**3. Epigenetic mechanisms in CNS tumors**

rodent choroid plexus epithelial cell line Z310 and observed that growth-inhibitory activity of vincristine, doxorubicin, carboplatin, etoposide, and TMZ was significantly impaired by silencing of *TP53*, showing the potential predictive role of p53 in choroid plexus carcinomas.

Few studies have examined the *TP53* gene directly for mutations in meningiomas, [133-135], and these studies typically have not observed mutations in this gene, although rare mutants have been described, mainly associated with malignant histology [134, 136]. One group working on specimens from Korean patients has documented a rate of nearly 40% of p53 overexpressing meningiomas as having mutations, and observed that the mutation rate was

In contrast the low frequency of *TP53* point mutations, expression of p53 was found in 10% to 90% of meningiomas [138], but their role in pathogenesis is still uncertain. Studies suggested the involvement of the p53 pathway in meningioma development: the correlation of p53 protein expression with histological tumor grade and meningioma recurrence [139]; methyl‐ ation of the *p14 ARF* gene in 8.6% of benign, 20% of atypical and in 50% of anaplastic meningi‐ omas and loss of detectable Mdm2 protein in high grade meningiomas [140]; defective p53 response to gamma ray stress in meningioma cells [141]. In addition, the *NF2* protein product was reported to increase p53 stability through downregulation of Mdm2 levels in mouse fibroblast [142]. It follows that loss of NF2 may increase the likelihood of p53 suppression, thus decreasing tumor suppression activity and providing a possible mechanism for the involve‐

Many studies have examined benign and atypical/malignant meningiomas for over-expres‐ sion of the p53 protein with diverse results: p53 over-expression has been reported in 0–10% of benign, 50–72.7% of atypical and 77–88.9% of anaplastic or malignant meningiomas [143]. Despite the differing rates, all of the studies are consistent, with atypical/malignant tumors showing higher rates of over-expression than benign meningiomas. However, studies on the biological significance of p53 over-expression are highly contradictory. While over-expression of p53 has been associated with recurrence in some studies [139, 144], no association has been found in others [133, 145]; and still other studies have suggested that expression of high levels

Terzi *et al.* [147] analyzed the immunohistochemical expression of Ki-67, p53, p21, p16, and *PTEN* proteins in 130 meningiomas (64 benign, 39 atypical, and 27 malignant meningiomas) using tissue microarray and demonstrated that Histological grade, p53, Ki-67 labeling indices, and overexpression of p16 were strongly associated with decreased event-free survival in univariate analysis and Ki-67 and p53 labeling indices are useful additional tools in discrimi‐

associated with both histological grade and recurrence [137].

140 Tumors of the Central Nervous System – Primary and Secondary

ment of the p53 pathway in meningiomas.

of p53 may be protective against recurrence [146].

nating atypical from benign or anaplastic meningiomas.

**2.4. Meningiomas**

Epigenetics is defined as mitotically heritable changes in gene expression that are not due to changes in the primary DNA sequence. The coordinated interaction of these changes regulates gene expression activity and several types of epigenetic marks work in concert to drive appropriate gene expression, like DNA methylation at CpG dinucleotides, covalent modifications of histone proteins, non-coding RNAs, and other complementary mechanisms controlling higher order chromatin organization within the cell nucleus. Epigenetic alterations have been recognized as important mechanisms in neoplastic transformation, malignant progression of cancer, and although epigenetic changes are somatically inheritable, they are reversible and hence may represent actionable targets for novel therapies [148, 149]

Epigenetic changes are often observed at the earliest stages of neoplasia within the altered tissue stem and progenitor cells. These observations have led to the epigenetic progenitor model [149]. This model explains that transformation to a malignant state occurs in three steps. First, there is an expansion of an epigenetically permissive population due to an essential early epigenetic disruption of stem/progenitor cells. Second, an initiating genetic alteration in an oncogene or tumor suppressor gene occurs. Finally, genetic and epigenetic plasticity resulting in an enhanced ability to stably evolve the phenotype is observed. An important difference to the clonal genetic model is that the epigenetic 'hits' occur early, and are necessary to create an appropriate expansion of a polyclonal population, that is the cellular substrate for subsequent genetic alterations and transformation [150].

