Clinical studies with abiraterone have demonstrated striking responses in men with CRPC, though not all patients respond and most patients eventually develop resistance. While abiraterone is known to inhibit CYP17A1 in vitro, the effectiveness of abiraterone in suppressing intratumoral androgens has not been established, and it remains unclear which CRPC phenotypes and genotypes are susceptible to CYP17A1 inhibition and how tumors develop resistance. Using two distinct CRPC models, we demonstrate that treatment with abiraterone significantly inhibited tumor growth, serum PSA, and intratumoral androgen levels, supporting the hypothesis that abiraterone’s primary mechanism of action is through effects on tissue androgens. Furthermore, both CRPC models responded to CYP17A1 inhibition with mechanisms that maintain AR signaling. This included upregulated expression of full length AR and ligand independent AR variants, as well as induction of steroidogenic genes (including the target gene, CYP17A1), several of which showed strong correlations with DHT levels in the recurrent tumors. Thus, in the setting of tumor progression on abiraterone, the rationale for focusing further therapeutic efforts on more potent AR antagonists and agents suppressing AR ligands remains strong.
Although specific mechanisms driving induction of alternative AR splicing have not been established, generation of AR splice variants following suppression of tumor androgens by abiraterone is consistent with the castration-mediated induction of AR splice variants observed in castration sensitive prostate cancer models (10
). Interestingly, studies of testosterone replacement in this setting (either in vitro,
or when given within days of castration in vivo)
have been shown to inhibit castration-associated increases in AR variant expression (10
). However, we did not observe lower levels of full length or variant AR expression in those tumors with higher levels of androgens at recurrence (data not shown). These observations suggest that factors regulating the initial induction of AR splice variant expression could differ from those maintaining variant expression at later time points of recurrent growth. Moreover, these observations demonstrate that certain tumors may simultaneously engage or accrue multiple resistance pathways directed at preserving the AR axis.
The molecular alterations occurring in CRPC tumors following abiraterone treatment suggest tumor-specific methods of addressing resistance, either through optimizing steroidogenic blockade or by inhibiting AR signaling. Although substantially suppressed, androgen levels remained detectable in abiraterone-treated tumors. Importantly, a 2 to 3 fold increase in expression of full length AR is known to render low androgen levels physiologically relevant in promoting AR driven growth (2
), suggesting clinical treatment of abiraterone-resistant patients with more stringent ligand inhibition may be of benefit. More complete reduction of steroidogenesis might be achieved through enhancing local concentrations of CYP17A1 inhibitors, or targeting transcriptional activation of the enzyme. CYP17A1 is regulated by SF-1 and other co-factors (31
), and its regulatory domains contain multiple cAMP responsive elements, providing several targets for modulation of enzyme expression, such as using phosphodiesterase inhibitors (32
). Combining CYP17A1 blockade with inhibitors of other critical components of the pathway such as HSD3B1 or SRD5A2 could also offset adaptive upregulation of CYP17A1 (33
Data regarding expression of C′ terminal truncated AR splice variants in CRPC continues to emerge, and will be a critical area of investigation as more potent ligand synthesis and AR inhibitors become utilized in the treatment of CRPC. AR splice variants may act by potentiating activity of full length AR as well as mediating constitutive AR transactivation (10
). Thus, increased expression of AR splice variants in abiraterone-treated tumors may be an important biomarker of resistance and target for therapy. Incorporation of potent AR inhibitors, such as MDV3100, or agents targeting the AR N-terminal domain, such as EPI-001, could be utilized for tumors adapting to CYP17A1 inhibition via induction of full-length AR and/or AR splice variants lacking the ligand binding C-terminal domain (13
). Studies to delineate whether sequential or concurrent use of these agents with abiraterone can improve tumor growth inhibition and/or suppress adaptation in xenograft models will be important to provide rationale for human studies evaluating these agents in the treatment of clinical disease.
The response to abiraterone in our study is most likely due to suppression of de novo
intratumoral steroidogenesis (due to the reported lack of CYP17A1 in rodent adrenal glands) (37
), whereas in human studies the response to abiraterone may reflect inhibition of both adrenal and/or intratumoral CYP17A1 activity. Importantly, the proposed mechanism driving clinical activity of abiraterone in both scenarios is a decrease in intra-tumoral androgens (whether from suppression of adrenal steroidogenesis, intratumoral steroidogenesis, or both). Our observations confirm this expected mechanism of activity. Furthermore, increases in expression of full length AR and ligand-independent AR splice variants following abiraterone treatment are likely to be clinically relevant mechanisms of resistance regardless of whether tumoral CYP17A1 activity is present.
Another consideration for translation of our results to the clinical setting is that men with CRPC are likely to be treated with abiraterone plus prednisone (or dexametha-sone) rather than abiraterone alone. In castrate men treated with abiraterone, glucocorti-coids are primarily used to inhibit pituitary-mediated secretion of ACTH (induced by adrenal CYP17A1 inhibition) which can exacerbate side effects of mineralocorticoid excess, but may potentially inhibit tumor growth directly (39
). ACTH may also drive clinically significant increases in production of androgenic precursors by the adrenal gland, as evidenced by PSA declines following the addition of dexamethasone in men who had progressed on abiraterone (15
). Thus, the inclusion of prednisone in men with CRPC is likely to accentuate any decrease in tumor androgens caused by abiraterone, and may accentuate the types of tumoral responses observed in the abiraterone-treated xeno-grafts.
Our hypothesis that tumoral androgens in these xenograft studies are derived from de-novo
steroidogenesis is consistent with previous observation that a subset of CRPC metastases have increased levels of transcripts for CYP17A1 and HSD3B1 (necessary for de novo
synthesis) and the demonstration by Locke et al that CRPC xenografts are capable of synthesizing DHT from acetate (5
). However, alternative androgenic precursors (of adrenal or other origin) may also be present in the circulation as potential substrates. Unfortunately, serum samples adequate to assess circulating androgen levels were not available for analysis in this study. In addition, the duration of abiraterone treatment in our study was 21 days. It is possible that more prolonged treatment would have resulted in a more robust induction of the adaptive changes already observed. Finally, me-tastatic CRPC is characterized by significant heterogeneity, while our conclusions reflect an analysis of only two CRPC phenotypes, and we had small numbers in some of the treatment groups. These limitations can be addressed by evaluating diverse panels of CRPC xenografts over a more prolonged time-course of therapy, and through studies of human tumor biopsies obtained at abiraterone resistance.
In conclusion, our finding that abiraterone suppresses intratumoral androgens and inhibits CRPC growth supports the hypothesis that tissue androgen levels are major contributors to AR signaling and mediators of CRPC progression. Though hypothesized, this is the first demonstration that the efficacy of abiraterone is related to its ability to suppress tumor androgen levels and complements previously published data from phase I studies regarding suppression of serum androgens (15
). Our results also identify potential mechanisms of adaptation to CYP17A1 blockade, including increased expression of AR, AR splice variants, and the steroidogenic transcriptome. Importantly, these adaptive mechanisms can potentially be targeted by using higher dose levels of abiraterone or combinations with potent AR antagonists currently in development. These data provide optimism that more effective suppression of AR signaling will continue to be an important means of effectively treating advanced prostate cancer.