Dual blockade of EGFR and PI3K signaling pathways offers a therapeutic strategy for glioblastoma.

BACKGROUND: Glioblastoma multiforme (GBM) is a devastating disease that lacks effective drugs for targeted therapy. Previously, we found that the third-generation epidermal growth factor receptor (EGFR) inhibitor AZD-9291 persistently blocked the activation of the ERK pathway but had no inhibitory effect on the phosphoinositide 3-kinase (PI3K)/Akt pathway. Given that the PI3K inhibitor GDC-0084 is being evaluated in phase I/II clinical trials of GBM treatment, we hypothesized that combined inhibition of the EGFR/ERK and PI3K/Akt pathways may have a synergistic effect in the treatment of GBM. METHODS: The synergistic effects of cotreatment with AZD-9291 and GDC-0084 were validated using cell viability assays in GBM and primary GBM cell lines. Moreover, the underlying inhibitory mechanisms were assessed through colony formation, EdU proliferation, and cell cycle assays, as well as RNA-seq analyses and western blot. The therapeutic effects of the drug combination on tumor growth and survival were investigated in mice bearing tumors using subcutaneously or intracranially injected LN229 xenografts. RESULTS: Combined treatment with AZD-9291 and GDC-0084 synergistically inhibited the proliferation and clonogenic survival, as well as induced cell cycle arrest of GBM cells and primary GBM cells, compared to monotherapy. Moreover, AZD-9291 plus GDC-0084 combination therapy significantly inhibited the growth of subcutaneous tumors and orthotopic brain tumor xenografts, thus prolonging the survival of tumor-bearing mice. More importantly, the combination of AZD-9291 and GDC-0084 simultaneously blocked the activation of the EGFR/MEK/ERK and PI3K/AKT/mTOR signaling pathways, thereby exerting significant antitumor activity. CONCLUSION: Our findings demonstrate that the combined blockade of the EGFR/MEK/ERK and PI3K/AKT/mTOR pathways is more effective against GBM than inhibition of each pathway alone, both in vitro and in vivo. Our results suggest that AZD-9291 combined with GDC-0084 may be considered as a potential treatment strategy in future clinical trials. Video Abstract.

Corrigendum: MELK Inhibition Effectively Suppresses Growth of Glioblastoma and Cancer Stem-Like Cells by Blocking AKT and FOXM1 Pathways.

[This corrects the article DOI: 10.3389/fonc.2020.608082.].

GRP78 blockade overcomes intrinsic resistance to UBA1 inhibitor TAK-243 in glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor of the central nervous system. Despite continuous progression in treatment options for GBM like surgery, radiotherapy, and chemotherapy, this disease still has a high rate of recurrence. The endoplasmic reticulum (ER) stress pathway is associated with chemotherapeutic drug resistance. The UBA1 inhibitor TAK-243 can induce strong ER stress. However, the sensitivity of TAK-243 varies greatly in different tumor cells. This study evaluated the antitumor effects of the GRP78 inhibitor, HA15, combined with TAK-243 on GBM in the preclinical models. HA15 synergistically enhanced the sensitivity of GBM cells to TAK-243. When compared with TAK-243 monotherapy, HA15 combined with TAK-243 significantly inhibited GBM cell proliferation. It also induced G2/M-phase arrest in the cell cycle. In vivo studies showed that HA15 combined with TAK-243 significantly inhibited the growth of intracranial GBM and prolonged survival of the tumor-bearing mice. Mechanistically, HA15 and TAK-243 synergistically activated the PERK/ATF4 and IRE1α/XBP1 signaling axes, thereby eventually activating PARP and the Caspase families, which induced cell apoptosis. Our data provided a new strategy for improving the sensitivity of GBM to TAK-243 treatment and experimental basis for further clinical trials to evaluate this combination therapy.

GRP78 determines glioblastoma sensitivity to UBA1 inhibition-induced UPR signaling and cell death.

Glioblastoma multiforme (GBM) is an extremely aggressive brain tumor for which new therapeutic approaches are urgently required. Unfolded protein response (UPR) plays an important role in the progression of GBM and is a promising target for developing novel therapeutic interventions. We identified ubiquitin-activating enzyme 1 (UBA1) inhibitor TAK-243 that can strongly induce UPR in GBM cells. In this study, we evaluated the functional activity and mechanism of TAK-243 in preclinical models of GBM. TAK-243 significantly inhibited the survival, proliferation, and colony formation of GBM cell lines and primary GBM cells. It also revealed a significant anti-tumor effect on a GBM PDX animal model and prolonged the survival time of tumor-bearing mice. Notably, TAK-243 more effectively inhibited the survival and self-renewal ability of glioblastoma stem cells (GSCs) than GBM cells. Importantly, we found that the expression level of GRP78 is a key factor in determining the sensitivity of differentiated GBM cells or GSCs to TAK-243. Mechanistically, UBA1 inhibition disrupts global protein ubiquitination in GBM cells, thereby inducing ER stress and UPR. UPR activates the PERK/ATF4 and IRE1α/XBP signaling axes. These findings indicate that UBA1 inhibition could be an attractive strategy that may be potentially used in the treatment of patients with GBM, and GRP78 can be used as a molecular marker for personalized treatment by targeting UBA1.

