Inhibition of Casein Kinase II by CX-4945, But Not Yes-associated protein (YAP) by Verteporfin,Enhances the Antitumor Efficacy of Temozolomide in Glioblastoma

Overcoming temozolomide (TMZ) resistance in glioma cancer cells remains a major challenge to the effective treatment of the disease. Increasing TMZ efficacy for patients with glioblastoma (GBM) is urgently needed because TMZ treatment is the standard chemotherapy protocol for adult patients with glioblastoma. O⁶-methylguanine-DNA-methyltransferase (MGMT) overexpression is associated with TMZ resistance, and low MGMT is a positive response marker for TMZ therapy. Here, we used 3 glioma cell lines (SF767, U373, and LN229), which had different levels of TMZ sensitivity. We found TMZ sensitivity is positively correlated with MGMT expression and multidrug-resistance protein ABC subfamily G member 2 (ABCG2) in these cells. CK2-STAT3 signaling and Hippo-YAP signaling are reported to regulate MGMT expression and ABCG2 expression, respectively. We combined CK2 inhibitor CX-4945 and YAP inhibitor verteporfin with TMZ treatment. We found that CX-4945 but not verteporfin can sensitize TMZ-resistant cells SF767 to TMZ and that CX-4945 and TMZ combinational treatment was effective for glioma treatment in mouse models compared with TMZ alone.ImplicationsA combination of CK2 inhibitor with TMZ may improve the therapeutic efficiency of TMZ toward GBM with acquired resistance.     

High expression of leptin receptor leads to temozolomide resistance with exhibiting stem/progenitor cell features in gliobalastoma

Glioblastoma is a highly aggressive malignant disease with notable resistance to chemotherapy. In this study, we found that leptin receptor (ObR)-positive glioblastoma cells were resistant to temozolomide (TMZ), and TMZ-resistant cells exhibited high expression of ObR. ObR can serve as a marker to enrich glioblastoma cells with some stem/progenitor cell traits, which explained the reason for TMZ resistance of ObR+ cells. STAT3-mediated SOX2/OCT4 signaling axis maintained the stem/progenitor cell properties of ObR+ cells, which indirectly regulated glioblastoma TMZ resistance. These findings gain insight into the molecular link between obesity and glioblastoma, and better understanding of this drug-resistant population may lead to the development of more effective therapeutic interventions for glioblastoma.     

Elimination of Cancer Stem-Like Cells and Potentiation of Temozolomide Sensitivity by Honokiol in Glioblastoma Multiforme Cells

Glioblastoma multiforme (GBM) is the most common adult malignant glioma with poor prognosis due to the resistance to radiotherapy and chemotherapy, which might be critically involved in the repopulation of cancer stem cells (CSCs) after treatment. We had investigated the characteristics of cancer stem-like side population (SP) cells sorted from GBM cells, and studied the effect of Honokiol targeting on CSCs. GBM8401 SP cells possessed the stem cell markers, such as nestin, CD133 and Oct4, and the expressions of self-renewal related stemness genes, such as SMO, Notch3 and IHH (Indian Hedgehog). Honokiol inhibited the proliferation of both GBM8401 parental cells and SP cells in a dose-dependent manner, the IC₅₀ were 5.3±0.72 and 11±1.1 μM, respectively. The proportions of SP in GBM8401 cells were diminished by Honokiol from 1.5±0.22% down to 0.3±0.02% and 0.2±0.01% at doses of 2.5 μM and 5 μM, respectively. The SP cells appeared to have higher expression of O ⁶-methylguanine-DNA methyltransferase (MGMT) and be more resistant to Temozolomide (TMZ). The resistance to TMZ could be only slightly reversed by MGMT inhibitor O ⁶-benzylguanine (O ⁶-BG), but markedly further enhanced by Honokiol addition. Such significant enhancement was accompanied with the higher induction of apoptosis, greater down-regulation of Notch3 as well as its downstream Hes1 expressions in SP cells. Our data indicate that Honokiol might have clinical benefits for the GBM patients who are refractory to TMZ treatment.     

