Pratheesh Sathyan

Competing Interactions between Micro-RNAs Determine Neural Progenitor Survival and Proliferation after Ethanol Exposure: Evidence from an Ex Vivo Model of the Fetal Cerebral Cortical Neuroepithelium

The fetal brain is sensitive to a variety of teratogens, including ethanol. We showed previously that ethanol induced mitosis and stem cell maturation, but not death, in fetal cerebral cortex-derived progenitors. We tested the hypothesis that micro-RNAs (miRNAs) could mediate the teratogenic effects of ethanol in a fetal mouse cerebral cortex-derived neurosphere culture model. Ethanol, at a level attained by alcoholics, significantly suppressed the expression of four miRNAs, miR-21, -335, -9, and -153, whereas a lower ethanol concentration, attainable during social drinking, induced miR-335 expression. A GABAA receptor-dependent mechanism mediated miR-21, but not miR-335 suppression, suggesting that divergent mechanisms regulate ethanol-sensitive miRNAs. Antisense-mediated suppression of miR-21 expression resulted in apoptosis, suggesting that miR-21 is an antiapoptotic factor. miR-335 knockdown promoted cell proliferation and prevented death induced by concurrently suppressing miR-21, indicating that miR-335 is a proapoptotic, antimitogenic factor whose actions are antagonistic to miR-21. Computational analyses identified two genes, Jagged-1, a Notch-receptor ligand, and embryonic-lethal abnormal vision, Drosophila-like 2 (ELAVL2), a brain-specific regulator of RNA stability, as presumptive targets of three of four ethanol-sensitive micro-RNAs. Combined knockdown of miR-335, -21, and -153 significantly increased Jagged-1 mRNA. Furthermore, ethanol induced both Jagged-1 and ELAVL2 mRNA. The collective suppression of micro-RNAs is consistent with ethanol induction of cell cycle and neuroepithelial maturation in the absence of apoptosis. These data identify a role for micro-RNAs as epigenetic intermediaries, which permit teratogens to shape complex, divergent developmental processes, and additionally demonstrate that coordinately regulated miRNAs exhibit both functional synergy and antagonism toward each other.

Radiogenomic Mapping of Edema/Cellular Invasion MRI-Phenotypes in Glioblastoma Multiforme

Background Despite recent discoveries of new molecular targets and pathways, the search for an effective therapy for Glioblastoma Multiforme (GBM) continues. A newly emerged field, radiogenomics, links gene expression profiles with MRI phenotypes. MRI-FLAIR is a noninvasive diagnostic modality and was previously found to correlate with cellular invasion in GBM. Thus, our radiogenomic screen has the potential to reveal novel molecular determinants of invasion. Here, we present the first comprehensive radiogenomic analysis using quantitative MRI volumetrics and large-scale gene- and microRNA expression profiling in GBM. Methods Based on The Cancer Genome Atlas (TCGA), discovery and validation sets with gene, microRNA, and quantitative MR-imaging data were created. Top concordant genes and microRNAs correlated with high FLAIR volumes from both sets were further characterized by Kaplan Meier survival statistics, microRNA-gene correlation analyses, and GBM molecular subtype-specific distribution. Results The top upregulated gene in both the discovery (4 fold) and validation (11 fold) sets was PERIOSTIN (POSTN). The top downregulated microRNA in both sets was miR-219, which is predicted to bind to POSTN. Kaplan Meier analysis demonstrated that above median expression of POSTN resulted in significantly decreased survival and shorter time to disease progression (P<0.001). High POSTN and low miR-219 expression were significantly associated with the mesenchymal GBM subtype (P<0.0001). Conclusion Here, we propose a novel diagnostic method to screen for molecular cancer subtypes and genomic correlates of cellular invasion. Our findings also have potential therapeutic significance since successful molecular inhibition of invasion will improve therapy and patient survival in GBM.

