The critical role of p53 in tumor development is well appreciated, but its role in PC is less clearly defined. Alterations in the p53 tumor suppressor are clearly associated with progressive disease, including “androgen-independent” growth of PCs and metastases to bone [6
]. Loss of chromosome 17p, on which p53 is located, occurs with moderate frequency in advanced PC, and loss of one allele accompanied by point mutations in the remaining copy of p53 leads to functional inactivation (reviewed in [24
]). In contrast to wild-type p53, the mutant protein can be stably expressed, and detection of high levels by immunohistochemistry is of prognostic significance. Our data indicate that inhibition of p53 promotes the proliferation of PC cells in the context of androgen deprivation. Future studies elucidating the details of the mechanism underlying p53's influence on CRPC growth are expected to lead to the identification of new molecular markers and therapeutic targets. Increasing evidence suggests that p53 directly regulates androgen signaling via AR, and that this regulation can exist at multiple levels due to the functional complexity of both proteins. It was previously suggested that the presence of wild-type p53 at basal physiological levels is necessary for AR signaling [25
]. In the context of this background, our findings support the concept of a need for balance between AR and p53 expression during androgen-dependent cell proliferation, and highlights the importance of p53 as an AR modulator in prostate cancer.
Our investigation of the role of p53 in AR signaling in PC took advantage of LNsip53 cells, which had been generated in our laboratory and were described previously [10
]. In support of previous work [8
], we observed that LNsip53 can proliferate in the absence of hormones (i.e., in CSS). In CRPC, AR activity is thought to be maintained abnormally through several mechanisms, including AR amplification, AR mutation, an increase in AR sensitivity, local androgen production, and growth-factor activation [2
Since eliminating p53 did not affect the binding of AR to specific short DNA sequences in vitro (EMSA, Fig. ) but affected occupancy of the PB promoter region by AR and FoxA1 (Fig. ), we suggest that p53 elimination affects binding of AR to DNA at the chromatin level. Indeed, analysis by ChIP-seq revealed differences in the binding of AR to chromatin in LNCaP vs. LNsip53. In general the LNsip53 peaks were less numerous and of lower amplitude, and a lower percentage of LNsip53-specific peaks had the expected AR motifs.
The AR-specific peaks found in LNCaP are in a good agreement with those reported previously. In spite of the fact that we found fewer peaks than Yu et al. [21
], probably due to our use of a monoclonal anti-AR antibody, 75% of these matched peaks found in the earlier study. Also, our finding that AR is preferentially recruited to non-promoter-regulatory elements is consistent with earlier findings [17
The fact that our ChIP-PCR validation was less successful for the LNsip53 peaks than for those in either the LNCaP-specific or shared groups suggests that some of these may either be transient or represent false positives – and thus may not be relevant. Further studies looking at additional samples, as well as analyses of peaks with putative AR binding sites, will be required to enumerate the true LNsip53-specific AR binding sites. Nevertheless, some validation was successful, a particularly notable example being our demonstration that Wig1 expression is reduced and that this has functional consequences. This finding supports the hypothesis that AR binding in the absence of p53 is physiologically relevant. Our further finding that Wig-1 overexpression suppressed the proliferation of LNsip53 cells partially explains why LNsip53 proliferation occurs in the absence of androgens, and supports the notion that p53 plays an important role in the progression of PC to a castration-resistant state.
Functional FoxA1 sites were identified in the PB and PSA promoters, as well as other prostatic enhancers, across a range of species, some of these sites were immediately adjacent to AREs, suggesting that the organization of cis-acting elements is well conserved, and that FoxA1 plays a fundamental role in prostate-specific gene expression. A direct interaction between FoxA1 and AR has been observed in these promoters, suggesting that these transcription factors cooperate in promoting the expression of androgen-regulated and prostate-specific genes [28
]. The binding of DNA by FoxA1 has been reported to disrupt nucleosome formation, and thus to contribute to chromatin-mediated transcriptional repression [29
]. We speculate that elimination of p53 changes the histone signature around AR-specific genes, directly affecting the priming of regulatory regions by FoxA1 binding and, consequently, binding of AR to the DNA. Several lines of evidence lead us to suggest that p53-regulated histone methylation involves lysine-specific demethylase 1 (LSD1). This demethylase co-localizes with the AR in the normal prostate as well as in prostate cancer, interacts with AR in vitro
and in vivo
, and stimulates AR-dependent transcription: LSD1 inhibition abrogates androgen-induced transcriptional activation and cell proliferation [30
]; LSD1 forms a chromatin-associated complex with ligand-bound AR; LSD1 relieves repressive histone marks by demethylating histone H3 at lysine 9 (H3K9) (Fig. ); and, complexes containing both p53 and LSD1 have been shown to regulate transcription in a gene-specific manner [31
]. Thus, elimination of p53 may exert its downstream effects on AR-mediated transcription by interfering with LSD1 activity and the histone methylation profile across the genome.
Schematic depicting model for LSD1 regulation by p53 and AR
In conclusion, our findings suggest that reduced p53 expression, which is frequently observed in CRPC cases, modifies the specificity of chromatin binding by AR, thereby leading to changes in AR-mediated signaling. This finding might be important for clinical prediction of CRPC development. Molecular markers are not routinely used to evaluate patients with PC because their relevance to important health-related outcomes has not been well defined. However, results of Concato et al., 2009 [32
] suggest that p53 dysfunction is associated with increased risk for death from prostate cancer. Taken together our data suggest that understanding the p53 status is important for managing patients with CRPC.