Downregulation of microRNAs (miRNAs) at the 14q32 locus stabilizes the expression of cMYC, thus significantly contributing to osteosarcoma (OS) pathobiology. Here, we show that downregulation of ...14q32 miRNAs is epigenetically regulated. The predicted promoter regions of miRNA clusters at 14q32 locus showed no recurrent patterns of differential methylation, but Saos2 cells showed elevated histone deacetylase (HDAC) activity. Treatment with 4-phenylbutyrate increased acetylation of histones associated with 14q32 miRNAs, but interestingly, robust restoration of 14q32 miRNA expression, attenuation of cMYC expression, and induction of apoptosis required concomitant treatment with 5-Azacytidine, an inhibitor of DNA methylation. These events were associated with genome-wide gene expression changes including induction of pro-apoptotic genes and downregulation of cell cycle genes. Comparable effects were achieved in human and canine OS cells using the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat) and the DNA methylation inhibitor Zebularine (Zeb), with significantly more pronounced cytotoxicity in cells whose molecular phenotypes were indicative of aggressive biological behavior. These results suggested that the combination of these chromatin-modifying drugs may be a useful adjuvant in the treatment of rapidly progressive OS.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Many somatic mutations have been detected in pancreatic ductal adenocarcinoma (PDAC), leading to the identification of some key drivers of disease progression, but the involvement of large genomic ...rearrangements has often been overlooked. In this study, we performed mate pair sequencing (MPseq) on genomic DNA from 24 PDAC tumors, including 15 laser-captured microdissected PDAC and 9 patient-derived xenografts, to identify genome-wide rearrangements. Large genomic rearrangements with intragenic breakpoints altering key regulatory genes involved in PDAC progression were detected in all tumors. SMAD4, ZNF521, and FHIT were among the most frequently hit genes. Conversely, commonly reported genes with copy number gains, including MYC and GATA6, were frequently observed in the absence of direct intragenic breakpoints, suggesting a requirement for sustaining oncogenic function during PDAC progression. Integration of data from MPseq, exome sequencing, and transcriptome analysis of primary PDAC cases identified limited overlap in genes affected by both rearrangements and point mutations. However, significant overlap was observed in major PDAC-associated signaling pathways, with all PDAC exhibiting reduced SMAD4 expression, reduced SMAD-dependent TGFβ signaling, and increased WNT and Hedgehog signaling. The frequent loss of SMAD4 and FHIT due to genomic rearrangements strongly implicates these genes as key drivers of PDAC, thus highlighting the strengths of an integrated genomic and transcriptomic approach for identifying mechanisms underlying disease initiation and progression.
Asthma is an inflammatory disease in which altered calcium regulation, contractility, and airway smooth muscle (ASM) proliferation contribute to airway hyper-responsiveness and airway wall ...remodeling. The enzymatic activity of CD38, a cell-surface protein expressed in human ASM cells, generates calcium mobilizing second messenger molecules such as cyclic ADP-ribose. CD38 expression in human ASM cells is augmented by cytokines (e.g., TNF-α) that requires the activation of MAP kinases and the transcription factors, NF-κB and AP-1, and is post-transcriptionally regulated by miR-140-3p and miR-708 by binding to 3' Untranslated Region of CD38 as well as by modulating the activation of signaling mechanisms involved in its regulation. Mice deficient in Cd38 exhibit reduced airway responsiveness to inhaled methacholine relative to the response in wild-type mice. Intranasal challenge of Cd38-deficient mice with TNF-α or IL-13, or the environmental fungus Alternaria alternata, causes significantly attenuated methacholine responsiveness compared with wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyper-responsiveness to inhaled methacholine in the Cd38-deficient mice. These studies provide evidence for CD38 involvement in the development of airway hyper-responsiveness; a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and (or) activity are warranted.
