Pre-clinical and clinical studies on the role of RBM3 in muscle-invasive bladder cancer: longitudinal expression, transcriptome-level effects and modulation of chemosensitivity

Abstract

Background: The response to neoadjuvant cisplatin-based chemotherapy (NAC) in muscle-invasive bladder cancer (MIBC) is impaired in up to 50% of patients due to chemoresistance, with no predictive biomarkers in clinical use. The proto-oncogene RNA-binding motif protein 3 (RBM3) has emerged as a putative modulator of chemotherapy response in several solid tumours but has a hitherto unrecognized role in MIBC. Methods: RBM3 protein expression level in tumour cells was assessed via immunohistochemistry in paired transurethral resection of the bladder (TURB) specimens, cystectomy specimens and lymph node metastases from a consecutive cohort of 145 patients, 65 of whom were treated with NAC. Kaplan-Meier and Cox regression analyses were applied to estimate the impact of RBM3 expression on time to recurrence (TTR), cancer-specific survival (CSS), and overall survival (OS) in strata according to NAC treatment. The effect of siRNA-mediated silencing of RBM3 on chemosensitivity was examined in RT4 and T24 human bladder carcinoma cells in vitro. Cellular functions of RBM3 were assessed using RNA-sequencing and gene ontology analysis, followed by investigation of cell cycle distribution using flow cytometry. Results: RBM3 protein expression was significantly higher in TURB compared to cystectomy specimens but showed consistency between primary tumours and lymph node metastases. Patients with high-tumour specific RBM3 expression treated with NAC had a significantly reduced risk of recurrence and a prolonged CSS and OS compared to NAC-untreated patients. In high-grade T24 carcinoma cells, which expressed higher RBM3 mRNA levels compared to RT4 cells, RBM3 silencing conferred a decreased sensitivity to cisplatin and gemcitabine. Transcriptomic analysis revealed potential involvement of RBM3 in facilitating cell cycle progression, in particular G1/S-phase transition, and initiation of DNA replication. Furthermore, siRBM3-transfected T24 cells displayed an accumulation of cells residing in the G1-phase as well as altered levels of recognised regulators of G1-phase progression, including Cyclin D1/CDK4 and CDK2. Conclusions: The presented data highlight the potential value of RBM3 as a predictive biomarker of chemotherapy response in MIBC, which could, if prospectively validated, improve treatment stratification of patients with this aggressive disease.