Abstract Understanding the molecular etiology and heterogeneity of disease has a direct effect on cancer therapeutics. To identify novel molecular changes associated with breast cancer progression, we conducted phosphoproteomics of the MCF10AT model comprising isogenic, ErbB2- and ErbB3-positive, xenograft-derived cell lines that mimic different stages of breast cancer. Using in vitro animal model and clinical breast samples, our study revealed a marked reduction of epidermal growth factor receptor (EGFR) expression with breast cancer progression. Such diminution of EGFR expression was associated with increased resistance to Gefitinib/Iressa in vitro. Fluorescence in situ hybridization showed that loss of EGFR gene copy number was one of the key mechanisms behind the low/null expression of EGFR in clinical breast tumors. Statistical analysis on the immunohistochemistry data of EGFR expression from 93 matched normal and breast tumor samples showed that (a) diminished EGFR expression could be detected as early as in the preneoplastic lesion (ductal carcinoma in situ) and this culminated in invasive carcinomas; (b) EGFR expression levels could distinguish between normal tissue versus carcinoma in situ and invasive carcinoma with high statistical significance (P < 0.001, n = 81). However, no significant correlation of EGFR expression with disease-free survival and overall survival was observed. This is the first time EGFR expression has been tracked meaningfully and developmentally from the normal condition through disease progression using in vitro, xenograft, and matched normal and tumor samples. Thus, our study provides a new insight into the role of EGFR in breast cancer development. Although no value of EGFR expression in prognosis was found, our findings are likely to have implications in the design of clinical trials targeting the EGFR family of proteins in breast cancer. [Mol Cancer Ther 2007;6(11):2828–42]