Glioblastoma Gene Research Articles

IMMU-12. TUMOR CELL IDO ENHANCES IMMUNE SUPPRESSION AND DECREASES SURVIVAL INDEPENDENT OF TRYPTOPHAN METABOLISM IN GLIOBLASTOMA

Abstract OBJECTIVE Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is an immune checkpoint that’s characterized as a potent immunosuppressive mediator through its ability to metabolize tryptophan and wild-type IDH patient-resected glioblastoma (GBM) expresses IDO in ≥ 95% of cases. Recent findings from our group led us to investigate the alternative hypothesis that IDO possesses immunosuppressive effects that are independent of its associated metabolic activity. METHODS Murine GBM cell lines that overexpress either wild-type or enzyme-null IDO were created for in vivo characterization of IDO enzyme-independent immunosuppressive function. Microarray was conducted to identify human IDO expression-correlated genes, which were further investigated in human GBM cell lines, patient GBM tissues and plasma, as well as the TCGA database. Ex vivo cell co-culture assays and syngeneic mouse orthotopic GBM models were employed to study immunosuppressive mechanisms. RESULTS Here, we demonstrate that non-enzymic IDO activity decreases survival in experimental animals and increases the expression of immunosuppressive complement factor H (CFH) in human GBM. CFH mRNA levels positively correlate with those of IDO and many other immunosuppressive genes in patient resected GBM and can be applied as a prognostic marker in both lower grade gliomas and GBM. Similar to IDO, the increased expression of CFH in patient-resected glioma was positively correlated with an increased signature for regulatory T cells (Tregs) and myeloid-derived suppressive cells (MDSCs). High expression of CFH in tumor cells increases intratumoral Tregs levels and decreases overall survival in mice with GBM, while inducing tumor associated macrophage cell differentiation. CONCLUSIONS Here, we demonstrated that glioblastoma (GBM) cell IDO promotes the accumulation of intratumoral FoxP3+ regulatory T cells (Tregs) and tumor progression while decreasing overall survival - independent of IDO enzyme activity. Our study reveals a targetable non-metabolic IDO-dependent mechanism for future therapeutic intervention in patients with GBM.

PATH-31. METHYLATION SUBCLASS RECEPTOR TYROSINE KINASE II AS A DRIVER FOR SEIZURES IN IDH-WILDTYPE GLIOBLASTOMA

Abstract BACKGROUND Seizures are a common symptom of patients suffering from glioblastoma and there is evidence that these tumours are morphologically different. However, the relationship of DNA methylation and glioblastoma-related seizures has not been well characterized. Only one study described an amplification of genes encoding receptor tyrosine kinases (RTK) as a positive predictor for intraoperative seizures during craniotomy. METHODS 59 patients who underwent surgery with confirmed IDH-wildtype glioblastoma, WHO grade 4, were included. Genome-wide DNA methylation profiling was performed using an 850k Illumina EPIC array and classified by the DKFZ brain tumor classifier. Methylation glioblastoma subclasses and gene alterations were correlated with clinical characteristics including preoperative and long-term seizures. RESULTS Overall, 18 of 59 patients (30.5%) presented with seizures of whom suffered 8 (44.4%) from a focal and 10 (55.6%) from a generalized seizure. Correlation of preoperative seizures with glioblastoma subclasses and DNA genes identified a higher incidence of preoperative seizures in patients with the glioblastoma subclass of receptor tyrosine kinase II (RTK; EGFR amplified) (p=0.031). In addition, these patients were more likely to present with generalized seizures (p=0.05). In multivariate analysis, temporal location (p=0.014, OR 5.785) but moreover RTK II (p=0.024, OR 7.052) was most predictive for preoperative seizures. Furthermore, recurrent seizures with an increased antiepileptic medication at last follow-up (mean: 9.2 months) occurred more often in RTK II-glioblastoma (p< 0.05). Kaplan-Meier curves showed no significant association between the presentation of seizures and overall or progression-free survival. In addition, further survival analyses did not reveal a correlation of methylation glioblastoma subgroups with the outcome. CONCLUSION Our study showed a strong correlation of the RTK II methylation subclass with preoperative and long-term seizures in patients suffering from glioblastoma.

