The present study shows that loss of the Brm ATPase is sufficient to induce a hyperplastic phenotype in murine models of the prostate and that Brm down-regulation is observed with high frequency in prostate cancer. Although, Brm function has been proposed to influence AR function in vitro, functional studies in murine models show that AR signaling is retained in Brm−/− animals. By contrast, and consistent with a role in prostate cancer progression, Brm loss is associated with the transition to castration resistance. These outcomes are underpinned by observations in both the Brm−/− prostates and analyses of gene expression profiles in human prostate cancer, wherein attenuation of Brm was associated with a high proliferative index and E2F1 deregulation. Combined, these data identify Brm as a critical modulator of E2F1 expression and growth control in this tissue, and suggest that loss of Brm in prostate cancer confers a proliferative advantage.
The demonstration that Brm down-regulation occurs with high frequency in prostate cancer was not expected, as Brm−/− mice have not been reported to develop tumors in tissues examined previously (16
). However, challenge of Brm−/− mice with lung carcinogens was reported to increase adenoma formation (37
), and loss of Brm expression has been decisively demonstrated in human lung cancer (38
). This latter event was shown to occur through epigenetic regulation and silencing of Brm mRNA expression. By contrast, recently reported Brm loss in gastric cancers occurs as a result of posttranscriptional Brm mRNA regulation (39
). Although the mechanism by which Brm is down-regulated in human prostate cancer remains unexplored, gene expression arrays are suggestive of mRNA regulation. While this study was in preparation, a report emerged which suggested that Brg1 mRNA levels are elevated in prostate cancer, especially under conditions wherein Brm expression levels were low (40
). Ectopic expression of Brg1 (but not Brm) in cultured cell models increased the invasive capacity of AR-negative PC3 cells, therefore further suggesting that Brg1 and Brm serve differential functions in cells of prostatic origin. In the present study, no significant alteration in Brg1 mRNA was observed in Brm-deficient prostate tissues, either in the murine epithelia or in human prostate cancer (data not shown). Thus, it is not anticipated that the proliferative phenotypes observed result from Brg1 deregulation.
The result of Brm down-regulation in both human disease and targeted deletion in murine prostates seems to be the induction of a hyperplastic phenotype. Although observed only in the VP and AP, lobe-specific hyperplasia is a frequently observed event after oncogenic insult in the murine prostate (30
). Although the process that contributes to lobe-specific outcomes are of interest, our data showing that Brm expression is inversely correlated to the proliferative index in prostate cancer and the murine prostate provide compelling evidence that Brm loss confers a growth advantage to this tissue. This outcome seems to be tissue-specific, as previous analyses of Brm−/− animals revealed both organ-specific increases and decreases in weight (16
). More detailed analyses of proliferative defects in Brm−/− have been previously investigated in MEFs, which retain serum dependence, show little change in cell cycle kinetics after serum stimulation, and do not form multilayers, but are partially compromised in the cellular response to contact inhibition or UV-induced DNA damage (16
). Whether these phenotypes contribute to the proliferative advantage observed in the prostate, and the cause of observed tissue-specific effects in Brm−/− animals, will be the focus of future studies.
Particular to prostate cancer, it was surprising to observe that AR signaling was refractory to Brm loss, both in the murine prostates and as correlated with Brm down-regulation in human disease. Based on studies performed in vitro
), it was expected that AR activity might be compromised upon perturbation of this SWI/SNF subunit. Whether retained AR signaling in the absence of Brm is attributed to developmental plasticity remains an unexplored but clinically relevant question because AR target gene expression was unaltered in tumors with reduced Brm expression. Although Brg1 expression was not enhanced in Brm−/− epithelia, it is possible that Brg1-containing complexes are sufficient to sustain AR activity, and/or that the chromatin remodeling needs of AR are satisfied by other chromatin remodeling complexes. Interestingly, down-regulation of SMARCA3
(HLTF) and SMARCA5
(ISWI/SNF2H) was significantly observed in conjunction with Brm down-regulation in cancer specimens (Supplementary Fig. S6
), thus indicating that loss of Brm likely induces the perturbation of other chromatin remodeling pathways that may facilitate AR signaling.
Although AR activity was retained in Brm−/− epithelia (evidenced by sustained probasin expression in unchallenged animals and uncompromised response to androgen re-supplementation), a major implication of the present study is that Brm loss was sufficient to induce castration-resistant proliferation in a subset of prostatic epithelia. This may be of clinical importance, as the transition to serum androgen independence typically represents the development of incurable disease. Based on gene expression profiling of Brm-deficient tumors, at least one underlying mechanism is hypothesized to occur through the regulation of the retinoblastoma tumor suppressor (RB)/E2F1 axis. Prior studies showed that RB directly requires SWI/SNF function to exert negative control over E2F target genes that are critical for Sphase progression (41
); indeed, E2F1 itself is a known E2F target gene. Moreover, pathway analyses from human specimens revealed that several upstream alterations that would result in RB inactivation (down-regulation of Smad2/3, Smad4, p27kip1, and RB itself) likely contribute to the observed induction of E2F1. In the prostate, loss of RB function has been shown to convey resistance to androgen ablation and AR antagonist therapies in multiple in vitro
), and targeted deletion of RB in prostatic epithelia provides a proliferative advantage in vivo
). Thus, Brm down-regulation may represent a mechanism to partially suppress RB function in this tissue. Because elevated E2F1 is associated with both the induction of cellular proliferation and apoptosis (46
), these data likely explain the observed outcomes in Brm−/− prostatic epithelia. Based on these findings, it is hypothesized that these downstream effects of E2F1 deregulation may actually hinder the progression of the hyperplastic phenotype, and the relevance of this supposition for human disease is being explored.
One additional mechanism that could contribute to the castration-resistant phenotype is alterations in local hormone synthesis. Although there are multiple mechanisms that contribute to resurgent AR activity in therapy-resistant tumors (25
), it is now apparent that a substantive percentage of castration-resistant prostate cancers may arise from the activation of intracrine de novo
androgen synthesis within the tumor microenvironment (47
). Indeed, analysis of patients with recurrent tumors after hormone therapy supports this contention, and the recent observation that an irreversible Cyp17 inhibitor, abiraterone, can reduce prostate-specific antigen in patients that failed hormone therapy highlights the clinical relevance of this supposition (50
). It is of interest that four effectors of C21 steroid hormone metabolism (Cyp11A1
, which generates pregnenolone from cholesterol; Cyp11B2
, which converts corticosterone to aldosterone; Cyp17A1
, which assists pregnenolone and DHEA production, but has also been recently implicated in a backdoor pathway for steroid production; and Cyp21A7
, which hydroxylates progesterone) were anticorrelated with Brm levels in gene expression arrays from human tumors. Thus, alterations in Brm may affect steroid hormone synthesis. However, serum testosterone levels were indistinguishable from Brm-positive animals in castration/re-supplementation studies (Supplementary Fig. S3B
). As such, the current data do not support the contention that Brm loss increases serum testosterone levels; however, the local effect within the prostate cannot be excluded and is the focus of ongoing study.
In summary, the data presented herein show that Brm loss results in prostate-specific hyperplasia, the transition to androgen independence, and deregulated E2F1 expression after targeted ablation in murine tissues. These events hold consequence in human disease, wherein down-regulation of Brm is associated with a high proliferative index. Together, these data reveal a putative tumor suppressor function for Brm in the prostate, and suggest that loss of this protein is sufficient to confer a proliferative advantage in this tissue.