An Antagomir to MicroRNA Let7f Promotes Neuroprotection in an Ischemic Stroke Model

We previously showed that middle-aged female rats sustain a larger infarct following experimental stroke as compared to younger female rats, and paradoxically, estrogen treatment to the older group is neurotoxic. Plasma and brain insulin-like growth factor-1 (IGF-1) levels decrease with age. However, IGF-1 infusion following stroke, prevents estrogen neurotoxicity in middle-aged female rats. IGF1 is neuroprotective and well tolerated, but also has potentially undesirable side effects. We hypothesized that microRNAs (miRNAs) that target the IGF-1 signaling family for translation repression could be alternatively suppressed to promote IGF-1-like neuroprotection. Here, we report that two conserved IGF pathway regulatory microRNAs, Let7f and miR1, can be inhibited to mimic and even extend the neuroprotection afforded by IGF-1. Anti-mir1 treatment, as late as 4 hours following ischemia, significantly reduced cortical infarct volume in adult female rats, while anti-Let7 robustly reduced both cortical and striatal infarcts, and preserved sensorimotor function and interhemispheric neural integration. No neuroprotection was observed in animals treated with a brain specific miRNA unrelated to IGF-1 (anti-miR124). Remarkably, anti-Let7f was only effective in intact females but not males or ovariectomized females indicating that the gonadal steroid environment critically modifies miRNA action. Let7f is preferentially expressed in microglia in the ischemic hemisphere and confirmed in ex vivo cultures of microglia obtained from the cortex. While IGF-1 was undetectable in microglia harvested from the non-ischemic hemisphere, IGF-1 was expressed by microglia obtained from the ischemic cortex and was further elevated by anti-Let7f treatment. Collectively these data support a novel miRNA-based therapeutic strategy for neuroprotection following stroke.

miR-218 opposes a critical RTK-HIF pathway in mesenchymal glioblastoma

Significance Despite measurable advances in cancer treatment, patients with glioblastoma multiforme (GBM) typically survive only 12–14 mo because of tumor recurrence. Tumor hypoxia has been associated with chemoresistance. We hypothesized that the pronounced repression of microRNA-218 (miR-218) observed in samples from patients with highly hypoxic and necrotic GBM contributes to this recurrent phenotype. We demonstrate here that mice harboring intracranial tumors with increased miR-218 expression exhibit significantly reduced tumor burden and increased survival when challenged with the chemotherapeutic agent temozolomide. Moreover, low miR-218 levels increase the expression of multiple components of the receptor tyrosine kinase signaling pathway, which promote the activation of hypoxia-inducible factor. The identification of the molecular bases for miR-218-mediated chemoresistance should promote the development of targeted therapies. Glioblastoma multiforme (GBM) and the mesenchymal GBM subtype in particular are highly malignant tumors that frequently exhibit regions of severe hypoxia and necrosis. Because these features correlate with poor prognosis, we investigated microRNAs whose expression might regulate hypoxic GBM cell survival and growth. We determined that the expression of microRNA-218 (miR-218) is decreased significantly in highly necrotic mesenchymal GBM, and orthotopic tumor studies revealed that reduced miR-218 levels confer GBM resistance to chemotherapy. Importantly, miR-218 targets multiple components of receptor tyrosine kinase (RTK) signaling pathways, and miR-218 repression increases the abundance and activity of multiple RTK effectors. This elevated RTK signaling also promotes the activation of hypoxia-inducible factor (HIF), most notably HIF2α. We further show that RTK-mediated HIF2α regulation is JNK dependent, via jun proto-oncogene. Collectively, our results identify an miR-218–RTK–HIF2α signaling axis that promotes GBM cell survival and tumor angiogenesis, particularly in necrotic mesenchymal tumors.

