Monocarboxylate transporters (MCTs) in gliomas: expression and exploitation as therapeutic targets

Background

Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD147 and to assess the therapeutic impact of MCT inhibition in gliomas.

Methods

MCTs and CD147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models.

Results

MCT1, MCT4, and CD147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed.

Conclusions

This is the most comprehensive study reporting the expression of MCTs and CD147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.     

MiR-328 promotes glioma cell invasion via SFRP1-dependent Wnt-signaling activation

Background

Diffusely infiltrative growth of human astrocytic gliomas is one of the major obstacles to successful tumor therapy. Thorough insights into the molecules and pathways signaling glioma cell invasion thus appear of major relevance for the development of targeted and individualized therapies. By miRNA expression profiling of microdissected human tumor biopsy specimens we identified miR-328 as one of the main miRNAs upregulated in invading glioma cells in vivo and further investigated its role in glioma pathogenesis.

Methods

We employed miRNA mimics and inhibitors to functionally characterize miR-328, 3′ untranslated region luciferase assays, and T-cell factor/lymphoid enhancer factor reporter assays to pinpoint miR-328 targets and signaling pathways, and analyzed miR-328 expression in a large panel of gliomas.

Results

First, we corroborated the invasion-promoting role of miR-328 in A172 and TP365MG glioma cells. Secreted Frizzled-related protein 1 (SFRP1), an inhibitor of Wnt signaling, was then pinpointed as a direct miR-328 target. SFRP1 expression is of prognostic relevance in gliomas with reduced expression, being associated with significantly lower overall patient survival in both the Repository of Molecular Brain Neoplasia Data (REMBRANDT) and The Cancer Genome Atlas. Of note, miR-328 regulated both SFRP1 protein expression levels and Wnt signaling pathway activity. Finally, in human glioma tissues miR-328 appeared to account for the downregulation of SFRP1 preferentially in lower-grade astrocytic gliomas and was inversely related to SFRP1 promoter hypermethylation.

Conclusion

Taken together, we report on a novel molecular miR-328–dependent mechanism that via SFRP1 inhibition and Wnt activation contributes to the infiltrative glioma phenotype at already early stages of glioma progression, with unfavorable prognostic implications for the final outcome of the disease.     

Downregulation of CDKN2A and suppression of cyclin D1 gene expressions in malignant gliomas

Background

Malignant gliomas are the most common in central nervous system cancer. Genome-wide association study identifies that CDKN2A was a susceptibility loci for glioma. The CDKN2A/cyclin-dependent kinase 4, 6/Retinoblastoma protein (Rb) pathway is thought to play a crucial role in malignant gliomas pathogenesis. We have investigated the expression of CDKN2A for potential correlations with malignant gliomas grade and potential role of CDKN2A on malignant gliomas pathogenesis.

Methods

Tumour tissue samples from 61 patients suffering from malignant gliomas were investigated. The expression levels of CDKN2A were detected using immunohistochemical staining and western blot. Overexpression and knockdown of CDKN2A were performed in human glioma cell lines. Subsequently, colony formation, growth curves and CDKN2A-Cyclin-Rb pathway were analyzed.

Results

Here we show that a lower expression of CDKN2A and a higher expression of cyclin D1 in the patients with high-grade malignant gliomas than low-grade gliomas, respectively. Moreover, overexpression of CDKN2A inhibits growth of glioma cell lines by suppression of cyclin D1 gene expression.

Conclusions

Our study suggests that CDKN2A as a malignant gliomas suppressor gene, appears to be useful for predicting behaviour of high-grade malignant gliomas. CDKN2A-Cyclin-Rb pathway plays a key role on malignant gliomas formation and that therapeutic targeting of this pathway may be useful in malignant gliomas treatment.     

Experimental validation of 5 in-silico predicted glioma biomarkers

Background

Glioblastoma multiforme (GBM) is a high-grade glioma with poor prognosis. Identification of new biomarkers specific to GBM could help in disease diagnosis. We have developed and validated a bioinformatics method to predict proteins likely to be suitable as glioma biomarkers via a global microarray meta-analysis to identify uncharacterized genes consistently coexpressed with known glioma-associated genes.

Methods

A novel bioinformatics method was implemented called global microarray meta-analysis, using ∼16 000 microarray experiments to identify uncharacterized genes consistently coexpressed with known glioma-associated genes. These novel biomarkers were validated as proteins highly expressed in human gliomas varying in tumor grades using immunohistochemistry. Glioma gene databases were used to assess delineation of expression of these markers in varying glioma grades and subtypes of GBM.

