastatic malignant melanoma who received pharmacologically active doses of 17-AAG as part of a phase I clinical trial. One patient with disease stabilization for 49 months had a (G13D)NRAS mutation and (WT)BRAF. A second patient who had stable disease for 15 months had a (V600E)BRAF mutation and (WT)NRAS. These preliminary results suggest that BRAF and NRAS mutation status should be determined in prospective phase II studies of HSP90 inhibitors in melanoma.

    Funded by: Cancer Research UK

    Molecular cancer therapeutics 2008;7;4;737-9

  • Human cutaneous melanoma; a review of NRAS and BRAF mutation frequencies in relation to histogenetic subclass and body site.

    Platz A, Egyhazi S, Ringborg U and Hansson J

    Department of Oncology-Pathology, Karolinska Institute, Cancer Centre Karolinska, Karolinska University Hospital Solna, Stockholm S-17176, Sweden.

    A majority of cutaneous melanomas show activating mutations in the NRAS or BRAF proto-oncogenes, components of the Ras-Raf-Mek-Erk signal transduction pathway. Consistent data demonstrate the early appearance, in a mutually exclusive manner, of these mutations. The purpose of this paper is to summarize the literature on NRAS and BRAF activating mutations in melanoma tumors with respect to available data on histogenetic classification as well as body site and presumed UV-exposure. Common alterations of the signal transducing network seem to represent molecular hallmarks of cutaneous melanomas and therefore should continue to strongly stimulate design and testing of targeted molecular interventions.

    Molecular oncology 2008;1;4;395-405

  • CDC25A phosphatase: a rate-limiting oncogene that determines genomic stability.

    Ray D and Kiyokawa H

    Department of Molecular Pharmacology and Biological Chemistry, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA.

    CDC25A is a critical regulator of cell cycle progression and checkpoint response. Overexpression of this cyclin-dependent kinase phosphatase occurs often in human cancers. Our recent genetic studies in the mouse indicate that restricting CDC25A can limit tumorigenesis induced by the HER2/neu-RAS oncogenic pathway without compromising normal cell division or viability. These findings offer a sound foundation to justify development of CDC25A inhibitors for antitumor therapy.

    Funded by: NCI NIH HHS: CA112282, R01-CA100204; NICHD NIH HHS: HD38085

    Cancer research 2008;68;5;1251-3

  • Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules.

    Wasylyk C, Zheng H, Castell C, Debussche L, Multon MC and Wasylyk B

    Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Sante et de la Recherche Medicale/Université Louis Pasteur, Illkirch Cedex, France. boh@igbmc.u-strasbg.fr

    Net (Elk-3/SAP-2/Erp) is a transcription factor that is phosphorylated and activated by the Ras-extracellular signal-regulated kinase (Erk) signaling pathway and is involved in wound healing, angiogenesis, and tumor growth. In a cell-based screen for small molecule inhibitors of Ras activation of Net transcriptional activity, we identified a novel pyrazole, XRP44X. XRP44X inhibits fibroblast growth factor 2 (FGF-2)-induced Net phosphorylation by the Ras-Erk signaling upstream from Ras. It also binds to the colchicine-binding site of tubulin, depolymerizes microtubules, stimulates cell membrane blebbing, and affects the morphology of the actin skeleton. Interestingly, Combretastin-A4, which produces similar effects on the cytoskeleton, also inhibits FGF-2 Ras-Net signaling. This differs from other classes of agents that target microtubules, which have either little effect (vincristine) or no effect (docetaxel and nocodazole) on the Ras-Net pathway. XRP44X inhibits various cellular properties, including cell growth, cell cycle progression, and aortal sprouting, similar to other molecules that bind to the tubulin colchicine site. XRP44X has the potentially interesting property of connecting two important pathways involved in cell transformation and may thereby represent an interesting class of molecules that could be developed for cancer treatment.

    Cancer research 2008;68;5;1275-83

  • Familial non-medullary thyroid carcinoma (FNMTC): analysis of fPTC/PRN, NMTC1, MNG1 and TCO susceptibility loci and identification of somatic BRAF and RAS mutations.

    Cavaco BM, Batista PF, Martins C, Banito A, do Rosário F, Limbert E, Sobrinho LG and Leite V

    Centro de Investigação de Patobiologia Molecular (CIPM) and Serviço de Endocrinologia, Instituto Português de Oncologia de Lisboa Francisco Gentil, Lisboa, Portugal. bcavaco@ipolisboa.min-saude.pt

    Linkage analysis has identified four familial non-medullary thyroid carcinoma (FNMTC) susceptibility loci: fPTC/PRN (1p13.2-1q22), NMTC1 (2q21), MNG1 (14q32) and TCO (19p13.2). To date, there is no evidence for the involvement of genes from the RAS/RAF signalling pathway in FNMTC. The aim of our study was to evaluate the role of the four susceptibility loci, and RAS/RAF signalling pathway genes, in FNMTC. In total, 8 FNMTC families, and 27 thyroid lesions from family members (22 papillary thyroid carcinomas (PTCs): 11 classic, 10 of the follicular variant and 1 of the mixed variant; 4 follicular thyroid adenomas (FTAs) and 1 nodular goitre (NG)), were evaluated for the involvement of the four susceptibility regions, using linkage and loss of heterozygosity (LOH) analyses. BRAF and H-, N- and K-RAS mutations were also screened in the 27 lesions and patients. Linkage analysis in seven informative families showed no evidence for the involvement of any of the four candidate regions, supporting a genetic heterogeneity for FNMTC. Twenty tumours (74%), of which 18 were PTCs, showed no LOH at the four susceptibility loci. The remaining seven tumours (four PTCs, two FTAs and one NG) showed variable patterns of LOH. Fourteen tumours (52%) had somatic mutations: BRAF-V600E mutation was observed in 9 out of the 22 PTCs (41%); and H-RAS and N-RAS mutations were detected in 5 out of the 22 PTCs (23%). Our data suggest that the four candidate regions are not frequently involved in FNMTC and that the somatic activation of BRAF and RAS plays a role in FNMTC tumourigenesis.

    Endocrine-related cancer 2008;15;1;207-15

  • [N-ras and fms gene mutation in idiopathic thrombocytopenic purpura and myelodysplasia].

    Zhao HY, Hou M, Li XF, Ma DX, Liu QJ and Wang P

    Department of Hematology, Qilu Hospital, Shandong University, Jinan 250012, China.

    Objective: To explore the pathogenesis of idiopathic thrombocytopenic purpura (ITP) and improve the differential diagnosis from myelodysplastic syndromes (MDS).

    Methods: Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) was performed to detect the point mutation of codon 12,13 in N-ras gene and codon 301, 969 in fms gene in adult and aged ITP and MDS patients.

    Results: In 25 ITP patients, N-ras mutation and fms mutation were detected in one each (4%). Mutations were found in 3 of 8 MDS patients: two (25%) with N-ras mutation and one (12.5%) with fms mutation.

    Conclusions: Patients with N-ras or fms gene mutation diagnosed as MDS rather than ITP.

    Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 2008;29;3;158-60

  • Clinicopathological analysis of papillary thyroid cancer with PIK3CA alterations in a Middle Eastern population.

    Abubaker J, Jehan Z, Bavi P, Sultana M, Al-Harbi S, Ibrahim M, Al-Nuaim A, Ahmed M, Amin T, Al-Fehaily M, Al-Sanea O, Al-Dayel F, Uddin S and Al-Kuraya KS

    Department of Human Cancer Genomic Research, King Fahad National Center for Children's Cancer and Research, King Faisal Specialist Hospital and Research Cancer, MBC#98-16, P.O. Box 3354, Riyadh 11211, Saudi Arabia.

    Context: Genetic aberration in phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been detected in numerous and diverse human cancers. PIK3CA, which encodes for the catalytic subunit of p110alpha of PI3K, is amplified in some cases of papillary thyroid cancer (PTC). Mutations in the PIK3CA have also been identified in thyroid cancers and, although relatively common in anaplastic thyroid carcinoma, are uncommon in PTC.

    Objective: The objective of the study was to investigate genetic alterations like PIK3CA gene mutation, PIK3CA amplification, RAS, and RAF mutations and to further explore the relationship of these genetic alterations with various clinicopathological characteristics in Middle Eastern PTC.

    Design: We used the fluorescence in situ hybridization technique for analysis of PIK3CA amplification from 536 PTC cases, and selected amplified samples were further validated by real-time quantitative PCR. Mutation analysis was done by direct DNA sequencing of PIK3CA, N2-RAS, and BRAF genes.

    Results: PIK3CA amplification was seen in 265 of 499 PTC cases analyzed (53.1%); PIK3CA gene mutations in four of 207 PTC (1.9%); N2-RAS mutations in 16 of 265 PTC (6%); and BRAF mutations in 153 of 296 PTC (51.7%). N-RAS mutations were-associated with an early stage (P = 0.0465) and lower incidence of extrathyroidal extension (P = 0.027), whereas BRAF mutations were-associated with metastasis (P = 0.0274) and poor disease-free survival (P = 0.0121) in PTCs.

    Conclusion: A higher incidence of PIK3CA alterations and the possible synergistic effect of PIK3CA alterations and BRAF mutations suggest their major role in Middle Eastern PTC tumorigenesis and argue for therapeutic targeting of PI3K/AKT and MAPK pathways.

    The Journal of clinical endocrinology and metabolism 2008;93;2;611-8

  • Cooperating mutations of receptor tyrosine kinases and Ras genes in childhood core-binding factor acute myeloid leukemia and a comparative analysis on paired diagnosis and relapse samples.

    Shih LY, Liang DC, Huang CF, Chang YT, Lai CL, Lin TH, Yang CP, Hung IJ, Liu HC, Jaing TH, Wang LY and Yeh TC

    Division of Hematology-Oncology, Chang Gung Memorial Hospital, Taipei, Taiwan.

    c-KIT mutations have been described in core-binding factor (CBF) acute myeloid leukemia (AML) at diagnosis. The role of c-KIT mutations in the relapse of CBF-AML is not clear. The role of CSF1R mutation in the pathogenesis of AML remains to be determined. We analyzed receptor tyrosine kinases (RTKs) and Ras mutations on 154 children with AML. Also, we examined the paired diagnosis and relapse samples in CBF-AML. CBF-AML accounted for 27% (41/154). c-KIT mutations were detected in 41.5% of CBF-AML at diagnosis (6 in exon 8, 10 in exon 17 and 1 in both exons 8 and 17) , FLT3-TKD 2.7%, N-Ras mutations 7.3% and K-Ras mutations 4.9%. FLT3-LM and CSF1R mutations were not found in CBF-AML. The mutations of RTKs and Ras were mutually exclusive except for one patient who had both c-KIT and N-Ras mutations. Eight of the 41 CBF-AML patients relapsed; four patients retained the identical c-KIT mutation patterns as those at diagnosis, the remaining four without c-KIT mutations at diagnosis did not acquire c-KIT mutations at relapse. Our study showed that 54% of childhood CBF-AML had RTKs and/or Ras mutations; c-KIT but not CSF1R mutations play a role in the leukemogenesis of childhood CBF-AML.

    Leukemia 2008;22;2;303-7

  • Mutation analysis of the EGFR-NRAS-BRAF pathway in melanomas from black Africans and other subgroups of cutaneous melanoma.

    Akslen LA, Puntervoll H, Bachmann IM, Straume O, Vuhahula E, Kumar R and Molven A

    Section for Pathology, The Gade Institute, University of Bergen, Haukeland University Hospital, Bergen, Norway. lars.akslen@gades.uib.no

    Earlier studies have shown frequent mutations in the BRAF and NRAS genes in cutaneous melanoma, but these alterations have not been examined in the rare category of melanoma from black Africans. Moreover, the frequency of epidermal growth factor receptor (EGFR) mutations in melanocytic tumors is not known. We therefore examined 165 benign and malignant melanocytic lesions (including 118 invasive melanomas and 18 metastases collected as consecutive cases from various time periods and from two different pathology departments; the 51 nodular melanomas were randomly selected from a larger, consecutive, population-based series of nodular melanomas) with respect to alterations in the EGFR, BRAF and NRAS genes. Mutations in EGFR (exons 18-21) were not detected. EGFR protein expression was observed in a subgroup of melanomas, but without associations with clinicopathologic phenotype or prognosis. Cytoplasmic EGFR expression was, however, significantly increased from benign nevi to melanomas. Mutations in BRAF and NRAS were detected in superficial melanoma (25 and 29%, respectively), nodular melanoma (29 and 28%, respectively) and lentigo maligna melanoma (15 and 16%, respectively). In a series of melanomas from black Africans (n=26), only two BRAF mutations (8%) were found, both being different from the common T1799A substitution. Moreover, melanomas from black Africans exhibited mutations in NRAS exon 1 only (12%), whereas NRAS exon 2 mutations were predominant in melanomas from Caucasians. Thus, the frequencies of BRAF and NRAS mutations were particularly low in melanomas from black Africans, supporting a different pathogenesis of these tumors.

    Melanoma research 2008;18;1;29-35

  • The CD155/poliovirus receptor enhances the proliferation of ras-mutated cells.

    Kono T, Imai Y, Yasuda S, Ohmori K, Fukui H, Ichikawa K, Tomita S, Imura J, Kuroda Y, Ueda Y and Fujimori T

    Department of Surgical and Molecular Pathology, Dokkyo Medical University School of Medicine, Tochigi, Japan.

    Stimulation of the CD155/poliovirus receptor, which localizes in the cell-matrix and at cell-cell junctions, inhibits cell adhesion and enhances cell migration. Necl-5, a mouse homolog of CD155, is implicated in the formation of adherence junctions. Recently, Necl-5 has also been found to enhance cell proliferation via the stimulation of serum and platelet-derived growth factor through the Ras-Raf-MEK-ERK signaling pathway. In our present study, we find that CD155 significantly enhances the serum-induced cell proliferation of NIH3T3 cells which have been transformed by an oncogenic Ras (V12Ras-NIH3T3), but not the parental cells. CD155 expression in V12Ras-NIH3T3 cells is also found to upregulate cyclin D2, downregulate p27(Kip1) and shorten the G0/G1 phase of the cell cycle. An inhibitor of focal adhesion kinase does not reduce this CD155-mediated enhancement of V12Ras-NIH3T3 cell proliferation. The expression of CD155DeltaCP, which lacks the cytoplasmic region including the immunoreceptor tyrosine-based inhibitory motif (ITIM), has a reduced ability to enhance the serum responsiveness of V12Ras-NIH3T3 cells, suggesting that the ITIM might be required for this effect of CD155. In addition, the overexpression of exogenous CD155 enhances the serum responsiveness of HT1080 cells, which harbor a mutant N-ras gene. On the other hand, siRNA-induced knockdown of endogenous CD155 and/or CD155DeltaCP expression significantly repress the serum responsiveness of DLD-1 cells, which express endogenous CD155 and harbor a mutant K-ras gene, suggesting that this mutant may function in a dominant negative manner. Taken together, our present data suggest that CD155, at least in part, enhances the proliferation of ras-mutated cells.

    International journal of cancer 2008;122;2;317-24

  • Mutations of FLT3, NRAS, KRAS, and PTPN11 are frequent and possibly mutually exclusive in high hyperdiploid childhood acute lymphoblastic leukemia.

    Paulsson K, Horvat A, Strömbeck B, Nilsson F, Heldrup J, Behrendtz M, Forestier E, Andersson A, Fioretos T and Johansson B

    Department of Clinical Genetics, University Hospital, Lund, Sweden.

    Although it has been suggested that mutations of the FLT3, NRAS, KRAS, and PTPN11 genes are particularly frequent in high hyperdiploid (>50 chromosomes) pediatric acute lymphoblastic leukemias (ALLs), this has as yet not been confirmed in a large patient cohort. Furthermore, it is unknown whether mutations of these genes coexist in hyperdiploid cases. We performed mutation analyses of FLT3, NRAS, KRAS, and PTPN11 in a consecutive series of 78 high hyperdiploid ALLs. Twenty-six (33%) of the cases harbored a mutation, comprising six activating point mutations and one internal tandem duplication of FLT3 (7/78 cases; 9.0%), eight codon 12, 13, or 61 NRAS mutations (8/78 cases; 10%), five codon 12 or 13 KRAS mutations (5/78 cases, 6.4%), and seven exon 3 or 13 PTPN11 mutations (7/78 cases; 9.0%). No association was seen between the presence of a mutation in FLT3, NRAS, KRAS, or PTPN11 and gender, age, white blood cell count, or relapse, suggesting that they do not confer a negative prognostic impact. Only one case harbored mutations in two different genes, suggesting that mutations of these four genes are generally mutually exclusive. In total, one third of the cases harbored a FLT3, NRAS, KRAS, or PTPN11 mutation, identifying the RTK-RAS signaling pathway as a potential target for novel therapies of high hyperdiploid pediatric ALLs.

    Genes, chromosomes & cancer 2008;47;1;26-33

  • Mutations of RAS gene family in specimens of bladder cancer.

    Karimianpour N, Mousavi-Shafaei P, Ziaee AA, Akbari MT, Pourmand G, Abedi A, Ahmadi A and Afshin Alavi H

    Institute of Biochemistry and Biophysics, Tehran University, Tehran, Iran.

