Epidermal growth factor receptor (EGFR)–related protein inhibits multiple members of the EGFR family in colon and breast cancer cells Hu Xu, Yingjie Yu, Dorota Marciniak, Arun K. Rishi, Fazlul H. Sarkar, Omer Kucuk, and Adhip P.N. Majumdar Veterans Affairs Medical Center, Karmanos Cancer Institute, Departments of Internal Medicine and Pathology, Wayne State University, Detroit, Michigan Abstract Inactivation of epidermal growth factor receptor (EGFR) family members represents a promising strategy for the development of selective therapies against epithelial cancers. Current anti-EGFR therapies, such as cetuximab (Erbitux), gefitinib (Iressa), or trastuzumab (Herceptin), target EGFR or HER-2 but not both. Because solid tumors express different EGFRs, identification of inhibitor(s) targeting multiple EGFR family members may provide a therapeutic benefit to a broader patient population. We have identified a natural inhibitor of EGFRs called EGFR- related protein (ERRP), a 53 to 55 kDa protein that is present in most, if not all, normal human epithelial cells. The growth of colon (HCT-116, Caco2, and HT-29) and breast (MDA-MB-468 and SKBR-3) cancer cells expressing varying levels of EGFR, HER-2, and/or HER-4 was inhibited by recombinant ERRP in a dose-dependent manner. In contrast, ERRP caused no inhibition of growth of normal mouse fibroblast cell lines (NIH-3T3, NIH-3T3/PT-67), and the growth of nontransformed rat small intestinal IEC-6 cells expressing relatively low levels of EGFRs was inhibited only at high doses of ERRP. Transforming growth factor-A or heparin-binding epidermal growth factor – induced activation of EGFR and HER-2 was inhibited by ERRP in colon and breast cancer cells expressing high levels of EGFR or HER-2. In contrast, cetuximab inhibited the growth- and ligand-induced activation of EGFR in cell lines expressing high levels of EGFR, whereas trastu- zumab was effective only in HER-2 – overexpressing cells. ERRP and trastuzumab, but not cetuximab, attenuated heregulin-A – induced activation of colon and breast cancer cells that expressed high levels of HER-2. Further- more, ERRP, but not cetuximab or trastuzumab, signifi- cantly induced apoptosis of colon and breast cancer cells. None of these agents induced apoptosis of either NIH-3T3 mouse fibroblast or normal rat small intestinal IEC cells. Our results suggest that ERRP is an effective pan-erbB inhibitor and, thus, may be a potential therapeutic agent for a wide variety of epithelial cancers expressing different levels and subclasses of EGFRs. [Mol Cancer Ther 2005;4(3):435 – 42] Introduction Members of the receptor tyrosine kinase family, which include epidermal growth factor receptor (EGFR), ErbB-2/ HER-2, ErbB-3/HER-3, and ErbB-4/HER-4, are frequently implicated in experimental models of epithelial cell neoplasia in animals as well as in human cancers (1 – 4). Interference with growth factor receptor activation and/or with intracellular growth factor – activated signal transduc- tion pathways represents a promising strategy for the development of novel and selective anticancer therapies (5 – 7). A large body of experimental evidence supports a key role for EGFR and HER-2 in a wide variety of human epithelial cancers (1, 3, 5, 7). Overexpression and/or activation of EGFR have been associated with resistance to cytotoxic drugs, and are generally considered indicators of poor prognosis (7 – 10). Indeed, small molecule inhibitors of EGFR, such as gefitinib (Iressa) and erlotinib (Tarceva, OSI-774), have progressed to large-scale randomized clinical trials, but with limited success (5). With respect to gefitinib, it has recently been approved for clinical use and showed that it is effective only in a subgroup of non – small cell lung cancer patients who have specific mutations in the EGFR gene (11, 12). Monoclonal antibodies to EGFR (cetuximab/IMC-C225/ Erbitux) and HER-2 (trastuzumab/Herceptin) have been approved by the Federal Drug Administration for treat- ment of tumors that express high levels of EGFR and HER- 2, respectively. Although Erbitux and Herceptin treatments showed signs of success, failure in some patients may partly be due to coexpression of multiple EGFR family members, which may lead to an enhanced transforming potential and worsened prognosis (7 – 10, 13, 14). Therefore, identification of inhibitor(s), targeting multiple members of the EGFR family, may provide a greater therapeutic benefit to a broader range of patient population. To this end, we investigated the effectiveness of EGFR- related peptide (ERRP) as a pan-erbB inhibitor that targets multiple members of the EGFR family. ERRP, a recently isolated negative regulator of EGFR, is a 53 to 55 kDa Received 10/12/04; revised 12/29/04; accepted 1/5/05. Grant support: NIH grant RO1 AG14343 and Department of Veterans Affairs VA Merit Review grant (A.P.N. Majumdar). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Adhip P.N. Majumdar, John D. Dingell Veterans Affairs Medical Center, 4646 John R, Room B-4238, Detroit, MI 48201. Phone: 313-576-4460; Fax: 313-576-111. E-mail: a.majumdar@wayne.edu Copyright C 2005 American Association for Cancer Research. Molecular Cancer Therapeutics 435 Mol Cancer Ther 2005;4(3). March 2005 Research. on January 22, 2022. © 2005 American Association for Cancer mct.aacrjournals.org Downloaded from