Abstract While therapeutic strategies have improved, ∼500,000 women worldwide die from breast cancer each year. Many proteins have been implicated in breast cancer, including the HER family of receptors. Overexpression of epidermal growth factor receptor (EGFR) and one of its family members, HER2, each occur in ∼30% of breast cancers and correlates with poor clinical prognosis. Herceptin, an inhibitor of HER2, has been approved for treatment of breast cancer patients; however small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have not shown efficacy in breast cancer. To understand why breast cancers do not respond to EGFR tyrosine kinase inhibition, a panel of twenty breast cancer cell lines was characterized for EGFR expression. Thirteen of the twenty cell lines expressed EGFR; seven of which were resistant to EGFR TKIs. Unlike the response of HER2 positive breast cancers to Herceptin, increased expression of EGFR correlated with resistance to EGFR TKIs. One possible mechanism for resistance to EGFR TKIs is that EGFR expression is not required for growth. However, when EGFR expression was knocked down, cell growth was significantly decreased in four of the seven cell lines suggesting that EGFR expression is important for cell growth in a portion of EGFR TKI resistant breast cancers. A second mechanism for resistance to EGFR may be elevated HER2 and HER3 expression as observed in lung cancer. Conversely, in these cell lines, HER2 and HER3 expression levels did not correlate with response to EGFR TKIs. In addition, an EGFR family kinase inhibitor also was unable to abrogate the growth of these cells suggesting that HER2 expression and kinase activity was not a mediator of cell growth in the presence of EGFR TKIs. EGFR is known to localize within many sub-cellular regions including the nucleus, mitochondria, endosomes, and plasma membrane. Localization of EGFR to membrane microdomains (lipid rafts) is known to modulate EGFR signaling. Therefore, a third potential mechanism for EGFR TKI resistance may be localization of EGFR within lipid rafts. Using biochemical raft isolation and immunofluorescence, EGFR was shown to localize to lipid rafts in all four EGFR-dependent, EGFR TKI resistant breast cancer cell lines. To determine if this localization mediates resistance to EGFR TKIs, cholesterol (a main structural component of lipid rafts) was depleted using cholesterol biosynthesis inhibitors. While such inhibitors had no effect on cell growth alone, combinatorial depletion of lipid rafts and inhibition of EGFR-kinase activity resulted in decreased cell growth. Further, the interaction between cholesterol biosynthesis inhibitors and EGFR inhibitors was synergistic. Thus, we have discovered that EGFR expression and localization to lipid rafts correlate with EGFR TKI resistance in breast cancer, and that depletion of lipid rafts can overcome such resistance in EGFR expression-dependent breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 269.