As Hegde

Novel Glioblastoma Markers with Diagnostic and Prognostic Value Identified through Transcriptome Analysis

Purpose: Current methods of classification of astrocytoma based on histopathologic methods are often subjective and less accurate. Although patients with glioblastoma have grave prognosis, significant variability in patient outcome is observed. Therefore, the aim of this study was to identify glioblastoma diagnostic and prognostic markers through microarray analysis. Experimental Design: We carried out transcriptome analysis of 25 diffusely infiltrating astrocytoma samples [WHO grade II—diffuse astrocytoma, grade III—anaplastic astrocytoma, and grade IV—glioblastoma (GBM)] using cDNA microarrays containing 18,981 genes. Several of the markers identified were also validated by real-time reverse transcription quantitative PCR and immunohistochemical analysis on an independent set of tumor samples (n = 100). Survival analysis was carried out for two markers on another independent set of retrospective cases (n = 51). Results: We identified several differentially regulated grade-specific genes. Independent validation by real-time reverse transcription quantitative PCR analysis found growth arrest and DNA-damage–inducible α (GADD45α) and follistatin-like 1 (FSTL1) to be up-regulated in most GBMs (both primary and secondary), whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 were up-regulated in the majority of primary GBM. Further, identification of the grade-specific expression of GADD45α and FSTL1 by immunohistochemical staining reinforced our findings. Analysis of retrospective GBM cases with known survival data revealed that cytoplasmic overexpression of GADD45α conferred better survival while the coexpression of FSTL1 with p53 was associated with poor survival. Conclusions: Our study reveals that GADD45α and FSTLI are GBM-specific whereas superoxide dismutase 2 and adipocyte enhancer binding protein 1 are primary GBM-specific diagnostic markers. Whereas GADD45α overexpression confers a favorable prognosis, FSTL1 overexpression is a hallmark of poor prognosis in GBM patients.

PBEF1/NAmPRTase/Visfatin: A potential malignant astrocytoma/glioblastoma serum marker with prognostic value

Malignant astrocytomas comprise anaplastic astrocytoma (AA; grade III) and Glioblastoma (GBM; grade IV). GBM is the most malignant with a median survival of 10-12 months in patients. Using cDNA microarray based expression profiling of different grades of astrocytomas, we identified several fold increased levels of PBEF1 transcripts in GBM samples. Pre-B-cell colony enhancing factor 1 gene (PBEF1) encodes Nicotinamide phosphoribosyltransferase (NAmPRTase), which catalyses the rate limiting step in the salvage pathway of NAD metabolism in mammalian cells. Further validation using real time RT-qPCR on an independent set of tumor samples (n=91) and normal brain samples (n=9), GBM specific higher expression of PBEF1 was confirmed. Immunohistochemical staining for PBEF1 on a subset of the above samples largely reinforced our finding. We carried out ELISA analysis on serum samples of astrocytoma patients to determine whether this protein levels would correlate with the presence of tumor and tumor grade. PBEF1 serum levels were substantially elevated in many of the AA and GBM patients. Statistical analysis of these data indicates that in patients with astrocytoma, serum PBEF1 levels correlate with tumor grade and is highest in GBM. Immunohistochemical analysis of an independent set of 51 retrospective GBM cases with known survival data revealed that PBEF1 expression in the tumor tissue along with its co-expression with p53 was associated with poor survival. Thus, we have identified PBEF1 as a potential malignant astrocytoma serum marker and prognostic indicator among GBMs.

Proteomic identification of Haptoglobin α2 as a glioblastoma serum biomarker: implications in cancer cell migration and tumor growth

Glioblastoma (GBM; grade IV astrocytoma) is the most malignant and common primary brain tumor in adults. Using combination of 2-DE and MALDI-TOF MS, we analyzed 14 GBM and 6 normal control sera and identified haptoglobin α2 chain as an up-regulated serum protein in GBM patients. GBM-specific up-regulation was confirmed by ELISA based quantitation of haptoglobin (Hp) in the serum of 99 GBM patients as against lower grades (49 grade III/AA; 26 grade II/DA) and 26 normal individuals (p = 0.0001). Further validation using RT-qPCR on an independent set (n = 78) of tumor and normal brain (n = 4) samples and immunohistochemcial staining on a subset (n = 42) of above samples showed increasing levels of transcript and protein with tumor grade and were highest in GBM (p = <0.0001 and <0.0001, respectively). Overexpression of Hp either by stable integration of Hp cDNA or exogenous addition of purified Hp to immortalized astrocytes resulted in increased cell migration. RNAi-mediated silencing of Hp in glioma cells decreased cell migration. Further, we demonstrate that both human glioma and mouse melanoma cells overexpressing Hp showed increased tumor growth. Thus, we have identified haptoglobin as a GBM-specific serum marker with a role on glioma tumor growth and migration.

