Survival Of Glioblastoma Multiforme Patients Research Articles

Glioblastoma: What can we do for these patients today and what will we be able to do in the future?

Glioblastoma multiforme (GBM) is an extremely aggressive primary human brain tumor. The median survival of GBM patients is 15 months in case of completing the modern complex treatment protocol. Chemotherapy can help to extend the life expectancy of patients. GBM treatment resistance is associated with cancer stem cells (CSCs). The present paper analyses the main reasons for ineffectiveness of the existing GBM treatment methods and suggests treating CSCs as a complex phenomenon, resulting from the coordinated interaction of normal stem cells and cancer cells (CCs) in immunosuppressive microsurroundings. The GBM treatment strategy is suggested not for only suppressing strategically important signaling pathways in CCs, but also for regulating interaction between normal stem cells and cancer cells. The paper considers the issue of controlling penetrability of the blood-brain barrier that is one of the main challenges in neuro-oncology. Also, the paper suggests the ways of extending life expectancy of GBM patients today and prospects for the near future.

Abstract 2038: Overall survival of glioblastoma patients treated by standard therapy in comparison to standard therapy plus tumor treating fields

Abstract Introduction: Glioblastoma multiforme (GBM) is the most common malignant primary intracranial tumor and traditionally has a median survival of only 10 to 14 months, with only 3 to 5% of patients surviving more than three years. Recurrence is nearly universal, and further decreases the median survival to only 5 to 7 months with optimal therapy. Tumor treating fields (TTFields; Optune, Novocure, Haifa, Israel) therapy is a novel treatment technique that has recently shown significant prolonged survival in GBM patients. This therapy is approved for the treatment of newly diagnosed and recurrent GBM and is based on the principle that low intensity, intermediate frequency alternating electric fields (100 to 300 kHz) have an anti-mitotic effect in specific cell types. The applied fields disrupt the mitotic spindle, microtubule assembly and the segregation of intracellular organelles during cell division, leading to apoptosis or mitotic arrest. We aimed to compare overall survival between patients treated with standard therapy and standard therapy plus TTFields at our institution. Materials and Methods: A total of 34 patients (22 male, 15 female) diagnosed with GBM and treated by standard therapy plus TTFields (STDTh-TTF) at our institution were included. Standard therapy (STDTh) consisted of surgical resection, followed by combined radiotherapy and chemotherapy (Temozolomide); in 2 cases biopsy was performed in place of resection. The date of resection or biopsy was considered the entry date and was used in calculating survival, and ranged from June 2015 to July 2019. A matching control group treated by STDTh alone at our institution was assembled from our database, based primarily on date of resection or biopsy, and secondarily by age (1 subject underwent biopsy in place of resection). When assembling the control group, the investigators were blinded to survival outcome. Results: Significantly greater overall survival was observed for the group treated by TTFields in addition to standard therapy (p = 0.005; Hazard ratio [HR] 0.28; 95% confidence interval [CI] 0.11-0.69). The groups were identical with respect to sex, and no differences with respect to age (p=0.12; STDTh-TTF mean 50.54 years, SD 10.3; STDTh mean 54.39 years, SD 9.3) or inclusion date (p=0.3) were detected. Conclusions: Our initial results appear promising with respect to overall survival in patients undergoing TTFields treatment in addition to standard therapy. Citation Format: Aaron Rulseh, Adam Derner, Jan Sroubek, Jan Klener, Josef Vymazal. Overall survival of glioblastoma patients treated by standard therapy in comparison to standard therapy plus tumor treating fields [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2038.

