3265

HRAS

C-BAS/HAS|C-H-RAS|C-HA-RAS1|CTLO|H-RASIDX|HAMSV|HRAS1|K-RAS|N-RAS|RASH1;v-Ha-ras Harvey rat sarcoma viral oncogene homolog;HRAS;v-Ha-ras Harvey rat sarcoma viral oncogene homolog

GTP- and GDP-binding peptide B|GTPase HRas|H-Ras-1|Ha-Ras1 proto-oncoprotein|Ras family small GTP binding protein H-Ras|c-has/bas p21 protein|c-ras-Ki-2 activated oncogene|ha-Ras|p19 H-RasIDX protein|p21ras|transformation gene: oncogene HAMSV|transforming

11p15.5

protein-coding

Neuroblastoma (NB) is the most common malignant solid tumor in childhood, and among ALL childhood malignancies is second in prevalence only to leukemia. In NBwe need to both make an accurate diagnosis and rapidly analyze the expression ofgenetic prognostic factors such as MYCN, H-ras, and trkA. Moreover, it has recently become important to analyze the expression of survivin mRNA, a member of the inhibitor of apoptosis protein family. expression of the survivin gene is related to tumorigenesis and inhibition of apoptosis in some malignant tumors. We investigated its expression by reverse transcription-polymerase chain reaction (RT-PCR) in NB cell lines (SK-N-SH, NB-39, and IMR-32), two normal blood cell samples, and 13 clinical NB tumor samples. ALL three NB cell lines had high levels of mRNA expression for this gene, but normal blood cells had no expression. We detected expression of survivin mRNA in 7 of the 13 NB tumor samples (54%). Two NB patients were in stage I disease, 6 in stage II, and 5 in stage IV(A). Quantitative analysis by RT-PCR revealed that the ratio between survivin mRNA and human glyceraldehyde-3-phosphate dehydrogenase (h-GAPDH) mRNA was very low in stages I and II (0-0.017). In contrast, in advanced NBs (stage IV(A)) the ratio was much higher (0-0.050). The prognoses of the three patients in the advanced stage who had high ratios of expression were poor. A high level of expression of survivin mRNA indicates a high grade of malignancy, high likelihood of recurrence, and poor prognosis.

BACKGROUND: Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessedPIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as thesehave not been previously reported. METHODS: Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutationswithin the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations toassess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. RESULTS: We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studiedto date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. CONCLUSION: These data suggest that activating mutationsin the Ras/Raf-MAPK/PI3K signaling cascades occur infrequently in neuroblastoma.Further, despite compelling evidence for MYC and RAS cooperation in vitro and invivo to promote tumourigenesis, activation of RAS signal transduction does not constitute a preferred secondary pathway in neuroblastomas with MYCN deregulation in either human tumors or murine models.

Neuroblastoma (NB) is the most common malignant solid tumor in childhood. There are well-recognized prognostic factors in NB such as age at diagnosis, organ of origin, stages, MYCN gene amplification, and expression of H-ras, trkA and survivin. Moreover, we investigated the expression of vascular endothelial growth factor (VEGF), tyrosine hydroxylase (TH), p53, stem cell factor (SCF) and c-kit of its receptor with quantitative real-time polymerase chain reaction (PCR) in 22 NBs and 4 other tumors (one malignant lymphoma, one malignant teratoma, and 2 rhabdomyosarcomas) samples. The correlation between patients' prognoses and the expression of TH or c-kit was newly recognized, particularly the good prognosis in patients in whom c-kit highly expressed and the poor prognosis contrarily associated with low or no expression, although the SCF of its ligand had no relationship with patient prognosis. It is possible that tumors without c-kit expression can not react with SCF (via the autocrine or paracrine system) and remain immature. It may be that this is a new critical clinical event in NB patients.

Rhabdomyosarcoma (RMS) is a common childhood solid tumor, resulting from dysregulation of the skeletal myogenesis program. Two major histological subtypes occur in childhood RMS, embryonal and alveolar. While chromosomal rearrangementsaccount for the majority of alveolar tumors, the genetic defects underlying the pathogenesis of embryonal RMS remain largely undetermined. A few studies performed on small series of embryonal tumors suggest that dysregulation of RAS function may be relevant to disease pathogenesis. To explore further the biological and clinical relevance of mutations with perturbing consequences on RAS signaling in embryonal RMS, we investigated the prevalence of PTPN11, HRAS, KRAS, NRAS, BRAF, MEK1, and MEK2 mutations in a relatively large cohort of primary tumors. While HRAS and KRAS were found to be rarely mutated, we identified somatic NRAS lesions in 20% of cases. ALL mutations were missense andaffected codon 61, with the introduction of a positive charged amino acid residue representing the most common event. PTPN11 was found mutated in one tumor specimen, confirming that somatic defects in this gene are relatively uncommon in RMS, while no mutation was observed in BRAF and MEK genes. Although no clear association of mutations with any clinical variable was observed, comparison of the outcome between mutation-positive and mutation-negative cases indicated a trend for a higher percentage of patients exhibiting a better outcome in the former. Our findings provide evidence that dysregulation of RAS signaling is a major event contributing to embryonal RMS pathogenesis.#CI- Copyright 2009 Wiley-Liss, Inc.

