Transmembrane Protein TMEM230, Regulator of Glial Cell Vascular Mimicry and Endothelial Cell Angiogenesis in High-Grade Heterogeneous Infiltrating Gliomas and Glioblastoma.

High-grade gliomas (HGGs) and glioblastoma multiforme (GBM) are characterized by a heterogeneous and aggressive population of tissue-infiltrating cells that promote both destructive tissue remodeling and aberrant vascularization of the brain. The formation of defective and permeable blood vessels and microchannels and destructive tissue remodeling prevent efficient vascular delivery of pharmacological agents to tumor cells and are the significant reason why therapeutic chemotherapy and immunotherapy intervention are primarily ineffective. Vessel-forming endothelial cells and microchannel-forming glial cells that recapitulate vascular mimicry have both infiltration and destructive remodeling tissue capacities. The transmembrane protein TMEM230 (C20orf30) is a master regulator of infiltration, sprouting of endothelial cells, and microchannel formation of glial and phagocytic cells. A high level of TMEM230 expression was identified in patients with HGG, GBM, and U87-MG cells. In this study, we identified candidate genes and molecular pathways that support that aberrantly elevated levels of TMEM230 play an important role in regulating genes associated with the initial stages of cell infiltration and blood vessel and microchannel (also referred to as tumor microtubule) formation in the progression from low-grade to high-grade gliomas. As TMEM230 regulates infiltration, vascularization, and tissue destruction capacities of diverse cell types in the brain, TMEM230 is a promising cancer target for heterogeneous HGG tumors.

Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy.

Glioblastomas (GBM) are the most aggressive tumors originating in the brain. Histopathologic features include circuitous, disorganized, and highly permeable blood vessels with intermittent blood flow. These features contribute to the inability to direct therapeutic agents to tumor cells. Known targets for anti-angiogenic therapies provide minimal or no effect in overall survival of 12-15 months following diagnosis. Identification of novel targets therefore remains an important goal for effective treatment of highly vascularized tumors such as GBM. We previously demonstrated in zebrafish that a balanced level of expression of the transmembrane protein TMEM230/C20ORF30 was required to maintain normal blood vessel structural integrity and promote proper vessel network formation. To investigate whether TMEM230 has a role in the pathogenesis of GBM, we analyzed its prognostic value in patient tumor gene expression datasets and performed cell functional analysis. TMEM230 was found necessary for growth of U87-MG cells, a model of human GBM. Downregulation of TMEM230 resulted in loss of U87 migration, substratum adhesion, and re-passaging capacity. Conditioned media from U87 expressing endogenous TMEM230 induced sprouting and tubule-like structure formation of HUVECs. Moreover, TMEM230 promoted vascular mimicry-like behavior of U87 cells. Gene expression analysis of 702 patients identified that TMEM230 expression levels distinguished high from low grade gliomas. Transcriptomic analysis of patients with gliomas revealed molecular pathways consistent with properties observed in U87 cell assays. Within low grade gliomas, elevated TMEM230 expression levels correlated with reduced overall survival independent from tumor subtype. Highest level of TMEM230 correlated with glioblastoma and ATP-dependent microtubule kinesin motor activity, providing a direction for future therapeutic intervention. Our studies support that TMEM230 has both glial tumor and endothelial cell intracellular and extracellular functions. Elevated levels of TMEM230 promote glial tumor cell migration, extracellular scaffold remodeling, and hypervascularization and abnormal formation of blood vessels. Downregulation of TMEM230 expression may inhibit both low grade glioma and glioblastoma tumor progression and promote normalization of abnormally formed blood vessels. TMEM230 therefore is both a promising anticancer and antiangiogenic therapeutic target for inhibiting GBM tumor cells and tumor-driven angiogenesis.

Integrating Microstructured Electrospun Scaffolds in an Open Microfluidic System for in Vitro Studies of Human Patient-Derived Primary Cells.

Recent studies have suggested that microenvironmental stimuli play a significant role in regulating cellular proliferation and migration, as well as in modulating self-renewal and differentiation processes of mammary cells with stem cell (SCs) properties. Recent advances in micro/nanotechnology and biomaterial synthesis/engineering currently enable the fabrication of innovative tissue culture platforms suitable for maintenance and differentiation of SCs in vitro. Here, we report the design and fabrication of an open microfluidic device (OMD) integrating removable poly(ε-caprolactone) (PCL) based electrospun scaffolds, and we demonstrate that the OMD allows investigation of the behavior of human cells during in vitro culture in real time. Electrospun scaffolds with modified surface topography and chemistry can influence attachment, proliferation, and differentiation of mammary SCs and epigenetic mechanisms that maintain luminal cell identity as a function of specific morphological or biochemical cues imparted by tailor-made fiber post-treatments. Meanwhile, the OMD architecture allows control of cell seeding and culture conditions to collect more accurate and informative in vitro assays. In perspective, integrated systems could be tailor-made to mimic specific physiological conditions of the local microenvironment and then analyze the response from screening specific drugs for more effective diagnostics, long-term prognostics, and disease intervention in personalized medicine.

Zebrafish Tmem230a cooperates with the Delta/Notch signaling pathway to modulate endothelial cell number in angiogenic vessels.

