Abstract 4005: The human Norrin/beta-catenin signaling enhancer TSPAN12 is expressed on colorectal tumors; evidence for cell-type specific responsiveness to Norrin signaling in the tumor microenvironment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction. While the majority of colorectal cancers (CRC) harbor mutations in Wnt pathway component APC, leading to constitutive pathway activation, tumor cells remain responsive to Wnt signals arising at the cell membrane through the interaction of extracellular Wnt ligands and cell surface frizzled (Fz) receptors. The strength of the Wnt signal can be modified through epigenetic modification of genes encoding for extracellular Wnt pathway inhibitors that produce proteins released into the tumor microenvironment. Norrin is an extracellular molecule that binds to Fz4 and induces beta-catenin (canonical Wnt) signaling. This signaling is also dependent on the expression of TSPAN12 which functions as a signal enhancer. Norrin promotes angiogenesis in the eye but the role of Norrin signaling in colorectal cancer is not known. In this study we have examined the expression of Norrin pathway components in CRC and other cell types in the tumor microenvironment. Methods. Human CRC and normal colon tissues, including mucosa, stroma and endothelial cells were analyzed by immunohistochemistry and reversed transcription polymerase chain reaction in order to define expression of Norrin, Fz4 and TSPAN12. In vitro, endothelial cells were grown on collagen in the presence of CRC cell line-produced conditioned medium (CM), with or without Norrin blocking antibody. Results. Norrin and Fz4 were expressed in normal colonic mucosa, CRC, and endothelial cells. TSPAN12, the component that usually determines what tissues will respond to the Norrin signaling, was expressed in CRC but not in normal colonic mucosa. Low amounts of endothelial cell TSPAN12 in tissue sections was difficult to quantitate due to insufficient sensitivity of the method. In vitro, elongation of endothelial cells treated with CM from CRC cell lines was partly abrogated by Norrin blocking antibodies, suggesting responsiveness of these cells to Norrin-mediated signals. Conclusions. Critical components of the Norrin/beta-catenin pathway are expressed in CRC and the colon tumor microenvironment but TSPAN12, the signal enhancer that promotes signaling through this pathway is found on CRC cells and not expressed on normal mucosa In vitro evidence suggests that Norrin signaling also affects endothelial cells. Implications. The distribution of TSPAN expression in the CRC tumor microenvironment, and the effect of Norrin blocking antibodies on CRC CM-treated endothelial cells in vitro, suggest that the predominant roles of Norrin are a direct action on CRC and potentially also promotion of tumor angiogenesis. Citation Format: Kestutis Planutis, Marina Planutiene, Randall F. Holcombe. The human Norrin/beta-catenin signaling enhancer TSPAN12 is expressed on colorectal tumors; evidence for cell-type specific responsiveness to Norrin signaling in the tumor microenvironment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4005. doi:10.1158/1538-7445.AM2013-4005