NIMG-58. CANONICAL CORRELATION ANALYSIS IN GLIOBLASTOMA REVEALS ASSOCIATIONS BETWEEN EXPRESSION OF RADIOMIC SIGNATURES AND GENOMICS

Abstract PURPOSE Understanding the molecular underpinnings of imaging signatures of glioblastoma can provide insights into the biologic basis of tumor formation and progression as well as in vivo surrogate markers of molecular events driving the tumor’s phenotype. Through machine learning (ML), this study demonstrates that distinct imaging subtypes of glioblastoma are related to specific molecular alterations. METHODS Pre-operative multi-parametric MRI (T1, T2, T1CE, T2-FLAIR, DSC-MRI, DTI-MRI) of 669 IDH-wildtype subjects with glioblastoma were retrospectively collected and radiomic features, including descriptors of morphology, intensity, histogram, and texture, were extracted. Imaging subtypes were identified by a feature selection and clustering approach. Genomic markers, obtained using next generation sequencing (NGS) panel of 27 key glioblastoma genes, were available in 358/669 patients. Canonical correlation analysis (CCA) was conducted within each imaging subtype between the selected imaging features and genetic variables to seek maximum correlations between combinations of variables in imaging and genomic sets, and hence elucidate the molecular drivers of respective subtypes. RESULTS Three distinct imaging subtypes were identified by clustering on 50 selected features, representing characteristics of morphology, tumor neo-angiogenesis (DSC-derived features), and cellular density (DTI-derived features). These subtypes yielded differentiable overall survival based on Kaplan-Meier analysis. The canonical coefficients of each subtype revealed the distinction of the underlying genomic characteristics: one exhibited frequently mutated [ARID2, NTRK1], another subtype showed increased frequency of mutation in [ATRX, EGFR, PIK3R1], while the third was associated with all these genes and [NF1, PIK3CA, RB1], additionally. CONCLUSION We derived three distinct radiomic MRI subtypes for glioblastoma that highly correlate with the patients' survival and molecular genetic characteristics. Investigating the relationship between imaging and genomic information may enable identification of molecularly- and phenotypically-consistent tumor subtypes, which would offer non-invasive approaches for characterizing heterogeneity of glioblastoma, further facilitating patient stratification and treatment planning.

Lack of Benefit of Extending Temozolomide Treatment in Patients with High Vascular Glioblastoma with Methylated MGMT.

Simple SummaryDespite the complete treatment with surgery, chemotherapy and radiotherapy, patients with glioblastoma have a devasting prognosis. Although the role of extending temozolomide treatment has been explored, the results are inconclusive. Recent evidence suggested that tumor vascularity may be a modulating factor in combination with methylation of O6-methylguanine-DNA methyltransferase (MGMT) promotor gene on the effect of temozolomide-based therapies, opening new possibilities for personalized treatments. Before proposing a prospective interventional clinical study, it is necessary to confirm the beneficial effect of the combined effect of MGMT methylation and moderate tumor vascularity, as well as the lack of benefit of temozolomide in patients with a highly vascular tumor.In this study, we evaluated the benefit on survival of the combination of methylation of O6-methylguanine-DNA methyltransferase (MGMT) promotor gene and moderate vascularity in glioblastoma using a retrospective dataset of 123 patients from a multicenter cohort. MRI processing and calculation of relative cerebral blood volume (rCBV), used to define moderate- and high-vascular groups, were performed with the automatic ONCOhabitats method. We assessed the previously proposed rCBV threshold (10.7) and the new calculated ones (9.1 and 9.8) to analyze the association with survival for different populations according to vascularity and MGMT methylation status. We found that patients included in the moderate-vascular group had longer survival when MGMT is methylated (significant median survival difference of 174 days, p = 0.0129*). However, we did not find significant differences depending on the MGMT methylation status for the high-vascular group (p = 0.9119). In addition, we investigated the combined correlation of MGMT methylation status and rCBV with the prognostic effect of the number of temozolomide cycles, and only significant results were found for the moderate-vascular group. In conclusion, there is a lack of benefit of extending temozolomide treatment for patients with high vascular glioblastomas, even presenting MGMT methylation. Preliminary results suggest that patients with moderate vascularity and methylated MGMT glioblastomas would benefit more from prolonged adjuvant chemotherapy.

