Glioma Resistance Research Articles

Exosomes Derived from Hypoxic Glioma Cells Reduce the Sensitivity of Glioma Cells to Temozolomide Through Carrying miR-106a-5p.

BackgroundHypoxia is a frequent feature of solid tumors which significantly affects the efficacy of treatments such as chemotherapy. In addition, exosomes from hypoxic cancer cells could contribute to the chemoresistance of tumor cells through carrying miRNAs. It has been shown that miR-106-5p level was upregulated in glioma. However, whether exosomes derived from hypoxic glioma cells could affect temozolomide (TMZ) resistance in glioma through carrying miR-106a-5p remains unexplored.MethodsExosomes were isolated from glioma cells under normoxia or hypoxia condition. EdU staining and flow cytometry assays were used to assess the cell proliferation and cell apoptosis. The relation between miR-106a-5p and PTEN was investigated by dual luciferase assay.ResultsMiR-106a-5p was enriched in exosomes derived from hypoxic glioma cells compared to exosomes from cells under normoxia condition. Additionally, hypoxic glioma cells were able to transfer exosomes to glioma cells, resulting in a significant increase of miR-106a-5p level in cells. TMZ remarkably suppressed glioma cell proliferation and triggered cell apoptosis. However, hypoxic glioma cell-derived exosomes markedly promoted the proliferation and suppressed the apoptosis in TMZ-treated glioma cells, and miR-106a-5p inhibitor was able to abolish these phenomena. Meanwhile, PTEN was verified to be a direct target of miR-106a-5p. Furthermore, TMZ elevated PTEN and Bax level and reduced p-Akt level in glioma cells, whereas these changes were reversed by hypoxia glioma cell-derived exosomes. Furthermore, hypoxia glioma cell-derived exosomes reduced the sensitivity of glioma cells to TMZ in vivo via downregulating PTEN.ConclusionCollectively, exosomal miR-106a-5p derived from hypoxia glioma cells could reduce the sensitivity of glioma cells to TMZ through downregulating PTEN. Thus, our study might provide new strategies for improving the clinical efficacy of TMZ on glioma.

From protein-protein interactions to immune modulation: Therapeutic prospects of targeting Neuropilin-1 in high-grade glioma.

In the past several years there has been a marked increase in our understanding of the pathophysiological hallmarks of glioblastoma development and progression, with specific respect to the contribution of the glioma tumor microenvironment to the rapid progression and treatment resistance of high-grade gliomas. Despite these strides, standard of care therapy still only targets rapidly dividing tumor cells in the glioma, and does little to curb the pro-tumorigenic functions of non-cancerous cells entrenched in the glioma microenvironment. This tumor promoting environment as well as the heterogeneity of high-grade gliomas contribute to the poor prognosis of this malignancy. The interaction of non-malignant cells in the microenvironment with the tumor cells accentuate phenotypes such as rapid proliferation or immunosuppression, so therapeutically modulating one target expressed on one cell type may be insufficient to restrain these rapidly developing neoplasias. With this in mind, identifying a target expressed on multiple cell types and understanding how it governs tumor-promoting functions in each cell type may have great utility in better managing this disease. Herein, we review the physiology and pathological effects of Neuropilin-1, a transmembrane co-receptor which mediates signal transduction pathways when associated with multiple other receptors. We discuss its effects on the properties of endothelial cells and on immune cell types within gliomas including glioma-associated macrophages, microglia, cytotoxic T cells and T regulatory cells. We also consider its effects when elaborated on the surface of tumor cells with respect to proliferation, stemness and treatment resistance, and review attempts to target Neuroplin-1 in the clinical setting.

Sequence-structure functional implications and molecular simulation of high deleterious nonsynonymous substitutions in IDH1 revealed the mechanism of drug resistance in glioma.

In the past few years, various somatic point mutations of isocitrate dehydrogenase (IDH) encoding genes (IDH1 and IDH2) have been identified in a broad range of cancers, including glioma. Despite the important function of IDH1 in tumorigenesis and its very polymorphic nature, it is not yet clear how different nsSNPs affect the structure and function of IDH1. In the present study, we employed different machine learning algorithms to screen nsSNPs in the IDH1 gene that are highly deleterious. From a total of 207 SNPs, all of the servers classified 80 mutations as deleterious. Among the 80 deleterious mutations, 14 were reported to be highly destabilizing using structure-based prediction methods. Three highly destabilizing mutations G15E, W92G, and I333S were further subjected to molecular docking and simulation validation. The docking results and molecular simulation analysis further displayed variation in dynamics features. The results from molecular docking and binding free energy demonstrated reduced binding of the drug in contrast to the wild type. This, consequently, shows the impact of these deleterious substitutions on the binding of the small molecule. PCA (principal component analysis) and FEL (free energy landscape) analysis revealed that these mutations had caused different arrangements to bind small molecules than the wild type where the total internal motion is decreased, thus consequently producing minimal binding effects. This study is the first extensive in silico analysis of the IDH1 gene that can narrow down the candidate mutations for further validation and targeting for therapeutic purposes.

