Beta-arrestins regulate basal cell and cancer stem cell phenotype in muscle-invasive bladder cancer

Background: The muscle-invasive bladder cancer (MIBC) is the most aggressive form of bladder cancer with a five-year survival of less than 15%. High metastatic potential, poor response to chemotherapy and a largely basal cell molecular signature characterize MIBC. We identified two cytoplasmic proteins, β-arrestin 1 and β-arrestin 2 (BARR1 and BARR2), that are unevenly expressed in bladder cancer cell lines. The function of BARR1 and BARR2 is unknown in bladder cancer although these proteins are well characterized in other systems, notably as regulators of G-protein coupled receptor activity in normal and abnormal cell physiology. We investigated the potential role of BARRs in established bladder cancer cells. Methods: Manipulations of expression of individual arrestins were performed by establishing stable cell lines with forced overexpression by cDNA transfection, or depletion by shRNA or CRISPR-Cas9 mediated gene editing. The effect of altering BARRs in bladder cancer cells was investigated for changes in clonogenic survival, expression of basal cell markers, such as cytokeratins (CK), stem-cell associated markers, alteration in tumor cell motility and invasion. Results: Established cell lines from MIBC origin (e.g., 253J and HT-1376) had either high-level expression of BARR1 and BARR2 or only high expression of BARR1. Normal bladder cells predominantly expressed BARR2. Depletion of BARR2 in 253J cells resulted in increased basal cell markers (e.g., CK5, CK14). Depletion of BARR2 elevated many cancer stem cell (CSC) markers (e.g., ALDH2). BARR2 overexpression reduced expression of stem cell markers, and increased sensitivity towards gemcitabine. Furthermore, when cells were grown as spheroids–a technique used to enrich CSCs–we observed decreased expression of BARR2, whereas expression of BARR1 increased. In 253J cells, BARR2 depletion significantly increased motility, chemotaxis, and invasive potential. CRISPR-Cas9 mediated gene knockout of BARR1 resulted in reversal of aggressive phenotype, including decrease in CD44, and phospho-STAT3. Conclusions: This is the first report of the role of BARR1 and BARR2 in MIBC. The results presented here strongly suggest a critical role of BARRs in bladder cancer metastasis and resistance to chemotherapy drugs, two key factors that determine the poor survival of bladder cancer patients. Further exploration of the roles of β-arrestins in bladder cancer has potential to improve therapy for patients with muscle-invasive bladder cancer. Citation Format: Georgios Kallifatidis, Diandra K. Smith, Jie Gao, Richard Pearce, Jiemin Li, Vinata Lokeshwar, Balakrishna L. Lokeshwar. Beta-arrestins regulate basal cell and cancer stem cell phenotype in muscle-invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 86.