Inhibitor of differentiation 4 (ID4), a member of the helix-loop-helix family of transcriptional regulators has emerged as a tumor suppressor in prostate cancer (PCa). Recent studies have shown that Id4 is highly expressed in the normal prostate and decreases in prostate cancer due to epigenetic silencing. Id4 knockdown in androgen sensitive LNCaP cells has been shown to lead to castration resistant prostate cancer (CRPC) in vitro and in vivo. Id4-/- mice leads to underdeveloped prostate with PIN like lesions without the loss of Androgen Receptor (AR) expression. In this study we demonstrate that the loss of ID4 expression in PCa cell line LNCaP and DU145 may promote tumorigenesis by promoting stemness. LNCaP cells with stably silenced ID4 ((-)ID4) using retroviral based shRNA and LNCaP transfected with non-specific shRNA were used to perform colony forming assay and prostatosphere formation using matrigel. Expression of cancer stem cell markers was determined using western blotting and immunocytochemistry (ICC). FACS analysis was used to sort stem cells and determine the ID4 expression. Xenograft study was performed on SCID mice using CD133 positive LNCaP cells. LNCaP(-)ID4 and DU145 cells lacking ID4 showed increased holoclone as well as decreased paraclone formation, which are believed to be derived from stem cells and differentiated cells respectively, as compared to non-silencing control in the colony forming assay. There was also an increase in prostatosphere development in the LNCaP (-) ID4 cells indicating that the loss of ID4 is responsible for promoting the LNCaP cells towards cancer stem cells. The results were further validated via western blotting and ICC using known cancer stem cell markers on the holoclones and paraclones formed by these cells. Xenograft study showed that 10,000 cells from CD133 positive LNCaP cells developed tumor on SCID mice. This study reports for the first time that loss of ID4 increases holoclone and prostatosphere formation indicating that Id4 may contribute to promoting stemness in prostate cancer cells.

Inhibitor of DNA binding/differentiation protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. ID4 shares the homology of HLH domain with other ID proteins (ID1, ID2, and ID3) and lack the basic DNA binding region. Evidence suggested that unlike ID1, ID2 and ID3, ID4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promoting apoptosis. Consistent with these observations ID4 is epigenetically silenced in DU145 prostate cancer cell line. In this study we investigated whether ID4 is also epigenetically silenced in prostate cancer. We also examined association between ID4 promoter hyper-methylation and its expression in prostate cancer cell lines. ID4 protein expression was analyzed in human prostate adenocarcinoma samples by Immunohistochemistry (IHC). ID4 promoter methylation pattern on prostate cancer cell lines was examined by methylation specific PCR. In addition, we performed methylation specific PCR on the human prostate tissues and genomic DNA to correlate cell line studies with clinical studies. IHC demonstrated decreased ID4 protein expression in human prostate tissue samples, whereas higher nuclear ID4 expression was found in normal prostate tissues. ID4 methylation specific PCR (MSP) on prostate cancer cell lines, showed ID4 methylation in DU145, but not in LNCaP and C33 cells. C81 and PC3 cells showed partial methylation. Increased ID4 methylation in C81 as compared to LNCaP suggests its epigenetic silencing as cells acquire androgen independence. Tumors with ID4 promoter hyper-methylation showed distinct loss of ID4 expression. However, the underlying mechanism involved in epigenetic silencing of ID4 is currently unknown. We hypothesized that ID4 promoter methylation is initiated by an EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27Me3). ID4 expressing (LNCaP) and non-expressing (DU145 and C81) prostate cancer cell lines were used to investigate EZH2, H3K27Me3 and DNMT1 enrichment on ID4 promoter by Chromatin immuno-precipitation (ChIP). Increased enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines was compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner. ChIP on prostate cancer tissue specimens and cell lines suggested EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased EZH2, but decreased ID4 expression in prostate cancer strongly supports this model.

Overexpression of tumor-derived mutant p53 is a common event in tumorigenesis, suggesting an advantageous selective pressure in cancer initiation and progression. Given that p53 is found to be mutated in 50% of all human cancers, restoration of mutant p53 to its wild type biological function has been a widely sought after avenue for cancer therapy. Most research efforts have largely focused on restoration of mutant p53 by artificial means given that p53 has some degree of conformational flexibility allowing for introduction of short peptides and artificial compounds. Recently, theoretical modeling and studies focused on restoration of mutant p53 by physiological means has raised the question of whether there are effective therapies worth exploring that focus on global physiological mechanisms of restoration of p53. Herein, we provide computational analysis of the thermodynamic stabilities of both wild-type and mutant p53 core domains by studying their respective minimum potential energies. Also, it is widely accepted that wild type p53 is modulated by various acetyl transferases as well as deactylases, but whether this mechanism of p53 modulation can be exploited for physiological restoration of mutant p53 remains under intense investigation. Using prostate cancer cell lines representative of varying stages of aggressiveness as a model, we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn regulating p53-dependent biological activities. Together, our data provides computational analysis of the core domain of certain mutant forms of p53 and a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320, critical sites of post translational modification of p53, that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function.

The inhibitor of DNA binding protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors.1 Recently, Patel et al., demonstrated that inhibitor of differentiation 4 (ID4) acts as a tumor suppressor and its loss, frequently observed in prostate cancer, promotes castration-resistant prostate cancer (CRPC) through constitutive androgen receptor (AR) activation.2 However, the mechanism by which loss of ID4 promotes constitutively active AR signaling in the CRPC conditions is unknown. The rationale of the present study was to unravel the underlying molecular mechanisms through which loss of ID4 potentiates AR signaling in this setting. Initially, chromatin immunoprecipitation (ChIP) assay results demonstrated a significant increase in binding of AR to its respective response elements on PSA, FKBP51, TMPRSS2, and ETV1 promoters in L(-)ID4 cells, further implicating constitutive AR activity. Among the notable findings, proteomic profiling between prostate cancer cell line LNCaP (L+ns) and LNCaP lacking ID4 (L(-)ID4) revealed elevated protein levels of Heat shock protein 27 (Hsp27) and the 52-kDa FK506-binding protein (FKBP52), suggesting a role for these AR-associated co-chaperones in promoting constitutively active AR signaling in L(-)ID4 cells. Interestingly, protein interaction studies further confirmed a direct interaction between ID4 and FKBP52 in vitro but not with AR. Recent evidences suggest that FKBP52 is a positive regulator of AR signaling in cellular and whole animal models.3-6 Thus, we hypothesized that ID4 acts as a tumor suppressor by selectively regulating AR activity through interaction with FKBP52. To address the underlying mechanism, we blocked the FKBP52-AR signaling using a specific inhibitory compound known as MJC13.4, 6-7 The results demonstrated that MJC13 effectively inhibited AR-dependent expression and activity in a dose-dependent manner. In addition, xenograft studies further confirmed that inhibiting FKBP52-regulated AR activity via MJC13 significantly attenuated the growth of subcutaneous L(-)ID4 xenografts in vivo. Collectively, our results suggested that ID4 selectively regulates AR activity through direct interaction with FKBP52 in vitro, and, its loss promotes CRPC through FKBP52-mediated AR signaling. Increased AR signaling along with a subsequent decrease in ID4 expression levels in prostate cancer strongly supports this model.