Abstract 4400: SRPK1: The dominant power of alternative splicing in tumor chemoresistance

Abstract Introduction: Abnormal pre-mRNA alternative splicing (AS) is critical in shaping tumor chemoresistance by modulating the function of genes involved in resistance mechanisms. The serine arginine protein kinase 1 (SRPK1) is a major regulator of AS by phosphorylating an important group of splice factors named SR-proteins. Our study aims to elucidate the molecular mechanisms by which SRPK1 promotes resistance to chemotherapeutic drugs. Methods: Two cell lines resistant to cisplatin (CDDP) (breast cancer MDA-MB-231-CDDP-R and colon cancer HCT-116-CDDP-R) and one cell line resistant to docetaxel (DTX) (prostate cancer PC-3-DTX-R) were established by derivation from their parental using the pulse selection method. The CDDP and DTX resistance and the expression of SRPK1 of cells were verified by the IC50 measurements, MTT assay and western blotting (WB). The molecular mechanisms were determined using WB, siRNA, immunoprecipitation, the JC-10 apoptosis assay, and RT-PCR. Results: The MTT assay revealed that resistant cell lines were about 5-17 times more resistant to chemotherapeutic agents than parental cell lines. SRPK1 protein was upregulated in MDA-MB-231-CDDP-R and PC-3-DTX-R cells; however, its expression in HCT-116-CDDP-R cells was markedly lower than that in parental cells. Using siRNA to knockdown SRPK1 expression lead to the re-sensitization of the MDA-MB-231-CDDP-R and PC-3-DTX-R cells to CDDP and DTX, respectively, but this effect was not observed in SRPK1-downregulated HCT-116-CDDP-R cells. Both WB and JC-10 assay demonstrated that co-treatment with CDDP, DTX and SRPK1 specific inhibitor, SPHINX31, remarkably upregulated pro-apoptotic markers (cleavage of caspase-8, caspase-3, and PARP) in MDA-MB-231-CDDP-R and PC-3-DTX-R cells, compared with CDDP, DTX and SPHINX31 alone conditions. Notably, in SRPK1 down-regulated HCT-116-CDDP-R cells, transient transfection of SRPK1 increased the sensitivity of the cells to the cytotoxicity of CDDP. JC-10 assay results also indicated an increase in apoptosis upon CDDP or a combination treatment with SPHINX31 in HCT-116-CDDP-R cells transfected with SRPK1. Moreover, immunoprecipitation assay showed that the level of SRSF1, phosphorylated by SRPK1, was elevated in CDDP and DTX-treated MDA-MB-231-CDDP-R and PC-3-DTX-R cells. Mechanistically, SRSF1 modulates various cancer-related splicing events, particularly the splicing of BCL2L1 and MCL-1. SPHINX31 dramatically promoted the pro-apoptotic Bcl-xS and Mcl-1S isoforms in MDA-MB-231-CDDP-R and PC-3-DTX-R cells as compared to control. Conclusion: Our research reveals a key role for SRPK1 in chemoresistance by modulation of apoptotic genes splicing, suggesting a potential therapeutic avenue for alleviating challenges posed by chemoresistance. Citation Format: Duygu Duzgun, Sebastian Oltean. SRPK1: The dominant power of alternative splicing in tumor chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4400.