Mechanism of Transcriptional Repression of GADD45a in Prostate Cancer and Its Potential Role as a Therapeutic Target.

Methylation-mediated repression of tumor-suppressor genes or genes involved in cell death pathway is a common event in most human malignancies. We examined the mechanism of regulation of a gene involved in cell-cycle and apoptotic pathways, Growth Arrest and DNA Damage inducible, alpha (GADD45a), in prostate cancer cells. GADD45a is expressed at a low level in LNCaP and Du145 prostate cancer cells as compared to PC3 prostate cancer cells. Bisulfite genomic sequencing was used to determine the methylation pattern of CpG dinucleotides in GADD45a promoter region. Transcriptional repression of GADD45a occurs by an atypical pattern of methylation. Unlike the commonly reported methylation pattern of tumor-suppressor genes in human malignancies, the CpG islands of the proximal promoter region are unmethylated in all three prostate cancer cell lines Du145, LNCaP and PC3. However, methylation of 4 CpGs located approximately 700 bp upstream of the transcriptional start site correlates inversely with the expression of GADD45a in Du145, LNCaP, and PC3 cells. Treatment of Du145 and LNCaP cells with DNA methyl transferase (DNMT) inhibitors, 5-Azacytidine and 5 Aza-2′deoxycytidine led to an enhanced GADD45a expression concomitant with demethylation of the 4 CpGs. Frequent methylation of the 4 CpGs was also seen in prostate cancer tissues. Methylation-mediated transcriptional repression involves binding of specific proteins, Methyl-CpG binding proteins (MBDs) at methylated CpG dinucleotides. Using chromatin immunoprecipitation (ChIP), we analyzed the interaction of GADD45a- 4-CpG region with MeCP2, the archetypical MBD protein. Cell specific interaction of MeCP2 was observed: MeCP2 was associated with the methylated four CpGs in Du145 whereas no binding was detected in LNCaP cells. Stable transfection of MeCP2 siRNA vector in Du145 (Du145-MeCP2-ve) resulted in down regulation of MeCP2, release of MeCP2 from GADD45a promoter and upregulation of GADD45a expression suggesting a role of MeCP2 in mediating transcriptional repression in Du145 cells. Since GADD45a has been implicated in cell cycle arrest and apoptosis, we hypothesized that induction of GADD45a expression in Du145 cells would lead to increased sensitivity to chemotherapeutic drugs. Consistent with this hypothesis, we observed that

• Docetaxel treatment resulted in an increased cytotoxicity in MeCP2-ve Du145 cells that exhibit enhanced GADD45a expression as compared to the wt Du145 cells,

• Induction of GADD45a expression by prior treatment with DNMT inhibitor, 5-Azacytidine resulted in an enhanced cytotoxicity in wt Du145 cells and

• wt Du145 cells that were transfected with GADD45a expression vector exhibited an enhanced cyotoxicity to Docetaxel as compared to the mock-transfected cells.

In conclusion, we show that methylation of CpG dinucleotides away from the transcriptional start site but not in the proximal promoter region correlates inversely with GADD45a expression in prostate cancer cells, cell-specific interaction of MeCP2 to the methylated GADD45a promoter, and a potential role of DNA methyltransferase inhibitors in enhancing sensitivity to cytotoxic agents through upregulation of GADD45a in prostate cancer cells.