Prognosis and Survival Study in Patients with Glioblastoma Multiform and Its Relationship with EGFR Expression

Background and Aim: Glioblastoma multiforme (GBM) is the most common malignant and invasive tumor of the brain. The relation between prognosis and survival of GBM patients with Epidermal Growth Factor Receptor (EGFR) expression is challenging. Thus, we aimed to evaluate the prognosis and survival of patients with GBM and its relationship with EGFR expression. Materials and Methods: This single-arm cohort study was conducted on 70 patients with GBM during 2012-2018 in Shahid Rahnemoon and Mortaz hospitals. The immunohistochemistry technique was applied to paraffin blocks of brain tumors for examining EGFR expression. Other data were extracted from medical records. To determine the survival rate, the Kaplan–Meier curves were used. A chi-square test was used for the analysis of data. Statistically, p-value <0.05 was assumed significant. Results: The mean survival of patients with GBM was 22.3 ± 2.5 months (95% CI=17.41 - 27.10). In addition, 1, 2- and 5-year survival rates were 90%, 30% and 5%, respectively. The mean survival of patients with negative and positive EGFR was 27.4±7.3 and 20.6±2.4 months, respectively. Besides, 11.1% and 14.3% of patients in negative and positive EGFR groups were alive. There was no significant difference in patient’s survival in terms of EGFR expression (p=0.36). No significant difference was seen between the two groups (EGFR positive and negative groups), regarding the frequency of age, sex, tumor’s anatomical location, and place of living (p>0.05). Conclusion: Based on our study, it seems that the GBM tumor was associated with poor prognosis and a low survival rate. It was also found that the expression of the EGFR gene did not affect the survival rate of patients with GBM. Therefore, its use as a predictor factor for survival and prognosis is questionable.

Downregulation of α-l-fucosidase 1 suppresses glioma progression by enhancing autophagy and inhibiting macrophage infiltration.

α‐l‐Fucosidase 1 (FUCA1), a lysosomal enzyme that catalyses the hydrolytic cleavage of the terminal fucose residue, has been reported to be involved in tumorigenesis. However, the clinical significance and biological roles of FUCA1 in glioma remain largely unknown. We analyzed FUCA1 expression according to data in Oncomine, The Cancer Genome Atlas, and Chinese Glioma Genome Atlas databases and further verified FUCA1 expression with immunohistochemistry and real‐time PCR analysis in glioma tissues. The results showed that FUCA1 overexpression was significantly associated with high‐grade glioma as well as high mortality rates in the survival analysis. Data analyzed in cBioPortal showed that alterations in FUCA1 (1.4%) were correlated with worse survival in glioblastoma multiforme patients. Functional experiments showed that downregulation of FUCA1 suppressed glioma growth in vitro and in vivo. Conversely, overexpression of FUCA1 had the opposite effects on glioma. Mechanistically, transient inhibition of FUCA1 promoted the formation of large acidic vacuoles, as revealed by staining with acridine orange, increased the ratio of LC3‐B/LC3‐A, and modified the expression of Beclin‐1 and Atg12, which are autophagic markers. Upregulation of FUCA1 attenuated starvation‐induced autophagy in glioma. In addition, lower levels of tumor‐infiltrating macrophages, including CD68+ (−30%), F4/80+ (−50%), and CD11c+ macrophages (−50%), were identified in FUCA1‐downregulated glioma tissues, and CCL2/CCL5 neutralizing Abs blocked this effect. These results show that FUCA1 could serve as a potential therapeutic target for the treatment of patients with glioma by enhancing autophagy and inhibiting macrophage infiltration.

The Role of miRNA for the Treatment of MGMT Unmethylated Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most common high-grade intracranial tumor in adults. It is characterized by uncontrolled proliferation, diffuse infiltration due to high invasive and migratory capacities, as well as intense resistance to chemo- and radiotherapy. With a five-year survival of less than 3% and an average survival rate of 12 months after diagnosis, GBM has become a focus of current research to urgently develop new therapeutic approaches in order to prolong survival of GBM patients. The methylation status of the promoter region of the O6-methylguanine–DNA methyltransferase (MGMT) is nowadays routinely analyzed since a methylated promoter region is beneficial for an effective response to temozolomide-based chemotherapy. Furthermore, several miRNAs were identified regulating MGMT expression, apart from promoter methylation, by degrading MGMT mRNA before protein translation. These miRNAs could be a promising innovative treatment approach to enhance Temozolomide (TMZ) sensitivity in MGMT unmethylated patients and to increase progression-free survival as well as long-term survival. In this review, the relevant miRNAs are systematically reviewed.

The inhibition of AGTR1 and Aromatase as a new potential therapeutic strategy for Glioblastoma treatment.

IntroductionGlioblastoma multiforme (GBM) is the most malignant form of glioma causing 3–4% of all cancer‐related deaths. Specific cancer treatment for GBM includes surgery, radiotherapy and chemotherapy with temozolomide. Despite the continuous development of new clinical therapies, the prognosis and survival of GBM patients remain dismal. Thus, the deregulated growth, the migratory and invasive capabilities of glioma cells remain the most important obstacles to the development of effective GBM treatments. Angiotensin II (Ang II)/Ang II receptor 1 (AGTR1) axis is expressed in normal and tumoral tissues, including human GBM. It has been reported in different experimental models that Ang II is able to stimulate aromatase expression through the AGTR1 activation. Different studies have demonstrated that aromatase enzyme is expressed in GBM cells and local estrogen production may act as an autocrine or paracrine factor in enhancing glioblastoma growth and progression. In the present study, we investigated if Ang II/AGTR1 signalling may modulate aromatase expression and consequently local estrogen production, thus influencing glioblastoma survival and growth.MethodsThe Aromatase expression role under treatment with Ang II has been assessed by RT‐PCR, Western blotting, Aromatase activity assay, Transient transfection assays, ELISA assay, cell proliferation assay (Thymidine incorporation), Boyden chamber migration and invasion assays, in the GBM cell line U‐87 MG.ResultsWe identified in U‐87 MG cells that the Ang II up‐regulates the aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. In order to evaluate if Ang II may activate aromatase at transcriptional level we transiently transfected U‐87 MG cells with a vector containing human aromatase promoter I.4, broadly distributed around the brain and widely expressed in glioblastoma cells. Ang II treatment induces an increased aromatase promoter transcriptional activity. Mutagenesis studies and chromatin immunoprecipitation analysis reveal that the integrity of specificity protein 1 (SP1) and interferon‐gamma activation site (GAS) binding sites, within the aromatase promoter is required for Ang II modulation of human aromatase I.4 promoter activity in U87‐MG cells. The enhanced aromatase activity was paralleled by an higher estradiol production concomitant with an increase in cell proliferation, migration and invasion in U‐87 MG cells. These effects are reversed by treatment with an AGTR1 antagonist and/or an aromatase inhibitor, such as Losartan and Anastrozole respectively.ConclusionsAng II/AGTR1 signalling through Aromatase enhances local estrogen production which in turn promotes tumor growth and invasiveness. Therefore, their combined inhibitory targeting may prospectively provide a novel therapeutic strategy for Glioblastoma treatment.

NKCC1 involvement in the epithelial-to-mesenchymal transition is a prognostic biomarker in gliomas.

BackgroundGliomas are the most prevalent type of intracranial tumors. NKCC1 is an important regulator in tumor cell volume. We noticed that abnormally high NKCC1 expression resulted in changes in the shape and adhesion of glioma cells. However, little is known about the role of NKCC1 in the epithelial-mesenchymal transition (EMT) of gliomas. This study aims to clarify the biological function of NKCC1 in glioblastoma multiforme (GBM) progression.MethodsUsing data from The Cancer Genome Atlas (TCGA), we performed a Kaplan–Meier analysis on NKCC1 expression levels to estimate the rate of survival of mesenchymal GBM patients. The correlation between NKCC1 and EMT-related proteins was analyzed from the Gene Expression Profiling Interactive Analysis (GEPIA) server. We conducted Gene Set Enrichment Analysis (GSEA) to verify molecular signatures and pathways. We then studied the expression of NKCC1 in grade I–IV glioma tissue samples collected from patients using immunohistochemistry (IHC). Finally, we evaluated the effects of NKCC1 migration and invasion on the cellular behaviors of U251 cells using the transwell assay and western blots.ResultsHigh NKCC1 expression was associated with poor prognoses in mesenchymal GBM. Our results suggest a correlation between NKCC1 and EMT-protein markers: CDH2 and VIM. GSEA showed that gliomas, TGF-beta signaling and EMT were enriched in the NKCC1 high expression phenotype. Higher expression levels of NKCC1 in gliomas correlate with higher glioma grades. Transwell assay and western blot results demonstrated that the knockdown of NKCC1 led to a reduction in migration and invasion, while also inhibiting MMP-2 and MMP-9 expression in U251.ConclusionThese results suggest that high expression of NKCC1 regulates EMT in gliomas, providing a new therapeutic strategy for addressing the spread of gliomas by inhibiting the spread of intracranial tumors.

RETRACTED ARTICLE: Inhibition of autophagic flux differently modulates cannabidiol-induced death in 2D and 3D glioblastoma cell cultures

Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.

An Online Calculator for the Prediction of Survival in Glioblastoma Patients Using Classical Statistics and Machine Learning.

BACKGROUNDAlthough survival statistics in patients with glioblastoma multiforme (GBM) are well-defined at the group level, predicting individual patient survival remains challenging because of significant variation within strata.OBJECTIVETo compare statistical and machine learning algorithms in their ability to predict survival in GBM patients and deploy the best performing model as an online survival calculator.METHODSPatients undergoing an operation for a histopathologically confirmed GBM were extracted from the Surveillance Epidemiology and End Results (SEER) database (2005-2015) and split into a training and hold-out test set in an 80/20 ratio. Fifteen statistical and machine learning algorithms were trained based on 13 demographic, socioeconomic, clinical, and radiographic features to predict overall survival, 1-yr survival status, and compute personalized survival curves.RESULTSIn total, 20 821 patients met our inclusion criteria. The accelerated failure time model demonstrated superior performance in terms of discrimination (concordance index = 0.70), calibration, interpretability, predictive applicability, and computational efficiency compared to Cox proportional hazards regression and other machine learning algorithms. This model was deployed through a free, publicly available software interface (https://cnoc-bwh.shinyapps.io/gbmsurvivalpredictor/).CONCLUSIONThe development and deployment of survival prediction tools require a multimodal assessment rather than a single metric comparison. This study provides a framework for the development of prediction tools in cancer patients, as well as an online survival calculator for patients with GBM. Future efforts should improve the interpretability, predictive applicability, and computational efficiency of existing machine learning algorithms, increase the granularity of population-based registries, and externally validate the proposed prediction tool.

MicroRNA miR-100 Decreases Glioblastoma Growth by Targeting SMARCA5 and ErbB3 in Tumor-Initiating Cells.

Glioblastoma multiforme (GBM) is the most aggressive and most frequently diagnosed malignant human glioma. Despite the best available standard of care (surgery, radiation, and chemotherapy), the median survival of GBM patients is less than 2 years. Many recent studies have indicated that microRNAs (miRNAs) are important for promoting or reducing/limiting GBM growth. In particular, we previously showed that GBMs express decreased levels of miR-100 relative to control tissue and that restoring miR-100 expression reduced GBM tumorigenicity by modulating SMRT/NCOR2 (Nuclear Receptor Corepressor 2). Here, we demonstrate that miR-100 overexpression decreases expression of the stem cell markers, nestin and L1CAM, and decreases proliferation of GBM tumor-initiating cells (cancer stem cells). We further show that miR-100-mediated anti-tumorigenic activity limits the activity of SMARCA5 and its downstream target STAT3 (known as mTOR-STAT3-Notch pathway). In addition, we report ErbB3 (Her3) as a putative miR-100 target, including inhibition of its downstream AKT and ERK signaling pathways.

Overexpression of microRNA-129-5p in glioblastoma inhibits cell proliferation, migration, and colony-forming ability by targeting ZFP36L1.

Glioblastoma multiforme (GBM) is a highly invasive cancer with a high recurrence rate. The prognosis of GBM patients remains poor, even after standard surgical resection combined with chemoradiotherapy. Thus, there is an urgent need for new therapeutic targets in GBM. In recent years, microRNAs have received considerable attention due to their important role in tumor development and progression. In this study, we investigated the role of miR-129-5p and miR-129-5p/ZFP36L1 axis in GBM tumorigenesis. Analysis of GSE103228 microarray data from the GEO database showed that miR-129-5p was significantly downregulated in GBM vs. normal brain tissues. Quantitative reverse transcription PCR analysis of miR-129-5p expression in seven GBM cell lines (LN229, A172, U87, T98G, U251, H4, and LN118) vs. normal human astrocytes (NHA) showed miR-129-5p was significantly downregulated in GBM cells. Overexpression of miR-129-5p in LN229 and A172 cells significantly suppressed cell proliferation, migration, invasion, and colony-forming ability. Target Scan analysis identified ZFP36L1 as the target of miR-129-5p. UALCAN dataset analysis found that ZFP36L1 was significantly upregulated in GBM vs. normal brain tissues, and high ZFP36L1 expression was positively associated with poor survival of GBM patients. Western blot analysis demonstrated that ZFP36L1 was significantly upregulated in seven GBM cell lines vs. NHA. Overexpression of miR-129-5p in LN229 and A172 cells significantly inhibited ZFP36L1 mRNA and protein expression, while overexpression of ZFP36L1 in LN229 and A172 cells reversed miR-129-5p-mediated inhibition on GBM tumorigenesis. Our results revealed an important role of miR-129-5p in the negative regulation of ZFP36L1 expression in GBM, suggesting new candidates for targeted therapy in GBM patients.

Temporalis muscle width as a measure of sarcopenia correlates with overall survival in patients with newly diagnosed glioblastoma

Sarcopenia has been shown to correlate with poor oncologic outcomes, but the optimal method to evaluate sarcopenia and its use as a prognostic tool in glioblastoma multiforme (GBM) remain unclear. We developed a method to reproducibly quantify sarcopenia using the temporalis muscle and correlated sarcopenia with overall survival (OS) in GBM patients. We measured the temporalis muscle width (TMW) on the postoperative radiotherapy CT simulation scan of 87 newly diagnosed GBM patients from 2011 to 2016. All patients successfully completed standard of care radiotherapy to a dose of 6000 cGy in 30 fractions with concurrent temozolomide without treatment breaks. TMW was measured bilaterally on four axial CT slices defined by bony orbit anatomy: orbit ceiling, superior quarter (equidistant between orbit and mid-orbit), mid-orbit, and inferior quarter (equidistant between mid-orbit and orbit floor). For analysis, TMW was dichotomized to either wide or narrow TMW by the median TMW and correlated with OS using Cox regression models. TMW at the orbit inferior quarter significantly correlated with OS. Median OS for wide TMW (> 1.583 cm) was greater than that of narrow TMW: 28.3 mo vs. 22.2 mo (HR (wide/narrow) = 0.42, 95% CI = (0.22, 0.82), p = 0.01), controlling for resection type and MGMT methylation. This effect was pronounced especially in the following patient subsets: females (HR (wide/narrow) = 0.26, 95% CI = (0.07, 0.92), p = 0.04) and patients without MGMT methylation (HR = 0.35, 95% CI = (0.15, 0.81), p = 0.01). Our results suggest sarcopenia and TMW correlate with GBM OS. Prospective validation of sarcopenia is needed to evaluate its potential in determining optimal patient treatment.