Results

We have identified 5 potential biomarkers—spondin1, Plexin-B2, SLIT3, fibulin-1, and LINGO1—that were validated as proteins highly expressed on the surface of human gliomas using immunohistochemistry. Expression of spondin1, Plexin-B2, and SLIT3 was significantly higher (P < .01) in high-grade gliomas than in low-grade gliomas. These biomarkers were significant discriminators in grade IV gliomas compared with either grade III or II tumors and also distinguished between GBM subclasses.

Conclusions

This study strongly suggests that this type of bioinformatics approach has high translational potential to rapidly discern which poorly characterized proteins may be of clinical relevance.     

Functional analysis of a novel glioma antigen,EFTUD1

Background

A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.

Methods

EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.

Results

EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.

Conclusion

These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.     

MiR-145 functions as a tumor-suppressive RNA by targeting Sox9 and adducin 3 in human glioma cells

Background

MicroRNAs (miRNAs) are increasingly being recognized as being involved in cancer development and progression in gliomas.

Methods

Using a model cell system developed in our lab to study glioma progression comprising human neuroglial culture (HNGC)–1 and HNGC-2 cells, we report here that miR-145 is one of the miRNAs significantly downregulated during malignant transformation in glioblastoma multiforme (GBM). In a study using tumor samples derived from various glioma grades, we show that expression of miR-145 is decreased in a graded manner, with GBM patients showing lowest expression relative to lower-grade gliomas (P < .05) and normal brain tissues (P < .0001). Functional studies involving ectopic expression of miR-145 in glioma cells had a negative impact on cell proliferation and tumor development, as well as invasion and induced apoptosis, providing further support to the concept that inactivation of miR-145 is important for glioma disease pathogenesis. More notably, these growth-suppressive effects of miR-145 are mediated through its target proteins Sox9 and the cell adhesion-associated molecule adducin 3 (ADD3).

Results

Inhibiting Sox9 and ADD3 rescued effects of miR-145 loss. Interestingly, miR-145 loss in glioma cells led to overexpression of molecules involved in cell proliferation, like cyclin D1, c-myc, and N-myc, as well as enhanced expression of cell adhesion- and invasion-related molecules N-cadherin and E-cadherin, an effect which was again restored upon miR-145 overexpression in glioma cells. The miR-145 promoter was methylated at its cytosine–phosphate–guanine (CpG) islands in the glioma cell lines studied.

Conclusion

Our study demonstrates that miR-145 has a tumor-suppressive function in glioblastoma in that it reduces proliferation, adhesion, and invasion of glioblastoma cells, apparently by suppressing the activity of oncogenic proteins Sox9 and ADD3. Reduced levels of miR-145 may lead to neoplastic transformation and malignant progression in glioma due to unregulated activity of these proteins.     

Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy

Background:

Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues.

Methods:

To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines.

Results:

Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells.

Conclusion:

These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.     

Efficacy of systemic adoptive transfer immunotherapy targeting NY-ESO-1 for glioblastoma

Background

Immunotherapy is an ideal treatment modality to specifically target the diffusely infiltrative tumor cells of malignant gliomas while sparing the normal brain parenchyma. However, progress in the development of these therapies for glioblastoma has been slow due to the lack of immunogenic antigen targets that are expressed uniformly and selectively by gliomas.

Methods

We utilized human glioblastoma cell cultures to induce expression of New York–esophageal squamous cell carcinoma (NY-ESO-1) following in vitro treatment with the demethylating agent decitabine. We then investigated the phenotype of lymphocytes specific for NY-ESO-1 using flow cytometry analysis and cytotoxicity against cells treated with decitabine using the xCelligence real-time cytotoxicity assay. Finally, we examined the in vivo application of this immune therapy using an intracranially implanted xenograft model for in situ T cell trafficking, survival, and tissue studies.

Results

Our studies showed that treatment of intracranial glioma–bearing mice with decitabine reliably and consistently induced the expression of an immunogenic tumor-rejection antigen, NY-ESO-1, specifically in glioma cells and not in normal brain tissue. The upregulation of NY-ESO-1 by intracranial gliomas was associated with the migration of adoptively transferred NY-ESO-1–specific lymphocytes along white matter tracts to these tumors in the brain. Similarly, NY-ESO-1–specific adoptive T cell therapy demonstrated antitumor activity after decitabine treatment and conferred a highly significant survival benefit to mice bearing established intracranial human glioma xenografts. Transfer of NY-ESO-1–specific T cells systemically was superior to intracranial administration and resulted in significantly extended and long-term survival of animals.

Conclusion

These results reveal an innovative, clinically feasible strategy for the treatment of glioblastoma.     

PTK7 regulates Id1 expression in CD44-high glioma cells

Background

CD44 is a molecular marker associated with molecular subtype and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting the CD44-high gliomas.