A Novel Volume-Age-KPS (VAK) Glioblastoma Classification Identifies a Prognostic Cognate microRNA-Gene Signature

Background Several studies have established Glioblastoma Multiforme (GBM) prognostic and predictive models based on age and Karnofsky Performance Status (KPS), while very few studies evaluated the prognostic and predictive significance of preoperative MR-imaging. However, to date, there is no simple preoperative GBM classification that also correlates with a highly prognostic genomic signature. Thus, we present for the first time a biologically relevant, and clinically applicable tumor Volume, patient Age, and KPS (VAK) GBM classification that can easily and non-invasively be determined upon patient admission. Methods We quantitatively analyzed the volumes of 78 GBM patient MRIs present in The Cancer Imaging Archive (TCIA) corresponding to patients in The Cancer Genome Atlas (TCGA) with VAK annotation. The variables were then combined using a simple 3-point scoring system to form the VAK classification. A validation set (N = 64) from both the TCGA and Rembrandt databases was used to confirm the classification. Transcription factor and genomic correlations were performed using the gene pattern suite and Ingenuity Pathway Analysis. Results VAK-A and VAK-B classes showed significant median survival differences in discovery (P = 0.007) and validation sets (P = 0.008). VAK-A is significantly associated with P53 activation, while VAK-B shows significant P53 inhibition. Furthermore, a molecular gene signature comprised of a total of 25 genes and microRNAs was significantly associated with the classes and predicted survival in an independent validation set (P = 0.001). A favorable MGMT promoter methylation status resulted in a 10.5 months additional survival benefit for VAK-A compared to VAK-B patients. Conclusions The non-invasively determined VAK classification with its implication of VAK-specific molecular regulatory networks, can serve as a very robust initial prognostic tool, clinical trial selection criteria, and important step toward the refinement of genomics-based personalized therapy for GBM patients.

REST Regulates Oncogenic Properties of Glioblastoma Stem Cells

Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. Although many GBM tumors are believed to be caused by self‐renewing, glioblastoma‐derived stem‐like cells (GSCs), the mechanisms that regulate self‐renewal and other oncogenic properties of GSCs are only now being unraveled. Here we showed that GSCs derived from GBM patient specimens express varying levels of the transcriptional repressor repressor element 1 silencing transcription factor (REST), suggesting heterogeneity across different GSC lines. Loss‐ and gain‐of‐function experiments indicated that REST maintains self‐renewal of GSCs. High REST‐expressing GSCs (HR‐GSCs) produced tumors histopathologically distinct from those generated by low REST‐expressing GSCs (LR‐GSCs) in orthotopic mouse brain tumor models. Knockdown of REST in HR‐GSCs resulted in increased survival in GSC‐transplanted mice and produced tumors with higher apoptotic and lower invasive properties. Conversely, forced expression of exogenous REST in LR‐GSCs produced decreased survival in mice and produced tumors with lower apoptotic and higher invasive properties, similar to HR‐GSCs. Thus, based on our results, we propose that a novel function of REST is to maintain self‐renewal and other oncogenic properties of GSCs and that REST can play a major role in mediating tumorigenicity in GBM. STEM CELLS 2012;30:405–414

Mir-21-Sox2 axis delineates glioblastoma subtypes with prognostic impact

Glioblastoma (GBM) is the most aggressive human brain tumor. Although several molecular subtypes of GBM are recognized, a robust molecular prognostic marker has yet to be identified. Here, we report that the stemness regulator Sox2 is a new, clinically important target of microRNA-21 (miR-21) in GBM, with implications for prognosis. Using the MiR-21–Sox2 regulatory axis, approximately half of all GBM tumors present in the Cancer Genome Atlas (TCGA) and in-house patient databases can be mathematically classified into high miR-21/low Sox2 (Class A) or low miR-21/high Sox2 (Class B) subtypes. This classification reflects phenotypically and molecularly distinct characteristics and is not captured by existing classifications. Supporting the distinct nature of the subtypes, gene set enrichment analysis of the TCGA dataset predicted that Class A and Class B tumors were significantly involved in immune/inflammatory response and in chromosome organization and nervous system development, respectively. Patients with Class B tumors had longer overall survival than those with Class A tumors. Analysis of both databases indicated that the Class A/Class B classification is a better predictor of patient survival than currently used parameters. Further, manipulation of MiR-21–Sox2 levels in orthotopic mouse models supported the longer survival of the Class B subtype. The MiR-21–Sox2 association was also found in mouse neural stem cells and in the mouse brain at different developmental stages, suggesting a role in normal development. Therefore, this mechanism-based classification suggests the presence of two distinct populations of GBM patients with distinguishable phenotypic characteristics and clinical outcomes. SIGNIFICANCE STATEMENT Molecular profiling-based classification of glioblastoma (GBM) into four subtypes has substantially increased our understanding of the biology of the disease and has pointed to the heterogeneous nature of GBM. However, this classification is not mechanism based and its prognostic value is limited. Here, we identify a new mechanism in GBM (the miR-21–Sox2 axis) that can classify ∼50% of patients into two subtypes with distinct molecular, radiological, and pathological characteristics. Importantly, this classification can predict patient survival better than the currently used parameters. Further, analysis of the miR-21-Sox2 relationship in mouse neural stem cells and in the mouse brain at different developmental stages indicates that miR-21 and Sox2 are predominantly expressed in mutually exclusive patterns, suggesting a role in normal neural development.

