Abstract 3136: C-Myc-mediated transcriptional repression of HPP1 in colon cancer by recruitment of HDACs

Background: Epigenetic gene silencing plays a critical role in tumorigenesis. HPP1, a putative tumor suppressor gene is downregulated in a number of tumor types including colon cancer. As a mechanism of suppression, promoter hypermethylation of HPP1 is known to be important, however the contributions and mechanisms of histone deacetylation have not been described. Additionally, the c-myc oncogene also contributes to the suppression of HPP1 via promoter binding to a cognate E-box site. We sought to elucidate the role of histone deacetylation and the potential interplay with c-myc in the transcriptional regulation of HPP1. Methods: The HPP1 non-expressing cell lines, HCT-116 and DLD-1, were treated with the DNA methylation inhibitor, 5-aza-2′-deoxycitidine (5-Aza-DC) and/or histone deacetylation (HDAC) inhibitors, including sodium butyrate (SB), Vorinostat (SAHA), trichostatin A (TSA) and valproic acid (VPA). Knockdowns of c-Myc and HDAC-1, 2, 3 and 4 were performed using siRNA. Protein and mRNA expression of HPP1 were determined by Western Blot and RT-PCR respectively. Furthermore, Chromatin immunoprecipitation (ChIP) and ChIP-re-ChIP experiments were used to demonstrate the binding of c-Myc and HDACs to the HPP1 promoter. Co-IP assays were used to test the physical interaction between c-Myc and HDAC3. After knockdown of HDAC3 using siRNA in HCT116, ChIP was also used to test the activity of acetylated histone 4 (AcH4). Results: 5-Aza-DC as well as HDAC inhibitors, including SB, TSA, SAHA and VPA, resulted in the re-expression of HPP1 in HCT-116 and DLD-1 cells. Individual HDAC inhibitors administered in combination with 5-Aza-DC resulted in a synergistic effect on HPP1 re-expression. Concordantly, knockdown of HDAC2 and HDAC3 in HCT116 cells, and HDAC1 and HDAC3 in DLD-1 cells by siRNA significantly enhanced HPP1 expression. Co-IP, ChIP and double ChIP experiments confirmed binding of c-myc to the HPP1 promoter as a complex with dominant recruitment of HDAC3. By ChIP assay, the levels of AcH4 increased significantly in HCT116 after knockdown of HDAC3. Conclusions: We have demonstrated that HDAC-1, -2 and -3 contribute to the epigenetic regulation of HPP1. Moreover, c-myc binds to the HPP1 promoter and mediates epigenetic suppression via the dominant recruitment of HDAC3 with subsequent enhancement of histone deacetylation. Our findings may lead to a greater biologic understanding for the application of the targeted use of HDAC inhibitors for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3136. doi:1538-7445.AM2012-3136