MicroRNAs as Regulators of Neural Stem Cell-Related Pathways in Glioblastoma Multiforme

MicroRNAs are endogenous non-coding small RNAs that have been described as highly conserved regulators of gene expression. They are involved in cancer and in the regulation of neural development and stem cell function. Recent studies suggest that a small subpopulation of cancer stem cells (CSCs) has the capacity to repopulate solid tumours such as glioblastoma (GBM), drive malignant progression and mediate radio- and chemoresistance. GBM-derived CSCs share the fundamental stem cell properties of self-renewal and multipotency with neural stem cells (NSCs) and may be regulated by miRNAs. In this review, we will summarize the current knowledge regarding the role of miRNAs in GBM development with a focus on the regulation of GBM-CSCs. We propose a list of miRNAs that could serve as molecular classifiers for GBMs and/or as promising therapeutic targets for such brain tumours.     

HOTAIR/miR-125 axis-mediated Hexokinase 2 expression promotes chemoresistance in human glioblastoma

Drug resistance is one of the major obstacles in glioblastoma (GBM) treatments using temozolomide (TMZ) based conventional chemotherapy. Recent studies revealed that Hexokinase 2 (HK2)-mediated glycolysis is one of the sources, as the association of chemoresistance and the expression of HK2 was confirmed in multiple cancers. However, there has been little knowledge of the functional contribution of HK2 to TMZ resistance in GBM. In our study, we found that HK2 expression is crucial for GBM proliferation and chemoresistance. In contrast to the healthy brain, HK2 expression is much higher in human GBM, especially in those patients with GBM recurrence. High HK2 expression is negatively related to the overall survival in GBM patients. HK2 depletion in GBM cells suppressed the GBM cell proliferation and increased sensitivity to TMZ-induced apoptosis. Both HK2-mediated glycolysis and mitochondria permeability transition pore opening (MPTP) were associated with its function in chemoresistance. Furthermore, we also revealed that the abnormal expression of HK2 was modulated by the expression of HOTAIR, a long non-coding RNA (lncRNA). The absence of HOTAIR in GBM cells suppressed the HK2 expression in protein and mRNA level and, therefore, inhibited the cell proliferation and enhanced the cytotoxicity of TMZ both in vivo and in vitro. HOTAIR promoted the expression of HK2 by targeting mir-125, which suppressed the GBM cell proliferation and increased the TMZ-induced apoptosis. These findings shed light on a new therapeutic strategy in modulating HOTAIR/miR-125, which may interfere with the expression of HK2, and enhance the therapeutic sensitivity of GBM to TMZ.     

BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1�� stability and MGMT expression

Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in particular BMP2, are known to promote differentiation and growth inhibition in GBM cells. For this reason, we investigated whether a BMP2-based treatment would increase TMZ response in hypoxic drug-resistant GBM-derived cells. Here we show that BMP2 induced strong differentiation of GBM stem-like cells and subsequent addition of TMZ caused dramatic increase of apoptosis. Importantly, we correlated these effects to a BMP2-induced downregulation of both hypoxia-inducible factor-1α (HIF-1α) and MGMT. We report here a novel mechanism involving the HIF-1α-dependent regulation of MGMT, highlighting the existence of a HIF-1α/MGMT axis supporting GBM resistance to therapy. As confirmed from this evidence, over-stabilization of HIF-1α in TMZ-sensitive GBM cells abolished their responsiveness to it. In conclusion, we describe a HIF-1α-dependent regulation of MGMT and suggest that BMP2, by down-modulating the HIF-1α/MGMT axis, should increase GBM responsiveness to chemotherapy, thus opening the way to the development of future strategies for GBM treatment.     

Targeting ROR1 inhibits the self‐renewal and invasive ability of glioblastoma stem cells

Glioblastoma is the most malignant of brain tumours and is difficult to cure because of interruption of drug delivery by the blood–brain barrier system, its high metastatic capacity and the existence of cancer stem cells (CSCs). Although CSCs are present as a small population in malignant tumours, CSCs have been studied as they are responsible for causing recurrence, metastasis and resistance to chemotherapy and radiotherapy for cancer. CSCs have self‐renewal characteristics like normal stem cells. The aim of this study was to investigate whether receptor tyrosine kinase‐like orphan receptor 1 (ROR1) is involved in stem cell maintenance and malignant properties in human glioblastoma. Knockdown of ROR1 caused reduction of stemness and sphere formation capacity. Moreover, down‐regulation of ROR1 suppressed the expression of epithelial‐mesenchymal transition‐related genes and the tumour migratory and invasive abilities. The results of this study indicate that targeting ROR1 can induce differentiation of CSCs and inhibit metastasis in glioblastoma. In addition, ROR1 may be used as a potential marker for glioblastoma stem cells as well as a potential target for glioblastoma stem cell therapy. Copyright © 2016 John Wiley & Sons, Ltd.     

