Prostate cancer usually responds to androgen deprivation therapy, although the response in metastatic disease is almost always transient and tumors eventually progress as castration-resistant prostate cancer (CRPC). CRPC continues to be driven by testosterone or dihydrotestosterone from intratumoral metabolism of 19-carbon adrenal steroids from circulation, and/or de novo intratumoral steroidogenesis. Both mechanisms require 3beta-hydroxysteroid dehydrogenase (3betaHSD) metabolism of Delta(5)-steroids, including dehydroepiandrosterone (DHEA) and Delta(5)-androstenediol (A5diol), to testosterone. In contrast, reports that DHEA and A5diol directly activate the androgen receptor (AR) suggest that 3betaHSD metabolism is not required and that 3betaHSD inhibitors would be ineffective in the treatment of CRPC. We hypothesized that activation of AR in prostate cancer by DHEA and A5diol requires their conversion via 3betaHSD to androstenedione and testosterone, respectively. Here, we show that DHEA and A5diol induce AR chromatin occupancy and AR-regulated genes. Furthermore, we show that Delta(5)-androgens undergo 3beta-dehydrogenation in prostate cancer and that induction of AR nuclear translocation, AR chromatin occupancy, transcription of PSA, TMPRSS2, and FKBP5, as well as cell proliferation by DHEA and A5diol, are all blocked by inhibitors of 3betaHSD. These findings demonstrate that DHEA and A5diol must be metabolized by 3betaHSD to activate AR in these models of CRPC. Furthermore, this work suggests that 3betaHSD may be exploited as a pharmacologic target in the treatment of CRPC.
Purpose: Treatment with abiraterone (abi) acetate prolongs survival in castration-resistant prostate cancer (CRPC). Resistance to abi invariably occurs, probably due in part to upregulation of steroidogenic enzymes and/or other mechanisms that sustain dihydrotestosterone (DHT) synthesis, which raises the possibility of reversing resistance by concomitant inhibition of other required steroidogenic enzymes. On the basis of the 3b-hydroxyl, D 5 -structure, we hypothesized that abi also inhibits 3b-hydroxysteroid dehydrogenase/isomerase (3bHSD), which is absolutely required for DHT synthesis in CRPC, regardless of origins or routes of synthesis. Experimental Design: We tested the effects of abi on 3bHSD activity, androgen receptor localization, expression of androgen receptor-responsive genes, and CRPC growth in vivo.Results: Abi inhibits recombinant 3bHSD activity in vitro and endogenous 3bHSD activity in LNCaP and LAPC4 cells, including conversion ofandrogen receptor nuclear translocation, expression of androgen receptor-responsive genes, and xenograft growth in orchiectomized mice supplemented with DHEA. Abi also blocks conversion of D 5 -androstenediol to testosterone by 3bHSD. Abi inhibits 3bHSD1 and 3bHSD2 enzymatic activity in vitro; blocks conversion from DHEA to androstenedione and DHT with an IC 50 value of less than 1 mmol/L in CRPC cell lines; inhibits androgen receptor nuclear translocation; expression of TMPRSS2, prostate-specific antigen, and FKBP5; and decreases CRPC xenograft growth in DHEA-supplemented mice. Conclusions: We conclude that abi inhibits 3bHSD-mediated conversion of DHEA to active androgens in CRPC. This second mode of action might be exploited to reverse resistance to CYP17A1 inhibition at the standard abi dose by dose-escalation or simply by administration with food to increase drug exposure. Clin Cancer Res; 18(13);
Gonadal steroid production is stimulated by gonadotropin binding to G protein-coupled receptors (GPCRs). Although GPCR-mediated increases in intracellular cAMP are known regulators of steroidogenesis, the roles of other signaling pathways in mediating steroid production are not well characterized. Recent studies suggest that luteinizing hormone (LH) receptor activation leads to trans-activation of epidermal growth factor (EGF) receptors in the testes and ovary. This pathway is critical for LH-induced steroid production in ovarian follicles, probably through matrix metalloproteinase (MMP)-mediated release of EGF receptor (EGFR) binding ectodomains. Here we examined LH and EGF receptor crosstalk in testicular steroidogenesis using mouse MLTC-1 Leydig cells. We demonstrated that, similar to the ovary, trans-activation of the EGF receptor was critical for gonadotropin-induced steroid production in Leydig cells. LH-induced increases in cAMP and cAMP-dependent protein kinase (PKA) activity mediated trans-activation of the EGF receptor and subsequent mitogen-activated protein kinase (MAPK) activation, ultimately leading to StAR phosphorylation and mitochondrial translocation. Steroidogenesis in Leydig cells was unaffected by MMP inhibitors, suggesting that cAMP and PKA trans-activated EGF receptors in an intracellular fashion. Interestingly, although cAMP was always needed for steroidogenesis, the EGFR/MAPK pathway was activated and necessary only for early (30 -60 min), but not late (120 min or more), LH-induced steroidogenesis in vitro. In contrast, 36-h EGF receptor inhibition in vivo significantly reduced serum testosterone levels in male mice, demonstrating the physiologic importance of this cross-talk. These results suggest that GPCR-EGF receptor cross-talk is a conserved regulator of gonadotropin-induced steroidogenesis in the gonads, although the mechanisms of EGF receptor trans-activation may vary.
Many transcription-independent (nongenomic) steroid effects are regulated by G proteins. A well-established, biologically relevant example of steroid/G protein interplay is steroid-triggered oocyte maturation, or meiotic resumption, in Xenopus laevis. Oocyte maturation is proposed to occur through a release of inhibition mechanism whereby constitutive signaling by Gbetagamma and other G proteins maintains oocytes in meiotic arrest. Steroids (androgens in vivo, and androgens and progesterone in vitro) overcome this inhibition to promote meiotic resumption. To test this model, we used G protein-regulated inward rectifying potassium channels (GIRKs) as markers of Gbetagamma activity. Overexpression of GIRKs 1 and 2 in Xenopus oocytes resulted in constitutive potassium influx, corroborating the presence of basal Gbetagamma signaling in resting oocytes. Testosterone and progesterone rapidly reduced potassium influx, validating that steroids attenuate Gbetagamma activity. Interestingly, reduction of classical androgen receptor (AR) expression by RNA interference abrogated testosterone's effects on GIRK activity at low, but not high, steroid concentrations. Accordingly, androgens bound to the Xenopus progesterone receptor (PR) at high concentrations, suggesting that, in addition to the AR, the PR might mediate G protein signaling when androgens levels are elevated. In contrast, progesterone bound with high affinity to both the Xenopus PR and AR, indicating that progesterone might signal and promote maturation through both receptors, regardless of its concentration. In sum, these studies introduce a novel method for detecting nongenomic steroid effects on G proteins in live cells in real time, and demonstrate that cross talk may occur between steroids and their receptors during Xenopus oocyte maturation.
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