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.

Characterization of HCT116 human colon cancer cells in an orthotopic model.

BACKGROUND: Colorectal cancer metastases result in a significant number of cancer related deaths. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. In the absence of any transgenic models of colon cancer metastases, an in vivo model system that fulfills the rate limiting steps of metastasis (local invasion and distant colony formation) is needed. The purpose of this study was to characterize the behavior of a human colon cancer cell line, HCT116 in an orthotopic model. MATERIALS AND METHODS: HCT116 cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into 32 other male BALB/c nude mice using microsurgical techniques. Animals were serially imaged and euthanized at 6-8 weeks post-implantation. Tissues were procured and processed for hematoxylin and eosin analysis. RESULTS: All 32 animals demonstrated primary tumor growth, invasion and peritoneal spread. Liver metastases were identified in 15/32 (47%), and lung metastases were confirmed in 13/32 (41%). In total, 19/32 (59%) animals demonstrated distant metastatic colony formation. CONCLUSIONS: This orthotopic model of colon cancer fulfills the rate limiting steps of local invasion and distant colony formation in the process of metastases. HCT116 human colon cancer cell line in this in vivo model system provides a tool to dissect the molecular mechanism involved in the metastatic cascade.

Mutant PIK3CA-bearing colon cancer cells display increased metastasis in an orthotopic model.

Mutations in the PIK3CA gene are common in human cancers, including colon cancer. We compared two pairs of colon cancer cells (HCT116 and DLD1) bearing only the wild-type (WT) or mutant (MUT) PIK3CA allele for their survival capacity under stress conditions in vitro as well as their metastatic properties in an in vivo orthotopic model. When subjected to growth factor deprivation stress (GFDS), the MUT PIK3CA cells displayed resistance to GFDS-induced apoptosis relative to the WT cells. Phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT were constitutively activated during stress conditions in the MUT PIK3CA cells but not in the WT cells. The MUT cells showed hypersensitivity to PI3K inhibition. Moreover, the proapoptotic protein Bax was expressed at a very high level in the WT PIK3CA cells, whereas it was almost undetectable in the MUT cells. Inhibition of Bax expression by small interfering RNA protected the WT PIK3CA cells from GFDS-induced apoptosis, suggesting an important role of Bax in GFDS-induced apoptosis. These results indicated that the MUT PI3K confers resistance to GFDS-induced apoptosis and that the MUT cells are more dependent on the PI3K pathway for survival. In vivo studies showed that the MUT PIK3CA-bearing cells were more metastatic than the WT cells in an orthotopic model of colon cancer. Taken together, these results suggest that MUT PI3K imparts a more aggressive phenotype in colon cancer cells and could be a potential therapeutic target for treatment of colon cancer patients bearing PIK3CA mutations.