Expression In Glioblastoma Multiforme Research Articles

The "Superoncogene" Myc at the Crossroad between Metabolism and Gene Expression in Glioblastoma Multiforme.

The concept of the Myc (c-myc, n-myc, l-myc) oncogene as a canonical, DNA-bound transcription factor has consistently changed over the past few years. Indeed, Myc controls gene expression programs at multiple levels: directly binding chromatin and recruiting transcriptional coregulators; modulating the activity of RNA polymerases (RNAPs); and drawing chromatin topology. Therefore, it is evident that Myc deregulation in cancer is a dramatic event. Glioblastoma multiforme (GBM) is the most lethal, still incurable, brain cancer in adults, and it is characterized in most cases by Myc deregulation. Metabolic rewiring typically occurs in cancer cells, and GBM undergoes profound metabolic changes to supply increased energy demand. In nontransformed cells, Myc tightly controls metabolic pathways to maintain cellular homeostasis. Consistently, in Myc-overexpressing cancer cells, including GBM cells, these highly controlled metabolic routes are affected by enhanced Myc activity and show substantial alterations. On the other hand, deregulated cancer metabolism impacts Myc expression and function, placing Myc at the intersection between metabolic pathway activation and gene expression. In this review paper, we summarize the available information on GBM metabolism with a specific focus on the control of the Myc oncogene that, in turn, rules the activation of metabolic signals, ensuring GBM growth.

Analysis of regulatory sequences in exosomal DNA of NANOGP8.

Exosomes participate in intercellular communication by transporting functionally active molecules. Such cargo from the original cells comprising proteins, micro-RNA, mRNA, single-stranded (ssDNA) and double-stranded DNA (dsDNA) molecules pleiotropically transforms the target cells. Although cancer cells secrete exosomes carrying a significant level of DNA capable of modulating oncogene expression in a recipient cell, the regulatory mechanism is unknown. We have previously reported that cancer cells produce exosomes containing NANOGP8 DNA. NANOGP8 is an oncogenic paralog of embryonic stem cell transcription factor NANOG and does not express in cells since it is a pseudogene. However, in this study, we evaluated NANOGP8 expression in glioblastoma multiforme (GBM) tissue from a surgically removed brain tumor of a patient. Significantly higher NANOGP8 transcription was observed in GBM cancer stem cells (CSCs) than in GBM cancer cells or neural stem cells (NSCs), despite identical sequences of NANOGP8-upstream genomic region in all the cell lines. This finding suggests that upstream genomic sequences of NANOGP8 may have environment-dependent promoter activity. We also found that the regulatory sequences upstream of exosomal NANOGP8 GBM DNA contain multiple core promoter elements, transcription factor binding sites, and segments of human viruses known for their oncogenic role. The exosomal sequence of NANOGP8-upstream GBM DNA is different from corresponding genomic sequences in CSCs, cancer cells, and NSCs as well as from the sequences reported by NCBI. These sequence dissimilarities suggest that exosomal NANOGP8 GBM DNA may not be a part of the genomic DNA. Exosomes possibly acquire this DNA from other sources where it is synthesized by an unknown mechanism. The significance of exosome-bestowed regulatory elements in the transcription of promoter-less retrogene such as NANOGP8 remains to be determined.

SOCS3 Acts as an Onco-immunological Biomarker With Value in Assessing the Tumor Microenvironment, Pathological Staging, Histological Subtypes, Therapeutic Effect, and Prognoses of Several Types of Cancer.

