Prostate adenocarcinoma is an androgen-sensitive, androgen receptor (AR)-dependent malignancy throughout its clinical course. Androgen deprivation therapy is active against metastatic prostate cancer and was one of the first successful "targeted" therapies for cancer treatment. However, clinical resistance eventually occurs almost uniformly. The latter clinical state, frequently mislabeled as "hormone-refractory" or "androgen-independent," should more appropriately be described as "castration-resistant" to emphasize the failure of the mode of therapy and, at the same time, to recognize the molecular mechanisms underlying treatment resistance. The AR signaling axis is very frequently reactivated in castration-resistant disease, as is clinically evident by the resumption of PSA production, and hypersensitized to even low levels of residual androgens, that arise from non-testicular sources, even in situ androgen metabolism in the tumor microenvironment. Despite recent advances, such as abiraterone (androgen synthesis inhibitor), MDV3100 (enzalutamide, second generation anti-androgen) and other similar agents, that open exciting therapeutic opportunities in castration-resistant prostate cancer (CRPC) management, disease progression eventually emerges, in the form of resistant tumors that retain PSA expression, suggesting that they still harbor an active AR axis. Efforts at a more comprehensive targeting of the AR axis offer opportunities for better patient outcomes in prostate cancer.
A Roadmap to Comprehensive Androgen Receptor (AR) Axis Targeting for Castration-Resistant Prostate Cancer
Gonadal androgen suppression (castration via orchiectomy or GnRH analogs) suppresses circulating testosterone levels but does not achieve adequate androgen ablation within the prostate cancer microenvironment because it does not address adrenal and intratumoral steroid contributions. These residual extragonadal sources of androgens allow prostate cancer cells to survive, adapt and evolve into castration-resistant prostate cancer (CRPC). The persistent significance of the androgen receptor (AR) axis in CRPC was recently validated by the clinical efficacy of androgen synthesis inhibitors (abiraterone) and novel second generation AR antagonists (enzalutamide). The appreciation that conventional therapeutic approaches achieve a suboptimal ablation of intratumoral androgens and AR axis signaling output opens transformative therapeutic opportunities. A treatment paradigm of comprehensive AR axis targeting at multiple levels (androgen synthesis, metabolism and action) and at all relevant sites (gonadal, adrenal, intratumoral) simultaneously at the time of initiation of endocrine therapy (instead of the current approach of sequentially adding one agent at a time and only after disease progression) deserves examination in clinical trials, in order to explore whether maximal frontline AR axis suppression via combination therapy can achieve maximal induction of cancer cell apoptosis (before they have the chance to adapt and evolve into CRPC) and, thus, improve patient outcomes.
AR and Steroid Receptor Coactivators in Prostate Cancer
AR is a nuclear hormone receptor harboring an N-terminal domain (NTD), capable of transcriptional activation; a DNA-binding domain (DBD); a hinge region; and a ligand-binding domain (LBD). Upon ligand binding, AR translocates to the nucleus, binds to androgen-response elements in the genome and recruits a plethora of molecules that enhance (coregulators) and or repress (corepressors) gene transcription. The p160 Steroid Receptor Coactivators: SRC-1 (NCOA1), SRC-2 (NCOA2/GRIP1/TIF2) and SRC-3 (AIB1/NCOA3) play a critical role in AR transcriptional activity in prostate cancer cells. Elevated expression in prostate cancer has been reported for all three SRCs, enhancing androgen-dependent AR transcriptional activity in vitro, and is associated with more aggressive disease, while knockdown of either one of them in prostate cancer cell lines impedes cell proliferation. Recent data suggest that SRC-3 is the preferred coregulator for androgen-activated AR. However, there appears to be significant overlap and redundancy between coactivators, that can compensate for loss of expression of each other. For example, in mice lacking Src-1, expression of Src-2 and Src-3 is upregulated. Moreover, the SRCs can integrate input from other signaling cascades, allowing for cross-talk with other growth and survival pathways (e.g. kinases/phosphatases, acetyltransferases/deacetylases, etc) that can regulate SRC protein stability and activity, allowing for enormous plasticity of the AR signal, but also for numerous mechanisms of amplification of the AR signal in a ligand-independent manner, that can be exploited by CRPC cells to survive in a castrate environment.
