Abstract ErbB2 expression is observed in 25 - 30 % of invasive breast cancers and is associated with reduced response to chemotherapy, and shorter time to relapse and reduced overall survival of breast cancer patients. Malignant mammary epithelial cells that overexpress ErbB2 exhibit enhanced proliferation, invasion of mammary tissue and metastasis. The stability of overexpressed mature ErbB2 at the plasma membrane of mammary epithelial cells is critically dependent on its interaction with the chaperone protein, heat shock protein 90 (Hsp90). Thus, Hsp90 promotes the proliferation and survival of malignant mammary epithelial cells and interruption of this interaction is expected to inhibit the growth of ErbB2-overexpressing tumors. 17-AminoAllyl-Geldanamycin (17-AAG), an Hsp90 inhibitor, is a semi-synthetic ansamycin antibiotic currently in phase II clinical trials for breast cancer treatment (www.clinicaltrials.gov). Because it preferentially binds to Hsp90 of the chaperone complex in malignant mammary epithelial cells, treatment of ErbB2-overexpressing breast cancer with 17-AAG is expected to destabilize ErbB2 and inhibit tumor growth. While a number of Hsp90 client proteins have been identified, ErbB2 is amongst the most sensitive to Hsp90 inhibition. These facts provide strong rationale for using 17-AAG as a part of the targeted therapy against ErbB2-overexpressing breast cancer. However the cellular machinery responsible for 17-AAG induced degradation of ErbB2 is not completely understood. At present it is unclear whether internalization of ErbB2 occurs via Clathrin-dependent or -independent endocytosis. Furthermore it is unresolved if the degradation of ErbB2 is dependent upon proteasomal or lysosomal proteolysis. And although C-terminus of Hsp70 Interacting Protein (CHIP) and Cullin 5 were identified as E3 ligases responsible for the ubiquitination of ErbB2, it is unknown if these ligases are the sole E3s responsible for the ubiquitnation of ErbB2. Also vague is if the rerouting of ErbB2 from the recycling pathway to the degradative pathway in response to 17-AAG is exclusively dependent upon ubiquitnation. In this current work we attempt to bridge these critical gaps in our knowledge of the mechanisms of 17-AAG induced degradation of ErbB2 using an automated microscopy-based analysis to identify genes whose siRNA-dependent suppression reduces 17AAG-induced ErbB2 degradation. We expect that this work will identify additional targets for therapeutic intervention and potential biomarkers of response to breast cancer drug therapy with Hsp90 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1221. doi:1538-7445.AM2012-1221