Marcelo B. Soares

Upregulation of mir-221 and mir-222 in atypical teratoid/rhabdoid tumors: potential therapeutic targets

PurposeThe aim of this study is to search for new therapeutic targets for atypical teratoid–rhabdoid tumors (ATRT).MethodsTo achieve this, we compared the expression of 365 microRNAs among ATRT, medulloblastomas, and normal brain.ResultsMiR-221 and miR-222 were within the top differentially expressed microRNAs. The deregulated expression of miR221/222 was demonstrated to inhibit the expression of the tumor suppressor and inhibitor of cell cycle p27Kip1. Here, we demonstrated the negative regulation of p27Kip1 by miR-221/222 in ATRT using microarray, real-time reverse transcriptase polymerase chain reaction, and immunohistochemistry.ConclusionAs anti-miR therapy was recently proposed as an alternative treatment for cancer, these findings suggest that anti-miR-221/222 therapy might have therapeutic potential in ATRT.

Extensive miRNA expression analysis in craniopharyngiomas

PurposeCraniopharyngiomas are benign tumors of the sellar or parasellar regions. They arise from the remnants of Rathke’s pouch and are considered a “developmental disease.” microRNAs are short non-coding RNAs that play a key regulatory role in the control of expression of entire gene networks. We performed an extensive analysis of miRNAs in craniopharyngiomas aiming to identify a miRNA expression signature that might aid in the prognosis of disease progression and outcome.MethodsThirty-seven craniopharyngioma samples from twenty-three patients, ten age-matched controls from autopsy, and ten infant controls from the developing pituitary from autopsy were evaluated for the expression of 754 miRNAs using TaqMan® Low Density Arrays (TLDAs) v2.0 (Applied Biosystems, Foster City, CA).ResultsAmong the most differentially expressed miRNAs, downregulation of miR-132 appears to be a marker of aggressiveness and also plays a role in epithelial–mesenchymal transition.ConclusionsThis is the first time that an extensive study of miRNA expression has been performed in craniopharyngiomas. Further research needs to be performed to investigate the potential role of miR-132 in the development and progression of craniopharyngiomas, and its value as a prognostic marker of aggressiveness.

Characterization of brain tumor initiating cells isolated from an animal model of CNS primitive neuroectodermal tumors

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC’s orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.

Stabilization of HIF-1α and HIF-2α, upregulation of MYCC and accumulation of stabilized p53 constitute hallmarks of CNS-PNET animal model

Recently, we described a new animal model of CNS primitive neuroectodermal tumors (CNS-PNET), which was generated by orthotopic transplantation of human Radial Glial (RG) cells into NOD-SCID mice’s brain sub-ventricular zone. In the current study we conducted comprehensive RNA-Seq analyses to gain insights on the mechanisms underlying tumorigenesis in this mouse model of CNS-PNET. Here we show that the RNA-Seq profiles derived from these tumors cluster with those reported for patients’ PNETs. Moreover, we found that (i) stabilization of HIF-1α and HIF-2α, which are involved in mediation of the hypoxic responses in the majority of cell types, (ii) up-regulation of MYCC, a key onco-protein whose dysregulation occurs in ~70% of human tumors, and (iii) accumulation of stabilized p53, which is commonly altered in human cancers, constitute hallmarks of our tumor model, and might represent the basis for CNS-PNET tumorigenesis in this model. We discuss the possibility that these three events might be interconnected. These results indicate that our model may prove invaluable to uncover the molecular events leading to MYCC and TP53 alterations, which would be of broader interest considering their relevance to many human malignancies. Lastly, this mouse model might prove useful for drug screening targeting MYCC and related members of its protein interaction network.

Abstract 3111: Identification of genes that frequently exhibit copy number alterations in pediatric ependymomas

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Ependymoma is a common pediatric central nervous system (CNS) tumor that is believed to originate from ependymal cells located in the lining of ventricular surfaces in the brain. Ependymoma presents great challenges in treatment despite the advances in neurosurgical techniques and adjuvant therapy. To uncover protein interaction networks that are consistently compromised in pediatric ependymomas, we conducted a computational analysis of the array CGH data derived from 203 primary ependymomas from the St. Jude Childrens Research Hospital cohort, published by Johnson and colleagues [Nature, 466(7306):632-6, 2010]. The analysis (Bischof and Soares, manuscript in preparation) revealed 15 regions of copy number variation (CNVs) that occurred in at least 50% of the tumors. Peak genes from these CNVs were selected for a confirmatory study using DNA digital PCR assays with an independent ependymoma cohort from the Ann & Robert H Lurie Childrens Hospital of Chicago. In this study, all ependymomas displayed copy number alterations in at least one of the 10 genes tested by digital PCR. The digital PCR data sheds light into the biology of ependymomas as the 10 candidate genes have a wide range of cellular functions; from neural stem cell development, to tumor suppression, to the pathogenesis of glioblastoma. In conclusion, we have confirmed that the occurrence of DNA copy number variation in these 10 genes prevail in ependymomas. Indeed, all tumors exhibited at least one of these CNVs. These alterations provide insights into the protein interaction networks underlying development of ependymomas while uncovering targets that may be exploited for therapeutic intervention. Citation Format: Christopher A. Hamm, Fabricio F. Costa, Jared M. Bischof, Elio F. Vanin, Maria de Fatima Bonaldo, Steve Iannaccone, Veena Rajaram, David George, Tadanori Tomita, Stewart Goldman, Lawrence J. Jennings, Richard J. Gilbertson, Marcelo B. Soares. Identification of genes that frequently exhibit copy number alterations in pediatric ependymomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3111. doi:10.1158/1538-7445.AM2014-3111

Abstract 3009: Genetic and epigenetic variations resulting from Alu retrotransposition

Alu repeats are the most prevalent retrotransposons, comprising approximately 10% of the human genome. Alu retrotransposons have amplified during primate evolution by an RNA-mediated copy and paste mechanism. Among the Alu subfamilies, AluY elements and its variants exhibit the highest rates of retrotransposition. De novo retrotransposition of active Alu elements have given rise to insertion polymorphisms in human populations. While generating methylation maps of Alu elements in human normal cerebellum and in ependymoma tumors, Xie and colleagues (2009, 2010) also identified putative new Alu element insertions. In this study, we explored these Alu libraries to identify recent Alu element insertions and to understand the (epi)genetic variations that result from such events. This study may also enable identification of DNA structural variation that may occur at the onset of tumorigenesis and/or during tumor progression. A total of 327 putative recent Alu element insertions represented by 1,762 sequence reads were identified, including 316 events that have not yet been documented in either dbSNP or dbRIP. Forty-seven out of a total of forty-nine randomly selected events representing nineteen genomic loci were sequence-verified. Alu element insertions remained heterozygous for sixteen out of nineteen genomic loci in one or more individuals. The polymorphic Alu elements are enriched for young Alu families and they present some characteristic sequence features, such as longer poly(A) tails. In addition, they also present the TT/AAAA consensus sequence at their 3’-flanking regions, and AT-rich target site duplications. Despite being homozygous or heterozygous insertions, these Alu elements are heavily methylated. Further methylation analysis on two genomic loci revealed no methylation difference in two heterozygous alleles and also no methylation difference at CpG dinucleotides flanking the Alu insertion sites. These results indicate that the recently integrated Alu elements have not influenced the methylation status of their neighboring CpG dinucleotides. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3009. doi:10.1158/1538-7445.AM2011-3009