During embryonic development, new arteries, and veins form from preexisting vessels in response to specific angiogenic signals. Angiogenic signaling is complex since not all endothelial cells exposed to angiogenic signals respond equally. Some cells will be selected to become tip cells and acquire migration and proliferation capacity necessary for vessel growth while others, the stalk cells become trailer cells that stay connected with pre-existing vessels and act as a linkage to new forming vessels. Additionally, stalk and tip cells have the capacity to interchange their roles. Stalk and tip cellular responses are mediated in part by the interactions of components of the Delta/Notch and Vegf signaling pathways. We have identified in zebrafish, that the transmembrane protein Tmem230a is a novel regulator of angiogenesis by its capacity to regulate the number of the endothelial cells in intersegmental vessels by co-operating with the Delta/Notch signaling pathway. Modulation of Tmem230a expression by itself is sufficient to rescue improper number of endothelial cells induced by aberrant expression or inhibition of the activity of genes associated with the Dll4/Notch pathway in zebrafish. Therefore, Tmem230a may have a modulatory role in vessel-network formation and growth. As the Tmem230 sequence is conserved in human, Tmem230 may represent a promising novel target for drug discovery and for disease therapy and regenerative medicine in promoting or restricting angiogenesis.

RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy.

Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human-induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogramming may prove to be more suitable for clinical applications and provide stable starting cell lines for gene-editing, isolation, and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene-editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA-based laboratory procedure for making and editing iPSCs. In the near-term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic, and translational research. J. Cell. Physiol. 232: 1262-1269, 2017. © 2016 Wiley Periodicals, Inc.

microRNA miR-200b affects proliferation, invasiveness and stemness of endometriotic cells by targeting ZEB1, ZEB2 and KLF4.

Endometriosis is characterized by growth of endometrial tissue at ectopic locations. Down-regulation of microRNA miR-200b is observed in endometriosis and malignant disease, driving tumour cells towards an invasive state by enhancing epithelial-to-mesenchymal transition (EMT). miR-200b up-regulation may inhibit EMT and invasive growth in endometriosis. To study its functional impact on the immortalized endometriotic cell line 12Z, the stromal cell line ST-T1b, and primary endometriotic stroma cells, a transient transfection approach with microRNA precursors was employed. Expression of bioinformatically predicted targets of miR-200b was analysed by qPCR. The cellular phenotype was monitored by Matrigel invasion assays, digital-holographic video microscopy and flow cytometry. qPCR revealed significant down-regulation of ZEB1 (P < 0.05) and ZEB2 (P < 0.01) and an increase in E-cadherin (P < 0.01). miR-200b overexpression decreased invasiveness (P < 0.0001) and cell motility (P < 0.05). In contrast, cell proliferation (P < 0.0001) and the stemness-associated side population phenotype (P < 0.01) were enhanced following miR-200b transfection. These properties were possibly due to up-regulation of the pluripotency-associated transcription factor KLF4 (P < 0.05) and require attention when considering therapeutic strategies. In conclusion, up-regulation of miR-200b reverts EMT, emerging as a potential therapeutic approach to inhibit endometriotic cell motility and invasiveness.

A Versatile Tool for Stable Inhibition of microRNA Activity.

MicroRNAs (miRNAs) are a class of small RNAs (18-22 nt) that post transcriptionally regulate gene expression by binding to complementary sequences on target mRNAs, resulting in translational repression or target degradation and gene silencing. As aberrant expression of miRNAs is implicated in important diseases including cancer miRNA-based therapies are under intensive investigation. We optimized strategies to stably or conditionally generate miRNA inhibitors for a continuous block of miRNA activity that allows for probing miRNA function in long-term cell culture experiments, cancer xenografts, 3D tissue models and for in vivo studies with transgenic organisms.

Identification and mapping of the leaf stripe resistance gene Rdg1a in Hordeum spontaneum.

Leaf stripe of barley, caused by Pyrenophora graminea, is an important seed-borne disease in organically grown as well as in conventionally grown Nordic and Mediterranean barley districts. Two barley segregating populations represented by 103 recombinant inbred lines (RILs) of the cross L94 (susceptible) x Vada (resistant) and 194 RILs of the cross Arta (susceptible) x Hordeum spontaneum 41-1 (resistant) were analysed with two highly virulent leaf stripe isolates, Dg2 and Dg5, to identify loci for P. graminea resistance. A major gene with its positive allele contributed by Vada and H. spontaneum 41-1 was detected in both populations and for both pathogen isolates on chromosome 2HL explaining 44.1 and 91.8% R (2), respectively for Dg2 and Dg5 in L94 x Vada and 97.8 and 96.1% R (2), respectively for Dg2 and Dg5 in Arta x H. spontaneum 41-1. Common markers in the gene region of the two populations enabled map comparison and highlighted an overlapping for the region of the resistance locus. Since the map position of the resistance locus identified in this report is the same as that for the leaf stripe resistance gene Rdg1a, mapped earlier in Alf and derived from the 'botanical' barley line H. laevigatum, we propose that leaf stripe resistance in Vada and H. spontaneum 41-1 is governed by the same gene, namely by Rdg1a, and that Rdg1a resistance could be traced back to H. spontaneum, the progenitor of cultivated barley. PCR-based molecular markers that can be used for marker-assisted selection (MAS) of Rdg1a were identified. An Rdg1a syntenic interval with the rice chromosome arm 4L was identified on the basis of rice orthologs of EST-based barley markers. Analysis of the rice genes annotated into the syntenic interval did not reveal sequences strictly belonging to the major class (nucleotide-binding site plus leucine-rich repeat) of the resistance genes. Nonetheless, four genes coding for domains that are present in the major disease-resistance genes, namely receptor-like protein kinase and ATP/GTP-binding proteins, were identified together with a homolog of the barley powdery mildew resistance gene mlo. Three (out of five) homologs of these genes were mapped in the Rdg1a region in barley and the mlo homolog map position was tightly associated with the LOD score peak in both populations.