Citations

Abstract The Cullin 3-based E3 ubiquitin ligase substrate-binding protein SPOP is inactivated in approximately 8-15% of human prostate cancers due to gene mutation. Intriguingly, SPOP mutations and inactivation of the PTEN tumor suppressor in human prostate cancers are mutually exclusive. However, it is unclear whether SPOP remains functionally active in PTEN-deficient prostate cancer cells. In the current study, we demonstrate that PTEN loss induces phosphorylation and nuclear exportation of SPOP. We identify a putative cyclin-dependent kinase (CDK) phosphorylation site (serine 222) in SPOP BTB domain, a motif interacting with Cullin 3 and demonstrate that PTEN loss induces SPOP cytoplasm accumulation in a manner dependent on CDK2-mediated phosphorylation of SPOP at serine 222. We further show that serine 222 phosphorylation impairs dimerization and the E3 ligase activity of SPOP without affecting its interaction with Cullin 3. We also identify a potential p300 acetylation site in the C-terminal nuclear localization sequence of SPOP. We demonstrate that acetylation by p300 promotes SPOP translocation from nucleus to cytoplasm and this effect is enhanced by CDK2-mediated phosphorylation of SPOP at serine 222. In contrast, Sirt 2 and Sirt 6 of the sirtuin family are verified to be responsible for deacetylation of SPOP. In the conditional Pten knockout prostate cancer mouse model, Pten deletion induces cytosolic accumulation of SPOP, but the process is reversed in Pten and p300 double knockout prostatic cells. Collectively, our findings reveal cytoplasmic localization and enzymatic inactivation of SPOP as a previously uncharacterized mechanism that contributes to PTEN loss-induced prostate tumorigenesis. They also suggest that blocking SPOP cytoplasm localization represents a viable therapeutic option for treatment of prostate cancers harboring PTEN inactivation. Citation Format: Jian An, Haojie Huang. PTEN loss promotes phosphorylation-dependent acetylation and inhibition of SPOP tumor suppressor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A25.

Abstract Reactivation of the androgen receptor (AR) is the hallmark of prostate cancer (PCa) progression to the castration resistant prostate cancer (CRPC) phenotype. Currently, treatment for CRPC strives to impede the full-length AR (AR-FL) from becoming activated through targeting the ligand binding domain (LBD) via androgen deprivation therapy or treatment with antiandrogens. However, these options can give rise to AR-variants (AR-Vs) that lack the LBD and are unresponsive to treatment. Therefore, targeting the N-terminus of AR, which is conserved between AR-FL and AR-Vs, is critical for identifying new treatment options. Phosphorylation of serine 81 (AR-pS81) in the N-terminus of AR is critical for AR activation, stability and recruitment to the chromatin. Similar phosphorylation sites can be observed in the N-terminus of both AR-FL and AR-Vs. Expression of Pin1, a proline isomerase, exhibits a strong correlation with progression and relapse of PCa. Also, suppression of Pin1 in PCa decreases cell proliferation rates, suggesting that Pin1 may play a pivotal role in PCa progression. Pin1 isomerizes proline residues from trans to cis when the adjacent serine/threonine residue is phosphorylated. Dephosphorylation by phosphatases occurs when a pS-P motif is in the trans conformation. Therefore, Pin1 isomerization of AR pS81-P82 from trans to cis could prevent dephosphorylation and result in constitutive or enhanced AR activity. Stable cell lines with Pin1 knockdown were generated via shRNA targeting the 5′ UTR of Pin1 that express either AR-FL and/or AR-Vs. Pin1 knockdown was confirmed by western blots, and AR activity was monitored by expression of several AR regulated genes. Additionally, AR recruitment to chromatin was assayed by ChIP and Re-Chip. Loss of Pin1 resulted in a 60% reduction of endogenous PSA expression, and AR was unable to induce PSA expression when treated with androgens. However, suppression of AR activity was not due to decreased recruitment of AR to chromatin. Phosphorylation of AR-pS81 was decreased by loss of Pin1, while protein stability and mRNA expression was not altered. These data suggest that Pin1 may play a critical role in regulating the transcriptional activity of AR by altering the accessibility of AR to interact with transcriptional machinery needed to maintain AR activity. (Funding for this project was provided by the T.J. Martell Foundation). Citation Format: Travis Van der Steen, Haojie Huang, Donald Tindall. Pin1 suppresses androgen receptor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 89. doi:10.1158/1538-7445.AM2015-89

BACKGROUNDProstate cancer (PCa) is the most commonly diagnosed male cancer in the United States and is a hormone‐driven disease. Androgens have been recognized as a major promoter of PCa development and progression. However, the mechanism of androgen action in PCa, especially in PCa cell invasion and metastasis remains largely unclear. SMAD ubiquitination regulatory factor‐1 (SMURF1) is a C2‐WW‐HECT‐domain E3 ubiquitin ligase that plays important roles in cancer cell metastasis. Whether there is a relationship between androgens and SMURF1 expression is not known.METHODSThe effect of androgens on the expression of SMURF1 in PCa cell lines was examined by Western blot analyses and reverse transcription‐polymerase chain reaction (RT‐PCR). Gene transfection was performed by electroporation to manipulate the expression levels of proteins studied. The binding of AR to the SMURF1 gene enhancer was determined by chromatin immunoprecipitation (ChIP) assay. Cell migration and invasion was measured by wound healing and Matrigel invasion assays, respectively.RESULTSWe found that expression of SMURF1 is upregulated by androgens in PCa cell lines and that this effect of androgens is mediated through the androgen receptor (AR). We further showed that androgens regulate SMURF1 expression at transcriptional level and provided evidence that AR transcriptionally activates SMURF1 by binding to its enhancer that contains a canonical half androgen responsive element (ARE). Finally, we demonstrated that SMURF1 is important for androgen‐induced invasion of PCa cells.CONCLUSIONSWe demonstrate for the first time that SMURF1 is a bona fide target gene of the AR. Our findings also suggest a potential role of SMURF1 in PCa metastasis. Prostate 75: 561–572, 2015. © 2015 Wiley Periodicals, Inc.

Abstract The Polycomb repressive complex 2 (PRC2) is important for maintenance of stem cell pluripotency and suppression of cell differentiation by promoting histone H3 lysine 27 trimethylation (H3K27me3) and transcriptional repression of differentiation regulatory genes. Here we show that the tumor suppressor protein BRCA1 interacts with EZH2 and other components of PRC2 in mouse embryonic stem (ES) and human breast cancer cells. The BRCA1-binding region in EZH2 overlaps with the non-coding RNA (ncRNA)-binding domain, and BRCA1 expression inhibits the binding of EZH2 to the HOTAIR ncRNA. Decreased expression of BRCA1 causes EZH2 re-targeting genome wide and elevates H3K27me3 levels at PRC2 target loci in both mouse ES and human breast cancer cells. BRCA1 deficiency blocks ES cell differentiation and enhances breast cancer migration and invasion in an EZH2-dependent manner. These results reveal that BRCA1 is a key negative modulator of PRC2 and that loss of BRCA1 inhibits ES cell differentiation and enhances cancer aggressive phenotypes via augmented PRC2 function. Citation Format: Xianzhou Zeng, Shuai Chen, Lan Wang, Haojie Huang. BRCA1 is a negative modulator of the PRC2 complex. [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 5358. doi:10.1158/1538-7445.AM2013-5358

Abstract Prostate carcinogenesis is a complicated process involving both genetic and epigenetic changes. Aberrant expression of LSD1 (Lysine-specific demethylase 1) has been reported in human prostate cancers. Although high expression of LSD1 is implicated in prostate tumorigenesis, the underlying molecular mechanisms are not fully understood. Here we reported that LSD1 promotes proliferation and migration of prostate cancer cells via up-regulation of SKP2 expression. Knockdown of LSD1 using small interfering RNA (siRNA) decreases SKP2 expression and inhibits prostate cancer cell proliferation and migration. Promoter luciferase reporter and chromatin immunoprecipitation (ChIP) experiments show that LSD1 regulates SKP2 expression by controlling the levels of H3K4 methylation in SKP2 promoter and this function of LSD1 is mediated through its demethylase activity. As demonstrated by immunohistochemistry, LSD1 and SKP2 proteins positively correlate with each other and their expression is associated with cell proliferation in human prostate cancer tissues. Our data not only identify SKP2 as a direct regulated target gene of LSD1 but also reveal that SKP2 plays an important role in contributing LSD1 mediated prostate cancer cell proliferation and migration, suggesting that the LSD1-SKP2 axis can serve as therapeutic targets for treatment of prostate cancers. Citation Format: Liya Ding, Haojie Huang. LSD1 regulation of SKP2 expression in prostate cancer. [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 1805. doi:10.1158/1538-7445.AM2013-1805