MicroRNA in Glioblastoma: An Overview Banelli, Barbara; Forlani, Alessandra; Allemanni, Giorgio ...
International journal of genomics,
01/2017, Volume:
2017
Journal Article
Peer reviewed
Open access
Glioblastoma is the most aggressive brain tumor and, even with the current multimodal therapy, is an invariably lethal cancer with a life expectancy that depends on the tumor subtype but, even in the ...most favorable cases, rarely exceeds 2 years. Epigenetic factors play an important role in gliomagenesis, are strong predictors of outcome, and are important determinants for the resistance to radio- and chemotherapy. The latest addition to the epigenetic machinery is the noncoding RNA (ncRNA), that is, RNA molecules that are not translated into a protein and that exert their function by base pairing with other nucleic acids in a reversible and nonmutational mode. MicroRNAs (miRNA) are a class of ncRNA of about 22 bp that regulate gene expression by binding to complementary sequences in the mRNA and silence its translation into proteins. MicroRNAs reversibly regulate transcription through nonmutational mechanisms; accordingly, they can be considered as epigenetic effectors. In this review, we will discuss the role of miRNA in glioma focusing on their role in drug resistance and on their potential applications in the therapy of this tumor.
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
In glioblastoma several histone demethylase genes (KDM) are overexpressed compared to normal brain tissue and the development of Temozolomide (TMZ) resistance is accompanied by the transient further ...increased expression of KDM5A and other KDMs following a mechanism that we defined as "epigenetic resilience". We hypothesized that targeting KDMs may kill the cells that survive the cytotoxic therapy.We determined the effect of JIB 04 and CPI-455, two KDM inhibitors, on glioblastoma cells and found that both molecules are more effective against TMZ-resistant rather than native cells.Because of its lower IC50, we focused on JIB 04 that targets KDM5A and other KDMs as well. We have shown that this molecule activates autophagic and apoptotic pathways, interferes with cell cycle progression, inhibits cell clonogenicity and dephosphorylates Akt thus inactivating a potent pro-survival pathway. We performed combination temozolomide/JIB 04 in vitro treatments showing that these two molecules, under certain conditions, have a strong synergic effect and we hypothesize that JIB 04 intercepts the cells that escape the G2 block exerted by TMZ. Finally we studied the permeability of JIB 04 across the blood-brain barrier and found that this molecule reaches bioactive concentration in the brain; furthermore a pilot in vivo experiment in an orthotopic GB xenograft model showed a trend toward longer survival in treated mice with an Hazard Ratio of 0.5.In conclusion we propose that the combination between cytotoxic drugs and molecules acting on the epigenetic landscape may offer the opportunity to develop new therapies for this invariably lethal disease.
Approximately 20% of stage 4 high-risk neuroblastoma patients are alive and disease-free 5 years after disease onset while the remaining experience rapid and fatal progression. Numerous findings ...underline the prognostic role of methylation of defined target genes in neuroblastoma without taking into account the clinical and biological heterogeneity of this disease. In this report we have investigated the methylation of the PCDHB cluster, the most informative member of the "Methylator Phenotype" in neuroblastoma, hypothesizing that if this epigenetic mark can predict overall and progression free survival in high-risk stage 4 neuroblastoma, it could be utilized to improve the risk stratification of the patients, alone or in conjunction with the previously identified methylation of the SFN gene (14.3.3sigma) that can accurately predict outcome in these patients. We have utilized univariate and multivariate models to compare the prognostic power of PCDHB methylation in terms of overall and progression free survival, quantitatively determined by pyrosequencing, with that of other markers utilized for the patients' stratification utilizing methylation thresholds calculated on neuroblastoma at stage 1-4 and only on stage 4, high-risk patients. Our results indicate that PCDHB accurately distinguishes between high- and intermediate/low risk stage 4 neuroblastoma in agreement with the established risk stratification criteria. However PCDHB cannot predict outcome in the subgroup of stage 4 patients at high-risk whereas methylation levels of SFN are suggestive of a "methylation gradient" associated with tumor aggressiveness as suggested by the finding of a higher threshold that defines a subset of patients with an extremely severe disease (OS <24 months). Because of the heterogeneity of neuroblastoma we believe that clinically relevant methylation markers should be selected and tested on homogeneous groups of patients rather than on patients at all stages.