Abstract 1988: A bi-directional feedback loop between Wnt and ERK is important in maintaining the proliferative phenotype of normal human urothelial cells

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Abstract Introduction: The urothelial lining of the bladder is ordinarily mitotically quiescent, but can rapidly regenerate in response to injury. This regenerative capacity is thought to be important in restoring urinary barrier function and offers a primary target for deregulation in cancer. Previous work in our laboratory has implicated autocrine EGFR activation in this process, but other regulatory pathways may be involved. Wnt signalling has been shown to be important in the renewal of a number of adult epithelial tissues, but to date, no conclusive evidence for a role for Wnt in urothelial regeneration has been demonstrated. In the absence of Wnt ligand, β-catenin is sequestered via the destruction complex. Activation of the canonical Wnt pathway by ligand, inactivates the destruction complex, allowing β-catenin to translocate to the nucleus and drive proliferation. The aim of this study was to assess whether Wnt signaling was functional in normal human urothelial (NHU) cells, and to analyze the effects of Wnt and downstream EGFR signaling on proliferation. Materials and Methods: NHU cells were grown as finite lifespan cell lines in proliferating cultures. Destruction complex antagonists were used to activate the Wnt pathway. Proliferation assays, immunofluorescence labeling and TOPFLASH luciferase reporter assays were used to assess NHU cell responses. Pathway crosstalk was analyzed using shRNA targeted to β-catenin, as well as antagonists of the EGFR signaling pathways. Results: Pharmacological inhibition of the destruction complex or activation of the Wnt pathway with exogenous Wnt ligand led to nuclear accumulation of β-catenin and an increase in TCF/LEF transcription factor activity. These effects were only apparent when autocrine EGFR signaling was blocked. Knockdown of β-catenin led to significant changes in p-ERK and p-AKT downstream signaling. Conclusion: Wnt signaling can be activated in NHU cells, verifying the presence of a functional pathway. Activation of the pathway is however masked by EGFR signaling, indicating pathway crosstalk. The effect of β-catenin knockdown on p-ERK and p-AKT indicates a bi-directional positive feedback loop between the Wnt and EGFR signaling cascades in NHU cells. This study identifies the Wnt signaling pathway as an important signaling cascade in regulating the regenerative phenotype of NHU cells and indicates at least two pathways that may be arrogated by urothelial cells during neoplastic transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2011-1988