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  • 1
    Abstract: Analysis of biological processes is frequently performed with the help of phenotypic assays where data is mostly acquired in single end-point analysis. Alternative phenotypic profiling techniques are desired where time-series information is essential to the biological question, for instance to differentiate early and late regulators of cell proliferation in loss-of-function studies. So far there is no study addressing this question despite of high unmet interests, mostly due to the limitation of conventional end-point assaying technologies. We present the first human kinome screen with a real-time cell analysis system (RTCA) to capture dynamic RNAi phenotypes, employing time-resolved monitoring of cell proliferation via electrical impedance. RTCA allowed us to investigate the dynamics of phenotypes of cell proliferation instead of using conventional end-point analysis. By introducing data transformation with first-order derivative, i.e. the cell-index growth rate, we demonstrate this system suitable for high-throughput screenings (HTS). The screen validated previously identified inhibitor genes and, additionally, identified activators of cell proliferation. With the information of time kinetics available, we could establish a network of mitotic-event related genes to be among the first displaying inhibiting effects after RNAi knockdown. The time-resolved screen captured kinetics of cell proliferation caused by RNAi targeting human kinome, serving as a resource for researchers. Our work establishes RTCA technology as a novel robust tool with biological and pharmacological relevance amenable for high-throughput screening.
    Type of Publication: Journal article published
    PubMed ID: 21765947
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  • 2
    Keywords: CANCER ; CELLS ; ACTIVATION ; PROGRESSION ; TERM-FOLLOW-UP ; OSTEOPONTIN ; KIT ; PDGFRA MUTATIONS ; EPIGENETICS ; RECEPTOR-ALPHA MUTATIONS
    Abstract: Gastrointestinal stromal tumors (GISTs) have distinct gene expression patterns according to localization, genotype and aggressiveness. DNA methylation at CpG dinucleotides is an important mechanism for regulation of gene expression. We performed targeted DNA methylation analysis of 1.505 CpG loci in 807 cancer-related genes in a cohort of 76 GISTs, combined with genome-wide mRNA expression analysis in 22 GISTs, to identify signatures associated with clinicopathological parameters and prognosis. Principal component analysis revealed distinct DNA methylation patterns associated with anatomical localization, genotype, mitotic counts and clinical follow-up. Methylation of a single CpG dinucleotide in the non-CpG island promoter of SPP1 was significantly correlated with shorter disease-free survival. Hypomethylation of this CpG was an independent prognostic parameter in a multivariate analysis compared to anatomical localization, genotype, tumor size and mitotic counts in a cohort of 141 GISTs with clinical follow-up. The epigenetic regulation of SPP1 was confirmed in vitro, and the functional impact of SPP1 protein on tumorigenesis-related signaling pathways was demonstrated. In summary, SPP1 promoter methylation is a novel and independent prognostic parameter in GISTs, and might be helpful in estimating the aggressiveness of GISTs from the intermediate-risk category.
    Type of Publication: Journal article published
    PubMed ID: 25046773
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  • 3
    Keywords: EXPRESSION ; LINES ; DYNAMICS ; breast cancer ; REQUIRES ; SERUM RESPONSE FACTOR ; EPITHELIAL-MESENCHYMAL TRANSITION ; miR-200 family ; FORMIN ; FIBER FORMATION ; ZEB1
    Abstract: MicroRNA-200c (miR-200c) has been shown to suppress epithelial-to-mesenchymal transition (EMT), which is mainly attributed to targeting of ZEB1/ZEB2, repressors of the cell-cell contact protein E-Cadherin. Here, we demonstrated that modulation of miR-200c in breast cancer cells regulates cell migration, cell elongation and TGF-beta-induced stress fiber formation, by impacting the re-organization of cytoskeleton that is independent of the ZEB/E-Cadherin-axis. We identified FHOD1 and PPM1F, direct regulators of the actin cytoskeleton, as novel targets of miR-200c. Remarkably, expression levels of FHOD1 and PPM1F were inversely correlated with miR-200c in breast cancer cell lines, breast cancer patient samples, as well as in 58 cancer cell lines of various origin. Furthermore, individual knockdown/overexpression of these target genes phenocopied the effects of miR-200c overexpression/inhibition on cell elongation, stress fiber formation, migration and invasion. Mechanistically, targeting of FHOD1 by miR-200c resulted in decreased expression and transcriptional activity of SRF mediated by interference with the translocation of SRF co-activator MRTF-A. This finally led to downregulation of the expression and phosphorylation of the SRF target gene MLC2 required for stress fiber formation and contractility. Thus, miR-200c impacts on metastasis by regulating several EMT-related processes, including a novel mechanism involving the direct targeting of actin-regulatory proteins.