Ferroptosis: A novel therapeutic strategy and mechanism of action in glioma.

Glioma is the most common malignant tumor of the central nervous system and resistance is easily developed to chemotherapy drugs during the treatment process, resulting in high mortality and short survival in glioma patients. Novel therapeutic approaches are urgently needed to improve the therapeutic efficacy of chemotherapeutic drugs and to improve the prognosis of patients with glioma. Ferroptosis is a novel regulatory cell death mechanism that plays a key role in cancer, neurodegenerative diseases, and other diseases. Studies have found that ferroptosis-related regulators are closely related to the survival of patients with glioma, and induction of ferroptosis can improve glioma resistance to chemotherapy drugs. Therefore, induction of tumor cell ferroptosis may be an effective therapeutic strategy for glioma. This review summarizes the relevant mechanisms of ferroptosis, systematically summarizes the key role of ferroptosis in the treatment of glioma and outlines the relationship between ferroptosis-related ncRNAs and the progression of glioma.

A novel hypoxia-driven gene signature that can predict the prognosis and drug resistance of gliomas.

Hypoxia spontaneously forms in the interior of glioma tissues and regulates the expression of various genes. However, the status of hypoxia-driven genes in glioma tissues is not completely known. In the current study, RNA-seq data of 695 glioma tissues in The Cancer Genome Atlas (TCGA) were set as a discovery cohort and were used to identify hypoxia-driven genes and construct a novel gene signature. The prognostic values of that signature were verified in data from the TCGA and the Chinese Glioma Genome Atlas (CGGA). The expression and diagnostic values of hypoxia-driven genes were analyzed using immunohistochemistry and receiver operator characteristic curves. Finally, the effects of hypoxia-driven genes on temozolomide (TMZ) resistance were analyzed by western blot, CCK-8 and colony formation assay. A total of 169 hypoxia-driven genes were identified, which were associated with a poor outcome in glioma patients. Among them, 22 genes had a degree score ≥10 and 6 genes (WT1, HOXA2, HOXC6, MMP9, SHOX2 and MYOD1) were selected to construct a signature to classify glioma patients into low- or high-risk groups. That signature had a remarkable prognostic value for glioma patients in TCGA and CGGA. The expression of HOXC6, MMP9, SHOX2 and MYOD1 was associated with hypoxia degree in glioma tissues and in recurrent cases, had a remarkable diagnostic value and a significant relationship with disease free survival in glioma patients. Moreover, SHOX2 was highly expressed in glioma tissues with O-6-methylguanine-DNA methyltransferase (MGMT)-unmethylation and temozolomide (TMZ) resistant glioma cell lines, and associated with MGMT expression. Knockdown the expression of SHOX2 significantly reduced the TMZ-resistance induced by hypoxia in glioma cells. Ultimately, we identified six novel hypoxia-driven genes for reliable prognostic prediction in gliomas and found that SHOX2 might be a potential target to overcome the TMZ resistance induced by hypoxia.

Anoikis resistance in diffuse glioma: The potential therapeutic targets in the future.

Glioma is the most common malignant intracranial tumor and exhibits diffuse metastasis and a high recurrence rate. The invasive property of glioma results from cell detachment. Anoikis is a special form of apoptosis that is activated upon cell detachment. Resistance to anoikis has proven to be a protumor factor. Therefore, it is suggested that anoikis resistance commonly occurs in glioma and promotes diffuse invasion. Several factors, such as integrin, E-cadherin, EGFR, IGFR, Trk, TGF-β, the Hippo pathway, NF-κB, eEF-2 kinase, MOB2, hypoxia, acidosis, ROS, Hsp and protective autophagy, have been shown to induce anoikis resistance in glioma. In our present review, we aim to summarize the underlying mechanism of resistance and the therapeutic potential of these molecules.

High expression of HOXA4 in patients with glioma indicates unfavorable clinical outcomes

ABSTRACT HOXA4 is a novel oncogene that has been observed in many kinds of tumors, but its role during glioma carcinogenesis and its clinical significance in diagnosing and prognosis human glioma remains unknown. In the present study, the Chinese Glioma Atlas (CGGA)-RNA sequencing database, CGGA microarray, and The Cancer Genome Atlas (TCGA)-RNA seq data from 1674 glioma patients were obtained from online databases and analyzed using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) to detect changes in the expression level of HOXA4 and characterize the relationship between HOXA4 and the clinical characteristics and prognosis of patients with glioma. Gene set enrichment analysis (GSEA) was used to reveal how HOXA4 regulates tumor-related pathways. HOXA4 mRNA levels in glioma tissue were higher than those in adjacent brain tissue. HOXA4 expression was also closely related to the clinical and molecular characteristics of gliomas, such as tumor grade and isocitrate dehydrogenase (IDH) mutation. Functional enrichment analysis revealed that HOXA4 could regulate cancer-related signal pathways, such as Cell cycle, Cell adhesion molecules cams, and JAK/STAT signaling pathway. Results of in vitro experiments confirmed that knockdown of HOXA4 blocks the cell cycle pathway and inhibits the proliferation, invasion and chemotherapy resistance in gliomas. We concluded that HOXA4 was an independent risk factor for glioma and may have clinical diagnostic potential. Meanwhile, our findings revealed that HOXA4 could be used as a biomarker for glioma diagnosis and treatment.

