Increase in hepatocyte growth factor receptor tyrosine kinase activity in renal carcinoma cells is associated with increased motility partly through phosphoinositide 3-kinase activation

Abstract

Dysregulated cell motility is one of the major characteristics of invasion and metastatic potentials of malignant tumor cells. Here, we examined the hepatocyte growth factor (HGF)-induced cell motility of two human renal carcinoma cell lines, ACHN and VMRC-RCW. Scattering and migration was induced in ACHN in an HGFdependent manner, whereas they were maintained in VMRC-RCW even in the absence of HGF. In VMRC-RCW, HGF receptor (HGFR) tyrosine kinase was constitutively active, and sequence analysis showed N375S, A1209G and V1290L mutations. However, transfection experiments using porcine aortic endothelial (PAE) cells demonstrated that no single mutation or combination of two or three mutations caused HGFindependent constitutive activation. Conversely, the expressed amount of receptor protein had a pivotal role in the basal kinase activity. With respect to downstream signaling molecules of HGFR in ACHN or VMRC-RCW, the Ras-MAPK pathway was downregulated, whereas phosphoinositide 3-kinase (PI3-kinase) was not further activated by HGF-treatment in VMRC-RCW cells. The PI3-kinase inhibitors, wortmannin and LY294002 strongly inhibited spontaneous migration of VMRC-RCW. One transfected PAE cell line with massive overexpression of HGFR demonstrated scattered morphology and increased PI3-kinase activity in association with increased motility, which was partially inhibited by LY294002. Taken together, our results indicate that the overexpression of HGFR causes increase in cellular motility and PI3-kinase shows the important contribution on the increased motility of renal carcinoma cells. Oncogene (2001) 20, 7610 ± 7623.

Hepatocyte Growth Factor-induced Ectodomain Shedding of Cell Adhesion Molecule L1

“…In PC12 cells, nerve growth factor (NGF) was found to increase shedding of the NILE glycoprotein (39), which was later identified as the rat homologue of L1 (31). We found that in renal carcinoma cells, basal shedding of L1 is stimulated by HGF, a growth factor important for proper kidney development (17), which is strongly expressed in 90% of renal cell carcinomas (10,11,12) and increases invasiveness in renal carcinoma cells in vitro and in vivo (13,14). HGF stimulated basal shedding of L1 at physiological concentrations with a maximum reached at 5 ng/ml HGF.…”

“…Changes in the normal expression patterns of HGF and GDNF are associated with abnormal proliferation in the human renal system. While in the normal human kidney, HGF and c-Met are expressed at very low levels, increased HGF and c-Met expression is found in about 90% of renal cell carcinomas (10 -12), and overexpression of HGF/c-Met enhances cellular motility of renal carcinoma cells in vitro and in vivo (13,14). GDNF is overexpressed in renal dysplasia (15) and in collecting duct cysts (16).…”

Long-term exposure of human renal carcinoma cells to PD98059 induces epithelial–mesenchymal transition-like phenotype and enhanced motility

“…ACHN cells grow as a compact seat with cell-cell contact, whereas VMRC-RCW cells grow without cell-cell contact (scattered morphology) [13]. We first examined the effect of short-term PD98059 treatment on serum-activated ERK in these cells.…”

“…To quantify cell migration, we performed the chemokinesis assays [13]. Briefly, cells were suspended in a serum-free medium and placed onto the upper surface of the membranes of Transwell inserts (6.7-mm in diameter, 8-µm pore) pre-coated with bovine type I collagen (30 mg/ml) at a density of 1.5  10 4 cells/membrane.…”

“…Stress fiber formation was determined as described previously [13]. In brief, cells (2  10 4 cells/well) were seeded onto cover slips in 24-well plates and cultured for 20 h. They were then incubated in a serum-free medium for 3 h and either stimulated with 10% FBS for 20 min or left unstimulated.…”