Glioblastoma Clinical Samples Research Articles

A novel nuclear RNA HSD52 scaffolding NONO/SFPQ complex modulates DNA damage repair to facilitate temozolomide resistance.

Temozolomide (TMZ) is used in the treatment of glioblastoma (GBM). However, the primary obstacle remains the emergence of TMZ chemotherapy resistance. NONO and SFPQ are multifunctional nuclear proteins involved in genome stability and gene regulation. However, the specific role of NONO and SFPQ in TMZ resistance of GBM remains to be explored. RIP-chip and RNA microarray of TMZ-resistant and parental cells were performed for the gain of HSD52. The effects of HSD52 on TMZ resistance were investigated through in vitro assays, intracranial xenograft and GBM organoid models. The underlying mechanisms were explored by DNA methylation chip, RIP, RNA pulldown assays, among others. GBM clinical samples were rolled in to investigate the clinical significance of HSD52. We identified a novel non-coding RNA, HSD52, that was highly expressed in TMZ-resistant GBM and facilitated the interaction between NONO and SFPQ. H3 ubiquitination attenuation and reduced DNMT1 recruitment increased HSD52 transcription via DNA hypo-methylation. HSD52 formed an RNA duplex with UFL1 mRNA, thereby promoting NONO/SFPQ complex binding to UFL1 mRNA and enhancing its stability, and then contributed to TMZ resistance through activating ATM signaling pathway. In vivo xenograft and GBM organoid models showed significant repression in tumor growth after HSD52 knockout with TMZ treatment. In GBM clinical samples, HSD52 was responsible for the malignant progression and TMZ resistance. Our results revealed that HSD52 could serve as a promising therapeutic target to overcome TMZ resistance, improving the clinical efficacy of TMZ chemotherapy in GBM.

Lnc-H19-derived protein shapes the immunosuppressive microenvironment of glioblastoma

The immunosuppressive tumor microenvironment (TME) is a prominent feature of glioblastoma (GBM), the most lethal primary brain cancer resistant to current immunotherapies. The mechanisms underlying GBM-TME remain to be explored. We report that long non-coding RNA (LncRNA) H19 encodes an immune-related protein called H19-IRP. Functionally separated from H19 RNA, H19-IRP promotes GBM immunosuppression by binding to the CCL2 and Galectin-9 promoters and activating their transcription, thereby recruiting myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), leading to Tcell exhaustion and an immunosuppressive GBM-TME. H19-IRP, overexpressed in clinical GBM samples, acts as a tumor-associated antigen (TAA) presented by major histocompatibility complex class I (MHC-I). A circular RNA vaccine targeting H19-IRP (circH19-vac) triggers a potent cytotoxic Tcell response against GBM and inhibits GBM growth. Our results highlight the unrevealed function of H19-IRP in creating immunosuppressive GBM-TME by recruiting MDSCs and TAMs, supporting the idea of targeting H19-IRP with cancer vaccine for GBM treatment.

DNA hypomethylator phenotype reprograms glutamatergic network in receptor tyrosine kinase gene-mutated glioblastoma

DNA methylation is crucial for chromatin structure and gene expression and its aberrancies, including the global “hypomethylator phenotype”, are associated with cancer. Here we show that an underlying mechanism for this phenotype in the large proportion of the highly lethal brain tumor glioblastoma (GBM) carrying receptor tyrosine kinase gene mutations, involves the mechanistic target of rapamycin complex 2 (mTORC2), that is critical for growth factor signaling. In this scenario, mTORC2 suppresses the expression of the de novo DNA methyltransferase (DNMT3A) thereby inducing genome-wide DNA hypomethylation. Mechanistically, mTORC2 facilitates a redistribution of EZH2 histone methyltransferase into the promoter region of DNMT3A, and epigenetically represses the expression of DNA methyltransferase. Integrated analyses in both orthotopic mouse models and clinical GBM samples indicate that the DNA hypomethylator phenotype consistently reprograms a glutamate metabolism network, eventually driving GBM cell invasion and survival. These results nominate mTORC2 as a novel regulator of DNA hypomethylation in cancer and an exploitable target against cancer-promoting epigenetics.

