| Research Abstract |
Phosphoinositide 3-kinases (PI3Ks) constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, transformation, differentiation and protection from apoptosis. We found that genetic inactivation of p110γ, the catalytic subunit of PI3Kγ, leads to development of invasive colorectal adenocarcinomas in mice. In humans, p110γ protein expression is lost in primary colorectal adenocarcinomas from patients and colon cancer ceil lines. Overexpression of p110γin human colon cancer cells resulted in suppression of their growth.
To elucidate the underlying mechanism for the tumor suppressive effect of p110γ, we studied the ability of various p110γ mutants to suppress growth of human colorectal cancer cell lines in vitro. I found that overexpression of a kinase-dead mutant of p110γ (R947P) in HCT116 and DLD1 inhibited cell growth as assessed by colony formation assay and focus assay. These results suggest that P13
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K activity is dispensable for the tumor suppressive effect of p110γ. As the growth suppressive effects did not depend on its enzymatic activity, we next examined which domain structure is sufficient and/or necessary for the effects. Recent structural studies have revealed that p110γpossesses five distinct domains, namely, N-terminal PH domain, Ras-binding domain, C2-domain, kinase domain and Helical domain. Expression of either the Ras-binding domain or the kinase domain led to growth suppression in the colon cancer cells. However, two Ras-binding mutants that cannot bind to Ras suppressed cell growth and wild type p110γ showed the effects in a DLD1 cell line that lacks endogenous Ki-Ras expression. In addition, expression of truncated mutants that lack the C-terminus kinase domain with intact Ras-binding domain had no effect on cell growth. Taken together, these results demonstrate that the kinase domain is necessary and sufficient for the growth suppression of the colon cancer cells by p110γ. Notably, the conclusion that the kinase activity is dispensable for the effects, provides a novel paradigm for p110γfunction and predict a unique signaling cascade that suppresses tumorigenesis. Less
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