Coordination of self-renewal in glioblastoma by integration of adhesion and microRNA signaling

BACKGROUND Cancer stem cells (CSCs) provide an additional layer of complexity for tumor models and targets for therapeutic development. The balance between CSC self-renewal and differentiation is driven by niche components including adhesion, which is a hallmark of stemness. While studies have demonstrated that the reduction of adhesion molecules, such as integrins and junctional adhesion molecule-A (JAM-A), decreases CSC maintenance. The molecular circuitry underlying these interactions has yet to be resolved. METHODS MicroRNA screening predicted that microRNA-145 (miR-145) would bind to JAM-A. JAM-A overexpression in CSCs was evaluated both in vitro (proliferation and self-renewal) and in vivo (intracranial tumor initiation). miR-145 introduction into CSCs was similarly assessed in vitro. Additionally, The Cancer Genome Atlas dataset was evaluated for expression levels of miR-145 and overall survival of the different molecular groups. RESULTS Using patient-derived glioblastoma CSCs, we confirmed that JAM-A is suppressed by miR-145. CSCs expressed low levels of miR-145, and its introduction decreased self-renewal through reductions in AKT signaling and stem cell marker (SOX2, OCT4, and NANOG) expression; JAM-A overexpression rescued these effects. These findings were predictive of patient survival, with a JAM-A/miR-145 signature robustly predicting poor patient prognosis. CONCLUSIONS Our results link CSC-specific niche signaling to a microRNA regulatory network that is altered in glioblastoma and can be targeted to attenuate CSC self-renewal.

REST-miR-21-SOX2 axis maintains pluripotency in E14Tg2a.4 embryonic stem cells

Our previous studies have shown that the regulatory network that maintains pluripotency in mouse embryonic stem cells (mESCs) is regulated in a context-dependent manner and can be modulated, at least in part, by re-calibration of an intracellular network of pluripotency factors as well as cues arising from the extracellular matrix. The transcriptional repressor REST represses miR-21 and, thus, regulates self-renewal in E14Tg2a.4 mESCs cultured in the absence of mouse embryonic fibroblast feeder cell effects. However, how miR-21 connects to the nuclear regulatory network has not been clear. Here, we show that miR-21, a direct target of REST-mediated repression, directly targets Sox2. Exogenously added miR-21 to mESCs decreases the expression of Sox2, decreasing mESC self-renewal, and this effect of miR-21 on mESC self-renewal can be blocked by expression of exogenous Sox2. Conversely, destabilization of Sox2 by miR-21 can be blocked by anti-miR-21. Thus, the REST-miR-21-Sox2 axis connects REST to the core nuclear pluripotency regulators in E14Tg2a.4 mESCs cultured in the absence of feeder cells.

188 Radiogenomic Mapping of MRI-FLAIR-Phenotypes Identifies a Novel Gene-microRNA Regulatory Axis to Target Glioblastoma Invasion

Introduction The search for an effective therapy of Glioblastoma Multiforme (GBM) continues. Imaging Genomics, a newly emerged field, links gene expression profiles with MRI phenotypes (Zinn et al, 2011). MRI-FLAIR was found to correlate with cellular invasion in GBM, thus, whole genome quantitative imaging analysis can reveal functional microRNA-gene regulatory networks as novel targets for cellular invasion in GBM.