ALKBH5 is a prognostic factor and promotes the angiogenesis of glioblastoma

Despite numerous reports indicating the significant impact of RNA modification on malignant glioblastoma (GBM) cell behaviors such as proliferation, invasion and therapy efficacy, its specific involvement in glioblastoma (GBM) angiogenesis is remains unclear and is currently under investigation. In this study, we aimed to investigate the relevance between RNA modification regulators and GBM angiogenesis. Our study employed bioinformatic analyses, including Gene Set Enrichment Analysis (GSEA), differential expression analysis, and Kaplan–Meier survival analysis, to identify regulators of angiogenesis-associated RNA modification (RM). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied to identify the enrichment of angiogenesis associated signatures in ALKBH5-high expression GBMs. We also utilized Western blot to verify the upregulation of ALKBH5 in clinical GBM samples. By a series of in vitro and in vivo assays, including plasmid transfection, wound healing, transwell invasion test, tube formation, RT-qPCR, ELISA assays and xenograft mice model, we validated the angiogenesis regulation ability of ALKBH5 in GBM. The N6-methyladenosine (m6A) modification “erase” ALKBH5 emerged as a candidate regulator associated with angiogenesis, demonstrating elevated expression and robust prognostic predictive ability in GBM patients. We also revealed enrichment of vasculature development biological process in GBMs with high ALKBH5 expression. Subsequently, we validated the elevated the expression of ALKBH5 in clinical GBM and paired adjacent tissues through western blot. Additionally, we knocked down the expression of ALKBH5 using sh-RNAs in U87 GBM cells to access the angiogenesis induction ability in U87 cells. In vitro experiments, Human Umbilical Vein Endothelial Cells (HUVECs) were used to perform wound healing, transwell migration and tube formation analysis, results indicated that ALKBH5 knock-down of U87 cells could decrease the pro-angiogenesis ability of U87 GBM cells. Further validation of our bioinformatic findings confirmed that ALKBH5 knockdown impaired VEGFA secretion in both in vitro and in vivo settings in U87 cells. These results comprehensively affirm the crucial role of ALKBH5 in regulating GBM-induced angiogenesis, both in vitro and in vivo. ALKBH5 not only emerges as a promising prognostic factor for GBM patients, but also plays a pivotal role in sustaining GBM progression by promoting angiogenesis.

Blockage of EGFR/AKT and mevalonate pathways synergize the antitumor effect of temozolomide by reprogramming energy metabolism in glioblastoma.

Metabolism reprogramming plays a vital role in glioblastoma (GBM) progression and recurrence by producing enough energy for highly proliferating tumor cells. In addition, metabolic reprogramming is crucial for tumor growth and immune-escape mechanisms. Epidermal growth factor receptor (EGFR) amplification and EGFR-vIII mutation are often detected in GBM cells, contributing to the malignant behavior. This study aimed to investigate the functional role of the EGFR pathway on fatty acid metabolism remodeling and energy generation. Clinical GBM specimens were selected for single-cell RNA sequencing and untargeted metabolomics analysis. A metabolism-associated RTK-fatty acid-gene signature was constructed and verified. MK-2206 and MK-803 were utilized to block the RTK pathway and mevalonate pathway induced abnormal metabolism. Energy metabolism in GBM with activated EGFR pathway was monitored. The antitumor effect of Osimertinib and Atorvastatin assisted by temozolomide (TMZ) was analyzed by an intracranial tumor model in vivo. GBM with high EGFR expression had characteristics of lipid remodeling and maintaining high cholesterol levels, supported by the single-cell RNA sequencing and metabolomics of clinical GBM samples. Inhibition of the EGFR/AKT and mevalonate pathways could remodel energy metabolism by repressing the tricarboxylic acid cycle and modulating ATP production. Mechanistically, the EGFR/AKT pathway upregulated the expressions of acyl-CoA synthetase short-chain family member 3 (ACSS3), acyl-CoA synthetase long-chain family member 3 (ACSL3), and long-chain fatty acid elongation-related gene ELOVL fatty acid elongase 2 (ELOVL2) in an NF-κB-dependent manner. Moreover, inhibition of the mevalonate pathway reduced the EGFR level on the cell membranes, thereby affecting the signal transduction of the EGFR/AKT pathway. Therefore, targeting the EGFR/AKT and mevalonate pathways enhanced the antitumor effect of TMZ in GBM cells and animal models. Our findings not only uncovered the mechanism of metabolic reprogramming in EGFR-activated GBM but also provided a combinatorial therapeutic strategy for clinical GBM management.

Deoxyhypusine hydroxylase: A novel therapeutic target differentially expressed in short-term vs long-term survivors of glioblastoma.

