Inhibiting Multiple Deubiquitinases to Reduce Androgen Receptor Expression in Prostate Cancer Cells

Prostate cancer (PCa), a leading cause of cancer-related death in men, becomes resistant to androgen deprivation therapy by inducing androgen receptor (AR) activity, which is known as castration-resistant PCa (CRPC). Enzalutamide is an approved drug that inhibits AR activity and increases overall survival. However, resistance to enzalutamide develops rapidly often by increasing AR activity, suggesting that new therapies are required for CRPC. We investigated whether betulinic acid (BA), a small molecule from plants that inhibits multiple deubiquitinases (DUBs), reduces AR, and selectively kills PCa cells, can provide an adjuvant strategy for CRPC. Our data indicated that BA reduced AR protein stability and mRNA expression, making it an attractive agent for CRPC. BA decreased AR mRNA possibly by inhibiting a histone 2A DUB thereby increasing ubiquitinated histone 2A, a transcriptional repressor. We identified multiple and specific DUBs inhibited by BA either in PCa cells or using recombinant DUBs. Similar results were obtained using another multi-DUB inhibitor WP1130, suggesting that these DUB inhibitors can decrease AR expression and increase PCa-specific death. Our results also suggest that combining multi-DUB inhibitors BA or WP1130 with enzalutamide may provide a novel strategy for CRPC by further decreasing AR expression and increasing apoptotic cell death.</p

Memory, Conflict and Reconciliation A Half-Day Conference

Join the Steven J. Green School of International and Public Affairs and the Vaclav Havel Program for Human Rights and Diplomacy for a half-day conference exploring the role of memory in conflict and reconciliation. A distinguished group of scholars and practitioners will articulate diverse perspectives on the nature of memory, its role in public discourse, and the ways it can both feed conflict and promote reconciliation. 1:15 PM-1:30 PM I OPENING REMARKS John F. Stack, Jr., Founding Dean, Steven J. Green School of International and Public Affairs Martin Palous, Director, Vaclav Havel Program for Human Rights and Diplomacy 1:30 PM-3:30 PM I PANEL 1: REMEMBRANCE OF THINGS PAST Henrik Syse, Research Professor, Peace Research Institute; Member, Norwegian Nobel Committee, Oslo, Norway Memory as a Means of Reconciliation and Dialogue: Philosophical and Practical Reflections on Memory and Conflict Glenn Hughes, St. Mary\u27s Chair in Catholic Philosophy, St. Mary\u27s University, San Antonio, Texas Uses and Abuses of Memory Aurora Morcillo, Professor, FIU Department of History; Director, Spanish and Mediterranean Studies Program Between Memory and History: A Proustian Intervention Ambassador Michael Zantovsky, Director of Vaclav Havel Library, Prague, the Czech Republic The Responsibility to Remember: Memory as an Attribute of Being 3:00 PM -3:20 PM I COFFEE BREAK 3:20 PM ~ 5:00 PM I PANEL 2: THE CURRENT USES OF MEMORY AND CLOSING REMARKS Marifeli Perez-Stable, Professor, FIU Department of Global & Sociocultural Studies Cuban National Reconciliation Liliana Trevizan, Professor of Modern Languages, State University of New York at Potsdam A Museum and Democratic Performance in Chile Carlos Gonzales, Independent Journalist and Filmmaker; Founder and CEO, Mentora News, Philadelphia Diary of Ukraine\u27s Forgotten War Marie Janouskova and Michal Smid, Representatives ofPostBellum, Prague, Czech Republic The Mission of the Non-Profit Organization Post Bellum Orlando Gutierrez-Boronat, Writer and Educator; Co-founder and Spokesperson, Cuban Democratic Directorate Beyond Narratives: Cuba\u27s Struggle for Memory 5:00 PM-6:00 PM I CLOSING RECEPTIONhttps://digitalcommons.fiu.edu/cri_events/1344/thumbnail.jp

Antitumor and antiangiogenic effect of the dual EGFR and HER-2 tyrosine kinase inhibitor lapatinib in a lung cancer model