Csnk1a1 inhibition modulates the inflammatory secretome and enhances response to radiotherapy in glioma.

Glioblastoma multiforme (GBM), a fatal brain tumour with no available targeted therapies, has a poor prognosis. At present, radiotherapy is one of the main methods to treat glioma, but it leads to an obvious increase in inflammatory factors in the tumour microenvironment, especially IL-6 and CXCL1, which plays a role in tumour to resistance radiotherapy and tumorigenesis. Casein kinase 1 alpha 1 (CK1α) (encoded on chromosome 5q by Csnk1a1) is considered an attractive target for Tp53 wild-type acute myeloid leukaemia (AML) treatment. In this study, we evaluated the anti-tumour effect of Csnk1a1 suppression in GBM cells in vitro and in vivo. We found that down-regulation of Csnk1a1 or inhibition by D4476, a Csnk1a1 inhibitor, reduced GBM cell proliferation efficiently in both Tp53 wild-type and Tp53-mutant GBM cells. On the contrary, overexpression of Csnk1a1 promoted cell proliferation and colony formation. Csnk1a1 inhibition improved the sensitivity to radiotherapy. Furthermore, down-regulation of Csnk1a1 reduced the production and secretion of pro-inflammatory factors. In the preclinical GBM model, treatment with D4476 significantly inhibited the increase in pro-inflammatory factors caused by radiotherapy and improved radiotherapy sensitivity, thus inhibiting tumour growth and prolonging animal survival time. These results suggest targeting Csnk1a1 exert an anti-tumour role as an inhibitor of inflammatory factors, providing a new strategy for the treatment of glioma.

MELK Inhibition Effectively Suppresses Growth of Glioblastoma and Cancer Stem-Like Cells by Blocking AKT and FOXM1 Pathways.

Glioblastoma multiforme (GBM) is a devastating disease yet no effective drug treatment has been established to date. Glioblastoma stem-like cells (GSCs) are insensitive to treatment and may be one of the reasons for the relapse of GBM. Maternal embryonic leucine zipper kinase gene (MELK) plays an important role in the malignant proliferation and the maintenance of GSC stemness properties of GBM. However, the therapeutic effect of targeted inhibition of MELK on GBM remains unclear. This study analyzed the effect of a MELK oral inhibitor, OTSSP167, on GBM proliferation and the maintenance of GSC stemness. OTSSP167 significantly inhibited cell proliferation, colony formation, invasion, and migration of GBM. OTSSP167 treatment reduced the expression of cell cycle G2/M phase-related proteins, Cyclin B1 and Cdc2, while up-regulation the expression of p21 and subsequently induced cell cycle arrest at the G2/M phase. OTSSP167 effectively prolonged the survival of tumor-bearing mice and inhibited tumor cell growth in in vivo mouse models. It also reduced protein kinase B (AKT) phosphorylation levels by OTSSP167 treatment, thereby disrupting the proliferation and invasion of GBM cells. Furthermore, OTSSP167 inhibited the proliferation, neurosphere formation and self-renewal capacity of GSCs by reducing forkhead box M1 (FOXM1) phosphorylation and transcriptional activity. Interestingly, the inhibitory effect of OTSSP167 on the proliferation of GSCs was 4-fold more effective than GBM cells. In conclusion, MELK inhibition suppresses the growth of GBM and GSCs by double-blocking AKT and FOXM1 signals. Targeted inhibition of MELK may thus be potentially used as a novel treatment for GBM.

[DESIGN AND CLINICAL APPLICATION OF LESSER TROCHANTERIC REDUCTION FIXATION SYSTEM].

