Downregulation of Mir-31, Mir-155, and Mir-564 in Chronic Myeloid Leukemia Cells

BACKGROUND/AIMS: MicroRNAs (miRNAs) are short non-coding regulatory RNAs that control gene expression and play an important role in cancer development and progression. However, little is known about the role of miRNAs in chronic myeloid leukemia (CML). Our objective is to decipher a miRNA expression signature associated with CML and to determine potential target genes and signaling pathways affected by these signature miRNAs. RESULTS: Using miRNA microarrays and miRNA real-time PCR we characterized the miRNAs expression profile of CML cell lines and patients in reference to non-CML cell lines and healthy blood. Of all miRNAs tested, miR-31, miR-155, and miR-564 were down-regulated in CML cells. Down-regulation of these miRNAs was dependent on BCR-ABL activity. We next analyzed predicted targets and affected pathways of the deregulated miRNAs. As expected, in K562 cells, the expression of several of these targets was inverted to that of the miRNA putatively regulating them. Reassuringly, the analysis identified CML as the main disease associated with these miRNAs. MAPK, ErbB, mammalian target of rapamycin (mTOR) and vascular endothelial growth factor (VEGF) were the main molecular pathways related with these expression patterns. Utilizing Venn diagrams we found appreciable overlap between the CML-related miRNAs and the signaling pathways-related miRNAs. CONCLUSIONS: The miRNAs identified in this study might offer a pivotal role in CML. Nevertheless, while these data point to a central disease, the precise molecular pathway/s targeted by these miRNAs is variable implying a high level of complexity of miRNA target selection and regulation. These deregulated miRNAs highlight new candidate gene targets allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment

Inter-Tumor Heterogeneity-Melanomas Respond Differently to GM-CSF-Mediated Activation.

Granulocyte-monocyte colony stimulating factor (GM-CSF) is used as an adjuvant in various clinical and preclinical studies with contradictory results. These were attributed to opposing effects of GM-CSF on the immune or myeloid systems of the treated patients or to lack of optimal dosing regimens. The results of the present study point to inter-tumor heterogeneity as a possible mechanism accounting for the contrasting responses to GM-CSF incorporating therapies. Employing xenograft models of human melanomas in nude mice developed in our lab, we detected differential functional responses of melanomas from different patients to GM-CSF both in vitro as well as in vivo. Whereas cells of one melanoma acquired pro metastatic features following exposure to GM-CSF, cells from another melanoma either did not respond or became less malignant. We propose that inter-melanoma heterogeneity as manifested by differential responses of melanoma cells (and perhaps also of other tumor) to GM-CSF may be developed into a predictive marker providing a tool to segregate melanoma patients who will benefit from GM-CSF therapy from those who will not

ANGPTL4 promotes the progression of cutaneous melanoma to brain metastasis.

In an ongoing effort to identify molecular determinants regulating melanoma brain metastasis, we previously identified Angiopoietin-like 4 (ANGPTL4) as a component of the molecular signature of such metastases. The aim of this study was to determine the functional significance of ANGPTL4 in the shaping of melanoma malignancy phenotype, especially in the establishment of brain metastasis. We confirmed that ANGPTL4 expression is significantly higher in cells metastasizing to the brain than in cells from the cutaneous (local) tumor from the same melanoma in a nude mouse xenograft model, and also in paired clinical specimens of melanoma metastases than in primary melanomas from the same patients. In vitro experiments indicated that brain-derived soluble factors and transforming growth factor β1 (TGFβ1) up-regulated ANGPTL4 expression by melanoma cells. Forced over-expression of ANGPTL4 in cutaneous melanoma cells promoted their ability to adhere and transmigrate brain endothelial cells. Over-expressing ANGPTL4 in cells derived from brain metastases resulted in the opposite effects. In vivo data indicated that forced overexpression of ANGPTL4 promoted the tumorigenicity of cutaneous melanoma cells but did not increase their ability to form brain metastasis. This finding can be explained by inhibitory activities of brain-derived soluble factors. Taken together these findings indicate that ANGPTL4 promotes the malignancy phenotype of primary melanomas of risk to metastasize to the brain

Insulin-Producing Cells Generated from Dedifferentiated Human Pancreatic Beta Cells Expanded In Vitro

Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening

Effects of IKAP/hELP1 Deficiency on Gene Expression in Differentiating Neuroblastoma Cells: Implications for Familial Dysautonomia

Familial dysautonomia (FD) is a developmental neuropathy of the sensory and autonomous nervous systems. The IKBKAP gene, encoding the IKAP/hELP1 subunit of the RNA polymerase II Elongator complex is mutated in FD patients, leading to a tissue-specific mis-splicing of the gene and to the absence of the protein in neuronal tissues. To elucidate the function of IKAP/hELP1 in the development of neuronal cells, we have downregulated IKBKAP expression in SHSY5Y cells, a neuroblastoma cell line of a neural crest origin. We have previously shown that these cells exhibit abnormal cell adhesion when allowed to differentiate under defined culture conditions on laminin substratum. Here, we report results of a microarray expression analysis of IKAP/hELP1 downregulated cells that were grown on laminin under differentiation or non-differentiation growth conditions. It is shown that under non-differentiation growth conditions, IKAP/hELP1 downregulation affects genes important for early developmental stages of the nervous system, including cell signaling, cell adhesion and neural crest migration. IKAP/hELP1 downregulation during differentiation affects the expression of genes that play a role in late neuronal development, in axonal projection and synapse formation and function. We also show that IKAP/hELP1 deficiency affects the expression of genes involved in calcium metabolism before and after differentiation of the neuroblastoma cells. Hence, our data support IKAP/hELP1 importance in the development and function of neuronal cells and contribute to the understanding of the FD phenotype

GEView (Gene Expression View) Tool for Intuitive and High Accessible Visualization of Expression Data for Non-Programmer Biologists

This article describes how the last decade has been characterized by the production of huge amounts of different types of biological data. Following that, a flood of bioinformatics tools have been published. However, many of these tools are commercial, or require computational skills. In addition, not all tools provide intuitive and highly accessible visualization of the results. The authors have developed GEView (Gene Expression View), which is a free, user-friendly tool harboring several existing algorithms and statistical methods for the analysis of high-throughput gene, microRNA or protein expression data. It can be used to perform basic analysis such as quality control, outlier detection, batch correction and differential expression analysis, through a single intuitive graphical user interface. GEView is unique in its simplicity and highly accessible visualization it provides. Together with its basic and intuitive functionality it allows Bio-Medical scientists with no computational skills to independently analyze and visualize high-throughput data produced in their own labs

Simulation and visualization of multiple KEGG pathways using BioNSi [version 2; referees: 2 approved, 1 approved with reservations]

Motivation: Many biologists are discouraged from using network simulation tools because these require manual, often tedious network construction. This situation calls for building new tools or extending existing ones with the ability to import biological pathways previously deposited in databases and analyze them, in order to produce novel biological insights at the pathway level. Results: We have extended a network simulation tool (BioNSi), which now allows merging of multiple pathways from the KEGG pathway database into a single, coherent network, and visualizing its properties. Furthermore, the enhanced tool enables loading experimental expression data into the network and simulating its dynamics under various biological conditions or perturbations. As a proof of concept, we tested two sets of published experimental data, one related to inflammatory bowel disease condition and the other to breast cancer treatment. We predict some of the major observations obtained following these laboratory experiments, and provide new insights that may shed additional light on these results. Tool requirements: Cytoscape 3.x, JAVA 8 Availability: The tool is freely available at http://bionsi.wix.com/bionsi, where a complete user guide and a step-by-step manual can also be found