    Introduction: Studies have shown different types of RAS mutations in human bladder tumors with a wide range of mutation frequencies in different patient populations. This study aimed to assess the frequency of specific-point mutations in the RAS gene family of a group of Iranian patients with bladder cancer.

    We examined the tumor specimens of 35 consecutive patients with transitional cell carcinoma. The DNA samples were evaluated for the occurrence of HRAS, KRAS, and NRAS activation using a polymerase chain reaction-restriction fragment length polymorphism technique.

    Results: None of the patients had mutations in the RAS gene family "hot spots" including codons 12, 13, and 61.

    Conclusion: We failed to find RAS mutations in our bladder tumor samples. These observations may reflect the involvement of different etiological factors in the induction of bladder tumor of which RAS mutation might not be present in all populations.

    Urology journal 2008;5;4;237-42

  • Phospholipase D provides a survival signal in human cancer cells with activated H-Ras or K-Ras.

    Shi M, Zheng Y, Garcia A, Xu L and Foster DA

    Department of Biological Sciences, Hunter College of The City University of New York, 695 Park Avenue, New York, NY 10021, USA .

    Phospholipase D (PLD) is elevated in rodent fibroblasts expressing activated H-Ras mutants. We therefore examined the PLD activity in human cancer cells with activating Ras mutations. T24 bladder carcinoma cells express an activated H-Ras gene and Calu-1 lung carcinoma cells express an activated K-Ras gene. We report here that both of these cancer cell lines express highly elevated levels of PLD activity and that the PLD activity is dependent upon Ras. We also show that the PLD activity is dependent upon the Ras effector molecules RalA and phosphatidylinositol-3-kinase (PI3K). PLD activity has been shown to provide a survival signal in breast cancer cell lines that suppressed stress-induced apoptosis. Suppression of PLD activity in the T24 and Calu-1 cells resulted in apoptotic cell death in the absence of serum, indicating that the elevated PLD activity provided a survival signal in these cancer cell lines. Suppression of Ras, RalA, or PI3K also led to apoptosis in the absence of serum. These data indicate that a critical component of Ras signaling in human cancer cells is the activation of PLD and that targeting PLD survival signals in cancer cells could be an effective strategy to induce apoptosis in human cancers with activating Ras mutations.

    Funded by: NCI NIH HHS: CA46677, R01 CA046677; NCRR NIH HHS: G12 RR003037, RR-03037; NIGMS NIH HHS: GM60654, S06 GM060654

    Cancer letters 2007;258;2;268-75

  • CD200 is induced by ERK and is a potential therapeutic target in melanoma.

    Petermann KB, Rozenberg GI, Zedek D, Groben P, McKinnon K, Buehler C, Kim WY, Shields JM, Penland S, Bear JE, Thomas NE, Serody JS and Sharpless NE

    Department of Genetics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7295, USA.

    Immune-mediated antitumor responses occur in patients with metastatic melanoma (MM), and therapies designed to augment such responses are clinically beneficial. Despite the immunogenicity of melanoma, immunomodulatory therapies fail in the majority of patients with MM. An inability of DCs to sufficiently activate effector cells may, in part, underlie this failure of the antitumor response seen in most patients. In this work, we show that mutation of N-RAS or B-RAF, signature genetic lesions present in most MMs, potently induced the expression of cell-surface CD200, a repressor of DC function. Employing 2 independent, genome-wide microarray analyses, we identified CD200 as a highly dynamic, downstream target of RAS/RAF/MEK/ERK activation in melanoma. CD200 protein was similarly overexpressed in human melanoma cell lines and primary tumors. CD200 mRNA expression correlated with progression and was higher in melanoma than in other solid tumors or acute leukemia. Melanoma cell lines expressing endogenous CD200 repressed primary T cell activation by DCs, while knockdown of CD200 by shRNA abrogated this immunosuppressive effect. These data indicate that in addition to its effects on growth, survival, and motility, ERK activation in MM attenuates a host antitumor immune response, implicating CD200 and its interaction with the CD200 receptor as a potential therapeutic target for MM.

    Funded by: NCI NIH HHS: CA105837, CA106991, CA90679, K08 CA090679, P50 CA106991, R01 CA112243, R01 CA112243-03, R21 CA105837; NIEHS NIH HHS: ES14635, P01 ES014635

    The Journal of clinical investigation 2007;117;12;3922-9

  • 16-kDa prolactin inhibits endothelial cell migration by down-regulating the Ras-Tiam1-Rac1-Pak1 signaling pathway.

    Lee SH, Kunz J, Lin SH and Yu-Lee LY

    Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA.

    Angiogenesis plays a key role in promoting tumorigenesis and metastasis. The 16-kDa fragment of prolactin (16k PRL) is an NH(2)-terminal natural breakdown fragment of the intact 23-kDa prolactin and has been shown to have potent antiangiogenic and antitumor activities. The mechanism(s) involved in the action of 16k PRL in endothelial cells remains unclear. In this study, we showed that 16k PRL reduced rat aortic endothelial cell (RAEC) migration in a wound-healing assay and in a Matrigel tube formation assay, suggesting that 16k PRL inhibits endothelial cell migration, an important activity involved in angiogenesis and tumorigenesis. We further investigated how 16k PRL attenuates endothelial cell migration. We first showed that RAEC migration is mediated through the Rho GTPase Rac1, as Rac1 inhibition by the Rac1-specific inhibitor NSC27366 or Rac1 knockdown by small interfering RNA both blocked RAEC migration. We next showed that 16k PRL reduced the activation of Rac1 in a concentration-dependent manner. Furthermore, 16k PRL inhibition of Rac1 is mediated through the suppression of T lymphoma invasion and metastasis 1 (Tiam1) and its upstream activator Ras in a phosphoinositide-3-kinase-independent manner. 16k PRL also down-regulated the phosphorylation of the downstream effector of Rac1, p21-activating kinase 1 (Pak1), and inhibited its translocation to the leading edge of migrating cells. Thus, 16k PRL inhibits cell migration by blocking the Ras-Tiam1-Rac1-Pak1 signaling pathway in endothelial cells.

    Funded by: NCI NIH HHS: R01-CA111479; NIDDK NIH HHS: R01-DK53176; NIGMS NIH HHS: R01-GM068098

    Cancer research 2007;67;22;11045-53

  • High prevalence of RET, RAS, and ERK expression in Hashimoto's thyroiditis and in papillary thyroid carcinoma in the Korean population.

    Kang DY, Kim KH, Kim JM, Kim SH, Kim JY, Baik HW and Kim YS

    Department of Pathology and Cancer Research Institute, Chungnam National University College of Medicine, Daejeon, Korea.

    Background: The RET/PTC-RAS-BRAF cascade is associated with papillary thyroid carcinoma (PTC).

    Objective: The relationship between PTC and Hashimoto's thyroiditis (HT) is still elusive. To determine whether thyrocytes showing oxyphil cell metaplasia in HT also express RET, RAS, and ERK proteins, which are associated with PTC.

    Design: We investigated the expression of RET, RAS, and ERK proteins in oxyphil cells in the vicinity of large lymphoid HT infiltrates and in malignant PTC cells. BRAF and N-RAS missense mutations were also examined in oxyphil cells of the HT. We used 47 PTC samples with no HT diagnosis, 28 PTC with HT, 39 HT with no PTC, and 36 HT with PTC. We also studied 75 normal portions of thyroid tissue from PTC specimens. Immunohistochemical analysis and polymerase chain reaction were used to determine activation of the RET/PTC-RAS-BRAF cascade in HT and PTC.

    In PTC cells, HT oxyphil cells, and normal thyrocytes, the frequency of high RET expression was 23/70 (32.9%), 36/57 (63.2%), and 1/57 (1.8%) (p = 0.000); that of high nuclear localized RAS expression (nuclearRAS) was 65/71 (91.5%), 52/58 (89.7%), and 5/58 (8.6%) (p = 0.000); and that of high ERK expression was 38/70 (54.3%), 34/61 (55.7%), and 0/61 (0.0%) (p = 0.000), respectively. Of 66 HT cases studied for BRAF mutation and 57 HT cases studied for N-RAS mutation, no BRAF exon 15 or N-RAS exon 2 mutations were found in the amplified DNA extracted from oxyphil cells excised by laser capture microdissection.

    Conclusion: The expression of RET, nuclearRAS, and ERK proteins is greatly enhanced in PTC cells and HT oxyphil cells. Thus, the RET/PTC-RAS-BRAF cascade may be involved in the development of PTC and oxyphil cell metaplasia in HT. Our results show the possibility of a molecular link between oxyphil cell metaplasia in HT and the progression of PTC.

    Thyroid : official journal of the American Thyroid Association 2007;17;11;1031-8

  • Comparative genomic hybridization, BRAF, RAS, RET, and oligo-array analysis in aneuploid papillary thyroid carcinomas.

    Rodrigues R, Roque L, Espadinha C, Pinto A, Domingues R, Dinis J, Catarino A, Pereira T and Leite V

    Cytogenetic Laboratory, CIPM, Portuguese Cancer Institute, 1099-023 Lisbon, Portugal.

    Aneuploidy in papillary thyroid carcinomas (PTCs) is considered a marker of worse prognosis. Multiple genetic surveys have been performed in PTCs, however, we are not aware of any such studies in aneuploid PTCs. In order to contribute to a better comprehension of the genetic basis of this neoplasm's more aggressive behaviour in 17 aneuploid PTCs we performed a comparative genomic hybridization (CGH) analysis, studied the BRAF and RAS mutational status, searched for RET/PTC1 and RET/PTC3 rearrangements and determined their expression profile. Array results were validated by TaqMan and immunohistochemistry. CGH revealed multiple non-random chromosomal abnormalities. BRAFV600E and RAS mutations were found in 41.2% and 33% of the carcinomas respectively. None of the studied cases presented RET/PTC1 or RET/PTC3 rearrangement. When comparing array data with the chromosomal, mutational and clinical data we found that: a) loss of control of cellular transcription was of major relevance in this group of neoplasms, HMGA2 being one of the most overexpressed genes; b) gene expression correlated with the mutational status of PTCs, as in BRAF+ cases cMET and FN1 were concomitantly overexpressed; and c) death from disease and distant metastasis was associated to the overexpression of DDR2 and to the down-regulation of genes involved in immune, inflammatory response, signal transduction and cell adhesion processes. In conclusion we have identified in aneuploid PTCs a group of significantly altered molecules that may represent preferential targets for the development of new more efficient therapies in this type of cancer.

    Oncology reports 2007;18;4;917-26

  • Prevalence of RAS point mutations in papillary thyroid carcinoma; a novel mutation at codon 31 of K-RAS.

    Cyniak-Magierska A, Brzeziańska E, Januszkiewicz-Caulier J, Jarzab B and Lewiński A

    Department of Endocrinology and Metabolic Diseases, Medical University of Lodz, Polish Mother's Memorial Hospital - Research Institute, Lodz, Poland.

    The aim of this study was to assess the incidence of point mutations in RAS oncogenes of papillary thyroid carcinoma (PTC). Tumour specimens were obtained from 29 PTCs. The fragments of exons 1 and 2 of RAS oncogenes family (H- RAS, K- RAS, N- RAS) were amplified and then, point mutations were detected by SSCP and/or by RFLP analysis. Several DNA samples were directly sequenced to confirm the results. Two mutations were found in this study (GAA/CAA at codon 31 of K- RAS and CAA/CAC at codon 61 of N- RAS oncogene). These data confirm the results of previous studies, showing that RAS mutations are more rarely found in PTC than in follicular neoplasms. The influence of a novel mutation at codon 31 of K- RAS oncogene on the development of PTC needs further studies.

    Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association 2007;115;9;594-9

  • Nras and Kras mutation in Japanese lung cancer patients: Genotyping analysis using LightCycler.

    Sasaki H, Okuda K, Kawano O, Endo K, Yukiue H, Yokoyama T, Yano M and Fujii Y

    Department of Surgery II, Nagoya City University Medical School, Nagoya 467-8601, Japan. hisasaki@med.nagoya-cu.ac.jp

    Activating mutations of Ras gene families have been found in a variety of human malignancies, including lung cancer, suggesting their dominant role in tumorigenesis. Many studies have showed that the Kras gene is activated by point mutations in approximately 15-20% of non-small cell lung cancers (NSCLCs), however, there are only a few reports on Nras mutations in NSCLC. We have genotyped Nras mutation status (n=195) and Kras mutation status (n=190) in surgically treated lung adenocarcinoma cases. The presence or absence of Nras and Kras mutations was analyzed by real-time quantitative polymerase chain reaction (PCR) with mutation-specific sensor and anchor probes. EGFR mutation status at kinase domain has already been reported. Nras mutation was found in 1 of 195 patients. This mutation was a G-to-T transversion, involving the substitution of the normal glycine (GGT) with cystein (TGT) and thought to be a somatic mutation. The patient was male and a smoker. Kras mutant patients (11.1%; 21/190) had a significantly worse prognosis than wild-type patients (p=0.0013). Eighty-two EGFR mutations at kinase domain had exclusively Nras or Kras mutations. Although Nras gene mutation might be one of the mechanisms of oncogenesis of lung adenocarcinoma, this was a very rare event. Further studies are needed to confirm the mechanisms of Nras mutations for the sensitivity of molecular target therapy for lung cancer.

    Oncology reports 2007;18;3;623-8

  • Differential oncogenic potential of activated RAS isoforms in melanocytes.

    Whitwam T, Vanbrocklin MW, Russo ME, Haak PT, Bilgili D, Resau JH, Koo HM and Holmen SL

    Molecular Medicine and Virology Group, Van Andel Research Institute, Grand Rapids, MI, USA.

    RAS genes are mutated in approximately 30% of all human cancers. Interestingly, there exists a strong bias in favor of mutation of only one of the three major RAS genes in tumors of different cellular origins. NRAS mutations occur in approximately 20% of human melanomas, whereas HRAS and KRAS mutations are rare in this disease. To define the mechanism(s) responsible for this preference in melanocytes, we compared the transformation efficiencies of mutant NRAS and KRAS in immortal, non-transformed Ink4a/Arf-deficient melanocytes. NRAS mutation leads to increased cellular proliferation and is potently tumorigenic. In contrast, KRAS mutation does not enhance melanocyte proliferation and is only weakly tumorigenic on its own. Although both NRAS and KRAS activate mitogen-activated protein kinase signaling, only NRAS enhances MYC activity in these cells. Our data suggest that the activity of specific RAS isoforms is context-dependent and provide a possible explanation for the prevalence of NRAS mutations in melanoma. In addition, understanding this mechanism will have important implications for cancer therapies targeting RAS pathways.

    Oncogene 2007;26;31;4563-70

  • Spontaneous improvement of hematologic abnormalities in patients having juvenile myelomonocytic leukemia with specific RAS mutations.

    Matsuda K, Shimada A, Yoshida N, Ogawa A, Watanabe A, Yajima S, Iizuka S, Koike K, Yanai F, Kawasaki K, Yanagimachi M, Kikuchi A, Ohtsuka Y, Hidaka E, Yamauchi K, Tanaka M, Yanagisawa R, Nakazawa Y, Shiohara M, Manabe A, Kojima S and Koike K

    Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan.

    Of 11 children with juvenile myelomonocytic leukemia (JMML) carrying RAS mutations (8 with NRAS mutations, 3 with KRAS2 mutations), 5 had a profound elevation in either or both the white blood cells and spleen size at diagnosis. Three patients had no or modest hepatosplenomegaly and mild leukocytosis at presentation but subsequently showed a marked increase in spleen size with or without hematologic exacerbation, for which nonintensive chemotherapy was initiated. The other three patients with NRAS or KRAS2 glycine to serine substitution received no chemotherapy, but hematologic improvement has been observed during a 2- to 4-year follow up. In the third group, all hematopoietic cell lineages analyzed had the RAS mutations at the time of hematologic improvement, whereas DNA obtained from the nails had the wild type. Additionally, numbers of circulating granulocyte-macrophage progenitors were significantly reduced during the clinical course. Thus, some patients with JMML with specific RAS mutations may have spontaneously improving disease.

    Blood 2007;109;12;5477-80

  • A comparative study of molecular mutations in 381 patients with myelodysplastic syndrome and in 4130 patients with acute myeloid leukemia.

    Bacher U, Haferlach T, Kern W, Haferlach C and Schnittger S

    Bone Marrow Transplant Unit, University Hospital of Hamburg-Eppendorf, Hamburg, Germany.

    The precise relationship between myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is unclear and the role of molecular mutations in leukemic transformation in MDS is controversial. The aim of this study was to clarify the relationship between AML and MDS by comparing the frequency of molecular mutations in the two conditions.

    We compared the frequency of FLT3-length mutations (FLT3-LM), FLT3-TKD, MLL-partial tandem duplications (MLL-PTD), NRAS, and KITD816 in 381 patients with MDS refractory anemia with excess blasts [RAEB] n=49; with ringed sideroblasts [RARS] n=310; chronic monomyelocytic leukemia [CMML] n=22) and in 4130 patients with AML (de novo: n=3139; secondary AML [s-AML] following MDS: n=397; therapy-related [t-AML]: n=233; relapsed: n=361).

