The Biological Characterization of Galeterone Analogs VNPT-178 and VNLG-74A for the Treatment of Prostate Cancer Item Type dissertation Authors McCarty, David Publication Date 2017 Abstract The expression of truncated androgen receptor spice-variants (AR-Vs) presents a significant clinical challenge for the treatment of advanced stages of prostate cancer and confers complete resistance to abiraterone and enzalutamide. Targeted depletion... Keywords galeterone; Prostate--Cancer; Receptors, Androgen; Unfolded Protein Response Download date 05/10/2021 16:14:55 Link to Item http://hdl.handle.net/10713/7073 CURRICULUM VITAE: David McCarty [email protected] Ph.D., July 25th, 2017 University of Maryland School of Medicine, August 2011-July 2017 Doctor of Philosophy, July 2017 Molecular Medicine – Cellular and Molecular Biology and Physiology Saint Cloud State University, August 2003-December 2009 Bachelor of Science, December 2009 Biomedical Sciences Associate of Arts, May 2005 General Education PENDING PUBLICATION: David J. McCarty, Weiliang Huang, Maureen A. Kane, Puranik Purushottamachar, Lalji K. Gediya. (2017) Novel Galeterone Analogs Act Independently of AR and AR-V7 for the Activation of the Unfolded Protein Response and Induction of Apoptosis in the CWR22Rv1 Prostate Cancer Cell Model. Oncotarget ISSN: 1949-2553 i ABSTRACT: Title of Dissertation: The Biological Characterization of Galeterone Analogs VNPT-178 and VNLG-74A for the Treatment of Prostate Cancer David J. McCarty, Doctor of Philosophy, 2017 Dissertation Directed by: Dr. Vincent C.O. Njar, Professor of Medicinal Chemistry and Pharmacology, Department of Pharmacology The expression of truncated androgen receptor spice-variants (AR-Vs) presents a significant clinical challenge for the treatment of advanced stages of prostate cancer and confers complete resistance to abiraterone and enzalutamide. Targeted depletion of these receptors in addition to antagonism of the androgen signaling axis has been actively evaluated as a superior treatment paradigm but has thus far been unsuccessful in clinical trials. Based on our studies of galeterone and its analogs, we believe these compounds possess a specific activity that is both independent and overlapping of their antagonism of the androgen receptor (AR). In this study, we perform basic biochemical techniques attempting to better delineate the actions of galeterone analogs VNPT-178 and VNLG- 74A and identify a rational, common target for improved study of their utility against all stages of prostate cancer development. Direct comparisons of VNPT-178 and VNLG-74A to vehicle or equimolar concentrations of abiraterone and enzalutamide reveal both compounds exhibit improved antiproliferative activities in multiple prostate cancer cell models of androgen and AR dependence. VNPT-178 and VNLG-74A directly antagonize the full-length AR in ii LNCaP and CWR22Rv1 (22Rv1) cells while promoting the depletion of full-length and truncated receptors with dose- and time-dependency. Activation of the unfolded protein response is rapid and sustained, long preceding appreciable antagonism of the AR in 22Rv1 cells, and followed by CHOP