ARTICLE ABSTRACTTriple-negative breast cancer (TNBC) is a subclass of breast cancers (i.e., estrogen receptor–negative, progesterone receptor–negative, and HER2-negative) that have poor prognosis and very few identified molecular targets. Strikingly, a high percentage of TNBCs overexpresses the EGF receptor (EGFR), yet EGFR inhibition has yielded little clinical benefit. Over the last decade, advances in EGFR biology have established that EGFR functions in two distinct signaling pathways: (i) classical membrane-bound signaling and (ii) nuclear signaling. Previous studies have demonstrated that nuclear EGFR (nEGFR) can enhance resistance to anti-EGFR therapies and is correlated with poor overall survival in breast cancer. On the basis of these findings, we hypothesized that nEGFR may promote intrinsic resistance to cetuximab in TNBC. To examine this question, a battery of TNBC cell lines and human tumors were screened and found to express nEGFR. Knockdown of EGFR expression demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab. Furthermore, Src Family Kinases (SFKs) influenced nEGFR translocation in TNBC cell lines and in vivo tumor models, where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab. Collectively, these data suggest that targeting both the nEGFR signaling pathway, through the inhibition of its nuclear transport, and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of patients with TNBC. Mol Cancer Ther; 13(5); 1356–68. ©2014 AACR.