Predicting Hub Genes of Glioblastomas Based on a Support Vector Machine Combined with CFS Algorithms

Aim: To search the genes related to the mechanisms of the occurrence of glioma and to try to build a prediction model for glioblastomas. Background: The morbidity and mortality of glioblastomas are very high, which seriously endanger human health. At present, the goals of many investigations on gliomas are mainly to understand the cause and mechanism of these tumors at the molecular level and to explore clinical diagnosis and treatment methods. However, there is no effective early diagnosis method for this disease, and there are no effective prevention, diagnosis, or treatment measures. Methods: Firstly, the gene expression profiles derived from GEO were downloaded. Then, differentially expressed genes (DEGs) in the disease samples and the control samples were identified. After that, GO and KEGG enrichment analyses of DEGs were performed by DAVID. Furthermore, the correlation- based feature subset (CFS) method was applied to the selection of key DEGs. In addition, the classification model between the glioblastoma samples and the controls was built by a Support Vector Machine (SVM) based on selected key genes. Results and Discussion: Thirty-six DEGs, including 17 upregulated and 19 downregulated genes, were selected as the feature genes to build the classification model between the glioma samples and the control samples by the CFS method. The accuracy of the classification model by using a 10-fold crossvalidation test and the independent set test was 76.25% and 70.3%, respectively. In addition, PPP2R2B and CYBB can also be found in the top 5 hub genes screened by the protein-protein interaction (PPI) network. Conclusions: This study indicated that the CFS method is a useful tool to identify key genes in glioblastomas. In addition, we also predicted that genes such as PPP2R2B and CYBB might be potential biomarkers for the diagnosis of glioblastomas.

The long non-coding RNA HOTAIRM1 promotes tumor aggressiveness and radiotherapy resistance in glioblastoma

Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, the role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach.

A novel patient stratification strategy to enhance the therapeutic efficacy of dasatinib in glioblastoma.

Glioblastoma is the most common primary malignancy of the central nervous system with a dismal prognosis. Genomic signatures classify isocitrate dehydrogenase 1 (IDH)-wildtype glioblastoma into three subtypes: proneural, mesenchymal, and classical. Dasatinib, an inhibitor of proto-oncogene kinase Src (SRC), is one of many therapeutics which, despite promising preclinical results, have failed to improve overall survival in glioblastoma patients in clinical trials. We examined whether glioblastoma subtypes differ in their response to dasatinib and could hence be evaluated for patient enrichment strategies in clinical trials. We carried out in silico analyses on glioblastoma gene expression (TCGA) and single-cell RNA-Seq data. In addition, in vitro experiments using glioblastoma stem-like cells (GSCs) derived from primary patient tumors were performed, with complementary gene expression profiling and immunohistochemistry analysis of tumor samples. Patients with the mesenchymal subtype of glioblastoma showed higher SRC pathway activation based on gene expression profiling. Accordingly, mesenchymal GSCs were more sensitive to SRC inhibition by dasatinib compared to proneural and classical GSCs. Notably, SRC phosphorylation status did not predict response to dasatinib treatment. Furthermore, serpin peptidase inhibitor clade H member 1 (SERPINH1), a collagen-related heat-shock protein associated with cancer progression, was shown to correlate with dasatinib response and with the mesenchymal subtype. This work highlights further molecular-based patient selection strategies in clinical trials and suggests the mesenchymal subtype as well as SERPINH1 to be associated with response to dasatinib. Our findings indicate that stratification based on gene expression subtyping should be considered in future dasatinib trials.