Methods

Protein tyrosine kinase 7 (PTK7) mRNA expression was analyzed based on The Cancer Genome Atlas glioblastoma dataset. PTK7 expression was depleted through lentivirus-mediated short hairpin RNA knockdown. Terminal deoxynucleotidyl transferase dUTP nick-end labeling was used to evaluate cell apoptosis following PTK7 knockdown. Gene expression analysis was performed on Affymetrix microarray. A nude mice orthotopic tumor model was used to evaluate the in vivo effect of PTK7 depletion.

Results

PTK7 is highly expressed in CD44-high glioblastoma and predicts unfavorable prognosis. PTK7 knockdown attenuated cell proliferation, impaired tumorigenic potential, and induced apoptosis in CD44-high glioma cell lines. Gene expression analysis identified inhibitor of DNA Binding 1 (Id1) gene as a potential downstream effector for PTK7. Overexpression of Id1 mostly restored the cell proliferation and colony formation attenuated by PTK7 depletion. PTK7 enhanced anchorage-independent growth in normal human astrocytes, which was attenuated by Id1 knockdown. Furthermore, PTK7 regulated Id1 expression through modulating TGF-β/Smad signaling, while pharmacological inhibition on TGF-β/Smad signaling or PTK7/Id1 depletion attenuated TGF-β–stimulated cell proliferation. PTK7 depletion consistently reduced Id1 expression, suppressed tumor growth, and induced apoptosis in a murine orthotopic tumor model, which could be translated into prolonged survival in tumor-bearing mice.

Conclusions

PTK7 regulates Id1 expression in CD44-high glioma cell lines. Targeting PTK7 could be an effective strategy for treating glioma with high CD44 expression.     

Activation of the NRF2 pathway and its impact on the prognosis of anaplastic glioma patients

Background

Nuclear factor erythroid 2–related factor 2 (NRF2) plays pivotal roles in cytoprotection. We aimed at clarifying the contribution of the NRF2 pathway to malignant glioma pathology.

Methods

NRF2 target gene expression and its association with prognosis were examined in 95 anaplastic gliomas with or without isocitrate dehydrogenase (IDH) 1/2 gene mutations and 52 glioblastomas. To explore mechanisms for the altered activity of the NRF2 pathway, we examined somatic mutations and expressions of the NRF2 gene and those encoding NRF2 regulators, Kelch-like ECH-associated protein 1 (KEAP1) and p62/SQSTSM. To clarify the functional interaction between IDH1 mutations and the NRF2 pathway, we introduced a mutant IDH1 to T98 glioblastoma-derived cells and examined the NRF2 activity in these cells.

Results

NRF2 target genes were elevated in 13.7% and 32.7% of anaplastic gliomas and glioblastomas, respectively. Upregulation of NRF2 target genes correlated with poor prognosis in anaplastic gliomas but not in glioblastomas. Neither somatic mutations of NRF2/KEAP1 nor dysregulated expression of KEAP1/p62 explained the increased expression of NRF2 target genes. In most cases of anaplastic glioma with mutated IDH1/2, NRF2 and its target genes were downregulated. This was reproducible in IDH1 R132H–expressing T98 cells. In minor cases of IDH1/2-mutant anaplastic gliomas with increased expression of NRF2 target genes, the clinical outcomes were significantly poor.

Conclusions

The NRF2 activity is increased in a significant proportion of malignant gliomas in general but decreased in the majority of IDH1/2-mutant anaplastic gliomas. It is plausible that the NRF2 pathway plays an important role in tumor progression of anaplastic gliomas with IDH1/2 mutations.     

Invasion and proliferation kinetics in enhancing gliomas predict IDH1 mutation status

Background

Glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than gliomas with wild-type IDH1.

Methods

Here we compare the IDH1 mutational status in 172 contrast-enhancing glioma patients with the invasion profile generated by a patient-specific mathematical model we developed based on MR imaging.

Results

We show that IDH1-mutated contrast-enhancing gliomas were relatively more invasive than wild-type IDH1 for all 172 contrast-enhancing gliomas as well as the subset of 158 histologically confirmed glioblastomas. The appearance of this relatively increased, model-predicted invasive profile appears to be determined more by a lower model-predicted net proliferation rate rather than an increased model-predicted dispersal rate of the glioma cells. Receiver operator curve analysis of the model-predicted MRI-based invasion profile revealed an area under the curve of 0.91, indicative of a predictive relationship. The robustness of this relationship was tested by cross-validation analysis of the invasion profile as a predictive metric for IDH1 status.