The suppressor of cytokine signaling (SOCS) family contains eight members, including SOCS1–7 and CIS, and SOCS3 has been shown to inhibit cytokine signal transduction in various signaling pathways. Although several studies have currently shown the correlations between SOCS3 and several types of cancer, no pan-cancer analysis is available to date. We used various computational tools to explore the expression and pathogenic roles of SOCS3 in several types of cancer, assessing its potential role in the pathogenesis of cancer, in tumor immune infiltration, tumor progression, immune evasion, therapeutic response, and prognostic. The results showed that SOCS3 was downregulated in most The Cancer Genome Atlas (TCGA) cancer datasets but was highly expressed in brain tumors, breast cancer, esophageal cancer, colorectal cancer, and lymphoma. High SOCS3 expression in glioblastoma multiforme (GBM) and brain lower-grade glioma (LGG) were verified through immunohistochemical experiments. GEPIA and Kaplan–Meier Plotter were used, and this bioinformatics analysis showed that high SOCS3 expression was associated with a poor prognosis in the majority of cancers, including LGG and GBM. Our analysis also indicated that SOCS3 may be involved in tumor immune evasion via immune cell infiltration or T-cell exclusion across different types of cancer. In addition, SOCS3 methylation was negatively correlated with mRNA expression levels, worse prognoses, and dysfunctional T-cell phenotypes in various types of cancer. Next, different analytical methods were used to select genes related to SOCS3 gene alterations and carcinogenic characteristics, such as STAT3, SNAI1, NFKBIA, BCL10, TK1, PGS1, BIRC5, TMC8, and AFMID, and several biological functions were identified between them. We found that SOCS3 was involved in cancer development primarily through the JAK/STAT signaling pathway and cytokine receptor activity. Furthermore, SOCS3 expression levels were associated with immunotherapy or chemotherapy for numerous types of cancer. In conclusion, this study showed that SOCS3 is an immune-oncogenic molecule that may possess value as a biomarker for diagnosis, treatment, and prognosis of several types of cancer in the future.

Centrin 2: A Novel Marker of Mature and Neoplastic Human Astrocytes.

As microtubule-organizing centers (MTOCs), centrosomes play a pivotal role in cell division, neurodevelopment and neuronal maturation. Among centrosomal proteins, centrin-2 (CETN2) also contributes to DNA repair mechanisms which are fundamental to prevent genomic instability during neural stem cell pool expansion. Nevertheless, the expression profile of CETN2 in human neural stem cells and their progeny is currently unknown. To address this question, we interrogated a platform of human neuroepithelial stem (NES) cells derived from post mortem developing brain or established from pluripotent cells and demonstrated that while CETN2 retains its centrosomal location in proliferating NES cells, its expression pattern changes upon differentiation. In particular, we found that CETN2 is selectively expressed in mature astrocytes with a broad cytoplasmic distribution. We then extended our findings on human autoptic nervous tissue samples. We investigated CETN2 distribution in diverse anatomical areas along the rostro-caudal neuraxis and pointed out a peculiar topography of CETN2-labeled astrocytes in humans which was not appreciable in murine tissues, where CETN2 was mostly confined to ependymal cells. As a prototypical condition with glial overproliferation, we also explored CETN2 expression in glioblastoma multiforme (GBM), reporting a focal concentration of CETN2 in neoplastic astrocytes. This study expands CETN2 localization beyond centrosomes and reveals a unique expression pattern that makes it eligible as a novel astrocytic molecular marker, thus opening new roads to glial biology and human neural conditions.

RPP25 as a Prognostic-Related Biomarker That Correlates With Tumor Metabolism in Glioblastoma.

RPP25, a 25 kDa protein subunit of ribonuclease P (RNase P), is a protein-coding gene. Disorders associated with RPP25 include chromosome 15Q24 deletion syndrome and diffuse scleroderma, while systemic sclerosis can be complicated by malignancy. However, the functional role of RPP25 expression in glioblastoma multiforme (GBM) is unclear. In this study, comprehensive bioinformatics analysis was used to evaluate the impact of RPP25 on GBM occurrence and prognosis. Differential analysis of multiple databases showed that RPP25 was commonly highly expressed in multiple cancers but lowly expressed in GBM. Survival prognostic results showed that RPP25 was prognostically relevant in six tumors (CESC, GBM, LAML, LUAD, SKCM, and UVM), but high RPP25 expression was significantly associated with poor patient prognosis except for CESC. Analysis of RPP25 expression in GBM alone revealed that RPP25 was significantly downregulated in GBM compared with normal tissue. Receiver operating characteristic (ROC) combined with Kaplan-Meier (KM) analysis and Cox regression analysis showed that high RPP25 expression was a prognostic risk factor for GBM and had a predictive value for the 1-year, 2-year, and 3-year survival of GBM patients. In addition, the expression of RPP25 was correlated with the level of immune cell infiltration. The gene set enrichment analysis (GSEA) results showed that RPP25 was mainly associated with signalling pathways related to tumor progression and tumor metabolism.

PD-L1-R: A MR based surrogate for PD-L1 expression in Glioblastoma multiforme.

