enhance results that will occur through a better mechanistic understanding of the signaling pathway [61]. A phase II study of using Erlotinib and Gemcitabine demonstrated lower than anticipated effects on patients with metastatic breast cancer [58] while a Phase I study applying Gefitinib and Docetaxel demonstrated encouraging anti-tumor activity as a first-line chemotherapy in metastatic breast cancer [60]. Abnormal expression of proteoglycans (PGs), such as versican, in cancer and stromal cells may serve as a biomarker for tumor progression and patient survival [62]. Enhanced understanding of the regulation and involvement of versican in cancer may offer a novel approach to cancer therapy by targeting the tumor microenvironment [62]. The effect of signaling pathways on versican synthesis can be reversed following treatment with various tyrosine kinase inhibitors [63]. The tyrosine kinase inhibitor genistein can block versican expression induced by growth factors in malignant mesothelioma cell lines [64]. Therefore, targeting versican synthesis may be a potential mechanism for reducing this powerful tumor-promoting agent. Genetic and preclinical studies support the targeting of growth factor signaling as a therapeutic strategy for combating cancer. Individuals with overexpression of versican in breast cancer may more likely benefit from anti-EGFR therapy given known effects of EGF-like motifs in versican, a scientific consideration that warrants further evaluation. However, there are no data to show that such approaches are effective in inhibiting the effects of versican in cancer cell models. The presence of two EGF-like domains in versican G3 and the importance of versican as a prognostic factor in breast cancer motivates further research in delineating the role of EGF receptors and the downstream signaling pathways in invasive breast cancer [19]. Versican G3 domain appears to be important in local and systemic invasiveness of human breast cancer [19]; our previous investigation demonstrated that versican G3 domain enhanced breast cancer cell growth, migration and systemic metastasis by up-regulating the EGFR-mediated signaling pathway [20]. Both selective EGFR inhibitor AG 1478 and selective MEK inhibitor PD 98059 were observed to be able to block this signaling pathway and prevent versican G3 induced effects on mammary cancer cell proliferation. In the present study, we have focused on the role of versican G3 domain in modulating breast cancer cell apoptosis. Breast cancer cell apoptosis appears to be a factor associated with cancer cell sensitivity or resistance to chemotherapy and mechanisms appear influenced by EGFR signaling. The particular activation or inhibition of downstream EGFR signaling appears to influence cancer cell apoptotic responses to versican mediated effects and appear variably modulated dependant on chemother- apeutic drug or EGFR inhibitor delivered. It has been reported that versican and its G3 domain possess properties that promote cell growth and survival in low serum and serum-free conditions in breast cancer cells [19,20]. Versican has also been described to contribute an important role in reducing oxidant injury through an enhancement of cell-matrix interactions [8]. Integrin-b1 was reported to reduce radical-induced apoptosis by binding to G3 domain [65]. In the current study, we demonstrated that versican G3-expressing breast cancer cells express enhanced cell survival in serum free medium and in response to certain chemotherapeutic drugs such as Doxorubicin and Epirubicin. G3 expressing cells demonstrated a greater viability in serum free medium and chemotherapeutic drugs such as Doxorubicin or Epirubicin, which expressed activated EGFR/ ERK signaling. pERK, GSK-3b (S9P) and CDK2 levels were continually recorded at high levels in G3-expressing cells. Recent advances in the mechanisms of oncogenesis have revealed that the constitutive activation of the EGFR/ERK pathway allows the Versican G3 Modulates Breast Cancer Cell Apoptosis tumor cells to bypass regulatory check points that normally balance cell growth and cell apoptosis thereby activating cell cycle entry. Effective chemotherapy may induce cellular damage on a massive scale because it can engage one or more of these check points or drive cancer cells towards apoptosis [66]. Activation of CDK2 and pERK, and that the bypass of regulatory controls in cell cycle progression and cell apoptosis appear to significantly influence tumor growth and survival [66]. Activated glycogen synthase kinase-3ß serine-9 phosphorylation (GSK-3b, S9P) is also required for tumor cell survival and anti-apoptosis [67]. Based on that the present study, enhanced expression of pERK, GSK-3b (S9P) and CDK2 in G3- expressing breast cancer cells favored cell survival and growth even in serum free conditions or when cultured in the environment of applied chemotherapeutic reagents. In particular, versican G3 enhanced cell survival was prevented by both selective EGFR inhibitor AG 1478 and selective MEK (ERK kinase) inhibitor PD 98059 through mechanisms blocking G3 activated expression of pERK and GSK-3 b (S9P). Versican G3-expressing breast cancer cells demonstrated enhanced cell survival in serum free medium and chemotherapy by activating EGFR/ERK signaling and its down- stream pathway proteins CDK2 and GSK-3b (S9P). To validate the roles of versican and G3 domain in modulating breast cancer cell apoptosis in response to applied chemotherapy, we transfected tumor cells with anti-versican siRNA as well as by linking versican G3 domain with versican 39-UTR (G3-UTR) that reduces versican and G3’s functionality. Prior study demonstrated that non-coding versican 39-UTR significantly down-regulates G3 protein expression [48]. Concordantly, we observed that both anti-versican siRNA and G3-UTR construct reduced G3 enhanced anti-apoptosis when treated with Doxorubicin and Epirubicin. The EGFR signaling pathway is indispensable for cell cycle progression while it may also efficiently enhance apoptosis [68]. Although activation of the EGFR/ERK signaling pathway is generally considered to lead to cell survival [69], there is evidence that in certain conditions it may also transmit pro-apoptotic signals [69,70]. In addition to its effects on proliferative capacity and increasing apoptotic resistance, over-expression of versican can be accompanied by selective sensitization to apoptosis [7]. Whereas V1-transfected cells have shown resistance to apoptosis, they also have become significantly sensitized to other apoptotic stimuli, including UV radiation, chemotherapeutics, hypoxic mimetics, and conjugated linoleic acid. Elevated resting levels of the tumor suppressor p53 play a key role in inducing apoptosis in response to various detrimental events, including DNA damage, hypoxia, and telomere erosion [7]. In this study we also noted that versican G3 expressing breast cancer cells showed enhanced apoptosis when treated with certain chemicals, such as C2-ceramide and Docetaxel. In this scenario, chemotherapy induced apoptosis may be enhanced due to the recruitment of enhanced efficiency of cellular signaling. We found that although high levels of pERK were observed in G3-expressing cells when treated with these chemicals, one of the other EGFR down-stream proteins p- SAPK/JNK was dramatically activated. The pro-death or pro- survival role of ERK can have both, survival or cell death activities [22,71]. Literature supports an effect of breast cancer cells on cellular SAPK/JNK activation in a pro-death capacity but a role of pro-survival was also observed [72]. In our study, both p-ERK and p-JNK was expressed in high levels in the G3-expressing cells after treatment with C2-ceramide and Docetaxel. To determine which factor played a key role in versican G3 enhanced cell apoptosis, we co-treated the G3-expressing cells with chemicals and AG 1478, PD 98059 or SP 600125; we observed that G3 PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e26396