Loss of GRLF1 induces aneuploidy and enhances bladder cancer cell migration and invasion by modulating actin dynamics

A previous RNA interference (RNAi) screening approach in our laboratory identified GRLF1, a GTPase-activating protein that regulates the Rho family of small GTPases, as a ploidy-control gene, which implied a potential function of GRLF1 in genome stability. GRLF1 has been studied across various cancer types, but previous studies revealed contradictory results as to whether GRLF1 is a tumor promoter or tumor suppressor protein. Mutation rates of up to 20% where observed in some cancers, further supporting the significance of GRLF1 in carcinogenesis. Since the relevance of GRLF1 in bladder cancer has not been addressed so far, we attempted to explore it. Evaluation of TCGA high-throughput sequencing datasets revealed GRLF1 mutations in up to 8% of bladder cancer samples across several studies. Employing immunohistochemistry on tissue microarray that included 202 BC patient samples, we observed that lower GRLF1 expression correlates with increased invasiveness and poorer survival outcomes. These findings suggested that GRLF1 may have a tumor suppressor function in bladder tissue, consistent with its anticipated function in ploidy-control. GRLF1 knockdown resulted in chromosomal instability in ureter-derived epithelial cells with otherwise normal karyotype, supporting its potential involvement in tumorigenesis. Loss-of-function studies in low-invasive bladder RT4 cell line and gain-of-function experiments in two highly invasive bladder cancer cell lines (T24 and BFTC) demonstrated that GRLF1 influences cell migration and invasion in vitro , as determined by scratch assay and Boyden chamber approaches. This conclusion was further validated by ex vivo porcine bladder invasion approach, invadopodia formation, and gelatin degradation assays. Pathway analysis revealed that altered GRLF1 expression influences both the Rho-ROCK-dependent LIMK1-cofilin pathway and the phosphorylation of cortactin by focal adhesion kinase, both of which regulate critical cellular processes such as actin network organization and polarization to facilitate efficient, coordinated cell migration and division. In summary, our results indicate that GRLF1 has a genuine role in controlling cell-ploidy and regulates actin dynamics in the bladder urothelium, while loss of GRLF1 results in genome instability and drives bladder cancer initiation and progression through deregulated actin dynamics.