upregulation and PARP cleavage—an effect also seen in PC-3 cells albeit with slower kinetics. Molecular docking of VNPT-178 and VNLG-74A reveals greater potential for binding the ATPase domains of BiP/Grp78 and Hsp70-1A compared to the AR’s ligand-binding domain. Severe or sustained activation of the unfolded protein response can induce apoptosis. Taken together, our data suggest that galeterone analogs VNPT-178 and VNLG-74A directly modulate the substrate interactions of BiP promoting endoplasmic reticulum stress and apoptosis independent of AR antagonism. Depletion of AR proteins is possibly the result of similar activities against Hsp70. The sensitivity of 22Rv1 cells to VNPT-178- and VNLG-74A-induced apoptosis may underlie a failure of this cell model to adequately represent the clinical challenge of AR-V-expressing prostate cancers. iii The Biological Characterization of Galeterone Analogs VNPT-178 and VNLG-74A for the Treatment of Prostate Cancer by David J. McCarty Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, Baltimore in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2017 iv TABLE OF CONTENTS: INTRODUCTION……………………………………………………………………………1 PROSTATE CANCER AT A GLANCE……………………………………………………...1 PROSTATE CANCER BIOLOGY AND CLINICAL PRESENTATION………………………….1 HORMONE THERAPY FOR PROSTATE CANCER………………………………………….3 THE ANDROGEN RECEPTOR (AR)……………………………………………………...7 MECHANISMS OF RESISTANCE TO AR-DIRECTED PROSTATE CANCER THERAPIES………………………………………………………………………..11 ANDROGEN RECEPTOR DEPLETING AGENTS (ARDAS) AND GALETERONE…………..12 THE ENDOPLASMIC RETICULUM STRESS/UNFOLDED PROTEIN RESPONSE (ERSR/UPR)……………………………………………………………………..15 PROJECT OVERVIEW AND SPECIFIC AIMS ……………………………………………...19 MATERIALS AND METHODS……………………………………………………………..22 SPECIFIC AIM 1…………………………………………………………………………..31 S1A: COMPARE THE ANTIPROLIFERATIVE ACTIVITY OF NOVEL GALETERONE ANALOGS TO ABIRATERONE AND ENZALUTAMIDE USING THE MTT ASSAY.....................................................................................................32 S1B: COMPARE THE ABILITY OF GALETERONE ANALOGS FOR THE INHIBITION OF FULL-LENGTH AR TRANSCRIPTIONAL ACTIVATION USING A TRANSIENTLY TRANSFECTED LUCIFERASE REPORTER SYSTEM…………...39 S1C: DETERMINE WHICH OF THE NOVEL ANALOGS CAN INHIBIT THE ENDOGENOUS EXPRESSION OF THE ANDROGEN RECEPTOR IN 22RV1 CELLS………41 SUMMARY AND CONCLUSIONS FROM SPECIFIC AIM 1………………………………...43 SPECIFIC AIM 2…………………………………………………………………………..45 S2A: DETERMINE IF LONGER TREATMENTS WITH LOWER DOSES THAN THE PREDICTED GI50 VALUES COULD IMPROVE RELATIVE ACTIVITY OF GALETERONE ANALOGS TO ESTABLISHED CONTROLS..............................................46 S2B: DETERMINE THE MINIMUM CONCENTRATION REQUIRED TO ELICIT AR ANTAGONISM AND DEPLETION WITHIN 24 HOURS IN LNCAP AND 22RV1 CELLS……………………………………………………………………..48 S2C: EVALUATE CELL DEATH ASSOCIATED WITH DOSE AND TIME OF TREATMENT WITH VNPT-178 AND VNLG-74A USING THE TRYPAN BLUE EXCLUSION ASSAY………………………………………………………….52 iii S2D: EVALUATE CHANGES IN CELL CYCLE PROGRESSION FOLLOWING TREATMENT WITH INCREASING DOSES OF VNPT-178 AND VNLG-7A …………...54 SUMMARY AND CONCLUSIONS FROM SPECIFIC AIM 2 ………………………………..58 SPECIFIC AIM 