Targeting the androgen receptor pathway in prostate cancer

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When prostate cancers progress following androgen depletion therapy, there are currently few treatment options with only one, docetaxel, that has been shown to prolong life. Recent work has shown that castration-resistant prostate cancers (CRPCs) continue to depend on androgen receptor (AR) signaling which is reactivated despite low serum androgen levels. Currently available AR-targeted therapy, including GnRH agonists and antiandrogens, cannot completely shut down AR signaling. Several mechanisms that enhance AR signaling in an androgen-depleted environment have been elucidated. These include AR mutations that allow activation by low androgen levels or by other endogenous steroids, AR overexpression, increased local intracrine synthesis of androgens, and upregulation of tyrosine kinase pathways. This has led to the development of a number of novel agents targeting the AR signaling pathway, including more effective antiandrogens, inhibitors of CYP17, an enzyme required for androgen synthesis, inhibitors of 5α-reductase, inhibitors of HSP90 which protects AR from degradation, inhibitors of histone deacetylases which is required for optimal AR-mediated transcription, as well as inhibitors of tyrosine kinase inhibitors. Many of these strategies are currently being tested in clinical trials in CRPC.

Introduction

The androgen receptor (AR), located on Xq11–12, is a 110 kDa nuclear receptor that, upon activation by androgens, mediates transcription of target genes that modulate growth and differentiation of prostate epithelial cells. AR signaling is crucial for the development and maintenance of male reproductive organs including the prostate gland, as genetic males harboring loss of function AR mutations and mice engineered with AR defects do not develop prostates or prostate cancer [1, 2]. This dependence of prostate cells on AR signaling continues even upon neoplastic transformation, leading to the seminal discovery by Huggins and Hodges in 1941 that orchiectomy produced prostate cancer regression [3]. Androgen depletion (now using GnRH agonists) continues to be the mainstay of prostate cancer treatment. However, androgen depletion is usually effective for a limited duration and prostate cancer evolves to regain the ability to grow despite low levels of circulating androgens [4]. Treatment options for castration-resistant prostate cancer (CRPC) are an unmet need with docetaxel being the only agent that has been shown to prolong survival [5, 6]. Interestingly, while a small minority of CRPC does bypass the requirement for AR signaling [7], the vast majority of CRPC, though frequently termed ‘androgen independent prostate cancer’ or ‘hormone refractory prostate cancer’, retains its lineage dependence on AR signaling. Over the past several years, several important mechanisms of enhanced AR signaling in low serum androgen levels in CRPC have been elucidated. This has led to novel therapeutic strategies targeting AR signaling which offer promising potential in future treatment of CRPC (Figure 1).

Several clinical observations have long offered clues that AR signaling is active and required in most CRPC. PSA, an exquisitely AR-dependent gene, is widely used as a marker for disease activity. PSA declines after the initiation of hormone depletion therapy and a subsequent rise is commonly the first sign of disease progression. This indicates that reactivation of AR signaling accompanies the development of CRPC. Both the relative and absolute level of PSA decline  markers of the degree of AR inhibition  after initial androgen depletion is predictive of outcome [8]. After the development of castration resistance, further hormonal manipulations targeting AR can elicit response while treatment with exogenous androgens usually results in tumor flare. First demonstrated for flutamide, treatment with any currently available antiandrogen may result in agonist conversion, and tumor response can be observed upon antiandrogen withdrawal (AAWD) [9]. Gene expression studies of clinical prostate cancer specimens show that AR-activated genes (defined as genes downregulated after neoadjuvant androgen deprivation before prostatectomy) were reactivated in CRPC despite continued androgen deprivation [10••]. In the laboratory, knockdown of AR results in cell death in both human and murine CRPC cell lines [11, 12••, 13].

Recently, it was discovered that up to 90% of all prostate cancers overexpress an ets oncogene, including ERG, ETV1, ETV5, and ETV6 via a variety of mechanisms. The most common mechanism of overexpression is fusion of the ets gene (particularly ERG) to the 5′-untranslated region of highly AR-regulated TMPRSS2 gene [14••, 15••]. Thus, in addition to the lineage dependence of prostate cells on AR signaling, prostate cancer has additional selection pressure to maintain TMPRSS2 expression and AR activity.

Numerous mechanisms have been implicated in the reactivation of AR in the castrate environment and have been extensively reviewed (Figure 1) [16, 17, 18]. Most directly, mutations of AR that allow other steroids such as corticosteroids and antiandrogens are detected in ∼10% of CRPC in a CALGB clinical trial, though the actual incidence may be more frequent [19]. Other mechanisms including activation of kinase pathways that can both stabilize AR and enhance its transcriptional activity and upregulation of AR coactivators that increase AR-mediated transcription. These sensitize AR to lower levels of ligand. Here, we focus on three crucial and druggable mechanisms  increased level of AR and increased level of ligand and activation of kinase pathways.

Hormone ablation therapy does not completely eliminate serum androgens. Serum testosterone is reduced to a mean of 15 ng/ml from a normal range of >200 ng/ml while serum levels of adrenal androgens such as dehydroepiandrosterone (DHEA) and androsteindione are unaffected. Intraprostatic androgen concentration is reduced much less dramatically by only ∼75%, and is sufficient to activate AR [20, 21]. Although this level of reduction is sufficient to induce response in untreated prostate cancer, cellular alterations that sensitize the AR pathway induce resistance and confer growth.