2041 Background: Efficacy of PD-L1 immune checkpoint inhibitor therapy in Glioblastoma multiforme (GBM) is limited and the prognostic value of PD-L1 in GBM is an active field of research. We therefore sought to identify a MR based surrogate for PD-L1 expression in GBM. Methods: T1 post contrast images (T1ce) acquired immediately before surgery of 121 subjects with primary GBM (RTK I, II and mesenchymal subtype, as determined from Illumina Human Methylation array data) were analyzed. Following standard pre-processing (bias filed correction, brain extraction), 1150 radiomics features were calculated from gross tumor volumes (GTV). The cohort was then divided into training/validation (70%/30%). Cross-validation and model-selection were applied to identify features associated with PD-L1-M expression (estimated from methylation data and highly correlated with PD-L1 RNA-sequencing based measure, as recently reported). Features were used to identify two groups of tumors differing in PD-L1-M expression (PD-L1-R high and low), for which a logistic regression model was trained. Overall survival was assessed between PD-L1-R high/low. Results: PD-L1-R high and low groups showed significant differences in PD-L1-M values (training: p=0.002, validation: p=0.04, full cohort: p<0.001). The same model was used to split tumors into 2 groups, using features from non-T1ce sequences. All of the tested MR modalities showed at least a trend in PD-L1-M values in the two groups (T2w: p=0.037, T1w: p=0.089, FLAIR: p=0.091). Further investigations on the whole cohort (121 subjects) showed that PD-L1-R low group was enriched for RTK II sub-type (48%), while PD-L1-R high for mesenchymal sub-type (48%). Refer to Table 1 for more information. In addition, evaluation of survival data showed a difference in overall survival (likelihood ratio test p value 0.048, OR 0.52, 95% CI [0.27; 0.98]), with the PD-L1-R high group having a better prognosis. Conclusions: We presented a radiomics model PD-L1-R which allowed to identify GBM with low and high PD-L1-M expression from T1 post contrast agent images. Future work should validate these findings in independent cohorts.[Table: see text]

Prognostic role of ALK-1 and h-TERT expression in glioblastoma multiforme: correlation with ALK gene alterations.

BackgroundAnaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is expressed in the developing central and peripheral nervous systems during embryogenesis. Human telomerase reverse transcriptase (h-TERT) protein resumption is the main process of preservation of telomeres that maintains DNA integrity. The present study aims to evaluate the prognostic role of ALK-1 and h-TERT protein expression and their correlation with ALK gene alterations in glioblastoma multiforme (GBM). MethodsThe current study is a retrospective study on a cohort of patients with GBM (n = 53) that attempted to detect ALK gene alterations using fluorescence in situ hybridization. ALK-1 and h-TERT proteins were evaluated using immunohistochemistry. ResultsScore 3 ALK-1 expression was significantly associated with male sex, tumor multiplicity, Ki labeling index (Ki LI), and type of therapeutic modality. Score 3 h-TERT expression exhibited a significant association with Ki LI. ALK gene amplifications (ALK-A) were significantly associated with increased Ki LI and therapeutic modalities. Score 3 ALK-1 protein expression, score 3 h-TERT protein expression, and ALK-A were associated with poor overall survival (OS) and progression-free survival (PFS). Multivariate analysis for OS revealed that ALK gene alterations were an independent prognostic factor for OS and PFS.ConclusionsHigh protein expression of both ALK-1 and h-TERT, as well as ALK-A had a poor impact on the prognosis of GBM. Further studies are needed to establish the underlying mechanisms.

The predictive capability of immunohistochemistry and DNA sequencing for determining TP53 functional mutation status: a comparative study of 41 glioblastoma patients.

Tumor protein 53 (p53) regulates fundamental pathways of cellular growth and differentiation. Aberrant p53 expression in glioblastoma multiforme, a terminal brain cancer, has been associated with worse patient outcomes and decreased chemosensitivity. Therefore, correctly identifying p53 status in glioblastoma is of great clinical significance. p53 immunohistochemistry is used to detect pathological presence of the TP53 gene product. Here, we examined the relationship between p53 immunoreactivity and TP53 mutation status by DNA Sanger sequencing in adult glioblastoma. Of 41 histologically confirmed samples, 27 (66%) were immunopositive for a p53 mutation via immunohistochemistry. Utilizing gene sequencing, we identified only eight samples (20%) with TP53 functional mutations and one sample with a silent mutation. Therefore, a ≥10% p53 immunohistochemistry threshold for predicting TP53 functional mutation status in glioma is insufficient. Implementing this ≥10% threshold, we demonstrated a remarkably low positive-predictive value (30%). Furthermore, the sensitivity and specificity with ≥10% p53 immunohistochemistry to predict TP53 functional mutation status were 100% and 42%, respectively. Our data suggests that unless reliable sequencing methodology is available for confirming TP53 status, raising the immunoreactivity threshold would increase positive and negative predictive values as well as the specificity without changing the sensitivity of the immunohistochemistry assay.

