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FULL LENGTH AND CONSTITUTIVELY ACTIVE LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN (LRP6) CAN DEGRADE BETA-CATENIN IN COLON CANCER CELL LINES

I Pacheco, M Hayes, RJ MacLeod
GIDRU, Department of Physiology, Queen's University, Kingston, Ontario

BACKGROUND: To understand how Ca²⁺-supplementation provides chemoprotection in colon cancer, we activated the colonic Extracellular calcium-sensing receptor (CaSR) on colonic epithelial cell lines and performed microarray analysis. Increases of Wnt5a and LRP6 were observed. LRP6 is a co-receptor of canonical Wnt which causes nuclear beta-catenin accumulation. Wnt5a antagonizes canonical Wnt signaling. The aim of our study was to assess the role of LRP6 in Wnt/B-catenin signaling in human colon cancer cell lines utilizing overexpression of several LRP6 constructs.
METHODS: HT-29, HT-29-APC (Zn²⁺-inducible WT APC), DLD-1, SW-48, SW-480 and COS-1 cells were transiently transfected with full length (FL)-LRP6, LRP6-C2 (extracellular domain truncation), or LRP6-C3 (extracellular and transmembrane truncation), in the presence of Tcf/TOPFLASH reporter and 24h later treated with low or high Ca²⁺ (0.005 vs 3 mM Ca²⁺) for 18h and luciferase activity measured. Western blot was utilized to determine cellular levels of beta-catenin.
RESULTS: In COS-1 cells an increase in Tcf reporter was observed with FL-LRP6 (2.5 fold), LRP6-C2 (4 fold) or LRP6-C3 (3 fold) transfection. These increases were all enhanced by co-transfection with canonical Wnts Wnt3a and Wnt1. In contrast, all colonic cells transfected with FL-LRP6 or LRP6-C2, which is constitutively active, showed a reduction of Tcf/TOPFLASH reporter activity of 50-95% when compared with respective controls. Western blot analysis of HT-29 cell lysates demonstrated reduction of cellular beta-catenin in cells transfected with FL-LRP6 or the constitutively active LRP6-C2 or LRP6-C3 constructs.
CONCLUSION: We conclude that in adenocarcinoma cells with truncated APC, increases in full length or constitutively active LRP6 substantially inhibit Wnt/beta-catenin signaling. CaSR activation increased the inhibitory effect of constitutively active LRP6. We speculate that the effects of LRP6 on Wnt/beta-catenin signaling may be distinctive in intestinal epithelial cells.

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