Malignant gliomas are the most common and the deadliest brain malignancies in adults. Despite the lack of a complete understanding of the biology of these tumors, significant advances have been made in the past decades. One of the key discoveries made in the area of malignant gliomas is that these tumors can be induced and maintained by aberrant signaling networks. In this context, the Ras pathway has been extensively exploited, from both basic and translational perspectives. Although somatic oncogenic mutations of Ras genes are frequent in several cancer types, early investigations on gliomas revealed disappointing facts that the Ras mutations are nearly absent in malignant gliomas and that theBRAF mutations are present in a very small percentage of gliomas. Therefore, theobserved deregulation of the Ras-RAF-ERK signaling pathway in gliomas is attributed to its upstream positive regulators, including, EGFR and PDGFR known to be highly active in the majority of malignant gliomas. In contrast to the initial negative results on the somatic mutations of H-Ras, K-Ras and BRAF, recent breakthrough studies on pediatric low-grade astrocytomas uncovered genetic alterations of the BRAF gene involving copy number gains and rearrangements. The7q34 rearrangements result in a novel in-frame KIAA1549:BRAF fusion gene that possesses constitutive BRAF kinase activity resembling oncogenic BRAF (V600E). In light of the earlier findings and recent breakthroughs, this review summarizes our current understanding of the Ras-RAF-ERK signaling pathway in gliomas and the outcome of preclinical and clinical studies that evaluated the efficacy of Ras-targeted therapy in malignant gliomas.

The recessive autosomal hereditary disease, xeroderma pigmentosum (XP), is characterized by a high incidence of tumors in sun-exposed skin. The defect in early steps of excision repair of XP cells leads to hypermutability towards UV-mimicking agents. DNA from eight XP tumors were screened for activated transforming genes using 3T3 transfection. In two skin tumors isolated from a XPchild, an activated N-ras oncogene was detected. Synthetic oligonucleotide probes were used to characterize the mutation in the ras gene. Both tumors were found to be mutated in the 61st N-ras codon from gln to his. The mutation was accompaniedby an increase in the level of N-ras specific mRNA and in one transformant, by the alteration of the p21 protein. In the same tumors, c-myc amplification and over transcription, and Ha-ras gene rearrangement and amplification were also detected. Analysis of other XP tumors with eleven different oncogene probes revealed an amplification of the Ha-ras gene in 6 out of 10 cases. The normal skin fibroblasts from XP patients show normal pattern levels of N-ras, c-myc andHa-ras sequences. The hypothesis is proposed that the presence of several oncogene alterations in the same tumor could be due to the high amount of UV-induced DNA lesions found in the exposed skin cells, in the absence of efficient repair.

Fusion of an auxotrophic mutant hamster cell with the skin fibroblasts of a child with the Wilms' tumor-aniridia association produced clones which, on the one hand, contained the child's normal chromosome 11 and, on the other, the chromosome 11 with the 11p13 deletion associated with the syndrome. Both hybridswere positive for human LDH-A by enzymatic assay. Clones containing the normal human chromosome 11 were killed by a cytotoxic monoclonal antibody to a cell surface antigen previously mapped to the 11p13----11pter region of chromosome 11. Clones with the abnormal 11 were not killed. Thus, we have produced hybrids fromthe same patient distinct from each other on the basis of their chromosome 11. These hybrids have been used to map the locus for a cell surface antigen to the deleted region on chromosome 11 of a patient with the Wilms tumor-aniridia association. The linkage between this antigen and the syndrome should be helpfulin further study of the genetics of this disease. In addition, we have found that the c-Ha-ras-1 oncogene is distal to the p13 region of chromosome 11 and the position of insulin and beta-globin on the chromosome. Finally, by producing segregants of the hybrids containing the abnormal chromosome 11, we have provided evidence that chromosome 11-associated c-Ha-ras-1 is syntenic with chromosome 11and not moved to a different portion of the genome.#FAU - Fisher, J H

Hepatoblastoma is the most common pediatric liver cancer, and complete resectionis required for long-term survival. There are few reported hepatoblastoma cell lines available for study. In order to develop in vitro and animal models, the authors isolated an additional cell line (HB1) from a human hepatoblastoma and here report its characteristics in culture. Cells were analyzed for growth rate,clonigenicity, and tumorigenicity, using electron microscopy. Immunocytochemistry and Northern analysis for the expression of specific surface markers and genes of interest were also performed. HB1 cells required fetal calf serum but grew well in culture (population doubling time of 2 days) and had an undifferentiated appearance under electron microscopy. Epidermal growth factor (EGF) and hydrocortisone stimulated growth with or without serum, but not autocrine growthstimulation via the epidermal growth factor receptor was detected. Proteins produced by HB1 cells under normal culture conditions included alpha-fetoprotein, cytokeratins 8 and 18, and lactate dehydrogenase (with an isozyme subunit composition similar to that of liver). HB1 cells showed chronic, high expressionof c-myc and Ha-ras oncogenes but no N-myc and apparently normal RB gene expression. This cell line shows characteristics in culture consistent with malignant, hepatic epithelial cells and is apparently EGF dependent. It may provide a model system for the future development of alternative therapies.

The role of tumor suppressor genes and oncogenes in the development of Ewing's sarcoma has not yet been fully clarified. In this study, we analyzed the frequency of p53 tumor suppressor gene mutation in exons 4-8 by PCR-SSCP and direct sequencing, and the expression of p53-protein in Ewing's sarcoma (ES) by using immunohistochemistry. The overexpression of MDM2, which acts as a functional inactivator of p53, was studied by immunohistochemistry. In addition,a screening for point mutations in the hot spot regions codon 12 and 13 of exon 1 and codon 61 of exon 2 of ras-genes (H-ras, N-ras, K-ras) was performed. In one case, a p53 gene mutation could be confirmed in codon 238 of exon 7 (1/24). Overexpression of MDM2 was found in five cases; in ras-genes, no mutations were detected. Compared with other highly malignant mesenchymal pediatric tumors suchas osteosarcomas, mutations of p53 and ras in Ewing's sarcomas are an extraordinarily rare event. However, their frequency is comparable to that of PNET, suggesting that the low incidence of these mutations in ES and PNET could be group-specific for tumors of neuroectodermal genesis.