To better understand the multiple cellular pathways involved in their development, estab‐ lishment markers of resistance to traditional therapies, and contribution to the development of targeted therapies, a comprehensive appreciation of the integrated genomics and epige‐ nomics of CNS tumors is needed [151].

#### **3.1. DNA methylation of gene** *TP53*

Hypermethylation of promoters usually occurs at CpG islands. Methylation of *TP53* was reported as a mechanism for its inactivation in neoplasias, such as acute lymphoblastic leukemia, multiple myeloma, malignant glioma cells, and brain metastases of solid tumors [152]. Since the promoter region of *TP53* does not contain a classic CpG island, methylation of one or two sites may produce a proportionately greater effect in downregulation of transcrip‐ tion compared to a tumor suppressor gene with a classic CpG island in the promoter [153]. The *TP53* promoter region has been sequenced and basal promoter activity localized to an 85 bp region (nucleotide 760–844) that is indispensable for full promoter activity and the *TP53* promoter has putative binding sites for transcriptional factors [154]. Schroeder and Mass [155] have shown that methylation in the promoter region of the p53 gene reduces reporter gene activity. They found down-regulation of p53 in cultured cells transfected with a plasmid incorporating a *TP53* promoter containing methylated CpG dinucleotides. Furthermore, this region has been shown to be methylated in several cancers [156].

Analyses of methylation of *TP53* promoter region are controversial. While some researchers reported low frequencies of *TP53* methylation in neuroblastic tumors (0/44), astrocytomas (2/24, 8%), GBM (1/43, 2%) [157], oligodendroglial tumors (0/41) and ependymomas (0/7) [158], other authors observed a higher frequency [159, 160]. The reason for this discrepancy remains to be clarified.

influence almost every cellular process [163]. Currently, 1, 048 human microRNAs are known to modulate approximately 3 % of all genes and up to 30 % of protein-coding genes. Vital for protein expression, microRNAs are integrally associated with both normal and abnormal

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miRNAs play important roles in the regulation of normal gene expression at developmental timing, cell proliferation and apoptosis [165]. As these processes are altered in cancer cells, there are in literature several studies that were undertaken to provide evidence for an involvement of miRNAs in cancer formation. miRNA-encoding genes as well as mRNAencoding genes have been meanwhile classified as oncogenic or tumor suppressive genes according to their function in cellular transformation and expression in tumors [166, 167]. Furthermore, tumor cells seem to undergo a general loss of miRNA expression, and forced reduction of global miRNA expression promotes transformation [168]. Interestingly, miRNAs cluster within fragiles sites and other genomic regions frequently altered in cancers [169]. Because of their role in tumor formation, miRNAs may be very useful for the classification,

Profiling miRNA provides an attractive, novel, and non-invasive biomarker for tumor diagnosis and prognosis. Molecular biology techniques, such as Northern blot, RNase protection assay, and primer extension assay can measure expression of a miRNA. The small size of miRNAs initially hampered polymerase chain reaction-based methods. However, PCRbased techniques have become very popular since the development of adaptor-mediated quantitative real-time PCR (qRT-PCR) due to their high sensitivity [170]. Microarray techni‐ ques are widely used to comprehensively assay the entire miRNome (the global miRNA expression profile) in tissues or in cell lines [171]. In addition to microarray and qRT-PCR, miRNomes are obtained by *in situ* hybridization [172] and serial analysis of gene expression adapted for small RNAs [173]. Overall, these technical improvements are expected to greatly

p53 is a transcription factor, so transactivates or represses many protein-encoding genes and this underlies much of its tumor suppressor function. Recently, it has been reported that p53 directly transactivates specifics miRNAs [174]. miRNA have also been shown to target p53 and/or components of p53 regulatory pathways affecting its activities directly and/or indirectly

Several reports shed light on the involvement of miRNAs in the p53 pathway. He *et al.* [177] profiled miRNA gene expression in wild-type (wt) and p53-deficient cells and found that the miR34s (miR-34 gene family, including miR-34a, b and c) was among the most upregulated in wt p53 cells. In addition, Luan *et al.* [178] analyzed the expression levels of miR-34a in human glioma cell lines (U251, A172 and SHG-44) using real time quantitative PCR and compared with that observed in normal brain and determined its role in cell proliferation, cycle distri‐ bution, apoptosis and capabilities of in vitro migration and invasion of p53-mutant glioma cells. The results showed that miR-34a is remarkably reduced in p53-mutant glioma cell line U251, that had a mutation of codon 273 (CGT/CAT; Arg/His) in exon 8 than other p53-wild

diagnosis, prognosis, and therapy of malignancies [166, 167].

widen the repertoire of miRNAs in a variety of biological systems.

glioma cell lines A172, SHG-44 and normal brains.

biological processes [164].

