Neelam Shirsat

Distinctive microRNA signature of medulloblastomas associated with the WNT signaling pathway

AIM Medulloblastoma is a malignant brain tumor that occurs predominantly in children. Current risk stratification based on clinical parameters is inadequate for accurate prognostication. MicroRNA expression is known to be deregulated in various cancers and has been found to be useful in predicting tumor behavior. In order to get a better understanding of medulloblastoma biology, miRNA profiling of medulloblastomas was carried out in parallel with expression profiling of protein-coding genes. MATERIALS AND METHODS miRNA profiling of medulloblastomas was carried out using Taqman Low Density Array v 1.0 having 365 human microRNAs. In parallel, genome-wide expression profiling of protein-coding genes was carried out using Affymetrix gene 1.0 ST arrays. RESULTS Both the profiling studies identified four molecular subtypes of medulloblastomas. Expression levels of select protein-coding genes and miRNAs could classify an independent set of medulloblastomas. Twelve of 31 medulloblastomas were found to overexpress genes belonging to the canonical WNT signaling pathway and carry a mutation in CTNNB1 gene. A number of miRNAs like miR-193a, miR-224/miR-452 cluster, miR-182/miR-183/miR-96 cluster, and miR-148a having potential tumor/metastasis suppressive activity were found to be overexpressed in the WNT signaling associated medulloblastomas. Exogenous expression of miR-193a and miR-224, two miRNAs that have the highest WNT pathway specific upregulation, was found to inhibit proliferation, increase radiation sensitivity and reduce anchorage-independent growth of medulloblastoma cells. CONCLUSION Expression level of tumor/metastasis suppressive miRNAs in the WNT signaling associated medulloblastomas is likely to determine their response to treatment, and thus, these miRNAs would be important biomarkers for risk stratification within the WNT signaling associated medulloblastomas.

Real-time PCR assay based on the differential expression of microRNAs and protein-coding genes for molecular classification of formalin-fixed paraffin embedded medulloblastomas

BACKGROUND Medulloblastoma has recently been found to consist of 4 molecularly and clinically distinct subgroups: WNT, Sonce hedgehog (SHH), Group 3, and Group 4. Deregulated microRNA expression is known to contribute to pathogenesis and has been shown to have diagnostic and prognostic potential in the classification of various cancers. METHODS Molecular subgrouping and microRNA expression analysis of 44 frozen and 59 formalin-fixed paraffin embedded medulloblastomas from an Indian cohort were carried out by real-time RT-PCR assay. RESULTS The differential expression of 9 microRNAs in the 4 molecular subgroups was validated in a set of 101 medulloblastomas. The tumors in the WNT subgroup showed significant (P < .0001) overexpression of miR-193a-3p, miR-224, miR-148a, miR-23b, and miR-365. Reliable classification of medulloblastomas into the 4 molecular subgroups was obtained using a set of 12 protein-coding genes and 9 microRNAs as markers in a real-time RT-PCR assay with an accuracy of 97% as judged by the Prediction Analysis of Microarrays. Age at diagnosis, histology, gender-related incidence, and the relative survival rates of the 4 molecular subgroups in the present Indian cohort were found to be similar to those reported for medulloblastomas from the American and European subcontinent. Non-WNT, non-SHH medulloblastomas underexpressing miR-592 or overexpressing miR-182 were found to have significantly inferior survival rates, indicating utility of these miRNAs as markers for risk stratification. CONCLUSIONS The microRNA based real-time PCR assay is rapid, simple, inexpensive, and useful for molecular classification and risk stratification of medulloblastomas, in particular formalin-fixed paraffin embedded tissues, wherein the expression profile of protein-coding genes is often less reliable due to RNA fragmentation.

Tamoxifen-Induced Cell Death of Malignant Glioma Cells Is Brought About by Oxidative-Stress-Mediated Alterations in the Expression of BCL2 Family Members and Is Enhanced on miR-21 Inhibition

High-grade gliomas are refractory to the current mode of treatment primarily due to their inherent resistance to cell death. Tamoxifen has been reported to inhibit growth and induce cell death of glioma cells in vitro, in an estrogen-receptor-independent manner. Delineating the molecular mechanism underlying tamoxifen-induced cell death of human glioma cells would help in identifying pathways/genes that could be targeted to induce tumor-cell-specific cell death. In the present study, tamoxifen was found to bring about autophagic cell death of human glioma cells that was accompanied by oxidative stress induction, JNK activation, downregulation of anti-autophagic BCL2 family members, viz. BCL2 and BCL-XL, and increased expression of the pro-autophagic members BCL-Xs and BAK. Oxidative stress induction appears to be primarily responsible for the tamoxifen-induced cell death since the cell death, JNK activation, and the alterations in the expression levels of BCL2 family members were abrogated on pretreatment with antioxidant vitamin E. MiR-21, an oncogenic miRNA, is known to be highly upregulated in malignant glioma. Inhibition of miR-21 activity was found to enhance tamoxifen-induced cell death of U87 MG malignant glioma cells. Tamoxifen treatment coupled with miR-21 inhibition could therefore be an effective strategy for the treatment of malignant gliomas.