Age-dependent prognostic effects of EGFR/p53 alterations in glioblastoma: study on a prospective cohort of 140 uniformly treated adult patients

Aims To assess the prognostic influence of EGFR amplification/overexpression, p53 immunoreactivity and their age-dependent prognostic effects in a large prospective cohort of uniformly treated adult patients with newly diagnosed glioblastoma. Methods Tumours from a uniformly treated prospective cohort of adult patients with newly diagnosed glioblastoma (n=140) were examined for EGFR amplification by fluorescence in situ hybridisation and EGFR/p53 expression by immunohistochemistry. Statistical methods were employed to assess the degree of association between EGFR amplification/overexpression and p53 immunopositivity. Survival analyses were performed by employing Cox proportional hazard models to assess the independent prognostic value of EGFR/p53 alterations and test the propensity for risk with age by assessing their interaction with patient age. Results A strong positive correlation between EGFR amplification and EGFR overexpression (ρ=0.5157; p<0.0001; CI 0.3783 to 0.6309) and a negative association of EGFR amplification (ρ=−0.3417; p<0.0001; CI −0.4842 to −0.1816) and EGFR overexpression (ρ=−0.3095; p<0.001; CI −0.4561 to −0.1465) with p53 immunopositivity was observed. Only patient age (HR: 1.029; p=0.004; CI 1.009 to 1.049) was associated with shorter survival by univariate Cox regression analysis. Multivariable Cox proportional hazards models revealed a statistically significant interaction between EGFR overexpression and age to be associated with shorter survival (HR: 1.001; p<0.0001; CI 1.000 to 1.002), thus predicting a higher hazard with increasing age. No age interaction of EGFR amplification status (HR: 1.001; p=0.642; CI 0.995 to 1.008) and p53 immunopositivity (HR: 1.000; p=0.841; CI 0.999 to 1.001) was noted in this cohort. Conclusions The prognostic value of EGFR overexpression is age-dependent, and there is a propensity for a higher hazard with increasing patient age. Identifying such groups of patients with more aggressive disease becomes mandatory, since they would benefit from intense therapeutic protocols targeting EGFR.

Essential role of METTL3-mediated m 6 A modification in glioma stem-like cells maintenance and radioresistance

Despite advances in biology and therapeutic modalities, existence of highly tumorigenic glioma stem-like cells (GSCs) makes glioblastomas (GBMs) invincible. N6-methyl adenosine (m6A), one of the abundant mRNA modifications catalyzed by methyltransferase-like 3 and 14 (METTL3/14), influences various events in RNA metabolism. Here, we report the crucial role of METTL3-mediated m6A modification in GSC (neurosphere) maintenance and dedifferentiation of glioma cells. METTL3 expression is elevated in GSC and attenuated during differentiation. RNA immunoprecipitation studies identified SOX2 as a bonafide m6A target of METTL3 and the m6A modification of SOX2 mRNA by METTL3 enhanced its stability. The exogenous overexpression of 3′UTR-less SOX2 significantly alleviated the inhibition of neurosphere formation observed in METTL3 silenced GSCs. METTL3 binding and m6A modification in vivo required intact three METTL3/m6A sites present in the SOX2-3′UTR. Further, we found that the recruitment of Human antigen R (HuR) to m6A-modified RNA is essential for SOX2 mRNA stabilization by METTL3. In addition, we found a preferential binding by HuR to the m6A-modified transcripts globally. METTL3 silenced GSCs showed enhanced sensitivity to γ-irradiation and reduced DNA repair as evidenced from the accumulation of γ-H2AX. Exogenous overexpression of 3′UTR-less SOX2 in METTL3 silenced GSCs showed efficient DNA repair and also resulted in the significant rescue of neurosphere formation from METTL3 silencing induced radiosensitivity. Silencing METTL3 inhibited RasV12 mediated transformation of mouse immortalized astrocytes. GBM tumors have elevated levels of METTL3 transcripts and silencing METTL3 in U87/TIC inhibited tumor growth in an intracranial orthotopic mouse model with prolonged mice survival. METTL3 transcript levels predicted poor survival in GBMs which are enriched for GSC-specific signature. Thus our study reports the importance of m6A modification in GSCs and uncovers METTL3 as a potential molecular target in GBM therapy.