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Epigenetic alterations are hallmarks of cancer and powerful biomarkers, whose clinical utilization is made difficult by the absence of standardization and of common methods of data interpretation. ...The coordinate methylation of many loci in cancer is defined as ‘CpG island methylator phenotype' (CIMP) and identifies clinically distinct groups of patients. In neuroblastoma (NB), CIMP is defined by a methylation signature, which includes different loci, but its predictive power on outcome is entirely recapitulated by the PCDHB cluster only. We have developed a robust and cost-effective pyrosequencing-based assay that could facilitate the clinical application of CIMP in NB. This assay permits the unbiased simultaneous amplification and sequencing of 17 out of 19 genes of the PCDHB cluster for quantitative methylation analysis, taking into account all the sequence variations. As some of these variations were at CpG doublets, we bypassed the data interpretation conducted by the methylation analysis software to assign the corrected methylation value at these sites. The final result of the assay is the mean methylation level of 17 gene fragments in the protocadherin B cluster (PCDHB) cluster. We have utilized this assay to compare the methylation levels of the PCDHB cluster between high-risk and very low-risk NB patients, confirming the predictive value of CIMP. Our results demonstrate that the pyrosequencing-based assay herein described is a powerful instrument for the analysis of this gene cluster that may simplify the data comparison between different laboratories and, in perspective, could facilitate its clinical application. Furthermore, our results demonstrate that, in principle, pyrosequencing can be efficiently utilized for the methylation analysis of gene clusters with high internal homologies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The hypermethylation of CpG islands within gene promoter regions is an epigenetic phenomenon that is often, but not always, associated with the transcriptional silencing of downstream genes and ...contributes to carcinogenesis. We have determined the pattern of methylation of several genes involved in distinct biological pathways, including cell proliferation and apoptosis, in neuroblastoma and in the nonmalignant ganglioneuroma. The purpose of this work was to search for epigenetic signatures that could be associated with defined clinical and biological parameters and that, in prospective, could identify specific risk categories among the patients. We have analysed 31 malignant neuroblastoma with or without MYCN amplification and 13 benign ganglioneuroma and we have observed dramatic differences in the methylation pattern of five genes (CASP8, 14.3.3sigma, DeltaN-p73, RASSF1A and DCR2) between these tumors indicating that this phenomenon is not tissue-specific and can be considered as cancer-dependent. Furthermore, the methylation pattern of 14.3.3sigma, RASSF1A and of an intragenic segment of CASP8 was significantly different between MYCN amplified and single copy neuroblastoma suggesting a specific role of epigenetic alterations in aggressive neuroblastoma.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The aim of our study was to identify threshold levels of DNA methylation predictive of the outcome to better define the risk group of stage 4 neuroblastic tumor patients. Quantitative pyrosequencing ...analysis was applied to a training set of 50 stage 4, high risk patients and to a validation cohort of 72 consecutive patients. Stage 4 patients at lower risk and ganglioneuroma patients were included as control groups. Predictive thresholds of methylation were identified by ROC curve analysis. The prognostic end points of the study were the overall and progression‐free survival at 60 months. Data were analyzed with the Cox proportional hazard model. In a multivariate model the methylation threshold identified for the SFN gene (14.3.3σ) distinguished the patients presenting favorable outcome from those with progressing disease, independently from all known predictors (Training set: Overall Survival HR 8.53, p = 0.001; Validation set: HR 4.07, p = 0.008). The level of methylation in the tumors of high‐risk patients surviving more than 60 months was comparable to that of tumors derived from lower risk patients and to that of benign ganglioneuroma. Methylation above the threshold level was associated with reduced SFN expression in comparison with samples below the threshold. Quantitative methylation is a promising tool to predict survival in neuroblastic tumor patients. Our results lead to the hypothesis that a subset of patients considered at high risk—but displaying low levels of methylation—could be assigned at a lower risk group.
