Sheng-Yu Ku

SOX2 promotes lineage plasticity and antiandrogen resistance in TP53- and RB1-deficient prostate cancer

Evading cancer drugs by identity fraud Prostate cancer growth is fueled by male hormones called androgens. Drugs targeting the androgen receptor (AR) are initially efficacious, but most tumors eventually become resistant (see the Perspective by Kelly and Balk). Mu et al. found that prostate cancer cells escaped the effects of androgen deprivation therapy through a change in lineage identity. Functional loss of the tumor suppressors TP53 and RB1 promoted a shift from AR-dependent luminal epithelial cells to AR-independent basal-like cells. In related work, Ku et al. found that prostate cancer metastasis, lineage switching, and drug resistance were driven by the combined loss of the same tumor suppressors and were accompanied by increased expression of the epigenetic regulator Ezh2. Ezh2 inhibitors reversed the lineage switch and restored sensitivity to androgen deprivation therapy in experimental models. Science, this issue p. 84, p. 78; see also p. 29 Prostate cancer cells escape androgen deprivation therapy by morphing into a cell type that does not require androgens. Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. We use in vitro and in vivo human prostate cancer models to show that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR)–dependent luminal epithelial cells to AR-independent basal-like cells. This lineage plasticity is enabled by the loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2, and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching.

Sunitinib Dose Escalation Overcomes Transient Resistance in Clear Cell Renal Cell Carcinoma and Is Associated with Epigenetic Modifications

Sunitinib is considered a first-line therapeutic option for patients with advanced clear cell renal cell carcinoma (ccRCC). Despite sunitinibs clinical efficacy, patients eventually develop drug resistance and disease progression. Herein, we tested the hypothesis whether initial sunitinib resistance may be transient and could be overcome by dose increase. In selected patients initially treated with 50 mg sunitinib and presenting with minimal toxicities, sunitinib dose was escalated to 62.5 mg and/or 75 mg at the time of tumor progression. Mice bearing two different patient-derived ccRCC xenografts (PDX) were treated 5 days per week with a dose-escalation schema (40–60–80 mg/kg sunitinib). Tumor tissues were collected before dose increments for immunohistochemistry analyses and drug levels. Selected intrapatient sunitinib dose escalation was safe and several patients had added progression-free survival. In parallel, our preclinical results showed that PDXs, although initially responsive to sunitinib at 40 mg/kg, eventually developed resistance. When the dose was incrementally increased, again we observed tumor response to sunitinib. A resistant phenotype was associated with transient increase of tumor vasculature despite intratumor sunitinib accumulation at higher dose. In addition, we observed associated changes in the expression of the methyltransferase EZH2 and histone marks at the time of resistance. Furthermore, specific EZH2 inhibition resulted in increased in vitro antitumor effect of sunitinib. Overall, our results suggest that initial sunitinib-induced resistance may be overcome, in part, by increasing the dose, and highlight the potential role of epigenetic changes associated with sunitinib resistance that can represent new targets for therapeutic intervention. Mol Cancer Ther; 14(2); 513–22. ©2014 AACR.

Epigenetics in Castration Resistant Prostate Cancer

Castration resistant prostate cancer (CRPC) remains a therapeutic challenge despite recent therapeutic advances. Epigenetic mechanisms, including DNA methylation, histone modifications, and microRNA (miRs), have been associated with the biology of CRPC and have been identified as potential targets for therapeutic interventions. Rapid development of inhibitors towards these epigenetic targets has given rise to promising novel therapeutic opportunities in cancer, including CRPC. The preclinical data on the role of histone deacetylases, methyltransferases, demethylases, and miRs in CRPC offer a scenario for potential clinical success of epigenetic therapies in rational combination strategies. The identification of predictors of response to agents targeting histone modifications will be critical to identify the CRPC patients who are most suitable for this therapeutic approach.

