Exploring pVHL Regulation of α5 Integrin by the Endocytic Pathway in Relation to Kidney Cancers Resulting From VHL Disease

PURPOSE The Von-Hippel Lindau (VHL) gene encodes for a tumor suppressor protein, pVHL, which is responsible for the regulation of a family cancer syndrome. Mutations in pVHL render the protein non-functional which can lead to VHL disease and the growth of benign and malignant tumors. pVHL is part of the ubiquitin E3 ligase complex which attaches ubiquitin to target substrates. Alpha 5 (α5) integrin levels have shown to decrease in the presence of functional pVHL, however, the mechanism by which the process occurs is unclear. It is hypothesized that mutations in pVHL could inhibit integrin degradation and allow some integrins to recycle to the cell membrane leading to tumor metastasis. METHODS Endocytic pathway proteins Rab5a and Rablla were used as vesicle markers to characterize the movement of integrins on the pathway. The 786-O cell line comes from a patient that had VHL disease. 786-O VHL– cells are the parental line that do not have a functional VHL gene. 786-O VHL+ cells are a cell line in which a functional copy of the VHL gene was reintroduced as a plasmid to the parental cell line. The 786-O cell lines will be immunostained to determine changes in abundance and location of vesicles containing these proteins. RESULTS Immunostaining revealed that Rab11a has been shown to localize closer to the surface of cancerous 786-O cells in comparison to non-cancerous 786-O cells. Immunostaining of the Rab5a dominant active form Q79L was shown to form large vesicles in VHL+ cells while the dominant negative Rab5a S34N was shown to form smaller vesicles in VHL+ cells. CONCLUSION Rab11a may play a role in the mechanism behind tumor development in VHL disease because of the distinct organization of the proteins to the cell surface in cancerous 786-O cells. Rab5a dominant active and dominant negative forms have been shown to affect the formation of vesicles between VHL– and VHL+ 786-O cells. Immunostaining and immunoprecipitation assays will be conducted to determine whether Rab11a and Rab5a colocalize with α5 integrin.