The fundamental role for mesenchymal change in promoting invasion, malignancy treatment response and even cancer stem cell function in human carcinoma and GBM invasion is increasingly recognized [8, 9]. TWIST1 is a central regulator of mesenchymal change in carcinoma  but its relevance to invasion and mesenchymal change in GBM models has not been studied. Since tumor invasion is perhaps the major obstacle to improved outcome for patients with carcinomas and gliomas the elucidation of TWIST1 function in GBMs is potentially of great clinical importance. Following on our previous observation in the SF767 GBM cell line  this study validated the pro-invasive function of TWIST1 in multiple cell lines in vitro and in vivo and demonstrated that TWIST1 promoted clinically relevant mesenchymal molecular and cellular phenotypes that partially recapitulated those associated with carcinoma EMT. These findings identify TWIST1 as a regulator of mesenchymal change and invasion in GBM that can be leveraged for further investigation of the clinical potential of subverting mesenchymal change as a therapeutic strategy in treating GBM.
Collectively TWIST1 promoted invasion in vitro of all GBM cells tested to date (including a GBM stem cell line). We further established that TWIST1 enhanced invasion in the more relevant settings of brain slice culture and orthotopic xenotransplant models using SNB19 and T98G GBM cell lines. Of interest, the patterns of enhanced invasion generated by TWIST1 over-expression were cell-line specific with SNB19 TW cells invading as single cells or small aggregates from a central core while T98G TW cells diffusely invaded throughout the brain. These extreme patterns of invasion are similar to those in cases of gliomatosis cerebri . These findings clearly demonstrate the generic pro-invasive function for TWIST1 in GBM and suggest that cell-intrinsic factors can modify TWIST1- mediated patterns of GBM invasion.
Consistent with this, TWIST1 over-expression generated cell-specific changes in gene expression with shared pro-invasive functional attributes. TWIST1-mediated changes in expression of specific genes in SNB19 and T98G were heterogeneous but overlapped at the functional level within five common categories related to the cellular requirements for glioma invasion and EMT including cell adhesion, extracellular matrix, cell motility and locomotion, cell migration and actin cytoskeleton organization. Importantly, TWIST1 over-expression generated cell phenotypes highly consistent with the over-representation of genes within these functional categories that reflect critical individual cellular features required for carcinoma and GBM invasion . We also determined that TWIST1 induced re-localization of activated FAK to sites of abundant lamellipodia formation, a significant finding given the association between FAK activation, cytoskeletal organization and its role in EMT and glioma malignancy (reviewed in [34–36]. Loss of apical-basal polarity (relative to a basement membrane) is an additional feature of EMT in carcinomas which was not tested here since assays of polarity for GBM cells in vitro are not well established. However, the recent description of polarized ciliated neural stem cells within the ventricular zone neuroepithelium [37–39] suggests that such studies could be attempted in situ or with novel co-culture systems or at earlier stages of glioma development. This approach could reveal polarity changes (analogous to carcinoma EMT) as fundamental steps in the process of gliomagenesis and acquisition of an invasive phenotype. Together gene expression analysis and cellular assays demonstrated that TWIST1 over-expression in glioma cells orchestrated the acquisition of a robust mesenchymal phenotype and cellular changes that closely mirror those of carcinoma cells undergoing mesenchymal transformation  and required for tumor invasion and metastasis .
TWIST1-mediated molecular changes also provided important insight into its role in mesenchymal change in GBM. Many genes related to carcinoma EMT were also up-regulated by TWIST1 in GBM indicating potential mechanistic overlap between the two processes. However, the lack of a TWIST1-mediated "cadherin switch" in GBM cells suggested that alternative mechanisms in nervous tissue and gliomas function to modulate cell adhesion and invasion. Alternatively, a cadherin switch could occur early in gliomagenesis or require specific anatomic or environmental interactions not present in our experimental system. The recent discovery that normal neural stem cells -- putative GBM cells of origin -- express E-cadherin supports this possibility [42, 43]. Further studies are warranted to examine the impact of TWIST1 and other factors related to mesenchymal change in normal GBM cells of origin (neural stem and progenitor cells) or in cells at early stages of gliomagenesis to better define how alterations in E-cadherin or other cell-cell adhesion molecules impact the acquisition of an invasive malignant phenotype.
The clinical relevance of identified putative TWIST1 targets was established through correlation between TWIST1, SNAI2 and FAP expression levels in 39 human gliomas of different grades. These studies demonstrated that the current in vitro model of TWIST1 pro-invasive function was capable of identifying clinically relevant pro-invasive targets and candidate downstream mechanisms of TWIST1-mediated glioma invasion. Our data also confirms prior reports that expression of SNAI2  and FAP  is directly linked to malignant glioma grade and further showed that they are coordinately upregulated in gliosarcoma, the grade IV glioma with the most overt mesenchymal differentiation. As regulators of invasiveness, TWIST1 and SNAI2 are potential targets for therapeutic modulation, a proposition further supported by their known functions to promote cell survival and treatment resistance in other cancer types [45–50]. FAP is expressed in wounds and fibrotic tissues as well as carcinoma-associated fibroblasts in multiple cancer types and is thought to degrade tumor matrix and facilitate carcinoma invasion . Further studies are needed to determine which cell type(s) express FAP and whether it serves a similar role of altering tumor stroma to promote invasion in GBM.
The significance of TWIST1 function to promote invasion through mesenchymal change in GBMs is underscored by recent reports of clinically relevant mesenchymal phenotypes in GBMs. Gene expression array studies identified a mesenchymal stem cell (MSC) phenotype in human GBMs  and distinct pro-neural, proliferative and mesenchymal gene expression signatures among malignant grade III and IV human gliomas . The mesenchymal signature is associated with poor prognosis, increased angiogenesis and tumor recurrence . Therefore, along with other transcription factors such as STAT3 and C/EBP which were recently identified as regulators of mesenchymal transformation in GBM cells  the correlation of TWIST1 with induction of mesenchymal changes, increased glioma grade and invasiveness implicate TWIST1 as an additional central regulator of this process in human GBM. Of note, STAT3 transcriptionally upregulates TWIST1 expression and promotes breast carcinoma cell migration  prompting speculation that STAT3-TWIST1 interactions in GBM may also contribute to invasion and mesenchymal change.
Inhibitors of TWIST1 are not available; therefore, to investigate the therapeutic relevance of inhibiting TWIST1 in GBM we knocked down TWIST1 expression using shRNA and assayed its effects on cell invasion and glioma stem cell properties. Specific inhibition of TWIST expression resulted in marked reductions in glioma cell invasion in vitro. These findings are consistent with the pro-invasive function of TWIST1 in GBM and support the therapeutic potential of inhibiting TWIST1 or TWIST1-mediated signaling to inhibit GBM invasion. Glioma stem cells are recognized as tumor-initiating cells that determine tumor malignancy and growth. Through activation of EMT, TWIST1 promotes the formation and maintenance of breast cancer stem cells  and TWIST1 over-expression is implicated in mesenchymal stem cell activity . Given these observations we propose that targeting TWIST1 may have additional therapeutic relevance in gliomas by abrogating glioma stem cell functions. Our data showed that inhibition of TWIST1 expression resulted in a dramatic reduction in GBM stem cell sphere formation and growth. These results suggest that a unique therapeutic potential of inhibiting TWIST1 may result from simultaneous targeting of glioma cell invasiveness and stem cell function -- hallmark GBM properties that both contribute to tumor growth, progression and treatment resistance. To address this potential, ongoing and future studies will address the effects of TWIST1 inhibition in GBM cells on tumor growth, invasion and response to therapy in vivo.