    Results: All mutations were more frequent in s-AML than in MDS and all but the FLT3-TKD were more frequent in RAEB than in RA/RARS. The higher incidences in s-AML were significant for FLT3-TKD (p=0.032), MLL-PTD (p=0.034), and FLT3-LM (RA/RARS: 0/45; RAEB: 8/293; 2.7%; s-AML: 45/389; 11.6%; p<0.0001). The incidence of NRAS-mutations increased from 17/272 (6.3%) in MDS to 41/343 in s-AML (12.0%) and that of KITD816-mutations from 2/290 (0.7%) to 5/341 (1.5%) (p=n.s.). FLT3-LM-acquisition occurred in 3/22 cases (13.6%) during MDS transformation; NRAS-acquisition occurred in 1/24 (4.2%). FLT3-LM and MLL-PTD were more frequent in AML relapse than in de novo AML or s-AML (p<0.0001).

    The increase of molecular mutations from low- to high-risk MDS, to s-AML, and to relapsed AML emphasizes the value of these mutations as markers of progressing disease. Finally, we found a low rate of 5q- in the molecularly mutated cases in MDS which might explain the stability of this subtype.

    Haematologica 2007;92;6;744-52

  • NRAS mutation causes a human autoimmune lymphoproliferative syndrome.

    Oliveira JB, Bidère N, Niemela JE, Zheng L, Sakai K, Nix CP, Danner RL, Barb J, Munson PJ, Puck JM, Dale J, Straus SE, Fleisher TA and Lenardo MJ

    Department of Laboratory Medicine, Clinical Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

    The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization, and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous, and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4(-), CD8(-) alphabeta T cells. Mutations in ALPS typically affect CD95 (Fas/APO-1)-mediated apoptosis, one of the extrinsic death pathways involving TNF receptor superfamily proteins, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair CD95-mediated apoptosis. The increase in active, GTP-bound NRAS augments RAF/MEK/ERK signaling, which markedly decreases the proapoptotic protein BIM and attenuates intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects. Our observations on the effects of NRAS activation indicate that RAS-inactivating drugs, such as farnesyltransferase inhibitors should be examined in human autoimmune and lymphocyte homeostasis disorders.

    Funded by: Intramural NIH HHS

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;21;8953-8

  • Number of nevi and early-life ambient UV exposure are associated with BRAF-mutant melanoma.

    Thomas NE, Edmiston SN, Alexander A, Millikan RC, Groben PA, Hao H, Tolbert D, Berwick M, Busam K, Begg CB, Mattingly D, Ollila DW, Tse CK, Hummer A, Lee-Taylor J and Conway K

    Department of Dermatology, University of North Carolina, Chapel Hill, NC 27599, USA. nthomas@med.unc.edu

    Malignant melanomas often contain BRAF or NRAS mutations, but the relationship of these mutations to ambient UV exposure in combination with phenotypic characteristics is unknown. In a population-based case series from North Carolina, 214 first primary invasive melanoma patients in the year 2000 were interviewed regarding their risk factors. Ambient solar UV exposures were estimated using residential histories and a satellite-based model. Cases were grouped on the basis of BRAF and NRAS somatic mutations, determined using single-strand conformation polymorphism analysis and radiolabeled DNA sequencing, and the risk profiles of these groups were compared. Mutually exclusive BRAF-mutant and NRAS-mutant cases occurred at frequencies of 43.0% and 13.6% with mean ages at diagnosis of 47.3 and 62.1 years, respectively. Tumors from patients with >14 back nevi were more likely to harbor either a BRAF mutation [age-adjusted odds ratio (OR), 3.2; 95% confidence interval (95% CI), 1.4-7.0] or an NRAS mutation (age-adjusted OR, 1.7; 95% CI, 0.6-4.8) compared with patients with 0 to 4 back nevi. However, BRAF-mutant and NRAS-mutant tumors were distinctive in that BRAF-mutant tumors were characteristic of patients with high early-life ambient UV exposure (adjusted OR, 2.6; 95% CI, 1.2-5.3). When ambient UV irradiance was analyzed by decadal age, high exposure at ages 0 to 20 years was associated with BRAF-mutant cases, whereas high exposure at ages 50 and 60 years was characteristic of NRAS-mutant cases. Our results suggest that although nevus propensity is important for the occurrence of both BRAF and NRAS-mutant melanomas, ambient UV irradiance influences risk differently based on the age of exposure. The association of BRAF mutations with early-life UV exposure provides evidence in support of childhood sun protection for melanoma prevention.

    Funded by: NCI NIH HHS: CA102096, CA103089, CA112243, CA83180, K07 CA102096, K07 CA102096-05, R01 CA112243, R01 CA112243-04, R01 CA112524, R03 CA103089, R03 CA103089-02

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2007;16;5;991-7

  • Further evidence for a somatic KRAS mutation in a pilocytic astrocytoma.

    Janzarik WG, Kratz CP, Loges NT, Olbrich H, Klein C, Schäfer T, Scheurlen W, Roggendorf W, Weiller C, Niemeyer C, Korinthenberg R, Pfister S and Omran H

    Department of Neurology, University Hospital Freiburg, Freiburg, Germany.

    Astrocytomas are the most common brain tumors of childhood. However, knowledge of the molecular etiology of astrocytomas WHO grade I and II is limited. Germline mutations in the Ras-guanosine triphosphatase-activating protein, neurofibromin, in individuals with neurofibromatosis type I predispose to pilocytic astrocytomas. This association suggests that constitutive activation of the Ras signaling pathway plays a fundamental role in astrocytoma development. We screened 25 WHO I and II astrocytomas for mutations of PTPN11, NRAS, KRAS, and HRAS genes and identified the somatic G12A KRAS mutation in one pilocytic astrocytoma. These data suggest that Ras is rarely mutated in these tumors. Analyzed astrocytomas without mutations in Ras or neurofibromin may harbor mutations in other proteins of this pathway leading to hyperactive Ras signaling.

    Neuropediatrics 2007;38;2;61-3

  • Relevance of Ras gene mutations in the context of the molecular heterogeneity of multiple myeloma.

    Intini D, Agnelli L, Ciceri G, Ronchetti D, Fabris S, Nobili L, Lambertenghi-Deliliers G, Lombardi L and Neri A

    Centro di Genetica Molecolare ed Espressione Genica, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy.

    Ras gene mutations are a recurrent genetic lesion in multiple myeloma (MM). Here, we report a mutation analysis of N- and K-Ras genes in purified plasma cell populations from a panel of 81 newly diagnosed MM patients stratified according to the most frequent genetic and molecular features associated with the neoplasia. Ras gene mutations, mostly involving the N-Ras gene, were detected in 20% of the patients. Ras mutations did not correlate with the presence of chromosome 13q deletion, trisomy of chromosome 11, 1q amplification or hyperdiploidy. In addition, despite an appreciable association with tumours overexpressing Cyclin D1, Ras mutations did not correlate at significant levels with any of the proposed groups in the TC classification, based on the presence of the major IgH chromosomal translocations and expression of Cyclin D genes. Finally, transcription analyses revealed the presence of differentially expressed transcripts in human multiple myeloma cell lines carrying the Ras gene mutations but not in primary tumours. Overall, these data suggest that Ras gene mutations are not likely to represent a master lesion in MM but its relevance needs to be considered in the context of other genetic abnormalities.

    Hematological oncology 2007;25;1;6-10

  • Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations.

    Zenker M, Lehmann K, Schulz AL, Barth H, Hansmann D, Koenig R, Korinthenberg R, Kreiss-Nachtsheim M, Meinecke P, Morlot S, Mundlos S, Quante AS, Raskin S, Schnabel D, Wehner LE, Kratz CP, Horn D and Kutsche K

    Institute of Human Genetics, University of Erlangen-Nuremberg, Germany.

    Background: Noonan syndrome, cardio-facio-cutaneous syndrome (CFC) and Costello syndrome constitute a group of developmental disorders with an overlapping pattern of congenital anomalies. Each of these conditions can be caused by germline mutations in key components of the highly conserved Ras-MAPK pathway, possibly reflecting a similar pathogenesis underlying the three disorders. Germline mutations in KRAS have recently been identified in a small number of patients with Noonan syndrome and CFC.

    260 patients were screened for KRAS mutations by direct sequencing. Overall, we detected KRAS mutations in 12 patients, including three known and eight novel sequence alterations. All mutations are predicted to cause single amino acid substitutions. Remarkably, our cohort of individuals with KRAS mutations showed a high clinical variability, ranging from Noonan syndrome to CFC, and also included two patients who met the clinical criteria of Costello syndrome.

    Conclusion: Our findings reinforce the picture of a clustered distribution of disease associated KRAS germline alterations. We further defined the phenotypic spectrum associated with KRAS missense mutations and provided the first evidence of clinical differences in patients with KRAS mutations compared with Noonan syndrome affected individuals with heterozygous PTPN11 mutations and CFC patients carrying a BRAF, MEK1 or MEK1 alteration, respectively. We speculate that the observed phenotypic variability may be related, at least in part, to specific genotypes and possibly reflects the central role of K-Ras in a number of different signalling pathways.

    Journal of medical genetics 2007;44;2;131-5

  • NRAS and BRAF mutations in melanoma tumours in relation to clinical characteristics: a study based on mutation screening by pyrosequencing.

    Edlundh-Rose E, Egyházi S, Omholt K, Månsson-Brahme E, Platz A, Hansson J and Lundeberg J

    Department of Gene Technology, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Center, Stockholm, Sweden.

    We have previously demonstrated the use of pyrosequencing to investigate NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog] mutations in melanoma biopsies. Here, we expanded the analysis to include BRAF (V-raf murine sarcoma viral oncogene homolog B1), another member of the Ras-Raf-mitogen-activated protein kinase (MAPK) signalling pathway, and analysed a total of 294 melanoma tumours from 219 patients. Mutations in BRAF exons 11 and 15 were identified in 156 (53%) tumours and NRAS exon 2 mutations in 86 (29%) tumours. Overall, mutations in NRAS or BRAF were found in 242 of 294 tumours (82%) and were found to be mutually exclusive in all but two cases (0.7%). Multiple metastases were analysed in 57 of the cases and mutations were identical in all except three, indicating that BRAF and NRAS mutations occur before metastasis. Association with preexisting nevi was significantly higher in BRAF mutated tumours (P=0.014). In addition, tumours with BRAF mutations showed a significantly more frequent moderate to pronounced infiltration of lymphocytes (P=0.013). NRAS mutations were associated with a significantly higher Clark level of invasion (P=0.022) than BRAF mutations. Age at diagnosis was significantly higher in tumours with NRAS mutations than in those with BRAF mutations (P=0.019). NRAS and BRAF mutations, however, did not influence the overall survival from time of diagnosis (P=0.7). In conclusion, the separate genotypes were associated with differences in several key clinical and pathological parameters, indicating differences in the biology of melanoma tumours with different proto-oncogene mutations.

    Melanoma research 2006;16;6;471-8

  • The expression of the homologue of the Caenorhabditis elegans lin-45 raf is regulated in the motile stages of the plant parasitic nematode Meloidogyne artiellia.

    Cortese MR, Di Vito M and De Giorgi C

    Dipartimento di Biochimica e Biologia Molecolare, Via Orabona 4, 70126 Bari, Italy.

    The Ras-MAPK signal transduction pathway controls multiple developmental events and is involved in the processing of olfactory information in the free living nematode Caenorhabditis elegans. We have studied the Ras-MAPK pathway in the plant parasitic nematode Meloidogyne artiellia. The genes Mt-let-60, Mt-lin-45, Mt-mek-2 and Mt-mpk-1 have been isolated and sequenced. Each of them shows a high level of sequence similarity to its presumed ortholog in C. elegans and key functional domains are structurally conserved. Furthermore, we show that the M. artiellia recombinant MEK-2 protein can phosphorylate and activate the M. artiellia recombinant MPK-1 and the recombinant MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. Surprisingly, the Mt-lin-45 message is not detectable in freshly emerged juveniles or in male specimens, suggesting that it may be quickly degraded in these life stages.

    Molecular and biochemical parasitology 2006;149;1;38-47

  • Structural model of the membrane-bound C terminus of lipid-modified human N-ras protein.

    Reuther G, Tan KT, Köhler J, Nowak C, Pampel A, Arnold K, Kuhlmann J, Waldmann H and Huster D

    Nachwuchsgruppe "Strukturbiologie von Membranproteinen", Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 3, 06120 Halle, Germany.

    Angewandte Chemie (International ed. in English) 2006;45;32;5387-90

  • BRAF and NRAS mutations in melanoma and melanocytic nevi.

    Poynter JN, Elder JT, Fullen DR, Nair RP, Soengas MS, Johnson TM, Redman B, Thomas NE and Gruber SB

    Department of Epidemiology, University of Michigan, Ann Arbor, Michigan 48109-2200, USA.

    In this report, we investigated BRAF/NRAS mutations in samples from a case-control study of melanoma and a series of benign melanocytic nevi. We evaluated potential associations between BRAF mutations and histopathologic and pigmentary characteristics of melanoma. Mutations in BRAF and NRAS were detected by sequencing microdissected/laser-captured DNA from 18 in-situ melanomas, 64 primary melanomas, and 51 nevi. Nevi showed the highest frequency of BRAF mutations (82%). BRAF mutations were identified in 29% of invasive melanomas and in only 5.6% of in-situ melanomas. Mutations in NRAS were found in 5.2% of primary melanomas, 5.9% of nevi and no NRAS mutations were seen in in-situ melanomas. A majority of the BRAF mutations observed in primary invasive melanoma were seen in superficial spreading melanoma (15/17), and melanomas with BRAF mutations were also more likely to be found on a body site that was likely to be exposed to intermittent sun exposure compared with chronic or no sun exposure (P=0.02). Tumors with BRAF mutations were also significantly more likely to occur in association with a contiguous nevus (odds ratio 3.49, 95% confidence interval 1.06-11.46), although a contiguous nevus was not found in all melanomas with a BRAF mutation. Our data support the evidence that the mitogen-activated protein kinase pathway is upregulated in a large percentage of melanocytic lesions, but these mutations are not sufficient for malignant transformation. We suggest that BRAF mutations contribute to benign melanocytic hyperplasia, but are likely to contribute to invasive melanoma only in conjunction with other mutations.

    Funded by: NCI NIH HHS: K07 CA102096, K07 CA102096-03, R01 CA112243, R01 CA112243-03, U01 CA83180; NHGRI NIH HHS: T32 HG 00040

    Melanoma research 2006;16;4;267-73

  • Frequency of RAS gene mutation and its cooperative genetic events in Southeast Asian adult acute myeloid leukemia.

    Auewarakul CU, Lauhakirti D and Tocharoentanaphol C

    Department of Medicine, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand. cicaw@mahidol.ac.th

    RAS gene as one of the most frequently mutated genes in acute myeloid leukemia (AML) has become an attractive target for molecular therapy. The role of oncogenic RAS and its associated genetic events in AML are not yet defined. We examined the frequency of RAS mutation in 239 Thai de novo adult AML patients using polymerase chain reaction-single-strand conformational polymorphism analysis. Thirty-five RAS mutations were found in 32 cases (13%) predominantly classified as M1/M2 (53%) followed by M4/M5 subtype (38%). Ten cases were positive for N-RAS codon 12, 11 cases for N-RAS codon 61, 13 cases for N-RAS codon 13, and one case for K-RAS codon 13. No mutation was found in K-RAS exon 2 or H-RAS. The most common base substitution was the G to A transition at codon 13. Most M1/M2 cases had mutations at codon 12 or 13, whereas M4/M5 cases preferentially affected codon 61. Half of the patients with RAS mutations had abnormal karyotypes with the majority involving chromosomes 21, 11 and 7. Four patients had core-binding factor leukemia and four additional patients had coexisting FLT3 or AML1 mutation. One patient had RAS, FLT3 and t(8;21) and the other had RAS, AML1 point mutation and del(9q). In conclusion, mutation of RAS gene was not as common in the Thais as in the western population. Several additional genetic abnormalities occurred in RAS-mutated patients. Future molecular-targeting approaches should take into account the multiple genetic events that coexist with RAS mutations in AML patients.

    European journal of haematology 2006;77;1;51-6

  • Mutually exclusive NRASQ61R and BRAFV600E mutations at the single-cell level in the same human melanoma.

    Sensi M, Nicolini G, Petti C, Bersani I, Lozupone F, Molla A, Vegetti C, Nonaka D, Mortarini R, Parmiani G, Fais S and Anichini A

    Human Tumor Immunobiology Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy. marialuisa.sensi@istitutotumori.mi.it

    Activating BRAF or NRAS mutations have been found in 80% of human sporadic melanomas, but only one of these genetic alterations could be detected in each tumour. This suggests that BRAF and NRAS 'double mutants' may not provide advantage for tumour growth, or may even be selected against during tumorigenesis. However, by applying mutant-allele-specific-amplification-PCR method to short-term melanoma lines, one out of 14 tumours was found to harbour both BRAFV600E and the activating NRASQ61R mutations. On the other hand, analysis of 21 melanoma clones isolated by growth in soft agar from this tumour indicated that 16/21 clones harboured a BRAFV600E, but were wild-type for NRAS, whereas the remaining had the opposite genotype (NRASQ61R/wild-type BRAF). When compared to BRAFV600E clones, NRASQ61R clones displayed reduced growth in soft agar, but higher proliferative ability in vitro in liquid medium and even in vivo after grafting into SCID/SCID mice. These data suggest that NRAS and BRAF activating mutations can coexist in the same melanoma, but are mutually exclusive at the single-cell level. Moreover, the presence of NRASQ61R or BRAFV600E is associated with distinct in vitro and in vivo growth properties of neoplastic cells.

