The effect of Wnt9A induced by lithium chloride on β-catenin and colorectal cancer cell proliferation.

Abstract

631 Background: Malignant tissues are characterized by uncontrolled proliferation and dysfunctional differentiation which often results from dysregulated Wnt signaling. β-catenin, a second messenger upregulated in neoplastic tissues is utilized by the canonical Wnt pathway (CWP) to promote cellular proliferation. In healthy tissues CWP is stringently controlled by components of the non-canonical Wnt pathway (NCWP). The NCWP, which does not utilize β-catenin for its activity, acts in concert with the CWP to maintain homeostasis in healthy tissues. Our recent studies in CRC cells indicate that LiCl increases expression of the NCWP ligand, Wnt9A leading to activation of the NCWP. Suppressing Wnt9A significantly attenuates LiCl-mediated inhibition of CRC cell proliferation. The present study investigated the capacity of Wnt9A to directly modulate expression of β-catenin mRNA and/or protein levels in CRC cells, and the effects on proliferation and apoptosis. Methods: Primary CRC lines established from resected tumors of 5 patients with metastatic disease were treated with LiCl and Wnt9A protein. Cellular proliferation and apoptosis were determined by MTS and caspase 3 assays. Total and activated β-catenin protein were determined by ELISA and Wnt pathway genes and β-catenin mRNA were determined with PCR arrays. Results: LiCl inhibited CRC proliferation (p<0.001) and increased apoptosis (p<0.01) which coincided with a 23-fold (p<0.025) increase of the NCWP ligand Wnt9A. LiCl decreased β-catenin mRNA (p<0.03) and suppressed protein levels of the total (p<0.025) and the activated form of β-catenin. Recombinant Wnt9A protein emulated LiCl effects by inhibiting proliferation (p<0.001), increasing apoptosis (p<0.03) and suppressing activated β-catenin protein (p<0.001) in CRC cells. Conclusions: This study demonstrates a novel mechanism for inhibiting CRC cell proliferation through suppression of activated β-catenin levels by the non-canonical Wnt ligand, Wnt9A. These results suggest that in CRC tumors that are driven by β-catenin, activation of the non-canonical pathway and/or direct induction of or treatment with Wnt9A protein may be therapeutically beneficial.