depletion generated polyclonal populations that either lost E- cadherin suppression over time (shEcad#24), or exhibited a partial reduction of E-cadherin levels for the duration of these studies (shEcad#23) (Figure 6A, B). Importantly, the shEcad#21 and shEcad#23 viruses were very effective (80–90% reduction) in reducing endogenous E-cadherin expression in MCF7 cells, without any evidence of a growth deficit in the cadherin-depleted MCF7 cells (Figure S3). Taken together, these results strongly suggest that SF767 cells are dependent on E-cadherin expression for growth. To follow up on this observation, we further examined the only stable SF767 E-cadherin knockdown line that we obtained, which was an incomplete knockdown produced by the shEcad#23 shRNA. Even this partial E-cadherin knockdown resulted in SF767 cells that grew more slowly in culture than did their control, non-target shRNA counterparts (Figure 6C). We also examined the ability of these E-cadherin depleted cells to migrate towards a gradient of FBS. To control for the growth effects of the E- cadherin reduction, cells in this transwell assay were only allowed to migrate for 6 hours. Figure 6D shows that the migration of E- cadherin-reduced SF767 cells was significantly slower than that of their non-target shRNA counterparts in this assay. Taken together, these results indicate that E-cadherin plays a positive role in the ability of the SF767 glioma cell line to grow and migrate in vitro. Discussion E-cadherin is usually assigned a tumor suppressor role in epithelial cells because it is lost in many carcinomas and its loss is associated with a less favorable prognosis [43,44]. Loss of E- cadherin in these tumors is associated with increased Ras, Rac1, and MAPK signaling, which induce tumor cell growth and invasion [45,46]. Conversely, re-expression of E-cadherin in such tumor cells suppresses tumor cell growth and invasion [45,46]. However, under some circumstances, E-cadherin is associated with increased tumorigenesis and tumor dispersion. Indeed, E- cadherin expression, which is absent from the normal, mesenchy- mally-derived ovary, is upregulated in the vast majority of malignant ovarian tumors and correlates with increased survival, proliferation, and metastatic spread of ovarian cancer cells [47,48]. Our data, presented here, are consistent with the idea that E- cadherin expression is also an important component of tumor growth and invasiveness in a rare subset of high-grade gliomas. We have shown that, in patients with GBM exhibiting an epithelial/ pseudo-epithelial morphology, E-cadherin expression correlates with a worse prognosis compared to patients that did not express E-cadherin. E-cadherin expression also correlates with increased invasiveness of glioma xenograft cell lines in an orthotopic mouse model of invasion. Finally, endogenous E-cadherin expression promotes the growth and migration of the conventional SF767 glioma cell line. Collectively, these results suggest an atypical role for E-cadherin in brain glial tumor biology that, while uncommon, may be of particular importance when it does occur. Our results suggest that the cadherin class of cell adhesion molecules in general, and E-cadherin in particular, may play a key role in the biology of malignant gliomas. The role and rarity of E-cadherin in gliomas contrasts with the expression of E-cadherin in another type of nervous system tumor: meningiomas. Meningiomas are derived from meningothelial/ arachnoid cells, which are some of the few normal nervous system cells that do express E-cadherin. E-cadherin is expressed in most meningiomas [21,22,49], and its loss may be associated with tumor progression [20]. In contrast, in the case of GBM, epithelial E-Cadherin Expression in GBM differentiation with E-cadherin expression is considered a metaplastic (conversion) process in a glial tumor, similar to the more commonly observed malignant mesenchymal component of gliosarcomas. This interpretation is supported by the finding that the glial and metaplastic components in either tumor type (gliosarcoma or GBM) both exhibit similar genetic alterations and apparently arise from a common precursor [33,50,51]. Our data on E-cadherin expression is consistent with the majority of previous studies and shows that E-cadherin is not expressed in normal brain, including neuronal cells, astrocytes, oligodendrocytes, or the majority of GBMs. As 18 different classical cadherins are thought to be expressed in brain, cadherin- specific antibodies should be carefully screened for non-specific effects, as indicated by our results with a widely used anti-E- cadherin antibody (clone 36). The lack of E-cadherin expression in normal brain also suggests that a classic EMT is not involved in glioma progression. However, the process of EMT includes the upregulation and downregulation of a variety of genes and proteins, not just cadherins, which together cause the tumorigenic transition [15,19]. The lack of classic epithelial-to-mesenchymal cadherin-switching in ordinary glial tumorigenesis does not mean that these other aspects of EMT are nonoperational. For example, all of the E-cadherin positive GBMs with epithelial or pseudo- epithelial differentiation were also positive for N-cadherin, a classic EMT marker (Rodriguez, data not shown). In addition, many conventional or serially transplanted GBM cell lines express cadherin 11 (Figure 2, 4), another classic EMT marker. Preliminary data suggest that cadherin 11 can promote GBM cell growth and migration (Lewis-Tuffin, unpublished observations). Indeed, a progression from a pro-neural to a mesenchymal phenotype has been suggested by genomic studies in GBM tumors, and is associated with poor prognosis [52]. E-cadherin expression in GBM appears to be an exception to the EMT rule: when E-cadherin expression is turned on in these tumors it is related to tumorigenesis and poor prognosis. The molecular mechanisms underlying the contribution of E-cadherin to growth and/or invasiveness in GBM are currently unknown. Previous studies in epithelial tumor cells have shown that mesenchymal cadherins (i.e. N-cadherin and cadherin 11) can promote tumor cell growth and migration via p120-mediated activation of Rac1 signaling [45,46]. Despite an initial activation of Rac1 upon epithelial cell-cell contact, the overall level of Rac1 activity is normally suppressed by E-cadherin expression [45]. However, this E-cadherin effect is thought to be mediated by the formation of mature cell-cell junctions [53,54]. In the SF767 glioma cell line, mature cell-cell junctions fail to form. It is therefore possible that deregulated E-cadherin signaling causes increased Rac1 activation and induces tumor cell growth and invasion of human glioma cells. Another possible mechanism by which E-cadherin could regulate proliferation and parenchymal infiltration involves adhesion-induced ligand-independent activation of the EGF receptor, leading to Akt and MAPK activation. The occurrence of such a mechanism has been demonstrated in the OVCAR-3 ovarian cancer cell line and is thought to underlie E-cadherin effects in ovarian cancer in general [47]. This mechanism is particularly intriguing to consider in the context of GBM, which is known for the frequency with which it displays EGFR overex- pression or expression of the constitutively-active EGFRvIII mutant. It is of note that GBMs with epithelial and ‘‘adenoid’’ morphology show a decreased frequency of EGFR amplification [33]. E-cadherin expression could represent an alternative mechanism for activating the EGF pathway in this subset of tumors. PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13665