Cancer stem cells render abnormal growth, frequency and radio-resistance in malignant glioblastoma. Therapeutic strategies in treating glioblastoma require a better understanding of the mechanisms that promote stem cell maintenance that sustain tumor growth. Elias, et al. [34
] proposed neural precursor cells as the likely targets for malignant transformation and showed that deregulation of the self-renewal capacity of normal stem cells resulted in stable augmentation of self-renewing cancer cells and tumor formation. Investigations to study this tumorigenic process will provide a powerful tool to uncover new targets for glioma therapeutic intervention. On par with the available evidence, it is understood that cancer stem cells (CSC) play an important role in tumor initiation, growth, invasion and recurrence. Therefore, identification and study of CSC will provide insights into better understanding of brain tumors and their cellular targets. In this study, we have obtained GSCs from U251, U87, 4910 and 5310 cell lines using the surface protein CD133 and FACS sorting as reported previously [37
In the present study, we show that malignant human grade IV gliomas display significant expression of Twist1 and Sox2. Twist1 expression was observed in hGBMs as well as in medulloblastoma, meningioma, lung and pancreatic cancer specimens, while Sox2 expression was observed in hGBM specimens along with that of meningioma, lung and pancreatic cancers. These results indicate the participation of both Twist1 and Sox2 in the genesis of a wide range of neuroectodermal tumors. Twist1 encourages cancer cell survival and tumor progression by increasing resistance to cytotoxic therapies resulting in increased cellular proliferation [40
]. Twist1 is considered to be a key player of EMT, a metastatic cascade in which epithelial cells take over a mesenchymal phenotype [10
]. EMT phenotype expresses cancer stem-like cells signatures associated with tumor recurrence and a drug-resistant phenotype [41
]. Immunoblotting analysis conducted on the hGBM specimens have shown reduced epithelial E-cadherin expression and upregulated mesenchymal N-cadherin, vimentin, β-catenin and Twist1, suggesting the EMT mode. High expression levels of other stem cell markers such as CD44, Msi-1, Stro-1, and EMT markers, such as Sox2, Twist1, vimentin suggested an invasive phenotype. The synergistic up-regulation of Twist1 and Sox2 expression in hGBM specimens may instigate E-cadherin replacement by N-cadherin expression triggering cadherin switching [42
Numerous lines of evidences suggest that the Twist1 regulates programmed cell death and induces apoptosis [44
]. Moreover, it has been observed that transfection of Bel7402 (highly expressing Twist1) cells with Twist1 shRNA plasmid resulted in suppression of Twist1 expression and subsequent inhibition of cell invasion and migration in vitro
, indicating its possible role in tumor cell plasticity [46
]. The widespread expression of Twist1 in various cancer phenotypes suggests its action upon a diverse set of downstream target genes, depending upon the tissue, to elicit a variety of cellular responses. The chimeric nature of beta-Helix loop Helix (bHLH) proteins, such as Twist1 are implicated to possibly combine heterodimerically with other bHLH proteins to activate or repress diverse downstream targets generating different types of biological responses [47
]. Cimadamore et al.
] reported that knockdown of Sox2, the SRY (sex-determining region)-box 2 gene, resulted in downregulation of proneural bHLH transcription factors.
Twist1-mediated molecular changes in our study provided important insight into its role in understanding mesenchymal change with genes related to EMT being upregulated in U251, U87, 4910 and 5310 GSCs. Since we observed the co-expression of Sox2 along with Twist1, further studies are warranted to examine their role in acquiring an invasive malignant phenotype. Knockdown of Twist1 reduced the expression levels of Sox2, while knockdown of Sox2 reduced the expression levels of Twist1. In both experiments, increased expression of E-cadherin was observed, which suggests that both Twist1 and Sox2 function in harmony and that their concurrence is essential for the maintenance of tumor plasticity. Inhibition of Twist1 or Sox2 expression by their specific siRNAs resulted in differentiation of GSC formation and growth and is suggestive of the above discussed mechanism.
Targeted migration of mesenchymal stem cells (MSCs) to tumors makes them a very promising strategy for anti-tumor therapy [49
]. Previously, we have demonstrated the potential therapeutic applications of hUCBSC in the treatment of gliomas [28
]. In addition, with increased understanding of the mechanistic underpinnings of MSC action, the potential for enhancing MSC-targeted therapies appears promising. This report provides a strong evidence that hUCBSC inhibit the metastatic and invasive potential of U251, U87, 4910 and 5310 GSCs. Co-culture of hUCBSC with these GSCs changed their morphology and clearly reduced their migration and invasion abilities. We also showed that hUCBSC induced MET in U251, U87, 4910 and 5310 GSCs, which might be responsible for inhibition of metastasis-related abilities.
It appears that hUCBSC counteract and repress Twist1 expression by increasing E-cadherin levels, a phenomenon generally observed at sites of EMT. Our present data demonstrated the ability of hUCBSC to inhibit GSC phenotypes in co-culture experiments both in vitro and in vivo. Immunoblotting experiments showed a decrease in the expression levels of both Twist1 and Sox2 along with other EMT markers, suggesting that hUCBSC treatment reversed the EMT to MET. Based on all our findings, we propose that hUCBSC inhibit GSC invasion, migration by suppressing key molecules involved in EMT. These actions resulted in induction of a MET state, which is a key step in reversal and treatment of the cellular metastatic process.
In conclusion, for the first time, we showed that interactive and co-regulatory role of Twist1 and Sox2 necessary to maintain glioma stemness was inhibited by hUCBSC. Here, hUCBSC was administered to study key aspects in treating the glioma stem cell machinery. We engineered hUCBSC near the tumor site and observed that hUCBSC are effective in regressing tumor growth in the intracranial xenograft mouse model. Based on our findings, we conclude that hUCBSC can be used as a means of therapy and will be of great interest for the clinical application of stem cell-based cancer therapy.