Colon carcinoma cells harboring PIK3CA mutations display resistance to growth factor deprivation induced apoptosis.

PIK3CA, encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is mutated in a variety of human cancers. We screened the colon cancer cell lines previously established in our laboratory for PIK3CA mutations and found that four of them harbored gain of function mutations. We have now compared a panel of mutant and wild-type cell lines for cell proliferation and survival in response to stress. There was little difference in PI3K activity between mutant PIK3CA-bearing cells (mutant cells) and wild-type PIK3CA-bearing cells (wild-type cells) under optimal growth conditions. However, the mutant cells showed constitutive PI3K activity during growth factor deprivation stress (GFDS), whereas PI3K activity decayed rapidly in the wild-type cells. Importantly, constitutively active PI3K rendered the mutant cells resistant to GFDS-induced apoptosis relative to the wild-type cells, indicating a biological advantage under stress conditions that is imparted by the mutant enzymes. Compared with the wild-type cells, the mutant cells were hypersensitive to the apoptosis induced by the PI3K inhibitor LY294002. In addition, PIK3CA small interfering RNA significantly decreased DNA synthesis and/or induced apoptosis in the mutant cells but not in the wild-type cells. Furthermore, ecotopic expression of a mutant PIK3CA in a nontumorigenic PIK3CA wild-type cell line resulted in resistance to GFDS-induced apoptosis, whereas transfection of wild-type PIK3CA or empty vector had little effect. Taken together, our studies show that mutant PIK3CA increases the capacity for proliferation and survival under environmental stresses, such as GFDS while also imparting greater dependency on the PI3K pathway for proliferation and survival

Knockdown of Ron Kinase Inhibits Mutant Phosphatidylinositol 3-Kinase and Reduces Metastasis in Human Colon Carcinoma*

Abnormal accumulation and activation of receptor tyrosine kinase Ron (recepteur d'origine nantais) has been demonstrated in a variety of primary human cancers. We show that RNA interference-mediated knockdown of Ron kinase in a highly tumorigenic colon cancer cell line led to reduced proliferation as compared with the control cells. Decreased Ron expression sensitized HCT116 cells to growth factor deprivation stress-induced apoptosis as reflected by increased DNA fragmentation and caspase 3 activation. In addition, cell motility was decreased in Ron knockdown cells as measured by wound healing assays and transwell assays. HCT116 cells are heterozygous for gain of function mutant PIK3CA H1047R. Analysis of signaling proteins that are affected by Ron knockdown revealed that phosphatidylinositol 3-kinase (PI3K) activity of the mutant PI3K as well as AKT phosphorylation was substantially reduced in the Ron knockdown cells compared with the control cells. Moreover, we demonstrated in vivo that knockdown of Ron expression significantly reduced lung metastasis as compared with the control cells in the orthotopic models. In summary, our results demonstrate that Ron plays an essential role in maintaining malignant phenotypes of colon cancer cells through regulating mutant PI3K activity. Therefore, targeting Ron kinase could be a potential strategy for colon cancer treatment, especially in patients bearing gain of function mutant PI3K activity