    Oncogene 2006;25;24;3357-64

  • Mutational analysis of K-ras and Ras protein expression in larynx squamous cell carcinoma.

    Ruíz-Godoy R LM, Garcia-Cuellar CM, Herrera González NE, Suchil BL, Pérez-Cárdenas E, Sácnchez-Pérez Y, Suárez-Roa ML and Meneses A

    Basic Research Subdirection, National Institute of Cancerology, Tlalpan, Mexico.

    The ras gene family (H, K and N-ras) encodes the Ras protein, a GTPase-activating protein that regulates several signal transduction pathways including cellular proliferation and differentiation. Mutations in codons 12, 13 and 61 of the ras genes constitute one of the most frequent alterations in human cancer. In the Western Hemisphere, a low frequency of mutations in these genes has been observed in head and neck carcinomas; a higher frequency has been found in countries such as India and Taiwan. Increased protein expression is a relatively frequent event in larynx carcinomas. This study was aimed to evaluate the participation of the k-ras gene and Ras expression in 20 Mexican patients with larynx squamous carcinoma, 2 with dysplasia and 4 with normal mucosa. Samples (of 26 patients) were embedded in paraffin and immunohistochemical analysis was performed for the Ras protein, as well as amplification of the k-ras gene exon 1 (108 bp) by laser capture microdissection. Then, DNA extraction, PCR and sequencing were performed looking for possible mutation in codons 12 and 13. All patients with larynx carcinoma were men, median age 62 years. Eighty-five percent of the patients had risk factors such as smoking and/or alcohol consumption, 25% were in clinical stages I and II, and 75% in stages III and IV; 45% of the patients presented tumor recurrence or persistence. In this study, no mutations were found in codons 12 or 13 of the k-ras gene; however, protein expression was observed in 95% of the samples and a higher expression of the protein was associated with tumor recurrence or persistence, although this was not statistically significant. Unexpectedly, well-differentiated carcinomas and dysplasias presented an increase in protein expression. These results suggest that ras may be involved in early stages of larynx carcinogenesis and may be activated by other mechanisms different from mutations, such as epigenetic events.

    Journal of experimental & clinical cancer research : CR 2006;25;1;73-8

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Nucleophosmin gene mutations are predictors of favorable prognosis in acute myelogenous leukemia with a normal karyotype.

    Schnittger S, Schoch C, Kern W, Mecucci C, Tschulik C, Martelli MF, Haferlach T, Hiddemann W and Falini B

    Department of Internal Medicine III, University Hospital Grosshadern, Ludwig Maximilian's University, Munich, Germany. susanne.schnittger@mllonline.com

    Nucleophosmin (NPM1) exon-12 gene mutations are the hallmark of a large acute myelogenous leukemia (AML) subgroup with normal karyotype, but their prognostic value in this AML subset has not yet been determined. We screened 401 AML patients with normal karyotype treated within the German AML Cooperative Group Protocol 99 (AMLCG99) study for NPM1 mutations. Results were related with partial tandem duplications within the MLL gene (MLL-PTD), Fms-like tyrosine kinase 3-length mutations (FLT3-LM), the tyrosine kinase domain of FLT3 (FLT3-TKD), NRAS, KIT, and CEBPA mutations and with clinical characteristics and outcome. NPM1 mutations were detected in 212 (52.9%) of 401 patients. Fourteen mutations, including 8 new variants, were identified. NPM1-mutated cases associated frequently with FLT3 mutations but rarely with other mutations. The NPM1-mutated group had a higher complete remission (CR) rate (70.5% vs 54.7%, P = .003), a trend to a longer overall survival (OS; median 1012 vs 549 days, P = .076), and significantly longer event-free survival (EFS; median 428 vs 336 days; P = .012). The favorable impact of NPM1 mutations on OS and EFS clearly emerged in the large group (264 [66.8%] of 395 cases) of normal-karyotype AML without FLT3-LM. This positive effect was lost in the presence of a concomitant FLT3-LM, since survival of the NPM1+/FLT3-LM+ double positive was similar to NPM1-/FLT3-LM+ cases. In conclusion, this study demonstrates that NPM1+/FLT3-LM- mutations are an independent predictor for a favorable outcome in AML with normal karyotype.

    Blood 2005;106;12;3733-9

  • RAS mutations are uncommon in multiple myeloma and other monoclonal gammopathies.

    Martín P, Santón A, García-Cosío M and Bellas C

    Department of Pathology, Hospital Universitario Puerta de Hierro, 28035 Madrid, Spain.

    Monoclonal gammopathies are a group of diseases characterised by the proliferation of a single clone of plasma cells that produce a homogeneous monoclonal protein (M protein or myeloma protein) that consist of two heavy polypeptide chains of the same class and subclass and two light polypeptide chains of the same type. Multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) are the most common monoclonal gammopathies. Despite advances in systemic and supportive therapies, MM is an incurable hematological malignancy with a median survival of between two and three years. Point mutations in the Ras genes can be detected in a variety of human malignancies, indicating that ras activation represents a widespread oncogenic event. Several studies have analysed the incidence of Ras mutation in MM and MGUS with great differences in their results. To date, the etiopathogenesis of these diseases is still unknown and the relevance of Ras mutation to the clinical and biological behaviour of monoclonal gammopathies remains to be elucidated. In this study, we have analysed K-ras codon 12 and N-ras codon 61 mutations on anti-CD138 sorted bone marrow plasma cell samples of 44 cases of monoclonal gammopathies: 30 MM, 13 MGUS and 1 plasma cell leukaemia, using polymerase chain reaction. No mutations within either codon 12 of K-ras or codon 61 of N-ras have been found in any of the samples. These results indicate that Ras mutations do not play a significant role in the pathogenesis of MM in the Spanish population.

    International journal of oncology 2005;27;4;1023-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras.

    Swarthout JT, Lobo S, Farh L, Croke MR, Greentree WK, Deschenes RJ and Linder ME

    Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H- and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 co-distribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2.Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9.GCP16 exhibits substrate specificity, palmitoylating H- and N-Ras but not myristoylated G (alphai1) or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9.GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.

    Funded by: NCI NIH HHS: CA050211, R01 CA050211, R01 CA050211-15A2; NIGMS NIH HHS: GM51466

    The Journal of biological chemistry 2005;280;35;31141-8

  • Pathway- and expression level-dependent effects of oncogenic N-Ras: p27(Kip1) mislocalization by the Ral-GEF pathway and Erk-mediated interference with Smad signaling.

    Kfir S, Ehrlich M, Goldshmid A, Liu X, Kloog Y and Henis YI

    Department of Neurobiochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

    Overactivation of Ras pathways contributes to oncogenesis and metastasis of epithelial cells in several ways, including interference with cell cycle regulation via the CDK inhibitor p27(Kip1) (p27) and disruption of transforming growth factor beta (TGF-beta) anti-proliferative activity. Here, we show that at high expression levels, constitutively active N-Ras induces cytoplasmic mislocalization of murine and human p27 via the Ral-GEF pathway and disrupts TGF-beta-mediated Smad nuclear translocation by activation of the Mek/Erk pathway. While human p27 could also be mislocalized via the phosphatidylinositol 3-kinase/Akt pathway, only Ral-GEF activation was effective for murine p27, which lacks the Thr157 Akt phosphorylation site of human p27. This establishes a novel role for the Ral-GEF pathway in regulating p27 localization. Interference with either Smad translocation or p27 nuclear localization was sufficient to disrupt TGF-beta growth inhibition. Moreover, expression of activated N-Ras or specific effector loop mutants at lower levels using retroviral vectors induced p27 mislocalization but did not inhibit Smad2/3 translocation, indicating that the effects on p27 localization occur at lower levels of activated Ras. These findings have important implications for the contribution of activated Ras to oncogenesis and for the conversion of TGF-beta from an inhibitory to a metastatic factor in some epithelial tumors.

    Molecular and cellular biology 2005;25;18;8239-50

  • BRAF and NRAS mutations are frequent in nodular melanoma but are not associated with tumor cell proliferation or patient survival.

    Akslen LA, Angelini S, Straume O, Bachmann IM, Molven A, Hemminki K and Kumar R

    The Gade Institute, Section of Pathology, University of Bergen, Haukeland University Hospital, Bergen, Norway. lars.akslen@gades.uib.no

    Previous studies have shown frequent mutations in the BRAF (V-raf murine sarcoma viral oncogene homolog B1) or NRAS (neuroblastoma RAS viral [V-ras] oncogene homolog) genes in cutaneous melanoma, but the relationship between these alterations and tumor cell proliferation has not been examined in human melanoma. In our study of 51 primary nodular melanomas and 18 paired metastases, we found mutations in BRAF (codon 600, previously denoted 599) in 15 primary tumors (29%) and eight metastases (44%). The figures for NRAS mutations were 27% and 22%, respectively. Mutations in BRAF and NRAS genes were mutually exclusive in all but one case, and were maintained from primary tumors through their metastases. Mutations, however, were not associated with tumor cell proliferation by Ki-67 expression, tumor thickness, microvessel density, or vascular invasion, and there were no differences in patient survival. Although BRAF and NRAS mutations are likely to be important for the initiation and maintenance of some melanomas, other factors might be more significant for proliferation and prognosis in subgroups of aggressive melanoma.

    The Journal of investigative dermatology 2005;125;2;312-7

  • Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells.

    Eskandarpour M, Kiaii S, Zhu C, Castro J, Sakko AJ and Hansson J

    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska University Hospital Solna and Karolinska Institute, Stockholm, Sweden.

    The majority of human melanomas harbor activating mutations in either the BRAF or NRAS gene. To date, the role of oncogenic NRAS in melanoma remains poorly defined and no current therapies are directed at specifically suppressing oncogenic NRAS in human melanoma tumors. The aim of our study, therefore, was to investigate the effects of suppressing oncogenic NRAS in human melanoma cell lines in vitro. Using both small interfering RNA- and plasmid based-RNA interference techniques, oncogenic NRAS was specifically suppressed in 2 human melanoma cell lines, 224 and BL, which harbor a codon 61 CAA (glutamine) to CGA (arginine) NRAS mutation. Suppression of oncogenic NRAS in these cell lines resulted in increased apoptosis. Furthermore, in 224 cells we demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and reduced expression of NF-kappaB and cyclin D1 in the N-Ras signaling pathway. In contrast, RNA interference directed at wild-type (WT) NRAS had no significant effect on apoptosis of 224 cells or 2 human melanoma cell lines (A375 and 397) containing WT NRAS but a codon 600 GTG (valine) to GAG (glutamate) mutation in BRAF. These data suggest that oncogenic NRAS is important for avoidance of apoptosis in melanomas that harbor the codon 61 NRAS mutation and emphasizes oncogenic NRAS as a therapeutic target in patients with tumors that harbor this mutation.

    International journal of cancer 2005;115;1;65-73

  • An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.

    Rocks O, Peyker A, Kahms M, Verveer PJ, Koerner C, Lumbierres M, Kuhlmann J, Waldmann H, Wittinghofer A and Bastiaens PI

    Department of Structural Biology, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.

    We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

    Science (New York, N.Y.) 2005;307;5716;1746-52

  • High-throughput mapping of a dynamic signaling network in mammalian cells.

    Barrios-Rodiles M, Brown KR, Ozdamar B, Bose R, Liu Z, Donovan RS, Shinjo F, Liu Y, Dembowy J, Taylor IW, Luga V, Przulj N, Robinson M, Suzuki H, Hayashizaki Y, Jurisica I and Wrana JL

    Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5.

    Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-beta (TGFbeta) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGFbeta pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFbeta type I receptor localization for efficient TGFbeta-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.

    Funded by: NIGMS NIH HHS: P50 GM-62413

    Science (New York, N.Y.) 2005;307;5715;1621-5

  • RAS is regulated by the let-7 microRNA family.

    Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, Labourier E, Reinert KL, Brown D and Slack FJ

    Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520, USA.

    MicroRNAs (miRNAs) are regulatory RNAs found in multicellular eukaryotes, including humans, where they are implicated in cancer. The let-7 miRNA times seam cell terminal differentiation in C. elegans. Here we show that the let-7 family negatively regulates let-60/RAS. Loss of let-60/RAS suppresses let-7, and the let-60/RAS 3'UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. mir-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1 degrees vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3'UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 in cancer.

    Funded by: NIGMS NIH HHS: GM62594, R01 GM062594

    Cell 2005;120;5;635-47

  • Distinct gene expression patterns associated with FLT3- and NRAS-activating mutations in acute myeloid leukemia with normal karyotype.

    Neben K, Schnittger S, Brors B, Tews B, Kokocinski F, Haferlach T, Müller J, Hahn M, Hiddemann W, Eils R, Lichter P and Schoch C

    Division of Molecular Genetics (B060), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.

    In acute myeloid leukemia (AML), constitutive activation of the FLT3 receptor tyrosine kinase, either by internal tandem duplications (FLT3-ITD) of the juxtamembrane region or by point mutations in the second tyrosine kinase domain (FLT3-TKD), as well as point mutations of the NRAS gene (NRAS-PM) are among the most frequent somatic gene mutations. To elucidate whether these mutations cause aberrant signal transduction in AML, we used gene expression profiling in a series of 110 newly diagnosed AML patients with normal karyotype. The different algorithms used for data analysis revealed highly concordant sets of genes, indicating that the identified gene signatures are specific for each analysed subgroup. Whereas samples with FLT3-ITD and FLT3-TKD could be separated with up to 100% accuracy, this did not apply for NRAS-PM and wild-type samples, suggesting that only FLT3-ITD and FLT3-TKD are associated with an apparent signature in AML. The set of discriminating genes included several known genes, which are involved in cell cycle control (CDC14A, WEE1), gene transcription (HOXB5, FOXA1), and signal transduction (SMG1). In conclusion, we showed that unique gene expression patterns can be correlated with FLT3-ITD and FLT3-TKD. This might lead to the identification of further pathogenetic relevant candidate genes particularly in AML with normal karyotype.

    Oncogene 2005;24;9;1580-8

  • Prognostic significance of N-RAS and K-RAS mutations in 232 patients with acute myeloid leukemia.

    Ritter M, Kim TD, Lisske P, Thiede C, Schaich M and Neubauer A

    Reports on the prognostic impact of mutations in the RAS proto-oncogenes in patients with acute myeloid leukemia (AML) are conflicting. A peptide nucleic acid (PNA)-based technique was used on 232 AML samples to detect point mutations of the hotspots in N-RAS and K-RAS. No significant correlations between RAS mutations and clinical features, karyotype or FLT3 were found.

    Haematologica 2004;89;11;1397-9

  • Low frequency of NRAS and KRAS2 gene mutations in childhood myelodysplastic syndromes.

    Jekic B, Novakovic I, Lukovic L, Kuzmanovic M, Popovic B, Pastar I, Milasin J, Bunjevacki G and Bunjevacki V

    Institute of Biology and Human Genetics, School of Medicine, 26, Visegradska Street, 11000 Belgrade, Serbia and Montenegro.

    In children, myelodysplastic syndromes (MDS) represent less then 10% of all hematological malignancies; consequently, molecular genetic studies dealing with this group of patients are scarce. We have analyzed 35 archival bone marrow samples of children with MDS for the presence of mutations in the first and second exons of the NRAS and KRAS2 genes. Mutations were detected with single-strand conformation polymorphism analysis in three patients. One patient harbored a mutation in the second exon of NRAS and two patients in the second exon of KRAS2. Sequencing was performed in two samples and novel mutations were found in both. One patient had a missense mutation in codon 45 of NRAS; the other had a silent mutation in codon 53 and a missense mutation in codon 55 of KRAS2.

    Cancer genetics and cytogenetics 2004;154;2;180-2

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Ras gene mutations in patients with acute myeloid leukaemia and exposure to chemical agents.

    Barletta E, Gorini G, Vineis P, Miligi L, Davico L, Mugnai G, Ciolli S, Leoni F, Bertini M, Matullo G and Costantini AS

    Department of Experimental Pathology and Oncology, University of Florence, Florence, Italy.