Secreted Frizzled-Related Protein 4 Inhibits Glioma Stem-Like Cells by Reversing Epithelial to Mesenchymal Transition,Inducing Apoptosis and Decreasing Cancer Stem Cell Properties

The Wnt pathway is integrally involved in regulating self-renewal, proliferation, and maintenance of cancer stem cells (CSCs). We explored the effect of the Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), in modulating epithelial to mesenchymal transition (EMT) in CSCs from human glioblastoma cells lines, U87 and U373. sFRP4 chemo-sensitized CSC-enriched cells to the most commonly used anti-glioblastoma drug, temozolomide (TMZ), by the reversal of EMT. Cell movement, colony formation, and invasion in vitro were suppressed by sFRP4+TMZ treatment, which correlated with the switch of expression of markers from mesenchymal (Twist, Snail, N-cadherin) to epithelial (E-cadherin). sFRP4 treatment elicited activation of the Wnt-Ca₂ ⁺ pathway, which antagonizes the Wnt/ß-catenin pathway. Significantly, the chemo-sensitization effect of sFRP4 was correlated with the reduction in the expression of drug resistance markers ABCG2, ABCC2, and ABCC4. The efficacy of sFRP4+TMZ treatment was demonstrated in vivo using nude mice, which showed minimum tumor engraftment using CSCs pretreated with sFRP4+TMZ. These studies indicate that sFRP4 treatment would help to improve response to commonly used chemotherapeutics in gliomas by modulating EMT via the Wnt/ß-catenin pathway. These findings could be exploited for designing better targeted strategies to improve chemo-response and eventually eliminate glioblastoma CSCs.     

Pharmacologic blockade of chloride channel synergistically enhances apoptosis of chemotherapeutic drug-resistant cancer stem cells

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is the most commonly used chemotherapeutic agent in the treatment of human glioblastoma multiforme (GBM). However, BCNU chemotherapy fails due to subpopulations of intrinsic resistant-cells within the tumor mass. In our previous study, we dissociated BCNU-resistant cancer stem cells (CSCs) and observed the over-expression of multiple ion channel genes related to drug efflux. In the present study, we identified chloride intracellular channel 1 (CLIC1) in dissociated-BCNU-resistant CSCs using 2-DE and MALDI-TOF/MS analysis. To develop the specific target therapy of BCNU-resistant CSCs, we evaluated the drug-sensitivity of these CSCs using the chloride channel blocker, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). When combined with BCNU, DIDS synergistically increased the apoptotic events of BCNU-resistant CSCs in vitro and augmented BCNU sensitivity ex vivo. These findings suggest that CLIC1 is involved in the resistance of BCNU-resistant CSCs and BCNU/DIDS combined-therapy can provide valuable insight for promoting apoptosis or sensitizing glioblastomas to BCNU chemotherapy.     

Temozolomide resistance in glioblastoma cells occurs partly through epidermal growth factor receptor-mediated induction of connexin 43

Glioblastoma Multiforme (GBM) is an aggressive adult primary brain tumor with poor prognosis. GBM patients develop resistance to the frontline chemotherapy, temozolomide (TMZ). As the connexins (Cx) have been shown to have a complex role in GBM, we investigated the role of Cx43 in TMZ resistance. Cx43 was increased in the TMZ-resistant low passage and cell lines. This correlated with the data in The Cancer Genome Atlas. Cx43 knockdown, reporter gene assays, chromatin immunoprecipitation assay, real-time PCR and western blots verified a role for Cx43 in TMZ resistance. This occurred by TMZ-resistant GBM cells being able to activate epidermal growth factor receptor (EGFR). In turn, EGFR activated the JNK-ERK1/2-AP-1 axis to induce Cx43. The increased Cx43 was functional as indicated by gap junctional intercellular communication among the resistant GBM cells. Cell therapy could be a potential method to deliver drugs, such as anti-EGF to tumor cells. Similar strategies could be used to reverse the expression of Cx43 to sensitize GBM cells to TMZ. The studies showed the potential for targeting EGF in immune therapy. These agents can be used in conjunction with stem cell therapy to treat GBM.     