Nonhematogenic circulating aneuploid cells confer inferior prognosis and therapeutic resistance in gliomas

Aneuploidy is the hallmark of malignancy. Our previous study successfully detected nonhematogenic circulating aneuploidy cells (CACs) in types of gliomas. The current prospective clinical study aims to further precisely subcategorize aneuploid CACs, including CD31− circulating tumor cells (CTCs) and CD31+ circulating tumor endothelial cells, and thoroughly investigate the clinical utilities of these different subtypes of cells. Co‐detection and analysis of CTCs and circulating tumor‐derived endothelial cells (CTECs) expressing CD133, glial fibrillary acidic protein (GFAP), or epidermal growth factor receptor variant III (EGFR vIII) were performed by integrated subtraction enrichment and immunostaining fluorescence in situ hybridization (SE‐iFISH) in 111 preoperative primary diffuse glioma patients. Aneuploid CACs could be detected in most de novo glioma patients. Among detected CACs, 45.6% were CD31−/CD45− aneuploid CTCs and the remaining 54.4% were CD31+/CD45− aneuploid CTECs. Positive detection of CTECs significantly correlated with disruption of the blood–brain barrier. The median number of large CTCs (LCTCs, >5 μm, 2) in low‐grade glioma (WHO grade 2) was less than high‐grade glioma (WHO grades 3 and 4) (3, p = 0.044), but this difference was not observed in small CTCs (SCTCs, ≤5 μm), CTECs or CACs (CTCs + CTECs). The numbers of CTCs, CTECs, or CACs in patients with contrast‐enhancing (CE) lesions considerably exceeded that of non‐CE lesions (p < 0.05). Receiver operating characteristic curves demonstrated that CD31+ CTECs, especially LCTECs, exhibited a close positive relationship with CE lesions. Survival analysis revealed that the high number of CD31− CTCs could be an adverse factor for compromised progression‐free survival and overall survival. Longitudinal surveillance of CD31− CTCs was suitable for evaluating the therapeutic response and for monitoring potential emerging treatment resistance.

Interferon-γ resistance and immune evasion in glioma develop via Notch-regulated co-evolution of malignant and immune cells.

Immune surveillance is critical to prevent tumorigenesis. Gliomas evade immune attack, but the underlying mechanisms remain poorly understood. We show that glioma cells can sustain growth independent of immune system constraint by reducing Notch signaling. Loss of Notch activity in a mouse model of glioma impairs MHC-I and cytokine expression and curtails the recruitment of anti-tumor immune cell populations in favor of immunosuppressive tumor-associated microglia/macrophages (TAMs). Depletion of Tcells simulates Notch inhibition and facilitates tumor initiation. Furthermore, Notch-depleted glioma cells acquire resistance to interferon-γ and TAMs re-educating therapy. Decreased interferon response and cytokine expression by human and mouse glioma cells correlate with low Notch activity. These effects are paralleled byupregulation of oncogenes anddownregulation of quiescence genes. Hence, suppression of Notch signaling enables gliomas to evade immune surveillance and increases aggressiveness. Our findings provide insights into how brain tumor cells shape their microenvironment to evade immune niche control.

Mechanisms for hypoxia-induced long non-coding small nucleolar RNA host gene 14 in promoting temozolomide resistance of glioma.