Glioblastoma (GB) is the most aggressive neoplasm of the brain. Poor prognosis is mainly attributed to tumor heterogeneity, invasiveness and drug resistance. Only a small fraction of GB patients survives longer than 24 months from the time of diagnosis (ie, long-term survivors [LTS]). In our study, we aimed to identify molecular markers associated with favorable GB prognosis as a basis to develop therapeutic applications to improve patients' outcome. We have recently assembled a proteogenomic dataset of 87 GB clinical samples of varying survival rates. Following RNA-seq and mass spectrometry (MS)-based proteomics analysis, we identified several differentially expressed genes and proteins, including some known cancer-related pathways and some less established that showed higher expression in short-term (<6 months) survivors (STS) compared to LTS. One such target found was deoxyhypusine hydroxylase (DOHH), which is known to be involved in the biosynthesis of hypusine, an unusual amino acid essential for the function of the eukaryotic translation initiation factor 5A (eIF5A), which promotes tumor growth. We consequently validated DOHH overexpression in STS samples by quantitative polymerase chain reaction (qPCR) and immunohistochemistry. We further showed robust inhibition of proliferation, migration and invasion of GB cells following silencing of DOHH with short hairpin RNA (shRNA) or inhibition of its activity with small molecules, ciclopirox and deferiprone. Moreover, DOHH silencing led to significant inhibition of tumor progression and prolonged survival in GB mouse models. Searching for a potential mechanism by which DOHH promotes tumor aggressiveness, we found that it supports the transition of GB cells to a more invasive phenotype via epithelial-mesenchymal transition (EMT)-related pathways.

Yes-Associated Protein Nuclear Translocation Is Regulated by Epidermal Growth Factor Receptor Activation Through Phosphatase and Tensin Homolog/AKT Axis in Glioblastomas

Gliomas are the most common and lethal primary brain tumors in adults. Glioblastomas, the most frequent and aggressive form of gliomas, represent a therapeutic challenge as no curative treatment exists to date, and the prognosis remains extremely poor. Recently, the transcriptional cofactors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) belonging to the Hippo pathway have emerged as a major determinant of malignancy in solid tumors, including gliomas. However, the mechanisms involved in its regulation, particularly in brain tumors, remain ill-defined. In glioblastomas, EGFR represents one of the most altered oncogenes affected by chromosomal rearrangements, mutations, amplifications, and overexpression. In this study, we investigated the potential link between epidermal growth factor receptor (EGFR) and the transcriptional cofactors YAP and TAZ by in situ and in vitro approaches. We first studied their activation on tissue microarray, including 137 patients from different glioma molecular subtypes. We observed that YAP and TAZ nuclear location was highly associated with isocitrate dehydrogenase 1/2 (IDH1/2) wild-type glioblastomas and poor patient outcomes. Interestingly, we found an association between EGFR activation and YAP nuclear location in glioblastoma clinical samples, suggesting a link between these 2 markers contrary to its ortholog TAZ. We tested this hypothesis in patient-derived glioblastoma cultures by pharmacologic inhibition of EGFR using gefinitib. We showed an increase of S397-YAP phosphorylation associated with decreased AKT phosphorylation after EGFR inhibition in phosphatase and tensin homolog (PTEN) wild-type cultures, unlike PTEN-mutated cell lines. Finally, we used bpV(HOpic), a potent PTEN inhibitor, to mimic the effect of PTEN mutations. We found that the inhibition of PTEN was sufficient to revert back the effect induced by Gefitinib in PTEN-wild-type cultures. Altogether, to our knowledge, these results show for the first time the regulation of pS397-YAP by the EGFR-AKT axis in a PTEN-dependent manner.

P12.03.B Ascorbate alters the hypoxic pathway in glioblastoma cellsin vitro and associates with improved patient survival

Abstract Background Glioblastomas are highly aggressive and hypoxic tumours. This environment activates the hypoxic pathway, driving glioma progression and treatment resistance. The hypoxic pathway is regulated by the hypoxia inducible factor (HIF) hydroxylases, which require oxygen as a substrate. Under normoxic conditions, the HIF hydroxylases are active, causing degradation and inhibition of HIF transcription factors. Under hypoxia, the activity of the hydroxylases reduces and HIF accumulates, activating the hypoxic response. HIF hydroxylases also require ascorbate as a cofactor for optimal function. The brain has one of the highest ascorbate levels in the human body, yet data on ascorbate levels in gliomas is scarce. Cellular ascorbate uptake occurs through solute carrier family 23 member 2 (SLC23A2). My aim is to understand the relationship between ascorbate, SLC23A2 and the hypoxic pathway in brain cancer using both in vitro cell culture and clinical samples. Material and Methods Ascorbate uptake was measured in human glioblastoma cell lines (T98G, U251MG, U87MG; ATCC) using reverse phase high performance liquid chromatography (HPLC-ECD). CRISPR-Cas was designed to knock-out SLC23A2. Clinical glioblastoma samples (n=37) and follow-up data were provided by the Cancer Society Tissue Bank and University of Otago Dunedin. Ethics and informed consent were obtained (H19/163, MEC/08/02/016). Ascorbate levels, measured by HPLC-ECD, and HIF-1α and downstream targets were measured using Western blotting or ELISA. A HIF score was calculated from HIF-1α and downstream target protein levels to estimate hypoxic pathway activity. Results In this study we have shown that T98G and U251 cells accumulate up to 15 nmol ascorbate/106 cells when exposed to 500 µM ascorbate for up to 24 hours, compared to U87MG cells with up to 3 nmol ascorbate/106 cells. Cancer Cell Line Encyclopaedia data shows that T98G and U251MG cells express higher levels of SLC23A2 compared to U87MG cells, aligning with our results. Clinical glioblastoma tissue contained a median of 7.6 µg ascorbate/100 mg tissue. Patients survival was significantly longer with above, vs below, median tumour ascorbate levels (Gehan-Breslow-Wilcoxon p = 0.027). The HIF score was negatively correlated with tumour ascorbate levels (Pearson r -0.327, p = 0.048). Patients with higher HIF-score had significantly shorter survival time compared to those with a lower HIF score (Gehan-Breslow-Wilcoxon p = 0.005). Conclusion Ascorbate uptake in glioblastoma cells varies between cell lines and appears reliant on the level of SLC23A2. Higher ascorbate content in clinical glioblastoma samples was associated with reduced hypoxic pathway activity and longer patient survival. Ongoing work, using SLC23A2 CRISPR-Cas knock-out cells, is investigating the effect of disrupting ascorbate uptake on hypoxic pathway signalling in glioblastoma cells.

