Knockdown of Ron kinase inhibits mutant phosphatidylinositol 3-kinase and reduces metastasis in human colon carcinoma

Abstract

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.

FIGURE 1.

Ron is highly expressed in colon, breast, pancreatic, and prostate cancers. RNA from cells in the NCI 60 panel was isolated and Ron mRNA expression was measured using Q-PCR. Values were normalized to glyceraldehyde-3-phosphate dehydrogenase control.

FIGURE 2.

Knockdown of Ron expression by small interfering RNA-induced apoptosis. HCT116 cells were transfected with either a nonspecific siRNA or Ron-specific siRNA (Smartpool, Dharmacon). A, message levels of Ron were determined at 24 h. B, Ron Smartpool and two individual siRNA duplexes were transfected into HCT116 cells and caspase 3 induction was measured 48 h later. Values are the means of three replicates. Error bars indicate S.E. C, protein expression of Ron and poly(ADP-ribose) polymerase (PARP) cleavage was determined in HCT116 cells 48 h post siRNA transfection. Actin was used to normalize sample loading. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 3.

Clonogenic and anchorage-independent growth was reduced in HCT116 cells stably transfected with Ron shRNA. A, Ron knockdown clones and the control cells were lysed and subjected to Western blot analyses with an antibody to Ron. Actin was used as a loading control. B and D, cells were plated in 24-well plates at 300 cells/well. Cell colonies were stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and visualized after 2 weeks of incubation (B). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide stain was dissolved in dimethyl sulfoxide. Relative cell numbers were then determined by the resultant absorbance at 595 nm. Values are the means of four replicates (D). Error bars indicate S.E. C, anchorage-independent colony formation in soft agarose by HCT116 control and Ron knockdown cells was determined as described under “Experimental Procedures.”

FIGURE 4.

Ron knockdown-sensitized HCT116 cells to GFDS-induced apoptosis and impaired their motility. Ron knockdown clones and control cells were grown to 80-90% confluence and the cells were then deprived of growth factors for 5 days. A, DNA fragmentation assays were performed to determine apoptosis as described under “Experimental Procedures.” Error bars indicate S.E. B, cells were harvested and Western blot analyses were performed with Ron or caspase 3 antibody. Actin was used as a loading control. C, Ron knockdown clones and the control cells were grown to confluence. Motility was examined in wound healing assays as described under “Experimental Procedures.” D, Ron knockdown clones and the control cells were starved in growth factor-deprived medium overnight. They were then plated in the transwells and transwell assays were performed as described under “Experimental Procedures.”

FIGURE 5.

Knockdown of Ron kinase inhibited the PI 3-kinase pathway. Ron knockdown clones and control cells were grown to 80-90% confluence and the cells were then deprived of growth factors for 5 days. A, cell lysates were harvested and subjected to PI 3-kinase assays as described under “Experimental Procedures.” B, cell lysates harvested in A were subjected to Western blot analysis with pAKT (S473) or AKT antibody. Actin was used as a loading control. C, cell lysates from A were subjected to Western blot (WB) analyses with a p85 antibody or immunoprecipitated (IP) with a Ron antibody followed by Western blot analyses with a p85 antibody.

FIGURE 6.

A, Ron was overexpressed in one of the Ron knockdown clones (Cl.9). The vector or Ron expressing cells were deprived of growth factors for 5 days. Western blot analyses were performed with antibodies to Ron, pAKT (S473), or caspase 3. Actin was used as a loading control. B, CBS and RKO cells were transfected with either a nonspecific scrambled shRNA (SC) or a Ron-specific shRNA (KD). Transfected cells were grown to 80-90% confluence and then deprived of growth factors for 5 days. Cell lysates were harvested and Western blot analyses were performed with antibodies to Ron, pAKT (S473), AKT, or caspase 3. Actin was used as a loading control.

FIGURE 7.

Exponentially growing cells (5 × 10⁶) were inoculated subcutaneously in athymic nude mice. The xenografts formed by the control cells and Ron knockdown clones were used for subsequent orthotopic implantation. A, fluorescence imaging of the animals on the indicated days. B, primary tumors established from the control and RON knockdown cells were processed for hematoxylin and eosin (left panel), TUNEL (middle panel), and Ki67 (right panel) staining. Both TUNEL and Ki67 images were captured at ×20 magnification. C, primary tumors established from the control and RON knockdown cells were analyzed to assess the proliferation and apoptotic rate. NIH Image J software was utilized to enumerate positive staining cells and total number of cells. The percentage of positive staining cells was calculated. Error bars indicate S.E. D, 38 days post-implantation, animals were euthanized. Organs were explanted and imaged. E, immunohistochemistry was performed for pAKT (S473) on primary tumors of control or Ron knockdown cells. The blocking peptide was used to show the specificity. The red arrows indicate tumor cells.