Endocrine Abstracts

Introduction: Prostate cancer (PC) is dependent on androgen receptor (AR) activation by its canonical ligands testosterone and 5α-dihydrotestosterone (DHT). Intratumoural androgens persisting after castration give rise to castration-resistant PC (CRPC). These intraprostatic androgen levels are hypothesized to result from either adrenal androgen conversion or intratumoural de novo DHT synthesis through the classic or alternative pathways. Quantifying the steroid fluxes responsible for CRPC development can help optimize current endocrine treatment strategies.

Methods: Five PC cell lines were incubated with 1 μM of 16 steroid intermediates of the classic and alternative androgen synthesis pathways. A PC steroid metabolome was constructed through measurement of steroid metabolite concentrations with liquid chromatography/tandem mass spectrometry (LC–MS/MS). Steroidogenic enzyme expression was estimated by quantitative PCR.

Results: Steroid metabolites could be detected for up to three enzymatic steps downstream of the employed steroid substrate. 17-hydroxylase/17,20-lyase activity was undetectable in all PC cell lines with eight different substrates tested, thereby excluding conversion above 0.03% of the substrate. In contrast, 3α-, 3β- and 17β-hydroxysteroid dehydrogenase and 5α-reductase activities were present in all cell lines to varying degrees. Steroid flux analysis confirmed conversion of adrenal androgen precursors and androgen metabolites into DHT. Furthermore, C21 steroids progesterone and 17hydroxy-progesterone were metabolised into intermediates of the alternative pathway without reaching androsterone. The highest steroidogenic activity was observed in C4-2B cells, a castration-resistant, bone-metastasizing clone of LNCaP. Expression profiles of steroidogenic enzymes matched the observed enzymatic activities.

Conclusion: For the first time, a quantitative steroid metabolome of PC cells has been generated through LC–MS/MS, providing strong evidence against intratumoural de novo steroid synthesis. Adrenal androgens can effectively be converted into DHT, whereas precursor C21 steroids divert towards the alternative pathway. The presence of these hormones in PC might suggest a role for activity or further metabolism of alternative pathway steroids in PC evolution.

Disclosure: This work was supported by the European Union (Marie-Curie Intra-European Fellowship, grant number PIEF-GA-2013-629745).