PTBP1 Facilitates Lymphatic Metastasis and Proliferation of Bladder Cancer Via Alternative Splicing of MEIS2 and PKM

Abstract

Background: Lymph node (LN) metastasis is the leading cause of bladder cancer-related mortality. Splicing factors facilitate cancer progression by modulating oncogenic variants, but it is unclear whether and how splicing factors regulate bladder cancer LN metastasis. Methods: In this study, qRT-PCR was used to identify differential expression of splicing factors between bladder cancer with and without LN metastasis. Associations between expression of the splicing factor polypyrimidine tract binding protein 1 (PTBP1) and clinicopathologic features and prognosis of bladder cancer were analyzed. In vitro and in vivo assays were performed to explore the biological role of PTBP1 in bladder cancer cells. RNA-sequencing, RNA immunoprecipitation, and RNA pull down assays were used to investigate the molecular mechanisms of PTBP1 in bladder cancer. Findings: PTBP1 expression was found to relate to bladder cancer LN metastasis, and was positively correlated with LN metastasis status, tumor stage, histological grade, and poor patient prognosis. Functional assays demonstrated that PTBP1 promoted bladder cancer cell migration, invasion, and proliferation in vitro, as well as LN metastasis and tumor growth in vivo. Mechanistic investigations revealed that PTBP1 upregulated MEIS2-L variant to promote metastasis and increased expression of a PKM2 variant to enhance proliferation by modulating alternative mRNA splicing. Moreover, overexpression of MEIS2-L or PKM2 could rescue the oncogenic abilities of bladder cancer cells after PTBP1 knockdown. Interpretation: Our data demonstrate that PTBP1 induces bladder cancer LN metastasis and proliferation through an alternative splicing mechanism. PTBP1 may serve as a novel prognostic marker and therapeutic target for LN-metastatic bladder cancer. Funding Statement: This study was supported by the National Natural Science Foundation of China (Grant No. 81702523, 81772719, 81772728, 81572514, U1301221, 81472384), National Natural Science Foundation of Guangdong (Grant No. 2016A030313321, 2016A030313244), Science and Technology Program of Guangzhou (Grant No. 201804010041, 201604020156, 201604020177), Project Supported by Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (for Tianxin Lin) , the Fundamental Research Funds for the Central Universities (for Xu Chen, 18ykpy18), Yat-Sen Scholarship for Young Scientist (for Xu Chen), Sun Yat-sen Initiative Program for Scientific Research (for Xu Chen, YXQH201708), and National Clinical Key Specialty Construction Project for Department of Urology and Department of Oncology. Grant KLB09001 from the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes, Sun-Yat-Sen University. Grant [2013]163 from Key Laboratory of Malignant Tumor Molecular Mechanism and Translational Medicine of Guangzhou Bureau of Science and Information Technology. Declaration of Interests: We declare that we have no conflicts of interest. Ethics Approval Statement: Ethical consent was approved by Sun Yat-sen University’s Committees for Ethical Review of Research involving Human Subjects