BET bromodomain proteins are required for glioblastoma cell proliferation

Epigenetic proteins have recently emerged as novel anticancer targets. Among these, bromodomain and extra terminal domain (BET) proteins recognize lysine-acetylated histones, thereby regulating gene expression. Newly described small molecules that inhibit BET proteins BRD2, BRD3, and BRD4 reduce proliferation of NUT (nuclear protein in testis)-midline carcinoma, multiple myeloma, and leukemia cells in vitro and in vivo. These findings prompted us to determine whether BET proteins may be therapeutic targets in the most common primary adult brain tumor, glioblastoma (GBM). We performed NanoString analysis of GBM tumor samples and controls to identify novel therapeutic targets. Several cell proliferation assays of GBM cell lines and stem cells were used to analyze the efficacy of the drug I-BET151 relative to temozolomide (TMZ) or cell cycle inhibitors. Lastly, we performed xenograft experiments to determine the efficacy of I-BET151 in vivo. We demonstrate that BRD2 and BRD4 RNA are significantly overexpressed in GBM, suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. I-BET151 treatment enriched cells at the G1/S cell cycle transition. Importantly, I-BET151 is as potent at inhibiting GBM cell proliferation as TMZ, the current chemotherapy treatment administered to GBM patients. Since I-BET151 inhibits GBM cell proliferation by arresting cell cycle progression, we propose that BET protein inhibition may be a viable therapeutic option for GBM patients suffering from TMZ resistant tumors.     

BET bromodomain proteins are required for glioblastoma cell proliferation

Epigenetic proteins have recently emerged as novel anticancer targets. Among these, bromodomain and extra terminal domain (BET) proteins recognize lysine-acetylated histones, thereby regulating gene expression. Newly described small molecules that inhibit BET proteins BRD2, BRD3, and BRD4 reduce proliferation of NUT (nuclear protein in testis)-midline carcinoma, multiple myeloma, and leukemia cells in vitro and in vivo. These findings prompted us to determine whether BET proteins may be therapeutic targets in the most common primary adult brain tumor, glioblastoma (GBM). We performed NanoString analysis of GBM tumor samples and controls to identify novel therapeutic targets. Several cell proliferation assays of GBM cell lines and stem cells were used to analyze the efficacy of the drug I-BET151 relative to temozolomide (TMZ) or cell cycle inhibitors. Lastly, we performed xenograft experiments to determine the efficacy of I-BET151 in vivo. We demonstrate that BRD2 and BRD4 RNA are significantly overexpressed in GBM, suggesting that BET protein inhibition may be an effective means of reducing GBM cell proliferation. Disruption of BRD4 expression in glioblastoma cells reduced cell cycle progression. Similarly, treatment with the BET protein inhibitor I-BET151 reduced GBM cell proliferation in vitro and in vivo. I-BET151 treatment enriched cells at the G1/S cell cycle transition. Importantly, I-BET151 is as potent at inhibiting GBM cell proliferation as TMZ, the current chemotherapy treatment administered to GBM patients. Since I-BET151 inhibits GBM cell proliferation by arresting cell cycle progression, we propose that BET protein inhibition may be a viable therapeutic option for GBM patients suffering from TMZ resistant tumors.     

Sonic hedgehog pathway is essential for maintenance of cancer stem-like cells in human gastric cancer

Abnormal activation of the Sonic hedgehog (SHH) pathway has been described in a wide variety of human cancers and in cancer stem cells (CSCs), however, the role of SHH pathway in gastric CSCs has not been reported. In this study, we investigated the possibility that abnormal activation of the SHH pathway maintained the characteristics of gastric CSCs. First, we identified cancer stem-like cells (CSLCs) from human gastric cancer cell lines (HGC-27, MGC-803 and MKN-45) using tumorsphere culture. Compared with adherent cells, the floating tumorsphere cells had more self-renewing capacity and chemoresistance. The cells expressing CSCs markers (CD44, CD24 and CD133) were also significantly more in tumorsphere cells than in adherent cells. More importantly, in vivo xenograft studies showed that tumors could be generated with 2×10⁴ tumorsphere cells, which was 100-fold less than those required for tumors seeding by adherent cells. Next, RT-PCR and Western blot showed that the expression levels of Ptch and Gli1 (SHH pathway target genes) were significantly higher in tumorsphere cells than in adherent cells. The results of quantitative real-time PCR were similar to those of RT-PCR and Western blot. Further analysis revealed that SHH pathway blocked by cyclopamine or 5E1 caused a higher reduction in self-renewing capacity of HGC-27 tumorsphere cells than that of adherent cells. We also found that SHH pathway blocking strongly enhanced the efficacy of chemotherapeutic drugs in HGC-27 tumorsphere cells in vitro and in vivo but had no significant effect in adherent cells. Finally, we isolated the tumorspheres from gastric cancer specimen, these cells also had chemoresistance and tumorigenic capacity, and SHH pathway maintained the gastric CSLCs characteristics of tumorsphere cells from primary tumor samples. In conclusion, our data suggested that SHH pathway was essential for maintenance of CSLCs in human gastric cancer.     

