Chromatin-directed proteomics-identified network of endogenous androgen receptor in prostate cancer cells

Abstract

Treatment of prostate cancer confronts resistance to androgen receptor (AR)-targeted therapies. AR-associated coregulators and chromatin proteins hold a great potential for novel therapy targets. Here, we employed a powerful chromatin-directed proteomics approach termed ChIP-SICAP to uncover the composition of chromatin protein network, the chromatome, around endogenous AR in castration resistant prostate cancer (CRPC) cells. In addition to several expected AR coregulators, the chromatome contained many nuclear proteins not previously associated with the AR. In the context of androgen signaling in CRPC cells, we further investigated the role of a known AR-associated protein, a chromatin remodeler SMARCA4 and that of SIM2, a transcription factor without a previous association with AR. To understand their role in chromatin accessibility and AR target gene expression, we integrated data from ChIP-seq, RNA-seq, ATAC-seq and functional experiments. Despite the wide co-occurrence of SMARCA4 and AR on chromatin, depletion of SMARCA4 influenced chromatin accessibility and expression of a restricted set of AR target genes, especially those involved in cell morphogenetic changes in epithelial-mesenchymal transition. The depletion also inhibited the CRPC cell growth, validating SMARCA4’s functional role in CRPC cells. Although silencing of SIM2 reduced chromatin accessibility similarly, it affected the expression of a much larger group of androgen-regulated genes, including those involved in cellular responses to external stimuli and steroid hormone stimulus. The silencing also reduced proliferation of CRPC cells and tumor size in chick embryo chorioallantoic membrane assay, further emphasizing the importance of SIM2 in CRPC cells and pointing to the functional relevance of this potential prostate cancer biomarker in CRPC cells. Overall, the chromatome of AR identified in this work is an important resource for the field focusing on this important drug target.

Proteomic Landscape of Prostate Cancer: The View Provided by Quantitative Proteomics, Integrative Analyses, and Protein Interactomes

“…Interestingly, three major subgroups of genomic subcomplexes of AR interaction partners were identified, where FOXA1 and HOXB13, known transcription factors interacting with AR and affecting the AR cistrome in prostate cancer, dictate selective gain of function for AR action [ 104 ]. Launonen et al [ 113 ] utilized chromatin immunoprecipitation coupled with selective isolation of chromatin-associated proteins (ChIP-SICAP) to study the protein interaction network of chromatin-bound endogenous AR in VCaP cells in response to R1881. Eighty-seven R1881-induced AR-interacting proteins were recognized.…”

“…In addition to corroborating AR interaction with many known co-regulators, they identified several previously unidentified AR interactions. They further studied how interactions with SMARCA4 and SIM2 affected AR target gene expression and cellular functions therein, finding that while the former affected cell morphogenetic changes and EMT, the latter influenced cellular responses to external and steroid hormone stimuli [ 113 ].…”

CREB5 reprograms FOXA1 nuclear interactions to promote resistance to androgen receptor-targeting therapies

“…This included binding elements (AR, FOXA1, HOXB13) that we previously demonstrated through ChIP-seq experiments ( Figure 1C ). In addition, we also identify that retained or gained sequences nominated well-studied regulators or co-factors of AR such as GRHL2 ( Paltoglou et al, 2017 ), EP300 ( Yu et al, 2020 ), and SMARCA4 ( Launonen et al, 2021 ; Marshall et al, 2003 ). In confirmation of previous findings, the genetic ablation of AR or FOXA1 reduced viability of CREB5-overexpressing cells ( Figure 1—figure supplement 1 ; Hwang et al, 2019 ).…”

From Omics to Multi-Omics Approaches for In-Depth Analysis of the Molecular Mechanisms of Prostate Cancer

“…AR occupancy is further altered by MED19 overexpression, consequently favoring androgen-independent growth in cooperation with ELK1 [ 112 ]. Interaction of SMARCA4 with AR-binding sites leads to the regulation of genes involved in cell adhesion and extracellular matrix organization [ 113 ]. ChIP-Seq analysis of prostate cancer cell lines revealed AR peaks in several genes involved in lipid synthesis [ 114 ].…”

“…In several cases, the correlation with genomic and transcriptomic data could be assessed, but was found to be limited, underlining the specific benefit of dedicated proteomic studies [ 203 , 204 , 205 ]. Additional interactome studies, mainly focusing on the AR, permitted a better understanding of the crosstalk with essential partners, such as FOXA1 and HOXB13 [ 105 , 113 ]. Additional interactomes important for prostate cancer growth involve N-Myc and ERG.…”