OBJECTIVE: To design and produce a lesser trochanteric reduction fixation system and verify its value and effectiveness. METHODS: A lesser trochanteric reduction fixation system was designed and produced according to the anatomical features of the lesser trochanteric fractures. Sixty-six patients with intertrochanteric fractures of Evans type III were included between January 2010 and July 2012. Of 66 patients, 32 were treated with dynamic hip screw (DHS) assisted with the lesser trochanteric reduction fixation system (study group), and 34 cases were treated with DHS only (control group). The 2 groups were comparable with no significant difference in gender, age, the reasons, and the types of the fractures (P > 0.05). The operation time, intraoperative blood loss, neck-shaft angle, bone healing time, ratio of successful fixations, and the functional evaluation of the hip joint after operation were compared between 2 groups. RESULTS: The study group had shorter operation time [(58.4 ± 5.3) minutes] and less intraoperative blood loss [(186.3 ± 6.6) mL than the control group [(78.5 ± 6.2)minutes and (246.2 ± 8.7) mL], showing significant differences (t = -14.040, P = 0.000; t = -31.145, P = 0.000). There was no significant difference in neck-shaft angle between study group [(138.6 ± 3.0)] and control group [(139.4 ± 2.9) degrees] (t = -1.044, P = 0.301). The wounds healed by first intention in both groups. The 30 and 31 patients were followed up 12 to 24 months (mean, 15 months) in the study group, and 13 to 25 months (mean, 16 months) in the control group, respectively. All fractures healed well in 2 groups. The study group had significantly shorter healing time [(8.8 ± 2.0) weeks] than the control group [(10.7 ± 3.4) weeks] (t = -2.871, P = 0.006). At 12 months after operation, coxa vara happened in 2 cases of the study group with a successful fixation ratio of 93.3% and in 10 cases of the control group with a successful fixation ratio of 67.7%, showing significant difference (Χ2 = 6.319, P = 0.022). According to Harris hip score, the excellent and good rate was 83.3% in the study group (25/30) and was 58.1% in the control group (18/31), showing significant difference (Χ2 = 4.680, P = 0.049). CONCLUSION: The application of the lesser trochanteric reduction fixation system can reduce stripping of the soft tissue around the fracture fragments, shorten the operation time and the healing time, and preserve the function of the hip joint maximumly.

Effects of myocardial transplantation of marrow mesenchymal stem cells transfected with vascular endothelial growth factor for the improvement of heart function and angiogenesis after myocardial infarction.

OBJECTIVE: To establish the transfection method of vascular endothelial growth factor (VEGF) gene into mesenchymal stem cells (MSCs), to investigate the effect of this gene-transfected MSCs for heart function restoration and angiogenesis after myocardial infarction, and to compare the therapeutic differences among cell therapy, gene therapy, and combined therapy. METHODS: Ischemic heart models were constructed in inbred Wistar rats by ligation of the left anterior descending coronary artery. MSCs of Wistar rats were isolated by density gradient centrifugation and purified on the basis of their ability to adhere to plastic, and identified by checking the surface markers and their differentiation capacity, and then followed by transfection of pcDNA(3.1)-hVEGF(165) using the liposome-mediated method. The expression of hVEGF(165) in the transfected cells was detected by Enzyme-Linked Immunosorbent Assay, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western Blot Analysis. The ligated animals were randomly divided into four groups (12 in each) and, after 2 weeks, were injected at the heart infarct zone with hVEGF(165)-transfected MSCs (Combo group), MSCs (Cell group), liposome-hVEGF gene plasmid (Gene group), or medium (Control group). And other six ligated rats (without any injection) were used as Model-assessment group for the baseline heart infarcted size evaluation, and other 12 non-ligated rats (Non-ischemic group) were used as the normal control. Four weeks after the injection, the rats' cardiac function was measured by the Buxco system. Brdu and Troponin-T double labeling and factor VIII were identified by immunohistochemical staining to demonstrate the survival and differentiation of engrafted cells or to evaluate the angiogenesis in the injured heart area; heart infarcted size was calculated by Evan's blue staining. VEGF expression was evaluated by RT-PCR. RESULTS: MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation. The cultured MSCs were CD34-, CD45-, CD44+ and SH+. They can differentiate into osteoblasts and adipocytes successfully. The expression of hVEGF(165) in the transfected MSCs was demonstrated with Enzyme-Linked Immunosorbent Assay, RT-PCR and Western Blot Assay. Four weeks after the cells were transplanted, among all groups but the Non-ischemic group, the Combo group had the smallest heart infarcted size and the best heart function. The capillary density of the Combo group was significantly greater than those of both Cell and Control groups. The heart infarcted size, heart function and capillary density of both Cell and Gene groups were similar with each other and smaller, better and greater than those of the Control group, respectively. Brdu and Troponin-T double staining detected a varied increase in the number of survived cardiomyocytes at the heart infarcted area, some of which were double stain positive. RT-PCR showed that the hVEGF(165) gene was expressed in the Combo and Gene groups, and that the former was higher than the latter. CONCLUSIONS: Eukaryotic expression vector pcDNA(3.1)-hVEGF(165) can effectively be expressed in MSCs. Transplantation of VEGF gene-transfected MSCs can bring better improvement in myocardial perfusion and in restoration of heart function than either cellular or gene therapy alone.