<p>Abstract</p> <p>Background</p> <p>There is strong evidence demonstrating that activation of epidermal growth factor receptors (EGFRs) leads to tumor growth, progression, invasion and metastasis. Erlotinib and gefitinib, two EGFR-targeted agents, have been shown to be relevant drugs for lung cancer treatment. Recent studies demonstrate that lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER-2 receptors, is clinically effective against HER-2-overexpressing metastatic breast cancer. In this report, we investigated the activity of lapatinib against non-small cell lung cancer (NSCLC).</p> <p>Methods</p> <p>We selected the lung cancer cell line A549, which harbors genomic amplification of EGFR and HER-2. Proliferation, cell cycle analysis, clonogenic assays, and signaling cascade analyses (by western blot) were performed <it>in vitro</it>. <it>In vivo </it>experiments with A549 cells xenotransplanted into nude mice treated with lapatinib (with or without radiotherapy) were also carried out.</p> <p>Results</p> <p>Lapatinib dramatically reduced cell proliferation (<it>P </it>< 0.0001), DNA synthesis (<it>P </it>< 0.006), and colony formation capacity (<it>P </it>< 0.0001) in A549 cells <it>in vitro</it>. Furthermore, lapatinib induced G1 cell cycle arrest (<it>P </it>< 0.0001) and apoptotic cell death (<it>P </it>< 0.0006) and reduced cyclin A and B1 levels, which are regulators of S and G2/M cell cycle stages, respectively. Stimulation of apoptosis in lapatinib-treated A549 cells was correlated with increased cleaved PARP, active caspase-3, and proapoptotic Bak-1 levels, and reduction in the antiapoptic IAP-2 and Bcl-xL protein levels. We also demonstrate that lapatinib altered EGFR/HER-2 signaling pathways reducing p-EGFR, p-HER-2, p-ERK1/2, p-AKT, c-Myc and PCNA levels. <it>In vivo </it>experiments revealed that A549 tumor-bearing mice treated with lapatinib had significantly less active tumors (as assessed by PET analysis) (<it>P </it>< 0.04) and smaller in size than controls. In addition, tumors from lapatinib-treated mice showed a dramatic reduction in angiogenesis (<it>P </it>< 0.0001).</p> <p>Conclusion</p> <p>Overall, these data suggest that lapatinib may be a clinically useful agent for the treatment of lung cancer.</p

2-Methoxyestradiol and paclitaxel have similar effects on the cell cycle and induction of apoptosis in prostate cancer cells

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with promise for cancer chemotherapy, including advanced prostate cancer. We have focused on events related to cell cycle arrest (G1 and G2/M) and induction of apoptosis in human prostate cancer cells. Treatment with 2-ME increased cyclin B1 protein and its associated kinase activity followed by later inhibition of cyclin A-dependent kinase activity and induction of apoptosis. Similar results were obtained with paclitaxel (taxol), a clinically relevant agent used to treat advanced prostate cancer. Cyclin-dependent kinase inhibitors prevented 2-ME and paclitaxel-mediated increase in cyclin B1-dependent kinase activity and blocked induction of apoptosis. Reduction of X-linked inhibitor of apoptosis (XIAP) protein by 2-ME and paclitaxel correlated with increased apoptosis. Lower doses of 2-ME and paclitaxel resulted in G1 (but not G2/M) cell cycle arrest in the p53 wild type LNCaP cell line, but with minimal induction of apoptosis. We suggest that 2-ME and paclitaxel-mediated induction of apoptosis in prostate cancer cells requires activation of cyclin B1-dependent kinase that arrests cells in G2/M and subsequently leads to the induction of apoptotic cell death

Sequential combination of flavopiridol and docetaxel reduces the levels of X-linked inhibitor of apoptosis and AKT proteins and stimulates apoptosis in human LNCaP prostate cancer cells

