Abstract Introduction Prostate cancer (CaP) will cause the deaths of more than 34,000 American men in 2022 because systemic treatments for metastatic CaP eventually fail. There is a need for actionable targets for novel treatments that work via entirely different mechanisms, bypass resistance to current therapies, and control the cellular processes that drive CaP progression to improve patient survival. We examine the contribution of citron kinase (CIT), a key mediator of cytokinesis, to CaP progression and explore the manner by which CIT impacts CaP cell behavior. Experimental procedures Cell proliferation and gene silencing, overexpression and mutation assays were performed in cell line models or PDX-derived organoids (PDXOs) reflecting treatment-resistant progression. Xenografting and tumorigenesis assays using cell lines that allow for CIT induction or repression were done in immunodeficient mice. Clinical relevance and translational potential of CIT was determined using immunohistochemistry and RNA-Seq assays combined with GSEA, MSigDB, rMATS and PCTA computational analyses. Mass spectrometry assays coupled with phospho-enrichment were done to identify CIT substrates; their interactions with CIT and RNA were confirmed via coIP and RNA-IP. Results We found that CIT controls CaP cell cycle progression and cell division and is overexpressed during clinical CaP progression. CIT overexpression was tumorigenic and overcame strategies to inhibit CaP growth. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of CaP progression and treatment resistance. Such silencing did not affect proliferation of benign epithelial prostate cells, indicating that CIT action is CaP-specific. CIT’s effects on CaP relied entirely on its kinase activity, which we isolated as druggable. Overexpression of kinase-dead CIT blunted CaP cell proliferation and pharmacological CIT inhibition decreased proliferation of treatment-resistant CaP cells and PDXOs, suggesting that CIT substrates mediate CaP growth. Using state-of-the-art proteomics approaches, we isolated the first CIT kinase substrates and found that over one-third function in RNA binding, metabolism and/or alternative splicing, a hallmark of CaP aggressiveness that is tightly controlled by phosphorylation. Combining RNA-Seq with bioinformatics analyses, we found that CIT induced alternative splicing patterns that were enriched in treatment-resistant castration-resistant CaP and neuroendocrine CaP. Moreover, putative CIT substrates were recruited to CIT-induced splicing sites, and CIT-controlled alternatively spliced transcripts differentially impacted CaP cell proliferation. Conclusion Collectively, these findings isolate CIT, and its kinase action, as a pivotal novel regulator of CaP progression. Moreover, CIT’s CaP driver function was mediated at least in part by clinically relevant alternative splicing events. These results suggest that blocking CIT or its dependent splicing events may overcome CaP-specific treatment resistance and progression. Citation Format: Chitra Rawat, Nidhi Singh, Gaurav Chauhan, Anja Rabljenovic, Salma Ben-Salem, Vishwa Vaghela, Varadha B. Venkadakrishnan, Jonathan MacDonald, Ujjwal Dahiya, Adam D. DePriest, Sanghee Lee, Michelle Muldong, Hyun-Tae Kim, Sangeeta Kumari, Qiang Hu, Eduardo Cortes, Scott M. Dehm, Amina Zoubeidi, Christina A.M. Jamieson, Marlo Nicolas, Jesse McKenney, Belinda Willard, Eric A. Klein, Cristina Magi-Galluzzi, Shaun R. Stauffer, Song Liu, Hannelore V. Heemers. Citron kinase is a novel driver of prostate cancer progression and controls alternative splicing associated with treatment-resistance [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 A015.
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Jun 1, 2024
Feng-Hao Zhang + 5
Open Access