Increased Ascorbate Content of Glioblastoma Is Associated With a Suppressed Hypoxic Response and Improved Patient Survival.

Glioblastoma multiforme is a challenging disease with limited treatment options and poor survival. Glioblastoma tumours are characterised by hypoxia that activates the hypoxia inducible factor (HIF) pathway and controls a myriad of genes that drive cancer progression. HIF transcription factors are regulated at the post-translation level via HIF-hydroxylases. These hydroxylases require oxygen and 2-oxoglutarate as substrates, and ferrous iron and ascorbate as cofactors. In this retrospective observational study, we aimed to determine whether ascorbate played a role in the hypoxic response of glioblastoma, and whether this affected patient outcome. We measured the ascorbate content and members of the HIF-pathway of clinical glioblastoma samples, and assessed their association with clinicopathological features and patient survival. In 37 samples (37 patients), median ascorbate content was 7.6 μg ascorbate/100 mg tissue, range 0.8 – 20.4 μg ascorbate/100 mg tissue. In tumours with above median ascorbate content, HIF-pathway activity as a whole was significantly suppressed (p = 0.005), and several members of the pathway showed decreased expression (carbonic anhydrase-9 and glucose transporter-1, both p < 0.01). Patients with either lower tumour HIF-pathway activity or higher tumour ascorbate content survived significantly longer than patients with higher HIF-pathway or lower ascorbate levels (p = 0.011, p = 0.043, respectively). Median survival for the low HIF-pathway score group was 362 days compared to 203 days for the high HIF-pathway score group, and median survival for the above median ascorbate group was 390 days, compared to the below median ascorbate group with 219 days. The apparent survival advantage associated with higher tumour ascorbate was more prominent for the first 8 months following surgery. These associations are promising, suggesting an important role for ascorbate-regulated HIF-pathway activity in glioblastoma that may impact on patient survival.

EPHA2 mediates PDGFA activity and functions together with PDGFRA as prognostic marker and therapeutic target in glioblastoma

Platelet-derived growth subunit A (PDGFA) plays critical roles in development of glioblastoma (GBM) with substantial evidence from TCGA database analyses and in vivo mouse models. So far, only platelet-derived growth receptor α (PDGFRA) has been identified as receptor for PDGFA. However, PDGFA and PDGFRA are categorized into different molecular subtypes of GBM in TCGA_GBM database. Our data herein further showed that activity or expression deficiency of PDGFRA did not effectively block PDGFA activity. Therefore, PDGFRA might be not necessary for PDGFA function.To profile proteins involved in PDGFA function, we performed co-immunoprecipitation (Co-IP) and Mass Spectrum (MS) and delineated the network of PDGFA-associated proteins for the first time. Unexpectedly, the data showed that EPHA2 could be temporally activated by PDGFA even without activation of PDGFRA and AKT. Furthermore, MS, Co-IP, in vitro binding thermodynamics, and proximity ligation assay consistently proved the interaction of EPHA2 and PDGFA. In addition, we observed that high expression of EPHA2 leaded to upregulation of PDGF signaling targets in TCGA_GBM database and clinical GBM samples. Co-upregulation of PDGFRA and EPHA2 leaded to worse patient prognosis and poorer therapeutic effects than other contexts, which might arise from expression elevation of genes related with malignant molecular subtypes and invasive growth. Due to PDGFA-induced EPHA2 activation, blocking PDGFRA by inhibitor could not effectively suppress proliferation of GBM cells, but simultaneous inhibition of both EPHA2 and PDGFRA showed synergetic inhibitory effects on GBM cells in vitro and in vivo. Taken together, our study provided new insights on PDGFA function and revealed EPHA2 as a potential receptor of PDGFA. EPHA2 might contribute to PDGFA signaling transduction in combination with PDGFRA and mediate the resistance of GBM cells to PDGFRA inhibitor. Therefore, combination of inhibitors targeting PDGFRA and EHA2 represented a promising therapeutic strategy for GBM treatment.