    Type of Publication: Journal article published
    PubMed ID: 22144583
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  • 4
    Keywords: INVASION ; proliferation ; microarray ; FAMILY ; GROWTH-FACTOR RECEPTOR ; SIGNAL-TRANSDUCTION ; leukemia ; TARGETS ; TUMOR-SUPPRESSOR ; EXPRESSION PROFILES
    Abstract: The EGFR-driven cell-cycle pathway has been extensively studied due to its pivotal role in breast cancer proliferation and pathogenesis. Although several studies reported regulation of individual pathway components by microRNAs (miRNAs), little is known about how miRNAs coordinate the EGFR protein network on a global miRNA (miRNome) level. Here, we combined a large-scale miRNA screening approach with a high-throughput proteomic readout and network-based data analysis to identify which miRNAs are involved, and to uncover potential regulatory patterns. Our results indicated that the regulation of proteins by miRNAs is dominated by the nucleotide matching mechanism between seed sequences of the miRNAs and 3'-UTR of target genes. Furthermore, the novel network-analysis methodology we developed implied the existence of consistent intrinsic regulatory patterns where miRNAs simultaneously co-regulate several proteins acting in the same functional module. Finally, our approach led us to identify and validate three miRNAs (miR-124, miR-147 and miR-193a-3p) as novel tumor suppressors that co-target EGFR-driven cell-cycle network proteins and inhibit cell-cycle progression and proliferation in breast cancer.
    Type of Publication: Journal article published
    PubMed ID: 22333974
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  • 5
    Keywords: ANGIOGENESIS ; EXPRESSION ; GROWTH-FACTOR ; INVASION ; proliferation ; ACTIVATION ; UP-REGULATION ; EMBRYONIC STEM-CELLS ; bone metastasis ; MicroRNAs
    Abstract: MicroRNAs (miRNAs) as modulators of gene expression have been described to display both tumor-promoting and tumor-suppressive functions. Although their role has been studied in different tumor types, little is known about how they regulate nuclear factor kappaB (NF-kappaB) signaling in breast cancer. Here, we performed an unbiased whole genome miRNA (miRome) screen to identify novel modulators of NF-kappaB pathway in breast cancer. The screen identified 13 miRNA families whose members induced consistent effects on NF-kappaB activity. Among those, the miR-520/373 family inhibited NF-kappaB signaling through direct targeting of RELA and thus strongly reduced expression and secretion of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8. With a combination of in vitro and in vivo approaches, we propose a metastasis-suppressive role of miR-520/373 family. miR-520c and miR-373 abrogated both in vitro cell invasion and in vivo intravasation of highly invasive MDA-MB-231 cells. However, knockdown of RELA did not affect their metastatic ability. mRNA profiling of MDA-MB-231 cells on overexpression of miR-520/373 members revealed a strong downregulation of transforming growth factor-beta (TGF-beta) signaling. Mechanistically, the metastasis-suppressive role of miR-520/373 can be attributed to direct suppression of TGFBR2, as the silencing of TGFBR2 phenocopied the effects of miR-520/373 overexpression on suppression of Smad-dependent expression of the metastasis-promoting genes parathyroid hormone-related protein, plasminogen activator inhibitor-1 and angiopoietin-like 4 as well as tumor cell invasion, in vitro and in vivo. A negative correlation between miR-520c and TGFBR2 expression was observed in estrogen receptor negative (ER(-)) breast cancer patients but not in the ER positive (ER(+)) subtype. Remarkably, decreased expression of miR-520c correlated with lymph node metastasis specifically in ER(-) tumors. Taken together, our findings reveal that miR-520/373 family has a tumor-suppressive role in ER(-) breast cancer by acting as a link between the NF-kappaB and TGF-beta pathways and may thus contribute to the interplay of tumor progression, metastasis and inflammation.Oncogene advance online publication, 12 December 2011; doi:10.1038/onc.2011.571.
    Type of Publication: Journal article published
    PubMed ID: 22158050
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  • 6
    Keywords: EXPRESSION ; DISEASE ; BREAST-CANCER ; Drosophila ; HUMAN GENOME ; PREDICTION ; MICROARRAY ANALYSIS ; MESSENGER-RNAS ; MicroRNAs ; GENE-COEXPRESSION
    Abstract: Recent development of high-throughput, multiplexing technology has initiated projects that systematically investigate interactions between two types of components in biological networks, for instance transcription factors and promoter sequences, or microRNAs (miRNAs) and mRNAs. In terms of network biology, such screening approaches primarily attempt to elucidate relations between biological components of two distinct types, which can be represented as edges between nodes in a bipartite graph. However, it is often desirable not only to determine regulatory relationships between nodes of different types, but also to understand the connection patterns of nodes of the same type. Especially interesting is the co-occurrence of two nodes of the same type, i.e., the number of their common neighbours, which current high-throughput screening analysis fails to address. The co-occurrence gives the number of circumstances under which both of the biological components are influenced in the same way. Here we present SICORE, a novel network-based method to detect pairs of nodes with a statistically significant co-occurrence. We first show the stability of the proposed method on artificial data sets: when randomly adding and deleting observations we obtain reliable results even with noise exceeding the expected level in large-scale experiments. Subsequently, we illustrate the viability of the method based on the analysis of a proteomic screening data set to reveal regulatory patterns of human microRNAs targeting proteins in the EGFR-driven cell cycle signalling system. Since statistically significant co-occurrence may indicate functional synergy and the mechanisms underlying canalization, and thus hold promise in drug target identification and therapeutic development, we provide a platform-independent implementation of SICORE with a graphical user interface as a novel tool in the arsenal of high-throughput screening analysis.
    Type of Publication: Journal article published
    PubMed ID: 24039936
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