[175, 176].

Amatya *et al.*[159] assessed whether promoter methylation was present in cells of six malignant gliomas and whether there is an association with reduced expression of *TP53* mRNA and protein. They also assessed the frequencies of disruption of the p53/p14 *ARF* pathway in 49 lowgrade astrocytomas (40 fibrillary astrocytomas and 9 gemistocytic astrocytomas), 42 oligoden‐ drogliomas and 18 oligoastrocytomas. The Methylation-specific PCR (MS-PCR) revealed methylation of the promoter region of the *TP53* gene in three (U87MG, LNT-229, T98G) out of six malignant glioma cell lines. Real time RT-PCR revealed that two malignant glioma cell lines (U87MG and T98G) led to up-regulated expression of *TP53* mRNA and protein after treatment with 5-aza-2'-deoxycytidine (5-aza-dC, an epigenetic modifier that results in DNA demethy‐ lation), suggesting that promoter methylation is associated with reduced expression in some malignant glioma cells. *TP53* promoter methylation in primary tissue of low-grade gliomas was observed in 29/48 (60%) low-grade astrocytomas, 11/18 (61%) oligoastrocytomas, and 31/42 (74%) oligodendrogliomas, while promoter methylation of the p14 ARF was detected by MS-PCR in 5/49 (10%) low-grade astrocytomas, 7/18 (39%) oligoastrocytomas, and 15/41 (37%) oligodendrogliomas. Briefly, alterations of at least one of *TP53* promoter methylation, p14 ARF promoter methylation, and *TP53* mutations were found in 43/49 (88%) of low-grade astrocy‐ tomas, 15/18 (83%) of oligoastrocytomas, and 35/42 (83%) oligodendrogliomas, suggesting that disruption of the p53/p14 *ARF* pathway is frequent in all histological types of low-grade glioma.

Almeida *et al.* [160] evaluated the promoter hypermethylation profile of the *TP53* gene in 90 extra-axial brain tumors (48 meningiomas, 23 schwannomas and 19 metastases) using MS-PCR and sequencing. The group showed that the methylation of the *TP53* gene is an important event associated with extra-axial brain tumors, since 37.5% of meningiomas, 30% of schwan‐ nomas and 52.6% of metastases were hypermethylated. When tumor grade was compared, 35.3% of benign tumors and 48% of malignant tumors were methylated, and these results suggested that *TP53* methylation can be involved in the progression of these tumors.

#### **3.2. The new insights of MicroRNAs/***TP53* **in cancer**

MicroRNAs (miRNA) are a large class of small, non-coding RNAs, 21 – 28 nucleotides long, produced naturally in cells after being cut into segments from larger strands of RNA by the enzyme Dicer. They function by binding to complementary sites on the 3'-untranslated region (3'-UTR) of genes and promoting the recruitment of protein complexes responsible for impairing translation and/or decreasing the stability of mRNA [161, 162]. A specific miRNA may simultaneously regulate multiple targets, thereby enabling complex changes in protein expression profiles. Furthermore, a single target can be regulated by multiple miRNAs, and upstream regulation of a given miRNA can involve multiple regulators at different steps of miRNA biogenesis. Thus, miRNAs take part in complex regulatory networks that may influence almost every cellular process [163]. Currently, 1, 048 human microRNAs are known to modulate approximately 3 % of all genes and up to 30 % of protein-coding genes. Vital for protein expression, microRNAs are integrally associated with both normal and abnormal biological processes [164].

