Polypyrimidine tract binding protein 1 promotes lymphatic metastasis and proliferation of bladder cancer via alternative splicing of MEIS2 and PKM

Highlights

• PTBP1 overexpression correlates with bladder cancer LN metastasis, tumor stage, histological grade and predicts disease prognosis outcome.

• PTBP1 facilitates bladder cancer cell migration, invasion, and proliferation both in vitro and in vivo.

• PTBP1 modulates alternative splicing of MEIS2 and PKM by directly interacting with pre-mRNA.

• MEIS2-L and PKM2 isoforms are required for PTBP1-induced metastasis and proliferation.

Abstract

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. In this study, Polypyrimidine tract binding protein 1 (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 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 and the expression of MMP9 or CCND1 respectively after PTBP1 knockdown. In conclusion, 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.

Introduction

Bladder cancer is one of the most common malignancies in the world, with approximately 429,800 newly diagnosed cases and 165,100 deaths annually [1]. Bladder cancer is a clinically heterogeneous malignancy represented by two subtypes, including non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC); NMIBC usually recurs but rarely progress, while MIBC often progress and is associated with poor long-term survival [2]. Compared to MIBC lacking lymph node (LN) metastasis, the mortality rate of MIBC with LN metastasis rises from 18.6% to 77.6% within 5 years, even when the MIBC is treated with radical cystectomy [3]. LN metastasis is a complex multistep process involving the spread, transportation, settlement, and colonization of tumor cells into and within the lymph nodes [4], and effective methods to diagnose LN metastasis in bladder cancer have been established in our previous study [5,6], but the molecular mechanisms of bladder cancer LN metastasis still remain largely unknown. Therefore, it is essential to explore novel molecular mechanisms underlying bladder cancer LN metastasis to identify new targets and solutions for bladder cancer diagnosis and therapy.

Alternative splicing (AS) is a critical step in the posttranscriptional regulation of gene expression; AS expands the proteomic complexity from a limited gene repertoire and plays a significant role in normal development and in various human diseases [7,8]. RNA–protein interactions with splicing factors, RNA–RNA base-pairing interactions, and chromatin-based effects that can change or determine splicing patterns are the three common mechanisms of alternative splicing [9]. The aberrant expression of splicing factors facilitates cancer progression by modulating expression of oncogenic variants [8]. Emerging evidence indicates that alternative splicing events can provide selective drug targets and can serve as biomarkers for cancer diagnosis [10].

PTBP1, a member of the heterogeneous nuclear ribonucleprotein (hnRNP) family that contains RNA Recognition Motif (RRM) domains, is a critical regulator of post-transcriptional gene expression by regulating mRNA splicing, RNA metabolism, stability, localization, and translation [11]. PTBP1 is elevated in several types of cancers, including glioblastoma [12], colorectal cancer [13,14], and renal cancer [15], and is involved in cancer progression by facilitating the alternative splicing of numerous gene variants. However, whether and how PTBP1 regulate the progression of bladder cancer remains largely unknown.

In this study, we identified PTBP1 as a splicing factor associated with LN metastatic bladder cancer. PTBP1 was overexpressed in LN-metastatic tumors and primary tumors tissues, and PTBP1 overexpression predicted poor prognosis. PTBP1 promoted metastasis and proliferation of bladder cancer cells both in vitro and in vivo. Moreover, PTBP1 regulated alternative splicing of MEIS2 and PKM, contributing to cancer progression. Therefore, our findings illustrated the significant role of PTBP1 in the malignant potential of bladder cancer, suggesting that PTBP1 could serve as prognostic biomarker and a promising therapeutic target.