Identifizierung und differentielle Expression von Notch1-Zielgenen in murinen embryonalen Stammzellen und während der frühen mesodermalen Differenzierung

Der Notch-Signaltransduktionsweg ist von essentieller Bedeutung für die Embryonalentwicklung vielzelliger Organismen. Er induziert und reguliert zahlreiche Zelllinienentscheidungen und ist für äußerst vielfältige kontextabhängige Effekte in den unterschiedlichsten Zell- und Gewebetypen verantwortlich. Diese Effekte betreffen sowohl Differenzierung als auch Proliferation und Apoptose. Die Regulation der Signaltransduktion durch Notch ist in ihren Grundzügen bekannt; die molekularen Mechanismen, die den Zelllinienentscheidungen zugrunde liegen, oft nicht. Eine Erklärung für die Vielfältigkeit der durch Notch vermittelten Effekte ist, dass bisher nicht alle generellen oder kontextabhängigen Zielgene von Notch identifiziert werden konnten. Ausgangspunkt der vorliegenden Arbeit war die Blockierung der mesodermalen Differenzierung durch Notch1, quantifiziert durch die Repression Flk-1+ mesodermaler Vorläuferzellen nach Aktivierung von Notch1 in vitro in der murinen embryonalen Stammzelllinie EB5. Neben der genaueren Charakterisierung dieser Blockierung durch Notch1 stand die Identifizierung der Zielgene von Notch1, welche wahrscheinlich den Mechanismus dieser Zelllinienentscheidung vermitteln, im Vordergrund. Durch Microarray-Analysen in embryonalen Stammzellen sowie in daraus differenzierten mesodermalen Vorläuferzellen konnten 484 bekannte Gene identifiziert werden, deren Expression nach Aktivierung von Notch1 in zumindest einer der untersuchten Kulturbedingungen mindestens zweifach reguliert worden war. 32 dieser Gene, deren biologische Funktion zu der von Notch1 passend war, wurden zur weiteren Untersuchung ausgewählt. Zu dieser Gruppe zählten bereits bekannte Zielgene von Notch1, aber auch Gene, deren Expression mit der Aktivierung von Notch1 bisher nicht im direkten Zusammenhang stand, z. B. Myf5, Sox9 oder Pax6. Neben der Analyse der differentiellen kontextabhängigen Expression dieser Gene unter verschiedenen Kulturbedingungen und in verschiedenen Zelltypen wurde ebenfalls überprüft, ob die durch Notch1 vermittelte Regulation der Expression dieser Gene direkt war. Abschließend wurde begonnen, mittels RNA-Interferenz ein System zur Charakterisierung der biologischen Funktion der Zielgene während der Inhibition der mesodermalen Differenzierung durch Notch1 zu entwickeln. Durch die Identifizierung der Zielgene von Notch1 in embryonalen Stammzellen und mesodermalen Vorläuferzellen wurde ein erster wichtiger Schritt getan, um die molekularen Mechanismen der kontextabhängigen Effekte von Notch1 während der frühen Embryonalentwicklung der Maus aufdecken zu können.Notch signalling is essential for the embryonic development of multicellular organisms. Depending on the cellular context, Notch induces and regulates a variety of cell lineage decisions and affects differentiation, proliferation and apoptosis in different types of cells or tissues. Up to now basic aspects of the regulation of Notch signalling are known, however in most cases little is known about the molecular mechanisms which are the basis of the cell lineage decisions. One explanation for the diversity of Notch regulated effects is maybe the presence of still not identified general or context dependent target genes. This study was based on the repression of mesodermal differentiation caused by Notch1. This effect was quantified by measuring the amount of Flk-1+ mesodermal progenitor cells generated in vitro in the embryonic stem cell line EB5 after activation of Notch1. Beside further characterization of this result, the main goal of this study was to identify the target genes of Notch1 which probably cause this cell lineage decision. 484 known genes - whose expression was at least two fold regulated after activation of Notch1 in at least one of the analysed culture conditions - were identified by microarray analysis in embryonic stem cells as well as in mesodermal progenitor cells. 32 genes out of this group were chosen for ongoing analysis. The known functions of these genes were comparable to the known functions of Notch1 during embryonic development. Belonging to this group of genes were known target genes of Notch1 as well as genes with so far unknown direct relation between their expression and the activation of Notch1, for example Myf5, Sox9 or Pax6. The differential context dependent expression of the identified Notch1 target genes was analysed in different culture conditions as well as in different cell types. In addition, it was investigated if regulation of gene expression after activation of Notch1 was induced directly or indirectly. Final part of this work was starting the development of an experimental system based on RNA interference to characterize the biological function of the identified target genes during inhibition of mesodermal differentiation caused by activation of Notch1. A first important step was taken to uncover the molecular mechanisms which manage the context dependent effects of Notch1. The identification of Notch1 target genes in embryonic stem cells and mesodermal progenitor cells and the analysis of their differential expression laid the foundation for future investigations