Analyses of methylation of *TP53* promoter region are controversial. While some researchers reported low frequencies of *TP53* methylation in neuroblastic tumors (0/44), astrocytomas (2/24, 8%), GBM (1/43, 2%) [157], oligodendroglial tumors (0/41) and ependymomas (0/7) [158], other authors observed a higher frequency [159, 160]. The reason for this discrepancy remains

Amatya *et al.*[159] assessed whether promoter methylation was present in cells of six malignant gliomas and whether there is an association with reduced expression of *TP53* mRNA and protein. They also assessed the frequencies of disruption of the p53/p14 *ARF* pathway in 49 lowgrade astrocytomas (40 fibrillary astrocytomas and 9 gemistocytic astrocytomas), 42 oligoden‐ drogliomas and 18 oligoastrocytomas. The Methylation-specific PCR (MS-PCR) revealed methylation of the promoter region of the *TP53* gene in three (U87MG, LNT-229, T98G) out of six malignant glioma cell lines. Real time RT-PCR revealed that two malignant glioma cell lines (U87MG and T98G) led to up-regulated expression of *TP53* mRNA and protein after treatment with 5-aza-2'-deoxycytidine (5-aza-dC, an epigenetic modifier that results in DNA demethy‐ lation), suggesting that promoter methylation is associated with reduced expression in some malignant glioma cells. *TP53* promoter methylation in primary tissue of low-grade gliomas was observed in 29/48 (60%) low-grade astrocytomas, 11/18 (61%) oligoastrocytomas, and 31/42 (74%) oligodendrogliomas, while promoter methylation of the p14 ARF was detected by MS-PCR in 5/49 (10%) low-grade astrocytomas, 7/18 (39%) oligoastrocytomas, and 15/41 (37%) oligodendrogliomas. Briefly, alterations of at least one of *TP53* promoter methylation, p14 ARF promoter methylation, and *TP53* mutations were found in 43/49 (88%) of low-grade astrocy‐ tomas, 15/18 (83%) of oligoastrocytomas, and 35/42 (83%) oligodendrogliomas, suggesting that disruption of the p53/p14 *ARF* pathway is frequent in all histological types of low-grade glioma.

Almeida *et al.* [160] evaluated the promoter hypermethylation profile of the *TP53* gene in 90 extra-axial brain tumors (48 meningiomas, 23 schwannomas and 19 metastases) using MS-PCR and sequencing. The group showed that the methylation of the *TP53* gene is an important event associated with extra-axial brain tumors, since 37.5% of meningiomas, 30% of schwan‐ nomas and 52.6% of metastases were hypermethylated. When tumor grade was compared, 35.3% of benign tumors and 48% of malignant tumors were methylated, and these results

MicroRNAs (miRNA) are a large class of small, non-coding RNAs, 21 – 28 nucleotides long, produced naturally in cells after being cut into segments from larger strands of RNA by the enzyme Dicer. They function by binding to complementary sites on the 3'-untranslated region (3'-UTR) of genes and promoting the recruitment of protein complexes responsible for impairing translation and/or decreasing the stability of mRNA [161, 162]. A specific miRNA may simultaneously regulate multiple targets, thereby enabling complex changes in protein expression profiles. Furthermore, a single target can be regulated by multiple miRNAs, and upstream regulation of a given miRNA can involve multiple regulators at different steps of miRNA biogenesis. Thus, miRNAs take part in complex regulatory networks that may

suggested that *TP53* methylation can be involved in the progression of these tumors.

**3.2. The new insights of MicroRNAs/***TP53* **in cancer**

to be clarified.

142 Tumors of the Central Nervous System – Primary and Secondary

miRNAs play important roles in the regulation of normal gene expression at developmental timing, cell proliferation and apoptosis [165]. As these processes are altered in cancer cells, there are in literature several studies that were undertaken to provide evidence for an involvement of miRNAs in cancer formation. miRNA-encoding genes as well as mRNAencoding genes have been meanwhile classified as oncogenic or tumor suppressive genes according to their function in cellular transformation and expression in tumors [166, 167]. Furthermore, tumor cells seem to undergo a general loss of miRNA expression, and forced reduction of global miRNA expression promotes transformation [168]. Interestingly, miRNAs cluster within fragiles sites and other genomic regions frequently altered in cancers [169]. Because of their role in tumor formation, miRNAs may be very useful for the classification, diagnosis, prognosis, and therapy of malignancies [166, 167].