PI3K Pathway Inhibition with NVP-BEZ235 Hinders Glycolytic Metabolism in Glioblastoma Multiforme Cells.

Glioblastoma (GBM) is the most lethal primary brain cancer that lacks effective molecular targeted therapies. The PI3K/AKT/mTOR pathway is activated in 90% of all Glioblastoma multiforme (GBM) tumors. To gain insight into the impact of the PI3K pathway on GBM metabolism, we treated U87MG GBM cells with NVP-BEZ235 (PI3K and mTOR a dual inhibitor) and identified differentially expressed genes with RNA-seq analysis. RNA-seq identified 7803 differentially regulated genes in response to NVP-BEZ235. Gene Set Enrichment Analysis (GSEA) identified two glycolysis-related gene sets that were significantly enriched (p < 0.05) in control samples compared to NVP-BEZ235-treated samples. We validated the inhibition of glycolytic genes by NVP-BEZ235 and examined the impact of the FOXO1 inhibitor (AS1842856) on these genes in a set of GBM cell lines. FOXO1 inhibition alone was associated with reduced LDHA expression, but not ENO1 or PKM2. Bioinformatics analyses revealed that PI3K-impacted glycolytic genes were over-expressed and co-expressed in GBM clinical samples. The elevated expression of PI3K-impacted glycolytic genes was associated with poor prognosis in GBM based on Kaplan–Meier survival analyses. Our results suggest novel insights into hallmark metabolic reprogramming associated with the PI3K-mTOR dual inhibition.

CircFLNA promotes glioblastoma proliferation and invasion by negatively regulating miR‑199‑3p expression.

Glioblastoma (GBM) is one of the most common and malignant types of primary cancer in the central nervous system; however, the clinical outcomes of patients with GBM remain poor. Circular RNAs (circRNAs) have been revealed to serve important roles in diverse biological processes, such as regulating cell proliferation, epithelial-mesenchymal transition and tumor development. However, the underlying biological function of circRNA filamin A (circFLNA) and its potential role in GBM remain to be determined. The present study aimed to identify differentially expressed circRNAs in GBM. Reverse transcription-quantitative PCR was used to analyze the expression levels of circFLNA. The results demonstrated that the expression levels of circFLNA were significantly upregulated in clinical GBM samples and GBM cells compared with adjacent healthy brain tissues and normal human astrocytes, respectively. The results of the Cell Counting Kit-8 and Transwell assays revealed that circFLNA knockdown significantly inhibited the proliferative and invasive abilities of GBM cell lines. Moreover, high circFLNA expression levels were associated with a worse prognosis in GBM. MicroRNA (miR)-199-3p was subsequently predicted to be target of circFLNA. The inhibitory effect of miR-199-3p on cell proliferation and invasion was partially reversed following circFLNA knockdown. In conclusion, the findings of the present study identified novel roles for circFLNA in GBM and indicated that the circFLNA/miR-199-3p signaling axis may serve an important role in GBM progression. Therefore, circFLNA may represent a novel target for the diagnosis and treatment of GBM.

Abstract 2419: Two-tiered inhibition of TGFBR2 signaling via ITD-1 and miR-149-3p targets CD44High glioma stem cells and non-stem-like GBM cells