3…………………………………………………………………………..62 S3A: DETERMINE WHETHER GALETERONE ANALOGS INDUCE NECROSIS OR APOPTOSIS …………………………………………………………………….63 S3B: DETERMINE THE AR-DEPENDENCY OF CELL DEATH INDUCTION IN PROSTATE CANCER CELLS ………………………………………………………...65 S3C: DETERMINE THE ERSR/UPR DEPENDENCY OF CELL DEATH INDUCTION IN PROSTATE CANCER CELLS………………………………………….69 S3D: IDENTIFY NOVEL TARGETS OF GALETERONE ANALOGS VNPT-178 AND VNLG-74A………………………………………………………………….74 SUMMARY AND CONCLUSIONS FROM SPECIFIC AIM 3………………………………...78 SPECIFIC AIM 4…………………………………………………………………………..80 S4A: DETERMINE THE GROWTH INHIBITORY EFFECTS OF GALETERONE ANALOGS AGAINST THE AR-V7-EXPRESSING 22RV1 XENOGRAFT MODEL………..81 S4B: EVALUATE THE SAFETY AND TOLERABILITY OF GALETERONE ANALOGS ADMINISTERED VIA ORAL GAVAGE IN MALE ATHYMIC NUDE MICE……..82 SUMMARY AND CONCLUSIONS FROM SPECIFIC AIM 4………………………………...85 SUMMARY AND FUTURE DIRECTIONS…………………………………………………...86 REFERENCES……………………………………………………………………………..93 iv LIST OF TABLES: TABLE 1: SUMMARY OF AR EXPRESSION AND ACTIVITY IN PROSTATE CANCER CELLS…...34 TABLE 2: SUMMARY OF MTT ASSAYS IN LNCAP CELLS...……………………………….35 TABLE 3: SUMMARY OF MTT ASSAYS IN 22RV1 CELLS….………………………………36 TABLE 4: SUMMARY OF MTT ASSAYS IN PC-3 CELLS....…………………………………37 v LIST OF FIGURES: FIGURE 1: THE PROSTATE GLAND………………………………………………………….2 FIGURE 2: THE HYPOTHALAMIC-PITUITARY-GONADAL/ADRENAL (HPG/A) AXIS…………5 FIGURE 3: SYNTHESIS OF STEROID HORMONES FROM CHOLESTEROL………………………6 FIGURE 4: THE STRUCTURE OF THE ANDROGEN RECEPTOR………………………………...8 FIGURE 5: ANDROGEN ACTION IN THE CELL………...……………………………………10 FIGURE 6: THE ANDROGEN RESPONSE ELEMENT (ARE) IS A PALINDROMIC SEQUENCE WITH TWO COMPLEMENTARY HEXAMERS SEPARATED BY THREE BASES ……………………………………………………...………11 FIGURE 7: THE ANDROGEN RECEPTOR (AR) GENE CONTAINS MULTIPLE CODING REGIONS THAT CAN BE ALTERNATIVELY INCORPORATED INTO PROTEIN…………………………………………………………………13 FIGURE 8: THE ENDOPLASMIC RETICULUM STRESS RESPONSE (ERSR)…………………...16 FIGURE 9: CHEMICAL STRUCTURES OF DIHYDROTESTOSTERONE (DHT), GALETERONE, AND GALETERONE ANALOGS…………………………………..33 FIGURE 10: DOSE-RESPONSE CURVES FOR ABIRATERONE, ENZALUTAMIDE, GALETERONE, AND THE TOP FIVE ANTIPROLIFERATIVE ANALOGS …………... 38 FIGURE 11: ANDROGEN RECEPTOR ANTAGONISTS INHIBIT EXPRESSION OF ARR2-LUCIFERASE REPORTER IN LNCAP AND 22RV1 CELLS……………….40 FIGURE 12: GALETERONE ANALOGS CAN REDUCE FULL-LENGTH AND TRUNCATED ANDROGEN RECEPTOR PROTEIN EXPRESSION IN 22RV1 CELLS………………………………………………………………...42 FIGURE 13: GALETERONE ANALOGS VNPT-178 AND VNLG-74A DOSE DEPENDENTLY INHIBIT FORMATION AND GROWTH OF ADHERENT 22RV1 COLONIES………………………………………………………...47-48 FIGURE 14: GALETERONE ANALOGS VNPT-178 AND VNLG-74A DOSE- DEPENDENTLY DECREASE AR AND PSA PROTEIN EXPRESSION IN LNCAP AND 22RV1 CELLS………………………………………………50-51 FIGURE 15: GALETERONE ANALOGS VNPT-178 AND VNLG-74A INHIBIT PROLIFERATION AND INDUCE CELL DEATH IN LNCAP CELLS………………...53 FIGURE 16: CELL CYCLE ANALYSIS OF LNCAP, 22RV1, AND PC-3 CELLS……………55-57 FIGURE 17: CONTINUOUS TREATMENT WITH ARDA ACIDIFIES MEDIA IN LNCAP AND 22RV1 CELLS AFTER 48 HOURS………………………………..59 vi FIGURE 18: REFRESHING MEDIA AFTER 24 HOURS IMPROVES THE AR- DEPLETIVE EFFECTS