Conclusions

The strong correlation between IDH1 mutation status and the MRI-based invasion profile suggests that use of our tumor growth model may lead to noninvasive clinical detection of IDH1 mutation status and thus lead to better treatment planning, particularly prior to surgical resection, for contrast-enhancing gliomas.     

Do relevant markers of cancer stem cells CD133 and Nestin indicate a poor prognosis in glioma patients? A systematic review and meta-analysis

Background

CD133 and Nestin, as the markers of cancer stem cells, have recently been reported frequently in the pathogenesis and development of human gliomas. However, the prognostic role of CD133 and Nestin in gliomas still remains controversial. In this study, we aimed to evaluate the association between the expression of CD133 and Nestin and the outcome of glioma patients by conducting a systematic review and meta-analysis.

Methods

We performed systematically electronic and manual searches through the database of Pubmed and embase (until to December 25, 2014) for titles and abstracts which investigated the relationships between CD133 and Nestin expression and outcome of glioma patients. A systematic review and meta-analysis was executed to generate Pooled hazard ratios (HRs) with 95 % confidence intervals (CIs) for overall survival (OS) and progression-free survival (PFS).

Results

A total of 1,490 patients from 32 studies (13 articles) were included in the analysis. 19 studies and 13 studies investigated correlation between CD133 expression or Nestin and survival in gliomas, respectively. Our results showed that high CD133 expression in patients with glioma was associated with poor prognosis in terms of OS (HR 1.69; 95 % CI, 1.16–2.47; P =0.0060) and PFS (HR, 1.64; 95 % CI, 1.12–2.39; P = 0.010). In addition, high Nestin expression were associated with worse OS (HR 1.751; 95 % CI, 1.19–2.58, p = 0.004) but has no significant association with PFS (HR 1.55; 95 % CI, 0.96–2.51, p = 0.074). Even more important, the results of the subgroup meta-analyses show that that high CD133 expression was associated with worse prognosis in terms of OS and PFS in patients with WHO IV glioma but not WHO II-III. On the other hand, Nestin high expression was associated with worse prognosis in terms of OS and PFS in patients with WHO II-III glioma but not WHO IV.

Conclusion

High level of CD133 expression trends to correlate with a worse OS and PFS in glioma patients, especially WHO IV gliomas and Nestin high expression trends to correlate with a worse OS in glioma patients especially WHO II–III, revealing both the markers of cancer stem cells may as the potential pathological prognostic markers for glioma patients.     

TGM2 inhibition attenuates ID1 expression in CD44-high glioma-initiating cells

Background

CD44 is a molecular marker associated with cancer stem cell populations and treatment resistance in glioma. More effective therapies will result from approaches aimed at targeting glioma cells high in CD44.

Methods

Glioma-initiating cell lines were derived from fresh surgical glioblastoma samples. Expression of tissue transglutaminase 2 (TGM2) was attenuated through lentivirus-mediated short hairpin RNA knockdown. MTT assay [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was used to evaluate the growth inhibition induced by TGM2 inhibitor. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling was used to evaluate cell apoptosis following TGM2 inhibition. CD44⁺ glioma stem cells were sorted by flow cytometry. A nude mice orthotopic xenograft model was used to evaluate the in vivo effect of TGM2 inhibitor.

Results

TGM2 was highly expressed in CD44-high glioblastoma tissues and tumor-derived glioma-initiating cell lines. TGM2 knockdown impaired cell proliferation and induced apoptosis in CD44-high glioma-initiating cell lines. Further studies indicated that expression of inhibitor of DNA binding 1 protein (ID1) is regulated by TGM2 and might be an important mediator for TGM2-regulated cell proliferation in CD44-high glioma-initiating cell lines. TGM2 inhibitor reduces ID1 expression, suppresses cell proliferation, and induces apoptosis in CD44-high glioma-initiating cell lines. Furthermore, TGM2 is highly expressed in CD44⁺ glioma stem cells, while pharmacological inhibition of TGM2 activity preferentially eliminates CD44⁺ glioma stem cells. Consistently, TGM2 inhibitor treatment reduced ID1 expression and induced apoptosis in our orthotopic mice xenograft model, which can be translated into prolonged median survival in tumor-bearing mice.

Conclusions

TGM2 regulates ID1 expression in glioma-initiating cell lines high in CD44. Targeting TGM2 could be an effective strategy to treat gliomas with high CD44 expression.     

Lactate dehydrogenase A silencing in IDH mutant gliomas

Background

Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects.

Methods

We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant (^mt) and IDH wild-type (^wt) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas.

Results

We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDH^mt gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDH^mt derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDH^wt), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDH^mt glioblastomas.

Conclusion

To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDH^mt gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis.