G protein-coupled lysophosphatidic acid receptors stimulate proliferation of colon cancer cells through the beta-catenin pathway

By: Ming Yang, Wendy W Zhong, Neelam Srivastava, Anthony Slavin, Jianxin Yang, Timothy Hoey and Songzhu An

Background: G coupled protein receptors LPA1, LPA2, and LPA3 have been shown to mediate lysophosphatidic acid stimulation, which is associated with the proliferation of colon cancer cells.  Two important oncogenic transformation genes related to colorectal cancer proliferation are c-myc and cyclin D1; both in which can be transcribed as a result of the beta-catenin pathway.  It has been observed that with the inhibition of beta-catenin degradation a nuclear summation of beta-catenin leads to increased levels of these deadly oncogenic proteins.  However, the mechanism in which LPA stimulation was involved with both the colorectal cancer proliferation and the beta-catenin pathway were both unknown.

Objective: Using HCT116 and LS174T adenocarcinoma cell lines, it was sought to identify which LPA receptors were responsible for colorectal cancer cell proliferation, and what mechanism was induced during this process.

Results:

• With the treatment of LPA to HCT116 and LS174T cells, colon cancer cells underwent proliferation in a dose dependent response over a concentration titration of  .01 micromolar-1.0micromolar LPA.  Cells were then analysed for the expression of LPA1, LPA2, and LPA3 mRNA, revealing significant levels of only LPA2 and LPA3, not LPA1.
• HCT116 and LS174T cells were than transfected with siRNA of LPA2 and LPA3 showing a direct decrease in cell proliferation and tumor volume. Beta-catenin siRNA similarly reduced colon cell proliferation, even though the treated cells were induced with LPA, corresponding to the belief that LPA induction stimulates the beta-catenin pathway increasing colon cancer growth.  
•  It was then shown with Western Blot analysis that when induced with LPA the phosphorylation of GSK3beta via cPKC, a multicomplex protein that stabalizes beta-catenin and prevents degradation, increases drastically.  From this it was deduced that the phosophorylation of GSK3beta was one of the ways that LPA stimulated the beta-catenin pathway.
• Lastly, the activation of the beta-catenin pathway via both LPA2 and LPA3 was research.  It was found using siRNA knockdowns for both receptors that both were efficient in increasing the beta-catenin pathway, which further increased the proliferation of both HCT116 and LS174T colorectal cancer cells.

-JI