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DOBA, FSPLJ, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs (miRNAs) are important players of post-transcriptional gene regulation. Individual miRNAs can target multiple mRNAs and a single mRNA can be targeted by many miRNAs. We hypothesized that ...miRNAs select and regulate their targets based on their own expression levels, those of their target mRNAs and triggered feedback loops. We studied the effects of varying concentrations of let-7a-7f and the miR-17-92 cluster plasmids on the reporter genes carrying either DICER- or cMYC -3′UTR in Huh-7 cells. We showed that let-7 significantly downregulated expression of DICER 3′UTR reporter at lower concentrations, but selectively downregulated expression of a cMYC 3′UTR reporter at higher dose. This miRNA dose-dependent target selection was also confirmed in other target genes, including CCND1, CDKN1 and E2F1. After overexpressing let-7a-7f or the miR-17-92 clusters at wide-ranging doses, the target genes displayed a nonlinear correlation to the transfected miRNA. Further, by comparing the expression levels of let-7a and miR-17-5p, along with their selected target genes in 3 different cell lines, we found that the knockdown dose of each miRNA was directly related to their baseline expression level, that of the target gene and feedback loops. These findings were supported by gene modulation studies using endogenous levels of miR-29, -1 and -206 and a luciferase reporter system in multiple cell lines. Finally, we determined that the miR-17-92 cluster affected cell viability in a dose-dependent manner. In conclusion, we have shown that miRNAs potentially select their targets in a dose-dependent and nonlinear fashion that affects biological function; and this represents a novel mechanism by which miRNAs orchestrate the finely tuned balance of cell function.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Worldwide, colorectal cancer (CRC) is one of the leading causes of cancer-related deaths. Recent advances in high-throughput technologies have shown that the gut microbiota may have a major influence ...on human health, including CRC. Nonetheless, how the gut microbiota interacts with tumor cells in CRC patients is largely unknown. Studies have shown that the microbiota fills in a variety of niche metabolic pathways that the host does not possess. For example, the microbiota produces butyrate, which provides the colon's epithelial cells with about 70% of their energy needs. The typically fast proliferation of tumor cells in CRC patients drastically alters the tumor's nutrient microenvironment. Those alterations correspond to the microbiota composition and functional changes. In tumor cells, a central mediator of metabolic changes is the aberrant expression of microRNAs (miRNAs). In this study, we explored recent insights into metabolic interactions between the microbiota and tumor cells in CRC pathobiology, focusing on the role of miRNAs. These observations support our view that miRNAs may also serve as mediators of the metabolites' effects.
MicroRNAs (miRNAs) have emerged as one of the major regulatory mechanisms of gene expression. A major function of miRNAs involves the post-transcriptional regulation of target mRNAs, which is ...reported to occur primarily in the cytoplasm. However, there is a significant amount of evidence demonstrating the existence of small non-coding RNAs, including small-interfering RNA (siRNA), miRNA, and Piwi-interacting RNA (piRNA) in the nucleus. In order to elucidate the potential subcellular localizations and functions of miRNAs, we have identified numerous miRNAs that are present in isolated nuclei from human colon cancer HCT116 cells. MicroRNA profiles were compared between cytoplasmic and nuclear fractions of the HCT116 cell line on the basis of multiple microarray analyses. MicroRNA species showing significant existence in isolated and highly purified populations of nuclei were selected and further tested with RT-PCR. The nuclear localization of the mature form of miRNAs was verified again by control RT-PCR excluding the detection of premature forms of miRNA, such as pri-miRNA or pre-miRNA. The elevated levels of representative miRNAs identified in purified nuclei were confirmed by Northern blot analysis, supporting the notion that significant numbers of mature miRNAs exist not only in the cytoplasm but also in the nucleus. These results will likely provide a basis for further studies concerning the intracellular trafficking and nuclear location of miRNAs.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
The discovery of microRNAs (miRNAs) has led to a paradigm shift in our basic understanding of gene regulation. Competing endogenous RNAs (ceRNAs) are the recent entrants adding to the complexities of ...miRNA mediated gene regulation. ceRNAs are RNAs that share miRNA recognition elements (MREs) thereby regulating each other. It is apparent that miRNAs act as rheostats that fine-tune gene expression and maintain the functional balance of various gene networks. Thus MREs in coding and non-coding transcripts have evolved to become the crosstalk hubs of gene interactions, affecting the expression levels and activities of different ceRNAs. Decoding the crosstalk between MREs mediated by ceRNAs is critical to delineate the intricacies in gene regulation, and we have just begun to unravel this complexity.