This study aims to investigate the role of hypoxia-induced long non-coding small nucleolar RNA host gene 14 (lncRNA SNHG14) in glioma temozolomide (TMZ) resistance and underlying mechanisms. According to different treatments, the experiment was divided into a normoxia group and a hypoxia group, a control group and a TMZ group. The lncRNA SNHG14 and O6-methylguanine DNA methyltransferase (MGMT) levels in glioma SNB19 and U251 cell line were detected by real-time PCR and Western blotting, respectively, and the association of lncRNA SNHG14 level with hypoxia and TMZ treatment was analyzed. siRNA was used to knockdown the lncRNA SNHG14 expression in glioma cells, and the transfected glioma cells were divided into a negative control group (si-NC group) and a si-SNHG14 group. The interference efficiency was examined by real-time PCR, the key factor MGMT of lncRNA SNHG14 sensitivity regulation was detected by Western blotting, and the cell apoptosis was detected by cytometry. In addition, MTT method was used to detect the cell viability of gliomas in the different groups under the different TMZ concentrations, and the effect of lncRNA SNHG14 on TMZ sensitivity of gliomas was analyzed. Online tools were used to predict miRNAs that could specifically bind to lncRNAs SNHG14 and MGMT. A si-NC group, a si-SNHG14 group, a normoxia group and a hypoxia group were set up, and the changes of miR-143 abundance in different environments were observed by real-time PCR. miR-143 mimics and inhibitor were used to change the level of miR-143 in glioma cells. A NC inhibitor group, a miR-143 inhibitor group, a NC mimics group and a miR-143 mimics group were set up, the interference efficiency was detected by real-time PCR, the expression level of MGMT was detected by Western blotting, and the effect of miR-143 on the level of MGMT were analyzed. The NC inhibitor group, the miR-143 inhibitor group, the NC mimics group and the miR-143 mimics group were treated with different interventions, and the dual luciferase reporter assay was used to observe the changes of lncRNA SNHG14 and MGMT luciferase activities, and to verify the relationship among lncRNA SNHG14, miR-143 and MGMT. Finally, a NC group and a lncRNA SNHG14 overexpression group were set up, and the changes in the abundance of miR-143 and MGMT in each group were detected by RNA-binding protein immunoprecipitation experiments, and the competitive binding relationship among lncRNA SNHG14, miR-143 and MGMT was analyzed. Compared with the normoxia group, the hypoxia group could promote the expression of lncRNA SNHG14 in glioma cells. Compared with the control group, the expression of lncRNA SNHG14 could be significantly inhibited in the TMZ group (P<0.05). Compared with the si-NC group, the expression of lncRNA SNHG14 in the si-SNHG14 group could be effectively inhibited, and the expression level of MGMT was significantly decreased, and the apoptosis rate was significantly increased (all P<0.05). With the increase of TMZ concentrations, the glioma cell viability in the si-SNHG14 group was significantly lower than that in the si-NC group, and the cell viability in the hypoxia group was significantly higher than that in the normoxia group (both P<0.05). Online tool prediction found that miR-143 had binding sites with lncRNA SNHG14 and MGMT. The abundance of miR-143 in the hypoxia group was significantly lower than that in the normoxic group, and the abundance of miR-143 in the si-SNHG14 group was significantly higher than that in the si-NC group (both P<0.05). The miR-143 mimics group or the miR-143 inhibitor group could significantly over-express or under-express miR-143 (both P<0.05). But there was no significant difference between the NC mimics group (or the NC inhibitor group) and the control group (both P>0.05). The level of MGMT protein could significantly up-regulate in the miR-143 inhibitor group, and on the contrary which could significantly down-regulate in the miR-143 mimics group (both P<0.01). The dual luciferase reporter assay showed that there was no significant difference between the NC mimics group (or the NC inhibitor group) and the control group (both P>0.05). The wild-type SNHG14 and MGMT luciferase activities were significantly down-regulated in the miR-143 mimics group, which were significantly up-regulated in the miR-143 inhibitor group (P<0.01 and P<0.05, respectively), but there was no significant change in the luciferase activities of mutant SNHG14 and MGMT (both P>0.05). The results of the RNA-binding protein immunoprecipitation experiment showed that: compared with the NC group, more lncRNA SNHG14 was bound to the precipitated argonaute 2 protein in the cells in the lncRNA SNHG14 overexpression group, but the abundance of MGMT mRNA was decreased significantly, and there were significant differences (both P<0.01). There was a targeting regulatory relationship among lncRNA SNHG14, miR-143 and MGMT. The up-regulated lncRNA SNHG14 can target miR-143, relieve the inhibition of miR-143 on MGMT, and promote the TMZ resistance in the hypoxia-induced glioma cells.

HECTD3 enhances cell radiation resistance and migration by regulating LKB1 mediated ZEB1 in glioma.

Homologous to the E6-associated protein carboxyl terminus domain containing 3 (HECTD3) has been reported to play a role in carcinogenesis. Here, we explored the role of HECTD3 in regulating the radiation resistance of glioma, and the underlying mechanism. HECTD3 expressions in glioma tissues were assessed using Western blotting, quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and immunohistochemistry. Glioma cells were exposed to 2-, 4-, 6- or 8-Gy X-ray to mimic the radiation treatment. Cell count kit-8 (CCK-8), clone formation assay, flow cytometry assay, transwell chambers and animal assay were used to test cell viability, apoptosis, migration, invasiveness and tumourigenesis, respectively. HECTD3 expression was increased in glioma tissues, especially from patients with radiation resistance. Knockdown of HECTD3 promoted cell apoptosis and inhibited cell viability under the condition of 8-Gy X-ray, as well as suppressed cell migration and invasiveness. In mechanism, HECTD3 positively regulated ZEB1 (zinc finger E-box binding hemeobox 1) expression through regulating the ubiquitination of liver kinase B1 (LKB1) protein. Overexpression of ZEB1 significantly abolished the effects of HECTD3 downregulation in inhibiting the radiation resistance and migration of glioma cells. Moreover, downregulation of HECTD3 further enhanced the anti-tumour effect of X-ray on glioma growth in vivo. In conclusion, HECTD3 was overexpressed in glioma patients with radiation resistance. Knockdown of HECTD3 sensitized glioma cells to radiation and inhibited cell migration by downregulating ZEB1 expression via regulating the ubiquitination of LKB1 protein. This study reveals that HECTD3 might be a potent target to enhance the radiation sensitivity of glioma.