Profiling miRNA provides an attractive, novel, and non-invasive biomarker for tumor diagnosis and prognosis. Molecular biology techniques, such as Northern blot, RNase protection assay, and primer extension assay can measure expression of a miRNA. The small size of miRNAs initially hampered polymerase chain reaction-based methods. However, PCRbased techniques have become very popular since the development of adaptor-mediated quantitative real-time PCR (qRT-PCR) due to their high sensitivity [170]. Microarray techni‐ ques are widely used to comprehensively assay the entire miRNome (the global miRNA expression profile) in tissues or in cell lines [171]. In addition to microarray and qRT-PCR, miRNomes are obtained by *in situ* hybridization [172] and serial analysis of gene expression adapted for small RNAs [173]. Overall, these technical improvements are expected to greatly widen the repertoire of miRNAs in a variety of biological systems.

p53 is a transcription factor, so transactivates or represses many protein-encoding genes and this underlies much of its tumor suppressor function. Recently, it has been reported that p53 directly transactivates specifics miRNAs [174]. miRNA have also been shown to target p53 and/or components of p53 regulatory pathways affecting its activities directly and/or indirectly [175, 176].

Several reports shed light on the involvement of miRNAs in the p53 pathway. He *et al.* [177] profiled miRNA gene expression in wild-type (wt) and p53-deficient cells and found that the miR34s (miR-34 gene family, including miR-34a, b and c) was among the most upregulated in wt p53 cells. In addition, Luan *et al.* [178] analyzed the expression levels of miR-34a in human glioma cell lines (U251, A172 and SHG-44) using real time quantitative PCR and compared with that observed in normal brain and determined its role in cell proliferation, cycle distri‐ bution, apoptosis and capabilities of in vitro migration and invasion of p53-mutant glioma cells. The results showed that miR-34a is remarkably reduced in p53-mutant glioma cell line U251, that had a mutation of codon 273 (CGT/CAT; Arg/His) in exon 8 than other p53-wild glioma cell lines A172, SHG-44 and normal brains.

miR34s are induced after genotoxic stress in a p53-dependent manner *in vitro* and *in vivo*. miR-34b and-34c are clustered at chromosome 11, whereas miR-34a is located in a separate genomic locus. p53 directly activates both pri-miRNAs. The miR-34s seem to be critical downstream effectors of p53, as ectopic expression of the miR-34s recapitulate the phenotype of p53 activation. The miR-34s promotes repression of several direct targets, such as Bcl-2, Cdk4, hepatocyte growth factor receptor (MET), and other, resulting in cell cycle arrest, apoptosis, and senescence [179] (Figure 3). Several other laboratories corroborated the finding that miR-34s are critical components of the p53 network [180-182]. Taken together, these results support a pivotal downstream role of miRNAs in the regulation of the p53 pathway.

neuroblastoma, which may be due to the relatively common deletion of a region on chromo‐ some 1p36, which encompasses miR-34a [183]. However, the mechanisms leading to decreased

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Other miRNAs may be important in the p53 network. miR-30c, -103, -26a, -107, and-182 were induced clearly, although less robustly, upon DNA damage in a p53-dependent manner [181]. In another approach, the searching for p53-binding elements in DNA sequences near miRNAs identified miR-129 as a good candidate for regulation by p53 [184]. miR-125b, a brain-enriched microRNA, was identified as a bona fide negative regulator of p53 in both zebrafish and humans [185]. Recently, Hu *et al.* [186] showed that miR-504 directly represses p53 expression and

Since recent studies have indicated that p53 enters into miRNA world [187], some researchers provided important insights into the central roles of miRNAs in a well-known tumor sup‐ pressor network, the p53 pathway, which may provide a route to therapeutic miRNA inter‐ vention in CNS tumors. Shyamal *et al.* [188] were the first to demonstrate that miR-34a directly targets the *MAGE-A* family of oncogenes, disengaging p53 from *MAGE-A*–mediated repres‐ sion. The group demonstrated that miR-34a directly targets the 3 ′ UTR of *MAGE-A* genes and decreases MAGE-A protein levels in medulloblastoma cell lines. This decreasing in *MAGE-A* results in a concomitant increasing in p53 and its associated transcriptional targets, p21/ WAF1/CIP1 and, importantly, miR-34a. This establishes a positive feedback loop where miR-34a is not only induced by p53 but increases p53 mRNA and protein levels through the modulation of *MAGE-A* genes and a consequence of this mechanism is that sensitizes medul‐ loblastoma cells to chemotherapeutic agents via delayed G2/M progression and increased

