Final Report Summary - ASPIRE-PC (A novel Androgen Synthesis Pathway in treatment-REsistant Prostate Cancer) The last 10 years have witnessed a paradigm shift in our understanding of advanced stages of prostate cancer (PC) biology. Disease progression following castration was deemed androgen-independent and as such non-hormonal therapies were pursued. Pre-clinical developments since 2004 have however demonstrated continued relevance of PC on androgen receptor (AR) activation as well as detected the presence of intratumoural androgens following castration. Dr. Hofland was actively involved in this field and demonstrated that intratumoural steroids were most likely the consequence of adrenal androgen to dihydrotestosterone conversion. Concurrently, the introduction of novel anti-hormonal therapies with the steroidogenic enzyme CYP17A1 inhibitor abiraterone and AR antagonist enzalutamide led to improved survival in patients with castration-resistance prostate cancer (CRPC). This undeniably confirmed the relevance of intratumoural androgens and AR activation as key drivers of castration-resistance disease. Following the introduction of abiraterone and enzalutamide in the treatment of PC, focus shifted to unravelling the causes of resistance to these novel anti-hormonal therapies. Among other mechanisms, preclinical and clinical studies pointed towards upregulation of intratumoural steroidogenic enzymes and steroid flux through an alternative androgen synthesis pathway as possible contributors to treatment resistance. Key data published by Prof. Arlt in collaboration with the Institute of Cancer Research, London, uncovered elevated levels of an alternative pathway intermediate in men treated with abiraterone. As a consequence PC cells might be capable of developing the ability to produce their own androgens from the common steroid precursor cholesterol and regain AR activation and cellular proliferation. The combination of Prof. Arlt’s track record as a world leader in the field of steroid diagnostics and the fellow’s expertise in endocrinology of PC was the ideal basis for this project on the alternative androgen synthesis pathway in PC as a novel mechanism of resistance to contemporary hormonal treatment.Dr. Hofland employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to map the steroid metabolome in PC cell lines, mice engrafted with human PC xenografts and ex vivo patient prostate samples. His studies comprised the first comprehensive steroid fluxes analysis in PC cells and was made possible through an optimized LC-MS/MS-based technique simultaneously measuring 21 steroid compounds at a nanomolar threshold. Collaboration with Dr. Wytske van Weerden from the Department of Urology at the Erasmus MC, Rotterdam, ensued the unique availability of a plethora of drug-resistant cell lines and xenografts as well as patient tissues resistant to hormonal therapy. On top of this, human adrenal and prostate tissues were collected at the University Hospital Birmingham for biobanking and primary culture.The results of these studies, which are currently prepared for publication, consistently demonstrated a persistent CYP17 block in PC samples throughout disease evolution. Our results showed for the first time that flux into the alternative adrenal androgen pathway occurs within PC cells. The low but detectable presence of CYP17A1 mRNA however is not accompanied by 17-hydroxylase or 17,20-lyase activities through either the classic or the alternative pathway. This effectively demonstrates the lack of de novo androgen synthesis at all stages of PC. Alternatively, conversion of adrenal androgens into active androgens occurred in all tested PC models. These enzyme activities were verified by demonstration of wildtype AR activation in PC cells by only steroids that were downstream of CYP17A1. In cells with mutant AR though alternative pathway steroids were also able to activate the AR. Furthermore, novel 20α/β-reduced and 6α-hydroxylated steroids were detected through an unbiased gas chromatography-mass spectrometry (GC-MS) approach. Together these findings suggest a lack of de novo steroid synthesis during PC evolution and emphasize the vital importance of circulating androgen precursors of adrenocortical origin for PC progression. These studies provide conclusive evidence on the debate of the source of intratumoural androgens as well as disprove a hypothesized and frequently cited mechanism of resistance to the novel anti-hormonal drugs. In addition, these findings open up new hypotheses for the role of alternative pathway steroids in PC. Presentation of these findings at the European Congress of Endocrinology resulted in a poster prize award for the fellow Dr. Hofland (top 10 poster of 1700 submissions).During his fellowship Dr. Hofland also studied the effects of abiraterone and enzalutamide on adrenal steroidogenesis. Abiraterone induced flux into the alternative pathway in both the adrenocortical H295R cell line and human primary adrenal cell cultures. Steroid flux from CYP17A1 substrate allopregnanolone to DHT however did not occur in the presence of abiraterone. Factors regulating entry into the alternative pathway were further investigated by siRNA-mediated knockdown of the steroidogenic enzymes CYP17A1 and CYP21A2 as well as co-factor POR in adrenal cells. These results revealed that steroid flux into the alternative pathway appears to primarily depend on CYP17A1 activity. Interestingly, we discovered a previously unreported effect of enzalutamide on adrenal steroidogenic enzyme expression, stimulating androgen production. Validation of this novel finding with possible clinically relevance is currently underway.This project was also instrumental in improving the scientific skills of the fellow. Besides in-depth knowledge on LC-MS/MS he further extended his competence in GC-MS, primary tissue isolation, yeast cell cultures, microsomal and nuclear receptor activation assays, RNA interference, transfection and protein blotting. Through collaboration with Dr. van Weerden at the Erasmus MC the fellow helped to develop and validate a novel culture technique of microtome-cut tissue slices, steroid extraction from tumour tissues and a humanized PC xenograft model in mice. These method developments are significant contributions to the field of steroid metabolome mapping in prostate and other cancers and will soon be submitted for publication.The international fellowship allowed Dr. Hofland to interact with key reseachers in the PC field in Europe and beyond. This led to new collaborations and project proposals with The Institute of Cancer Research in London, the Cambridge Research Institute, Center for Liver, Digestive and Metabolic Diseases at University Medical Center Groningen and Stellenbosch University, South Africa. During this period he also became part of the research initiatives European Task Group for Endocrine Cancers (ETEC) and the Sunrise group (Erasmus Prostate Cancer Research Group). As a clinician scientist Dr. Hofland was also actively involved in clinics by attending PC clinics at the department of Urology and Oncology as well as perform his own out-patient clinics for patient with endocrine diseases at the University Hospital Birmingham.