Regulation of temozolomide resistance via lncRNAs: Clinical and biological properties of lncRNAs in gliomas (Review).

Gliomas are a primary types of intracranial malignancies and are characterized by a poor prognosis due to aggressive recurrence profiles. Temozolomide (TMZ) is an auxiliary alkylating agent that is extensively used in conjunction with surgical resection and forms the mainstay of clinical treatment strategies for gliomas. However, the frequent occurrence of TMZ resistance in clinical practice limits its therapeutic efficacy. Accumulating evidence has demonstrated that long non-coding RNAs (lncRNAs) can play key and varied roles in glioma progression. lncRNAs have been reported to inhibit glioma progression by targeting various signaling pathways. In addition, the differential expression of lncRNAs has also been found to mediate the resistance of glioma to several chemotherapeutic agents, particularly to TMZ. The present review article therefore summarizes the findings of previous studies in an aim to report the significance and function of lncRNAs in regulating the chemoresistance of gliomas. The present review may provide further insight into the clinical treatment of gliomas.

High levels of NRF2 sensitize temozolomide-resistant glioblastoma cells to ferroptosis via ABCC1/MRP1 upregulation

Glioblastoma patients have a poor prognosis mainly due to temozolomide (TMZ) resistance. NRF2 is an important transcript factor involved in chemotherapy resistance due to its protective role in the transcription of genes involved in cellular detoxification and prevention of cell death processes, such as ferroptosis. However, the relation between NRF2 and iron-dependent cell death in glioma is still poorly understood. Therefore, in this study, we analyzed the role of NRF2 in ferroptosis modulation in glioblastoma cells. Two human glioblastoma cell lines (U251MG and T98G) were examined after treatment with TMZ, ferroptosis inducers (Erastin, RSL3), and ferroptosis inhibitor (Ferrostatin-1). Our results demonstrated that T98G was more resistant to chemotherapy compared to U251MG and showed elevated levels of NRF2 expression. Interestingly, T98G revealed higher sensitivity to ferroptosis, and significant GSH depletion upon system xc− blockage. NRF2 silencing in T98G cells (T98G-shNRF2) significantly reduced the viability upon TMZ treatment. On the other hand, T98G-shNRF2 was resistant to ferroptosis and reverted intracellular GSH levels, indicating that NRF2 plays a key role in ferroptosis induction through GSH modulation. Moreover, silencing of ABCC1, a well-known NRF2 target that diminishes GSH levels, has demonstrated a similar collateral sensitivity. T98G-siABCC1 cells were more sensitive to TMZ and resistant to Erastin. Furthermore, we found that NRF2 positively correlates with ABCC1 expression in tumor tissues of glioma patients, which can be associated with tumor aggressiveness, drug resistance, and poor overall survival. Altogether, our data indicate that high levels of NRF2 result in collateral sensitivity on glioblastoma via the expression of its pro-ferroptotic target ABCC1, which contributes to GSH depletion when the system xc− is blocked by Erastin. Thus, ferroptosis induction could be an important therapeutic strategy to reverse drug resistance in gliomas with high NRF2 and ABCC1 expression.

Inhibitory effects of temozolomide on glioma cells is sensitized by RSL3-induced ferroptosis but negatively correlated with expression of ferritin heavy chain 1 and ferritin light chain

