Cancer Biology and Signal Transduction Nuclear Epidermal Growth Factor Receptor Is a Functional Molecular Target in Triple-Negative Breast Cancer

Triple-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 correlatedwith 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 tumormodels,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. Introduction Approximately 15% to 20% of all breast cancers lack expression of the estrogen receptor, progesterone receptor, and HER2, and are thus considered to be triplenegative breast cancers (TNBC; refs. 1, 2). Although a high percentage of patients with TNBC initially respond to conventional chemotherapy, they tend to have a higher rate of relapse and worse prognosis as compared with other breast cancer subtypes (1, 2). In efforts to identify new molecular targets in TNBC, various groups have performed gene expression profiling studies and identified that the EGF receptor (EGFR) is commonly overexpressed (3–6). Although inhibition of EGFR activity has yieldedmodest clinical success inTNBC, substantial gains in clinical response rates have not been achieved (7, 8). Thus, improving the efficacy of anti-EGFR therapy in TNBC is imperative. Classically, EGFR functions as a plasma membranebound receptor tyrosine kinase that initiates growth and survival signals (9). However, studies over the last 15 years have identified that EGFR can be localized and function from intracellular organelles, one of which includes the nucleus (10, 11). Within the nucleus, EGFR can function as a cotranscription factor to regulate genes involved in tumor progression (10, 11), in addition to functioning as a nuclear kinase to enhance DNA replication and repair (12–14). These nuclear functions have been linked to three parameters of tumor biology: (i) inverse correlation with overall survival innumerous cancers (15–20), (ii) resistance to therapeutic agents including radiation (12, 21–24), chemotherapy (12, 13, 24), and anti-EGFR therapies gefitinib (25) and cetuximab (26), and (iii) enhanced tumor growth (27, 28). These findings suggest that tumors rely on two distinct compartments of EGFR signaling to sustain their oncogenicphenotype: (i) classicalmembrane-boundEGFR signaling, and (ii) nuclear EGFR (nEGFR) signaling. Previous work from our laboratory has identified that non–small cell lung cancer (NSCLC) cells that have acquired resistance to cetuximab express increased nEGFR and Src Family Kinase (SFK) activity (26, 29). SFK inhibition blocked nEGFR translocation in cetuximab-resistant Authors' Affiliations: Departments of Human Oncology and Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Department of Medicine, University of Wisconsin Carbone Cancer Center, Madison, Wisconsin; and Division of Hematology/Oncology, Department of Medicine, University of Chicago, Chicago, Illinois Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Corresponding Author: Deric L. Wheeler, University of Wisconsin, 1111 Highland Ave, WIMR 3159, Madison, WI 53705. Phone: 608-262-7837; Fax: 608-263-9947; E-mail: dlwheeler@wisc.edu doi: 10.1158/1535-7163.MCT-13-1021 2014 American Association for Cancer Research. Molecular Cancer Therapeutics Mol Cancer Ther; 13(5) May 2014 1356 on January 5, 2018. © 2014 American Association for Cancer Research. mct.aacrjournals. org Downloaded from Published OnlineFirst March 14, 2014; DOI: 10.1158/1535-7163.MCT-13-1021