Abstract Epidermal growth factor receptor (EGFR) is frequently overexpressed or aberrantly activated in various malignancies. As a result, EGFR is considered an important target in cancer therapeutics. Strategies of EGFR targeting include blockade of its activation, inhibition of downstream signaling or induction of EGFR downregulation and/or degradation. Histone deacetylases (HDACs) inhibitors were previously shown to deplete EGFR through numerous mechanisms varying from suppression of EGFR transcription, attenuation of its mRNA stability and disruption of chaperone activity of HSP90. In this work, we are studying alteration of EGFR vesicular trafficking as another mechanism that may be involved in HDAC inhibitors-induced EGFR depletion. Cytotoxic and EGFR-degrading effects of vorinostat, a pan-HDAC inhibitor, were investigated in the EGFR-overexpressing cell lines, DU-145 (prostate cancer cells) and MDA-MB-231 (breast cancer cells). Using different experimental designs, dose- and time- effects of vorinostat were studied in both cell lines. Proliferation assay, annexin V staining, PARP and caspase 7 cleavage were used to evaluate overall cellular toxicity. Effects on total EGFR protein level and relevant ERK phosphorylation were also investigated by immunoblotting. Moreover, accumulation of acetyl-α-tubulin was verified through immunoblotting and immunofluorescence imaging. Finally, serum-starved vorinostat-treated cells were stimulated with EGF in order to elucidate effect of vorinostat on EGF-induced EGFR degradation, an endocytic trafficking-dependent process. As presented in this study, vorinostat exerted potent cytotoxic effects on DU-145 and MDA-MB-231 cells manifested by a dose-dependent decrease in proliferation and increase in percentage of apoptotic cells. In addition, vorinostat caused a dose-dependent cleavage of both PARP and caspase 7, two markers of apoptotic cascade activation. Furthermore, vorinostat elicited a dose- and a time-dependent depletion of EGFR total protein in both cell lines with a corresponding decrease in ERK activation. Add to that, vorinostat caused accumulation of acetyl-α-tubulin and promoted EGFR degradation upon EGF stimulation denoting accelerated endocytic trafficking. In conclusion, vorinostat demonstrated a potent anti-tumor activity against EGFR-overexpressing cell lines. Such activity was accompanied by EGFR depletion that may be contributed in part by vorinostat-induced deregulated vesicular trafficking. Citation Format: Asmaa E. Elkenawi, John K. Cowell. Involvement of endocytic pathway in vorinostat-induced EGFR degradation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1029. doi:10.1158/1538-7445.AM2013-1029