The metabolism of 11β-hydroxyandrostenedione by steroidogenic enzymes yields metabolites contributing to the androgen pool in prostate cancer by Therina du Toit Dissertation presented for the degree of Doctor of Philosophy (Biochemistry) in the Faculty of Science at Stellenbosch University Promoter: Prof Amanda C. Swart March 2018 Stellenbosch University https://scholar.sun.ac.za Declaration By submitting this thesis electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly otherwise stated), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. This thesis includes 4 original papers published in peer-reviewed journals. The development and writing of the papers were the principal responsibility of myself and, for each of the cases where this is not the case, a declaration is included in the thesis indicating the nature and extent of the contributions of co-authors. T du Toit March 2018 Copyright © 2018 Stellenbosch University All rights reserved ii Stellenbosch University https://scholar.sun.ac.za Ter herinnering aan Katherina Helena (Ina) Theron Dankie vir my naam, ek mis Ouma iii Stellenbosch University https://scholar.sun.ac.za Summary This study describes: • The development and validation of three ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS) analytical methods which were applied in the detection and quantification of C19 and C21 steroids, including C11-oxy C19 and C11-oxy C21 steroids; • The investigation into the contribution of adrenal 11β-hydroxyandrostenedione (11OHA4) and 11β-hydroxytestosterone (11OHT) to the pool of active androgens in the prostate, by following androgen metabolism in normal epithelial prostate PNT2, benign prostatic hyperplasia (BPH-1) and prostate cancer LNCaP, C4-2B and VCaP cell models; Steroid profiles revealed 11β-hydroxysteroid dehydrogenase type 2 (11βHSD2) activity in all the cell models, confirmed in the conversion of 11OHA4 to 11keto-androstenedione (11KA4), with reductive 17β-hydroxysteroid dehydrogenase (17βHSD) enzymes metabolising 11KA4, ultimately yielding 11keto-testosterone (11KT). • The in vitro investigation into the inactivation, reactivation, glucuronidation and sulfation of 11OHA4, 11OHT and their downstream metabolites; In prostate cancer (PCa) cell models, the conjugation of 11KT and 11keto- dihydrotestosterone (11KDHT) were hampered compared to testosterone (T) and dihydrotestosterone (DHT), while the inactivation and reactivation of the C11-oxy C19 steroids were less efficient than the C19 steroids in BPH-1 cells. • The in vivo steroid profiles in PCa, BPH and castration-resistant prostate cancer (CRPC) tissue and plasma of healthy and PCa patients; Analyses of the C19 and C11-oxy C19 steroids, together with glucuronide and sulfate conjugates, showed increased unconjugated levels of 11KT and 11KDHT in plasma of PCa patients compared to a healthy subject, and 11OHA4, 11KT and 11KDHT levels were prominent in PCa tissue, while downstream inactive C11-oxy 3α-reduced metabolites were identified in BPH and CRPC tissue. iv Stellenbosch University https://scholar.sun.ac.za Opsomming Hierdie studie beskryf die volgende: • Die ontwikkeling en validering van drie UPC2-MS/MS analitiese metodes wat vervolgens toegepas is in die skeiding en kwantifisering van steroïedmetaboliete. • Die ondersoek na 11OHA4 and 11OHT se bydrae tot die aktiewe androgeen poel in die prostaat; Androgeen metabolisme is in normale epiteel prostaat PNT2, BPH-1 en prostaat kanker LNCaP, C4-2B en VCaP selmodelle ondersoek. Die steroïdprofiele bevestig die teenwoordigheid van 11βHSD2 aktiwiteit in al die modelle in die omsetting van 11OHA4 na 11KA4, sowel as die aktiwiteit van 17βHSD wat vervolgens die omsetting van 11KA4 na 11KT gekataliseer het. • Die in vitro inaktivering, heraktivering en konjugering van 11OHA4 en 11OHT metaboliete; In prostaatkanker selmodelle was 11KT en 11KDHT konjugering oneffektief in vergelyking met T en DHT, en die inaktivering van die C11-oksie C19 steroïede was nie optimaal in vergelyking met die C19 steroïede in BPH-1 selmodelle nie. • Die in vivo steroïedprofiele in prostaatkankerweefsel, BPH weefsel en kastrasie- weerstandige prostaatkankerweefsel en in plasma van gesonde normale individuë en prostaatkanker pasïente, asook die teenwoordigheid van ongekonjugeerde en gekonjugeerde steroïedmetaboliete in sirkulasie; Analiese toon dat ongekonjugeerde 11KT en 11KDHT hoër was in die plasma van pasïente met prostaatkanker; 11OHA4, 11KT en 11KDHT vlakke was abnormaal hoog in die prostaatkankerweefsel, terwyl onaktiewe C11-oksie 3α-gereduseerde metaboliete geïdentifiseer was in BPH weefsel en in kastrasie-weerstandige