Abstract LB045: Identification of plasma-derived, EV-based biomarkers for glioblastoma

Abstract Background: Glioblastoma (GBM) is the most malignant and aggressive primary brain cancer in adults, with an incidence of 3.2 per 100,000 people. Currently, diagnosis is only performed via histopathological investigation of a tissue sample from a GBM lesion, complemented with molecular diagnostics for identification of select biomarkers. MRI is the standard of care for follow-up and monitoring of treatment response. Therefore, development of a “liquid biopsy” to obtain disease-relevant information from body fluids is highly desirable.Methods: We present the results from a clinical study in which extracellular vesicle (EV)-derived mRNAs were profiled from the plasma of GBM patients and control individuals for a total of 32 individual RNAseq libraries. Initial biomarker discovery was performed on plasma from 16 patients (8 GBM patients at the time of initial diagnosis and 8 age and sex matched controls). Hybrid capture and RNA-seq were performed via our proprietary pipeline on EV-associated mRNA isolated from 1-2mL plasma. Sequencing data was analyzed for differential gene expression. Validation was done with plasma samples from a new cohort of 8 patients and 8 control individuals.Results: We observed 98 mRNAs as differentially detected between GBM and control samples, with 83 mRNAs enriched in GBM samples and 15 mRNAs decreased (p < 0.05). Correlation based on differentially detected mRNAs separated GBM and control samples into two unique populations. We were able to use the differentially expressed gene signature from the first cohort to separate GBM patients and control individuals in the second cohort. We are currently following up these results with a larger patient population. These data, while preliminary, provide a potential basis for the further development of a GBM gene panel test.Conclusions: We have identified a novel gene signature for GBM in plasma-derived EVs, which differs from previously identified biomarkers isolated from tissue. Further work will refine this signature to enable detection, characterization, and patient monitoring for GBM with less invasive and intensive methods. Citation Format: Yevgenia L. Khodor, Sudipto K. Chakrabortty, Christian Fischer, Martina Rauscher, Leonora Balaj, Bob S. Carter, Seth Yu, Johan Skog. Identification of plasma-derived, EV-based biomarkers for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB045.

PLEK2, RRM2, GCSH: A Novel WWOX-Dependent Biomarker Triad of Glioblastoma at the Crossroads of Cytoskeleton Reorganization and Metabolism Alterations.

Simple SummaryCytoskeleton reorganization affects the malignancy of glioblastoma. The WWOX gene is a tumor suppressor in glioblastoma and was found to modulate the cytoskeletal machinery in neural progenitor cells. To date, the role of this gene in the cytoskeleton or glioblastoma has been studied separately. Therefore, the purpose of this study was to investigate WWOX-dependent genes in glioblastoma and indicate cytoskeleton-related processes they are involved in. The most relevant WWOX-dependent genes were found to be PLEK2, RRM2, and GCSH, which have been proposed as novel biomarkers. Their biological functions suggest that there is an important link between cytoskeleton and metabolism, orchestrating tumor proliferation, metastasis, and resistance. Searching for such new therapeutic targets is important due to the constant lack of effective treatment for glioblastoma patients.Glioblastoma is one of the deadliest human cancers. Its malignancy depends on cytoskeleton reorganization, which is related to, e.g., epithelial-to-mesenchymal transition and metastasis. The malignant phenotype of glioblastoma is also affected by the WWOX gene, which is lost in nearly a quarter of gliomas. Although the role of WWOX in the cytoskeleton rearrangement has been found in neural progenitor cells, its function as a modulator of cytoskeleton in gliomas was not investigated. Therefore, this study aimed to investigate the role of WWOX and its collaborators in cytoskeleton dynamics of glioblastoma. Methodology on RNA-seq data integrated the use of databases, bioinformatics tools, web-based platforms, and machine learning algorithm, and the obtained results were validated through microarray data. PLEK2, RRM2, and GCSH were the most relevant WWOX-dependent genes that could serve as novel biomarkers. Other genes important in the context of cytoskeleton (BMP4, CCL11, CUX2, DUSP7, FAM92B, GRIN2B, HOXA1, HOXA10, KIF20A, NF2, SPOCK1, TTR, UHRF1, and WT1), metabolism (MTHFD2), or correlation with WWOX (COL3A1, KIF20A, RNF141, and RXRG) were also discovered. For the first time, we propose that changes in WWOX expression dictate a myriad of alterations that affect both glioblastoma cytoskeleton and metabolism, rendering new therapeutic possibilities.