Abstract 4061: Evidence for hdac6 and er-α association in a subset of clear cell renal cell carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Histone deacetylases are overexpressed in several tumors including prostate, breast and clear cell renal cell carcinoma (ccRCC). Our group has previously reported that class II HDACs, HDAC4 and HDAC6 regulate HIF-α stability in ccRCC cell lines. Interestingly, HDAC6 overexpression in ER-α positive breast cancer has been shown to correlate with overall and cancer specific survival in response to tamoxifen treatment. In addition, HDAC6 increases cell motility by deacetylating α-tubulin, and HDAC6 interaction with ER-α on the cell membrane increases its deacetylating activity. The objective of our study was to assess whether HDAC6 associates with ER-α in a subset of ccRCC and whether this association can be targeted therapeutically. Methods: Radical nephrectomy tumor samples (n=14) with matched adjacent non tumor tissues were collected and analyzed for HDAC6 expression by Western blot analysis. HDAC6 expression was also assessed in C2, C2VHL and 786-O ccRCC cell lines by Western blot and immunofluorescence analysis. HDAC6 and ER-α colocalization was examined by immunoprecipitation and immunofluorescence. HDAC 6 was overexpressed in cell lines and investigated for cell motility by scratch assay. Cell lines were also treated with hydroxy tamoxifen in short term (4 hours) and long term (24, 48 and 72 hours) culture experiments for evaluation of effects on acetylated α-tubulin and cell proliferation, respectively. Results: Analysis of matched patient tumor samples revealed that a subset of ccRCC had higher HDAC6 expression as compared to the adjacent non tumor tissue. HDAC6 and ER-α examined in ccRCC tumors (n=44) by immunofluorescence showed overexpression in 10% of tumor samples. Immunoprecipitation of HDAC 6 in ccRCC cell lines showed that ER-α is present in the same complex as HDAC 6 as confirmed also by fluorescence microscopy. HDAC6 overexpression in ccRCC cell lines increased cell motility, although overexpression did not affect cell proliferation. Cells treated for short term experiments with hydroxy tamoxifen showed an increase in acetylated α-tubulin when examined by immunofluorescence. Upon long term hydroxy tamoxifen treatment in regular DMEM medium with serum, ccRCC cell proliferation was affected at high concentrations (10-20µM), similar to MCF 7 cells treated under similar conditions. Conclusions: HDAC 6 and ER-α are overexpressed in a subset of ccRCC. HDAC6 overexpression affects cell motility but not proliferation. HDAC6 and ER-α are present in the same immunocomplex and this association may be targeted with therapeutic interventions. Ongoing studies are testing concomitant, either genetic or pharmacological, inhibition of both HDAC6 and ER-α in ccRCC and will provide the rationale for novel targeted therapies for a selected group of patients with ccRCC. Citation Format: Swathi Ramakrishnan, Sheng-Yu Ku, Wendy Swetzig, Dylan Conroy, Li Shen, Sreenivasulu Chintala, Paula Sotomayor, Kiersten M. Miles, Remi Adelaiye, Eric Ciamporcero, Ashley Orillion, Leigh Ellis, Gokul Das, Roberto Pili. Evidence for hdac6 and er-α association in a subset of clear cell renal cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4061. doi:10.1158/1538-7445.AM2014-4061

Abstract 1462: Characterization of patient-derived bladder cancer xenografts: role of xCT in response to cisplatin

Background: A major challenge in cancer drug development has been largely attributed to the inability of cell lines to recapitulate the heterogeneity of human tumors. Patient derived xenografts (PDX) represent a major advance as they are more representative of the clinical setting. However, appropriate characterization of PDXs is necessary to guide biomarker driven drug discovery research. Methods: HE 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1462. doi:10.1158/1538-7445.AM2015-1462

Abstract 686: Regulation of intracellular sequestration of sunitinib by cystine transporter xCT in renal cancer