The reversible effects of glial cell line-derived neurotrophic factor (GDNF) in the human brain.

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor, and a member of the transforming growth factor β (TGF-β) superfamily acting on different neuronal activities. GDNF was originally identified as a neurotrophic factor crucially involved in the survival of dopaminergic neurons of the nigrostriatal pathway and is currently an established therapeutic target in Parkinson's disease. However, GDNF was later reported to be highly expressed in gliomas, especially in glioblastomas, and was demonstrated as a potent proliferation factor involved in the development and migration of gliomas. Here, we review our current understanding and progress made so far by researchers in our laboratories with references to relevant articles to support our discoveries. We present past and recent discoveries on the mechanisms involved in the protection of neurons by GDNF and examine its emerging roles in gliomas, as well as reasons for the abnormal expression in Glioblastoma Multiforme (GBM). Collectively, our work establishes a paradigm by which the ability of GDNF to protect dopaminergic neurons from degradation and its corresponding effects on glioma cells points to an underlying biological vulnerability in the effects of GDNF in the normal brain which can be subverted for use by cancer cells. Hence, presenting novel opportunities for intervention in glioma therapies.

Abstract 3835: Targeting loss of MTAP expression in glioblastoma multiforme with a combination of 2-fluoroadenine and methylthioadenosine

Abstract Methylthioadenosine Phosphorylase (MTAP) is a key enzyme in the purine and methionine salvage pathways that convert the polyamine byproduct 5′-deoxy-5′-methylthioadenosine (MTA) into adenine and methylthioribose-1-phosphate. Homozygous deletion of the MTAP gene is observed in 55% of all glioblastoma multiforme (GBM) tumors. Since MTAP is a house-keeping enzyme found in all normal tissues, the lack of MTAP in GBM makes MTAP-loss a potential therapeutic target. In this study, we explore a novel strategy to target MTAP-loss in GBM by combining 2-fluoroadenine (2FA), a highly toxic adenine analogue, with MTA. Because MTAP is expressed in all normal tissues, MTA will be converted to adenine, which will protect against 2FA toxicity. However, in tumor cells, the effects of 2FA will not be ameliorated because they lack MTAP. Here we show that there is undetectable expression of MTAP in A172 and U87 GBM cell lines by both western blot and MTAP enzyme activity assays. In vitro toxicity studies in which cells were cultured with either 2FA alone or 2FA plus MTA revealed that both cell lines were sensitive to 2FA with or without MTA. The LD50 of A172 cells was 110 nM (2FA) and 127 nM (2FA + 5 μM MTA), while for U87 cells it was 397 nM (2FA) and 323 nM (2FA + 5 μM MTA). This is in sharp contrast to MTAP+ NIH3T3 cells in which we observed a 44 fold shift in the ratio of the LD50 concentration in the presence and absence of MTA (160 nM versus 7000 nM). Addition of MTA protected normal tissues against 2FA toxicity in SCID mice. SCID mice subcutaneously injected with either A172 or U87 cells and then treated with 2FA(10 mg/kg) + MTA(50 mg/kg) showed significantly reduced tumor growth compared to vehicle treated controls. Our results indicated that 2FA+MTA might be effective in treating GBM. Citation Format: Baiqing Tang. Targeting loss of MTAP expression in glioblastoma multiforme with a combination of 2-fluoroadenine and methylthioadenosine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3835.

Comprehensive analysis of PD-L1 expression in glioblastoma multiforme.