One possible source of increased intratumoral androgens is the tumor cells. Two expression profiling studies comparing metastatic CRPC with primary tumors show that enzymes involved in androgen synthesis are upregulated in CRPC. Holzbeierlein et al. found overexpression of enzymes involved in the synthesis of cholesterol, the common steroid precursor, from acetyl-CoA [10••] and Stanbrough et al. found overexpression of enzymes involved in the synthesis of testosterone and the more potent androgen DHT from cholesterol [22] (Figure 2). Using mass spectrometry to directly measure intratumoral androgens, Montgomery and colleagues found that castration-resistant metastatic tumors in men treated with GnRH have higher levels of testosterone but not dihydrotestosterone when compared with primary tumors in untreated men. They also corroborated overexpression of enzymes in androgen synthesis measured by real-time PCR. Castration-resistant xenografts similarly maintained elevated intratumoral testosterone levels in castrate mice [23••]. These data suggest that intracrine androgen synthesis may allow tumors to grow despite low serum androgen levels.

Overexpression of AR is common in CRPC. Compared to localized disease, CRPC has higher expression of AR based on immunochemical staining with genomic amplification seen in ∼20% of cases [24, 25]. Expression profiling studies consistently identify AR to be overexpressed in CRPC [10••, 22•, 26, 27].

Laboratory data indicate that AR overexpression is necessary and sufficient to induce CRPC in xenograft models. To identify genes important for the development of castration resistance, a panel of seven prostate cancer xenografts was selected for castration resistance by passage in castrated mice. Comparison of the expression profiles between the seven isogenic pairs reveals that AR is the only gene overexpressed in all resistant xenografts. Further, forced modest overexpression of AR in the parental LnCaP and LaPC4 xenografts conferred castration resistance by sensitizing cells to residual levels of androgens restoring expression of AR-regulated genes. Intriguingly, overexpression of AR also converted the antiandrogen bicalutamide into a weak agonist, indicating that AR overexpression can underlie AAWD [12••].

The HER2 receptor tyrosine kinase is progressively overexpressed in more advanced, castrate resistance prostate cancers, though it is seldom, if ever, amplified as seen in breast cancers [28]. In experimental systems, forced overexpression of HER2 results in increased AR activity and stability while pharmacologic inhibition or knockdown of the protein results in growth suppression [29]. One possible downstream target of HER2 activation is the Cdc42-associated tyrosine kinase Ack1. Activated Ack1 mediates AR activation through tyrosine phosphorylation of Y267 and knockdown of Ack1 or mutation of Y267 to phenylalanine abrogates HER2-mediated AR activation and growth [30].

In addition to HER2, increased signaling by a number of other growth factor receptors (e.g. EGFR, IGF-1R and IL-6R) can enhance AR signaling and confer castration resistance in preclinical models [31, 32, 33]. These receptors induce downstream activation of critical growth and survival pathways, including the AKT, MAPK, and STAT pathways. Expression of both activated AKT and BRAF also results in castration resistance [13]. Although these mechanisms of CRPC are frequently referred to as ‘ligand independent’, it is unknown whether AR is truly activated without ligand binding or whether AR is sensitized to lower levels of ligands since experimental systems to decrease AR ligands such as in vitro growth in charcoal stripped serum or castration of mice in vivo leave residual ligands. This distinction between true ligand independent and hypersensitized AR is not just semantic since therapies designed to further reduce AR ligands would be active only if ligand is still required. Furthermore, AR alleles containing mutations that impair ligand binding can no longer confer resistance to castration.

Another kinase implicated in AR crosstalk is SRC. Upon ligand binding, AR binds and activates SRC and downstream events within 5 min in a ‘nongenomic’ mechanism [34]. SRC can in turn tyrosine phosphorylate AR augmenting its transcriptional activity. SRC activity is substantially increased in models of CRPC [35, 36]. A 10-amino acid peptide that blocks the AR–SRC interaction inhibits androgen-mediated proliferation in tissue culture and xenografts [37].

Section snippets

Antiandrogens

Upon development of castration resistance, an antiandrogen, such as flutamide, bicalutamide, nilutamide, and cyproterone acetate, is typically added if it was not included in initial treatment. In some cases, this can result in prolonged disease control. However, in the majority of patients who have progressed despite androgen ablation and especially in symptomatic patients, the time to progression is usually short [38, 39]. In addition, all these compounds have clinically been observed to

Conclusion

With increasing appreciation that AR signaling remains crucial in CRPC, a number of new therapeutic strategies have evolved. Phases I–II clinical data show that some have very promising activity while others have been disappointing. As expected from the fact that multiple mechanisms can underlie AR activation, no single therapeutic agent is active in all patients. The ability to dissect AR pathway aberrations in patients would allow individualized therapy targeting the particular aberration.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

HIS would like to thank the MSKCC Prostate Cancer SPORE grant, the Prostate Cancer Foundation. CLS is a Doris Duke Distinguished Clinical Scientist and an Investigator of the Howard Hughes Medical Institute and would like to thank grants from the US National Cancer Institute and the UCLA Prostate SPORE seed grant. YC would like to thank the Ruth L. Kirschstein National Research Service Awards (5T32CA009207), the AACR BMS Oncology Fellowship, and the ASCO YIA.

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