Characterization of the receptor tyrosine kinase EphB6 in breast cancer: Its potential as a diagnostic marker and its role in invasiveness
In the United States, breast cancer is the second leading cause of death in women. The identification of genes responsible for the progression of this disease could lead to improved diagnosis of breast cancer or even additional therapeutic targets. This study has identified EphB6, a kinase-deficient receptor tyrosine kinase, as a molecule that may be involved in the progression of breast cancer. The EphB6 transcript and protein were not detected in three out of four invasive breast cell lines, but were detected in all the non-invasive cell lines tested. Following the identification of the EphB6 promoter, I have shown that it is sensitive to methylation and has unique methylation patterns in breast carcinoma cell lines. The three cell lines where EphB6 was not detected have considerable levels of methylation within the EphB6 promoter, while those cell lines where EphB6 is more abundant have no methylation in this region. Growth of MDA-MB231 cells in media containing the methyltransferase inhibitor 5-Aza-2' deoxycytidine leads to partial re-expression of the transcript and protein. Two distinct functions for EphB6 are described in MCF-7 and MDA-MB-231 cells. Native MCF-7 cells have intermediate amounts of the EphB6 protein compared to MCF-10A and MDA-MB-231 cells. Overexpression of EphB6 in MCF-7 cells led to a decrease in doubling time (faster growth) and an increase in anchorage-independent growth, whereas silencing of the endogenous levels of EphB6 led to an increase in doubling time (slower growth) and a decrease in anchorage-independent growth. In MDA-MB-231 cells, on the other hand, overexpression of EphB6 had no effect on doubling time but was capable of significantly decreasing both anchorage-independent-growth and invasiveness. Following transfection with the full length EphB6 construct, these cells also showed a decrease in MMP7, MMP19, TIMP3 transcript levels and an increase in TIMP2 transcript levels. In this study, I have identified a new set of EphB6 interacting proteins by screening a human brain cDNA library using the yeast-two hybrid assay. These interactions were confirmed using a co-immunoprecipitation approach in mammalian cells. The confirmed interactors in mammalian cells are: aldolaseA, clusterin, dynactin, EphA2, EphB2, plekstrin homology domain-containing family B member 1 and TMEM25. The development of an methylation sensitive PCR assay capable of recognizing a methylated EphB6 promoter and the determination of its sensitivity in detecting circulating tumor cells are also described. This assay is capable of detecting as few as 5 tumor cells in 200 μl of blood or as few as 50 tumor cells in 5 mL of blood. These investigations point to the involvement of EphB6 in the invasiveness of breast carcinoma cells, demonstrate a set of proteins as EphB6 interactors and implicate EphB6 as a potential biomarker for breast cancer.
Fox, Brian P, "Characterization of the receptor tyrosine kinase EphB6 in breast cancer: Its potential as a diagnostic marker and its role in invasiveness" (2007). ETD Collection for Fordham University. AAI3301436.