Abstract The remarkable heterogeneity of glioblastoma (GBM), its therapy resistance and high recurrence rate present formidable challenges for effective treatment. A critical component of GBM malignancy derives from the heterogeneous population of glioma stem-like cells (GSCs) that are especially efficient in promoting tumorigenicity and chemoradiation resistance. Previous findings by our lab and others have established Oct4 and Sox2 transcription factors as key drivers of the GSC phenotype. Development of effective GBM therapeutics requires a deeper knowledge of the genetic and epigenetic drivers of GSCs, particularly the Oct4/Sox2-mediated mechanisms that promote and maintain more tumorigenic and resistant GSC subsets. In GBM, TGF-β has well-established oncogenic functions and is tied to multiple phenotypic aspects of GSCs. However, little has been done to elucidate the relevance of TGF-β type II receptor (TGFBR2) in GSCs and GBM as a whole. Here, we show that TGFBR2 is a direct transcriptional target of Oct4 and Sox2 and is upregulated in both newly diagnosed and recurrent GBM. Multidimensional analysis identified overactivation of canonical TGF-β signaling in Oct4/Sox2-expressing GSCs. Furthermore, transcriptional targets of Smad2/3 induced by Oct4 and Sox2 are upregulated in TGFBR2High clinical GBM samples, a subpopulation also shown to associate with poor patient outcome. Notably, scRNA-Seq analysis revealed enrichment of TGFBR2 in CD44High, but not CD133/SSEA-1High, GSCs, a subset known to be highly proliferative and resistant to standard therapy. Likewise, a strong correlation between TGFBR2 and CD44 expression was found in clinical GBM samples. Despite promising results in preclinical studies, inhibition of TGF-β signaling through ligand or receptor targeting has been ineffective against clinical GBM, due to multiple potential reasons including activation of compensatory signaling pathways. Therefore, we apply a multidimensional approach, incorporating direct inhibition of both TGFBR2 and its oncogenic downstream effectors, in an effort to produce a more robust response and minimize the potential for compensatory resistance. Bioinformatic analysis predicted miR-149-3p to target a collection of Oct4/Sox2-induced Smad2/3-regulated transcripts, most of which, including CD44, have well-established oncogenic function and/or overexpression in GBM. Transient expression of miR-149-3p reduced self-renewal capacity of GSC spheres, a necessary ability for in vivo tumor propagation. Furthermore, miR-149-3p enhanced the growth-inhibitory effects of ITD-1, a small molecule TGFBR2 inhibitor, in both GSCs and non-stem-like GBM cells. These findings demonstrate that Oct4/Sox2-induced TGFBR2 signaling is clinically relevant and has the potential to be effectively combated using a carefully structured two-tiered therapeutic approach in GBM. Citation Format: Amanda L. Johnson, Jack Korleski, Hernando Lopez-Bertoni, John Laterra. Two-tiered inhibition of TGFBR2 signaling via ITD-1 and miR-149-3p targets CD44High glioma stem cells and non-stem-like GBM cells [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 2419.

Coronin 3 Promotes the Development of Oncogenic Properties in Glioma Through the Wnt/β-Catenin Signaling Pathway.

PurposeEvidence indicates that the actin-binding protein Coronin 3, which is aberrantly expressed in various cancers, is associated with cancer development and progression. However, little is known about the role of Coronin 3 in glioma tumorigenesis. Here, we aimed to explore the biological function and regulatory mechanism of Coronin 3 in glioblastoma (GBM).Materials and MethodsCoronin 3 level in human GBM clinical samples and cell lines was investigated. The shRNA knockdown strategy was used to assess the tumor characteristics of GBM cell lines. The role of β-catenin in Coronin 3-mediated oncogenic phenotypes was evaluated.ResultsCoronin 3 was found to be highly upregulated in glioma cell lines. Furthermore, knockdown of Coronin 3 significantly inhibited the growth of glioma cells both in vivo and in vitro and suppressed the expression of Wnt/β-catenin pathway genes, including β-catenin, Cyclin D1, and c-Myc. Moreover, we demonstrated that Coronin 3 regulates the expression of β-catenin in glioma. Our results revealed that Coronin 3-stimulated tumor growth was β-catenin-dependent.ConclusionOur study reveals a new molecular mechanism of Coronin 3 in promoting glioma growth and development through regulating the Wnt/β-catenin signaling pathway.

Epidermal growth factor receptor expression as a molecular determinant of glioblastoma response to the dopamine receptor D2 antagonist, ONC201.

e14552 Background: Durable response in glioblastoma patients have been reported in phase I/II clinical trials for the blood-brain penetrant dopamine receptor D2 (DRD2) antagonist, ONC201. Here we examine potential molecular determinants of response to DRD2 inhibition. Methods: The Cancer Genome Atlas (TCGA) glioblastoma database and other published mRNA profiles were used to analyze the DRD2 expression pattern. In vitro and in vivo responses to ONC201 were determined using patient derived xenograft glioblastoma models. Immunohistochemical studies were performed on clinically annotated glioblastoma samples derived from phase I/II clinical trials involving ONC201. Results: For the majority of clinical glioblastoma specimens in both the TCGA and non-TCGA dataset, epidermal growth factor receptor (EGFR) expression was inversely correlated with DRD2. This observation was recapitulated in a panel of patient-derived glioblastoma lines. In this panel of DRD2 expressing lines, high EGFR expression was associated with poor response to ONC201 in vitro and in vivo. Moreover, ectopic expression of EGFR reduced DRD2 expression and ONC201 sensitivity, suggesting functional redundancy between DRD2 and EGFR. In cell lines and clinical glioblastoma samples, DRD2 expression closely associated with the expression of rate-limiting enzymes for dopamine synthesis, suggesting dependency of a subset of glioblastomas on autocrine DRD2 signaling. Analysis of specimens from patients treated with ONC201 (n = 15) showed an inverse correlation between the intensity of EGFR staining and clinical response. The median overall survival for patients with high and low EGFR staining was 162 and 373 days, respectively (p = 0.037). All patients who exhibited progression free survival beyond 200 days showed low to no EGFR expression. Conclusions: Our results suggest EGFR expression as a determinant of response to ONC201 in glioblastoma patients and should inform the design of future clinical trials.

