Functional Characterization of CCCTC-binding Factor (CTCF) in the Pathogenesis of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the sixth most common neoplasm and the third leading cause of cancer related mortality worldwide. Treatment of HCC is complicated by the fact that the existing treatment strategies are of limited efficacy when the tumor is at an advanced stage that is no longer amendable to curative surgery. Therefore, there is an urgent need to understand HCC carcinogenesis at the molecular level and to identify new therapeutic approaches for the disease. Sirtuins are mammalian homologs of the yeast SIR2 histone acetylases that requires NAD+ as cofactor. Earlier studies revealed a positive association between the expression of sirtuin 1 (SIRT1) and HCC. Mechanistic studies further revealed that SIRT1 might promote tumorigenesis by maintaining telomeric integrity via regulating the expression of telomere-capping proteins and telomerase reverse transcriptase (TERT). Therefore the original goal of this study was to elucidate the underlying mechanisms of SIRT1-regulated TERT expression. However, molecular analysis revealed that SIRT1 does not mediate TERT expression via transcriptional and epigenetic regulations, mRNA stability, and miRNA regulation. Nevertheless, subsequent investigations have led to identification of the role of CCCTC-binding factor (CTCF) in the regulation of TERT expression and in the pathogenesis of HCC. CTCF expression was low in nontumorigenic livers, but it was up-regulated in HCC cell lines and a subset of HCCs (46.8%). Gene silencing of CTCF suppressed HCC cell growth that was associated with cellular senescence and the induction of G1 cell cycle arrest. In addition, depletion of CTCF promoted the formation of telomere dysfunction-induced foci (TIF) and nuclear abnormalities. Knockdown of CTCF was also associated with substantial reduction in the expression of TERT and TRF1 gene, both of which are essential for the maintenance of telomere integrity. Gene expression profiling analysis further revealed that CTCF might regulate the expression of genes involved in tumor metastasis. Among others, FoxM1, which is implicated in tumor cell motility and invasion, was significantly downregulated upon knockdown of CTCF. Luciferase reporter gene and chromatin immunoprecipitation analysis revealed that CTCF was recruited to the proximal promoter and positively regulate the expression of TERT and FoxM1 gene respectively. On the other hand, investigation into the potential relationship between CTCF and SIRT1 revealed that CTCF may be a transcriptional regulator of SIRT1. Concordantly, in primary HCC, a positive correlation between the expression of CTCF and TERT, CTCF and FoxM1, as well as CTCF and SIRT1, was revealed. Together my study discovered a novel role of CTCF in the pathogenesis of HCC via directing a genetic program important for tumor cell growth and metastasis.