The RB/E2F pathway regulates expression of genes that play crucial roles during cellular proliferation and growth and has a well-established role in controlling tumorigenesis. However, the contribution of RB to tumor progression remains largely unexplored. The current study is the first to our knowledge to provide a clinically relevant function for RB in mediating tumor progression by impinging on nuclear receptor expression and output. Our data strongly support a model wherein RB perturbation occurs during PCa progression and promotes lethal tumor phenotypes. This conclusion is supported by 5 key observations. (a) RB loss of function was overrepresented in CRPC and metastatic PCa and was associated with poor outcome. (b) Loss of the RB1 gene locus contributed significantly to RB inactivation in CRPC. (c) RB depletion was sufficient to induce hormone therapy resistance via AR deregulation, exemplified by resistance to hormone therapy in vitro and reduced time to restored AR activity, tumor progression, and castration resistance in vivo. (d) AR gene expression was under stringent E2F1-mediated regulation, and this process was deregulated by RB dysfunction. (e) Perturbation of the RB/E2F/AR axis was frequently observed in CRPC, resulting in enhanced AR expression. Based on these findings, we propose what we believe to be a new paradigm for RB function in protecting against the progression to lethal tumor phenotypes (Figure ), wherein RB loss promotes PCa progression through a pathway linked to nuclear receptor expression and output. These data underscore the relevance of E2F deregulation for controlling progression to lethal phenotypes in PCa and reveal critical facets of RB function in protecting against tumor progression.
Model for RB loss in controlling tumor progression through nuclear receptor expression and output.
Identification of the RB/E2F/AR network is significant, as AR-directed therapeutics are the first line of intervention for patients with non–organ-confined PCa, and restored AR activity is the major mechanism of transition to the incurable CRPC stage (7
). While incompletely understood, resurgent AR activity after hormone therapy can be induced by multiple mechanisms, which include somatic AR mutation, ligand-independent AR activation, alternative splicing of the AR
transcript, deregulation of AR cofactors, aberrant AR posttranslational modification, and intratumor androgen production (7
). Each has disease relevance, but the most common AR pathway alteration in CRPC is via heightened accumulation of AR itself, as is observed in approximately 30% of CRPCs. Only a fraction can be accounted for by amplification of the AR
), indicating that amplification-independent mechanisms of AR deregulation must exist. The data herein provide a mechanism by which AR overexpression is achieved in CRPC, as mediated by loss of RB function and/or deregulation of E2F1. This is of significance, as forced AR deregulation alone is sufficient to induce CRPC transition in tumor models (23
), and high AR levels are strongly correlated with increased risk of death from PCa (24
). Thus, the present findings suggest that aberrations in RB promote PCa progression and is associated with lethal tumor phenotypes. Supporting this view, the RB loss signature was significantly overrepresented in CRPC and PCa metastases. These data provide the impetus for future studies linking RB pathway status to PCa morbidity and suggest that RB could be developed as a metric for predicting the response to hormone therapy. Combined, these data establish a paradigm for RB in controlling tumor progression through regulation of nuclear receptor expression and signaling.
Copy number analyses revealed that the RB1
gene locus is lost in significant percent of CRPC tissues, suggesting that RB1
deletion may significantly contribute to the CRPC transition. While the present manuscript was under review, a genome-wide CGH analyses study by Taylor et al. reported that loss of RB1
is enriched in advanced PCa (25
). In their study, deletion of the 13q14.2 locus was observed in only 5% of primary tumors but in 37% of the metastatic cases, consistent with loss of RB1
as highly overrepresented in CRPC. Moreover, these data are highly concordant with copy number analyses from the LuCaP series reported here, wherein significant number of the cases demonstrated loss of at least 1 RB1
copy. Notably, loss of RB expression occurred with much higher frequency at the transcript and protein levels, and significant variability in expression was observed in tumors retaining only a single RB1
allele (Figure and Table ). These data indicate that multiple mechanisms are employed in CRPC to ablate RB expression and function, and ongoing studies are in progress to discern the underlying mechanisms.