ACTR-18. A PHASE IB/II CLINICAL TRIAL OF DODECAFLUOROPENTANE EMULSION (DDFPE) AS A RADIOSENSITIZER IN GLIOBLASTOMA

Abstract BACKGROUND Tumor hypoxia decreases the response of glioblastoma multiforme (GBM) to radiotherapy (RT) and chemotherapy (temozolomide[TMZ]). The purpose of this study was to evaluate the safety and efficacy of a novel oxygen therapeutic, dodecafluoropentane emulsion (DDFPe), in chemoradiation treatment of GBM and stratify the results based on predicted TMZ response. METHODS With ethics approval and informed consent, 11 adult GBM patients were enrolled. Patients were administered DDFPe via IV infusion (2% w/vol at doses of 0.05, 0.1 or 0.17 mL/kg) within 5–30 minutes prior to each 2 Gy fraction of RT (30 fractions over 6-weeks) while breathing supplemental oxygen. Patients also received standard concurrent and adjuvant TMZ. To evaluate the reversal of tumor hypoxia, patients underwent oxygen-sensitive (TOLD) MRI before and after DDFPe administration. Patients were also studied with serial MRI scans per standard of care and followed for survival. RESULTS The non-serious adverse events considered by the investigator to be possibly, probably, or definitely related to DDFPe administration included fatigue (n=4), headache (n=2) and decrease in platelet count (n=2). Serious adverse events included two patients with symptomatic radiation necrosis: one patient at each of dose levels 0.1 and 0.17 mL/kg. Enrollment continued at the 0.1 mL/kg dose without additional significant DDFPe-related toxicity. Historically, the average overall survival for GBM patients is about 14.6 months. The median overall survival for the study was 591 days, or 19.4 months. According to independent review of the serial MR images, the median time to progression was 555 days, or 18 months, compared to a historical control of 6.9 months. TOLD MRI showed a trend in improved tumor oxygenation. CONCLUSION Although small, this trial shows that DDFPe used as a radiosensitizer appears to be safe and may provide some survival benefit. The FDA has allowed a Phase II clinical trial to assess its effectiveness.

381P - XKR8 is a promising potential prognostic marker in glioblastoma multiforme patients

BackgroundThe most frequent and aggressive primary brain malignancy in adults is a glioblastoma multiforme (GBM) which is frequently resistant to apoptosis-inducing chemotherapeutic agents. Surface exposure of phosphatidylserine (PS) is a universally recognized apoptotic phenomenon. Although in acute myeloid leukemia and T-cell lymphoma cells Xkr8 mediates PS exposure in response to apoptotic stimuli, the mechanisms underlying the regulation of Xkr8 expression remain unclear. Nevertheless, the loss of Xkr8 in blood cancer cells blocks cell-surface exposure of PS and causes dramatic decreasing of engulfment by phagocytes. Whether Xkr8 is dysregulated in specific cancers and contribute to the survival of patients has not been investigated. This study was aimed to investigate the association of overall survival (OS) and disease-free survival (DFS) with expression and copy number alterations (CNA) of XKR8 gene in GBM patients. MethodsData of gene expression, CNA and clinical information from 619 GBM tumors were obtained from TCGA Glioblastoma Multiforme study using the open platform CBioPortal. Among all cases, 572 GBM samples were with CNA (GISTIC 2.0. Values) data and 162 samples were with mRNA expression data (RNA Seq V2 RSEM). Patients were divided into two groups taking into account CNA or expression of mRNA. XKR8 expression was scored as downregulated when mRNA value was lower than the mean expression + standard deviation. XKR8 gene was marked as amplified when CNA value was equal 1 or 2. Kaplan-Meier and log-rank analyses were performed using RStudio. ResultsSurvival analysis in GBM patients showed that the OS (p-value=0,015) of the group with downregulated XKR8 mRNA expression (n = 22) was better than of group with normal and upregulated expression (n = 140). Equivalent results have been shown for DFS, where the patients with low XKR8 expression (n = 15) had a weaker risk of disease recurrence (p-value=0,003) than other GBM patients (n = 103). Also, the OS of GBM patients with amplified XKR8 gene (n = 83) was worse (p-value=0,049) than of patients with other CNAs (n = 489). ConclusionsThus XKR8 gene can be characterized as prognostic biomarker of survival and disease progression in GBM patients. Legal entity responsible for the studyResearch Laboratory "Biomarker" IFMB KFU. FundingRussian Government Program of Competitive Growth of Kazan Federal University. DisclosureAll authors have declared no conflicts of interest.

