Jared M. Bischof

Identification of Differentially Expressed MicroRNAs in Osteosarcoma

A limited number of reports have investigated the role of microRNAs in osteosarcoma. In this study, we performed miRNA expression profiling of osteosarcoma cell lines, tumor samples, and normal human osteoblasts. Twenty-two differentially expressed microRNAs were identified using high throughput real-time PCR analysis, and 4 (miR-135b, miR-150, miR-542-5p, and miR-652) were confirmed and validated in a different group of tumors. Both miR-135b and miR-150 have been previously shown to be important in cancer. We hypothesize that dysregulation of differentially expressed microRNAs may contribute to tumorigenesis. They might also represent molecular biomarkers or targets for drug development in osteosarcoma.

Identification of MicroRNAs as Potential Prognostic Markers in Ependymoma

Introduction We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers. Materials and Methods We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features. Results We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival. Conclusion We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.

Folic Acid Remodels Chromatin on Hes1 and Neurog2 Promoters during Caudal Neural Tube Development

The mechanism(s) behind folate rescue of neural tube closure are not well understood. In this study we show that maternal intake of folate prior to conception reverses the proliferation potential of neural crest stem cells in homozygous Splotch embryos (Sp−/−) via epigenetic mechanisms. It is also shown that the pattern of differentiation seen in these cells is similar to wild-type (WT). Cells from open caudal neural tubes of Sp−/− embryos exhibit increased H3K27 methylation and decreased expression of KDM6B possibly due to up-regulation of KDM6B targeting micro-RNAs such as miR-138, miR-148a, miR-185, and miR-339-5p. In our model, folate reversed these epigenetic marks in folate-rescued Sp−/− embryos. Using tissue from caudal neural tubes of murine embryos we also examined H3K27me2 and KDM6B association with Hes1 and Neurog2 promoters at embryonic day E10.5, the proliferative stage, and E12.5, when neural differentiation begins. In Sp−/− embryos compared with WT, levels of H3K27me2 associated with the Hes1 promoter were increased at E10.5, and levels associated with the Neurog2 promoter were increased at E12.5. KDM6B association with Hes1 and Neurog2 promoters was inversely related to H3K27me2 levels. These epigenetic changes were reversed in folate-rescued Sp−/− embryos. Thus, one of the mechanisms by which folate may rescue the Sp−/− phenotype is by increasing the expression of KDM6B, which in turn decreases H3K27 methylation marks on Hes1 and Neurog2 promoters thereby affecting gene transcription.

Epigenetically reprogramming metastatic tumor cells with an embryonic microenvironment

UNLABELLED We have previously shown that the microenvironment of human embryonic stem cells (hESCs) is able to change and reprogram aggressive cancer cells to a less aggressive state. Some mechanisms implicated in the phenotypic changes observed after this exposure are mainly associated with the Nodal signaling pathway, which plays a key role in tumor cell plasticity. However, several other molecular mechanisms might be related directly and/or indirectly to these changes, including microRNA (miRNA) regulation and DNA methylation. AIM To further explore the epigenetic mechanisms potentially underlying the phenotypic changes that occur after exposing metastatic melanoma cells to a hESC microenvironment. MATERIALS & METHODS A total of 365 miRNAs were screened using the TaqMan® Low Density Arrays. We also evaluated whether DNA methylation could be one of the factors regulating the expression of the inhibitor of Nodal, Lefty, in hESCs (where it is highly expressed) vs melanoma cells (where it is not expressed). RESULTS Using these experimental approaches, we identified miRNAs that are up- and down-regulated in melanoma cells exposed to a hESC microenvironment, such as miR-302a and miR-27b, respectively. We also demonstrate that Notch4 is one of the targets of miR-302a, which is upstream of Nodal. Additionally, one of the mechanisms that might explain the absence of the inhibitor of Nodal, Lefty, in cancer cells is silencing by DNA methylation, which provides new insights into the unregulated expression of Nodal in melanoma. CONCLUSION These findings suggest that epigenetic changes such as DNA methylation and regulation by microRNAs might play a significant role in tumor cell plasticity and the metastatic phenotype.