Alterations in tumour suppressor gene p53 in human gliomas from Indian patients

Alterations in the tumour suppressorp53 gene are among the most common defects seen in a variety of human cancers. In order to study the significance of thep53 gene in the genesis and development of human glioma from Indian patients, we checked 44 untreated primary gliomas for mutations in exons 5–9 of thep53 gene by PCR-SSCP and DNA sequencing. Sequencing analysis revealed six missense mutations. The incidence of p53 mutations was 13⋅6% (6 of 44). All the six mutations were found to be located in the central core domain of p53, which carries the sequence-specific DNA-binding domain. These results suggest a rather low incidence but a definite involvement of p53 mutations in the gliomas of Indian patients.

Shift in AP-2α localization characterizes astrocytoma progression

Activator protein 2α (AP-2α) has been shown to be lost in the advanced stages of many cancers, including gliomas. In this study, we wanted to analyze the expression of AP-2α in astrocytoma samples of different grades both at the RNA level, by real-time qPCR and at the protein level, by immunohistochemistry, and to examine its correlation, if any, with patient outcome. Five Grade I, 14 Grade II, 18 Grade III, 72 Grade IV samples and 13 normal brain controls were included. We did not find any clear pattern of regulation at the RNA level with tumor grade. The RNA expression levels however, correlated to a large extent with the nuclear AP-2α staining in these samples (72.09%; 31/43). Further, we did not find a complete loss of nuclear AP-2α expression in the higher grades, in contrast to previous reports. Interestingly, we found cytoplasmic AP-2α expression in a majority of higher grade astrocytomas (Grade IV - 85%; 33/39 and Grade III- 74%; 14/19) in comparison to lower grades (Grade I - 0%; 0/5 and Grade II - 37.5%; 3/8) suggesting that the translocation of this protein from the nucleus to the cytoplasm may be responsible for the increased malignancy. The nuclear expression in these grades was found to be concomitantly reduced. Within GBMs, we found that decreased nuclear expression was indicative of a better prognosis. The striking observation was the shift in localization of this protein from the nucleus to the cytoplasm with increasing tumor grade, pointing to a crucial role for this transcription factor in the progression of astrocytomas.

Integrative functional genomic analysis identifies epigenetically regulated fibromodulin as an essential gene for glioma cell migration

An integrative functional genomics study of multiple forms of data are vital for discovering molecular drivers of cancer development and progression. Here, we present an integrated genomic strategy utilizing DNA methylation and transcriptome profile data to discover epigenetically regulated genes implicated in cancer development and invasive progression. More specifically, this analysis identified fibromodulin (FMOD) as a glioblastoma (GBM) upregulated gene because of the loss of promoter methylation. Secreted FMOD promotes glioma cell migration through its ability to induce filamentous actin stress fiber formation. Treatment with cytochalasin D, an actin polymerization inhibitor, significantly reduced the FMOD-induced glioma cell migration. Small interfering RNA and small molecule inhibitor-based studies identified that FMOD-induced glioma cell migration is dependent on integrin-FAK-Src-Rho-ROCK signaling pathway. FMOD lacking C-terminus LRR11 domain (ΔFMOD), which does not bind collagen type I, failed to induce integrin and promote glioma cell migration. Further, FMOD-induced integrin activation and migration was abrogated by a 9-mer wild-type peptide from the FMOD C-terminus. However, the same peptide with mutation in two residues essential for FMOD interaction with collagen type I failed to compete with FMOD, thus signifying the importance of collagen type I–FMOD interaction in integrin activation. Chromatin immunoprecipitation–PCR experiments revealed that transforming growth factor beta-1 (TGF-β1) regulates FMOD expression through epigenetic remodeling of FMOD promoter that involved demethylation and gain of active histone marks with a simultaneous loss of DNMT3A and EZH2 occupancy, but enrichment of Sma- and Mad-related protein-2 (SMAD2) and CBP. FMOD silencing inhibited the TGF-β1-mediated glioma cell migration significantly. In univariate and multivariate Cox regression analysis, both FMOD promoter methylation and transcript levels predicted prognosis in GBM. Thus, this study identified several epigenetically regulated alterations responsible for cancer development and progression. Specifically, we found that secreted FMOD as an important regulator of glioma cell migration downstream of TGF-β1 pathway and forms a potential basis for therapeutic intervention in GBM.