Mechanisms of Persistent AR Signaling Activity in Castration-Resistant Prostate Cancer
Mechanisms of persistent AR signaling activity in castration-resistant prostate cancer, include:
- Maintenance of residual intratumoral testosterone and DHT concentrations via local androgen synthesis and metabolism
- AR overexpression (frequently due to AR gene amplification) can increase sensitivity to low androgen levels, and even convert first generation anti-androgens to agonists
- AR mutations that can broaden ligand specificity leading to promiscuous activation with alternative ligands
- Presence of alternatively spliced AR isoforms that can be constitutively active even in the absence of ligand
- Changes in coregulatory molecules including coactivators and corepressors that modulate AR stability and ligand sensitivity (for example, overexpression/gene amplification and activating somatic mutations have been reported in CRPC)
- Inactivation of the E3 ubiquitin ligase adaptor speckle-type POZ protein (SPOP) via point mutations or decreased expression
- Activation of the AR complex via “cross-talk” with other signaling pathways, such as HER2, IGF1R, IL-6 receptor, Src, and Akt pathways
These mechanisms are not mutually exclusive, and castration-resistant prostate cancer cells may simultaneously harbor several of these aberrations, which may also cooperate to enhance the AR axis signaling output. For example, the presence of residual tissue androgens may enhance most of the other mechanisms of AR activation. Moreover, aberrant expression or post-translational modification of coregulators may enhance both ligand-dependent and ligand-independent AR signaling.
See illustration Mechanisms of persistent AR transcriptional activity in CRPC cells and target sites of therapeutic agents: Extragonadal (adrenal and/or intratumoral) steroidogenesis can serve as a source of residual intratumoral androgens (A); AR overexpression (frequently due to AR gene amplification) and/or AR LBD mutations (B) can increase sensitivity to low androgen levels and/or broaden ligand specificity leading to promiscuous activation with alternative ligands; constitutively-active AR variants lacking the ligand-binding domain (LBD), e.g. alternatively spliced variants, can signal in a ligand-independent manner (C). Other mechanisms include changes in expression and posttranslational modification of AR coactivators and corepressors; and enhanced activation of the AR complex via “cross-talk” with other growth and survival pathways (e.g. kinases/phosphatases, acetyltransferases/deacetylases, etc).
Mechanisms of Dysregulated Androgen Metabolism in Prostate Cancer
Does acute adaptation precede (and allow for) clonal selection?
The mechanism(s) underlying the aberrant expression in CRPC of transcripts involved in androgen metabolism remain(s) to be fully characterized. Obviously, the full elucidation of these mechanisms could reveal novel therapeutic targets for inhibition of the AR axis in CRPC. In our study of integrated gene expression and comparative genomic hybridization datasets, these aberrant expression patterns were only rarely associated with respective copy-number alterations (CNAs) (Mitsiades et al. Cancer Res. 2012). On the contrary, AR overexpression was frequently associated with AR gene amplification, an event that possibly would require a process of clonal selection. In the absence of frequent CNAs, we examined whether the dysregulation of androgen metabolism enzymes occurs at the mRNA level. Expression of several enzyme transcripts, in particular of the AKR1C family, is induced by androgen deprivation within a timeframe (24-48 hrs) that is too fast for clonal selection. Conversely, androgen treatment can suppress the expression of steroidogenic enzymes. The finding that androgen deprivation rapidly upregulates the mRNA levels of AKR1C3, an enzyme that can convert androstenedione to testosterone, raises the hypothesis that androgen deprivation triggers an acute adaptation feedback loop that enhances the ability of the PC cell to metabolize adrenal precursors into testosterone and DHT, thus sustaining tissue androgen levels and AR stimulation.
This hypothesis is also consistent with studies of neoadjuvant medical castration therapy in men with localized PC that exhibited suboptimal suppression of intratumoral androgens (by ~only 70-80%, in contrast to the >90% concomitant reduction in serum androgens) and AR-dependent gene expression, suboptimal induction of PC apoptosis and disappointingly low rates of pathologic complete response (<3%). Once again, the term "androgen deprivation therapy" may be misleading, as it overestimates the impact of the systemic treatment on androgen levels and the AR axis within the tumor microenvironment. Instead, noncommittal terms, such as "medical castration therapy," would be more appropriate.
The "Androgenic Set Point" of PC Cells and Maintenance of Cell Survival
In studies of neoadjuvant medical castration therapy, the probability of early biochemical recurrence was inversely correlated to the degree of pathological effect and positively correlated to the residual expression of AR-dependent genes, including PSA, as identified as early as 3 months after the initiation of hormonal therapy. Therefore, failure to adequately suppress the AR axis (or early reactivation of AR signaling) is associated with inferior clinical outcomes.
Short-term (24-48 hours) exposure of PC cells to androgen-depleted medium in vitro stimulates the expression of steroidogenic enzymes, AR itself and its coactivators. In the case of the AR gene, it has been found that agonist-bound AR protein negatively regulates gene transcription via recruitment of the histone demethylase and transcriptional corepressor Lysine-Specific Demethylase 1 (LSD1) to a highly conserved site in the second AR gene intron. Similar LSD1-dependent mechanisms have been reported for the rapid androgen-mediated down-regulation of the steroidogenic enzyme AKR1C3 by agonist-bound AR. Collectively, these data suggest that agonist-bound AR directly mediates a physiological intracellular feedback loop to negatively regulate AR axis activity. Conversely, androgen withdrawal is proposed to trigger an acute adaptive response as a pre-programmed attempt of the PC cells to retain AR transcriptional activity, restoring it towards a predetermined "androgenic set point" which is critical for their survival. According to this hypothesis, these adaptive cellular responses would allow a sizeable pool of PC cells to maintain adequate intratumoral androgen levels and AR axis activity and survive, despite peripheral castrate androgen levels, eventually leading to the emergence of castration-resistant disease (possibly after later acquiring clonal genetic lesions, such as AR gene amplification).
Therefore, as current approaches achieve a suboptimal inhibition of AR signaling output, a more comprehensive AR axis targeting at multiple levels (androgen synthesis, metabolism and action) and at all relevant sites (gonadal, adrenal, intratumoral) simultaneously at the time of initiation of endocrine therapy, deserves examination in clinical trials, in order to explore whether it can improve patient outcomes over the current treatment paradigm of sequentially adding one agent at a time and only after disease progression.
Combination-Based Endocrine Therapy for PC: Finally Moving Beyond Proof-of-Concept
Recently, the CYP17 enzymatic inhibitor abiraterone prolonged median overall survival in men with chemotherapy-refractory CRPC by 3.9 months and also demonstrated clinical activity in chemotherapy-naïve CRPC, thus validating the importance of residual intratumoral androgens in CRPC pathophysiology. The second-generation AR antagonist enzalutamide (MDV3100), which was rationally designed to overcome the antagonist-to-agonist conversion of first-generation AR antagonists, prolonged median overall survival in men with chemotherapy-refractory CRPC by 4.8 months and is currently being tested in chemotherapy-naïve CRPC.
The appreciation of the importance of extragonadal contributions to AR signaling in PC is not recent. Second- and third-line hormonal manipulations have previously been used in CRPC, yielding small successes that provided proof-of-principle. Efforts to suppress adrenal steroids by surgical adrenalectomy and hypophysectomy date back five decades, with anecdotal responses reported. Glucocorticoids, via suppression of ACTH and adrenal androgen synthesis, also have documented activity in CRPC, with reported PSA responses as high as ~60% in small Phase II studies. Chemical adrenalectomy with aminoglutethimide or ketoconazole has provided PSA responses, but a survival benefit was never formally demonstrated. The concept of adding an anti-androgen to gonadal suppression for "combined androgen blockade" or "maximal androgen blockade" has been proposed before. However, clinical trials performed in the pre-abiraterone/pre-enzalutamide era had failed to demonstrate a consistent, clinically meaningful benefit from the addition of a first generation anti-androgen (or any other second-line hormonal agent) to gonadal suppression (a meta-analysis of 27 randomized trials including a total of 8275 patients revealed, at best, a non-significant gain of 1.8% in 5-year survival). Similarly, despite a very strong preclinical rationale, 5α-reductase inhibitors have not found yet a use in CRPC (although the combination of dutasteride with ketoconazole and hydrocortisone provided promising PSA responses in a recent study).
So why did abiraterone and enzalutamide achieve a survival benefit, while earlier second-line hormonal approaches did not? This could be because of more effective targeting of androgen synthesis and AR LBD, respectively, with fewer side-effects. Abiraterone is a more specific and better tolerated inhibitor of steroidogenesis than aminoglutethimide and ketoconazole. Enzalutamide appears to lack the AR agonistic effects of first-generation anti-androgens. With these advanced therapeutic agents in our armamentarium now, and with a galvanized interest in the AR axis as a therapeutic target in PC, the next steps will be to optimize their use for maximal therapeutic benefit. For example, the best timing, setting and sequence for using these novel agents, in order to maximize clinical benefit and minimize the risk of cross-resistance, remain to be defined.