Integrated genomic and transcriptomic analysis identified KRT18 mutation and MTAP loss as key genetic alterations related to the prognosis of astrocytoma and glioblastoma.

e14039 Background: Astrocytoma and glioblastoma (GBM) are two main subtypes of glioma classified by WHO guide of center nervous system (CNS) tumors to different grades. GBM is the most malignant among all CNS tumors with a 5-year survival rate of less than 5%. Although the prognosis of patients with astrocytoma is better than that of GBM in general, astrocytoma patients with IDH wild-type have a similar prognosis as GBM. Exploring the molecular driving force behind the malignant phenotype of astrocytoma and GBM will help explain the diversity of glioma and discover new drug targets. Methods: We enrolled 12 patients with astrocytoma and 12 patients with GBM and performed whole-exome sequencing (WES) and RNA-seq analysis on tumor samples from the patients. Comparative analysis of somatic mutation, copy number variation, cytoband alteration and differently expressed genes were performed between astrocytoma and GBM. Immune cells infiltration was analyzed by CIBERSORT Algorithm with LM22 immune subsets. Results: This study includes 14 male patients and 10 female patients, and the median age was 45.5 (range, 18-68) years old. Seven patients had IDH1 mutation, including 6 patients with astrocytoma and 1 patient with GBM. Somatic mutation of KRT18, which is associated with cell apoptosis and adhesion by interacting with TRADD and Pinin , was significantly enriched in astrocytoma, but rare in GBM. Copy number loss of MTAP, which is a potential drug target and closely related to a poor prognosis of glioma, was found significantly enriched in GBM. Pathway enrichment analysis found that GBM was mainly enriched in tumor-related pathways, such as cell adhesion, cell division, and angiogenesis, but astrocytoma was mainly enriched in pathways with ion transport, cell-cell signaling and neurogenesis. Immune cells infiltration analysis showed that macrophages M0 was significantly enriched in GBM, and activated mast cells, monocytes and plasma cells were significantly enriched in astrocytoma. Conclusions: This study revealed the distinct characteristics of astrocytoma and GBM at the DNA and RNA levels. Somatic mutation of KRT18, which was first reported in glioma, may be closely related to the malignant phenotype of astrocytoma. Copy number loss of MTAP, which was associated with the susceptibility to purine starvation and specific chemotherapy, was a key genetic alterative gene in GBM and had the potential to become the therapeutic target of GBM.

Arginine-tocopherol bioconjugated lipid vesicles for selective pTRAIL delivery and subsequent apoptosis induction in glioblastoma cells

The incorporation of specific therapeutic gene into glioblastoma offers potent therapeutic strategy to treat the disease. Non-viral gene delivery vectors are of particular interest due to their tuneable transfection efficiency and easy scale-up. Herein, we demonstrate successful delivery of plasmid encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (pTRAIL) using arginine-conjugated tocopherol lipid (AT) nanovesicles into glioblastoma cell lines. Another cationic lipid, glycine-conjugated tocopherol lipid (GT) having glycine in the head group region is also synthesized as a control lipid. Both lipid-derived liposomes effectively condensed the pDNA and the corresponding biomacromolecular assemblies (lipoplexes) are efficiently transfected into different cell lines. AT-based liposomes exhibit higher transfection efficacy in various cell lines, particularly selective in glioma cell lines. At an optimized N/P ratio, both the liposomal formulations show low cytotoxicity. AT-based lipoplexes have superior cellular uptake in U87 than the control lipid GT. The expression of TRAIL protein regulated death receptor and apoptosis signaling pathway is assayed by western blot using transfection of AT-based/pTRAIL into U87 cell lines. Induction of apoptosis in U87 cells exposed to AT-based/pTRAIL plasmid is evaluated by MTT assay as well as Annexin V-propidium iodide dual-staining assay. All results indicate that the developed AT-based/pTRAIL system offers a potentially safe and efficient therapeutic strategy for glioblastoma gene therapy.

