Androgen receptor splice variants drive castration-resistant prostate cancer metastasis by activating distinct transcriptional programs
Dong Han, … , Xiaohong Li, Changmeng Cai
Dong Han, … , Xiaohong Li, Changmeng Cai
Published April 30, 2024
Citation Information: J Clin Invest. 2024;134(11):e168649. https://doi.org/10.1172/JCI168649.
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Research Article Oncology

Androgen receptor splice variants drive castration-resistant prostate cancer metastasis by activating distinct transcriptional programs

Abstract

One critical mechanism through which prostate cancer (PCa) adapts to treatments targeting androgen receptor (AR) signaling is the emergence of ligand-binding domain–truncated and constitutively active AR splice variants, particularly AR-V7. While AR-V7 has been intensively studied, its ability to activate distinct biological functions compared with the full-length AR (AR-FL), and its role in regulating the metastatic progression of castration-resistant PCa (CRPC), remain unclear. Our study found that, under castrated conditions, AR-V7 strongly induced osteoblastic bone lesions, a response not observed with AR-FL overexpression. Through combined ChIP-seq, ATAC-seq, and RNA-seq analyses, we demonstrated that AR-V7 uniquely accesses the androgen-responsive elements in compact chromatin regions, activating a distinct transcription program. This program was highly enriched for genes involved in epithelial-mesenchymal transition and metastasis. Notably, we discovered that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7. Its protein expression was dramatically upregulated in AR-V7–induced bone lesions. Moreover, we found that Ser81 phosphorylation enhanced AR-V7’s pro-metastasis function by selectively altering its specific transcription program. Blocking this phosphorylation with CDK9 inhibitors impaired the AR-V7–mediated metastasis program. Overall, our study has provided molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC.

Authors

Dong Han, Maryam Labaf, Yawei Zhao, Jude Owiredu, Songqi Zhang, Krishna Patel, Kavita Venkataramani, Jocelyn S. Steinfeld, Wanting Han, Muqing Li, Mingyu Liu, Zifeng Wang, Anna Besschetnova, Susan Patalano, Michaela J. Mulhearn, Jill A. Macoska, Xin Yuan, Steven P. Balk, Peter S. Nelson, Stephen R. Plymate, Shuai Gao, Kellee R. Siegfried, Ruihua Liu, Mary M. Stangis, Gabrielle Foxa, Piotr J. Czernik, Bart O. Williams, Kourosh Zarringhalam, Xiaohong Li, Changmeng Cai

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Figure 6

AR-V7 transcriptionally activates SOX9.

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AR-V7 transcriptionally activates SOX9. (A) Genome browser view for indi...
(A) Genome browser view for indicated protein binding at the S2 site of the SOX9 gene (Note: AR-FL binding indicates AR ChIP-seq peaks in LN-tet-ARFL cells treated with doxycycline; other tracks are for LN-tet-ARV7 cells). (B) qRT-PCR for SOX9 mRNA in LN-tet-ARFL cells (0.1 nM DHT for 24 hours, 0.25 μg/mL doxycycline for 48 hours) and in LN-tet-ARV7 (0.25 μg/mL doxycycline for 48 hours). (C) ChIP-qPCR for V5 (AR-V7), FOXA1, H3K4me2, H3K27ac, and C-terminal AR (AR-FL) at the S2 site in LN-tet-ARV7 cells treated with/out 0.25 μg/mL doxycycline for 48 hours or 0.1 nM DHT for 4 hours. (D) Spearman’s correlation of SOX9 expression with AR-V7 or AR-FL expression in the SU2C mCRPC data set. (E) Matrigel invasion assay in LN-tet-ARV7 cells (doxycycline) compared with LN-tet-ARFL cells (0–10 nM DHT, 0.1 μg/mL doxycycline for 3 days). (F and G) Immunoblotting for SOX9 (F) and Matrigel invasion assay (G) in LN-tet-ARV7 cells transfected with siNTC or siSOX9 for 3 days. (H) Immunoblotting for SOX9 in GFP-labeled C4-2-tet-ARV7 cells transfected with siNTC or siSOX9 for 3 days. (I) GFP-labeled C4-2-tet-ARFL (grown under 0.1 nM DHT) or C4-2-tet-ARV7 stable cells, pretreated with or with out 0.25 μg/mL doxycycline and transfected with siNTC or siSOX9 for 3 days, were injected into the zebrafish embryos. AR-V7–mediated tumor cell intravasation process was observed within 1 hour (indicated by red arrow). The proportion of invaded embryos relative to the total number of embryos injected is displayed. (J) C4-2 cells stably expressing doxycycline-regulated AR-V7 together with doxycycline-regulated shRNA against SOX9 (LN-tet-ARV7/shSOX9) were established. Immunoblotting for AR-V7 and SOX9 was performed (right panel). LN-tet-ARV7/shSOX9 or control LN-tet-ARV7 cells were then injected into the tibias of castrated male mice, which were then fed with a doxycycline-supplemented diet. The bone lesion area was monitored and quantified (left panel). (K and L) Immunoblotting for AR-V7 and SOX9 (K) and zebrafish embryo metastasis assay (L) in GFP-labeled LNCaP-95 cells transfected with siNTC or siARV7 for 3 days. (M and N) Immunoblotting for AR-V7 and SOX9 (M) and zebrafish embryo metastasis assay (N) in GFP-labeled 35CR cells transfected with siNTC or siARV7 for 3 days. All the cell lines were hormone depleted prior to the experiments. ***P < 0.001; ****P < 0.0001 by unpaired, 2-sided Student’s t test (B, C, E, G, and J), Fisher’s exact test (K and L), or χ2 test (N). Data are represented as mean ± SD.