Personal Opinion: What Will the Landscape of Advanced PC Treatment Be in the Next 5 Years?
The Roadmap to Comprehensive AR Axis Targeting
The above concepts lay the framework for new directions in the treatment of advanced PC. Both CYP17 inhibitors and second-generation AR antagonists are being tested in pre-CRPC disease states (hormone-naïve advanced PC and in the neoadjuvant setting) and it is reasonable to anticipate that they will be active there as well, by enhancing the efficacy of conventional medical castration therapy. In addition, the consecutive use of these agents, including identifying the optimal sequencing approach to augment clinical benefit and minimize the risk of cross-resistance, remains to be established by clinical evidence (so far, the Phase III clinical trials of both abiraterone and enzalutamide have excluded patients previously treated with the other agent).
It is my personal opinion that the most promising approach would be the early application of combination systemic therapy, at the initiation of medical castration, rather than after the onset of CRPC. Combinations of both classes of agents with GnRH analogs are being explored in clinical trials, and I am optimistic that, as part of a frontline comprehensive AR axis targeting approach, they could move us closer to our goal of a completely androgen-free PC microenvironment. Further towards that goal, additional steroidogenic enzymes are being explored as therapeutic targets: inhibitors of AKR1C3, 3beta-hydroxysteroid dehydrogenase (HSD3B1 and HSD3B2) and 5α-reductase (SRD5A1) would also be interesting choices to be included in future clinical trials of multi-drug combination regimens aiming at maximal frontline inhibition of the AR axis, in order to augment PC cell apoptosis and deplete the pool of surviving PC cells that can later accumulate additional genetic events and resurge as CRPC.
Based on the clinical activity of CYP17 inhibitors and enzalutamide, AR is now a validated therapeutic target in CRPC. Still, however, current treatment approaches do not achieve complete inhibition of AR signaling in PC, because they do not address all aspects of this axis (androgen synthesis, metabolism and action) and all relevant sites (gonadal, adrenal, intratumoral) early enough in the course of hormonal therapy. Failure to adequately and comprehensively inhibit the AR axis may allow PC cells to survive, adapt and evolve into CRPC. There is a need and opportunity to explore in clinical trials a new paradigm in the management of advanced PC: frontline regimens based on combinations of new generation inhibitors of all these AR axis components.
Modified from: Mitsiades N. A Road Map to Comprehensive Androgen Receptor Axis Targeting for Castration-Resistant Prostate Cancer. Cancer Res. 2013 Aug 1;73(15):4599-605.
Our Translational Research Program
Our endocrine cancer developmental therapeutics program aims to identify novel clinically applicable treatment approaches for castration-resistant prostate cancer by targeting the AR axis at the level of:
a) androgen synthesis (e.g. with the novel agent abiraterone)
b) AR expression and stability
c) Ligand binding to AR (e.g. with the novel anti-androgen MDV3100)
d) AR cofactor expression and function. The steroid receptor coactivators (SRC-1, -3 and -3) are key coactivators of AR and other transcription factors and are necessary for androgen signaling in health and disease, thus representing an important therapeutic target in endocrine-related neoplasia (including prostate cancer). The E3 ubiquitin ligase adaptor speckle-type POZ protein (SPOP) plays a critical tumor suppressor role in prostate cancer cells, promoting the turnover of SRC-3 protein and suppressing AR transcriptional activity. This tumor suppressor effect is abrogated by the prostate cancer-associated SPOP mutations (read more about role of SPOP in prostate cancer). We have hypothesized that targeting the AR axis at the level of expression of SRCs may overcome resistance caused by multiple upstream mechanisms, and, thus, have wide therapeutic implications in CRPC.
Under the leadership of Dr. Bert W. O'Malley, the Department of Molecular and Cellular Biology has unique experience and expertise in the molecular biology of nuclear hormone receptors and steroid receptor coactivators. Our goal is to translate this exceptional scientific background into novel therapies for endocrine cancers and overcome resistance to current treatments.
Residents and fellows with an interest in translational research, prostate cancer, oncology or endocrinology are encouraged to contact Dr. Mitsiades (email@example.com) for more details and to discuss ongoing and available future projects.