AR is the critical therapeutic target for disseminated prostate cancer and is effectively targeted through manipulation of the receptor LBD. However, recurrent tumors invariably overcome this point of intervention, and alternative strategies to target AR function therapeutically remain elusive (9
). The present study identifies abrogation of BAF57 recruitment as a novel means to inhibit AR activity based on several key observations herein. First, BAF57 is expressed in prostate cancer and elevated in a subset of tumors, indicating that the protein may continue to support AR activity in all stages of disease progression. Second, BAF57 is required specifically for the response to AR agonists but not therapeutic antagonists and is transiently recruited to sites of AR activity after androgen stimulation. These observations suggest that BAF57 is required at the early stages of AR function. Third, BAF57 impinges on a region of AR not currently targeted by cancer therapeutics and interacts with a motif known to be essential for transactivation potential. Fourth, the AR-binding site of BAF57 was used to provide proof of principle that abrogation of BAF57-AR association can block receptor function. Expression of this inhibitory peptide (BIPep) caused destabilization of AR residence on chromatin and suppressed AR function on clinically relevant target genes. BIPep was also sufficient to inhibit proliferation in AR-positive (but not AR-negative) prostate cancer cells. Together, these data provide conceptual evidence of a new means to hinder AR function and AR-dependent cellular proliferation in prostate cancer.
The observation that BAF57 expression is retained in human prostate cancer specimens and seems to be elevated in a subset of these tumors is critical to the development of this AR effector as a potential therapeutic target. Several AR cofactors are induced as a function of prostate cancer progression, including SRC1, SRC2, and ARA70, and it is thought that these events may contribute to the development of recurrent disease (1
). Whether BAF57 belongs to this class of AR modulator can now be determined, as the antisera described are effective for immunohistochemical studies. Intriguingly, several reports have been published, which support the contention that tissue-specific alteration of SWI/SNF subunits confers differential effects in cancer. Although it is well established that the SNF5 subunit is a rhabdoid-specific tumor suppressor (37
), recent studies have implicated loss of the ATPase subunit Brm in gastric cancer (38
), and depletion of Brm facilitates carcinogen-induced lung adenoma formation (39
). Although Brm can support AR activity in prostate cancer cells, it has been observed that it is frequently down-regulated in prostate cancer6
and is associated with the acquisition of proliferative advantage. By contrast, Brg1 may be induced in prostate cancer and is associated with invasive disease (40
). These collective observations underscore the growing appreciation that SWI/SNF complexes seem to exist in a delicate balance and that alterations in the expression of individual subunits can result in significant cellular outcomes. The observation herein that BAF57 levels seem to be induced in a subset of cancers suggests that altered BAF57 expression may be required to support or trigger alterations in cooperating SWI/SNF subunits. Although this hypothesis remains to be tested, it is apparent from the present study that BAF57 is critical for agonist-induced AR function in the context of androgen-dependent tumor cells.
The observed early recruitment of BAF57 to the sites of AR activity and its subsequent displacement indicate that SWI/SNF may be necessary to facilitate initial events in transcriptional activation. This supposition agrees with the proposed mechanisms of active SWI/SNF remodeling function in that the complex is thought to progress along the nucleosome-bound DNA (away from the AREs; refs. 7
). Importantly, AR is able to induce recruitment of the ATPase (42
), presumably through the BAF57 interaction motif as shown herein. It has been proposed that retention of AR at the PSA enhancer permits looping and contact with the proximal promoter, thus augmenting transcriptional output (33
). The observation of BAF57 dismissal indicates that SWI/SNF may no longer be required after this looping event occurs, and future investigations will test this hypothesis directly. Given the dynamic interplay between AR and BAF57, the biochemical mechanisms that underpin BAF57 recruitment were delineated.