Recently, Suh *et al.* [189] identified two miRNAs (miR-25 and-32) as p53-repressed miRNAs in glioblastoma multiforme cells through p53-dependent negative regulation of their transcrip‐ tional regulators, E2F1 and MYC. The study provided compelling evidence that expression of these miRNAs causes tumor suppression through mechanisms that lead to accumulation of p53 protein, by directly targeting Mdm2 and TSC1, leading to inhibition of cellular prolifera‐ tion through cell cycle arrest. Thus, there is a recurrent autoregulatory circuit involving expression of p53, E2F1, and MYC to regulate the expression of miR-25 and-32, which are miRNAs that, in turn, control p53 accumulation. Significantly, overexpression of transfected miR-25 and-32 in cells of GBM inhibited growth of these cells in mouse brain *in vivo*. The results define miR-25 and-32 as positive regulators of p53, underscoring their role in tumorigenesis

Until a few years ago, the brain was thought to lack a stem cell population, but actually, it is now known that there are two regions of the adult human brain that contain neural stem cells (NSCs) (a group of self-renewing cells in the nervous system that can generate both neurons

expression of miR-34s require further exploration.

function in human cell lines.

apoptosis.

in glioblastoma.

**4. The cancer stem cell model**

**Figure 3.** Representation of p53 and the miR-34 family interactions. The p53 protein stimulates the transcription of miR34s, which inhibits oncoproteins and leads to cell senescence, apoptosis and cell cycle arrest.

As cell cycle arrest, senescence, and apoptosis are tumor suppressive mechanisms, the inactivation of members of the miR-34 family, which induce these cellular responses, may be a selective advantage for cancer cells. Besides decreased expression of MiR-34 due to inacti‐ vating mutations of p53, the miR-34 encoding genes themselves may be targets for mutational or epigenetic inactivation in cancer. For example, loss of miR34a expression was observed in neuroblastoma, which may be due to the relatively common deletion of a region on chromo‐ some 1p36, which encompasses miR-34a [183]. However, the mechanisms leading to decreased expression of miR-34s require further exploration.

Other miRNAs may be important in the p53 network. miR-30c, -103, -26a, -107, and-182 were induced clearly, although less robustly, upon DNA damage in a p53-dependent manner [181]. In another approach, the searching for p53-binding elements in DNA sequences near miRNAs identified miR-129 as a good candidate for regulation by p53 [184]. miR-125b, a brain-enriched microRNA, was identified as a bona fide negative regulator of p53 in both zebrafish and humans [185]. Recently, Hu *et al.* [186] showed that miR-504 directly represses p53 expression and function in human cell lines.

Since recent studies have indicated that p53 enters into miRNA world [187], some researchers provided important insights into the central roles of miRNAs in a well-known tumor sup‐ pressor network, the p53 pathway, which may provide a route to therapeutic miRNA inter‐ vention in CNS tumors. Shyamal *et al.* [188] were the first to demonstrate that miR-34a directly targets the *MAGE-A* family of oncogenes, disengaging p53 from *MAGE-A*–mediated repres‐ sion. The group demonstrated that miR-34a directly targets the 3 ′ UTR of *MAGE-A* genes and decreases MAGE-A protein levels in medulloblastoma cell lines. This decreasing in *MAGE-A* results in a concomitant increasing in p53 and its associated transcriptional targets, p21/ WAF1/CIP1 and, importantly, miR-34a. This establishes a positive feedback loop where miR-34a is not only induced by p53 but increases p53 mRNA and protein levels through the modulation of *MAGE-A* genes and a consequence of this mechanism is that sensitizes medul‐ loblastoma cells to chemotherapeutic agents via delayed G2/M progression and increased apoptosis.

Recently, Suh *et al.* [189] identified two miRNAs (miR-25 and-32) as p53-repressed miRNAs in glioblastoma multiforme cells through p53-dependent negative regulation of their transcrip‐ tional regulators, E2F1 and MYC. The study provided compelling evidence that expression of these miRNAs causes tumor suppression through mechanisms that lead to accumulation of p53 protein, by directly targeting Mdm2 and TSC1, leading to inhibition of cellular prolifera‐ tion through cell cycle arrest. Thus, there is a recurrent autoregulatory circuit involving expression of p53, E2F1, and MYC to regulate the expression of miR-25 and-32, which are miRNAs that, in turn, control p53 accumulation. Significantly, overexpression of transfected miR-25 and-32 in cells of GBM inhibited growth of these cells in mouse brain *in vivo*. The results define miR-25 and-32 as positive regulators of p53, underscoring their role in tumorigenesis in glioblastoma.
