Swathi Chinaranagari

Inhibitor of differentiation 4 (ID4) acts as an inhibitor of ID-1, -2 and -3 and promotes basic helix loop helix (bHLH) E47 DNA binding and transcriptional activity.

The four known ID proteins (ID1-4, Inhibitor of Differentiation) share a homologous helix loop helix (HLH) domain and act as dominant negative regulators of basic-HLH transcription factors. ID proteins also interact with many non-bHLH proteins in complex networks. The expression of ID proteins is increasingly observed in many cancers. Whereas ID-1, ID-2 and ID-3, are generally considered as tumor promoters, ID4 on the contrary has emerged as a tumor suppressor. In this study we demonstrate that ID4 heterodimerizes with ID-1, -2 and -3 and promote bHLH DNA binding, essentially acting as an inhibitor of inhibitors of differentiation proteins. Interaction of ID4 was observed with ID1, ID2 and ID3 that was dependent on intact HLH domain of ID4. Interaction with bHLH protein E47 required almost 3 fold higher concentration of ID4 as compared to ID1. Furthermore, inhibition of E47 DNA binding by ID1 was restored by ID4 in an EMSA binding assay. ID4 and ID1 were also colocalized in prostate cancer cell line LNCaP. The alpha helix forming alanine stretch N-terminal, unique to HLH ID4 domain was required for optimum interaction. Ectopic expression of ID4 in DU145 prostate cancer line promoted E47 dependent expression of CDKNI p21. Thus counteracting the biological activities of ID-1, -2 and -3 by forming inactive heterodimers appears to be a novel mechanism of action of ID4. These results could have far reaching consequences in developing strategies to target ID proteins for cancer therapy and understanding biologically relevant ID-interactions.

Inhibitor of Differentiation 4 (ID4) Inactivation Promotes De Novo Steroidogenesis and Castration-Resistant Prostate Cancer

Prostate cancer (PCa) is the most commonly diagnosed cancer in men in the Western world. The transition of androgen-dependent PCa to castration-resistant (CRPC) is a major clinical manifestation during disease progression and presents a therapeutic challenge. Our studies have shown that genetic ablation of inhibitor of differentiation 4 (Id4), a dominant-negative helix loop helix protein, in mice results in prostatic intraepithelial neoplasia lesions and decreased Nkx3.1 expression without the loss of androgen receptor (Ar) expression. ID4 is also epigenetically silenced in the majority of PCa. However, the clinical relevance and molecular pathways altered by ID4 inactivation in PCa are not known. This study investigates the effect of loss of ID4 in PCa cell lines on tumorigenicity and addresses the underlying mechanism. Stable silencing of ID4 in LNCaP cells (L-ID4) resulted in increased proliferation, migration, invasion, and anchorage-independent growth. An increase in the rate of tumor growth, weight, and volume was observed in L-ID4 xenografts compared with that in the LNCaP cells transfected with nonspecific short hairpin RNA (L+ns) in noncastrated mice. Interestingly, tumors were also observed in castrated mice, suggesting that loss of ID4 promotes CRPC. RNA sequence analysis revealed a gene signature mimicking that of constitutively active AR in L-ID4, which was consistent with gain of de novo steroidogenesis. Prostate-specific antigen expression as a result of persistent AR activation was observed in L-ID4 cells but not in L+ns cells. The results demonstrate that ID4 acts as a tumor suppressor in PCa, and its loss, frequently observed in PCa, promotes CRPC through constitutive AR activation.

Prostate cancer epigenome.

Prostate cancer is a major health burden within the ever-increasingly aging US population. The molecular mechanisms involved in prostate cancer are diverse and heterogeneous. In this context, epigenetic changes, both global and gene specific, are now an emerging alternate mechanism in disease initiation and progression. The three major risk factors in prostate cancer: age, geographic ancestry, and environment are all influenced by epigenetics and additional significant insight is required to gain an understanding of the underlying mechanisms. The androgen receptor and its downstream effector pathways, central to prostate cancer initiation and progression, are subject to a multitude of epigenetic alterations. In this review we focus on the global perspective of epigenetics and the use of recent next-generation sequencing platforms to interrogate epigenetic changes in the prostate cancer genome.