The oncogenic role of both lncRNA PANDA and BCL2 gene in glioblastoma

Glioblastoma multiform (GBM) is the most prevalent primary tumor of the brain with aggressive behavior. This type of cancer accounts for 60% of all brain tumors in humans. More recently, the participation of long non-coding RNAs (lncRNAs) in the regulation of many biological processes in tumors has been evidenced. In the present study, we measured expression levels of lncRNA PANDA (P21 associated non-coding RNA DNA damage activated, hereafter termed PANDA) and anti-apoptotic gene B-cell lymphoma 2 (BCL2) in tissue samples of 50 GBM patients and 50 healthy-matched subjects. According to our data, both lncRNA PANDA and BCl2 mRNA were significantly overexpressed in tumor tissues of patients with GBM in comparison to healthy control specimens. Furthermore, a significant correlation was observed between the expression of lncRNA PANDA and BCL2 transcripts in the patients group. Additionally, the results of receiver operating characteristic (ROC) curve analysis and the area under the curve (AUC) unraveled that PANDA had a significant value in the diagnosis of GBM. The present data uncovered the up-regulated expressions of PANDA and BCL2 transcripts in patients with GBM, which are positively correlated with tumor grade. This may suggest the oncogenic function for these two markers and propose them for the interpretation of prognostic and diagnostic data as well as the promising potential treatment target in GBM patients.

Integrated genomic and transcriptomic analysis suggests KRT18 mutation and MTAP are key genetic alterations related to the prognosis between astrocytoma and glioblastoma

BackgroundAstrocytoma and glioblastoma (GBM) are the two main subtypes of glioma, with the 2016 World Health Organization Classification of Tumors of the Central Nervous System (CNS WHO) classifying them into different grades. GBM is the most malignant among all CNS tumors with a 5-year survival rate of less than 5%. Although the prognosis of patients with astrocytoma is better than that of GBM in general, patients with anaplastic astrocytoma (AA) and isocitrate dehydrogenase (IDH) wild type have a similar prognosis as GBM and entail a high risk of progression. Exploring the molecular driving force behind the malignant phenotype of astrocytoma and GBM will help explain the diversity of glioma and discover new drug targets.MethodsWe enrolled 12 patients with astrocytoma and 12 patients with GBM and performed whole-exome sequencing (WES) and RNA sequencing analysis on tumor samples from the patients.ResultsWe found that the somatic mutation of KRT18, which is associated with cell apoptosis and adhesion by interacting with receptor 1-associated protein (TRADD) and pinin, was significantly enriched in astrocytoma, but rare in GBM. Copy number loss of MTAP, which is closely related to a poor prognosis of glioma, was found to be significantly enriched in GBM. In addition, different somatic copy number alteration (SCNA), gene expression, and immune cell infiltration patterns between astrocytoma and GBM were found.ConclusionsThis study revealed the distinct characteristics of astrocytoma and GBM at the DNA and RNA level. Somatic mutation of KRT18 and copy number loss of MTAP, two key genetic alterative genes in astrocytoma and GBM, have the potential to become therapeutic targets in glioma.

Deletions on Chromosome Y and Downregulation of the SRY Gene in Tumor Tissue Are Associated with Worse Survival of Glioblastoma Patients.

Simple SummaryGlioblastoma (GBM) is one of the most common and most aggressive brain tumors with higher prevalence among men than women. Loss of chromosome Y (LOY) in the peripheral blood cells has been associated with increased risk of developing cancer. However, there is a lack of data about LOY in GBM tumor tissue and the potential impact on patients’ prognosis. Through droplet digital PCR (ddPCR) analysis of 10 markers spread throughout chromosome Y in 105 male GBM patients, we were able to identify deletion of SRY gene as a factor strongly correlating with reduced overall survival. This finding was later corroborated by the analysis of GBM gene expression data collected in TCGA, showing correlation between decreased SRY expression and shortened overall survival.Background: Biological causes of sex disparity seen in the prevalence of cancer, including glioblastoma (GBM), remain poorly understood. One of the considered aspects is the involvement of the sex chromosomes, especially loss of chromosome Y (LOY). Methods: Tumors from 105 isocitrate dehydrogenase (IDH) wild type male GBM patients were tested with droplet digital PCR for copy number changes of ten genes on chromosome Y. Decreased gene expression, a proxy of gene loss, was then analyzed in 225 IDH wild type GBM derived from TCGA and overall survival in both cohorts was tested with Kaplan–Meier log-rank analysis and maximally selected rank statistics for cut-off determination. Results: LOY was associated with significantly shorter overall survival (7 vs. 14.6 months, p = 0.0016), and among investigated individual genes survival correlated most prominently with loss of the sex-determining region Y gene (SRY) (10.8 vs. 14.8 months, p = 0.0031). Gene set enrichment analysis revealed that epidermal growth factor receptor, platelet-derived growth factor receptor, and MYC proto-oncogene signaling pathways are associated with low SRY expression. Conclusion: Our data show that deletions and reduced gene expression of chromosome Y genes, especially SRY, are associated with reduced survival of male GBM patients and connected to major susceptibility pathways of gliomagenesis.