MiR-224 expression increases radiation sensitivity of glioblastoma cells.

Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effect of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30-55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85-90% on irradiation at a dose of 6Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55-65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy, therefore appear as promising options for the treatment of glioblastoma, which is refractory to the existing treatment strategies.

ETV/Pea3 family transcription factor-encoding genes are overexpressed in CIC-mutant oligodendrogliomas

Oligodendrogliomas with combined loss of chromosome arms 1p and 19q are known to be particularly sensitive to chemotherapy, and the CIC gene located on 19q is known to be mutated in over 50% of the 1p/19q codeleted oligodendrogliomas. However, the role of CIC in the oligodendroglioma pathogenesis is not known. Exome sequencing of 11 oligodendroglial tumors identified 9 tumors with combined loss of 1p and 19q. Somatic mutations were found in the CIC and FUBP1 genes. Recurrent somatic mutations were also identified in the Notch signaling pathway genes NOTCH1 and MAML3, the chromatin modifying gene ARID1A and in KRAS. Comparison of the transcriptome profiles of CIC‐mutant and CIC‐wild type oligodendrogliomas from the study cohort as well as 65 1p/19q codeleted oligodendrogliomas from the TCGA cohort identified genes encoding the ETV transcription factor family to be significantly upregulated in the CIC‐mutant tumors. Upregulation of a number of negative regulators of the receptor tyrosine kinase signaling pathway like Sprouty and SPRED family members in the CIC‐mutant oligodendrogliomas is likely due to the constitutive activation of the pathway resulting from inactive CIC protein. Higher expression of the oncogenic ETV transcription factors in the CIC‐mutant oligodendrogliomas may make these tumors more aggressive than the CIC‐wild type tumors.

MiR-206, a Cerebellum Enriched miRNA Is Downregulated in All Medulloblastoma Subgroups and Its Overexpression Is Necessary for Growth Inhibition of Medulloblastoma Cells

Medulloblastoma is the most common and a highly malignant pediatric brain tumor located in the cerebellar region of the brain. Medulloblastomas have recently been shown to consist of four distinct molecular subgroups, viz., WNT, SHH, group 3, and group 4. MiR-206, a miRNA first identified as a myomiR due to its enriched expression in skeletal muscle was found to be expressed specifically in the cerebellum, the site of medulloblastoma occurrence. MiR-206 expression was found to be downregulated in medulloblastomas belonging to all the four molecular subgroups as well as in established medulloblastoma cell lines. Further, the expression of murine homolog of miR-206 was also found to be downregulated in SHH subgroup medulloblastomas from the Smo+/+ transgenic mice and the Ptch1+/− knockout mice. MiR-206 downregulation in all the four medulloblastoma subgroups suggests tumor-suppressive role for miR-206 in medulloblastoma pathogenesis. The effect of miR-206 expression was analyzed in three established medulloblastoma cell lines, viz., Daoy, D425, and D283 belonging to distinct molecular subgroups. Restoration of miR-206 expression to the levels comparable to those in the normal cerebellum, however, was found to be insufficient to inhibit the growth of established medulloblastoma cell lines. OTX2, an oncogenic miR-206 target, overexpressed in all non-SHH medulloblastomas, is known to inhibit myogenic differentiation of medulloblastoma cells. Overexpression of miR-206 was necessary to downregulate OTX2 expression and inhibit growth of medulloblastoma cell lines.

Indian Society of Neuro-Oncology consensus guidelines for the contemporary management of medulloblastoma

Introduction: The high success rate in the management medulloblastoma achieved in the western world is not exactly mirrored in developing countries including India. Socio-demographic differences, health-care disparity, and lack in uniformity of care with resultant widespread variations in the clinical practice are some of the reasons that may partly explain this difference in outcomes. Patients with medulloblastoma require a multi-disciplinary team approach involving but not limited to neuro-radiology, neurosurgery; neuropathology, molecular biology, radiation oncology, pediatric medical oncology and rehabilitative services for optimizing outcomes. Methods: The Indian Society of Neuro-Oncology (ISNO) constituted an expert multi-disciplinary panel with adequate representation from all stakeholders to prepare national consensus guidelines for the contemporary management of medulloblastoma. Results: Minimum desirable, as well as preferable though optional recommendations (as appropriate), were developed and adopted for the pre-surgical work-up including neuroimaging; neurosurgical management including surgical principles, techniques, and complications; neuropathology reporting and molecular testing; contemporary risk-stratification in the molecular era; appropriate adjuvant therapy (radiotherapy and chemotherapy); and follow-up schedule in medulloblastoma. Conclusions: The current document represents a broad consensus reached amongst various stakeholders within the neuro-oncology community involved in the contemporary curative-intent management of children with medulloblastoma. It provides both general as well as specific guidelines and recommendations to be adopted by physicians and health care providers across India to achieve uniformity of care, improve disease-related outcomes, and compare results between institutions within the country.