Molecular profiling of single circulating tumor cells with diagnostic intention

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of >90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance

Molecular profiling of single circulating tumor cells with diagnostic intention

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of >90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance

The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper

BACKGROUND: The heterogeneity of metastatic breast cancer (MBC) necessitates novel biomarkers allowing stratification of patients for treatment selection and drug development. We propose to use the prognostic utility of circulating tumor cells (CTCs) for stratification of patients with stage IV disease. METHODS: In a retrospective, pooled analysis of individual patient data from 18 cohorts, including 2436 MBC patients, a CTC threshold of 5 cells per 7.5\u2009ml was used for stratification based on molecular subtypes, disease location, and prior treatments. Patients with 65 5 CTCs were classified as Stage IVaggressive, those with < 5 CTCs as Stage IVindolent. Survival was analyzed using Kaplan-Meier curves and the log rank test. RESULTS: For all patients, Stage IVindolent patients had longer median overall survival than those with Stage IVaggressive (36.3 months vs. 16.0 months, P\u2009<\u20090.0001) and similarly for de novo MBC patients (41.4 months Stage IVindolent vs. 18.7 months Stage IVaggressive, p\u2009<\u20090.0001). Moreover, patients with Stage IVindolent disease had significantly longer overall survival across all disease subtypes compared to the aggressive cohort: hormone receptor-positive (44 months vs. 17.3 months, P\u2009<\u20090.0001), HER2-positive (36.7 months vs. 20.4 months, P\u2009<\u20090.0001), and triple negative (23.8 months vs. 9.0 months, P\u2009<\u20090.0001). Similar results were obtained regardless of prior treatment or disease location. CONCLUSIONS: We confirm the identification of two subgroups of MBC, Stage IVindolent and Stage IVaggressive, independent of clinical and molecular variables. Thus, CTC count should be considered an important tool for staging of advanced disease and for disease stratification in prospective clinical trials

Articles Clinical validity of circulating tumour cells in patients with metastatic breast cancer: a pooled analysis of individual patient data

Summary Background We aimed to assess the clinical validity of circulating tumour cell (CTC) quantifi cation for prognostication of patients with metastatic breast cancer by undertaking a pooled analysis of individual patient data

Expression of Stem Cell and Epithelial-Mesenchymal Transition Markers in Circulating Tumor Cells of Breast Cancer Patients

Evaluation and characterization of circulating tumor cells (CTCs) have become a major focus of translational cancer research. Presence of CTCs predicts worse clinical outcome in early and metastatic breast cancer. Whether all cells from the primary tumor have potential to disseminate and form subsequent metastasis remains unclear. As part of the metastatic cascade, tumor cells lose their cell-to-cell adhesion and undergo epithelial-mesenchymal transition (EMT) in order to enter blood circulation. During EMT epithelial antigens are downregulated; thus, such tumor cells might elude classical epithelial marker-based detection. Several researchers postulated that some CTCs express stem cell-like phenotype; this might lead to chemoresistance and enhanced metastatic potential of such cells. In the present review, we discuss current data on EMT and stem cell markers in CTCs of breast cancer and their clinical significance

Activated Notch1 target genes during embryonic cell differentiation depend on the cellular context and include lineage determinants and inhibitors

BACKGROUND: Notch receptor signaling controls developmental cell fates in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the target genes that are directly activated by Notch in the respective tissues. METHODOLOGY/PRINCIPAL FINDINGS: To analyze how Notch signaling mediates its context dependent function(s), we utilized a Tamoxifen-inducible system to activate Notch1 in murine embryonic stem cells at different stages of mesodermal differentiation and performed global transcriptional analyses. We find that the majority of genes regulated by Notch1 are unique for the cell type and vary widely dependent on other signals. We further show that Notch1 signaling regulates expression of genes playing key roles in cell differentiation, cell cycle control and apoptosis in a context dependent manner. In addition to the known Notch1 targets of the Hes and Hey families of transcriptional repressors, Notch1 activates the expression of regulatory transcription factors such as Sox9, Pax6, Runx1, Myf5 and Id proteins that are critically involved in lineage decisions in the absence of protein synthesis. CONCLUSION/SIGNIFICANCE: We suggest that Notch signaling determines lineage decisions and expansion of stem cells by directly activating both key lineage specific transcription factors and their repressors (Id and Hes/Hey proteins) and propose a model by which Notch signaling regulates cell fate commitment and self renewal in dependence of the intrinsic and extrinsic cellular context