Primary prostate cancer (PCa) cells can disseminate early and stay dormant in distant organs but reactivate later, causing lethal metastasis and recurrence. The mechanisms of PCa cell dissemination, dormancy, and reactivation are overlooked, especially whether and how various treatments affect these processes. Using C4-2B PCa subcutaneous xenograft mouse model, we found that in mice with xenograft tumor removed, the disseminated tumor cells (DTC) were exclusively enriched in the bones, more specifically, in the bone cortex instead of the bone marrow. We hypothesize that DTC are dormant in the bone cortex and proliferative in the bone marrow and developed an in vitro mixed co-culture model to determine the bone stromal cell type inducing the dormancy. We found that osteoblasts, but not other stromal cells induced C4-2B PCa cell into a dormancy-like state, i.e., slowed proliferation without increased apoptosis, G0/G1 arrest, increased expression of dormancy marker, and decreased expression of proliferation markers. Through RNA-sequencing, gene ontology, and public dataset analyses, we found a reverse correlation between mitochondrial gene expressions and enrichment of mitochondria-related functions and PCa progression. We further predicted dormancy-mimicking drugs using a novel artificial intelligence (AI) platform. One of the candidates, PF-562271, a focal adhesion kinase (FAK) inhibitor, was validated as dormancy-mimicking in vitro. Altogether, our study revealed for the first time the effects of primary tumor removal on PCa cell dissemination, profiled the osteoblasts-induced C4-2B cell dormancy, and induced PCa cell dormancy in vitro using AI predicted drugs, which could potentially inhibit PCa bone metastasis progression and recurrence clinically.

Introduction: Constitutively active androgen receptor splice variant 7 (AR-V7) correlates with the adaptation to androgen receptor signaling inhibition treatment in prostate cancer (PCa) patients. Despite intensive studies of AR-V7, it is still unclear how the distinct genomic and epigenomic characteristics of AR-V7-dependent cistrome and transcriptome are distinct from full-length AR (AR-FL) and whether AR-V7 may play a role in promoting the metastatic progression of castration-resistant PCa (CRPC). Experiments: To accurately compare the activity of AR-V7 with AR-FL, we generated lentiviral stable cell lines with inducible overexpression of AR-V7 or AR-FL using AR-V7 negative LNCaP and C4-2 cell lines. We assessed the in vivo impact of AR-V7 or AR-FL expression on PCa metastasis by injecting cells in zebrafish embryos and the tibias of castrated male mice. ChIP-seq analyses of AR-V7, AR-FL, FOXA1, and H3K27ac, ATAC-seq analysis, and RNA-seq analysis were conducted in to characterize the global cistrome and transcriptome activity of AR-V7 in comparison with AR-FL. In addition, we also examined how a posttranslational modification, Ser81 phosphorylation, affects AR-V7 activity by generating an S81A mutant of AR-V7 and using S81-phosphorylation inhibitors. Results: Our in vivo study revealed that overexpressed-AR-V7, but not AR-FL, strongly promotes tumor cell invasion into blood vessels and induces osteoblastic bone lesions. Through integrated ChIP-, ATAC- and RNA-seq analysis, we determined that AR-V7 has a “pioneer factor-like” ability to bind to androgen-responsive elements (AREs) located at compact chromatin regions and activates the enhancers by increasing the chromatin accessibility. This mechanism leads to a unique AR-V7-regulated transcriptional program that is completely independent of AR-FL activity and is highly enriched for genes mediating epithelial-mesenchymal transition and metastatic functions. We then identified a subset of AR-V7-specific targets with significant overexpression in CRPC tumor samples and associated with poor clinical outcomes. In particular, we found that SOX9, a critical metastasis driver gene, was a direct target and downstream effector of AR-V7, and its protein expression was dramatically upregulated at AR-V7-induced bone lesions. Furthermore, we found that the pro-metastasis function of AR-V7 can be enhanced by Ser81 phosphorylation through chromatin-binding-independent mechanisms, and that targeting this phosphorylation by CDK9 inhibitors can block AR-V7-induced SOX9 expression and impair AR-V7-mediated metastasis function. Conclusion: Our study provides novel molecular insights into the role of AR splice variants in driving the metastatic progression of CRPC. In specific, these data have demonstrated the distinctive transcriptional program of AR-V7 from AR-FL, identified critical AR-V7 targets, and determined the function of Ser81 phosphorylation on AR-V7. Our data also suggest a new therapeutic strategy to target AR-V7-induced metastasis cascade in CRPC by using CDK9 inhibitor treatment. Citation Format: Maryam Labaf, Dong Han, Yawei Zhao, Jude Owiredu, Songqi Zhang, Kavita Venkataramani, Jocelyn S. Steinfeld, Wanting Han, Muqing Li, Mingyu Liu, Zifeng Wang, Anna Besschetnova, Susan C. Patalano-Salsman, Jill A. Macoska, Xin Yuan, Steven P. Balk, Stephen R. Plymate, Shuai Gao, Kellee R. Siegfried-Harris, Ruihua Liu, Gabrielle Foxa, Mary M. Stangis, Piotr J. Czernik, Bart Williams, Kourosh Zarringhalam, Xiaohong Li, Changmeng Cai. Distinct activity of androgen receptor splice variants in promoting prostate cancer metastasis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A009.