Serum-derived extracellular vesicles facilitate temozolomide resistance in glioblastoma through a HOTAIR-dependent mechanism

Extracellular vesicle (EV)-mediated transfer of long non-coding RNAs (lncRNAs) has been reported to regulate chemoresistance in various cancers. We herein investigate the therapeutic potential of bioinformatically identified HOTAIR transferred by serum-derived EVs (serum-EVs) in temozolomide (TMZ) resistance of glioblastoma (GBM) and the downstream mechanisms. EVs were isolated from the serum of GBM patients. Expression of HOTAIR was examined in the clinical tissue samples and serum-EVs of GBM patients. The downstream miRNAs of HOTAIR and its target genes were predicted in silico. The effects of the HOTAIR transmitted by serum-EVs in malignant phenotypes, tumor growth, and TMZ resistance were assessed in vitro and in vivo. HOTAIR expression was upregulated in clinical tissues, cells, and serum-EVs of GBM. Co-culture data showed that GBM-serum-EVs facilitated GBM cell proliferative and invasive phenotypes and TMZ resistance by elevating HOTAIR. In GBM cells, HOTAIR competitively bound to miR-526b-3p and weakened miR-526b-3p’s binding ability to EVA1, thus increasing the expression of EVA1. Furthermore, HOTAIR carried by serum-EVs promoted tumor growth and TMZ resistance in vivo by suppressing miR-526b-3p-mediated EVA1 inhibition. GBM-serum-EV-enclosed HOTAIR may augment GBM progression and chemoresistance through miR-526b-3p downregulation and EVA1 upregulation. These results provide a strategy to reduce TMZ resistance in GBM treatment.Subject terms: Neuroscience, Diseases     

Assessment of genetic markers and glioblastoma stem-like cells in activation of dendritic cells

Glioblastoma (GBM) is the most common and aggressive intraparenchymal primary brain tumor in adults. The principal reasons for the poor outcomes of GBM are the high rates of recurrence and resistance to chemotherapy. The aim of this study was to determine the role of tailored cellular therapy for GBM with a poor prognosis and compare the activity of dendritic cells (DCs) that have encountered GBM cells. Detecting the correlations between methylation and expression of MGMT and PTEN genes and GBM cancer stem cells (CSCs) markers after co-cultures with a mononuclear cell cocktail are also aims for this study. Allogenic umbilical cord blood (UCB)-derived DCs were labeled with the CD11a and CD123 for immature DCs, and CD80 and CD11c for mature DCs. CD34, CD45, and CD56 cells were isolated from allogenic UCB for using in DCs maturation. GBM CSCs were detected with CD133/1 and CD111 antibodies after co-culture studies. DC activation was carried out via GBM cells including CD133 and CD111 cells and a mononuclear cells cocktail including CD34, CD45, and CD56 natural killer cells. Real-time PCR was performed to detect the expression and promoter methylation status of PTEN and MGMT genes. The expression of CSCs markers was found in all GBM cases, and a statistically significant correlation was found among them after co-culture studies. The most pronounced affinity of DCs to GBM cells was observed at dilutions between 1/4 and 1/256 in co-cultures. There was a statistically significant correlation between cellularity and granularity ratios for CD123 and CD11c. PTEN and MGMT gene expression and methylation values were evaluated with respect to CSCs expression and no statistical significance was found. Activation of DCs might associate with CSCs and the mononuclear cells cocktail including CD34, CD45, and CD56 cells which were obtained from allogenic UCB.