Versican G3 Domain Modulates Breast Cancer Cell Apoptosis: A Mechanism for Breast Cancer Cell Response to Chemotherapy and EGFR Therapy William Weidong Du1,2, Burton B. Yang2,3, Bing L. Yang1,2, Zhaoqun Deng2,3, Ling Fang2,3, Sze Wan Shan2,3, Zina Jeyapalan2,3, Yaou Zhang 4 , Arun Seth2,3, Albert J. Yee1* 1 Department of Surgery, Sunnybrook Health Sciences Centre and Centre for the Study of Bone Metastasis, Odette Cancer Centre, University of Toronto, Toronto, Canada, 2 Sunnybrook Research Institute, Toronto, Canada, 3 Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada, 4 Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen, China Abstract Overexpression of EGFR and versican has been reported in association with breast cancers. Considered oncogenic, these molecules may be attractive therapeutic targets. Possessing anti-apoptotic and drug resistant properties, overexpression of these molecules is accompanied by selective sensitization to the process of apoptosis. In this study, we exogenously expressed a versican G3 construct in breast cancer cell lines and analyzed the effects of G3 on cell viability in fetal bovine serum free conditioned media and evaluated the effects of apoptotic agent C2-ceramide, and chemotherapeutic agents including Docetaxel, Doxorubicin, and Epirubicin. Versican G3 domain enhanced tumor cell resistance to apoptosis when cultured in serum free medium, Doxorubicin, or Epirubicin by up-regulating pERK and GSK-3b (S9P). However, it could be prevented by selective EGFR inhibitor AG 1478 and selective MEK inhibitor PD 98059. Both AG 1478 and PD 98059 enhanced expression of pSAPK/JNK, while selective JNK inhibitor SP 600125 enhanced expression of GSK-3b (S9P). Versican G3 promoted cell apoptosis induced by C2-ceramide or Docetaxel by enhancing expression of pSAPK/JNK and decreasing expression of GSK-3b (S9P), an observation blocked by AG 1478 or SP 6000125. Inhibition of endogenous versican expression by siRNA or reduction of versican G3’s expression by linking G3 with 39UTR prevented G3 modulated cell apoptosis. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents may in part explain a potential mechanism for breast cancer cell resistance to chemotherapy and EGFR therapy. The apoptotic effects of chemotherapeutics depend upon the activation and balance of down stream signals in the EGFR pathway. GSK-3b (S9P) appears to function as a key checkpoint in this balance of apoptosis and anti-apoptosis. Investigation and potential consideration of targeting GSK-3b (S9P) merits further study. Citation: Du WW, Yang BB, Yang BL, Deng Z, Fang L, et al. (2011) Versican G3 Domain Modulates Breast Cancer Cell Apoptosis: A Mechanism for Breast Cancer Cell Response to Chemotherapy and EGFR Therapy. PLoS ONE 6(11): e26396. doi:10.1371/journal.pone.0026396 Editor: Ilya Ulasov, University of Chicago, United States of America Received March 7, 2011; Accepted September 26, 2011; Published November 9, 2011 Copyright: 2011 Du et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from National Sciences and Engineering Research Council of Canada (227937-01) to Dr. Yang who is the recipient of a Career Investigator Award (CI5958) from the Heart and Stroke Foundation of Ontario. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: albert.yee@sunnybrook.ca Introduction Chemotherapeutic drugs exhibit varied selectivity for tumour cells dependent on cell origins and are capable of inducing tumour cell death [1,2]. Additionally many of the commonly used chemotherapeutic drugs also appear to influence cellular signaling pathways that induce apoptosis in susceptible cancer cells [1,3]. Apoptosis seems to be one of the major physiologic safeguards against uncontrolled proliferation [4]. Growth and apoptosis are two diametrically opposed biological processes that ensure that multi-cellular organisms can cope with the normal physiologic yet mutagenic environment that generates millions of potential cancer cells every day [5]. With its effects on tumor cell proliferation and migration, versican has been shown to increase the resistance of cancer cells to apoptosis [6]. Our previous research demonstrated that versican appeared to confer cell resistance to apoptosis following treatment with low serum medium or hydrogen peroxide [7,8]. The combination of selective apoptotic resistance and sensitivity has been reported in overexpression of the V1 versican isoform [7]; the intimate relationship between proliferation and apoptosis cannot be separated and cancer cells often express either hypersensitivity or resistance to apoptosis that is dependent upon tissue conditions. As a member of the large aggregating chondroitin sulfate proteoglycan family, versican is structurally composed of a N- terminal G1 domain, a glycosaminoglycan (GAG) attachment region, and a C terminus (or G3) selectin-like domain [9,10]. The G3 domain interacts with different ECM proteins [11] and binds to certain cell surface proteins including epidermal growth factor receptor (EGFR) [12,13]. Extracellular versican has been observed to be elevated in a variety of human tumors including breast carcinoma [14,15,16]. High expression has been observed in the interstitial tissues at the invasive margins of breast carcinoma and appears prognositic being predictive of cancer relapse in patients PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e26396