    Mutations of the N- and K-ras genes occur in approximately 15-30% of acute myeloid leukaemia patients. The role of the oncogenic ras in leukaemogenesis remains unclear. Few studies have revealed that mutations in the ras oncogene family are more probably found in acute myeloid leukaemia patients with previous exposure to toxic agents. A case-case study was conducted in the areas of Florence and Turin, Italy, to investigate whether the presence of N- and K-ras mutations in acute myeloid leukaemia patients was related to a higher frequency of exposure to chemicals. During a 3-year period, 111 acute myeloid leukaemia patients were enrolled. All the patients were interviewed using a semi-structured questionnaire collecting data on residential history, occupation, personal habits and pathological history. The presence of N- and K-ras mutations was analysed by amplification and synthetic oligonucleotide probes and by the so-called polymerase chain reaction amplification for specific alleles technique. A total of 34 (30.6%) patients were found to harbour ras mutations in N-ras and/or K-ras. Fourteen patients (12.6%) had a single ras mutation and 20 patients (18%) had two ras mutations. A positive association between a priori at risk jobs and ras mutations was found, based on nine exposed cases; the odds ratio, adjusted by age, sex and previous X-ray and/or chemotherapy was 2.8 (95% confidence intervals: 0.9-9.0). When considering only subjects with two ras mutations the odds ratio was 4.8 (95% confidence intervals: 1.2-18.8). The odds ratio for a previous X-ray and/or chemotherapy was 16.2 (95% confidence intervals: 1.8-755.9); when only subjects with two ras mutations were considered, the odds ratio was 26.1 (95% confidence intervals: 2.5-1248.9). In conclusion, our data suggest that ras oncogene mutations might identify a group of leukaemia in people with previous X-ray/chemotherapy or with exposure to chemical agents in the work environment.

    Carcinogenesis 2004;25;5;749-55

  • Frequent alterations of Ras signaling pathway genes in sporadic malignant melanomas.

    Reifenberger J, Knobbe CB, Sterzinger AA, Blaschke B, Schulte KW, Ruzicka T and Reifenberger G

    Department of Dermatology, Heinrich-Heine-University, Düsseldorf, Germany. reifenbergerj@med.uni-duesseldorf.de

    Ras signaling is important for the intracellular transduction of mitogenic stimuli from activated growth factor receptors. We have investigated 37 sporadic malignant melanomas (15 primary cutaneous melanomas and 22 melanoma metastases) and 6 melanoma cell lines for mutations in the 3 Ras genes NRAS, KRAS and HRAS. All tumors and cell lines were additionally analyzed for mutation and expression of BRAF, which encodes a Ras-regulated serine/threonine kinase with oncogenic properties, as well as for expression of RASSF1A, which encodes a Ras-binding protein with tumor suppressor properties. Mutational analyses identified somatic NRAS mutations in 2 primary melanomas, 4 melanoma metastases and 2 cell lines. One melanoma metastasis showed a somatic KRAS mutation whereas HRAS mutations were not detected. Eight primary melanomas, 6 melanoma metastases and 4 melanoma cell lines carried BRAF mutations affecting the known hot-spot codon 599. None of the tumors or cell lines with BRAF mutation demonstrated NRAS or KRAS mutations. Real-time reverse transcription-PCR showed that 8 melanomas (3 primary tumors, 5 melanoma metastases) had reduced RASSF1A transcript levels of < or =50% relative to benign melanocytic nevi and normal skin. Three melanoma cell lines lacked detectable RASSF1A transcripts. The RASSF1A gene promoter was hypermethylated in these 3 cell lines as well as in 6 of 8 melanomas with reduced RASSF1A mRNA levels. Treatment of the cell lines with 5-aza-2'-deoxycytidine and trichostatin A resulted in demethylation of the RASSF1A promoter and re-expression of RASSF1A transcripts. Most tumors and all cell lines with RASSF1A promoter methylation additionally carried BRAF or NRAS mutations, suggesting a synergistic effect of these aberrations on melanoma growth. Taken together, 57% of the investigated melanomas and 100% of the melanoma cell lines carried mutations in either NRAS, KRAS or BRAF. In addition, 22% of the melanomas and 50% of the cell lines showed reduced RASSF1A transcript levels. Thus, alterations of Ras pathway genes are of paramount importance in the pathogenesis of sporadic melanomas.

    International journal of cancer 2004;109;3;377-84

  • Ras activation in Jurkat T cells following low-grade stimulation of the T-cell receptor is specific to N-Ras and occurs only on the Golgi apparatus.

    Perez de Castro I, Bivona TG, Philips MR and Pellicer A

    Department of Pathology and New York University Cancer Institute, New York, New York 10016, USA.

    Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells.

    Funded by: NCI NIH HHS: CA36327, CA50434, R01 CA036327, R01 CA050434; NCRR NIH HHS: M01 RR000096, M01RR00096; NIAID NIH HHS: AI36224, R01 AI036224, R29 AI036224; NIGMS NIH HHS: GM55279, R01 GM055279

    Molecular and cellular biology 2004;24;8;3485-96

  • Acquisition of FLT3 or N-ras mutations is frequently associated with progression of myelodysplastic syndrome to acute myeloid leukemia.

    Shih LY, Huang CF, Wang PN, Wu JH, Lin TL, Dunn P and Kuo MC

    Department of Internal Medicine, Division of Hematology-Oncology, Chang Gung Memorial Hospital, Taipei, Taiwan. sly7012@adm.cgmh.org.tw

    The role of internal tandem duplication of fms-like tyrosine kinase 3 (FLT3/ITD), mutations at tyrosine kinase domain (FLT3/TKD) and N-ras mutations in the transformation of myelodysplastic syndrome (MDS) to AML was investigated in 82 MDS patients who later progressed to AML; 70 of them had paired marrow samples at diagnosis of MDS and AML available for comparative analysis. Five of the 82 patients had FLT3/ITD at presentation. Of the 70 paired samples, seven patients acquired FLT3/ITD during AML evolution. The incidence of FLT3/ITD at diagnosis of MDS was significantly lower than that at AML transformation (3/70 vs 10/70, P<0.001). FLT3/ITD(+) patients progressed to AML more rapidly than FLT3/ITD(-) patients (2.5+/-0.5 vs 11.9+/-1.5 months, P=0.114). FLT3/ITD(+) patients had a significantly shorter survival than FLT3/ITD(-) patients (5.6+/-1.3 vs 18.0+/-1.7 months, P=0.0008). After AML transformation, FLT3/ITD was also associated with an adverse prognosis. One patient had FLT3/TKD mutation (D835Y) at both MDS and AML stages. Additional three acquired FLT3/TKD (one each with D835 H, D835F and I836S) at AML transformation. Five of the 70 matched samples had N-ras mutation at diagnosis of MDS compared to 15 at AML transformation (P<0.001), one lost and 11 gained N-ras mutations at AML progression. Coexistence of FLT3/TKD and N-ras mutations was found in two AML samples. N-ras mutations had no prognostic impact either at the MDS or AML stage. Our results show that one-third of MDS patients acquire activating mutations of FLT3 or N-ras gene during AML evolution and FLT3/ITD predicts a poor outcome in MDS.

    Leukemia 2004;18;3;466-75

  • NRAS and BRAF mutations arise early during melanoma pathogenesis and are preserved throughout tumor progression.

    Omholt K, Platz A, Kanter L, Ringborg U and Hansson J

    Cancer Centre Karolinska, Department of Oncology-Pathology, Radiumhemmet, Karolinska Hospital and Institute, Stockholm, Sweden.

    Purpose: Recently, it was reported that BRAF mutations are frequent in melanoma. Previously, we analyzed a large series of paired primary and metastatic melanomas for NRAS codon 61 mutations and showed that they arise early and are preserved during tumor progression. Here, we have screened the same tumor samples for BRAF mutations.

    Primary melanomas (n = 71) and corresponding metastases (n = 88) from 71 patients were screened for BRAF exon 11 and exon 15 mutations using single-strand conformational polymorphism and nucleotide sequence analysis

    Results: BRAF mutations were found in 42 of 71 patients (59%). Thirty-seven patients had mutations that lead to a Val599Glu change, whereas mutations resulting in Gly468Ser, Val599Arg, Val599Lys, and Lys600Glu changes were detected in one patient each. Furthermore, one patient had a 6-bp insertion between codons 598 and 599, encoding two threonine residues. In most cases, paired primary and metastatic lesions had the same BRAF genotype (i.e., mutations present in the primary tumors were preserved in the corresponding metastases, and mutations did not arise at the metastatic stage if they were not present in the primary lesion). Using laser-capture microdissection, BRAF mutations were found in the radial growth phase of the primary lesions. BRAF mutations occurred exclusively in tumors that were wild type for NRAS, and in total, 89% of the patients analyzed (63 of 71) had mutations in either of these two genes.

    Conclusions: The Ras-Raf-mitogen-activated protein kinase/extracellular signal-regulated kinase-extracellular signal-regulated kinase signaling pathway is activated in the vast majority of melanomas. Activation occurs through either NRAS or BRAF mutations, both of which arise early during melanoma pathogenesis and are preserved throughout tumor progression.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2003;9;17;6483-8

  • The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain.

    Radziwill G, Erdmann RA, Margelisch U and Moelling K

    Institute of Medical Virology, University of Zurich, CH-8028 Zurich, Switzerland.

    The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state.

    Molecular and cellular biology 2003;23;13;4663-72

  • Frequency of UV-inducible NRAS mutations in melanomas of patients with germline CDKN2A mutations.

    Eskandarpour M, Hashemi J, Kanter L, Ringborg U, Platz A and Hansson J

    Department of Oncology-Pathology, Cancer Center Karolinska, Karolinska Hospital and Karolinska Institute, Stockholm, Sweden.

    Background: Germline alterations in cyclin-dependent kinase inhibitor 2A (CDKN2A) are important genetic factors in familial predisposition to melanoma. Activating mutations of the NRAS proto-oncogene are among the most common somatic genetic alterations in cutaneous malignant melanomas. We investigated the occurrence of NRAS mutations in melanomas and dysplastic nevi in individuals with germline CDKN2A mutations.

    Methods: Genomic DNA was extracted from 39 biopsy samples (including primary melanomas, metastatic melanomas, and dysplastic nevi) from 25 patients in six Swedish families with a hereditary predisposition to melanoma who carried germline CDKN2A mutations. DNA was also extracted from 10 biopsy samples from patients with sporadic melanomas. NRAS was analyzed using polymerase chain reaction, single-strand conformation polymorphism analysis, and nucleotide sequence analysis. Differences in NRAS mutation frequency between hereditary and sporadic melanomas were analyzed by the chi-square test. All statistical tests were two-sided.

    Results: Activating mutations in NRAS codon 61, all of which were either CAA(Gln)-AAA(Lys) or CAA(Gln)-CGA(Arg) mutations, were found in 95% (20/21) of primary hereditary melanomas but in only 10% (1/10) of sporadic melanomas (P<.001). Multiple activating NRAS mutations were detected in tumor cells from different regions of individual primary melanomas in nine patients. Activating mutations that were detected in the primary melanomas of these patients were also retained in their metastases. NRAS mutations at sites other than codon 61 were also present in the primary melanomas, indicating genetic instability of this locus. NRAS codon 61 mutations were also detected in dysplastic nevi and in an in situ melanoma, suggesting a role for such mutations during early melanoma development.

    Conclusions: The high frequency of NRAS codon 61 mutations detected in these hereditary melanomas may be the result of a hypermutability phenotype associated with a hereditary predisposition for melanoma development in patients with germline CDKN2A mutations.

    Journal of the National Cancer Institute 2003;95;11;790-8

  • High density lipoprotein-induced angiogenesis requires the activation of Ras/MAP kinase in human coronary artery endothelial cells.

    Miura S, Fujino M, Matsuo Y, Kawamura A, Tanigawa H, Nishikawa H and Saku K

    Department of Cardiology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Jonan-Ku, Fukuoka, 814-0180, Japan. miuras@cis.fukuoka-u.ac.jp

    Objective: Plasma high density lipoprotein (HDL) levels have been shown to be inversely correlated with coronary artery disease, but the mechanisms of the direct protective effect of HDL on endothelial cells (ECs) are not fully understood. In this study, we investigated the role of the HDL-mediated promotion of angiogenesis in human coronary artery ECs (HCECs).

    We developed an in vitro model of HCEC tube formation on a matrix gel. We optimized the maximum dose of HDL required to induce tube formation in initial experiments, in which the dose response showed that the maximum effective dose of HDL was 100 microg/mL. PD98059, an inhibitor of p42/44 mitogen-activated protein kinase (MAPK) activity, but not SB203580, an inhibitor of p38 MAPK activity, suppressed HDL-induced tube formation. Dominant-negative Ras N17 inhibited HDL-induced tube formation. HDL activated Ras according to a ras pull-down assay, and this effect was inhibited by pertussis toxin. Moreover, HDL activated phospho(p)-p42/44 MAPK, whereas Ras N17 blocked HDL-induced pp42/44 MAPK.

    Conclusions: These results indicate that HDL induced a potent signal through a Ras/MAPK pathway mediated by a pertussis toxin-sensitive G-protein coupled receptor to the angiogenic phenotype in HCECs.

    Arteriosclerosis, thrombosis, and vascular biology 2003;23;5;802-8

  • RAS point mutations and PAX8-PPAR gamma rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma.

    Nikiforova MN, Lynch RA, Biddinger PW, Alexander EK, Dorn GW, Tallini G, Kroll TG and Nikiforov YE

    Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0529, USA.

    A series of 88 conventional follicular and Hürthle cell thyroid tumors were analyzed for RAS mutations and PAX8-PPAR gamma rearrangements using molecular methods and for galectin-3 and HBME-1 expression by immunohistochemistry. A novel LightCycler technology-based method was developed to detect point mutations in codons 12/13 and 61 of the H-RAS, K-RAS, and N-RAS genes. Forty-nine percent of conventional follicular carcinomas had RAS mutations, 36% had PAX8-PPAR gamma rearrangement, and only one (3%) had both. In follicular adenomas, 48% had RAS mutations, 4% had PAX8-PPAR gamma rearrangement, and 48% had neither. Follicular carcinomas with PAX8-PPAR gamma typically showed immunoreactivity for galectin-3 but not for HBME-1, tended to present at a younger patient age and be smaller size, and were almost always overtly invasive. In contrast, follicular carcinomas with RAS mutations most often displayed an HBME-1-positive/galectin-3-negative immunophenotype and were either minimally or overtly invasive. Hürthle cell tumors infrequently had PAX8-PPAR gamma rearrangement or RAS mutations. These results suggest that conventional follicular thyroid carcinomas develop through at least two distinct and virtually nonoverlapping molecular pathways initiated by either RAS point mutation or PAX8-PPAR gamma rearrangement.

    Funded by: NCI NIH HHS: CA75425, R01-CA88041; NCRR NIH HHS: M01 RR08084

    The Journal of clinical endocrinology and metabolism 2003;88;5;2318-26

  • Activation of N-ras and K-ras induced by interleukin-6 in a myeloma cell line: implications for disease progression and therapeutic response.

    Rowley M and Van Ness B

    Graduate Program in Molecular, Cellular, Developmental Biology and Genetics, The University of Minnesota, Minneapolis, Minnesota, MN 55455, USA.

    Ras is a major signaling molecule activated by interleukin-6. There have been no published reports, however, that specifically examine the kinetics and percentage of Ras activation in response to IL-6. Model cell lines were used to study activation of N- and K-ras induced by IL-6. All of the myeloma cell lines we tested express both N-ras and K-ras, but not H-ras. GTP-bound Ras was measured and the percentage of the total Ras pool that was activated in response to IL-6 was calculated. IL-6 is able to transiently activate both N- and K-ras in the ANBL6 cell line. In addition, increasing concentrations of IL-6 are able to activate increasing levels of both N- and K-ras. One ng/ml of IL-6 is able to activate approximately 10% of the N-ras pool and 18% of the K-ras pool. The amount of Ras-GTP in the cells correlates with the level of proliferation at low levels, but proliferation plateaus when higher levels of Ras-GTP are present. Protection from dexamethasone-induced apoptosis correlates with IL-6 concentration and Ras activation. However, IL-6 enhances apoptosis induced by doxorubicin. Interestingly, the ANBL6 cell line transfected with an N-ras12 or a K-ras12 gene is protected from doxorubicin-induced apoptosis.

    Funded by: NCI NIH HHS: P01CA62242

    Oncogene 2002;21;57;8769-75

  • Protein kinase C mediates mutant N-Ras-induced developmental abnormalities in normal human erythroid cells.

    Darley RL, Pearn L, Omidvar N, Sweeney M, Fisher J, Phillips S, Hoy T and Burnett AK

    Leukaemia Research Fund Differentiation Group, Department of Haematology, University of Wales College of Medicine, Cardiff, United Kingdom. darley@cf.ac.uk

    RAS mutations are one of the most frequent molecular abnormalities associated with myeloid leukemia and preleukemia, yet there is a poor understanding of how they contribute to the pathogenesis of these conditions. Here, we describe the consequences of ectopic mutant N-Ras (N-Ras*) expression on normal human erythropoiesis. We show that during early (erythropoietin [EPO]-independent) erythropoiesis, N-Ras* promoted the amplification of a phenotypically primitive but functionally defective subpopulation of CD34(+) erythroblasts. N-Ras* also up-regulated the expression of megakaryocyte antigens on human erythroblasts. Although early erythroblasts expressing N-Ras* were able to respond to erythropoietin and generate mature progeny, this occurred with greatly reduced efficiency, probably explaining the poor colony growth characteristics of these cells. We further report that this oncogene promoted the expression and activation of protein kinase C (PKC) and that the effects of N-Ras* on erythropoiesis could be abrogated or attenuated by inhibition of PKC. Similarly, the effects of this oncogene could be partially mimicked by treatment with PKC agonist. Together, these data suggest that expression of N-Ras* is able to subvert the normal developmental cues that regulate erythropoiesis by activating PKC. This gives rise to phenotypic and functional abnormalities commonly observed in preleukemia, suggesting a direct link between RAS mutations and the pathogenesis of preleukemia.