As has been well documented, AR contains a zinc finger DBD that requires the CTE domain to permit binding to DNA (3
). A closer examination of the AR CTE uncovered this region as an important region for BAF57 binding and activation of AR. Interestingly, there is precedence for a requirement of the CTE to confer the binding of HMG DBD-containing proteins (HMGB1 and SOX9) to AR (43
). Moreover, this region of AR can serve as a platform for interaction with Ubc9, a SUMO-1 conjugating enzyme (45
). As previous studies showed that BAF57 can cooperate with Ubc9 for AR activation (13
), it will be intriguing to determine how Ubc9 may influence the ability of SWI/SNF to bolster AR activity. The DBD/hinge domain within the estrogen receptor (ER) has also been shown to be a primary region for BAF57 interaction, indicating a possible trend in BAF57-nuclear receptor binding (46
). Because our data show that the AR-binding region of BAF57 (amino acids 1–145) is able to reduce AR residence at AREs, it is possible that binding of the BAF57 fragment to the CTE is sufficient to block either DNA binding or agonist-induced stabilization on DNA. Although additional investigation will be necessary to challenge this hypothesis, the current data strongly support ablation of the AR-BAF57 interaction as a means to disrupt AR-DNA association.
To move further toward the goal of inhibiting the AR-BAF57 interaction, the region of AR binding within BAF57 was defined. The PR and HMG domains of BAF57 emerged as the primary binding sites for AR, as deletion of these regions elicited a loss in AR binding. Interestingly, the observed binding to AR differs from that of ER association with BAF57, wherein truncation of either the NH2
or COOH terminus of BAF57 caused significant disruption of ER binding (46
). Similarly, the PR/HMG domain of BAF57 is also dispensable for association with BRG1/BRM (47
) and the BAF155 subunit of SWI/SNF, as this interaction was dependent on the coiled-coil and NHRLI domains of BAF57 (30
). This latter observation may have implications for AR, as BAF155 (and its murine homologue, SRG3) has been shown to regulate expression of specific SWI/SNF components, including BAF57, and is thought to serve as a critical regulator of SWI/SNF subunit levels (30
). As would be expected, therefore, SRG3 is important for AR regulation (15
Given the necessary role for the BAF57 NH2
terminus in AR binding and the ability of this region to disrupt AR recruitment, the utility of the AR-binding motif (BIPep) as a means to suppress AR activity was examined. As shown, AR activity was significantly attenuated and proliferation of AR-positive prostate cancer cells was diminished. Importantly, introduction of BIPep caused no reduction of endogenous full-length BAF57 protein expression, as shown in . This is in contrast to previous observations that showed a decrease in wild-type BAF57 (wtBAF57) expression on BAF57 mutant introduction (30
). Therefore, effects observed on addition of this AR-binding region are not a result of decreased wtBAF57 expression. Taken together, these data present the BAF57-AR interface as a viable medium for disrupting AR signaling. These findings gain significance, as recent research has validated the feasibility of developing small molecules to effectively disrupt protein-protein interaction in cancer. For example, the Nutlin compounds were developed to block MDM2 interaction with p53, consequently stabilizing the p53 tumor suppressor and inducing cell cycle arrest and apoptosis (49
). Furthermore, expression of the NH2
-terminal region of AR has been shown to block the growth of prostate cancer (50
). Additionally, targeting BAF57 could potentially be useful in combination with the commonly used therapy of AR antagonists (e.g., bicalutamide), as examination of BAF57 and SWI/SNF function on antagonist treatment revealed no functional requirement for the complex in antagonist-mediated repression (Supplementary Fig. S2
). These findings are also consistent with analyses of SWI/SNF function in living cells, wherein the Brm ATPase was shown to be recruited to sites of AR function in the presence of agonist but not antagonist (42
). The present observations provide much impetus to explore disruption of the AR-BAF57 interaction through small-molecule inhibitors and/or peptide exposure to impede prostate cancer proliferation.
In summary, the robust expression of BAF57 in a multitude of prostate cancer specimens and its dependence on the SWI/SNF complex strongly suggest that the integrity of BAF57 function is maintained in prostate cancer as a means to support AR activity. Specificity of action was revealed in that BAF57 seems selectively required for the response to agonist (but not antagonist) and acts rapidly at AR target sites on receptor activation. Disruption of this event, made possible through dissection of the AR-BAF57 interaction surfaces, proved sufficient to disrupt AR residence on chromatin, AR-dependent gene expression, and AR-positive prostate cancer cell proliferation. Combined, these data show that disruption of the BAF57-AR interaction may prove an effective, novel means to assist in suppressing AR function and cellular outcome in prostate cancer cells.