LncRNA MIR4435-2HG potentiates the proliferation and invasion of glioblastoma cells via modulating miR-1224-5p/TGFBR2 axis.

Glioblastoma (GBM) belongs to the high‐grade (IV) gliomas with extremely poor prognosis. Accumulating evidence uncovered the key roles of long non‐coding RNAs (lncRNAs) in GBM development. This study aimed to determine the biological actions and the clinical relevance of lncRNA MIR4435‐2 Host Gene (MIR4435‐2HG) in GBM. Data from GEPIA database showed that MIR4435‐2HG was up‐regulated in GBM tissues and high expression of MIR4435‐2HG correlated with shorter overall survival of GBM patients. Further experimental assays verified the up‐regulation of MIR4435‐2HG in GBM tissues and cell lines. In vitro cell studies and in vivo animal studies showed that knockdown of MIR4435‐2HG resulted in the inhibition of GBM cell proliferation and invasion and in vivo tumour growth, while MIR4435‐2HG overexpression driven GBM progression. Furthermore, MIR44435‐2HG was found to sponge miR‐1224‐5p and suppress miR‐1224‐5p expression; overexpression of miR‐1224‐5p attenuated the enhancement in GBM cell proliferation and invasion induced by MIR4435‐2HG overexpression. In a subsequent study, miR‐1224‐5p was found to target transforming growth factor‐beta receptor type 2 (TGFBR2) and repressed TGFBR2 expression, and in vitro assays showed that miR‐1224‐5p exerted tumour‐suppressive effects via targeting TGFBR2. More importantly, TGFRB2 knockdown antagonized hyper‐proliferation and invasion of GBM cells with MIR4435‐2HG overexpression. Clinically, the down‐regulation of miR‐1224‐5p and up‐regulation of TGFBR2 were verified in the GBM clinical samples. Taken together, the present study suggests the oncogenic role of MIR4435‐2HG in GBM and underlies the key function of MIR4435‐2HG‐driven GBM progression via targeting miR‐1224‐5p/TGFBR2 axis.

MTORC2 links growth factor signaling with epigenetic regulation of iron metabolism in glioblastoma

In cancer, aberrant growth factor receptor signaling reprograms cellular metabolism and global gene transcription to drive aggressive growth, but the underlying mechanisms are not well-understood. Here we show that in the highly lethal brain tumor glioblastoma (GBM), mTOR complex 2 (mTORC2), a critical core component of the growth factor signaling system, couples acetyl-CoA production with nuclear translocation of histone-modifying enzymes including pyruvate dehydrogenase and class IIa histone deacetylases to globally alter histone acetylation. Integrated analyses in orthotopic mouse models and in clinical GBM samples reveal that mTORC2 controls iron metabolisms via histone H3 acetylation of the iron-related gene promoter, promoting tumor cell survival. These results nominate mTORC2 as a critical epigenetic regulator of iron metabolism in cancer.

Abstract 253: Protein-tyrosine phosphatase 1B (PTP1B) as a therapeutic target in glioblastoma

Abstract New targeted therapies are urgently needed in glioblastoma (GBM). IL13Rα2 receptor is considered a highly specific receptor for GBM cells and has been proposed for therapeutic purposes. Here, we aimed to identify the protein interaction network of IL13Rα2 and its mediators in GBM searching for novel associated therapeutic targets. We investigated the IL13Rα2 protein interaction network in U251 GBM cells using immunoprecipitation followed by mass spectrometry. Different cell lines were used for validation. Two glioblastoma cell lines were used for testing proliferation, adhesion, migration and invasion. Claramine, a PTP1B inhibitor, was tested to study inhibition of GBM cells growth in cultured cells and in animal experiments. In vivo experiments with NSG mice were performed after treatment with PTP1B inhibitors. Using a proteomic approach, we identified the association of the PTP1B phosphatase with IL13Rα2 in U251 GBM cells. Although GBM cell lines were positive for PTP1B, the highest PTP1B expression was found in GBM clinical samples. Moreover, “in silico” dataset analysis showed PTP1B association with poor outcome in GBM patients. PTP1B silencing inhibited the IL-13-promoted activation of Src (Tyr419), AKT and ERK1/2 in all the cell lines tested causing a significant effect on IL-13-mediated migration and invasion, and proliferation at a lower extent, of GBM cells. Therefore, PTP1B-silencing reverted most of the pro-metastatic capacities of the IL-13/ IL13Rα2 signaling axis. Further immunoprecipitation of PTP1B showed its association with a large number of NF-κB and NOTCH-related proteins in GBM cells. Both pathways are highly relevant for cancer proliferation and invasion. Claramine, a selective inhibitor of PTP1B, caused similar results to PTP1B silencing, inhibiting the dephosphorylation of Src Tyr527, migration and invasion in GBM. Moreover, treatment with Claramine caused a complete regression of GBM xenografts in NSG mice and total inhibition of liver metastatic growth in nude mice inoculated with colorectal cancer cells. In conclusion, our results provide new insights into the mechanisms underlying IL13Rα2-driven GBM aggressiveness after identifying PTP1B as a critical mediator of IL-13/IL13Rα2 signaling. A PTP1B inhibitor such as Claramine showed a great value as a therapeutic candidate in GBM. PTP1B inhibitors might be highly effective to prevent GBM progression and invasion. Citation Format: Ruben Bartolomé, Marta Jaén, Miranda Burdiel, Irina V. Balyasnikova, Ignacio Casal. Protein-tyrosine phosphatase 1B (PTP1B) as a therapeutic target in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 253.

