Cunli Xiang

The induction of autophagy by γ-radiation contributes to the radioresistance of glioma stem cells

Malignant gliomas are characterized by a short median survival which is largely impacted by the resistance of these tumors tochemo‐ and radiotherapy. Recent studies suggest that a small subpopulation of cancer stem cells, which are highly resistant to γ‐radiation, has the capacity to repopulate the tumors and contribute to their malignant progression. γ‐radiation activates the process of autophagy and inhibition of this process increases the radiosensitivity of glioma cells; however, the role of autophagy in the resistance of glioma stem cells (GSCs) to radiation has not been yet reported. In this study we examined the induction of autophagy by γ‐radiation in CD133+ GSCs. Irradiation of CD133+ cells induced autophagy within 24–48 hr and slightly decreased the viability of the cells. γ‐radiation induced a larger degree of autophagy in the CD133+ cells as compared with CD133− cells and the CD133+ cells expressed higher levels of the autophagy‐related proteins LC3, ATG5 and ATG12. The autophagy inhibitor bafilomycin A1 and silencing of ATG5 and beclin1 sensitized the CD133+ cells to γ‐radiation and significantly decreased the viability of the irradiated cells and their ability to form neurospheres. Collectively, these results indicate that the induction of autophagy contributes to the radioresistance of these cells and autophagy inhibitors may be employed to increase the sensitivity of CD133+ GSCs to γ‐radiation.

MicroRNA-145 Is Downregulated in Glial Tumors and Regulates Glioma Cell Migration by Targeting Connective Tissue Growth Factor

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3′-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3′-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.

Proteasome inhibitors sensitize glioma cells and glioma stem cells to TRAIL-induced apoptosis by PKCε-dependent downregulation of AKT and XIAP expressions☆

In this study we examined the effects of proteasome inhibitors on cell apoptosis in TRAIL-resistant glioma cells and glioma stem cells (GSCs). Treatment with proteasome inhibitors and TRAIL induced apoptosis in all the resistant glioma cells and GSCs, but not in astrocytes and neural progenitor cells. Since PKCε has been implicated in the resistance of glioma cells to TRAIL, we examined its role in TRAIL and proteasome inhibitor-induced apoptosis. We found that TRAIL did not induce significant changes in the expression of PKCε, whereas a partial decrease in PKCε expression was obtained by proteasome inhibitors. A combined treatment of TRAIL and proteasome inhibitors induced accumulation of the catalytic fragment of PKCε and significantly and selectively decreased its protein and mRNA levels in the cancer but not in normal cells. Overexpression of PKCε partially inhibited the apoptotic effect of the proteasome inhibitors and TRAIL, and the caspase-resistant PKCεD383A mutant exerted a stronger inhibitory effect. Silencing of PKCε induced cell apoptosis in both glioma cells and GSCs, further supporting its role in cell survival. TRAIL and the proteasome inhibitors decreased the expression of AKT and XIAP in a PKCε-dependent manner and overexpression of these proteins abolished the apoptotic effect of this treatment. Moreover, silencing of XIAP sensitized glioma cells to TRAIL. Our results indicate that proteasome inhibitors sensitize glioma cells and GSCs to TRAIL by decreasing the expression of PKCε, AKT and XIAP. Combining proteasome inhibitors with TRAIL may be useful therapeutically in the treatment of gliomas and the eradication of GSCs.

RTVP-1 expression is regulated by SRF downstream of protein kinase C and contributes to the effect of SRF on glioma cell migration.

Gliomas are characterized by increased infiltration into the surrounding normal brain tissue. We recently reported that RTVP-1 is highly expressed in gliomas and plays a role in the migration of these cells, however the regulation of RTVP-1 expression in these cells is not yet described. In this study we examined the role of PKC in the regulation of RTVP-1 expression and found that PMA and overexpression of PKCα and PKCε increased the expression of RTVP-1, whereas PKCδ exerted an opposite effect. Using the MatInspector software, we identified a SRF binding site on the RTVP-1 promoter. Chromatin immunoprecipitation (ChIP) assay revealed that SRF binds to the RTVP-1 promoter in U87 cells, and that this binding was significantly increased in response to serum addition. Moreover, silencing of SRF blocked the induction of RTVP-1 expression in response to serum. We found that overexpression of PKCα and PKCε increased the activity of the RTVP-1 promoter and the binding of SRF to the promoter. In contrast, overexpression of PKCδ blocked the increase in RTVP-1 expression in response to serum and the inhibitory effect of PKCδ was abrogated in cells expressing a SRFT160A mutant. SRF regulated the migration of glioma cells and its effect was partially mediated by RTVP-1. We conclude that RTVP-1 is a PKC-regulated gene and that this regulation is at least partly mediated by SRF. Moreover, RTVP-1 plays a role in the effect of SRF on glioma cell migration.

PKCδ as a moelcular switch in the apoptosis and survival of glioma cells

tion what happens if the cells with self-renewal capacity are exposed to viral oncogenes. We studied how oncogenes and tumor suppressors affect the differentiation capacity, proportion and characteristics of progenitor cells in a model tissue. For this we used neural progenitor cells (NPCs) transduced with modified retroviruses carrying Human Papilloma Virus E6, E7 or E6/E7 oncogenes. HPV 16 E6/E7 is a classical oncogene complex, which degrades tumor suppressors p53 and pRb/p107/p130 and interferes with several other cellular activities. To examine further p53 and pRb involvement in NPC maintenance, we used p53−/− and E7 and E6/E7− expressing NPCs with restored pRb expression, respectively. E6/E7 expressing or p53−/− NPCs were able to differentiate, but simultaneously retained high capacity for self-renewal, proliferation, ability to remain multipotent in conditions promoting differentiation, and showed delayed cell cycle exit. These functions were mediated through p53 and pRb family and involved MEK-ERK signaling. Low amount of p53 increased self-renewal and proliferation, where as pRb affected only proliferation. This suggests that the oncogenes increase, while p53 and pRb-family tumor suppressors decrease the number and proportion of progenitor cells. These findings provide one explanation how oncogenes and tumor suppressors control tissue homeostasis and highlight their importance in stem cell selfrenewal, linked both to cancer and life-long tissue turnover.