Herceptin-resistance in breast cancer cells: a proteomic study.

Abstract

HER-2 is a cell membrane protein that belongs to the ErbB family of receptor tyrosine kinases (HER-1, HER-2, HER-3, HER-4). The over-expression of HER-2, which results in the 25-30% of breast cancer patients, is considered a predictive and prognostic marker for breast cancer malignancy and invasiveness and makes HER-2 an excellent therapeutic target. In the last years new therapeutic strategies have been improved in order to better deal tumor diseases an to minimize collateral effects due to classic chemotherapy in patients. In this way, a new approach was the somministration of humanized antibodies directed against tumor-associated molecular targets. Among these ones Herceptin, an anti-neoplastic humanized monoclonal antibody (Herceptin®, Roche, CH), has been shown to be active against breast cancer cells over-expressing HER-2 receptor. Unfortunately the initial response to Herceptin-based regimens is followed by drug-resistance occurring within 1 year in the majority of patients. Thus it becomes critical to understand the molecular mechanisms underlying primary or acquired Herceptin resistance in order to improve the survival of breast cancer patients whose tumors overexpress HER2. On these bases, we focused our attention to investigate the effects caused by Herceptin treatment on SK-BR-3 breast cancer cells, which overexpress HER-2. To obtain drug-resistant cells, SK-BR-3 were treated with 10 µgr/ml of drug, until cells growth rate became synchronous compared to untreated parental cells. Parental and resistant cells were then properly collected and subjected to cell-lysis to obtain protein extracts, while respective conditioned media were harvested to perform analyses of cell-released gelatinolic activities, such as MMP-2 and MMP-9. These investigations are justified by previous observation of our group, showing a certain degree of correlation between gelatinase expression and HER-2 overexpression in vivo [1]. Protein extracts were subjected to IPG-2D electrophoresis, firstly to evaluate proteomic profiles of both resistant and parental cells, secondly to detect differentially expressed proteins in two above mentioned experimental conditions and thirdly to identify new Herceptin-resistance related proteins by means of MALDI-TOF mass spectrometry. The results so far obtained, have shown that prolonged Herceptin treatment induces dramatic changes in malignant resistant cells, such as rearrangement of cytoskeletal structures, evident effects on expression of proteins involved in cytoskeleyton remodelling, metabolic processes, stress responses and cell cycle and proliferation control. We suggest that present data may contribute significantly to the knowledge of the mechanisms underlying Herceptin drug resistance on breast cancer cells. [1]La Rocca, G., Pucci-Minafra, I., Marrazzo, A., Taormina, P. & Minafra, S. 2004. British Journal of Cancer, 90: 1414-1421.