Abstract 4518: PCL/Maltodextrin delivered ID4 maintains its tumor suppressor role.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC INTRODUCTION: The Inhibitor of differentiation protein (Id) family has generated much attention in cancer cell biology due to its multifaceted role. These proteins have a helix–loop– helix (HLH) domain required for association with transcriptional factors but lack the basic amino acids required for DNA-binding therefore act as dominant-negative regulators of transcription. Tumor suppressor roles have been strongly associated with Id4 one of the four different isoforms identified so far. A recent study by Chaudhary et al indicated that Id4 was a potential tumor suppressor of prostate cancer with ectopic expression of Id4 in DU145 leading to increased apoptosis and decreased cell proliferation due in part by an S-phase arrest. Methylation mediated silencing of the Id4 gene has been associated with development of prostate cancer. In this study we evaluated the pro-apoptotic potential of intracellularly delivered Id4 using PCL/Maltodextrin nanoparticles as the delivery vector. METHOD: PCL/Maltodextrin nanoparticles were formulated using the double emulsion solvent evaporation technique with a Nano Debee high-pressure homogenizer. Nanoparticles were lyophilized and characterized for their size, zeta potential, and encapsulation efficiency. Western blot analysis was conducted on Id4 loaded nanoparticles and nanoparticle treated DU 145 cell lines to confirm the presence and delivery of intact protein. Immuno-cytochemical analysis of Id4 expression was conducted on DU-145, LNCaP and LNCaP cells silenced with Id4 (LNCaP-Id4) pre and post treatment with Id4 loaded nanoparticles.The effect of nanoparticle delivered Id4 on cellular processes such as apoptosis, proliferation and transwell migration was also determined. RESULTS: Id4 loaded PCL/Maltodextrin nanoparticles was successfully formulated with an average particle size of about 200nm. The formulation had encapsulation efficiency of 78.83±7.40% and percentage yield over 98%. Western blot analysis reveals that intact Id4 protein was successfully loaded and delivered to the cells. Immuno-cytochemical analysis of the three cell lines showed an efficient intracellular delivery of Id4 using PL/Maltodextrin with the intracellular amount increasing with time. The ICC analysis also showed possible intra- nuclear localization of Id4. Intracellular delivered Id4 increased the levels of Caspase 3/9 by about three folds associated with decreased mitochondrial membrane potential and plasma membrane asymmetry. The transwell migration assay also showed a significant decrease in cell migration after treatment with Id4 loaded nanoparticles in comparison to the untreated and blank nanoparticle group. CONCLUSION: PCL/Maltodextrin nanocarrier can successfully deliver Id4 to the cytoplasm as well as nucleus of LNCaP-Id4, DU145 and LNCaP. Intracellular delivered Id4 maintains it pro apoptotic potential. Citation Format: Maxwell Korang-Yeboah, Pankaj Sharma, Ashley Evans Knowell, Divya Patel, Yamini Gorantla, Swathi Chinaranagari, Ravi Palaniappa, Jaideep Chaudhary. PCL/Maltodextrin delivered ID4 maintains its tumor suppressor role. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4518. doi:10.1158/1538-7445.AM2013-4518

Abstract A23: Repression of Id4 (inhibitor of DNA binding 4) by the polycomb group protein EZH2 in prostate cancer

Introduction: Inhibitor of DNA binding/differentiation protein 4 (Id4) is dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. Id4 shares the homology of HLH domain with other Ids and lack the basic DNA binding region. Unlike Id1, Id2 and Id3, Id4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promote apoptosis. We have previously shown that decreased Id4 expression in prostate cancer is due to its promoter hypermethylation. In this study we investigated the molecular mechanism by which Id4 promoter is hypermethylated. Here, we demonstrate that Id4 promoter methylation is initiated by EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27me3). Experimental procedures: Id4 protein expression was analyzed on human prostate adenocarcinoma samples by Immunohistochemistry and prostate cancer cell lines (LNCaP, C81 and DU145) by Western blot. EZH2 was silenced in DU145 cells and confirmed through western blot and Immunocytochemistry. Chromatin Immunoprecipitation (ChIP) was performed with IgG, RNA polymerase, EZH2 and H3K27Me3 antibodies in both prostate cancer cell lines and prostate cancer tissue samples. Results: IHC demonstrated decreased Id4 protein expression in human prostate tissue samples whereas higher nuclear Id4 expression was found in normal prostate tissues. Re-expression of Id4 in EZH2 silenced Du145 demonstrated that Id4 is regulated in an EZH2 dependent manner. ChIP data on prostate cancer samples and cell lines suggested EZH2 occupancy and H3K27Me3 marks in the Id4 promoter region in cell lines and prostate cancer tissue specimens. Conclusions: Id4 appears as a potential tumor suppressor gene that is epigenetically silenced during prostate cancer development. H3K27Me3 marks and EZH2 occupancy suggest a PRC2 dependent mechanism in Id4 promoter silencing in prostate cancer. Acknowledgement: The research was supported by NIH/NCI-RO1CA128914 and in part by NIH/NCRR/RCMI G12RR03062 Citation Format: Swathi Chinaranagari, Pankaj Sharma, Jaideep Chaudhary. Repression of Id4 (inhibitor of DNA binding 4) by the polycomb group protein EZH2 in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A23.