HERC3-Mediated SMAD7 Ubiquitination Degradation Promotes Autophagy-Induced EMT and Chemoresistance in Glioblastoma.

Glioblastoma, a common malignant intracranial tumor, has the most dismal prognosis. Autophagy was reported to act as a survival-promoting mechanism in gliomas by inducing epithelial-to-mesenchymal transition (EMT). Here, we determined the critical molecules involved in autophagy-induced EMT and elucidated the possible mechanism of chemoradiotherapy resistance and tumor recurrence. We used isobaric tags for relative and absolute quantitation to identify the critical proteins and pathway mediating EMT via autophagy inducer treatment, and tested the expression of these proteins using tissue microarray of gliomas and clinical glioblastoma samples as well as tissues and cells separated from the core lesion and tumor-peripheral region. Analysis of the Cancer Genome Atlas database and 110 glioblastoma cases revealed the prognostic value of these molecules. The functional role of these critical molecules was further confirmed by in vitro experiments and intracranial xenograft in nude mice. Autophagy inducers significantly upregulated the expression of HERC3, which promotes ubiquitination-mediated degradation of SMAD7 in an autolysosome-dependent manner. The corresponding increase in p-SMAD2/3 level and TGFβ pathway activation finally induced EMT in cell lines and primary glioblastoma cells. Moreover, HERC3 overexpression was observed in pseudo-palisade cells surrounding tumor necrosis and in tumor-adjacent tissue; high HERC3 and low SMAD7 levels predicted poor clinical outcome in glioblastoma; xenograft of nude mice and in vitro experiments confirmed these findings. Together, our findings reveal the indispensable role of HERC3 in regulating canonical SMAD2/3-dependent TGFβ pathway involvement in autophagy-induced EMT, providing insights toward a better understanding of the mechanism of resistance to temozolomide and peripheral recurrence of glioblastoma.

Delineation of crosstalk between HSP27 and MMP-2/MMP-9: A synergistic therapeutic avenue for glioblastoma management

BackgroundEpithelial to mesenchymal transition (EMT) and extracellular matrix (ECM) remodeling, are the two elemental processes promoting glioblastoma (GBM). In the present work we propose a mechanistic modelling of GBM and in process establish a hypothesis elucidating critical crosstalk between heat shock proteins (HSPs) and matrix metalloproteinases (MMPs) with synergistic upregulation of EMT-like process and ECM remodeling. MethodsThe interaction and the precise binding site between the HSP and MMP proteins was assayed computationally, in-vitro and in GBM clinical samples. ResultsA positive crosstalk of HSP27 with MMP-2 and MMP-9 was established in both GBM patient tissues and cell-lines. This association was found to be of prime significance for ECM remodeling and promotion of EMT-like characteristics. In-silico predictions revealed 3 plausible interaction sites of HSP27 interacting with MMP-2 and MMP-9. Site-directed mutagenesis followed by in-vitro immunoprecipitation assay (IP) with 3 mutated recombinant HSP27, confirmed an interface stretch containing residues 29–40 of HSP27 to be a common interaction site for both MMP-2 and MMP-9. This was further validated with in-vitro IP of truncated (sans AA 29–40) recombinant HSP27 with MMP-2 and MMP-9. ConclusionThe association of HSP27 with MMP-2 and MMP-9 proteins along with the identified interacting stretch has the potential to contribute towards drug development to inhibit GBM infiltration and migration. General significanceCurrent findings provide a novel therapeutic target for GBM opening a new horizon in the field of GBM management.