Invasive growth of glioblastoma makes residual tumor unremovable by surgery and leads to disease relapse. Temozolomide is widely used first-line chemotherapy drug to treat glioma patients, but development of temozolomide resistance is almost inevitable. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is found to be related to temozolomide response of gliomas. However, whether inducing ferroptosis could affect invasive growth of glioblastoma cells and which ferroptosis-related regulators were involved in temozolomide resistance are still unclear. In this study, we treated glioblastoma cells with RSL3, a ferroptosis inducer, in vitro (cell lines) and in vivo (subcutaneous and orthotopic animal models). The treated glioblastoma cells with wild-type or mutant IDH1 were subjected to RNA sequencing for transcriptomic profiling. We then analyze data from our RNA sequencing and public TCGA glioma database to identify ferroptosis-related biomarkers for prediction of prognosis and temozolomide resistance in gliomas. Analysis of transcriptome data from RSL3-treated glioblastoma cells suggested that RSL3 could inhibit glioblastoma cell growth and suppress expression of genes involved in cell cycle. RSL3 effectively reduced mobility of glioblastoma cells through downregulation of critical genes involved in epithelial-mesenchymal transition. Moreover, RSL3 in combination with temozolomide showed suppressive efficacy on glioblastoma cell growth, providing a promising therapeutic strategy for glioblastoma treatment. Although temozolomide attenuated invasion of glioblastoma cells with mutant IDH1 more than those with wild-type IDH1, the combination of RSL3 and temozolomide similarly impaired invasive ability of glioblastoma cells in spite of IDH1 status. Finally, we noticed that both ferritin heavy chain 1 and ferritin light chain predicted unfavorable prognosis of glioma patients and were significantly correlated with mRNA levels of methylguanine methyltransferase as well as temozolomide resistance. Altogether, our study provided rationale for combination of RSL3 with temozolomide to suppress glioblastoma cells and revealed ferritin heavy chain 1 and ferritin light chain as biomarkers to predict prognosis and temozolomide resistance of glioma patients.

Exosomal circWDR62 promotes temozolomide resistance and malignant progression through regulation of the miR-370-3p/MGMT axis in glioma

Exosome-mediated delivery of circular RNAs (circRNAs) is implicated in cancer progression. However, the role of exosomal circRNAs in the chemotherapy resistance of tumours remains poorly understood. Here we identified a novel circRNA, circWDR62. It was found that circWDR62 expression was upregulated in TMZ-resistant glioma cells and TMZ-resistant glioma cell-derived exosomes compared with their controls by using high-throughput microarray analysis and quantitative real-time polymerase chain reaction, and high circWDR62 expression was associated with poor prognosis of glioma. Functionally, downregulation of circWDR62 expression could significantly inhibit the TMZ resistance and malignant progression of glioma. Further mechanistic studies showed that circWDR62 plays a role by sponging miR-370-3p as a competing endogenous RNA. Rescue experiments confirmed that MGMT is the downstream target of the circWDR62/miR-370-3p axis in glioma. In addition, circWDR62 could be transported between TMZ-resistant and TMZ-sensitive glioma cells via exosomes. Exosomal circWDR62 from TMZ-resistant cells conferred TMZ resistance in recipient sensitive cells while also enhancing the proliferation, migration and invasion of these cells. A series of clinical and in vivo trials corroborated that exosomal circWDR62 could promote TMZ chemoresistance and malignant progression of glioma. Our results demonstrate for the first time that exosome-mediated delivery of circWDR62 can promote TMZ resistance and malignant progression via targeting of the miR-370-3p/MGMT axis in vitro and in vivo in glioma, providing a new therapeutic strategy. Moreover, exosomal circWDR62 in human serum may serve as a promising therapeutic target and prognostic marker for glioma therapy.

The Network of Tumor Microtubes: An Improperly Reactivated Neural Cell Network With Stemness Feature for Resistance and Recurrence in Gliomas.

Gliomas are known as an incurable brain tumor for the poor prognosis and robust recurrence. In recent years, a cellular subpopulation with tumor microtubes (TMs) was identified in brain tumors, which may provide a new angle to explain the invasion, resistance, recurrence, and heterogeneity of gliomas. Recently, it was demonstrated that the cell subpopulation also expresses neural stem cell markers and shares a lot of features with both immature neurons and cancer stem cells and may be seen as an improperly reactivated neural cell network with a stemness feature at later time points of life. TMs may also provide a new angle to understand the resistance and recurrence mechanisms of glioma stem cells. In this review, we innovatively focus on the common features between TMs and sprouting axons in morphology, formation, and function. Additionally, we summarized the recent progress in the resistance and recurrence mechanisms of gliomas with TMs and explained the incurability and heterogeneity in gliomas with TMs. Moreover, we discussed the recently discovered overlap between cancer stem cells and TM-positive glioma cells, which may contribute to the understanding of resistant glioma cell subpopulation and the exploration of the new potential therapeutic target for gliomas.

In Vivo Quantitative Imaging of Glioma Heterogeneity Employing Positron Emission Tomography.

