key mediators of mammalian cell apoptosis (i.e. Caspase-3), which consequently led to cell death. This hypothesis was supported by the fact that both AG 1478 and SP 600125 blocked G3 enhanced expression of Caspase-3 and cell apoptosis while PD 98059 did not. Reduction in expression of versican and versican G3 domain by anti-versican siRNA and G3-39UTR construct significantly reduced G3 enhanced effects on cell apoptosis induced by chemotherapeutics and confirmed that versican G3-expressing breast cancer cells promoted cell apoptosis induced by chemo- therapeutics through G3 dependant mechanisms. An interesting observation of our study is the apparent dual roles of versican G3 domain in modulating breast cancer cell resistance to chemotherapy and EGFR targeting therapy. EGFR signaling appears crucial to the sensitivity or resistance of versican expressing breast cancer cells to chemotherapy. The apoptotic effects of chemotherapeutics on these cells depend on the activation and balance of EGFR signaling and its effects down- stream. Certain chemicals such as Doxorubicin and Epirubicin activate versican G3-expressing cells’ endogenous EGFR/ERK/ GSK-3b (S9P) signaling promoting chemical resistance while others chemicals appear to enhance these cells’ sensitivity to chemotherapy through increased expression of EGFR/JNK signaling and subsequent effects on apoptosis. Our study has identified a key EGFR down stream proteins, GSK-3b (S9P) that appears critically important as a regulatory check-point in the balance of apoptosis and anti-apoptosis (Fig. 8a). Results demonstrated that G3-expressing cells enhanced GSK-3b (S9P) expression when treated with a serum free medium, Doxorubicin or Epirubicin; they also expressed decreased GSK-3b (S9P) and activated pSAPK/JNK when treated with C2-ceramide or Docetaxel. The pERK expression remained at high levels when these cells were treated with different chemicals (Fig. 8b). The increased expression of GSK-3b (S9P) inhibits the expression of pSAPK/JNK, enhancing G3-cell survival. Chemicals such as C2- ceramide and Docetaxel reduce G3-cells expression of GSK-3b (S9P), which alleviates inhibition of pSAPK/JNK activity encouraging the survival system favor cell apoptosis. On the other hand, expression of pSAPK/JNK may also inhibit expression of GSK-3b (S9P), and enhance cell apoptosis (Fig. 8a). Selective JNK inhibitor SP 600125 enhanced G3 cells expression of GSK-3b (S9P) when treated with serum free or C2-ceramide medium suggesting that expression of pSAPK/JNK inhibits expression of GSK-3b (S9P), a pathway leading to cell apoptosis (Fig. 2c, Fig. 4c). A model based on this study of versican G3 modulating breast cancer cell apoptosis in response to chemotherapy and EGFR targeting therapy is shown in Fig. 8a. Although a large number of new agents targeting the EGFR pathways are being tested and have shown certain efficacy through greater survival in clinical and pre-clinical models, it remains unclear as to how combination EGFR therapy with chemotherapy will impact breast cancer patients. Literature is varied with some clinical trials demonstrating that EGFR targeting agents synergize with cytotoxic chemotherapies [59,60], while others have failed to show any survival advantage of combination over single-agent therapy in advanced breast cancer patients [58]. These varied effects could potentially be explained by the interaction of EGFR targeting and chemotherapeutics on EGFR signaling and effects of cell cycle entry as well as apoptosis. We have identified that key downstream pathway EGFR signaling proteins such as GSK-3b (S9P) may appear to play a role in how cells respond to treatment. Ongoing study on the mechanisms of cancer invasiveness and cellular signaling will further advance our knowledge on how extracellular matrix and cellular factors such as versican and EGFR signaling impact patient outcomes and can be modulated in response to treatment. Versican G3 Modulates Breast Cancer Cell Apoptosis Our study has clinical relevance and motivates additional pre- clinical study towards the development of new clinical agents that can be tested in the treatment of breast cancer. Our mechanistic study on EGFR related signaling demonstrates that chemother- apeutic drugs can have varying effects on signaling that may either positively or negatively impact cancer cell survival through mechanisms that influence apoptosis. Although there are several clinical agents that broadly target EGFR, downstream effects appear to critically influence cellular apoptosis and the develop- ment of more specific drugs that can modulate downstream targets such as GSK-3b (S9P) expression as demonstrated by this study is desirable. The field of breast cancer chemotherapeutics is also evolving with recent interest in neoadjuvant approaches to treatment which serves as a valuable research platform to test patient specific primary tumor response to systemic therapies prior to surgery in early disease thereby helping to refine patient selection for therapy limiting treatment specifically to those that are most likely to benefit from systemic agents many of which possess significant toxicity profiles. Supporting Information Figure S1 Silencing versican expression using siRNA. a) MT-1, MDA-MB-231, MCF-7, MDA-MB-468 cell lysates were subjected to immunoblotting and RT-PCR. b) MT-1 cells were stably transfected with anti-versican siRNA. Versican V1 expres- sion was analyzed by immunoblot and RT-PCR. c) The expression of pERK, ERK, pSAPK/JNK, SAPK/JNK of vector-expressing and anti-versican siRNA- expressing MT-1 cells was analyzed by immunoblotting, after treatment with 2 mM Docetaxel, 8 mM Doxorubicin, or 8 mM Epirubicin for 6 hours. d) WST-1 assays were used to test cell viability of vector-, versican G3-transfected, and anti-versican siRNA- transfected MT-1 cells, which were treated with 40 mM C2-ceramide, 2 mM Docetaxel, 8 mM Doxorubicin, or 10 mM Epirubicin for 24 hours. (TIF) Figure S2 Reduction of versican G3’s function using versican G3-UTR. a) Versican G3 domain was linked with or without the 39UTR of versican, producing G3 and G3-UTR constructs. b) Cell lysates prepared from 66c14 cells stably transfected with versican G3 and G3-UTR construct were subjected to immunoblotting. c) Vector-transfected, G3-UTR- transfected, and G3- transfected 66c14 cells (16105) were inoculated in 12 well culture dishes. After culture for 12 hours, all samples were treated with 40 mM C2-ceramide, 2 mM Docetaxel, 8 mM Doxorubicin, or 10 mM Epirubicin for 24 hours. Cell viability was analyzed by light microscopy. d) Vector, G3- UTR, and G3 transfected 66c14 cells (16104) were inoculated and cultured in 10% FBS/DMEM medium in 96 well culture dishes for 12 hours. After cell attachment, cells were treated with 40 mM, C2-ceramide, 2 mM Docetaxel, 8 mM Doxorubicin, or 10 mM Epirubicin for 24 hours. Cell viability was analyzed by WST-1 assays. Compared with vector control group, n = 6, * p,0.05, **p,0.01, analyzed with t-test. (TIF) Acknowledgments The authors wish to thank Ms. Gisele Knowles (Cytometry and Microscopy Core Facility of Sunnybrook Health Sciences Centre) for her assistance with the cell cycle assays, and Miss Sarah Davies (Division of Orthopaedics, Sunnybrook Health Sciences Centre) for her assistance with the manuscript. The mouse mammary tumor cell lines 67NR, 66c14, 4T07, 4T1 were kindly provided by Dr. Fred Miller (Karmanos Cancer Institute, Detroit, MI, USA). PLoS ONE | www.plosone.org 13 November 2011 | Volume 6 | Issue 11 | e26396