Identification, Epigenetic and Functional Characterization of Novel Tumor Suppressor Genes in Human Cancers

Tumor suppressor genes (TSGs) can be inactivated by genetic and/or epigenetic alterations during carcinogenesis. Identification and characterization of novel TSGs are critical for the understanding of cancer development, and provide potential targets for clinical treatment and biomarkers for tumor diagnosis. In this study, I aimed to identify novel TSGs epigenetically silenced in human cancers and further characterize the molecular basis of their anti-tumorigenic functions.;Emerging evidence highlights the importance of epigenetic modifiers as cancer genes. Here, I characterized two epigenetic modifier genes as functional TSGs. First, I found PRDM5, a candidate TSG methylated and downregulated in multiple cancers, suppressed tumor cell proliferation through inhibiting TCF/LEF-dependent transcription and inducing epigenetic repression of multiple oncogenes. Second, through expression profiling of 24 epigenetic modifiers, I identified a novel candidate TSG, TUSC12, showing frequent silencing in tumor cell lines. TUSC12 was broadly expressed in human normal tissues, but downregulated by promoter CpG methylation in tumor cell lines. Frequent TUSC12 methylation was detected in primary tumors as well. TUSC12 dramatically inhibited tumor cell clonogenicity, but this growth inhibitory effect was partially impaired by disrupting its PHD domain. TUSC12 colocalized with the transcription corepressor KAP1 in the nucleus and is likely to repress gene transcription through recruit HDAC-associated complex.;I also studied a novel 3p14.2 TSG, ZNF312, identified from previous aCGH profiling of nasopharyngeal carcinoma (NPC). Frequent promoter methylation silenced ZNF312 in NPC cell lines and tissues. Restored ZNF312 expression strongly suppressed NPC cell growth through inducing cell cycle arrest and apoptosis. Further, ZNF312 acted as a transcription repressor targeting the promoter regions of EZH2 and MDM2 and downregulating their expression.;Moreover, previous digital gene expression subtraction from cDNA libraries revealed a list of genes possibly downregulated in tumors compared to normal tissues. I characterized a novel TSG, TUSC45, initially isolated from this strategy. The expression of TUSC45 was significantly reduced in tumor tissues compared to normal tissues, with lower TUSC45 expression associated with poorer patient survival. Downregulation of TUSC45 in multiple tumor cell lines was also observed, while only infrequent genomic deletion was detected. In contrast, promoter methylation was responsible for TUSC45 silencing in most cell lines, in which pharmacologic or genetic demethylation can dramatically restore its expression. Remarkably, TUSC45 was frequently methylated in primary tumors but not in normal tissues. Further, TUSC45 suppressed anchorage-dependent and -independent tumor cell growth. Induced TUSC45 expression inhibited cell proliferation, induced apoptosis, cell cycle arrest and senescence, and lead to the upregulation of a key tumor suppressor p53. Moreover, TUSC45 activated p53 target genes in a p53- dependent manner. Forced TUSC45 expression in p53-null H1299 and HCT116/p53KO (knock out) cells showed no inhibitory effect on cell growth. Finally, TUSC45 interacted with p53/MDM2 complex and positively regulated p53 protein stability. The protein half-life of MDM2 was shortened by TUSC45, indicating a possible mechanism for TUSC45 modulation on p53 signaling.;In conclusion, this study showed the tumor-specific methylation and silencing of the four TSGs lead to the epigenetic disruption of multiple cell signalings during tumorigenesis and could potentially be used as biomarkers for cancer detection.