TGF-beta Induced Epithelial-Mesenchymal Transition Promotes Bladder Cancer-Cell Migration in a Nonmuscle Myosin IIb Dependant Manner
Sounak Gupta, Harwalkar Jyoti, Aaron Shoskes, Thomas T Egelhoff, Donna E Hansel. Cleveland Clinic Foundation, Cleveland, OH; University of Denver, Denver, CO
Background: Cancer of the urinary bladder is ranked as the 5th leading cause of cancer worldwide with invasive behavior being the primary driver of poor outcomes. Transforming growth factor beta (TGF-beta) induced epithelial-mesenchymal transition (EMT) has been demonstrated to promote the acquisition of invasive behavior in cancer cells. Recent studies have shown that TGF-beta induced switching of the nonmuscle myosin heavy chain isoform from IIc to IIb is associated with EMT and invasive behaviour. We therefore investigated baseline TGF-beta production, markers suggestive of EMT, nonmuscle myosin isoform expression and its relevance to bladder cancer cell migration in an in vitro setting.
Design: Benign papilloma derived RT4 cells and high grade muscle invasive UCa derived: UMUC3, T24 and J82 cells were profiled for TGF-beta expression (ELISA), markers of EMT and multiple nonmuscle myosin isoform expression (quantitative PCRs, immunoblotting). Relative rates of migration in these cells were assessed using transwell assays and a bladder slice assay. Finally, the myosin IIb isoform was ablated using siRNA and resultant changes in cell migration was assessed using transwell assays.
Results: The RT4 cells exhibited an “epithelial” phenotype characterized by the absence of TGF-beta production, a lack of EMT-promoting transcription factor expression as well as the presence of E-cadherin–dependent intercellular contacts. In contrast, the aggressive UCa derived cells demonstrated a “mesenchymal” phenotype that was correlated with increased TGF-beta production and EMT-promoting transcription factor expression such as the epithelial repressors: Snail, Twist1, Zeb1 and the mesenchymal activator: FoxC2, as well as a lack of E-Cadherin expression. Unlike the RT4 cells, the latter group showed a lack of the myosin heavy chain isoform IIc and concurrent enrichment for the IIb isoform which was correlated with increased migration by both the transwell and bladder slice assays. Finally, knocking down the IIb isoform led to statistically significant reductions in transwell migration of bladder cancer derived cells.
Conclusions: Our results suggest that the absence of the myosin IIc isoform coupled with enrichment for the IIb isoform is a signature that is suggestive of cells that have undergone EMT. Furthermore, the IIb isoform directly contributes to increased invasive behavior. Current efforts are being directed at identifying phosphorylation events that regulate nonmuscle myosin isoform organization.
Category: Genitourinary (including renal tumors)
Monday, March 4, 2013 1:00 PM
Poster Session II # 177, Monday Afternoon