Background: Acquired and/or intrinsic resistance to sunitinib, a multikinase inhibitor, is one of the major obstacles for the treatment of renal cell carcinoma (RCC). Recently, lysosomal sequestration of sunitinib has been shown as one of the potential mechanisms of resistance in renal cancer. The current study was initiated to understand the role of the Cystine Transporter xCT (encoded product of Solute Carrier family 7A member 11, SLC7A11 gene), a lysosome regulatory gene, on intracellular sequestration of sunitinib and to evaluate the cytotoxic effects of the xCT inhibitor sulfasalazine alone and in combination with sunitinib on RCC cells. Methods: Intracellular sequestration of sunitinib was evaluated using the fluorescent microscopy technique by determining the autofluorescence of sunitinib. Cytotoxic effects of sulfasalazine alone and in combination with sunitinib were assessed by sulforhodamine B assay. Quantitative RT-PCR analysis and Western blot analysis were utilized to determine the expression of xCT in normal renal tubular HK2 cells, human umbilical vein endothelial cells (HUVEC), and RCC 786-O cells, and to determine the association of sunitinib sequestration and xCT expression. Expression of xCT interacting partner CD44s and its variant forms CD44v6 and CD44v8, which influence the expression of xCT, was determined in 786-O and HK2 cells. Additionally, we utilized The Cancer Genome Atlas (TCGA) data to determine the expression of xCT in ccRCC patients and evaluated the significance of its expression and correlation of patient survival. Results: Our results revealed higher intracellular sequestration of sunitinib in RCC 786-O cells compared to HK2 and HUVEC cells. Furthermore, we found higher expression of xCT in 786-O cells compared to HK2 and HUVEC cells, which was associated with high intracellular sequestration of sunitinib. The pharmacological inhibition of xCT by sulfasalazine revealed the reversal of intracellular sequestration of sunitinib in 786-O cells. The SRB assay showed the enhanced cytotoxic effects of sunitinib in combination with sulfasalazine, which indicates that, the reversal of intracellular sequestration of sunitinib may led to the enhanced cytotoxic effects. Moreover, there was a significant decrease of xCT stabilizing isoform CD44v6 mRNA in 786-O cells compared to HK2 and no change was observed in CD44s and CD44v8. Bioinformatics analysis using cBioPortal Cancer Genomics from MSKCC data of TCGA revealed the significant up regulation of xCT in ccRCC patients correlated with poor prognosis. Conclusions: Our results indicate that lysosome regulatory protein xCT inhibition by sulfasalazine leads to the reversal of intracellular sequestration of sunitinib and enhances the efficacy of sunitinib in renal cancer. These results provide the rationale for the preclinical and clinical testing of sulfasalazine in combination with sunitinib in the treatment of ccRCC. Citation Format: Sreenivasulu Chintala, Hillary Nguyen, Swathi Ramakrishnan, Sheng-Yu Ku, Eric Ciamporcero, Kiersten M. Miles, Roberto Pili. Regulation of intracellular sequestration of sunitinib by cystine transporter xCT in renal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 686. doi:10.1158/1538-7445.AM2014-686