Identification of the RB/E2F pathway as a critical regulator of AR
gene expression is clinically important, as there is scant knowledge of the factors that control increased AR expression during transition to CRPC. Negative regulation of AR
gene expression can be mediated by ErbB kinases (26
) or the Purα transcriptional repressor (27
). Among the remaining known factors, androgen itself plays a complex role in regulating AR levels. Androgen increases overall AR protein levels and stabilizes the AR
transcript, whereas androgen-activated AR can attenuate or enhance AR
mRNA levels, dependent on cell context (28
). Potential positive regulation of AR
mRNA expression was reported as a consequence of Sp1, cAMP, NF-κB, and Twist1 (31
). It is intriguing to speculate that these mechanisms may crosstalk with the RB/E2F1-mediated AR regulation identified here. For example, RB can suppress NF-κB signaling (35
), and E2F1 binds to and promotes activity of the Rel-A subunit (36
). Twist1 is a candidate RB/E2F1 target gene (37
) and has been implicated in PCa progression (38
). Sp1 can associate with and enhance E2F1 activity (39
), which suggests that Sp1 may assist in E2F1-mediated deregulation of nuclear receptor signaling. Finally, the action of RB in controlling AR appears to be specific, as depletion of RB family members p107 and p130 yielded no effect on AR
transcript levels. While the underlying basis of specificity should be discerned, there is little evidence for p107 or p130 alteration in PCa, and to date, no studies have rigorously addressed the impact of RB family member alteration in CRPC. Based on the present data, it will be imperative to determine what factor(s) cooperate with RB loss and/or E2F1 deregulation in promoting aberrant AR expression in CRPC.
The observation that not all activator E2Fs can support AR deregulation was unexpected, and is a basis of current investigation. While E2F1 and E2F3 were each sufficient to enhance AR in vitro, E2F2 proved deficient in this function, and only E2F1 significantly associated with AR
mRNA in clinical CRPC. Accordingly, these data suggest that E2F1 is the primary effector of AR
gene deregulation mediated by RB loss and is essential for castration-resistant cell proliferation after RB depletion. Previous studies supported the contention that the activator E2Fs harbor overlapping but distinct functions (40
). The present study identified a selective function for E2F1 in mediating crosstalk between the RB axis and nuclear receptor signaling in tumor progression. Interestingly, a previous report showed that forced E2F1 overexpression in vitro using a single PCa cell line or 293T cells resulted in reduced AR
mRNA expression (41
). It is not clear how these results relate to the effects of E2F deregulation as mediated by RB loss, and downstream AR target gene expression was not considered. The same study suggested that E2F1 was associated in tissue samples with a subset of tumors that show low AR immunoreactivity, suggesting that in a subgroup of tumors, the RB/E2F-AR axis may not apply (41
); however, abundant evidence demonstrates that the majority of CRPCs are AR positive and express high levels of the receptor (42
). Herein, data obtained using multiple in vitro model systems, analysis of E2F1 binding at the endogenous AR
locus, in vivo xenograft models of tumor progression, and analysis of human CRPC showed that gain of E2F1 function (as mediated by either E2F1 deregulation or RB depletion) strongly induced AR induction and resultant CRPC progression. Moreover, our findings are consistent with previous studies wherein E2F1 was associated with AR-positive, androgen-independent proliferation in vitro (43
). Interestingly, although the RB loss signature only partially overlapped with E2F1-responsive gene expression profiles identified in other cell types, the 2 signatures were highly coregulated in tumor specimens (Supplemental Figure 8). It will be important to discern whether E2F1-independent functions of RB may also contribute to CRPC phenotypes. Nonetheless, the study herein clearly identified RB-loss mediated E2F1 deregulation as sufficient to drive AR-dependent castration resistance.
Finally, it is critical to consider that impairment of RB function and/or E2F1 activation resulted in AR deregulation sufficient to alter AR output and confer castration resistance. Enhanced recruitment of AR to target gene loci was observed in both the presence and the absence of ligand. While elevated AR may be sufficient to explain the gain of AR function, several findings suggest the existence of a possible feed-forward loop between E2F1 and AR. First, androgens induce G1 progression and E2F1 expression in vitro (44
). Second, we demonstrated that AR function was altered in CRPC, as the receptor was found to occupy what we believe to be new sites in CRPC cells, many of which are associated with mitosis (15
). As reported herein, RB depletion also promoted enhanced occupancy at CPRC-specific sites of AR action. Strikingly, several of these (e.g., UBE2C
, and CDK1
) have previously been identified as regulated by E2F1 (46
), which suggests the provocative hypothesis that E2F1 may both control AR expression and act in concert with AR to alter downstream gene regulation. Consistent with this hypothesis, AR is known to bind the second intron of the AR
gene through an ARE half-site (15
). Preliminary studies indicate that E2F1 may co-occupy this site under selected conditions (our unpublished observations), providing some indication that E2F1 may not only regulate AR levels, but could modify AR function on chromatin. Ongoing studies will address the impact of cell cycle position on AR function, the relevance of E2F1 cooperation for this event, and the interplay between AR and E2F1 on regulatory loci of cancer relevance.
In summary, the findings herein present a paradigm for RB function in protecting against tumor progression and suggest that disruption of the RB/E2F1-AR network identified herein is sufficient to promote lethal tumor phenotypes of clinical relevance. These findings identify unexpected actions of E2F1 as a regulator of therapeutic resistance and unmask a mechanism to explain AR deregulation in human disease. Together, the present results provide understanding of RB/E2F1 function, demonstrate the importance of discerning RB function outside canonical cell cycle control, and reveal potential new avenues of therapeutic intervention.