High Mutability of the Tumor Suppressor Genes RASSF1 and RBSP3 (CTDSPL) in Cancer

BACKGROUND:Many different genetic alterations are observed in cancer cells. Individual cancer genes display point mutations such as base changes, insertions and deletions that initiate and promote cancer growth and spread. Somatic hypermutation is a powerful mechanism for generation of different mutations. It was shown previously that somatic hypermutability of proto-oncogenes can induce development of lymphomas. METHODOLOGY/PRINCIPAL FINDINGS:We found an exceptionally high incidence of single-base mutations in the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) both located in 3p21.3 regions, LUCA and AP20 respectively. These regions contain clusters of tumor suppressor genes involved in multiple cancer types such as lung, kidney, breast, cervical, head and neck, nasopharyngeal, prostate and other carcinomas. Altogether in 144 sequenced RASSF1A clones (exons 1-2), 129 mutations were detected (mutation frequency, MF = 0.23 per 100 bp) and in 98 clones of exons 3-5 we found 146 mutations (MF = 0.29). In 85 sequenced RBSP3 clones, 89 mutations were found (MF = 0.10). The mutations were not cytidine-specific, as would be expected from alterations generated by AID/APOBEC family enzymes, and appeared de novo during cell proliferation. They diminished the ability of corresponding transgenes to suppress cell and tumor growth implying a loss of function. These high levels of somatic mutations were found both in cancer biopsies and cancer cell lines. CONCLUSIONS/SIGNIFICANCE:This is the first report of high frequencies of somatic mutations in RASSF1 and RBSP3 in different cancers suggesting it may underlay the mutator phenotype of cancer. Somatic hypermutations in tumor suppressor genes involved in major human malignancies offer a novel insight in cancer development, progression and spread

NotI genome scanning to identify unknown cancer associated genes in major human epithelial malignancies

Epithelial cancers cause many deaths every year. Changes in the genes of human chromosome 3 are particularly common in epithelial cancers in several organs. Alterations in DNA methylation is one of the best known epigenetic changes in cancer. The abnormal epigenetic landscape of the cancer cell is characterized by a massive genomic hypomethylation and hypermethylation of CpG islands in the promoter regions of tumor suppressor genes. Microarrays is a powerful tool for studying the molecular basis of diseases that are not possible with conventional methods. Being able to predict who will develop cancer and how the disease will behave and respond to treatment after diagnosis are some uses for this technology. NotI microarrays is a novel technology that makes it possible to simultaneously detect changes in methylation, amplification and deletions in cancer. The NotI microarrays technology based on restriction enzyme NotI is methylation sensitive, and therefore makes it possible to detect if methylation causes a gene to be altered in cancer. Cancer samples are hybridized with NotI microarrays and are evaluated bioinformatically. Using NotI microarray technology, 181 NotI loci in human chromosome 3 have been analyzed for methylation, amplification and deletions in different epithelial cancers; lung cancer, renal cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer and colon cancer. The genes that were shown to be altered in cancer samples compared with normal tissue samples were analyzed further with more accurate methods; bisulfitesequencing of cloned PCR products was the method used to control and verify methylation status. NotI loci that were found to have relevant changes include genes MINT24, BHLHB2, RPL15, RARbeta1, ITGA9, RBSP3, VHL, ZIC4, NKIRAS1, LRRC3B, suggesting that they probably are involved in cancer development. It was found that NPRL2/G21 gene has growth inhibitory activity for renal and lung cell lines when tested under controlled physiologic conditions of gene expression both in vitro and in vivo. Mutations were found in experimental tumors and intragenic homozygous deletions in renal, lung, and other cancer cell lines. It was also observed that NPRL2 could participate in mismatch repair. Further, it was shown that somatic hypermutations in tumor suppressor genes involved in major human malignancies is a mechanism for the development, progression and spread of cancer. RASSF1 and RBSP3 were demonstrated to have a high incidence of somatic hypermutations in several cancer types. Methylation and/or deletions of LRRC3B gene were detected in more than 50% of checked renal, cervical, ovarian, colon and breast cancer samples, and it was found to inhibit renal cell cancer cell line KRC/Y cell survival and replication in vitro. Altered genes found with the NotI microarrays technology may potentially be used as biomarkers or targets for epigenetic therapy. A set of 18 markers was suggested (BHLHB2, FBLN2, FLJ44898 (EPHB1), GATA2, GORASP1, Hmm210782 (PRICKLE2), Hmm61490, ITGA9, LOC285205, LRRC3B, MINA, MITF, MRPS17P3, NKIRAS1, PLCL2, TRH, UBE2E2, WNT7A) that allow to discriminate/diagnose different types of lung cancer