Variants in the Signaling Protein TSAd are Associated with Susceptibility to Ovarian Cancer in BRCA1/2 Negative High Risk Families.

A substantial fraction of familial ovarian cancer cases cannot be attributed to specific genetic factors. The discovery of additional susceptibility genes will permit a more accurate assessment of hereditary cancer risk and allow for monitoring of predisposed women in order to intervene at the earliest possible stage. We focused on a population with elevated familial breast and ovarian cancer risk. In this study, we identified a SNP rs926103 whose minor allele is associated with predisposition to ovarian but not breast cancer in a Caucasian high-risk population without BRCA1/BRCA2 mutations. We have found that the allelic variation of rs926103, which alters amino acid 52 of the encoded protein SH2D2A/TSAd, results in differences in the activity of this protein involved in multiple signal transduction pathways, including regulation of immune response, tumor vascularization, cell growth, and differentiation. Our observation provides a novel candidate genetic biomarker of elevated ovarian cancer risk in members of high-risk families without BRCA1/2 mutations, as well as a potential therapeutic target, TSAd.

Interferon regulatory factors IRF5 and IRF7 inhibit growth and induce senescence in immortal Li-Fraumeni fibroblasts.

Cellular immortalization is one of the prerequisite steps in carcinogenesis. By gene expression profiling, we have found that genes in the interferon (IFN) pathway were dysregulated during the spontaneous cellular immortalization of fibroblasts from Li-Fraumeni syndrome (LFS) patients with germ-line mutations in p53. IFN signaling pathway genes were down-regulated by epigenetic silencing during immortalization, and some of these same IFN-regulated genes were activated during replicative senescence. Bisulfite sequencing of the promoter regions of two IFN regulatory transcription factors (IRF5 and IRF7) revealed that IRF7, but not IRF5, was epigenetically silenced by methylation of CpG islands in immortal LFS cells. The induction of IRF7 gene by IFNalpha in immortal LFS cells was potentiated by pretreatment with the demethylation agent 5-aza-2'-deoxycytidine. Overexpression of IRF5 and IRF7 revealed that they can act either alone or in tandem to activate other IFN-regulated genes. In addition, they serve to inhibit the proliferation rate and induce a senescence-related phenotype in immortal LFS cells. Furthermore, polyinosinic:polycytidylic acid treatment of the IRF-overexpressing cells showed a more rapid induction of several IFN-regulated genes. We conclude that the epigenetic inactivation of the IFN pathway plays a critical role in cellular immortalization, and the reactivation of IFN-regulated genes by transcription factors IRF5 and/or IRF7 is sufficient to induce cellular senescence. The IFN pathway may provide valuable molecular targets for therapeutic interventions at early stages of cancer development.

Epigenetic and functional analysis of IGFBP3 and IGFBPrP1 in cellular immortalization.

Carcinogenic transformation of a cell requires bypassing senescence and becoming immortalized. A cellular senescence-like phenotype can be induced in immortal Li-Fraumeni syndrome (LFS) cells by treating them with the DNA methyltransferase inhibitor 5-aza-deoxycytidine. Our microarray-based expression profiling studies of spontaneously immortalized LFS cell lines identified genes that may provide the growth advantage required for the cells to become immortal. Several members of the IGFBP superfamily of genes fit the profile of genes involved in immortalization: silenced during immortalization and reactivated by 5-aza-deoxycytidine. Overexpression of IGFBP3 or IGFBPrP1 in the immortal LFS cell lines suppressed cell growth and inhibited colony formation. Both genes have the expression pattern of an epigenetically regulated gene and contain CpG islands suitable for methylation-dependent silencing. Analysis of how IGFBPs regulate immortalization will lead to a better understanding of this process and may lead to novel methods for the prevention and treatment of cancer.

Expression profiling identifies three pathways altered in cellular immortalization: interferon, cell cycle, and cytoskeleton.

Abrogation of cellular senescence, resulting in immortalization, is a necessary step in the tumorigenic transformation of a cell. Four independent, spontaneously immortalized Li-Fraumeni syndrome (LFS) cell lines were used to analyze the gene expression changes that may have given these cell lines the growth advantage required to become immortal. A cellular senescence-like phenotype can be induced in immortal LFS cells by treating them with the DNA methyltransferase (DNMT) inhibitor 5-aza-deoxycytidine. We hypothesized, therefore, that genes epigenetically silenced by promoter methylation are potentially key regulators of senescence. We used microarrays to compare the epigenetic gene expression profiles of precrisis LFS cells with immortal LFS cells. Gene ontology analysis of the expression data revealed a statistically significant contribution of interferon pathway, cell cycle, and cytoskeletal genes in the process of immortalization. The identification of the genes and pathways regulating immortalization will lead to a better understanding of cellular immortalization and molecular targets in cancer and aging.