The Role of Kinase Signaling in Resistance to Bevacizumab Therapy for Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most malignant primary brain tumor and is characterized by vascular hyperplasia, necrosis, and high cell proliferation. Despite current standard therapies, including surgical resection and chemoradiotherapy, GBM patients survive for only about 15 months after diagnosis. Recently, the U.S. Food and Drug Administration (FDA) has approved an antiangiogenesis medication for recurrent GBM-bevacizumab-which has improved progression-free survival in GBM patients. Although bevacizumab has resulted in significant early clinical benefit, it inescapably predisposes tumor to relapse that can be represented as an infiltrative phenotype. Fundamentally, bevacizumab antagonizes the vascular endothelial growth factor A (VEGFA), which is consistently released on both endothelial cells (ECs) and GBM cells. Actually, VEGFA inhibition on the ECs leads to the suppression of vascular progression, permeability, and the vasogenic edema. However, the consequence of the VEGFA pathway blockage on the GBM cells remains controversial. Nevertheless, a piece of evidence supports the relationship between bevacizumab application and compensatory activation of kinase signaling within GBM cells, leading to a tumor cell invasion known as the main mechanism of bevacizumab-induced tumor resistance. A complete understanding of kinase responses associated with tumor invasion in bevacizumab-resistant GBMs offers new therapeutic opportunities. Thus, this study aimed at presenting a brief overview of preclinical and clinical data of the tumor invasion and resistance induced by bevacizumab administration in GBMs, with a focus on the kinase responses during treatment. The novel therapeutic strategies to overcome this resistance by targeting protein kinases have also been summarized.

Abstract 774: Regulation of autophagy and epigenetic modulator micro RNA by transient receptor potential melastatin 7 (TRPM7) channel in glioma

Abstract High grade malignant gliomas are devastating, uniformly fatal cancers for which no effective therapies currently exist. Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. We have reported that TRPM7 channels, a subfamily member of the transient receptor potential (TRP), regulate glioma stem cell (GSC) growth/proliferation through STAT3 and Notch signaling pathways. Autophagy is a catabolic process that occurs during both physiological and pathological process which has been reported to have dual roles, functioning either as a tumor-suppressor or tumor-promoter in cancer, including glioma. In addition, because of the limited success of the traditional treatment in prolonging the overall survival in GBM patients, epigenetics and epigenetic modulators have been attracting an increasing amount of attention for their influence in many aspects of cancer. microRNAs (miRNA), which can be considered as epigenetic effectors, reversibly regulate transcription through binding to complementary sequences of mRNA and silencing its translation into proteins. Therefore, we first determined the role of autophagy process in regulating glioma by TRPM7 using real time RT2 Profile PCR arrays (SABioscience, PAHS-084Z). We found that ATG16L1, ATG9A, CDKN1B, CLN3, CXCR4, and RGS19 were upregulated in response to TRPM7 silencing with the fold change greater than 1.6 using the A172 cell line of human glioma. The gene changes were further validated in other three glioma cell lines U87MG, U373MG and SNB19, which demonstrated different glioma tumorigenicity. miroRNA microarray analysis were then were performed upon TRPM7 silencing in A172 cells (www.LCsciences.com). In-depth data analysis from miRNA microarray data showed a list of 16 downregulated and 10 upregulated miRNA whose transcripts are statistically significant with fold change greater than 2 by TRPM7 knock-down. Among these, the micro RNA of hsa-miR-26b-5p, has-miR4530, and has-miR-28-5p have shown to exert crucial influence on tumor growth and migration by modulating AKT, ERK and IGF-1 signaling pathways. Stem-loop RT-qPCR were then used to detect and quantify miRNAs identified in miRNA microarray data from U87MG, U373MG and SNB19 cell lines. Our data indicate that autophagy effects serve as tumor suppressors in gliomagenesis and are reversely regulated by TRPM7; while hsa-miR-26b-5p, has-miR4530, and has-miR-28-5p are regulated by TRPM7 and participate in glioma progression. Citation Format: Mingli Liu. Regulation of autophagy and epigenetic modulator micro RNA by transient receptor potential melastatin 7 (TRPM7) channel in glioma [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 774.