We summarize recent findings linking inflammatory hypoxia to chromatin modifications, in particular to repressive histone signatures. We focus on the role of Hypoxia-Induced Factor-1 in promoting the ...activity of specific histone demethylases thus deeply modifying chromatin configuration. The consequences of these changes are depicted in terms of gene expression and cellular phenotypes. We finally integrate available data to introduce novel speculations on the relationship between inflammation, histones, and DNA function and integrity.
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DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
9.
Caspase-8 Gene Expression in Neuroblastoma CASCIANO, IDA; BANELLI, BARBARA; CROCE, MICHELA ...
Annals of the New York Academy of Sciences,
December 2004, Volume:
1028, Issue:
1
Journal Article
Peer reviewed
: Neuroblastoma (NB) is a solid tumor of infancy that presents a high rate of spontaneous regression, a phenomenon that likely reflects the activation of an apoptotic/differentiation program. Indeed, ...the level of expression of molecules involved in the regulation of apoptosis, such as p73 or survivin, is a prognostic factor in NB patients. The caspase‐8 gene (CASP8) encodes a key enzyme at the top of the apoptotic cascade. Although methylation of a putative regulatory region of the CASP8 gene reportedly inhibits its transcription in some MYCN‐amplified NB, our results indicate that the transcriptional inactivation of caspase‐8 occurs in a subset of primary NB independently of MYCN amplification or CpG methylation. In addition, the apoptotic agent fenretinide (4HPR) and interferon‐γ (IFN‐γ) induce caspase‐8 expression without modifying the methylation status of this gene. Nevertheless, the methylation level of CASP8 intragenic and promoter regions is higher in MYCN‐amplified tumors as compared to nonamplified samples. This phenomenon might reflect the existence of distinct DNA methylation errors in MYCN‐amplified and MYCN‐single copy tumors. To gain information on the mechanisms that regulate the expression of this crucial apoptotic gene, we searched for potential CASP8 regulatory regions and cloned a DNA element at the 5′ terminus of this gene that functionally acts as a promoter only in NB cell lines that express caspase‐8. The retinoic acid analogue 4HPR, IFN‐γ, and the demethylating agent 5‐aza‐cytidine activate this promoter in NB cells that lack endogenous caspase‐8, indicating that this element may regulate both constitutive and inducible CASP8 expression. These results indicate also that demethylation of the cellular genome may upregulate CASP8 through the action of trans‐acting factors. Our results provide new insights to the regulation of CASP8, a gene with an essential role in a variety of physiologic and pathologic conditions.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Two different oncofetal fibronectins (FN) have been reported: one, generated by O-glycosylation in the splicing region IIICS that is recognized by monoclonal antibody (MAb) FDC-6, and another, ...recognized by MAb BC-I, generated by the alternative splicing of the FN pre-mRNA which includes an extra type-III repeat called ED-B. Using these and 2 other MAbs (IST-4 which recognizes all different FN isoforms and IST-6 which recognizes only the FN molecules that do not include the ED-B sequence) we have immunohistochemically studied 171 normal, hyperplastic and neoplastic breast-tissue specimens. Although all normal specimens reacted strongly with MAbs IST-4 and IST-6, they did not show the presence of oncofetal FNs as established by the use of BC-I and FDC-6. In contrast, out of the 97 cases of invasive ductal carcinomas studied, 90 (93%) and 96 (99%) reacted positively with BC-I and FDC-6, respectively, the reaction being observed in the tumoral stroma connective tissue and in tumoral vessels. Furthermore, invasive lobular carcinoma showed less intense and less frequent staining with BC-1 and FDC-6 (10 and 11 out of 14, respectively). We found differences in the distribution of the 2 oncofetal fibronectin isoforms within the same specimens. The most remarkable difference was observed in the tumoral vessels: in invasive ductal carcinoma MAb BC-1 revealed a positive reaction with vessels in 78% of cases while FDC-6 showed such a reaction in only 59% of cases.
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