prostaatkankerweefsel. v Stellenbosch University https://scholar.sun.ac.za Acknowledgements I hereby wish to express my sincere gratitude and appreciation to: Prof Amanda C Swart, a remarkable woman, who has helped me discover myself, my inquisitive nature for science and the world. Without you none of this would have been possible, thank you. You are an amazing supervisor and role-model, I hope that we will always find the time to talk about our day and have a G&T. Prof Pieter Swart, thank you for teaching me to appreciate the ‘everyday’. I will always cherish our conversations around the dinner table. Ralie Louw, a wonderful laboratory manager, who was always willing to help me and order the products needed for my project – thank you! The technical and administrative staff of the Biochemistry department, especially Reggie Brandt for all the nitrogen canisters you had to carry for me. Dr. Maria ‘Marietjie’ Stander and her CAF team at Stellenbosch University for their technical expertise during UPC2-MS/MS and UPLC-MS/MS analyses. The Harry Crossley foundation and trustees, the National Research Foundation, the South African Rooibos Council and Stellenbosch University for funding this project. My fellow students – this journey was worth it because of all of you! Colin – you are my best friend and my favourite person to talk to, you mean the world to me, never forget that. My mother – your support and unconditional love is what motivates me, keep me safe, allows me to dream and has provided me with a home and an amazing life. vi Stellenbosch University https://scholar.sun.ac.za Contents Chapter 1 ............................................................................................................................................................. 1 General introduction ....................................................................................................................................... 1 Chapter 2 ............................................................................................................................................................. 7 The biosynthesis of the C11-oxy C19 steroids and their contribution to prostate cancer ............................... 7 2.1 Introduction ........................................................................................................................................... 7 2.2 Adrenal steroidogenesis ........................................................................................................................ 8 2.2.1 The production of C11-oxy steroids by cytochrome P450 11β-hydroxylase and aldosterone synthase ................................................................................................................................................. 13 2.3 Steroid metabolic pathways in the prostate ....................................................................................... 17 2.3.1 The classic and alternative pathway to DHT production .............................................................. 17 2.3.2 The 11OHA4-pathway to 11KT and 11KDHT production ............................................................. 19 2.3.3 The backdoor pathway to DHT and 11KDHT production ............................................................. 22 2.4 Steroidogenic enzymes involved in steroid metabolic pathways in the prostate ............................... 24 2.4.1 11β-hydroxysteroid dehydrogenases ........................................................................................... 25 2.4.2 17β-hydroxysteroid dehydrogenases ........................................................................................... 26 2.4.3 5α-reductases ............................................................................................................................... 26 2.4.4 3α-hydroxysteroid dehydrogenases ............................................................................................. 27 2.4.5 Retinol-like hydroxysteroid dehydrogenases ............................................................................... 28 2.4.6 Uridine-diphosphate glucuronosyltransferases ........................................................................... 30 2.4.7 Sulfotransferases and sulfatases .................................................................................................. 31 2.5 C11-oxy C19 steroids and their implications in disease ........................................................................ 35 2.5.1 Androgen receptor action ............................................................................................................ 35 2.5.2