The commonality of plasticity underlying multipotent tumor cells and embryonic stem cells

Aggressive cancer cells and pluripotent stem cells converge in their capacity for self‐renewal, proliferation and plasticity. Recent studies have capitalized on these similarities by demonstrating that tumors arise from specific cancer stem cell populations that, in a manner reminiscent of normal stem cells, are able to both self‐renew and give rise to a heterogeneous tumor population. This stem cell like function of aggressive cancer cells is likely attributable to the ectopic expression of embryonic factors such as Nodal and Cancer Testis Specific Antigens (CTAs), which maintain a functional plasticity by promoting pluripotency and immortality. During development, the expression of these embryonic factors is tightly regulated by a dynamic array of mediators, including the spatial and temporal expression of inhibitors such as Lefty, and the epigenetic modulation of the genome. In aggressive cancer cells, particularly melanoma, this balance of regulatory mediators is disrupted, leading to the aberrant expression of pluripotency‐associated genes. By exposing aggressive cancer cells to embryonic microenvironments, this balance of regulatory mediators is restored, thereby reprogramming tumor cells to a more benign phenotype. These stem cell‐derived mediators, as well as the genes they regulate, provide therapeutic targets designed to specifically differentiate and eradicate aggressive cancers. J. Cell. Biochem. 101: 908–917, 2007.

Global Demethylation of Rat Chondrosarcoma Cells after Treatment with 5-Aza-2′-Deoxycytidine Results in Increased Tumorigenicity

Abnormal patterns of DNA methylation are observed in several types of human cancer. While localized DNA methylation of CpG islands has been associated with gene silencing, the effect that genome-wide loss of methylation has on tumorigenesis is not completely known. To examine its effect on tumorigenesis, we induced DNA demethylation in a rat model of human chondrosarcoma using 5-aza-2-deoxycytidine. Rat specific pyrosequencing assays were utilized to assess the methylation levels in both LINEs and satellite DNA sequences following 5-aza-2-deoxycytidine treatment. Loss of DNA methylation was accompanied by an increase in invasiveness of the rat chondrosarcoma cells, in vitro, as well as by an increase in tumor growth in vivo. Subsequent microarray analysis provided insight into the gene expression changes that result from 5-aza-2-deoxycytidine induced DNA demethylation. In particular, two genes that may function in tumorigenesis, sox-2 and midkine, were expressed at low levels in control cells but upon 5-aza-2-deoxycytidine treatment these genes became overexpressed. Promoter region DNA analysis revealed that these genes were methylated in control cells but became demethylated following 5-aza-2-deoxycytidine treatment. Following withdrawal of 5-aza-2-deoxycytidine, the rat chondrosarcoma cells reestablished global DNA methylation levels that were comparable to that of control cells. Concurrently, invasiveness of the rat chondrosarcoma cells, in vitro, decreased to a level indistinguishable to that of control cells. Taken together these experiments demonstrate that global DNA hypomethylation induced by 5-aza-2-deoxycytidine may promote specific aspects of tumorigenesis in rat chondrosarcoma cells.

Transcriptional Profiling of Polycythemia Vera Identifies Gene Expression Patterns Both Dependent and Independent From the Action of JAK2V617F