    Blood 2002;100;12;4185-92

  • Regulation of Fas-mediated apoptosis by N-ras in melanoma.

    Urquhart JL, Meech SJ, Marr DG, Shellman YG, Duke RC and Norris DA

    University of Colorado Health Sciences Center, Department of Dermatology, Denver, Colorado 80262, USA. jean.urquhart@uchsc.edu

    Oncogenic ras has been shown to downregulate Fas receptor expression and increase Fas ligand expression and thus contribute to resistance to Fas-mediated cell death in several cell types. The effects of ras on Fas-mediated apoptosis have not been studied in melanoma. We studied the effects of activated N-ras by measuring Fas, Fas ligand, and FLIP expression as well as susceptibility to Fas-ligand-induced cell death in transfectants of WM35, a radial growth phase human melanoma cell line. Based on quantitative polymerase chain reaction and fluorescence-activated cell sorter analysis, we found that the ras transfectants expressed less Fas mRNA and surface Fas receptor. Cr51 release cytotoxicity assays demonstrated less susceptibility to Fas-mediated apoptosis in ras transfectants, correlating with the Fas mRNA and protein expression results. Ras inhibition with the specific inhibitor FTI-277 showed that downregulation of Fas in the ras transfectants could be reversed. This correlates with cytotoxicity experiments showing that ras inhibition increases susceptibility to Fas-mediated apoptosis. The control transfectants expressed FLIP but ras did not affect FLIP expression. The control and ras transfectants did not express Fas ligand as demonstrated by reverse transcriptase polymerase chain reaction and fluorescence-activated cell sorter analysis. Cytotoxicity assays further confirmed that these melanoma ras transfectants do not express functional Fas ligand. These results suggest that ras contributes to tumor progression by decreasing susceptibility to Fas-mediated cell death at least in part through downregulation of Fas receptor at the transcriptional level.

    Funded by: NCI NIH HHS: CA46934; NIAMS NIH HHS: R01 AR26427-16, T32 AR07411-20

    The Journal of investigative dermatology 2002;119;3;556-61

  • Multiple stages of malignant transformation of human endothelial cells modelled by co-expression of telomerase reverse transcriptase, SV40 T antigen and oncogenic N-ras.

    MacKenzie KL, Franco S, Naiyer AJ, May C, Sadelain M, Rafii S and Moore MA

    James Ewing Laboratory of Developmental Haematopoiesis and Department of Human Genetics, Sloan-Kettering Cancer Institute, New York, NY, USA. k.mackenzie@unsw.edu.au

    We have modelled multiple stages of malignant transformation of human endothelial cells (ECs) by overexpressing the catalytic subunit of human telomerase (hTERT), together with SV40 T antigen (SV40T) and oncogenic N-ras. Transfection with hTERT alone, led to the immortalization of two out of three cultures of bone marrow-derived ECs (BMECs). One hTERT transduced BMEC culture underwent a long proliferative lag before resuming proliferation. BMECs transfected with hTERT alone were functionally and phenotypically normal. BMECs transfected with SV40T (BMSVTs) had an extended lifespan, but eventually succumbed to crisis. BMSVTs exhibited a partially transformed phenotype, demonstrating growth factor independence, altered antigen expression and forming tiny, infrequent colonies in vitro. Transduction of BMSVTs with hTERT resulted in immortalization of 4 out of 4 cultures. BMSVTs immortalized with hTERT formed large colonies in vitro and small transient tumours in vivo. BMECs co-expressing SV40T, hTERT and N-ras exhibited an overtly transformed phenotype; forming very large colonies with an altered morphology and generating rapidly growing tumours in vivo. These investigations demonstrate transformation of human ECs to an overtly malignant phenotype. This model will be useful for understanding mechanisms underlying vascular and angiogenic neoplasias, as well as for testing drugs designed to curtail aberrant EC growth.

    Funded by: NCI NIH HHS: CA59350; NHLBI NIH HHS: HL 61401

    Oncogene 2002;21;27;4200-11

  • SmgGDS displays differential binding and exchange activity towards different Ras isoforms.

    Vikis HG, Stewart S and Guan KL

    Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, MI 48109-0606, USA.

    Ras family GTPases play central roles in a wide variety of biological responses, including cell proliferation, differentiation, and oncogenic transformation. We searched for novel guanine nucleotide exchange factors of HRas and isolated small G-protein dissociation stimulator (smgGDS), a guanine nucleotide exchange factor known to act on numerous Ras and Rho family GTPases. SmgGDS specifically interacts with both dominant negative and nucleotide free forms of H and NRas, but not with the corresponding oncogenic forms. An effector domain mutant of HRas, HRasN17G37, selectively lost the ability to bind smgGDS. However, smgGDS does not catalyze guanine nucleotide exchange on either H or NRas in vitro. In contrast, substrates of smgGDS, such as KRas, Rac1, and RhoA, bind to smgGDS in both active and inactive forms which requires the presence of poly-basic residues in the C-termini of the GTPases. Our data suggest that the C-terminal poly-basic region of small GTPases is important for both binding and nucleotide exchange by smgGDS. Furthermore, these data underscore the idea that mammalian Ras isoforms are not functionally equivalent.

    Oncogene 2002;21;15;2425-32

  • High incidence of N and K-Ras activating mutations in multiple myeloma and primary plasma cell leukemia at diagnosis.

    Bezieau S, Devilder MC, Avet-Loiseau H, Mellerin MP, Puthier D, Pennarun E, Rapp MJ, Harousseau JL, Moisan JP and Bataille R

    Laboratoire de Génétique Moléculaire, Institut de Biologie, Nantes, France.

    Using allele-specific amplification method (ARMS), a highly sensitive one-stage allele-specific PCR, we have evaluated the incidence of NRAS and KRAS2 activating mutations (codons 12, 13, and 61) in 62 patients with either monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM), primary plasma-cell leukemia (P-PCL), and also in human myeloma cell lines (HMCL). NRAS and/or KRAS2 mutations were found in 54.5% of MM at diagnosis (but in 81% at the time of relapse), in 50% of P-PCL, and in 50% of 16 HMCL. In contrast, the occurrence of such mutations was very low in MGUS and indolent MM (12.50%). Of note, KRAS2 mutations were always more frequent than NRAS. The validity of the technique was assessed by direct sequencing of cell lines and of some patients. Multiple mutations found in two patients were confirmed by subcloning exon PCR amplification products, testing clones with our method, and sequencing them. Thus, these early mutations could play a major role in the oncogenesis of MM and P-PCL.

    Human mutation 2001;18;3;212-24

  • Isolation and characterization of a symbiosis-regulated ras from the ectomycorrhizal fungus Laccaria bicolor.

    Sundaram S, Kim SJ, Suzuki H, Mcquattie CJ, Hiremah ST and Podila GK

    Department of Biological Sciences, Michigan Technological University, Houghton 49931, USA.

    Ectomycorrhizae formed by the symbiotic interaction between ectomycorrhizal fungi and plant roots play a key role in maintaining and improving the health of a wide range of plants. Mycorrhizal initiation, development, and functional maintenance involve morphological changes that are mediated by activation and suppression of several fungal and plant genes. We identified a gene, Lbras, in the ectomycorrhizal fungus Laccaria bicolor that belongs to the ras family of genes, which has been shown in other systems to be associated with signaling pathways controlling cell growth and proliferation. The Lbras cDNA complemented ras2 function in Saccharomyces cerevisiae and had the ability to transform mammalian cells. Expression of Lbras, present as a single copy in the genome, was dependent upon interaction with host roots. Northern analysis showed that expression was detectable in L bicolor 48 h after interaction as well as in the established mycorrhizal tissue. Phylogenetic analysis with other Ras proteins showed that Lbras is related most closely to Aras of Aspergillus nidulans.

    Molecular plant-microbe interactions : MPMI 2001;14;5;618-28

  • Nerve growth factor signaling, neuroprotection, and neural repair.

    Sofroniew MV, Howe CL and Mobley WC

    Department of Neurobiology and Brain Research Institute, University of California Los Angeles, Los Angeles, California 90095-1763, USA. sofroniew@mednet.ucla.edu

    Nerve growth factor (NGF) was discovered 50 years ago as a molecule that promoted the survival and differentiation of sensory and sympathetic neurons. Its roles in neural development have been characterized extensively, but recent findings point to an unexpected diversity of NGF actions and indicate that developmental effects are only one aspect of the biology of NGF. This article considers expanded roles for NGF that are associated with the dynamically regulated production of NGF and its receptors that begins in development, extends throughout adult life and aging, and involves a surprising variety of neurons, glia, and nonneural cells. Particular attention is given to a growing body of evidence that suggests that among other roles, endogenous NGF signaling subserves neuroprotective and repair functions. The analysis points to many interesting unanswered questions and to the potential for continuing research on NGF to substantially enhance our understanding of the mechanisms and treatment of neurological disorders.

    Funded by: NINDS NIH HHS: NS24054

    Annual review of neuroscience 2001;24;1217-81

  • Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma.

    Pacold ME, Suire S, Perisic O, Lara-Gonzalez S, Davis CT, Walker EH, Hawkins PT, Stephens L, Eccleston JF and Williams RL

    MRC Laboratory of Molecular Biology Hills Road CB2 2QH, Cambridge, United Kingdom.

    Ras activation of phosphoinositide 3-kinase (PI3K) is important for survival of transformed cells. We find that PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPgammaS-loaded H-Ras in vitro. We have determined a crystal structure of a PI3Kgamma/Ras.GMPPNP complex. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3K conformation that may represent an allosteric component of Ras activation.

    Funded by: Medical Research Council: MC_U105184308

    Cell 2000;103;6;931-43

  • Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor.

    Clyde-Smith J, Silins G, Gartside M, Grimmond S, Etheridge M, Apolloni A, Hayward N and Hancock JF

    Queensland Cancer Fund Laboratory of Experimental Oncology, Department of Pathology, University of Queensland Medical School, Herston Road, Brisbane 4006, Queensland, Australia.

    Ras proteins operate as molecular switches in signal transduction pathways downstream of tyrosine kinases and G-protein-coupled receptors. Ras is switched from the inactive GDP-bound state to the active GTP-bound state by guanine nucleotide exchange factors (GEFs). We report here the cloning and characterization of RasGRP2, a longer alternatively spliced form of the recently cloned RapGEF, CalDAG-GEFI. A unique feature of RasGRP2 is that it is targeted to the plasma membrane by a combination of N-terminal myristoylation and palmitoylation. In vivo, RasGRP2 selectively catalyzes nucleotide exchange on N- and Ki-Ras, but not Ha-Ras. RasGRP2 also catalyzes nucleotide exchange on Rap1, but this RapGEF activity is less potent than that associated with CalDAG-GEFI. The nucleotide exchange activity of RasGRP2 toward N-Ras is stimulated by diacylglycerol and inhibited by calcium. The effects of diacylglycerol and calcium are additive but are not accompanied by any detectable change in the subcellular localization of RasGRP2. In contrast, CalDAG-GEFI is localized predominantly to the cytosol and lacks Ras exchange activity in vivo. However, prolonged exposure to phorbol esters, or growth in serum, results in localization of CalDAG-GEFI to the cell membrane and restoration of Ras exchange activity. Expression of RasGRP2 or CalDAG-GEFI in NIH3T3 cells transfected with wild type N-Ras results in an accelerated growth rate but not morphologic transformation. Thus, under appropriate growth conditions, CalDAG-GEFI and RasGRP2 are dual specificity Ras and Rap exchange factors.

    The Journal of biological chemistry 2000;275;41;32260-7

  • Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1.

    Kiyono M, Kato J, Kataoka T, Kaziro Y and Satoh T

    Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

    Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.

    The Journal of biological chemistry 2000;275;38;29788-93

  • The leucine-rich repeat protein SUR-8 enhances MAP kinase activation and forms a complex with Ras and Raf.

    Li W, Han M and Guan KL

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 USA.

    Caenorhabditis elegans sur-8 encodes a positive regulator of Ras signaling. We investigated the mechanism by which the human Sur-8 homolog can positively regulate Ras-MAP kinase signaling in mammalian cells. Sur-8 expression enhances Ras- or EGF-induced Raf and ERK activation but has no effect on ERK activation induced by active Raf or MEK. Furthermore, Sur-8 expression does not increase AKT or JNK activation. Sur-8 interacts with Ras and Raf and is able to form a ternary complex with the two proteins. Thus, Sur-8 may function as a scaffold that enhances Ras-MAP kinase signal transduction by facilitating the interaction between Ras and Raf.

    Genes & development 2000;14;8;895-900

  • The Ras branch of small GTPases: Ras family members don't fall far from the tree.

    Reuther GW and Der CJ

    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, North Carolina 27599-7295, USA. greuther@med.unc.edu

    The Ras branch of the Ras superfamily consists of small GTPases most closely related to Ras and include the R-Ras, Rap, Ral, Rheb, Rin and Rit proteins. Although our understanding of Ras signaling and biology is now considerable, recent observations suggest that Ras function is more complex than previously believed. First, the three Ras proteins may not be functionally identical. Second, Ras function involves functional cross-talk with their close relatives.

    Funded by: NCI NIH HHS: CA42978, CA55008, CA63071

    Current opinion in cell biology 2000;12;2;157-65

  • A genome-wide survey of RAS transformation targets.

    Zuber J, Tchernitsa OI, Hinzmann B, Schmitz AC, Grips M, Hellriegel M, Sers C, Rosenthal A and Schäfer R

    [1] Laboratory of Molecular Tumour Pathology, Institute of Pathology, Charité, Humboldt-University D-10117, Berlin, Germany.

    An important aspect of multi-step tumorigenesis is the mutational activation of genes of the RAS family, particularly in sporadic cancers of the pancreas, colon, lung and myeloid system. RAS genes encode small GTP-binding proteins that affect gene expression in a global way by acting as major switches in signal transduction processes, coupling extracellular signals with transcription factors. Oncogenic forms of RAS are locked in their active state and transduce signals essential for transformation, angiogenesis, invasion and metastasis via downstream pathways involving the RAF/MEK/ERK cascade of cytoplasmic kinases, the small GTP-binding proteins RAC and RHO, phosphatidylinositol 3-kinase and others. We have used subtractive suppression hybridization (SSH), a PCR-based cDNA subtraction technique, to contrast differential gene expression profiles in immortalized, non-tumorigenic rat embryo fibroblasts and in HRAS- transformed cells. Sequence and expression analysis of more than 1,200 subtracted cDNA fragments revealed transcriptional stimulation or repression of 104 ESTs, 45 novel sequences and 244 known genes in HRAS- transformed cells compared with normal cells. Furthermore, we identified common and distinct targets in cells transformed by mutant HRAS, KRAS and NRAS, as well as 61 putative target genes controlled by the RAF/MEK/ERK pathway in reverted cells treated with the MEK-specific inhibitor PD 98059.

    Nature genetics 2000;24;2;144-52

  • Bcl-2 differentially targets K-, N-, and H-Ras to mitochondria in IL-2 supplemented or deprived cells: implications in prevention of apoptosis.

    Rebollo A, Pérez-Sala D and Martínez-A C

    Department of Immunology and Oncology, Centro Nacional de Biotecnología, Universidad Autónoma, Campus de Cantoblanco, E-28049 Madrid, Spain.

    IL-2 deprivation triggers apoptosis in the murine T cell line TS1alphabeta, a process that can be blocked by overexpression of Bcl-2. Here we show that Bcl-2 and Ras proteins interact in mitochondria from TS1alphabeta cells in the presence or absence of IL-2, as evidenced by co-immunoprecipitation. All three Ras proteins, K-, N- and H-Ras, interact with Bcl-2; however, their mitochondrial localization is differentially regulated in IL-2-supplemented or -deprived cells. K-Ras is found in mitochondria only in IL-2-supplemented cells, whereas H-Ras is observed in mitochondria only after IL-2 withdrawal. N-Ras is detected in mitochondria under both experimental conditions. Bcl-2 transfection partially restored K- and N-Ras association with mitochondria in IL-2-deprived cells and rendered H-Ras association independent of IL-2 withdrawal. Inhibitors of Ras posttranslational processing did not alter the IL-2-induced differential pattern of mitochondrial localization. The processed forms of K- and N-Ras associated with mitochondria, although unprocessed H-Ras was also detected in mitochondria from mevastatin-treated cells. These results evidence a distinct behavior among the three Ras proteins in TS1alphabeta cells, depending on IL-2 supply, and suggest homologue-specific roles for Ras proteins in IL-2-dependent events.

    Oncogene 1999;18;35;4930-9

  • Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi.

    Choy E, Chiu VK, Silletti J, Feoktistov M, Morimoto T, Michaelson D, Ivanov IE and Philips MR

    Department of Medicine, New York University School of Medicine, New York 10016, USA.