Abstract A24: Epigenetic silencing of ID4 (inhibitor of DNA binding 4) is associated with prostate cancer

Introduction: Inhibitor of DNA binding/differentiation protein 4 (Id4) is dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. Id4 shares the homology of HLH domain with other Ids and lack the basic DNA binding region. Previous studies from our lab had shown that unlike Id1, Id2 and Id3, Id4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promote 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 hypermethylation and its expression in prostate cancer cell lines. Experimental procedures: Id4 protein expression was analyzed on human prostate adenocarcinoma samples by Immunhistochemistry. Id4 promoter methylation pattern on prostate cancer cell lines (C33, C81, DU145, LNCaP, DU145+ID4, and PC3) was examined. In addition, we performed Laser capture microdisection (LCMD) on the human prostate tissues and methylation specific PCR to correlate our cell line studies with clinical studies. Results: 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 cell and but not in LNCaP and C33 cells. In C81 and PC3 cells it showed partial methylation. C81 are derivatives of LNCaP cell lines and are Androgen independent and are more metastatic. Increased Id4 methylation in C81 as compared to LNCaP suggests its epigenetic silencing as cells acquire androgen independence. Tumors with Id4 promoter hypermethylation showed distinct loss of Id4 expression. Conclusions: Id4 appears as a potential tumor suppressor gene that is epigenetically silenced during cancer development.Id4 promoter hypermethylation had been reported in breast, colorectal cancer and chronic lymphocytic leukemia Thus, loss of Id4 expression due to promoter hypermethylation. Id4 methylation status could serve as a prognostic biomarker in human prostate cancer. Acknowledgement: The research was supported by NIH/NCI-RO1CA128914 and in part by NIH/NCRR/RCMI G12RR03062. Citation Format: Pankaj Sharma, Swathi Chinaranagari, Divya Patel, Jaideep Chaudhary. Epigenetic silencing of ID4 (inhibitor of DNA binding 4) is associated with prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A24.

Abstract LB-141: Expression and localization of Id3 in human prostate cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: The inhibitor of differentiation (Id) proteins are dominant negative basic helix-loop-helix family of transcription factors that lack the DNA-binding basic domain but have intact HLH domain. The four members of the Id protein family (Id1-Id4) are known to play a critical role in the coordinated regulation of gene expression during cell growth, differentiation and tumorigenesis. Although in vitro studies have demonstrated the role of Id1 in regulating prostate cancer (PCa) cell proliferation, apoptosis and androgen independence, its clinical significance in PCa remains controversial. On the other hand there is lack of evidence on the expression of Id2 and Id3 in PCa progression. Studies have demonstrated that silencing of Id3 is significantly more effective in blocking proliferation of PCa cells as compared to Id1 whereas Id2 silencing has been primarily shown to be involved in apoptotic pathway. Hence in this study we re-evaluated the role of Id1 in PCa and extended our study to show that Id3 protein expression has better clinical relevance compared to Id1 and Id2 in PCa. Experimental design: The expression of Id-1, -2 and -3 was determined by real time and Western blotting along with their sub-cellular localization by ICC in PCa cell lines LNCaP, DU145 and PC3. To assess their significance in prostate cancer progression, the protein expression was investigated in clinical prostate tumor samples on high density tissue microarray by IHC. Results: The expression of Id1 and Id3 mRNA was low in LNCaP and DU145 as compared to PC3 whereas the level of Id2 expression was similar between all the PCa cell lines. The protein expression data was consistent with the mRNA levels in the PCa cell lines. All three Ids were primarily cytoplasmic with some nuclear presence. The IHC data showed a significant increase in the expression of Id1 and Id3 in clinical prostate tumor tissue samples compared to normal prostate tissue whereas Id2 showed elevated levels but was not significant as compared to Id1 and Id3. The expression of Id3 protein correlated with the Gleason grading and stage of PCa. Conclusions: This is the first study demonstrating a correlation between increased expression of Id3 and prostate cancer progression. It also reconfirms and establishes the association of Id1with PCa. The results warrant further investigation into isoform specific effects of Id1and Id3 on prostate tumorigenesis. (Research Support: NIH/NCI RO1 [CA128914][1] and NIH/NCRR/RCMI G12RR03062. The first two authors contributed equally to this work. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2011-LB-141 [1]: /lookup/external-ref?link_type=GEN&access_num=CA128914&atom=%2Fcanres%2F71%2F8_Supplement%2FLB-141.atom