Clinical trials have shown that chemotherapy with docetaxel combined with prednisone can improve survival of patients with androgen-independent prostate cancer. It is likely that the combination of docetaxel with other novel chemotherapeutic agents would also improve the survival of androgen-independent prostate cancer patients. We investigated whether the combination of docetaxel and flavopiridol, a broad cyclin-dependent kinase inhibitor, can increase apoptotic cell death in prostate cancer cells. Treatment of DU 145 prostate cancer cells with 500 nmol/L flavopiridol and 10 nmol/L docetaxel inhibited apoptosis probably because of their opposing effects on cyclin B1-dependent kinase activity. In contrast, when LNCaP prostate cancer cells were treated with flavopiridol for 24 hours followed by docetaxel for another 24 hours (FD), there was a maximal induction of apoptosis. However, there was greater induction of apoptosis in DU 145 cells when docetaxel was followed by flavopiridol or docetaxel. These findings indicate a heterogeneous response depending on the type of prostate cancer cell. Substantial decreases in X-linked inhibitor of apoptosis (XIAP) protein but not survivin, both being members of the IAP family, were required for FD enhanced apoptosis in LNCaP cells. Androgen ablation in androgen-independent LNCaP cells increased activated AKT and chemoresistance to apoptosis after treatment with FD. The proteasome inhibitor MG-132 blocked FD-mediated reduction of XIAP and AKT and antagonized apoptosis, suggesting that the activation of the proteasome pathway is one of the mechanisms involved. Overall, our data suggest that the docetaxel and flavopiridol combination requires a maximal effect on cyclin B1-dependent kinase activity and a reduction of XIAP and AKT prosurvival proteins for augmentation of apoptosis in LNCaP cells

Abstract 334: Mcl-1 protects prostate cancer cells from chemotherapy-induced DNA damage

Abstract Mcl-1 (myeloid cell leukemia sequence 1 [Bcl2-related]) is predominantly localized in the cytoplasm and protects cells from apoptosis by blocking mitochondrial outer membrane permeabilization. There is evidence that nuclear localization of Mcl-1 has pleiotropic effects independent of apoptosis, including functional roles in DNA damage response. Mcl-1 is highly expressed in castration-resistant prostate cancer (CRPC), resulting in resistance to apoptosis and association with poor prognosis. The role of nuclear Mcl-1 in mediating sensitivity to chemotherapy-induced DNA damage and apoptotic cell death in prostate cancer (PCa) is not known. The novel antimitotic agent ENMD-1198 is a more stable and potent derivative of 2-methoxyestradiol (2ME2). We confirmed that 1198 was a more potent inhibitor of androgen-dependent LNCaP and castration-resistant DU145 and PC3 PCa cell viability compared to 2ME2 and induced apoptotic cell death in docetaxel-resistant PC3 cells that overexpress the multi-drug resistance protein (MDR). The ability of 1198 to increase the proteasomal-mediated degradation of Mcl-1 correlated with enhanced apoptotic cell death and shRNA knockdown of Mcl-1 further sensitized LNCaP and PC3 cells to 1198-mediated cell death. We then showed that the combination of 1198 with betulinic acid (BA, a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells) reduced nuclear Mcl-1, increased a pro-death Mcl-1 isoform, increased gamma histone 2AX (marker of DNA damage), and enhanced apoptotic cell death in human PC3 CRPC cells. In the TRAMP mouse model of PCa, the 1198 (75 mg/kg) + BA (10 mg/kg) combination significantly reduced PCa weights compared to 1198, BA alone, and vehicle controls. However, the reduced PCa weights between the 1198 + BA combination and BA alone was more associated with decreased Mcl-1 and increased gamma-H2AX (DNA damage) immunostaining compared to cleaved caspase-3 (apoptosis), CD31(angiogenesis), or Ki67 (proliferation). Furthermore, our results showed that blocking the DNA damage response signaling pathway with an ATM kinase (KU55933) or DNA-PK (NU7441) inhibitor lowered apoptotic cell death induced by the 1198 + BA combination in PC3 cells. Overall, our results demonstrate that nuclear Mcl-1 has an important role in protecting PCa cells from DNA damage and that agents such as 1198 and BA that reduce total/nuclear Mcl-1 and increase pro-death Mcl-1 isoforms will sensitize PCa cells to antimitotic DNA damaging agents. Citation Format: Carlos Perez-Stable, Teresita Reiner, Alicia de las Pozas, Ricardo Parrondo. Mcl-1 protects prostate cancer cells from chemotherapy-induced DNA damage. [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 334. doi:10.1158/1538-7445.AM2014-33