Endocrine-Related Cancer (2009) 16 1139–1155 Prostate cancer stromal cells and LNCaP cells coordinately activate the androgen receptor through synthesis of testosterone and dihydrotestosterone from dehydroepiandrosterone Atsushi Mizokami, Eitetsu Koh, Kouji Izumi, Kazutaka Narimoto, Masashi Takeda, Seijiro Honma1, Jinlu Dai 2, Evan T Keller 2 and Mikio Namiki Department of Integrative Cancer Therapy and Urology, Kanazawa University Graduate School of Medical Sciences, 13-1 Takaramachi, Kanazawa City 920-8640, Japan 1Teikoku Hormone MFG. Co., 1604 Shimosakunobe, Takatsu-ku, Kawasaki, Kanagawa 213, Japan 2Departments of Urology and Pathology, University of Michigan, Ann Arbor, Michigan, USA (Correspondence should be addressed to A Mizokami; Email: [email protected]) Abstract One of the mechanisms through which advanced prostate cancer (PCa) usually relapses after androgen deprivation therapy (ADT) is the adaptation to residual androgens in PCa tissue. It has been observed that androgen biosynthesis in PCa tissue plays an important role in this adaptation. In the present study, we investigated how stromal cells affect adrenal androgen dehydroepian- drosterone (DHEA) metabolism in androgen-sensitive PCa LNCaP cells. DHEA alone had little effect on prostate-specific antigen (PSA) promoter activity and the proliferation of LNCaP cells. However, the addition of prostate stromal cells or PCa-derived stromal cells (PCaSC) increased DHEA-induced PSA promoter activity via androgen receptor activation in the LNCaP cells. Moreover, PCaSC stimulated the proliferation of LNCaP cells under physiological concentrations of DHEA. Biosynthesis of testosterone or dihydrotestosterone from DHEA in stromal cells and LNCaP cells was involved in this stimulation of LNCaP cell proliferation. Androgen biosynthesis from DHEA depended upon the activity of various steroidogenic enzymes present in stromal cells. Finally, the dual 5a-reductase inhibitor dutasteride appears to function not only as a 5a-reductase inhibitor but also as a 3b-hydroxysteroid dehydrogenase inhibitor in LNCaP cells. Taken together, this coculture assay system provides new insights of coordinate androgen biosynthesis under the microenvironment of PCa cells before and after ADT, and offers a model system for the identification of important steroidogenic enzymes involved in PCa progression and for the development of the corresponding inhibitors of androgen biosynthesis. Endocrine-Related Cancer (2009) 16 1139–1155 Introduction progresses into what is termed an androgen non- Prostate cancer (PCa) is the most common malig- responsive phenotype. nancy and the second leading cause of cancer-related Multiple molecular mechanisms that could account death of men in the United States (Jemal et al. 2008). for the development of resistance to ADT have been Since advanced PCa is initially dependent upon proposed (Feldman & Feldman 2001), which typically androgens, androgen deprivation therapy (ADT) is invoke the androgen receptor (AR) as a key mediator in the first choice for advanced PCa. Unfortunately, after the progression of PCa (Takeda et al. 1996, Taplin & an initial response to ADT, PCa eventually loses Balk 2004). Moreover, alterations of AR itself, which responsiveness to the androgen blockade and are either absent or at low frequency in the original Endocrine-Related Cancer (2009) 16 1139–1155 Downloaded from Bioscientifica.comDOI: 10.1677/ERC-09-0070 at 09/25/2021 04:31:42AM 1351–0088/09/016–001139 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.orgvia free access A Mizokami et al.: Acceleration of DHEA activity androgen-dependent state, result in an androgen- expression of AKR1C3 (type 5 17b-HSD) in PCa hypersensitive situation where stimulation of PCa tissue, while Stanbrough et al. (2006) confirmed that growth occurs at castrate levels of androgens (Taplin ADT-resistant PCa and bone marrow metastases & Balk 2004). One of the AR alterations that occur is expressed increased levels of multiple genes respon- AR mutation that results in promiscuous ligand sible for androgen metabolism (HSD3B2, AKR1C3, specificity (Veldscholte et al. 1990). Therefore, in SRD5A1, AKR1C2, AKR1C1, and UGT2B15). These addition to its normal ligands, testosterone and studies provide support for the concept that PCa tissues dihydrotestosterone (DHT), both androstenediol, a can perform local biosynthesis of testosterone and precursor of testosterone, and estradiol can activate DHT resulting in activation of the AR (Labrie 1991). the AR and stimulate the proliferation of LNCaP cells, It remains unclear; however, in which cell types which have a mutated AR (Mizokami et al. 2004, testosterone and DHT are converted from DHEA to Arnold et al. 2005). Testosterone and the more active other androgens in PCa tissue, although the products androgen DHT are important factors in PCa pro- from DHEA and the relevant steroidogenic enzymes gression. These hormones are still present in PCa tissue are definitively present in the prostate. In this study, after