Abstract 1008: Concomitant HDAC and PI3K-AKT pathway inhibition as a new therapeutic strategy for prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Prostate cancer (PCa) is the most prevalent cancer in men in the United States. Prostate cancer patients that progress to a lethal phenotype are currently treated with chemotherapy, second generation androgen therapies and immunotherapy. Unfortunately, patient response is not often durable; therefore novel treatments are urgently needed. Histone deacetylases (HDACs) have shown to be up-regulated in PCa. Preclinical data indicates that HDAC inhibitors induce apoptosis in androgen receptor (AR)-positive cells. Unfortunately, HDACi display minimal clinical benefit. From this, we propose that combination of HDACi with novel targeted therapies will result in greater therapeutic efficacy. Methods: PC3, PC3AR and PtenCaP8 PCa cell lines treated with panobinostat (HDACi) and BEZ235 (PI3K-mTORC1 inhibitor) as single treatment or in combination for 48 hours. Cell death/apoptosis was assessed by loss of cell membrane integrity, phosphotidylserine exposure, cell cycle and caspase activation by flow cytometry. Expression levels of activated AKT were assessed by immunoblot. Results: Panobinostat induces cell death/apoptosis greater in PC3AR than parental PC3 cells by increasing subG1 population (47.8±9.3 vs. 11.1±2.8,), phosphotidylserine exposeure (41.4±3.0 vs. 21±7.2,) and caspase activation (57.3±0.9 vs. 12.0 ±4.1,) respectively. Moreover, PC3AR cells treated with panobinostat results in loss of AKT activation, but not in PC3 cells. Further, combination of panobinostat with BEZ235 increases greater cell apoptosis in PC3AR cells compared to panobinostat single agent (30.6±1.9 vs. 19.9±1.4) and in PtenCaP8 cells (42.0±6.5 vs. 29.4±2.2). Western blotting also shows that combination treatment leads to loss of AKT activation compared to panobinostat and BEZ235 as a single agent. Conclusions: Overall, our results suggest that inhibition of HDACs induces greater cell death associated with loss of AKT activation in AR-positive cells, but not in AR-null cells. Also, combination of HDAC inhibitor and PI3K-mTORC1 inhibitor has more effective cell cytotoxicity compared to single agent, suggesting that inhibition of HDACs and PI3K-mTORC1 will exhibit greater therapeutic efficacy. In future work, combination of panobinostat with BEZ235 will be investigated in preclinical animal models to propose this therapy combination for clinical trials in patients with lethal PCa. Citation Format: Sheng-Yu Ku, Roberto Pili, Leigh Ellis. Concomitant HDAC and PI3K-AKT pathway inhibition as a new therapeutic strategy for 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 1008. doi:10.1158/1538-7445.AM2013-1008

Abstract B5: Epigenetic modification of AR in PC3 cells negatively regulates mTORC1/2 activity

Abstract Wild type androgen receptor (AR) acts as a tumor suppressor and mediates the differentiation of non-malignant prostate epithelial cells. However, in the development of prostate cancer AR activity becomes critical for the development and progression of disease. Previous studies demonstrate that re-expressing wild type AR within an AR negative cell line (PC3) possesses tumor suppressor quality by reducing cell proliferation and tumorgenicity in mice. Further, it is documented that acetylation of AR in non-malignant cells is a post-translational modification associated with increased transcriptional activity of AR, whereas acetylation of AR in tumor cells results in negative regulation of AR transcriptional activity. We therefor are seeking to investigate the regulation of re-expressed AR within PC3 cells by induced acetylation post HDAC inhibition. Our preliminary data reveals that sensitivity to HDAC inhibition is increased with the re-expression of AR in PC3 cells. Further, epigenetic modification of AR in this model negatively regulates mTORC1 and mTORC2 expression and activity, independent of AR transcriptional activity. Ongoing experiments are being conducted to investigate further the role of epigenetic post-translational modification of AR and its negative regulation of mTORC1/2 signaling. Citation Format: Leigh Ellis, Remi Adelaiye, Shengyu Ku, Alejandro Godoy, Roberto Pili. Epigenetic modification of AR in PC3 cells negatively regulates mTORC1/2 activity [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 B5.

Abstract C7: Targeting the androgen receptor with novel combination therapeutic strategies in metastatic/castrate-resistant prostate cancer

Abstract While recent advances have resulted in the FDA approval of novel therapies for metastatic/castrate resistant prostate cancer, responses are not often durable and novel therapeutic interventions are still required. Recent work within our laboratory has resulted in the development of a novel orthotopic syngeneic transplant mouse model of prostate cancer. Further, treatment with a HDAC and mTORC1 inhibitor in combination resulted in greater anti-tumor and increased overall survival in tumor bearing animals. HDAC/mTORC1 combination therapy also resulted in decreased androgen receptor (AR) transcriptional activity without loss of AR protein expression. Current investigations are underway to delineate molecular mechanisms underlying sustained AR expression. Through this understanding, novel therapeutic interventions which will augment combined HDAC/mTORC1 inhibitor therapy are under investigation. Citation Format: Leigh Ellis, Swathi Ramakrishnan, Shengyu Ku, Roberto Pili. Targeting the androgen receptor with novel combination therapeutic strategies in metastatic/castrate-resistant 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 C7.