FSMP-11. TARGETING CHOLESTEROL HOMEOSTASIS DYSREGULATION FOR THE TREATMENT OF GLIOBLASTOMA

Dysregulated cholesterol metabolism is a hallmark of many cancers, including glioblastoma (GBM), but its role in disease progression is not well understood. Here, we identified cholesterol 24-hydroxylase (CYP46A1), a brain-specific enzyme responsible for elimination of cholesterol through conversion of cholesterol to 24(S)-hydroxycholesterol (24OHC), as one of the most dramatically dysregulated cholesterol metabolism genes in GBM. CYP46A1 was significantly decreased in GBM samples compared to normal brain tissue. In gliomas, a reduction in CYP46A1 expression was associated with increasing tumour grade and poor prognosis. Functionally, ectopic expression of CYP46A1 suppressed cell proliferation and in vivo tumour growth by increasing 24OHC levels. RNA-seq revealed that treatment of GBM cells with 24OHC suppressed tumour growth through regulation of LXR and SREBP signaling. Efavirenz (EFV), an activator of CYP46A1 that is known to penetrate the blood-brain barrier (BBB), inhibited GBM growth in vivo. Our findings demonstrate that CYP46A1 is a critical regulator of cellular cholesterol in GBM and that the CYP46A1/24OHC axis is a potential therapeutic target.

In vitro biomimetic models for glioblastoma-a promising tool for drug response studies.

The poor response of glioblastoma to current treatment protocols is a consequence of its intrinsic drug resistance. Resistance to chemotherapy is primarily associated with considerable cellular heterogeneity, and plasticity of glioblastoma cells, alterations in gene expression, presence of specific tumor microenvironment conditions and blood-brain barrier. In an attempt to successfully overcome chemoresistance and better understand the biological behavior of glioblastoma, numerous tri-dimensional (3D) biomimetic models were developed in the past decade. These novel advanced models are able to better recapitulate the spatial organization of glioblastoma in a real time, therefore providing more realistic and reliable evidence to the response of glioblastoma to therapy. Moreover, these models enable the fine-tuning of different tumor microenvironment conditions and facilitate studies on the effects of the tumor microenvironment on glioblastoma chemoresistance. This review outlines current knowledge on the essence of glioblastoma chemoresistance and describes the progress achieved by 3D biomimetic models. Moreover, comprehensive literature assessment regarding the influence of 3D culturing and microenvironment mimicking on glioblastoma gene expression and biological behavior is also provided. The contribution of the blood-brain barrier as well as the blood-tumor barrier to glioblastoma chemoresistance is also reviewed from the perspective of 3D biomimetic models. Finally, the role of mathematical models in predicting 3D glioblastoma behavior and drug response is elaborated. In the future, technological innovations along with mathematical simulations should create reliable 3D biomimetic systems for glioblastoma research that should facilitate the identification and possibly application in preclinical drug testing and precision medicine.

Blood-brain barrier alterations in human brain tumors revealed by genome-wide transcriptomic profiling.

Brain tumors, whether primary or secondary, have limited therapeutic options despite advances in understanding driver gene mutations and heterogeneity within tumor cells. The cellular and molecular composition of brain tumor stroma, an important modifier of tumor growth, has been less investigated to date. Only few studies have focused on the vasculature of human brain tumors despite the fact that the blood-brain barrier (BBB) represents the major obstacle for efficient drug delivery. In this study, we employed RNA sequencing to characterize transcriptional alterations of endothelial cells (EC) isolated from primary and secondary human brain tumors. We used an immunoprecipitation approach to enrich for EC from normal brain, glioblastoma (GBM), and lung cancer brain metastasis (BM). Analysis of the endothelial transcriptome showed deregulation of genes implicated in cell proliferation, angiogenesis, and deposition of extracellular matrix (ECM) in the vasculature of GBM and BM. Deregulation of genes defining the BBB dysfunction module was found in both tumor types. We identified deregulated expression of genes in vessel-associated fibroblasts in GBM. We characterize alterations in BBB genes in GBM and BM vasculature and identify proteins that might be exploited for developing drug delivery platforms. In addition, our analysis on vessel-associated fibroblasts in GBM shows that the cellular composition of brain tumor stroma merits further investigation.