MicroRNA miR-335 has been reported to have both tumor suppressor and oncogenic activities. In order to determine possible tissue and cell type differences in response to miR-335, we examined the ...effect of miR-335 on cell expression of MT1-MMP, a proteinase commonly expressed in tumors and associated with cell proliferation and migration. miR-335 increased cell surface expression of MT1-MMP in fibrosarcoma HT-1080 and benign prostate BPH-1 cells, but not in prostate LNCaP or breast MCF-7 tumor cells. miR-335 stimulated proliferation and cell migration in a wound healing in vitro assay in HT-1080, BPH-1, and U87 glioblastoma cells, cells which demonstrated significant cell surface expression of MT1-MMP. In contrast, miR-335 did not affect proliferation or migration in cells without a prominent plasma membrane associated MT1-MMP activity. Our data suggest that differences in response to miR-335 by tumor cells may lie in part in the mechanism of regulation of MT1-MMP production.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Molecular cytogenetic evaluation of human osteosarcoma (OS) has revealed the characteristically high degree of genomic reorganization that is the hallmark of this cancer. The extent of genomic ...disorder in OS has hindered identification of the genomic aberrations driving disease progression. With pathophysiological similarities to its human counterpart, canine OS represents an ideal model for comparison of conserved regions of genomic instability that may be disease-associated rather than genomic passengers. This study used high-resolution oligonucleotide array comparative genomic hybridization and a variety of informatics tools to aid in the identification of disease-associated genome-wide DNA copy number aberrations in canine and human OS. Our findings support and build upon the high level of cytogenetic complexity, through the identification of shared regions of microaberration (<500 kb) and functional analysis of possible orthologous OS-associated genes to pinpoint the cellular processes most commonly affected by aberration in human and canine OS. Aberrant regions contained previously reported genes such as CDC5L , MYC , RUNX2 , and CDKN2A / CDKN2B , while expanding the gene of interest list to include ADAM15 , CTC1 , MEN1 , CDK7 , and others. Such regions of instability may thus have functional significance in the etiology of OS, the most common primary bone tumor in both species.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Synovial sarcoma is a soft tissue malignancy characterized by the fusion of SS18 to either SSX1, SSX2, or SSX4 genes. SS18 and SSX are transcriptional cofactors involved in activation and repression ...of gene transcription, respectively. SS18 interacts with SWI/SNF, whereas SSX associates with the polycomb chromatin remodeling complex. Thus, fusion of these two proteins brings together two opposing effects on gene expression and chromatin structure. Recent studies have shown that a significant number of genes are down-regulated by the SS18-SSX fusion protein and that the clinically applicable histone deacetylase (HDAC) inhibitor romidepsin inhibits synovial sarcoma growth. Therefore, we set out to identify direct targets of SS18-SSX among genes down-regulated in synovial sarcoma and investigated if romidepsin can specifically counteract SS18-SSX-mediated transcriptional dysregulation. Here, we report that the tumor suppressor early growth response 1 (EGR1) is repressed by the SS18-SSX protein through a direct association with the EGR1 promoter. This SS18-SSX binding correlates with trimethylation of Lys(27) of histone H3 (H3K27-M3) and recruitment of polycomb group proteins to this promoter. In addition, we found that romidepsin treatment reverts these modifications and reactivates EGR1 expression in synovial sarcoma cell models. Our data implicate polycomb-mediated epigenetic gene repression as a mechanism of oncogenesis in synovial sarcoma. Furthermore, our work highlights a possible mechanism behind the efficacy of a clinically applicable HDAC inhibitor in synovial sarcoma treatment.
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