The regulation of elevated EphA2 protein levels in metastatic cancer cells: Roles for ligand binding, protein degradation, and c -Cbl
Abstract
A number of studies have shown that elevated protein tyrosine kinase activity is required for cancer metastasis. Knowledge of the specific tyrosine kinases that promote metastatic behaviors is therefore critical to understanding and treating cancer. One tyrosine kinase that is increasingly recognized to be a critical regulator of metastasis is the EphA2 receptor tyrosine kinase. EphA2 protein levels in human and canine models of prostate cancer were elevated in the most aggressive cancer cells. EphA2 immunoreactivity in vivo also was significantly greater in human prostate cancers as compared with benign prostate epithelium. Despite its elevated levels in cancer, the EphA2 on malignant cells demonstrated lower levels of ligand binding and tyrosine phosphorylation than the EphA2 on non-transformed epithelial cells. Metastatic cancer cells often have weak cell-cell adhesions, which prevent EphA2 from binding its cell-bound ligands on the surface of neighboring cells. Thus, we hypothesized that decreased EphA2-ligand binding may elevate EphA2 protein levels in metastatic cancer cells by preventing EphA2 protein degradation. Decreased EphA2-ligand binding is important because conditions that restore or mimic ligand binding decrease metastatic cell growth and invasiveness. EphA2 on metastatic cancer cells was stimulated, which caused the rapid internalization of EphA2 into endosomal vesicles as detected by immunofluorescence and electron microscopies. Western blot and pulse-chase analyses revealed a rapid and dramatic decrease in EphA2 protein levels following EphA2 stimulation. EphA2 stimulation also promoted an association between EphA2 and the c-Cbl adaptor protein as detected by co-immunoprecipitation and co-localization studies. These results demonstrate that stimulated EphA2 is internalized and degraded, and provide insight into the basic biochemical mechanism of stimulation induced EphA2 protein degradation. This information provides insight into the causes of elevated EphA2 protein levels in metastatic cancer cells and may assist in the design of therapeutic approaches to target the large number of metastatic human cancer cells that have elevated levels of EphA2 protein. EphA2 protein levels are also elevated following transformation of epithelial cells by oncogenic Ras. The mechanism through which Ras transformation increases EphA2 protein levels is not known but appears to be dependent upon the geranylgeranylation of certain cellular proteins.
Degree
Ph.D.
Advisors
Kinch, Purdue University.
Subject Area
Cellular biology|Oncology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.