VHL Decreases FGFR1 Activity to Control Cell Motility

Abstract

Tumors form in multiple organs in people with von Hippel-Lindau disease, and the cause is mutations in the VHL gene. Renal cell carcinomas (RCCs) with VHL mutations are frequently metastatic, and Hsu et al. provide evidence that this may be due to increased localization of fibroblast growth factor receptor 1 (FGFR1) at the cell surface and increased activation of the receptor, which may increase the migratory potential of RCCs. Hsu et al. examined the migratory potential of human RCC cell line 786-O (VHL-null) or 786-VHL, which was transfected to stably express wild-type VHL. VHL is an E3 ubiquitin ligase best known for its role in hypoxia signaling through transcription factors of the HIF family. 786-VHL cells exhibited decreased motility in two different assays (wound healing assay and Boyden chamber chemotaxis assay) compared with the parent 786-O cells. Compared with the 786-O cells, 786-VHL cells also exhibited decreased surface abundance of FGFR1 based on immunofluorescence and biotinylation assays and decreased activation of FGFR1 based on tyrosine phosphorylation of the receptor and phosphorylation of the downstream targets extracellular signal-regulated kinases 1 and 2 (ERK1/2). Pharmacological inhibitors of FGFR1 or expression of a dominant-negative FGFR1 demonstrated that activation of FGFR1 was responsible for the increased migratory potential of the 786-O cells. Human embryonic kidney (HEK) 293 cells were used to show that HIF was not involved in mediating the increased FGFR1 activity by VHL. RNA interference assays showed that the knockdown of VHL increased FGFR1 activity and abundance in the HEK293 cells and increased their motility. Because loss of VHL would be expected to stabilize HIF transcription factors, overexpression of HIF-2α in HEK293 was used to determine whether loss of VHL was acting through HIF to influence FGFR1. Overexpression of HIF-2α did not increase FGFR1 at the cell surface and, indeed, in the HIF-2α overexpressing cells, FGFR1 phosphorylation was modestly decreased. FGFR1 endocytosis was inhibited in 786-O cells, and in the 786-VHL cells, VHL was associated with FGFR1-containing endosomes early in the internalization process. However, VHL and FGFR1 occupied different microdomains of the endosome, suggesting that VHL and FGFR1 were not interacting directly. VHL interacts with the metastasis suppressor Nm23, and in the 786-VHL cells, these two proteins colocalized in FGFR1-containing endosomes. Furthermore, in the 786-O cells, Nm23 did not translocate to the cell periphery in response to application of bFGF, the ligand for FGFR1, and FGFR1 was not internalized in those cells. Thus, VHL appears to be involved in FGFR1 endocytosis and regulation of FGFR1 abundance and thus activation, which contributes to migratory potential and may contribute to the metastatic potential of tumors in VHL patients. T. Hsu, Y. Adereth, N. Kose, V. Dammai, Endocytic function of von Hippel-Lindau tumor suppressor protein regulates surface localization of fibroblast growth factor receptor 1 and cell motility. J. Biol. Chem. 281 , 12069-12080 (2006). [Abstract] [Full Text]