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Evidence of Limited Contributions for Intratumoral Steroidogenesis in Prostate Cancer

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Evidence of Limited Contributions for Intratumoral Steroidogenesis in Prostate Cancer Published OnlineFirst January 19, 2010; DOI: 10.1158/0008-5472.CAN-09-2092 Tumor and Stem Cell Biology Cancer Research Evidence of Limited Contributions for Intratumoral Steroidogenesis in Prostate Cancer Johannes Hofland1, Wytske M. van Weerden2, Natasja F.J. Dits2, Jacobie Steenbergen1, Geert J.L.H. van Leenders3, Guido Jenster2, Fritz H. Schröder2, and Frank H. de Jong1 Abstract Androgen-deprivation therapy for prostate cancer (PC) eventually leads to castration-resistant PC (CRPC). Intratumoral androgen production might contribute to tumor progression despite suppressed serum andro- gen concentrations. In the present study, we investigated whether PC or CRPC tissue may be capable of in- tratumoral androgen synthesis. Steroidogenic enzyme mRNAs were quantified in hormonally manipulated human PC cell lines and xenografts as well as in human samples of normal prostate, locally confined and advanced PC, local nonmetastatic CRPC, and lymph node metastases. Overall, the majority of samples showed low or absent mRNA expression of steroidogenic enzymes required for de novo steroid synthesis. Simultaneous but low expression of the enzymes CYP17A1 and HSD3B1, essential for the synthesis of androgens from preg- nenolone, could be detected in 19 of 88 patient samples. Of 19 CRPC tissues examined, only 5 samples ex- pressed both enzymes. Enzymes that convert androstenedione to testosterone (AKR1C3) and testosterone to dihydrotestosterone (DHT; SRD5A1) were abundantly expressed. AKR1C3 expression was negatively regulated by androgens in the experimental models and was increased in CRPC samples. Expression of SRD5A1 was upregulated in locally advanced cancer, CRPC, and lymph node metastases. We concluded that intratumoral steroid biosynthesis contributes less than circulating adrenal androgens, implying that blocking androgen pro- duction and its intraprostatic conversion into DHT, such as via CYP17A1 inhibition, may represent favorable therapeutic options in patients with CRPC. Cancer Res; 70(3); 1256–64. ©2010 AACR. Introduction docetaxel chemotherapy combined with prednisone is only temporarily effective with a small survival benefit (3). Due to Despite current early detection methods and surgical and this untreatable stage of disease, PC is still estimated to be the radiotherapeutic treatment options, many prostate cancer cause of 28,660 deaths in the United States in 2008 alone (4). (PC) patients still present with unresectable stages of disease Several hypotheses underlie the occurrence of CRPC, such (1). Since 1941, the mainstay of treatment of advanced PC as the selective outgrowth of androgen-independent clonal is focused on suppression of intraprostatic testosterone and cell populations caused by activation, suppression, or fusion dihydrotestosterone (DHT) actions. Nowadays, androgen of genes and AR signaling pathway–related causes, encom- deprivation therapy is based on lowering the luteinizing hor- passing ligand-independent AR activation, AR hypersensitiv- mone (LH)–induced testicular testosterone production ity due to AR overexpression, and ligand promiscuity due to through LH-releasing hormone agonists (chemical castration) AR mutations (5, 6). More recently, intratumoral conversion with or without antiandrogens that block the androgen re- of adrenal androgens and de novo steroid synthesis have been ceptor (AR). Growth inhibition is initially achieved in the ma- brought forward as potential causes of tumor progression jority of patients. However, eventually all patients develop (7–9). The presence of active AR in CRPC samples and the hormone-refractory or castration-resistant PC (CRPC), reported high intratumoral testosterone and DHT concentra- marked by an increase in prostate-specific antigen (PSA) tions in CRPC patients with castrate serum androgen levels and progression of the tumor (2). Second-line treatment with support the concept of intratumoral conversion of steroidal precursors (8, 10). Recent publications have put renewed em- Authors' Affiliations: Departments of 1Internal Medicine, 2Urology, and phasis on this intratumoral steroidogenesis by showing the 3Pathology, Erasmus MC, Rotterdam, the Netherlands previously unknown expression of steroidogenic enzymes in Note: Supplementary data for this article are available at Cancer normal prostate and PC tissue (8, 9, 11, 12), as well as a dif- Research Online (http://cancerres.aacrjournals.org/). ferential expression pattern between the various tumor types J. Hofland and W.M. van Weerden contributed equally to the study and in and the normal prostate gland (8, 9). Furthermore, conversion the preparation of the manuscript. of the radiolabeled steroid precursor acetic acid into DHT Corresponding Author: Johannes Hofland, Room Ee-532, Department of Internal Medicine, Erasmus MC, P.O. Box 2040, 3000 CA Rotterdam, has been shown to occur in vitro in LNCaP cells and ex vivo the Netherlands. Phone: 31-10-4073577; Fax: 31-10-7035430; E-mail: in CRPC cells (7). The potential upregulation of steroido- [email protected]. genic enzymes in CRPC and the resulting local testosterone doi: 10.1158/0008-5472.CAN-09-2092 and DHT production may account for the observed intratu- ©2010 American Association for Cancer Research. moral androgens in levels sufficient to activate the AR (7, 8, 1256 Cancer Res; 70(3) February 1, 2010 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 19, 2010; DOI: 10.1158/0008-5472.CAN-09-2092 Intraprostatic Steroidogenic Enzymes Figure 1. Simplified scheme of the classic steroid biosynthetic pathway. The depicted steroidogenic enzymes and cofactors are necessary for de novo synthesis of androgens from cholesterol and were investigated in this study. CYP, cytochrome P450; HSD, hydroxysteroid-dehydrogenase; CYB5, cytochrome b5; AKR, aldo-keto-reductase; SRD, 5α-reductase. 10, 13). Consequently, such a mechanism would require androstenedione were abundantly expressed. Their expres- new therapeutic modalities that aim to block intratumoral sion was affected by androgen levels and could be related steroidogenesis. to PC progression. De novo steroid production has been thought to be con- fined to a few organs: the gonads, the adrenal cortex, and Materials and Methods the placenta. Steroidogenesis is initiated by the transport of cholesterol through the mitochondrial membrane, for which In vitro cultures and tumor-bearing mice. The in- the steroid acute regulatory protein (STAR) is obligatory vestigated cell lines and xenografts have been characterized (Fig. 1). Once cholesterol has entered the mitochondria, it previously (17–21), as summarized in Table 1. The PC cell can be converted into pregnenolone by desmolase, also known as cytochrome P450 side-chain cleavage enzyme (CYP11A1). The Δ5-steroid pregnenolone can subsequently be converted through 17-hydroxylase and 17,20-lyase activi- Table 1. Prostate cancer cell line and xenograft ties [both encoded by CYP17A1, together with cofactors cyto- characteristics chrome P450 oxidoreductase (POR) and cytochrome b5 (CYB5A1)] and through 3β-hydroxysteroid dehydrogenase ac- Androgen AR status Origin tivity (by either of the isoenzymes HSD3B1 or HSD3B2) to dependence form androstenedione (14, 15). Further metabolism of andros- tenedione can take place in peripheral tissues, such as the Cell lines male reproductive tract. Here, several 17β-hydroxysteroid LNCaP Responsive AR+ Lymph node dehydrogenase enzymes are able to convert androstenedione VCaP Responsive AR+ Bone to testosterone, of which the type 5 enzyme (AKR1C3 or DuCaP Responsive AR+ Dura HSD17B5) is the most important isoform in the prostate gland PC346C Responsive AR+ PC346 (16). Androstenedione and testosterone can be aromatized in- Xenografts to estrogens by CYP19A1. The two 5α-reductase isoforms, PC82 Dependent AR+ Prostate SRD5A1 and SRD5A2, are able to metabolize testosterone to PC295 Dependent AR+ Lymph node DHT. The type 2 reductase is thought to be the most impor- PC310 Dependent AR+ Prostate tant isoform for intraprostatic conversion (16). PC346 Responsive AR+ TURP To study the capability of human PC to synthesize andro- PC346B Responsive AR+ TURP gens de novo as well as to convert adrenal androgens locally, PC374 Responsive AR+ Skin we measured steroidogenic enzyme expression in a large set PC133 Independent AR− Bone of hormonally manipulated experimental models of PC and PC135 Independent AR− Prostate in patient material from normal prostate, local PC, lymph PC324 Independent AR− TURP node metastases, and transurethral resection of the prostate PC339 Independent AR− TURP (TURP) tissues. In all samples, the expression of key enzymes PC346I Unresponsive AR+ PC346 required for de novo synthesis of androgens was low or ab- PC346BI Unresponsive AR+ PC346B sent and was not affected by androgen ablation therapy. The PC374F Unresponsive AR+ PC374 enzymes required for testosterone and DHT production from www.aacrjournals.org Cancer Res; 70(3) February 1, 2010 1257 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst January 19, 2010; DOI: 10.1158/0008-5472.CAN-09-2092 Hofland et al. Table 2. Patient characteristics First series Second series Normal Locally Lymph node Locally CRPC Locally CRPC prostate confined PC metastases advanced PC advanced PC No. patients, unique 17 11 16 21 10 4 9 Age at diagnosis 62 (54–72) 62 (56–70) 66 (52–71) 63 (46–68) 62 (53–68) 73 (67–81) 68 (50–86)
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