    We show that Nras is transiently localized in the Golgi prior to the plasma membrane (PM). Moreover, green fluorescent protein (GFP)-tagged Nras illuminated motile, peri-Golgi vesicles, and prolonged BFA treatment blocked PM expression. GFP-Hras colocalized with GFP-Nras, but GFP-Kras4B revealed less Golgi and no vesicular fluorescence. Whereas a secondary membrane targeting signal was required for PM expression, the CAAX motif alone was necessary and sufficient to target proteins to the endomembrane where they were methylated, a modification required for efficient membrane association. Thus, prenylated CAAX proteins do not associate directly with the PM but instead associate with the endomembrane and are subsequently transported to the PM, a process that requires a secondary targeting motif.

    Funded by: NCRR NIH HHS: M01 RR00096; NIAID NIH HHS: AI36224, R01 AI036224; NIGMS NIH HHS: GM55279

    Cell 1999;98;1;69-80

  • The structural basis of the activation of Ras by Sos.

    Boriack-Sjodin PA, Margarit SM, Bar-Sagi D and Kuriyan J

    Laboratory of Molecular Biophysics, The Rockefeller University, New York, New York 10021, USA.

    The crystal structure of human H-Ras complexed with the Ras guanine-nucleotide-exchange-factor region of the Son of sevenless (Sos) protein has been determined at 2.8 A resolution. The normally tight interaction of nucleotides with Ras is disrupted by Sos in two ways. First, the insertion into Ras of an alpha-helix from Sos results in the displacement of the Switch 1 region of Ras, opening up the nucleotide-binding site. Second, side chains presented by this helix and by a distorted conformation of the Switch 2 region of Ras alter the chemical environment of the binding site for the phosphate groups of the nucleotide and the associated magnesium ion, so that their binding is no longer favoured. Sos does not impede the binding sites for the base and the ribose of GTP or GDP, so the Ras-Sos complex adopts a structure that allows nucleotide release and rebinding.

    Nature 1998;394;6691;337-43

  • SUR-8, a conserved Ras-binding protein with leucine-rich repeats, positively regulates Ras-mediated signaling in C. elegans.

    Sieburth DS, Sun Q and Han M

    Howard Hughes Medical Institute, Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, 80309-0347, USA.

    We describe the identification and characterization of a novel gene, sur-8, that positively regulates Ras-mediated signal transduction during C. elegans vulval development. Reduction of sur-8 function suppresses an activated ras mutation and dramatically enhances phenotypes of mpk-1 MAP kinase and ksr-1 mutations, while increase of sur-8 dosage enhances an activated ras mutation. sur-8 appears to act downstream of or in parallel to ras but upstream of raf. sur-8 encodes a conserved protein that is composed predominantly of leucine-rich repeats. The SUR-8 protein interacts directly with Ras but not with the Ras(P34G) mutant protein, suggesting that SUR-8 may mediate its effects through Ras binding. A structural and functional SUR-8 homolog in humans specifically binds K-Ras and N-Ras but not H-Ras in vitro.

    Funded by: NIGMS NIH HHS: GM47869

    Cell 1998;94;1;119-30

  • Identification of Nore1 as a potential Ras effector.

    Vavvas D, Li X, Avruch J and Zhang XF

    The Diabetes Unit and Medical Services and the Department of Medicine, Harvard Medical School, Massachusetts General Hospital East, Charlestown, Massachusetts 02129, USA.

    The small GTP-binding protein Ras is pivotal in transmitting growth and differentiation signals downstream of cell surface receptors. Many observations have indicated that Ras transmits signals from cell surface receptors into multiple pathways via direct interaction with different effectors in mammalian cells. We have identified a novel potential Ras effector or target named Nore1. Nore1 has no significant sequence similarity to known mammalian proteins and lacks an identifiable catalytic domain, but contains sequence motifs that predict DAG_PE binding and SH3 domain binding. We show that Nore1 directly interacts with Ras in vitro in a GTP-dependent manner, and the interaction requires an intact Ras effector domain. Nore1 becomes associated with Ras in situ following activation of epidermal growth factor receptor in COS-7 and in KB cells.

    Funded by: NIGMS NIH HHS: GM51281

    The Journal of biological chemistry 1998;273;10;5439-42

  • Signals transduced through the CD4 molecule interfere with TCR/CD3-mediated ras activation leading to T cell anergy/apoptosis.

    Tamma SM, Chirmule N, McCloskey TW, Oyaizu N, Kalyanaraman VS and Pahwa S

    Department of Pediatrics, North Shore University Hospital-New York University School of Medicine, Manhasset, New York 11030, USA.

    It has been previously demonstrated that the occupancy of CD4 molecules by the HIV-1 envelope glycoprotein gp120 results in marked inhibition of T cell receptor-CD3 complex (TCR/CD3) activation-induced IL-2 secretion. To elucidate the mechanism of inhibitory effects of gp160 on T cell signaling, we have investigated the intracellular biochemical events and biological output in response to anti-CD3 mAb activation of purified peripheral blood CD4+ T cells from healthy donors with and without prior exposure to HIV-1 gp160. Pretreatment with gp160 resulted in marked inhibition of tyrosine phosphorylation of p59(fyn), PLC-gamma1, ras activation, and TNF-alpha secretion in anti-CD3 mAb activated CD4+ T cells, and a subset of CD4+ cells underwent activation-induced cell death. The data presented here provide insight into the mechanism by which the interaction of HIV-1 envelope glycoproteins with CD4 molecules may alter TCR/CD3-activation-induced signal transduction resulting in anergy and apoptosis with consequent functional deficiency of CD4+ T cells.

    Funded by: NIAID NIH HHS: AI28281; NIDA NIH HHS: DA 05161

    Clinical immunology and immunopathology 1997;85;2;195-201

  • CD4 cross-linking (CD4XL) induces RAS activation and tumor necrosis factor-alpha secretion in CD4+ T cells.

    Tamma SM, Chirmule N, Yagura H, Oyaizu N, Kalyanaraman V and Pahwa S

    Department of Pediatrics, North Shore University Hospital, New York University School of Medicine, Manhasset, NY 11030, USA.

    CD4 molecules are the primary receptors for human immunodeficiency virus (HIV) and bind the envelope glycoprotein gp120 of HIV with high-affinity. We have previously shown that cross-linking of CD4 molecules (CD4XL) in normal peripheral blood mononuclear cells (PBMC) results in secretion of cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not of interleukin-2 (IL-2) or IL-4. To investigate the intracellular signaling events associated with CD4-gp120 interaction, we incubated CD4+ T cells from peripheral blood of HIV-negative healthy donors with HIV envelope protein gp160 alone or performed CD4XL with gp160 and anti-gp160 antibody. This procedure resulted in tyrosine phosphorylation of intracellular substrates p59fyn, zap 70, and p95vav and also led to ras activation, as assessed by conversion of rasGDP to rasGTP. The role of ras in CD4 signaling was further investigated using CD4+ Jurkat cells transfected with a dominant negative ras mutant. CD4+ T cells expressing dn-ras secreted significantly reduced levels of TNF-alpha in response to CD4XL. These studies indicate that interaction of HIV gp160 with CD4 molecules activates the ras pathway in T cells, which may result in the cells becoming unresponsive to subsequent stimulation.

    Funded by: NIAID NIH HHS: AI 128281; NIDA NIH HHS: DA 05161

    Blood 1997;90;4;1588-93

  • Ras effectors and their role in mitogenesis and oncogenesis.

    Joneson T and Bar-Sagi D

    Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook 11794, USA.

    Ras proteins are membrane-bound GTP-binding proteins that play a critical role in the control of cell growth. Through a large number of genetic and biochemical studies it is becoming increasingly evident that the biological activity of Ras proteins is mediated by multiple signaling pathways. This review provides an account of the target proteins that interact with Ras and the functional consequences of these interactions. The relative contribution of the different Ras effector pathways to the mitogenic and oncogenic effects of Ras are discussed.

    Journal of molecular medicine (Berlin, Germany) 1997;75;8;587-93

  • UV-induced N-ras mutations are T-cell targets in human melanoma.

    van Elsas A, Scheibenbogen C, van der Minne C, Zerp SF, Keilholz U and Schrier PI

    Department of Clinical Oncology, University Hospital Leiden, The Netherlands.

    Human cutaneous melanoma is heterogeneous with respect to the genetic aberrations involved and the genes altered are potential targets for the immune system. The incidence of cutaneous melanoma is known to be linked to UV peak exposure, and the N-ras oncogene is clearly one of the genes involved in the UV carcinogenesis in melanoma. It is mutated in a significant proportion of melanomas and therefore may serve as a target for T cells. Here, we report that an human leukocyte antigen-A2 binding peptide CLLDILDTAGL, encompassing the frequently found 61-Leu mutation in N-ras, induces cytotoxic T lymphocytes from healthy donor blood that lyse 61-Leu N-ras transfected melanoma cells. Furthermore, we have found an association between the presence of N-ras mutations and clinical response to immunotherapy with interleukin-2 plus interferon in a group of stage IV melanoma patients. Although the overall survival of these patients was not affected by the N-ras status of their melanomas, these studies suggest that mutated N-ras may provide a target for cytotoxic T lymphocytes in melanoma patients.

    Melanoma research 1997;7 Suppl 2;S107-13

  • The complexity of Raf-1 regulation.

    Morrison DK and Cutler RE

    Molecular Basis of Carcinogenesis Laboratory, ABL-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.

    The activation of the serine/threonine kinase Raf-1 is proving to be an intricate multistep process. Recent advances in elucidating how Raf-1 becomes activated in response to signaling events have emphasized the role of phosphorylation and protein interactions in Raf-1 regulation. The picture clearly emerging is that Raf-1 activity can be regulated by multiple mechanisms.

    Current opinion in cell biology 1997;9;2;174-9

  • Signal transduction by the neurotrophin receptors.

    Kaplan DR and Miller FD

    Brain Tumor Research Centre, Montreal Neurological Institute, 3801 University Street, Montreal, PQ, Canada, H3A 2B4. mcdv@musica.mcgill.ca

    The neurotrophins signal cell survival, differentiation, growth cessation, and apoptosis through two cell surface receptors, the Trks and p75NTR (p75 neurotrophin receptor). Recent advances indicate that the particular events that are mediated by neurotrophins are dependent upon the cell type and the expression pattern of each neurotrophin receptor. For example, TrkA activation induces cell death of neural tumor cells, and survival and differentiation of neurons. Likewise, p75NTR, when activated in the absence of a strong Trk signal, induces apoptosis of neurons, while in the presence of Trk it enhances responses to neurotrophin. These differing responses point to a complex interplay between neurotrophin-stimulated survival, differentiation, and apoptosis pathways.

    Current opinion in cell biology 1997;9;2;213-21

  • Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation.

    Rodriguez-Viciana P, Warne PH, Vanhaesebroeck B, Waterfield MD and Downward J

    Imperial Cancer Research Fund, London.

    We have reported previously that Ras interacts with the catalytic subunit of phosphoinositide 3-kinase (PI 3-kinase) in a GTP-dependent manner. The affinity of the interaction of Ras-GTP with p85alpha/p110alpha is shown here to be approximately 150 nM. The site of interaction on the p110alpha and beta isoforms of PI 3-kinase lies between amino acid residues 133 and 314. A point mutation in this region, K227E, blocks the GTP-dependent interaction of PI 3-kinase p110alpha with Ras in vitro and the ability of Ras to activate PI 3-kinase in intact cells. In addition, this mutation elevates the basal activity of PI 3-kinase in intact cells, suggesting a direct influence of the Ras binding site on the catalytic activity of PI 3-kinase. Using an in vitro reconstitution assay, it is shown that the interaction of Ras-GTP, but not Ras-GDP, with PI 3-kinase leads to an increase in its enzymatic activity. This stimulation is synergistic with the effect of tyrosine phosphopeptide binding to p85, particularly at suboptimal peptide concentrations. These data show that PI 3-kinase is regulated by a number of mechanisms, and that Ras contributes to the activation of this lipid kinase synergistically with tyrosine kinases.

    The EMBO journal 1996;15;10;2442-51

  • Ras-related proteins in signal transduction and growth control.

    McCormick F

    Onyx Pharmaceuticals, Richmond, California 94806, USA.

    Ras proteins are members of a superfamily of small GTPases that are involved in many aspects of cell growth control. The ras p21 protooncogene products, H-ras, K-ras, and N-ras, transmit signals from growth factor receptors to a cascade of protein kinases that begins with the Raf protooncogene product, and leads to alterations in transcription factors and cell cycle proteins in the nucleus. This cascade is controlled at several points: Ras p21 proteins are regulated by GAPs and by exchange factors, whose activities are altered by growth factor receptor activation (Boguski and McCormick, 1993: Nature 366:643-654). Transmission of signals from Ras to Raf is regulated by the Ras-related protein Rap1 (a protein capable of reverting cell transformation) and by cAMP. Other aspects of Ras p21 regulation will be discussed, including the existence of RasGDl proteins that inhibit GDP dissociation from Ras, and may thus regulate the level of active Ras in the cell. The role of Ras in activation of Raf kinase appears to be limited to the recruitment of Raf to the plasma membrane, at which time Raf becomes stably modified to render it active (Leevers et al., 1994: Nature 369:411-414; Stokoe et al., 1994: Science 264:1463-1467). The nature of these modifications is unclear. Raf in the plasma membrane becomes associated with insoluble structural cell components that may be part of the activation. Furthermore, Raf is associated with proteins of the 14-3-3 family that appear necessary for kinase activation. The 14-3-3 proteins interact with all three conserved regions of Raf, including the kinase domain. In addition to Raf, Ras proteins interact with two known classes of proteins in a manner consistent with effector functions: these are the GAPs and regulators of the Ras-related protein Ral referred to as RalGDS. These biochemical data suggest that other functional pathways are regulated by Ras, including, perhaps, pathways involved in regulating cell shape and motility. The protein R-Ras p21 is about 50% identical to the Ras p21 protooncogene product. This protein is incapable of transforming cells, even though it interacts with Raf and other putative Ras effectors (Fernandez-Sarabia and Bischoff, 1993: Nature 366:274-275). On the other hand, it has recently been shown that R-Ras binds to the protooncogene product Bcl-2, a protein that transforms B cells by blocking apoptosis. R-Ras is regulated by the same GAP molecules as H-Ras and the other Ras protooncogene products, and may therefore be activated in a manner co-ordinate with these growth-promoting proteins. The possible connection between R-Ras and apoptosis will be discussed.

    Molecular reproduction and development 1995;42;4;500-6

  • Determination of the gene order of the three loci CD2, NGFB, and NRAS at human chromosome band 1p13 and refinement of their localisation at the subband level by fluorescence in situ hybridisation.

    Mitchell EL, Jones D, White GR, Varley JM and Santibanez Koref MF

    Cancer Research Campaign Department of Cancer Genetics, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK.

    The three loci NRAS, NGFB, and CD2 map to human chromosome band 1p13. Using fluorescence in situ hybridisation (FISH) to simultaneously DAPI-banded metaphase chromosomes, we have further refined the localisation of these three genes to specific subbands. NRAS localises to subband 1p13.2 and CD2 and NGFB to 1p13.1. Also, with the use of multicolour FISH, we have determined the order and orientation of the three loci in relation to the centromere. The order is cen-CD2-NGFB-NRAS.

    Cytogenetics and cell genetics 1995;70;3-4;183-5

  • Activated Ras interacts with the Ral guanine nucleotide dissociation stimulator.

    Hofer F, Fields S, Schneider C and Martin GS

    Department of Molecular and Cell Biology, University of California, Berkeley 94720-3204.

    The yeast two-hybrid system was used to identify proteins that interact with Ras. The H-Ras protein was found to interact with a guanine nucleotide dissociation stimulator (GDS) that has been previously shown to regulate guanine nucleotide exchange on another member of the Ras protein family, Ral. The interaction is mediated by the C-terminal, noncatalytic segment of the RalGDS and can be detected both in vivo, using the two-hybrid system, and in vitro, with purified recombinant proteins. The interaction of the RalGDS C-terminal segment with Ras is specific, dependent on activation of Ras by GTP, and blocked by a mutation that affects Ras effector function. These characteristics are similar to those previously demonstrated for the interaction between Ras and its putative effector, Raf, suggesting that the RalGDS may also be a Ras effector. Consistent with this idea, the RalGDS was found to inhibit the binding of Raf to Ras.

    Funded by: NCI NIH HHS: CA17542, CA28146

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;23;11089-93

  • The activation of phosphatidylinositol 3-kinase by Ras.

    Kodaki T, Woscholski R, Hallberg B, Rodriguez-Viciana P, Downward J and Parker PJ

    Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, London, UK.

    Background: Activation of the mammalian phosphatidylinositol 3-kinase complex can play a critical role in transducing growth factor responses. The lipid kinase complex, which is made up of p85 alpha and p110 alpha regulatory and catalytic subunits, becomes associated with a number of activated receptor protein tyrosine kinases, but the mechanism of its activation has not yet been defined. Recent evidence indicates that Ras can bind to the p85 alpha/p110 alpha complex. We describe here the functional regulation of the mammalian phosphatidylinositol 3-kinase complex by Ras.