Role of Klotho Protein in Tumor Genesis, Cancer Progression, and Prognosis in Patients with High-Grade Glioma

Klotho, a single-pass transmembrane protein associated with premature aging, acts as a tumor suppressor gene by inhibiting insulin/insulin-like growth factor-1 and fibroblast growth factor pathways. Downregulated Klotho expression is reported in melanoma, mesothelioma, bladder, breast, gastric, cervix, lung, and kidney cancers and is associated with a poor prognosis. Klotho expression and Klotho promoter hypermethylation are predictive factors for patient prognosis. To investigate the potential role of Klotho in glioblastoma-multiforme (GBM), 22 GBM samples were collected from the Sheba Tumor Bank and examined. We found that increased Klotho messenger ribonucleic acid (RNA) expression predicted longer survival (P= 0.03) of GBM patients. Methylation analysis was performed on bisulfite-treated deoxyribonucleic acid from the GBM patient samples using ionization time-of-flight mass spectrometry according to the Sequenom EpiTYPER protocols. Klotho promoter hypermethylation was detected in 65% of the GBM samples and correlated significantly with improved survival (P < 0.04). We found 3 major Klotho promotor hypermethylation sites located 585-579 bp, 540-533 bp, and 537-534 bp upstream of the transcription start site. Methylated deoxyribonucleic acid immunoprecipitation studies confirmed these results. Notably, the messenger RNA expression in these GBM samples revealed an unexpected linear correlation with methylation of these 3 hypermethylation sites identified in the Klotho promotor. Thus Klotho expression and methylation could predict prognosis in patients with GBM. Epigenetic regulation in GBM appears to be complicated. Specific CpG islands affect genes or micro RNAs that interact to control Klotho expression. The diverse effects of these islands may be due to unique factors of GBM.

Symbiotic Macrophage-Glioma Cell Interactions Reveal Synthetic Lethality in PTEN-Null Glioma

Heterotypic interactions across diverse cell types can enable tumor progression and hold the potential to expand therapeutic interventions. Here, combined profiling and functional studies of glioma cells in glioblastoma multiforme (GBM) models establish that PTEN deficiency activates YAP1, which directly upregulates lysyl oxidase (LOX) expression. Mechanistically, secreted LOX functions as a potent macrophage chemoattractant via activation of the β1 integrin-PYK2 pathway in macrophages. These infiltrating macrophages secrete SPP1, which sustains glioma cell survival and stimulates angiogenesis. In PTEN-null GBM models, LOX inhibition markedly suppresses macrophage infiltration and tumor progression. Correspondingly, YAP1-LOX and β1 integrin-SPP1 signaling correlates positively with higher macrophage density and lower overall survival in GBM patients. This symbiotic glioma-macrophage interplay provides therapeutic targets specifically for PTEN-deficient GBM.

Expression Profile Analysis Identifies a Novel Five-Gene Signature to Improve Prognosis Prediction of Glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive primary central nervous system malignant tumor. The median survival of GBM patients is 12–15 months, and the 5 years survival rate is less than 5%. More novel molecular biomarkers are still urgently required to elucidate the mechanisms or improve the prognosis of GBM. This study aimed to explore novel biomarkers for GBM prognosis prediction. The gene expression profiles from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets of GBM were downloaded. A total of 2241 overlapping differentially expressed genes (DEGs) were identified from TCGA and GSE7696 datasets. By univariate COX regression survival analysis, 292 survival-related genes were found among these DEGs (p < 0.05). Functional enrichment analysis was performed based on these survival-related genes. A five-gene signature (PTPRN, RGS14, G6PC3, IGFBP2, and TIMP4) was further selected by multivariable Cox regression analysis and a prognostic model of this five-gene signature was constructed. Based on this risk score system, patients in the high-risk group had significantly poorer survival results than those in the low-risk group. Moreover, with the assistance of GEPIA http://gepia.cancer-pku.cn/, all five genes were found to be differentially expressed in GBM tissues compared with normal brain tissues. Furthermore, the co-expression network of the five genes was constructed based on weighted gene co-expression network analysis (WGCNA). Finally, this five-gene signature was further validated in other datasets. In conclusion, our study identified five novel biomarkers that have potential in the prognosis prediction of GBM.

Blockade of a Laminin-411-Notch Axis with CRISPR/Cas9 or a Nanobioconjugate Inhibits Glioblastoma Growth through Tumor-Microenvironment Cross-talk.

There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (α4β1γ1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 α4 and β1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma.Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth.