Abstract 4959: Klotho suppresses colon cancer through modulation of the Wnt pathway and unfolded protein response

Aberrant activation of the canonical Wnt signal transduction pathway is implicated in the development and progression of colorectal cancer (CC). The key effector protein of this pathway is β-catenin, which functions with T-cell factor/lymphoid enhancer factor (TCF/LEF) to activate expression of Wnt target genes. Klotho is a transmembrane protein, which can be cleaved, shed and act as a circulating hormone. Klotho-deficient mice manifest a syndrome resembling accelerated aging, while klotho overexpression extends life span. We have previously identified klotho as a potent tumor suppressor in breast and pancreatic cancer, and recent data indicate it as a potent tumor suppressor in a wide array of malignancies, including CC. The activities of klotho include inhibition of the insulin like growth factor (IGF)-1 and the Wnt pathways. We aimed to decipher the role of klotho as a tumor suppressor and modulator of the Wnt pathway in CC. We first tested the effect of klotho on proliferation of CC cells and noted that overexpression of klotho inhibited colony formation of the HCT 116, SW480, HT-29, Colo-320 and RKO CC cells. We next examined the effects of klotho in vivo, using the azoxymethane (AOM) model, and found that daily administration of klotho to mice inhibited formation of colon polyps induced by AOM. While we did not see an effect of klotho on the IGF-1 signaling pathway, klotho did affect the Wnt pathway. Klotho reduced Wnt3A and β-catenin protein levels as measured by Western blotting, and inhibited transcriptional activity of the Wnt pathway as evidenced in luciferase assay. Moreover, this transcriptional inhibition was abrogated by overexpression of a constitutively active β-catenin. We, therefore, suspected that klotho inhibited the pathway upstream of β-catenin. Indeed, co-immunoprecipitation analyses indicated direct interaction between klotho and Wnt3A. As the inhibitory effect of klotho on colony formation of CC cells was only partially rescued by transfection with constitutively active β-catenin, we concluded that additional mechanisms may mediate the effects of klotho. In order to elucidate these mechanisms, we conducted a cDNA expression microarray and involvement of klotho in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) was observed. Taken together, our data indicate klotho as a potent tumor suppressor in CC and suggest its role already at the early stages of polyp formation. Klotho effects in CC are partially medi