Extraneuraxial metastases in medulloblastoma: is histology and molecular biology important?

with ENM from medulloblastoma, this report is limited to 7 of 300 (2.3%) patients with known molecular subgroup affiliation. All relevant patient, disease, and treatment-characteristics were retrieved by retrospective review of medical case records and are summarized in Table 1. Median age of the study cohort was 16.5 years (range 3–25 years) at initial diagnosis. 4 (57%) patients had desmoplastic medulloblastoma, while 3 (43%) patients had large-cell/anaplastic histology. Five of 7 (71%) patients belonged to SHH-subgroup, while the remaining 2 (29%) had subgroup 3 medulloblastoma. Using conventional risk-stratification, 3 (43%) patients were classified as average-risk disease, while 4 (57%) patients demonstrated high-risk features such as post-operative residual tumor (>1.5 × 1.5 cm2) and/or presence of CNS metastases. All patients had been treated with standard radiotherapy (craniospinal irradiation plus posterior fossa boost) followed by adjuvant systemic chemotherapy at initial diagnosis. Most patients were symptomatic at the time of ENM with bone pain and swelling being predominant symptoms. Whole-body 18F-flouro-deoxy-glucose positron emission tomography/computed tomography (FDG-PET/CT) was done in all patients at first suspicion of ENM. None of the patients presented with ENM; it was detected metachronously at a median of 10 months (range 4–33 months) from initial diagnosis with common sites of involvement being bone (71%), bone marrow (43%), and lymph nodes (29%). Liver metastases was detected in two patients (one incidentally on imaging), while pleural, peritoneal, pancreatic, and subcutaneous metastases were detected in a single patient each. Pathologic confirmation of ENM was obtained in all patients but one, to reliably differentiate it from second malignant neoplasm (myelodysplasia, leukemia, or lymphoma). Further therapy comprising of palliative and supportive care was individualized based on symptomatology, evolution of disease, prior To the Editor,

Abstract B41: miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1

Purpose: Medulloblastoma, a most common pediatric malignant brain tumor consists of four molecular subgroups viz. WNT, SHH, Group 3 and Group 4. MiR-148a is over-expressed in the WNT subgroup tumors, which have the lowest incidence of metastasis and excellent survival among all medulloblastomas. Therefore, the role of miR-148a in medulloblastoma biology was investigated. Experimental Design: MiR-148a was expressed either in a transient manner using synthetic mimic or in a stable doxycycline inducible manner using a lentiviral vector. Effect of miR-148a expression on the growth and malignant behavior of medulloblastoma cell lines was investigated. CpG Methylation status of region upstream to pri-miR-148a was determined by performing bisulphite sequencing of this region in medulloblastoma cell lines and tissues. Results: Expression of miR-148a to the levels comparable to that in the WNT subgroup tumors was found to inhibit proliferation, clonogenic potential and in vitro invasion potential of medulloblastoma cells. In vivo tumorigenicity of medulloblastoma cells Daoy, D425 and D283, as seen by either generating subcutaneous xenografts or orthotopic xenografts in immunodeficient mouse, was found to be reduced significantly upon miR-148a expression. NRP1, ROCK1 and DNMT1 were identified as direct targets of miR-148a with NRP1 being novel target. Restoration of NRP1 expression in medulloblastoma cells was found to rescue the reduction in the invasion potential and tumorigenicity brought about by miR-148a expression. NRP1expression in medulloblastomas was found to be associated with poor survival, with little or no expression in majority of the WNT tumors. This observation is consistent with high miR-148a expression and low incidence of metastasis and excellent survival of the WNT subgroup tumors. miR-148a is known to be down-regulated as a result of promoter hypermethylation in colon, lung, breast, head and neck carcinomas and melanoma. Bisulphite sequencing of the region upstream to pri-miR-148a revealed presence of CpG methylation in four medulloblastoma cell lines Daoy, D283, D341 and D425 and non WNT medulloblastoma tissues, while it was found to be un-methylated in WNT medulloblastomas. Conclusions: The tumor suppressive effect of miR-148a expression in medulloblastoma cells accompanied by the down-regulation of NRP1, ROCK1 and DNMT1 makes miR-148a an attractive therapeutic agent for the treatment of medulloblastomas. Preliminary findings provided by bisulphite sequencing suggest that under-expression of miR-148a may be as a result of CpG methylation of upstream region to pri-miR-148a sequence. Citation Format: Kedar Narsinha Yogi, Epari Sridhar, Naina Goel, Rakesh Jalali, Atul Goel, Aliasgar Moiyadi, Rahul Thorat, Pooja Panwalkar, Atul Khire, Archya Dasgupta, Prakash Shetty, Neelam Shirsat. miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B41.