Transfection of human VEGF165 gene into bone marrow mesenchymal stem cells in rats.

OBJECTIVE: To create a method for transfecting human vascular endothelial growth factor165 (hVEGF165) gene into bone marrow mesenchymal stem cells (MSCs) in rats. METHODS: MSCs of Wistar rats were isolated by density gradient centrifugation and purified based on their ability of adhesion to plastic. Detections of cell surface antigens, including CD34, CD45, CD44, and SH3, were performed using flow cytometry. MSCs' potential of differentiating into osteoblast and lipoblast in vitro was tested. The vector pcDNA(3.1)-hVEGF165 was transfected into MSCs with the liposome mediated method. The expression of hVEGF165 in the transfected cells was detected by enzyme linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analysis. RESULTS: The cultured MSCs were CD34-, CD45-, CD44+ , and SH+, which were differentiated into osteoblasts and lipocytes successfully. The expressed hVEGF165 in the transfected rat MSCs was demonstrated. CONCLUSION: The vector pcDNA(3.1)-hVEGF165 is successfully expressed in MSCs.

[Effects of myocardial transplantation of mesenchymal stem cells transfected with vascular endothelial factor gene on improvement of heart function and angiogenesis after myocardial infarction: experiment with rats].

UNLABELLED: To establish a method to transfect vascular endothelial factor (VEGF) gene into mesenchymal stem cells ( MSCs) , to investigate the effects of the gene-transfected MSCs on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences among cell therapy, gene therapy, and combined therapy. METHODS: Seventy-one Wistar rats underwent ligation of the left anterior descending coronary artery so as to establish heart ischemia models. Fifteen rats underwent sham operation. MSCs were isolated from several Wistar rats by density gradient centrifugation, purified, and transfected with pcDNA3.1-hVEGF165 or blank plasmid pcDNA3.1 respectively using the liposome mediated method. ELISA, Western blotting, and RT-PCR were used to detect the protein and mRNA expression of hVEG in these MSCs Forty-eight surviving rats that underwent ligation were randomly divided into 4 equal groups: combination group (Combo group) to be injected into the heart infarct zone with suspension of hVEGF165-transfected MSCs 2 weeks after the establishment of the model, cell group to be injected with suspension of MSCs not transfected with VEGF, gene group to be injected with suspension of DNA-liposome containing pcDNA3.1-VEGF165 and control group to be injected with cold culture fluid only. Twelve surviving rats that underwent sham operation were used as normal non-ischemic group. Four weeks after the injection the surviving rats underwent examination of heart functions by the Buxco system. The rats were killed and their hearts were taken out to undergo immunohistochemistry with 5-bromodeoxyuridine (Brdu) and troponin T and factor VIII to measure the area of cardiac infarction and the capillary density. RT-PCR was used to examine the mRNA expression of VEGF. The heart infarcted size was calculated by Evan's blue staining. RESULTS: (1) MSCs can be successfully isolated and cultured by density gradient centrifugation followed by adherence-separation. The expression of hVEGF165 in the transfected MSCs was demonstrated with ELISA, RT-PCR and Western Blot Assay. (2) Four weeks after the cells were transplanted, among all groups but the nonischemic group, the heart infarcted size of the Combo group was 27.8% +/- 3. 0% ,significantly less than those of the cell group (37.0% +/- 10. 1% ) and gene group (37.1% +/- 5.2%, both P <0.05). The heart function of the Combo group was better than those of other groups. (3) The capillary density of the Combo group was 40. 2 +/- 5.5/visual field, significantly greater than those of both the cell group (27.2 +/- 6. 3/visual field, P <0. 01) and that of the control group (18.5 +/- 5.8/visual field, P <0. 01) , and greater to some degree than that of the gene group (35. 8 +/-7.7/visual field, P =0. 189). (4)The heart infarcted size, heart function and capillary density of the cell and gene groups were similar and were smaller, better and greater than those of the control group. (5) Brdu and troponin T double staining detected a varied increase in the number of surviving cardiomyocytes at the heart infarcted area, some of which were double stain positive. RT-PCR showed mRNA expression of hVEGF165 in the Combo and gene groups, that in the Combo group being higher than that in the gene group. CONCLUSION: Eukaryotic expression vector pcDNA3.1-hVEGF165 can effectively be expressed in MSCs. Transplantation of VEGF gene by means of transfected MSCs brings better improvement in myocardial perfusion and in restoration of heart function than either cellular or gene therapy alone.