Targeting FOXA1 Pathways in Prostate Cancer

Park, Su Hong

doi:https://doi.org/10.21985/n2-amgw-6303

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Targeting FOXA1 Pathways in Prostate Cancer

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Prostate cancer (PCa) is the second most common cancer in American men and has led to approximately 29K deaths in 2018. The androgen signaling pathway plays a pivotal role in the pathogenesis of PCa, and androgen deprivation therapy (ADT) has been the main therapeutic approach for PCa. However, almost all patients develop resistance to ADT within 18-24 months, and approximately 30% of advanced PCa progress to castration-resistant prostate cancer (CRPC). Growing evidence suggests that, in addition to the androgen signaling, FOXA1 transcription factor is closely involved in PCa progression. Thus, understanding FOXA1 pathway will both improve current therapeutic strategies and identify new targeted therapies in PCa. FOXA1, a forkhead (FKHD) family transcription factor, is essential for the development and maturation of prostate luminal epithelial cells. FOXA1 gene is frequently mutated and its expression is deregulated during PCa progression. FOXA1 plays at least two major roles in PCa: repression of cell de-differentiation and induction of cell proliferation. In chapter 2, we first investigated FOXA1 regulation of de-differentiation via the TGF-B signaling pathway. We showed that FOXA1 directly binds to the regulatory elements of transforming growth factor-beta 3 (TGFB3) gene to repress its transcription. As such, FOXA1 loss led to increased TGFB3 expression and activation of the TGF-B signaling pathway. Functional studies in chapter 2 suggested that small molecule inhibitors targeting the TGF-B signaling pathway rescue FOXA1-loss-induced PCa cell invasions. In line with this, TGF-B inhibition also rescued PCa metastasis induced upon inhibition of the androgen signaling pathway. FOXA1 induces cell growth by transcriptionally activating genes related to the cell cycle process, DNA replication, cell division, and apoptosis pathways. In chapter 3, we investigated upstream regulators of the FOXA1 cell cycle pathway in PCa. We reported FOXA1 regulation by enhancer of zeste homolog 2 (EZH2), the enzymatic subunit of the polycomb repressive complex 2 (PRC2) whose canonical function is gene suppression by trimethylation of histone H3 at lysine 27 (H3K27me3). We demonstrated that EZH2 stabilizes FOXA1 protein by preventing FOXA1 degradation process through the ubiquitin-proteasome system. This was dependent on EZH2 methyltransferase (MTase) activity as we found that EZH2 directly methylates FOXA1 at K295 to decrease FOXA1 ubiquitination. Furthermore, we found that FOXA1 re-expression can rescue PCa cell growth from EZH2 inhibition, indicating FOXA1 as one of the major components of EZH2 oncogenic function in PCa. In the final chapter, we investigated the molecular mechanism responsible for FOXA1 protein stabilization in PCa cells. Through mass spectrometry and subsequence analysis by co-IP, we demonstrated that the USP7/BUB3 WD40-repreat protein-containing deubiquitinase complex deubiquitinates and stabilizes FOXA1 proteins in PCa. Indeed, we confirmed that both USP7 and BUB3 are required for FOXA1 deubiquitination induced by EZH2 MTase activity. We found that EZH2, BUB3, and USP7 cooperate together, where BUB3 binds specifically to K295 methylation and recruits USP7. Finally, our evidence suggests that targeting EZH2 and USP7 together significantly slowed PCa tumor growths by reducing FOXA1 levels and suppressing FOXA1-dependent cell cycle pathways. In summary, these findings described in three chapters highlight that inhibitions of downstream and upstream pathways of FOXA1 are attractive and alternatives strategies for targeting FOXA1-dependent pathways in PCa.

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