Inhibition of EGFR Localization to Mitochondria Figure 5. Inhibition of cell proliferation by hUCBSC treatments. (A) Control and hUCBSC-treated glioma cells at different time points were analyzed for cell proliferation by incorporation of BrdU. *Significant at p,0.05 compared to respective control cells. **Significant at p,0.01 compared to respective control cells (by t-test). (B) Control, EGF-treated and EGF+hUCBSC treated glioma cells were analyzed for cell proliferation. *Significant at p,0.05 both for control vs. EGF+hUCBSC treatment and EGF vs. EGF+hUCBSC treatments (one-way ANOVA followed by Bonferroni’s post hoc test). (C) Control, TMZ treated, TMZ+hUCBSC treated glioma cells were subjected to cell proliferation analysis by BrdU incorporation. *Significant at p,0.05; **Significant at p,0.01 both for control vs. TMZ treatment and control vs. TMZ+hUCBSC treatments (one-way ANOVA followed by Bonferroni’s post hoc test). For all these tests, raw data was taken into consideration to perform statistical analyses and then results were presented as % control values. Each bar represents n$6. doi:10.1371/journal.pone.0031884.g005 downregulated in hUCBSC treatments (Fig. S1). These results confirm that hUCBSC are efficient in inhibiting the phosphory- lation of EGFR, FAK and c-Src in glioma cells and tissues, thereby preventing the translocation of EGFR to mitochondria as well as interaction of EGFR with FAK and c-Src. Finally, we assessed the expression of EGFR signaling molecules in glioma cells in vitro and in vivo tumors after treatment with hUCBSC. The main pathways stimulated by the EGFR are the mitogen-activated protein kinases (MAPK), the phosphatidylino- sitol 3-kinase–AKT, and signal transducer and activator of transcription proteins (STAT), such as STAT3 [27]. In addition, the activation of STAT3 also relies on the recruitment of the Src kinase to the EGFR [28,29]. Upon nuclear translocation, the STAT3 transcription factor up-regulates several genes involved in cell cycle progression [30,31], cell survival [32] and metastasis [33]. Another molecule of EGFR signaling cascade RAF1 protein, can phosphorylate to activate the dual specificity protein kinases MEK1 and MEK2, which in turn phosphorylate to activate the serine/threonine specific protein kinases, ERK1 and ERK2. These key molecules of EGFR signaling MEK1, RAF1, STAT3 and PI3K were downregulated in hUCBSC treated glioma cells (Fig. 7G). Quantitative analysis shows that both RAF1 and PI3k (p,0.01) show significant downregulation compared to MEK1 and STAT3 (p,0.05) (Fig. 7H). We also checked the expression of these molecules in hUCBSC-treated glioma tumors in vivo and observed that these EGFR signaling molecules are downregulated compared to untreated tumors (Fig. 7I). Similar to our in vitro results, RAF1 and PI3k (p,0.01) molecules show significant downregulation compared to MEK1 and STAT3 (p,0.05) by hUCBSC treatments (Fig. 7J). Finally, we observed tumor size regression by about 75% in hUCBSC-treated in vivo nude mice brains of both U251 and 5310 xenografts (Fig. 7K). Our results show that hUCBSC has the ability to downregulate the expression of multiple members of EGFR signaling cascades, in both in vitro and in vivo conditions. Discussion Overexpression of EGFR is one of the most frequently diagnosed genetic aberrations of glioblastoma multiforme (GBM). EGFR signaling is involved in diverse cellular functions and is dependent on the type of preferred receptor complexes. EGFR activation stimulates many complex intracellular signaling pathways that are tightly regulated by the presence and identity of the ligand, heterodimer composition, and the availability of phosphotyrosine-binding proteins. The two primary signaling pathways activated by EGFR include the RAS/RAF/MEK/ERK and the PI3K/AKT axes; however, Src tyrosine kinases, PlCc, PKC, and STAT activation and downstream signaling have also been well documented [16,34,35]. EGFR gene amplification is one of the most common genetic changes in GBM and can lead to the activation of various downstream signaling molecules, including STAT3, MAPK, and AKT [36]. Receptor phosphor- ylation leads to the recruitment of multiple effector proteins through recognition and binding of Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains on the effector proteins to phosphotyrosine motifs on the receptor [37]. The formation of this signaling complex results in the initiation of various downstream signaling cascades, including the phosphoinositide-3-kinase (PI3K), mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 3 (STAT3) pathways, which regulate a multitude of cellular responses. To evaluate the signaling pathways mediated by EGFR, we determined the expression of EGFR in GBM-patient derived specimens. Our results confirm that immunohistochemically detectable EGFR is overexpressed in some grade IV glioblastoma samples. These results are in correlation with transcriptional and translational status of EGFR expression. However, in regards to the clinical outcome, EGFR expression did not correlate with other molecules like FAK. EGFR staining was negative in the normal brain samples and these results suggest that EGFR expression is an indicator of a malignant phenotype of the majority of GBM. Co- overexpression of EGFR, FAK and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth [38,39]. In the present study, we also observed co-localization of EGFR with FAK in mitochondria of GBM-patient derived specimens, which is indicative of the roles of EGFR, FAK and mitochondria in enhanced tumor growth. In recent years, EGFR has become a promising target for therapies for different tumors. The poor prognosis of GBM patients is due in part to classical chemo- and radioresistance properties demonstrated by these tumors. Newly diagnosed GBM patients typically undergo surgical resection of the tumor followed by concomitant temozolamide (TMZ) treatment and radiotherapy and subsequent administration of TMZ for 6 months as adjuvant chemotherapy [40]. Because EGFRvIII has been shown to contribute to tumor progression and chemo-radioresistance, an attractive strategy would be to develop targeted therapeutics against this receptor for use as a monotherapy, thereby minimizing the systemic side effects of whole-brain irradiation and TMZ administration, especially because genetic manipulation of EGFRvIII has resulted in tumor shrinkage in a preclinical setting [8]. The monoclonal anti-EGFR antibody cetuximab and the EGFR tyrosine kinase inhibitor erlotinib led (in monotherapy or in combination with cytotoxic drugs) to improved survival in patients with colorectal cancer, non-small cell lung cancer and advanced pancreatic cancer [41–43]. Based on these earlier reports and our successful results with GBM tumor regression by hUCBSC, we hypothesized that hUCBSC could efficiently downregulate the expression of EGFR and its signaling molecules. Previously, we reported that hUCBSC inhibit FAK expression and its related angiogenesis in GBM in vitro and in vivo [22]. In the present study, we observed downregulation of EGFR expression in glioma cells. Even when we supplied exogenous EGF, which increases EGFR expression of glioma cells in a drastic manner, treatment with hUCBSC efficiently inhibited the expression of both EGFR and FAK in these cells. Although generally represented as a plasma membrane protein, EGFR has been found in the nucleus and sub-cellular organelles. Recently, the mitochondrial localization of the EGFR was PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31884