Simple SummaryDefining glioma heterogeneity represents a promising strategy to unravel the mechanisms behind therapy resistance and tumor recurrence. The current review provides a comprehensive overview of experimental and clinical data concerning the visualization and quantification of the tumor microenvironment heterogeneity using molecular imaging, with a special emphasis on positron emission tomography (PET).Glioblastoma is the most common primary brain tumor, highly aggressive by being proliferative, neovascularized and invasive, heavily infiltrated by immunosuppressive glioma-associated myeloid cells (GAMs), including glioma-associated microglia/macrophages (GAMM) and myeloid-derived suppressor cells (MDSCs). Quantifying GAMs by molecular imaging could support patient selection for GAMs-targeting immunotherapy, drug target engagement and further assessment of clinical response. Magnetic resonance imaging (MRI) and amino acid positron emission tomography (PET) are clinically established imaging methods informing on tumor size, localization and secondary phenomena but remain quite limited in defining tumor heterogeneity, a key feature of glioma resistance mechanisms. The combination of different imaging modalities improved the in vivo characterization of the tumor mass by defining functionally distinct tissues probably linked to tumor regression, progression and infiltration. In-depth image validation on tracer specificity, biological function and quantification is critical for clinical decision making. The current review provides a comprehensive overview of the relevant experimental and clinical data concerning the spatiotemporal relationship between tumor cells and GAMs using PET imaging, with a special interest in the combination of amino acid and translocator protein (TSPO) PET imaging to define heterogeneity and as therapy readouts.

Abstract 5786: Mitochondrial genome evolution in gliomas under therapy

Abstract Gliomas are the most common malignant brain tumors and characterized by high recurrence rate and therapeutic resistance. Gliomas are clinically separated by mutations in isocitrate dehydrogenase (IDH) genes, but all subtypes have an altered energy metabolism. Mitochondria are essential cellular organelles that mediate many biological processes associated with tumorigenesis, such as energy metabolism and cell death, that can be impacted by mitochondrial mutations and copy number. As part of the Glioma Longitudinal AnalyiSiS consortium, we characterized mitochondrial genome evolution in response to treatment, using whole-genome sequencing data and associated RNA sequencing data from 152 paired tumor samples from 76 patients. Mitochondrial mutations are unequally distributed across the mitochondrial glioma genome, with only 4 of 13 mtDNA genes harboring a mutation across the cohort. Previous consortium analyses have investigated nuclear DNA hypermutation and changes in aneuploidy in response to chemotherapy and radiation treatment. Previous findings have identified hypermutation and copy number alterations as mechanisms of therapeutic resistance in glioma, but did not include analyses on mitochondrial DNA. Our analysis identified key differences between mitochondrial genome evolution and nuclear genome evolution. The mitochondrial mutational burden was not correlated with an increase in nuclear mutational burden. Unlike the nuclear DNA, we did not observe any samples with treatment-associated hypermutation. Mitochondrial copy number varied greatly but was increased at recurrence in IDH wild-type tumors. In tumors with a mutation in the IDH1 gene, mitochondrial copy number decreases were associated with an increase in aneuploidy score in samples that received radiation therapy. Expression analyses highlighted key differences in the active repair mechanisms between mtDNA and nuclear DNA. Our findings shed light on the variations in DNA repair mechanisms between the nucleus and the cytoplasm in glioma and lay the foundation for further analyses on the co-evolution of the mitochondrial and nuclear genomes in treatment-resistant tumors. Citation Format: Taylor E. Wade, Frederick S. Varn, Kevin C. Johnson, Roel G. Verhaak. Mitochondrial genome evolution in gliomas under therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5786.

Abstract 2168: Longitudinal analysis of diffuse glioma reveals cell state dynamics at recurrence associated with changes in genetics and the microenvironment

Abstract Diffuse glioma is characterized by a poor prognosis and a universal resistance to therapy, though the evolutionary processes behind this resistance remain unclear. The Glioma Longitudinal Analysis (GLASS) Consortium has previously demonstrated that therapy-induced selective pressures shape the genetic evolution of glioma in a stochastic manner. However, single-cell studies have revealed that malignant glioma cells are highly plastic and transition their cell state in response to diverse challenges, including changes in the microenvironment and the administration of standard-of-care therapy. To interrogate the factors driving therapy resistance in diffuse glioma, we collected and analyzed RNA- and/or DNA-sequencing data from temporally separated tumor pairs of over 300 adult patients with IDH-wild-type or IDH-mutant glioma. In a subset of these tumor pairs, we additionally performed multiplex immunofluorescence to capture the spatial relationship between tumor cells and their microenvironment. Recurrent tumors exhibited diverse changes that were attributable to changes in histological features, somatic alterations, and microenvironment interactions. IDH-wild-type tumors overall were more invasive at recurrence and exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. In contrast, recurrent IDH-mutant tumors exhibited a significant increase in proliferative expression programs that correlated with discrete genetic changes. Hypermutation and acquired CDKN2A homozygous deletions associated with an increase in proliferating stem-like malignant cells at recurrence in both glioma subtypes, reflecting active tumor expansion. A transition to the mesenchymal phenotype was associated with the presence of a specific myeloid cell state defined by unique ligand-receptor interactions with malignant cells, providing opportunities to target this transition through therapy. Collectively, our results uncover recurrence-associated changes in genetics and the microenvironment that can be targeted to shape disease progression following initial diagnosis. Citation Format: Frederick S. Varn, Kevin C. Johnson, Jan Martinek, Jason T. Huse, MacLean P. Nasrallah, Pieter Wesseling, Lee A. Cooper, Tathiane M. Malta, Taylor E. Wade, Thais S. Sabedot, Daniel J. Brat, Peter V. Gould, Adelheid Wöehrer, Kenneth Aldape, Azzam Ismail, Floris P. Barthel, Hoon Kim, Emre Kocakavuk, Nazia Ahmed, Kieron White, Santhosh Sivajothi, Indrani Datta, Jill S. Barnholtz-Sloan, Spyridon Bakas, Fulvio D'Angelo, Hui K. Gan, Luciano Garofano, Mustafa Khasraw, Simona Migliozzi, D. Ryan Ormond, Sun Ha Paek, Erwin G. Van Meir, Annemiek M. Walenkamp, Colin Watts, Michael Weller, Tobias Weiss, Karolina Palucka, Lucy F. Stead, Laila M. Poisson, Houtan Noushmehr, Antonio Iavarone, Roel G. Verhaak, The GLASS Consortium. Longitudinal analysis of diffuse glioma reveals cell state dynamics at recurrence associated with changes in genetics and the microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2168.

