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Proteomic analyses identify ANP32A and CAND1 as androgen receptor and splice variant coactivators, whose interactions are regulated by DNA-dependent protein kinase

ABSTRACT: ARv7, the most prevalent androgen receptor (AR) variant in castration resistant prostate cancer (CRPC), lacks the AR ligand binding domain (LBD) making it resistant to LBD-targeting pharmacological inhibitors, such as enzalutamide. Identifying new prostate cancer therapeutic targets by defining key coregulators (CoRs) and associated regulatory enzymes that govern AR and ARv7 transcriptional activity is critically needed. Here, we have developed a cell-free DNA pulldown assay employing androgen response elements (AREs) to isolate and characterize the AR/ARv7 CoR complexes formed on DNA. Mass spectrometry analyses of ARE pulldowns revealed previously undescribed AR and ARv7 interacting coregulators. ARv7 has enhanced recruitment of a subset of AR interacting CoRs, such as ANP32A and CAND1. SMRT, a known AR corepressor, is recruited with AR in the presence of androgens, but not with ARv7. Knockdown of ANP32A or CAND1 in prostate cancer cells reduced the expression of AR and ARv7 target gene expression, suggesting they function as coactivators. Bioinformatic analyses in human prostate cancer clinical datasets revealed that ANP32A and CAND1 expressions correlated with metastatic prostate cancer, poorer patient prognosis, and increased risk of recurrence. Additionally, DNA-dependent protein kinase (DNA-PK) was identified as an enzyme that directly phosphorylates AR and ARv7 and coactivates their transcriptional activities. DNA-PK action also stabilizes the interactions of ANP32A and CAND1 with AR and ARv7 complexes. Collectively, these findings suggest that DNA-PK coactivates the AR and ARv7 CoR complexes through its enzymatic function and by stabilizing the interaction of coactivators such as ANP32A and CAND1. Loss of SMRT recruitment combined with enhanced interaction of coactivators may contribute to unregulated ARv7 hyperactivity. Therefore, combination therapeutic approaches targeting AR/ARv7, DNA-PK, and ANP32A/CAND1 may lead to improved patient prognosis and novel prostate cancer treatment strategies.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Hela Cell

DISEASE(S): Prostate Adenocarcinoma

SUBMITTER: Bhoomi Bhatt

LAB HEAD: Ross Hamilton

PROVIDER: PXD021462 | Pride | 2026-04-06

REPOSITORIES: Pride

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			Action	DRS
	31335_1_Q_Charles_Human_onethird_1_3_5.msf	Msf
	31335_1_Q_Charles_Human_onethird_1_3_5.raw	Raw
	31335_1_Q_Charles_Human_onethird_2_4_6.msf	Msf
	31335_1_Q_Charles_Human_onethird_2_4_6.raw	Raw
	31336_1_Q_Charles_Human_onethird_1_3_5.msf	Msf

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Project description:Continued androgen receptor (AR) signaling is an established mechanism underlying castration-resistant prostate cancer (CRPC), and suppression of AR signaling remains a therapeutic goal of CRPC therapy. Constitutively active androgen receptor splicing variants (AR-Vs) lack the AR ligand-binding domain (AR-LBD), the intended target of androgen deprivation therapies (ADT) including new CRPC therapies such as abiraterone and MDV3100. While the canonical full-length AR (AR-FL) and AR-Vs are both increased in CRPC, their expression regulation, associated transcriptional programs, functional relationships, and respective roles in mediating responses to endocrine therapies have not been dissected. In this study, we show that suppression of canonical AR-FL signaling by targeting AR-LBD leads to increased AR-V expression in two cell line models of CRPC. Importantly, treatment-induced AR-Vs activate a distinct expression signature enriched for cell cycle genes without requiring the presence of AR-FL. Conversely, activation of AR-FL signaling suppresses the AR-V signature but activates expression programs mainly associated with macromolecular synthesis, metabolism, and differentiation. In prostate cancer cells and CRPC xenografts treated with MDV3100 and abiraterone, increased expression of two constitutively active AR-Vs, AR-V7 and ARV567ES, but not AR-FL, parallels increased expression of the AR-driven cell cycle gene UBE2C. In addition, protein expression of AR-V7, but not AR-FL, is positively correlated with UBE2C in clinical CRPC specimens. The cumulative in vitro and in vivo evidence support an adaptive shift toward AR-V-mediated signaling in at least a subset of CRPC tumors as the AR-LBD is rendered inactive, suggesting an important mechanism contributing to drug resistance to CRPC therapies. LNCaP cells lacking AR-V were transfected with AR-V7 with or without androgen stimulation of AR-FL (4 conditions); LNCaP95 cells expressing AR-Vs were treated with control siRNA or siRNA trageting AR-LBD or AR-DBD, with or without androgen stimulation of AR-FL (6 conditions); VCaP cells expressing AR-Vs in the presence of androgen were treated with control siRNA, siRNA trageting AR-LBD, DMSO or MDV3100 to inhibit AR-FL (4 conditions). Total 14 arrays with no replicates.

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Proteomic analyses identify ANP32A and CAND1 as androgen receptor and splice variant coactivators, whose interactions are regulated by DNA-dependent protein kinase

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