    Results: Expression of p110 alpha, the catalytic subunit of phosphatidylinositol 3-kinase, in the fission yeast, Schizosaccharomyces pombe, has been used to demonstrate an inhibitory effect of p85 alpha on p110 alpha activity in intact cells; inhibition did not result from a decrease in p110 alpha expression. In this cellular context, we have investigated the effect of a constitutively active mutant of Ras, v-Ras, either on p85 alpha or p110 alpha-alone, or on the p85 alpha/p110 alpha complex. In the presence of the p85 alpha/p110 alpha complex, v-Ras suppressed cell growth, but an effector-domain mutant of v-Ras did not. The growth-suppressive effect of v-Ras was not seen for any other combination of expressed proteins. The phenotype induced by v-Ras was consistent with activation of the p85 alpha/p110 alpha complex: it was sensitive to the phosphatidylinositol 3-kinase inhibitor, wortmannin, and the cells accumulated 3-phosphorylated polyphosphoinositides. Activation of purified p85 alpha/p110 alpha by purified recombinant Ras in vitro was also demonstrated.

    Conclusions: The phosphatidylinositol 3-kinase complex, p85 alpha/p110 alpha, shows a suppressed catalytic function in vivo when compared with free p110 alpha. This complex can, however, be activated by Ras. We suggest that the phosphatidylinositol 3-kinase p85 alpha/p110 alpha complex is a downstream effector of Ras.

    Current biology : CB 1994;4;9;798-806

  • Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.

    Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH and Bar-Sagi D

    Institut de Pharmacologie Moleculaire et Cellulaire, CNRS, Valbonne, France.

    A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.

    Funded by: NCI NIH HHS: CA46370, CA55360

    Science (New York, N.Y.) 1993;260;5112;1338-43

  • Mutations of the p53 tumor suppressor gene and the ras gene family in intrahepatic cholangiocellular carcinomas in Japan and Thailand.

    Kiba T, Tsuda H, Pairojkul C, Inoue S, Sugimura T and Hirohashi S

    Pathology Division, National Cancer Center Research Institute, Tokyo, Japan.

    The incidence and pattern of mutations of the ras oncogenes and the p53 tumor suppressor gene have been shown to differ among different cancer types and even among the same cancer types with different etiological backgrounds. For example, in a previous study we showed that not only the etiology but also the incidence of point mutation of the c-Ki-ras oncogene in cholangiocellular carcinomas (CCCs) differ between Japanese and Thai patients. In the study presented here, we examined the incidence of mutations in the ras gene family and the p53 gene in CCCs of both Japanese and Thai patients by single-strand conformation polymorphism and direct sequencing analyses and compared the pattern of p53 mutation between these two CCC groups. Although the incidence of ras mutation differed markedly between Japanese (seven of 12, 58%) and Thai (two of 26, 8%) cases, the incidence of p53 mutation was similar: four of 12 (33%) and nine of 26 (35%), respectively. Except for one case in which deletion-insertion was detected in the second exon of the N-ras gene, all ras mutations occurred at codon 12 or 13 of the c-Ki-ras gene. All p53 mutations but one were detected in a highly conserved region, and the predominant form of the mutations was G:C-->A:T transition at CpG sites in both Japanese and Thai cases, similar to that reported for colorectal cancers. Therefore, in contrast to the ras oncogenes, mutation of the p53 gene was frequently involved in the development of CCCs in both Japanese and Thai patients, irrespective of any difference in etiology.

    Molecular carcinogenesis 1993;8;4;312-8

  • Mapping the human amylase gene cluster on the proximal short arm of chromosome 1 using a highly informative (CA)n repeat.

    Dracopoli NC and Meisler MH

    Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.

    The human amylase gene cluster includes a (CA)n repeat sequence immediately upstream of the gamma-actin pseudogene associated with the AMY2B gene. Analysis of this (CA)n repeat by PCR amplification of genomic DNA from the 40 families of the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel revealed extensive polymorphism. A total of six alleles with (CA)n lengths of 16-21 repeats were found. The average heterozygosity for this polymorphism was 0.70. Multipoint linkage analysis showed that the amylase gene cluster is located distal to the nerve growth factor beta-subunit gene (NGFB) and is within 1 cM of the anonymous locus D1S10. The amylase (CA)n repeat provides a convenient marker for both the physical and the genetic maps of human chromosome 1p.

    Funded by: NCI NIH HHS: CA-44176; NIGMS NIH HHS: GM-24872

    Genomics 1990;7;1;97-102

  • Transforming genes from familial adenomatous polyposis patient cells detected by a tumorigenicity assay.

    Yuasa Y, Kamiyama T, Kato M, Iwama T, Ikeuchi T and Tonomura A

    Department of Hygiene and Oncology, School of Medicine, Tokyo Medical and Dental University, Japan.

    We tried to detect oncogenes associated with familial adenomatous polyposis by a tumorigenicity assay in nude mice. One polyp and two peripheral blood lymphocyte DNAs out of 12 samples from patients induced Alu-positive tumors. Lymphocyte DNAs from one of 5 healthy people also showed tumorigenic activity. The transforming genes of polyps from a patient and lymphocytes from a normal person were found to be the human N-ras gene. Since these N-ras genes were amplified in nude mouse tumors and did not show any alterations in the nucleotide sequences around codons 12 and 61, it is likely that the tumors were induced by the amplified normal N-ras genes. The transforming sequences from two patients' lymphocytes did not hybridize with 12 known oncogene probes, suggesting that these two genes are novel oncogenes or genes for which we have not yet examined the homology. One oncogene derived from a patient's lymphocytes was partially cloned and shown to be located on human chromosome 7. This gene did not hybridize with the met and erbB1 genes, which are potential oncogenes located on chromosome 7. These data indicate that this gene is a new oncogene.

    Oncogene 1990;5;4;589-96

  • All ras proteins are polyisoprenylated but only some are palmitoylated.

    Hancock JF, Magee AI, Childs JE and Marshall CJ

    Section of Cell and Molecular Biology, Royal Cancer Hospital, Chester Beatty Laboratories, London, England.

    The C-terminal CAAX motif of the yeast mating factors is modified by proteolysis to remove the three terminal amino acids (-AAX) leaving a C-terminal cysteine residue that is polyisoprenylated and carboxyl-methylated. Here we show that all ras proteins are polyisoprenylated on their C-terminal cysteine (Cys186). Mutational analysis shows palmitoylation does not take place on Cys186 as previously thought but on cysteine residues contained in the hypervariable domain of some ras proteins. The major expressed form of c-K-ras (exon 4B) does not have a cysteine residue immediately upstream of Cys186 and is not palmitoylated. Polyisoprenylated but nonpalmitoylated H-ras proteins are biologically active and associate weakly with cell membranes. Palmitoylation increases the avidity of this binding and enhances their transforming activity. Polyisoprenylation is essential for biological activity as inhibiting the biosynthesis of polyisoprenoids abolishes membrane association of p21ras.

    Cell 1989;57;7;1167-77

  • Analysis of RAS oncogene mutations in human lymphoid malignancies.

    Neri A, Knowles DM, Greco A, McCormick F and Dalla-Favera R

    Department of Pathology, New York University School of Medicine, NY 10016.

    We investigated the frequency of mutations activating RAS oncogenes in human lymphoid malignancies, including B- and T-cell-derived acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma. By the polymerase chain reaction/oligonucleotide hybridization method, DNA from 178 cases was analyzed for activating mutations involving codons 12 and 61 of the HRAS, KRAS and NRAS genes and codon 13 of the NRAS gene. Mutations involving codons 12 or 13 of the NRAS gene were detected in 6 of 33 cases of acute lymphoblastic leukemia (6/33, 18%), whereas no mutations were found in non-Hodgkin lymphoma or chronic lymphocytic leukemia. Direct nucleotide sequence analysis of polymerase chain reaction products showed that the mutations involved a G----A transition in five of the six cases of acute lymphocytic leukemia. In four cases the mutations seemed to occur in only a fraction of the neoplastic cells, and one case displayed two distinct NRAS mutations, most likely present in two distinct cell populations. These results indicate the following: (i) RAS oncogenes are not found in all types of human malignancies, (ii) significant differences in the frequency of RAS mutations can be found among subtypes of neoplasms derived from the same tissue, (iii) in lymphoid neoplasms the NRAS mutation correlates with the most undifferentiated acute lymphocytic leukemia phenotype, and (iv) NRAS mutations present in only a fraction of malignant cells may result from either the selective loss or the acquisition of mutated alleles during tumor development.

    Funded by: NCI NIH HHS: CA 37165, CA 37295, CA 44029

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;23;9268-72

  • Detection of a low frequency of activated ras genes in human melanomas using a tumorigenicity assay.

    Raybaud F, Noguchi T, Marics I, Adelaide J, Planche J, Batoz M, Aubert C, de Lapeyriere O and Birnbaum D

    Unité 119 de l'INSERM, Marseille, France.

    We have used an assay combining DNA-mediated gene transfer and tumorigenicity in Swiss athymic mice to look for activated ras genes in solid human sporadic melanomas. This assay can detect ras oncogenes mutated at codons 12, 13, or 61. We examined a panel of 13 independent surgical specimens of primary tumors and metastases. No H- or K-ras oncogenes were detected; an N-ras oncogene, mutated at codon 61, was identified in one of the 13 samples. No N-ras genes mutated at codon 13 were detected. Thus, the tumorigenicity assay detects a low frequency of ras gene activation in melanomas.

    Cancer research 1988;48;4;950-3

  • A point mutation at codon 13 of the N-ras oncogene in myelodysplastic syndrome.

    Hirai H, Kobayashi Y, Mano H, Hagiwara K, Maru Y, Omine M, Mizoguchi H, Nishida J and Takaku F

    Patients with a myelodysplastic syndrome (MDS) which has a risk of leukaemic change exhibit a variable clinical course. It has been suggested that the development of leukaemia in patients with MDS may be related to chromosomal abnormalities or genetic alterations: somatic mutation of the N-ras gene is now considered to be a critical step in the genetic basis of human leukaemogenesis. Here we report that DNAs of bone-marrow cells from three out of eight patients with MDS contained an activated N-ras oncogene, as detected by an in vivo selection assay in nude mice with transfected NIH 3T3 cells. Molecular analysis revealed the same single nucleotide substitution at codon 13 in all three transforming N-ras genes. Each of the three patients showed a progression of the disease and a resulting leukaemic change within the following year. Our observation of the mutation at codon 13 in leukaemic cell DNAs from all three cases suggests that activation of the N-ras gene is important in the development of leukaemia in some MDS cases.

    Nature 1987;327;6121;430-2

  • Amino-acid substitution at codon 13 of the N-ras oncogene in rectal cancer in a Japanese patient.

    Nitta N, Ochiai M, Nagao M and Sugimura T

    The activation of proto-oncogenes in colorectal cancers in Japanese patients was studied using a mouse NIH3T3 cell transfection assay system. Of thirty-five colorectal cancers examined, one rectal cancer showed an unusually high transformation efficiency and, in this rectal cancer, the N-ras oncogene was found to be activated. Nucleotide sequence analysis of the activated N-ras showed a single G----C point mutation at the first letter of codon 13, resulting in the coding of arginine instead of glycine. This amino-acid substitution at codon 13 may be responsible for the efficient induction of transformants of NIH3T3 cells in vitro.

    Japanese journal of cancer research : Gann 1987;78;1;21-6

  • Transforming genes in human leukemia cells.

    Hirai H, Tanaka S, Azuma M, Anraku Y, Kobayashi Y, Fujisawa M, Okabe T, Urabe A and Takaku F

    High-molecular weight DNAs of fresh bone marrow cells from 32 patients with fresh leukemia were assayed for the presence of transmissible activated transforming genes by a DNA-mediated gene transfer technique using NIH/3T3 cells. DNAs of bone marrow cells from four of the 32 patients induced transformation of NIH/3T3 cells. Two of the four cases, a chronic myelogenous leukemia and an acute lymphocytic leukemia, contained activated N-ras oncogenes. Molecular cloning and nucleotide sequence analysis revealed that the lesion responsible for the transforming activity was localized to a single nucleotide transition from guanine to thymine in codon 12 of the predicted protein in each of the two cases. These observations indicate that activation of N-ras oncogenes is independent of the specific stage of cell differentiation or the leukemia phenotype. The other two transforming genes associated with an acute myelogenous leukemia and an acute lymphocytic leukemia showed homology neither with members of the ras gene family nor with the human Blym-1 gene. Thus, the NIH/3T3 transfection assay frequently detects activated N-ras oncogenes in human leukemias, while other transforming genes, distinct from the ras gene family, can be detected in some leukemias by the transfection assay.

    Blood 1985;66;6;1371-8

  • Human N-ras: cDNA cloning and gene structure.

    Hall A and Brown R

    The structure and organisation of the human N-ras gene has been determined by analysing cDNA clones derived from the two main mRNA transcripts. One clone in particular is 4.1 Kb long and originates from the larger (4.3 Kb) message. Sequence analysis of this clone has revealed that the N-ras gene consists of seven exons. A second clone deriving from the smaller (2 Kb) message shows that the difference between the two transcripts is a simple extension through the termination site of the 2 Kb transcript. Using S1 analysis, two transcriptional starts have been mapped, 10 bp apart. There is no obvious TATA box in the expected promoter region of the gene, though there are 4 GGGCGG sequences surrounding the start sites. The 5' untranslated sequence contains 2 ATGs upstream of the initiation codon.

    Nucleic acids research 1985;13;14;5255-68

  • Activation of N-ras in a human melanoma cell line.

    Padua RA, Barrass NC and Currie GA

    DNA isolated from cell line Mel Swift, a human melanoma cell line, transforms NIH3T3 cells. Southern blot analysis of DNA from secondary foci revealed conserved 8.8- and 7.8-kilobase EcoRI fragments which hybridized with a human repetitive sequence clone, blur 8. The activated transforming gene was identified as N-ras, and the 8.8-kilobase EcoRI fragment from a secondary transformant was cloned. Synthetic 17-mer oligonucleotides which spanned either the normal codon 61 (CAA) or a mutant codon 61 (AAA) were used for hybridization. Cloned N-ras from melanoma cell line Mel Swift hybridized to the mutant (AAA) oligonucleotide. From this we predicted a glutamine-to-lysine substitution in amino acid 61, a change confirmed by conventional sequencing of the first and second exons of N-ras from cell line Mel Swift. Transfection experiments showed that only those recombinant clones with the mutation in position 61 were biologically active.

    Molecular and cellular biology 1985;5;3;582-5

  • Activation of an N-ras gene in acute myeloblastic leukemia through somatic mutation in the first exon.

    Gambke C, Hall A and Moroni C

    A transforming N-ras gene has been cloned from acute myeloblastic leukemia bone marrow cells, in parallel with the N-ras gene derived from fibroblasts of the same patient. N-ras derived from fibroblasts lacked focus-forming activity in NIH/3T3 cells, indicating that gene activation in the leukemia cells must have occurred by a somatic event. Construction of chimeric molecules between the transforming and the normal N-ras genes and subsequent biological and sequence analysis of these constructs revealed that the transforming gene was altered by a point mutation changing amino acid 12 of the N-ras protein from glycine to aspartic acid.

    Proceedings of the National Academy of Sciences of the United States of America 1985;82;3;879-82

  • Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080.

    Brown R, Marshall CJ, Pennie SG and Hall A

    A full length N-ras gene has been cloned from both the human fibrosarcoma cell line HT1080 and from normal human DNA. N-ras isolated from HT1080 will efficiently induce morphological transformation of NIH/3T3 cells in a transfection assay, whereas N-ras isolated from normal human DNA has no effect on NIH/3T3 cells. The coding regions of the normal N-ras gene have been sequenced and the predicted amino acid sequence of the N-ras product is very similar to that of the c-Ha-ras1 and c-Ki-ras2 products. By making chimeric molecules between the two cloned genes the activating alteration in the HT1080 N-ras gene has been localised to a single base change that results in an amino acid alteration at position 61 of the p21 N-ras product.

    The EMBO journal 1984;3;6;1321-6

  • Mechanism of activation of an N-ras oncogene of SW-1271 human lung carcinoma cells.

    Yuasa Y, Gol RA, Chang A, Chiu IM, Reddy EP, Tronick SR and Aaronson SA

    An N-ras-related transforming gene was detected in the human lung carcinoma cell line SW-1271 and molecularly cloned. The lesion responsible for its acquisition of transforming activity was localized to a single nucleotide transition from A to G in codon 61 of the predicted protein. This lesion in the second exon results in the substitution of arginine for glutamine at this position. These findings, together with previous studies, indicate that the activation of ras oncogenes in human tumors is most commonly due to point mutations at one of two major "hot spots" in the ras coding sequence.

    Proceedings of the National Academy of Sciences of the United States of America 1984;81;12;3670-4

  • Structure and activation of the human N-ras gene.

    Taparowsky E, Shimizu K, Goldfarb M and Wigler M

    The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.

    Funded by: NCI NIH HHS: CA20569

    Cell 1983;34;2;581-6

Gene lists (3)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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