Glioblastoma multiforme are highly malignant brain tumours with frequent genetic and epigenetic alterations. The poor clinical outcome of these tumours necessitates the development of new treatment options. Immunotherapies for glioblastoma multiforme including PD1/PD-L1 inhibition are currently tested in ongoing clinical trials. The purpose of this study was to investigate the molecular background of PD-L1 expression in glioblastoma multiforme and to find associated pathway activation and genetic alterations. We show that PD-L1 is up-regulated in IDH1/2 wildtype glioblastoma multiforme compared to lower-grade gliomas. In addition, a strong association of PD-L1 with the mesenchymal expression subgroup was observed. Consistent with that, NF1 mutation and corresponding activation of the MAPK pathway was strongly connected to PD-L1 expression. Our findings may explain different response to PD-L1 inhibition of patients in ongoing trials and may help to select patients that may profit of immunotherapy in the future.

Integrative Network-based Analysis of Magnetic Resonance Spectroscopy and Genome Wide Expression in Glioblastoma multiforme.

The goal of this study was to identify correlations between metabolites from proton MR spectroscopy and genetic pathway activity in glioblastoma multiforme (GBM). Twenty patients with primary GBM were analysed by short echo-time chemical shift imaging and genome-wide expression analyses. Weighed Gene Co-Expression Analysis was used for an integrative analysis of imaging and genetic data. N-acetylaspartate, normalised to the contralateral healthy side (nNAA), was significantly correlated to oligodendrocytic and neural development. For normalised creatine (nCr), a group with low nCr was linked to the mesenchymal subtype, while high nCr could be assigned to the proneural subtype. Moreover, clustering of normalised glutamine and glutamate (nGlx) revealed two groups, one with high nGlx being attributed to the neural subtype, and one with low nGlx associated with the classical subtype. Hence, the metabolites nNAA, nCr, and nGlx correlate with a specific gene expression pattern reflecting the previously described subtypes of GBM. Moreover high nNAA was associated with better clinical prognosis, whereas patients with lower nNAA revealed a shorter progression-free survival (PFS).

Loss of miR-124 and miR-137 expression in glioblastoma multiforme and their roles in glioma cell proliferation and differentiation

Background: Glioblastoma Multiforme (GBM) is a common malignant brain tumor. It is characterized by rapid growth and high tumor heterogeneity. The molecular mechanisms driving GBM tumorigenesis and progression are not yet fully elucidated.Methods: miR-124, and miR-137, expression in 43 high-grade GBM tumor tissues was measured by real-time PCR and compared with expression in 24 normal brain tissues. The effects of miR-124, and miR-137, overexpression on glioma U251 cell proliferation and differentiation were analyzed. Tuj1 and GFAP expression was detected using immunofluorescence staining. Cell proliferation was detected with flow cytometry.Results: miR-124, and miR-137, expression was significantly decreased in GBM tumor tissues compared to normal brain tissues (p < 0.01). Immunofluorescence showed, after miR-124 and miR-137 transfection, normal synapse growth structure in GBM, and that neuronal differentiation factor expression significantly increased, including Tuj1 and GFAP. Flow cytometry analysis showed that GBM cell cycle extension and differentiation was repressed.Conclusion: miR-124, and miR-137, function as tumor suppressors to inhibit cell proliferation and differentiation and in GBM, their expression was significantly decreased.

MRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

Abstract 511: EGFR-initated NADPH oxidase activity regulates Fyn expression in glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) constitute the most aggressive and common form of primary brain tumors, conferring the worst clinical prognosis. Epidermal growth factor receptor (EGFR) amplification, mutation and re-arrangement are commonly observed genetic lesions in GBM. The most frequently occurring EGFR variant in GBM, EGFRvIII, is characterized by a truncated extracellular domain, leading to a receptor which is unable to bind ligand yet rendered constitutively active. In addition to increasing proliferation and survival signaling, EGFRvIII increases the production of reactive oxygen species (ROS); redox signaling in GBM, however, remains an understudied oncogenic feature. Consistent with published findings, our studies indicate that EGFRvIII elevates ROS levels in comparison to wild-type EGFR or vector-transfected control in U87-MG and the observed increase in ROS levels is alleviated by pharmacological and genetic means of EGFR inhibition. ROS increases were determined to occur independent of augmented spare mitochondrial respiratory capacity, but did coincide with elevated basal levels of non-mitochondrial oxygen consumption as measured by Seahorse extracellular flux analysis. Given this finding, our studies focused on the NADPH oxidase (NOX), where chemical inhibition of NOX by apocynin effectively reduced ROS by greater than 50% in EGFRvIII expressing cells. Likewise, EGFRvIII increased the consumption of NADPH greater than 2.0-fold relative to vector control, as measured by cellular NADP/NADPH ratios, and the resulting increases in NADPH consumption were diminished by chemical inhibition of EGFRvIII as well as apocynin inhibition of NOX. These findings indicated that EGFRvIII-induced ROS increases might be related to increased activity of the NOX complex. To this end, siRNA knockdown of p47phox, an essential component of the NOX-2 complex, markedly reduced ROS content as well as cellular proliferation in these cells, thus reinforcing a possible link between aberrant EGFR signaling and ROS production through NOX. Our studies additionally indicate that EGFRvIII-mediated elevations in ROS up-regulate mRNA and protein level expression (&amp;gt;3.0-fold) of the SRC family kinase member Fyn, as Fyn expression is up-regulated in an EGFRvIII-dependent manner and concordantly down-regulated via chemical and genetic means of NOX inhibition. Moreover, siRNA knockdown of Fyn effectively lowers cellular proliferation, survival and increases sensitivity to EGFR kinase inhibition in EGFRvIII, thus indicating that redox-initiated signaling through Fyn, in part, mediates GBM proliferation. Taken together, our results suggest that EGFRvIII increases NOX-2 generated ROS through p47phox, enhancing the proliferative capacity of GBM, in part, via increased Fyn expression, suggesting that targeting redox alterations may prove beneficial in improving clinical outcomes in GBM. Citation Format: Blake Johnson, Joya Chandra. EGFR-initated NADPH oxidase activity regulates Fyn expression in glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 511. doi:10.1158/1538-7445.AM2014-511

Higher LRRFIP1 expression in glioblastoma multiforme is associated with better response to teniposide, a type II topoisomerase inhibitor

Previous studies from this laboratory indicated that microRNA-21 (miR-21) contributes to chemoresistance of glioblastoma multiforme (GBM) cells to teniposide, a type II topoisomerase inhibitor. We also showed that LRRFIP1 is a target of miR-21. In this study, we found that higher baseline LRRFIP1 expression in human GBM tissue (n=60) is associated with better prognosis upon later treatment with teniposide. Experiments in cultured U373MG cells showed enhanced toxicity of teniposide against U373MG cells transfected with a vector that resulted in LRRFIP1 overexpression (vs. cells transfected with control vector). Experiments in nude mice demonstrated better response of LRRFIP1 overexpressing xenografts to teniposide. These findings indicate that high baseline LRRFIP1 expression in GBM is associated with better response to teniposide, and encourage exploring LRRFIP1 as a target for GBM treatment.

The Effect of Antineoplastons A10 and AS2-1 and Metabolites of Sodium Phenylbutyrate on Gene Expression in Glioblastoma Multiforme

Antineoplastons are peptide and amino acid derivatives that occur naturally in the human body. They inhibit the growth of neoplastic cells without growth inhibition of normal cells. Phenylacetylglutaminate (PG) is an active ingredient of antineoplastons A10 and AS2-1 (ANP) and is also a metabolic by-product of phenylbutyrate (PB). The formulation of antineoplaston AS2-1 is a 4:1 mixture of phenylacetate (PN) and PG. Antineoplaston A10 is a 4:1 mixture of PG and isoPG. This study investigates the molecular mechanism of action of PG and PN. The Human U87 glioblastoma (GBM) cell line was used as the model system in this study. A total human gene array screen using the Affymetrix Human Genome plus 2.0 oligonucleotide arrays was performed using mRNA derived from U87 cells exposed to PG and PN. Pathway analysis was performed to allow the visualization of effect on metabolic pathways and gene interaction networks. Our preliminary results indicate that PG and PN interrupt signal transduction in RAS/MAPK/ERK and PI3K/AKT/PTEN pathways, interfere with cell cycle, decrease metabolism and promote apoptosis in human U87 GBM cells. The effect on multiple cellular pathways and targets, suggests that ANP and PB are promising candidates for clinical studies in GBM.

Circadian pathway genes in relation to glioma risk and outcome.

