| Breast cancer is a devastating disease, with an annual death rate of 43,000 in the United States. Twenty-five percent of breast tumors overexpress the ErbB2 receptor tyrosine kinase, which is an independent predictor of poor prognosis. The anti-ErbB2 monoclonal antibody, Herceptin, is routinely used to treat ErbB2-overexpressing metastatic breast cancer, with only 50% of patients responding. Our laboratory previously identified an interaction between ErbB2 and two alternatively spliced variants of the transforming growth factor alpha (TGF-alpha) precursor, variants 1 (V1) and 2 (V2), with exogenous expression of the TGF-alpha V2 precursor protein resulting in autonomous growth and formation of foci in CHO cells. In this study, we investigated the effects of pro-TGF-alpha wild type (WT) and variant precursor expression on the responsiveness of breast cancer cells to the growth inhibitory effects of Herceptin. Six breast cancer cell lines expressing different levels of the ErbB receptors were characterized for their endogenous levels of TGF-alpha WT and V2 mRNA, and for their growth inhibition by Herceptin, and the two seem to be inversely correlated. In the ErbB2-positive MCF7 cell line, exogenous expression of TGF-alpha V2, but not WT or V1, eliminated their growth-inhibition by Herceptin, while only V2 colocalized with ErbB2. Colocalization of V2 with ErbB2 was also observed in the SKBr3 cell line. The Herceptin-sensitive SKBr3 and MDA453 cell lines would not tolerate stable overexpression of TGF-alpha WT or variants, even when using an inducible system. We also investigated differences in ErbB2 internalization in response to Herceptin, and discovered that internalization was not observed in the Herceptin-sensitive SKBr3 and MDA453 cell lines, while significant internalization of ErbB2 was observed in the less sensitive T47D and MCF7 cell lines. The effect of Herceptin on the MAPK and PI3K pathways was also assessed. Strong induction of ERK1/2 was observed at later time points in the Herceptin-insensitive (MDA468) and growth-stimulated (MDA231) breast cancer cell lines, while AKT phosphorylation was unaffected. In SKBr3 and MDA453 cells, sporadic activation of ERK1/2 was observed across time points, but phosphorylation of AKT was not detected. These studies indicate the involvement of multiple factors in determining the responsiveness of breast cancer cells to Herceptin. |