Abstract 103: Knockout of cellular prion protein in glioblastoma affects the expression of Wnt signaling genes

Abstract Glioblastoma (GBM) is an aggressive and therapy resistant central nervous system tumor. Wnts are involved in a plethora of cellular functions such as cell proliferation, differentiation, and migration. Deregulation of Wnt/β-catenin signaling is frequently observed in GBM, leading to glioma stem cell (GSC) maintenance, invasion, and resistance to therapy. Moreover, the cellular prion protein (PrPC) may interact with β-catenin and activate transcriptional activity of the β-catenin/TCF complex. PrPC plays essential roles in proliferation and differentiation, and was shown to upregulate the activity of Wnt/β-catenin signaling on intestinal progenitors. Our group has demonstrated that PrPC is overexpressed in GBM and that this leads to a higher proliferation rate and stemness maintenance. We have also demonstrated that silencing PrPC decreases the expression of stem cell markers, self-renewal, and tumorigenesis of GSCs. To further explore this connection between Wnt signaling and PrPC, we generated PrPC KO human U87 and U251 GBM cells with the CRISPR-Cas9 system. We performed RNA sequencing and subsequently validated differentially expressed genes (DEGs) by RT-qPCR. DESeq2 analysis comparing PrPC-KO GBM cells with wild-type controls resulted in 1,295 DEGs for U87 cells and 363 DEGs for U251 cells. Of those U87 and U251 DEGs, 28 and 10, respectively, are part of signaling pathways regulated by Wnt proteins. We chose to validate the upregulated DEGs CAMK2A, NFATC4, WISP1, PPP2R2B, SFRP2; and the downregulated DEGs RAC3 and SHH. In U87 GBM cells, expression patterns of CAMK2A, NFATC4, WISP1, PPP2R2B and SHH matched what we found on the RNAseq analysis, while RAC3 expression did not differ. In addition, SFRP2 was downregulated in U87 cells but appeared as an upregulated DEG. In U251 cells, expression of NFATC4 and CAMK2A was downregulated, and expression of RAC3 was upregulated. Interestingly, NFATC4, CAMK2A and RAC3 were not U251 DEGs in our RNAseq analysis, but their expression is altered in the PrPC-KO group. We will analyze the expression of SFRP2, PPP2R2B, WISP1 and SHH on U251 GBM cells. Our results indicate that the Wnt-Ca2+ pathway may be activated in U87 PrPC-KO cells, due to the increased expression of NFATC4 and CAMK2A. Regarding the canonical Wnt signaling, it appears to be a balance in the expression of genes that activate or inhibit these pathways in PrPC-KO cells, since WISP1 and PPP2R2B were upregulated and SFRP2 was downregulated. Importantly, expression of SHH was downregulated in U87 PrPC-KO cells. Aberrant activation of the Sonic Hedgehog signaling is implicated in the invasive capacity of GBM. We will also evaluate the status of Wnt signaling on PrPC-KO cells through Western blotting. Nevertheless, our results demonstrate that the Wnt signaling is affected by the KO of PrPC in GBM and further corroborate the importance of PrPC in Wnt signaling for GBM biology. Supported by FAPESP (2019/14952-0) and Capes. Citation Format: Barbara Paranhos Coelho, Mariana Brandão Prado, Rebeca Iglesia, Maria Isabel M. Escobar, Frederico M. Ferreira, Tiago G. dos Santos, Helder I. Nakaya, Marilene H. Lopes. Knockout of cellular prion protein in glioblastoma affects the expression of Wnt signaling genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 103.