Abstract #3421: Her-2 over expression induces metabolic transformation resulting in insulin-independence in human breast cancer cells

Normal human mammary epithelial cells have an absolute requirement for both insulin and EGF for growth in vitro in serum-free media. We previously demonstrated that over expression of a number of breast cancer oncogenes can transform MCF-10A cells to an insulin-independent phenotype. Additionally, we found that that most human breast cancer cell lines are also insulin-independent for growth. In this study, we investigated the mechanism by which HER-2 over expression transforms MCF-10A cells to an insulin-independent phenotype. We analyzed the effects of various concentrations of insulin and/or IGF-1 on the proliferation of MCF-10A cells and demonstrated that the effects of insulin were independent from, and did not substitute for IGF-1, which suggests that oncogene over expression drives a true insulin-independence phenotype, and not IGF-1 independence. To test this hypothesis, we examined metabolic functions of insulin signaling in insulin-dependent and insulin independent cells. HER-2 over expression resulted in glucose uptake in the absence of insulin at a rate equal to insulin-induced glucose uptake in non-transduced cells. Immunoblot analysis for GLUT4 showed that it was constitutively expressed at the plasma membrane in HER-2 over expressing cells. Previously, we identified two genes likely to play a role in the transformation to insulin-independence: VAMP8 and PHGDH. VAMP8 is a vesicle-associated membrane protein required for GLUT4 plasma membrane endocytosis. HER-2 over expression in MCF-10A cells significantly down-regulated VAMP8 gene expression, and inhibition of HER-2 signaling caused a rapid up-regulation of VAMP8. Analysis of VAMP8 mRNA in a panel of 8 cancer cell lines showed that VAMP8 was markedly down-regulated in all of them compared to MCF-10A cells. We also observed that over expression of VAMP8 partially restored insulin-dependence to the HER-2 over expressing MCF-10A cells. PHGDH catalyzes a rate-limiting reaction that commits an intermediate of the glycolytic pathway to L-serine biosynthesis, which is a substrate for biosynthesis of lipids, proteins, and nucleotides. HER-2 over expression in MCF-10A cells significantly up-regulated PHGDH gene expression and inhibition of HER-2 signaling caused down-regulation of PHGDH. Based on these observations, we hypothesize that VAMP8 and PHGDH play complimentary roles in the metabolic transformation of HER-2 over expressing cells. Considering that we observed expression changes for VAMP8 and PHGDH in other breast cancer cells and oncogene transformed MCF-10A cells, the transition to insulin-independence and the metabolic transformation observed in insulin-independent cells may point to a metabolic alteration common among breast cancer cells.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3421.