Identifying the miRNA Signature Association with Aging-Related Senescence in Glioblastoma.

Glioblastoma (GBM) is the most common malignant brain tumor and its malignant phenotypic characteristics are classified as grade IV tumors. Molecular interactions, such as protein–protein, protein–ncRNA, and protein–peptide interactions are crucial to transfer the signaling communications in cellular signaling pathways. Evidences suggest that signaling pathways of stem cells are also activated, which helps the propagation of GBM. Hence, it is important to identify a common signaling pathway that could be visible from multiple GBM gene expression data. microRNA signaling is considered important in GBM signaling, which needs further validation. We performed a high-throughput analysis using micro array expression profiles from 574 samples to explore the role of non-coding RNAs in the disease progression and unique signaling communication in GBM. A series of computational methods involving miRNA expression, gene ontology (GO) based gene enrichment, pathway mapping, and annotation from metabolic pathways databases, and network analysis were used for the analysis. Our study revealed the physiological roles of many known and novel miRNAs in cancer signaling, especially concerning signaling in cancer progression and proliferation. Overall, the results revealed a strong connection with stress induced senescence, significant miRNA targets for cell cycle arrest, and many common signaling pathways to GBM in the network.

Multi-Omics Analysis Reveals Novel Subtypes and Driver Genes in Glioblastoma

Glioblastoma is the most lethal malignant primary brain tumor; nevertheless, there remains a lack of accurate prognostic markers and drug targets. In this study, we analyzed 117 primary glioblastoma patients’ data that contained SNP, DNA copy, DNA methylation, mRNA expression, and clinical information. After the quality of control examination, we conducted the single nucleotide polymorphism (SNP) analysis, copy number variation (CNV) analysis, and infiltrated immune cells estimate. And moreover, by using the cluster of cluster analysis (CoCA) methods, we finally divided these GBM patients into two novel subtypes, HX-1 (Cluster 1) and HX-2 (Cluster 2), which could be co-characterized by 3 methylation variable positions [cg16957313(DUSP1), cg17783509(PHOX2B), cg23432345(HOXA7)] and 15 (PCDH1, CYP27B1, LPIN3, GPR32, BCL6, OR4Q3, MAGI3, SKIV2L, PCSK5, AKAP12, UBE3B, MAP4, TP53BP1, F5, RHOBTB1) gene mutations pattern. Compared to HX-1 subtype, the HX-2 subtype was identified with higher gene co-occurring events, tumor mutation burden (TBM), and poor median overall survival [231.5 days (HX-2) vs. 445 days (HX-1), P-value = 0.00053]. We believe that HX-1 and HX-2 subtypes may make sense as the potential prognostic biomarkers for patients with glioblastoma.

Overexpression of rhophilin 2 promotes pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer and is the seventh leading cause of global cancer deaths. In recent years, targeted therapy has been used for pancreatic cancer; however, the drugs available for use in targeted therapy for pancreatic cancer are still very limited. Hence, identification of novel targeted molecules for PDAC is required. Rhophilin 2 (RHPN2) was proven to be a driver gene in glioblastoma. However, the function of RHPN2 in PDAC remains unknown. In the present study, the function of RHPN2 was investigated. The RHPN2 levels were overexpressed by pcDNA3.1-RHPN2 and downregulated by si-RHPN2. Cell proliferation was assessed using the MTT assay and apoptosis was assessed using flow cytometry. The results revealed that high RHPN2 levels in PDAC tissue were correlated with a low overall survival rate of patients with PDAC. Inhibition of RHPN2 reduced SW1990 and PANC1 proliferation and increased the rate of apoptosis. Network analysis demonstrated that centrosomal protein 78 expression was negatively correlated with RHPN2 expression. In conclusion, the present study demonstrated that RHPN2 may promote PDAC making it a potential candidate for targeted therapy.