There is growing evidence that circadian disruption may alter risk and aggressiveness of cancer. We evaluated common genetic variants in the circadian gene pathway for associations with glioma risk and patient outcome in a US clinic-based case-control study. Subjects were genotyped for 17 candidate single nucleotide polymorphisms in ARNTL, CRY1, CRY2, CSNK1E, KLHL30, NPAS2, PER1, PER3, CLOCK, and MYRIP. Unconditional logistic regression was used to estimate age and gender-adjusted odds ratios (OR) and 95% confidence intervals (CI) for glioma risk under three inheritance models (additive, dominant, and recessive). Proportional hazards regression was used to estimate hazard ratios for glioma-related death among 441 patients with high-grade tumors. Survival associations were validated using The Cancer Genome Atlas (TCGA) dataset. A variant in PER1 (rs2289591) was significantly associated with overall glioma risk (per variant allele OR 0.80; 95% CI 0.66-0.97; p trend=0.027). The variant allele for CLOCK rs11133391 under a recessive model increased risk of oligodendroglioma (OR 2.41; 95% CI 1.31-4.42; p=0.005), though not other glioma subtypes (p for heterogeneity=0.0033). The association remained significant after false discovery rate adjustment (p=0.008). Differential associations by gender were observed for MYRIP rs6599077 and CSNK1E rs1534891 though differences were not significant after adjustment for multiple testing. No consistent mortality associations were identified. Several of the examined genes exhibited differential expression in glioblastoma multiforme versus normal brain in TCGA data (MYRIP, ARNTL, CRY1, KLHL30, PER1, CLOCK, and PER3), and expression of NPAS2 was significantly associated with a poor patient outcome in TCGA patients. This exploratory analysis provides some evidence supporting a role for circadian genes in the onset of glioma and possibly the outcome of glioma.

Abstract 5417: HIF1α is a master regulator with the facilitate of demethylation for the hexokinase 2 expression during cancer progression.

Abstract Hexokinase 2 (HK2) is a key enzyme of aerobic glycolysis, known as the Warburg effect. A recent report showed that the HK2 expression in glioblastoma multiforme correlates with the progression of cancer and the survival of patients. The regulation of HK2 expression in cancer cells has been explored for many decades, however the precise mechanism is not yet identified. HK2 gene has a 4-kb promoter, which contains the potential responsive elements for glucose, insulin, glucagon, TPA, cAMP and HIF1 α. However, methylation of the promoter region makes normal cells nearly silent in HK2 expression. A study showed the potential contribution of demethylation of promoter for the HK2 expression during cancer progression. In this study, we tried to identify the master regulator of HK2 expression. We performed methylation beadchip array for the comparison of methylation status of HK2 gene between normal tissues and hepatocellular carcinoma (HCC). The 5’-UTR and 3’-UTR are densely methylated in normal tissues, but significantly demethylated in HCC. Moreover, we identified new hypoxia responsive element (HRE) through the deletion and site-directed mutagenesis study with 2-kb promoter of human HK2. The mutation of new HRE diminished the luciferase activity. Specific interaction between HIF1 α and HRE were identified by ChIP and EMSA. Then, we showed that demethylation by 5-Aza-deoxycytidine could contribute to the HK2 expression in hypoxic condition. In conclusion, we revealed the mechanism of HK2 expression and hypoxia is one of the major regulator among pre-known regulators. The sequential events, demethylation and exposure to hypoxic environments are the main requisite for the HK2 expression during the cancer progression. Citation Format: Hyemi Kim, Hyun G. Lee, Jae Myun Lee, Jeon Han Park. HIF1α is a master regulator with the facilitate of demethylation for the hexokinase 2 expression during cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5417. doi:10.1158/1538-7445.AM2013-5417

Portraying the expression landscapes of cancer subtypes

Self-organizing maps (SOM) portray molecular phenotypes with individual resolution. We present an analysis pipeline based on SOM machine learning which allows the comprehensive study of large scale clinical data. The potency of the method is demonstrated in selected applications studying the diversity of gene expression in Glioblastoma Multiforme (GBM) and prostate cancer progression. Our method characterizes relationships between the samples, disentangles the expression patterns into well separated groups of co-regulated genes, extracts their functional contexts using enrichment techniques, and enables the detection of contaminations and outliers in the samples. We found that the four GBM subtypes can be divided into two “localized” and two “intermediate” ones. The localized subtypes are characterized by the antagonistic activation of processes related to immune response and cell division, commonly observed also in other cancers. In contrast, each of the “intermediate” subtypes forms a heterogeneous cont...