Investigating Receptor Expression in Response to Wnt5a Isoforms in Urothelial Carcinoma Cell Lines

Abstract

Urothelial carcinoma (UC), the most common form of bladder cancer, has a high rate of recurrence and a low five‐year survival rate for high grade and metastatic tumors. Expression of Wnt5a, a protein that plays a role in many critical processes in cells and is involved in many different molecular signaling pathways, has been shown to correlate with UC development and pathological stage. Recently the Wnt5a gene has been shown to encode two different isoforms (Wnt5a‐L and Wnt5a‐S). Correlation of isoform expression with tumor cell phenotype suggests opposing roles, either as tumor‐promoting or tumor‐suppressing, depending on the cancer type. The effects of each Wnt5a isoform in UC, however, are not well understood.In order to understand how the Wnt5a isoforms function in the development of UC, it is important to understand which signaling pathways are being activated or inhibited by each isoform. This project aims to identify Wnt5a receptors that are differentially upregulated or downregulated in response to treatment with either Wnt5a‐L or Wnt5a‐S in several different UC cell lines derived from different stages of tumors, as well as in a primary human bladder cell line. The project will include identification and characterization of the Wnt5a receptors normally expressed in the untreated, basal state, information which is currently unknown. Wnt5a isoform treatment will be conducted using conditioned media collected from Chinese Hamster Ovary (CHO) cells that have been genetically modified to express either Wnt5a‐L or Wnt5a‐S. Using western blot analysis, we have already quantified the amount of each isoform present in the conditioned media so that equal amounts of each isoform can be used for cell treatments. We have also used western blot analysis to demonstrate that we can isolate membrane proteins from cell lysates. We will now be treating the cell lines with equal concentrations of Wnt5a‐L, Wnt5a‐S or commercially available Wnt5a as a positive control; isolating the membrane proteins from each cell line after treatment; and applying the isolated membrane proteins to custom antibody arrays in order to examine the expression of 17 different Wnt5a receptors. Results from these experiments will show what receptors are upregulated or downregulated in response to each Wnt5a isoform, which will help elucidate the signaling pathways regulated by each isoform in the pathogenesis of UC.Support or Funding InformationThis project was supported in part by funding from the Honors Tutorial College, Student Enhancement Award, and Heritage College of Osteopathic Medicine Research and Scholarly Activities Committee, all at Ohio University. Special thanks is given to Dr. Karl Willert, who provided the CHO cell lines expressing each Wnt5a isoform.