Purpose: To understand the changes in gene expression in polycythemia vera (PV) progenitor cells and their relationship to JAK2V617F. Experimental Design: Messenger RNA isolated from CD34+ cells from nine PV patients and normal controls was profiled using Affymetrix arrays. Gene expression change mediated by JAK2V617F was determined by profiling CD34+ cells transduced with the kinase and by analysis of leukemia cell lines harboring JAK2V617F, treated with an inhibitor. Results: A PV expression signature was enriched for genes involved in hematopoietic development, inflammatory responses, and cell proliferation. By quantitative reverse transcription-PCR, 23 genes were consistently deregulated in all patient samples. Several of these genes such as WT1 and KLF4 were regulated by JAK2, whereas others such as NFIB and EVI1 seemed to be deregulated in PV by a JAK2-independent mechanism. Using cell line models and comparing gene expression profiles of cell lines and PV CD34+ PV specimens, we have identified panels of 14 JAK2-dependent genes and 12 JAK2-independent genes. These two 14- and 12-gene sets could separate not only PV from normal CD34+ specimens, but also other MPN such as essential thrombocytosis and primary myelofibrosis from their normal counterparts. Conclusions: A subset of the aberrant gene expression in PV progenitor cells can be attributed to the action of the mutant kinase, but there remain a significant number of genes characteristic of the disease but deregulated by as yet unknown mechanisms. Genes deregulated in PV as a result of the action of JAK2V617F or independent of the kinase may represent other targets for therapy. Clin Cancer Res; 16(17); 4339–52. ©2010 AACR.

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.

Microenvironment alters epigenetic and gene expression profiles in Swarm rat chondrosarcoma tumors

BackgroundChondrosarcomas are malignant cartilage tumors that do not respond to traditional chemotherapy or radiation. The 5-year survival rate of histologic grade III chondrosarcoma is less than 30%. An animal model of chondrosarcoma has been established - namely, the Swarm Rat Chondrosarcoma (SRC) - and shown to resemble the human disease. Previous studies with this model revealed that tumor microenvironment could significantly influence chondrosarcoma malignancy.MethodsTo examine the effect of the microenvironment, SRC tumors were initiated at different transplantation sites. Pyrosequencing assays were utilized to assess the DNA methylation of the tumors, and SAGE libraries were constructed and sequenced to determine the gene expression profiles of the tumors. Based on the gene expression analysis, subsequent functional assays were designed to determine the relevancy of the specific genes in the development and progression of the SRC.ResultsThe site of transplantation had a significant impact on the epigenetic and gene expression profiles of SRC tumors. Our analyses revealed that SRC tumors were hypomethylated compared to control tissue, and that tumors at each transplantation site had a unique expression profile. Subsequent functional analysis of differentially expressed genes, albeit preliminary, provided some insight into the role that thymosin-β4, c-fos, and CTGF may play in chondrosarcoma development and progression.ConclusionThis report describes the first global molecular characterization of the SRC model, and it demonstrates that the tumor microenvironment can induce epigenetic alterations and changes in gene expression in the SRC tumors. We documented changes in gene expression that accompany changes in tumor phenotype, and these gene expression changes provide insight into the pathways that may play a role in the development and progression of chondrosarcoma. Furthermore, specific functional analysis indicates that thymosin-β4 may have a role in chondrosarcoma metastasis.

Nucleosome mapping across the CFTR locus identifies novel regulatory factors

Nucleosome positioning on the chromatin strand plays a critical role in regulating accessibility of DNA to transcription factors and chromatin modifying enzymes. Hence, detailed information on nucleosome depletion or movement at cis-acting regulatory elements has the potential to identify predicted binding sites for trans-acting factors. Using a novel method based on enrichment of mononucleosomal DNA by bacterial artificial chromosome hybridization, we mapped nucleosome positions by deep sequencing across 250 kb, encompassing the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR shows tight tissue-specific regulation of expression, which is largely determined by cis-regulatory elements that lie outside the gene promoter. Although multiple elements are known, the repertoire of transcription factors that interact with these sites to activate or repress CFTR expression remains incomplete. Here, we show that specific nucleosome depletion corresponds to well-characterized binding sites for known trans-acting factors, including hepatocyte nuclear factor 1, Forkhead box A1 and CCCTC-binding factor. Moreover, the cell-type selective nucleosome positioning is effective in predicting binding sites for novel interacting factors, such as BAF155. Finally, we identify transcription factor binding sites that are overrepresented in regions where nucleosomes are depleted in a cell-specific manner. This approach recognizes the glucocorticoid receptor as a novel trans-acting factor that regulates CFTR expression in vivo.