ADT. Specifically, when PCa patients are treated we explored the hypothesis that PCa stromal cells with ADT, serum testosterone and DHT decrease to contribute to the biosynthesis of testosterone and less than one-tenth of pretreatment levels (Labrie et al. DHT in PCa. We demonstrated that testosterone 1985). However, testosterone and DHT in PCa tissue and DHT synthesized from DHEA in stromal cells are still present at 20–40% of pretreatment values activated AR in PCa epithelial cells in a paracrine (Labrie et al. 1985, Belanger et al. 1989, Forti et al. fashion and thus contribute to the development of ADT 1989, Mizokami et al. 2004, Nishiyama et al. 2004, resistance in PCa. Titus et al. 2005). These remaining androgens that are still present post-therapy may continue to promote AR activation and account for the observation that Materials and methods combination therapy with a LHRH agonist, to block Isolation of stromal cells from prostate androgen production, and an antiandrogen, to block carcinoma tissue ligand binding to the AR, is more effective for PCa treatment than either therapy alone (Labrie et al. 1985, All studies were approved by the Institutional Review Akaza 2007, Labrie 2007). Board. We obtained informed consent for experimental Testosterone and DHT in PCa tissue after medical or use of all specimens obtained from prostate needle surgical castration are synthesized locally in the biopsy or surgical procedure. The characteristics of prostate from dehydroepiandrosterone (DHEA) of PCa patients are described in Table 1. Stromal cells adrenal origin (Labrie et al. 1985, Belanger et al. were isolated using a modification of a previously 1989, Forti et al. 1989, Mizokami et al. 2004, described method (Krill et al. 1997). Briefly, small Nishiyama et al.2004, Titus et al. 2005). The pieces of PCa tissue were minced with scissors and metabolism from DHEA to DHT in peripheral target washed twice with PBS. The fragments were then tissues depends upon the level of expression of various digested in 0.25% trypsin–EDTA (Invitrogen) for steroidogenic enzymes in the specific cell types of 30 min at 37 8C. After digestion, the dispersed stromal these tissues (Labrie et al. 2005). Adrenal DHEA is cells were cultured in RPMI supplemented with converted to testosterone by 17b-hydroxysteroid 1% penicillin/streptomycin and 10% FCS (Sigma; dehydrogenase (17b-HSD) and 3b-HSD. Testosterone RPMI–10% FCS) on 6 cm dishes. Bone-derived is then converted to DHT by 5a-steroid reductase (SRD5A) in the prostate (Andersson & Russell 1990, Table 1 Characteristics of prostate cancer (PCa) patients on Andersson et al. 1991, Labrie et al. 2005, Luu-The diagnosis et al. 2008). Currently, 2 types of 3b-HSD, 15 types of Stromal PSA (ng/ml) Gleason Stage on 17b-HSDs, and 3 types of SRD5A have been identified cells on diagnosis score diagnosis and localized in various peripheral tissues, including PCaSC-1 4799 5C5 T3a, N0, M1 the prostate, with specific expression patterns in each PCaSC-2 263 4C4 T3b, N0, M0 tissue (Luu-The et al. 2008, Uemura et al. 2008). For PCaSC-5 76.9 4C3 T3a, N0, M1 example, 3b-HSD and type 5 17b-HSD were localized PCaSC-6 46.4 4C4 T3a, N1, M0 in basal cells of alveoli, stromal cells, and endothelial PCaSC-7 648 4C3 T4, N1, M1 C cells of blood vessels of the prostate (Pelletier et al. PCaSC-8 246 4 5 T4, N0, M1 PCaSC-9 180 5C4 T3b, N0, M0 2001). Fung et al. (2006) have observed increased Downloaded from Bioscientifica.com at 09/25/2021 04:31:42AM via free access 1140 www.endocrinology-journals.org Endocrine-Related Cancer (2009) 16 1139–1155 stromal cells 1 (BDSC-1) were obtained from the 11th with 20 nM non-target (NT) siRNA, AR siRNA-1, or rib of a 48-year-old man during left adrenalectomy for AR siRNA-2 (Invitrogen) using RNAiMAX (Invi- pheochromocytoma. BDSC-2 were obtained from the trogen) for 12 h, and LNCaP cells were transfected 11th rib of a 58-year-old man during left nephrour- with pGL3PSAp-5.8 for 12 h. Then after changing the eterectomy for localized ureteral cancer. These bone medium, transfected LNCaP cells were cocultured samples were cut into bone chips and further processed with or without PrSC. Consequently, cells were treated with a bone grinder (Lu et al. 2004). Bone chips were with or without 100 nM DHEA (C) for 24 h. As a then cultured in RPMI–10% FCS like prostate-derived positive control, cells were treated with 0.1 nM DHT stromal cells. for 24 h. For knockdown of AR expression in PrSC by RNA interference, we transfected 3!105 PrSC on a 0 Cell culture and cell proliferation assay 6 cm dish with 50 nM RNAi or AR RNAi (5 -CAUA- GUGACACCCAGAAGCUUCAUC-30; Invitrogen) LNCaP cells were cultured in DMEM including phenol for 48 h with Lipofectamine RNAiMAX. Transfected red–5% FCS (DMEM–5% FCS). Normal prostate- cells were counted, and 5!104 siRNA-transfected derived stromal cells, PrSC, commercially available PrSC were cocultured with 5!104 LNCaP cells (Cambrex, East Rutherford, NJ, USA) were cultured transfected with pGL3PSAp-5.8 12 h before coculture.