Astrid Becker

Lysine-specific demethylase 1 (LSD1) is highly expressed in ER-negative breast cancers and a biomarker predicting aggressive biology

Breast carcinogenesis is a multistep process involving both genetic and epigenetic changes. Since epigenetic changes like histone modifications are potentially reversible processes, much effort has been directed toward understanding this mechanism with the goal of finding novel therapies as well as more refined diagnostic and prognostic tools in breast cancer. Lysine-specific demethylase 1 (LSD1) plays a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 of histone H3 and lysine 9 of histone H3. LSD1 is essential for mammalian development and involved in many biological processes. Considering recent evidence that LSD1 is involved in carcinogenesis, we investigated the role of LSD1 in breast cancer. Therefore, we developed an enzyme-linked immunosorbent assay to determine LSD1 protein levels in tissue specimens of breast cancer and measured very high LSD1 levels in estrogen receptor (ER)-negative tumors. Pharmacological LSD1 inhibition resulted in growth inhibition of breast cancer cells. Knockdown of LSD1 using small interfering RNA approach induced regulation of several proliferation-associated genes like p21, ERBB2 and CCNA2. Additionally, we found that LSD1 is recruited to the promoters of these genes. In summary, our data indicate that LSD1 may provide a predictive marker for aggressive biology and a novel attractive therapeutic target for treatment of ER-negative breast cancers.

SNAI1-Mediated Epithelial-Mesenchymal Transition Confers Chemoresistance and Cellular Plasticity by Regulating Genes Involved in Cell Death and Stem Cell Maintenance

Tumor cells at the tumor margin lose epithelial properties and acquire features of mesenchymal cells, a process called epithelial-to-mesenchymal transition (EMT). Recently, features of EMT were shown to be linked to cells with tumor-founding capability, so-called cancer stem cells (CSCs). Inducers of the EMT include several transcription factors, such as Snail (SNAI1) and Slug (SNAI2), as well as the secreted transforming growth factor (TGFß). In the present study, we found that EMT induction in MCF10A cells by stably expressing SNAI1 contributed to drug resistance and acquisition of stem/progenitor-like character as shown by increased cell population for surface marker CD44+/CD24− and mammosphere forming capacity. Using a microarray approach, we demonstrate that SNAI1 overexpression results in a dramatic change in signaling pathways involved in the regulation of cell death and stem cell maintenance. We showed that NF-κB/MAPK signaling pathways are highly activated in MCF10A-SNAI1 cells by IL1ß stimulation, leading to the robust induction in IL6 and IL8. Furthermore, MCF10A-SNAI1 cells showed enhanced TCF/ß-catenin activity responding to the exogenous Wnt3a treatment. However, EMT-induced stem/progenitor cell activation process is tightly regulated in non-transformed MCF10A cells, as WNT5A and TGFB2 are strongly upregulated in MCF10A-SNAI1 cells antagonizing canonical Wnt pathway. In summary, our data provide new molecular findings how EMT contributes to the enhanced chemoresistance and the acquisition of stem/progenitor-like character by regulating signaling pathways.

Derivation and Maintenance of Murine Trophoblast Stem Cells under Defined Conditions

Summary Trophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage.

Lineage Conversion of Murine Extraembryonic Trophoblast Stem Cells to Pluripotent Stem Cells

ABSTRACT In mammals, the first cell fate decision is initialized by cell polarization at the 8- to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals.

Atheroprotection via cannabinoid receptor-2 is mediated by circulating and vascular cells in vivo

Low-dose oral tetrahydrocannabinol (THC) reduces progression of atherosclerosis in mice. THC activates central cannabinoid-1 receptors (CB1) with subsequent psychoactive effects as well as peripheral cannabinoid-2 receptors (CB2). In order to dissect the underlying mechanisms, we performed experiments under selective CB2 stimulation as well as after genetic disruption of the CB2 receptor. Atherosclerosis prone apolipoprotein E-deficient mice were crossed with cannabinoid receptor-2 deficient mice to obtain ApoE -/- CB2 -/- double knockout mice. After 8weeks of a high-cholesterol diet, immunohistochemical stainings of the aortic root revealed that vascular leukocyte infiltration in atherosclerotic plaques was accelerated in ApoE -/- CB2 -/- mice compared with ApoE -/- mice. This was accompanied by increased release of reactive oxygen species as measured using L012-enhanced chemiluminescence, and by decreased endothelial function as assessed in isolated aortic rings in organ chamber experiments. ApoE -/- mice treated with the selective CB2 agonist JWH 133 during a high-cholesterol diet showed decreased atherosclerotic lesion formation, improved endothelial function and reduced levels of reactive oxygen species. To assess whether CB2 expression in circulating cells influences atherosclerosis, irradiated ApoE -/- mice were repopulated with bone marrow-derived cells from ApoE -/- and ApoE -/- CB2 -/- mice and were fed a high-cholesterol diet for 8weeks. CB2 deficiency in bone marrow-derived cells increased leukocyte infiltration into the vessel wall, but had no impact on plaque formation. Cell culture experiments revealed that CB2 activation diminishes ROS generation in vascular cells. Selective CB2 receptor stimulation modulates atherogenesis via impact on both circulating proinflammatory and vascular cells.

The H3K4me3 histone demethylase Fbxl10 is a regulator of chemokine expression, cellular morphology and the metabolome of fibroblasts

Background: Fbxl10 is a member of the JHDM family. Results: We show that Fbxl10 functions as an H3K4me3 demethylase. The PHD domain recognizes H3K4me3 and H3K36me2 and shows E3 ligase activity. Using a microarray approach we identified target genes for Fbxl10. Conclusion: Our data reveal a regulatory role of Fbxl10 in cell morphology, chemokine expression, and the metabolic control. Significance: Fbxl10 plays a novel role of in the regulation of target genes. Fbxl10 (Jhdm1b/Kdm2b) is a conserved and ubiquitously expressed member of the JHDM (JmjC domain-containing histone demethylase) family. Fbxl10 was implicated in the demethylation of H3K4me3 or H3K36me2 thereby removing active chromatin marks and inhibiting gene transcription. Apart from the JmjC domain, Fbxl10 consists of a CxxC domain, a PHD domain, and an Fbox domain. By purifying the JmjC and the PHD domain of Fbxl10 and using different approaches we were able to characterize the properties of these domains in vitro. Our results suggest that Fbxl10 is rather a H3K4me3 than a H3K36me2 histone demethylase. The PHD domain exerts a dual function in binding H3K4me3 and H3K36me2 and exhibiting E3 ubiquitin ligase activity. We generated mouse embryonic fibroblasts stably overexpressing Fbxl10. These cells reveal an increase in cell size but no changes in proliferation, mitosis, or apoptosis. Using a microarray approach we were able to identify potentially new target genes for Fbxl10 including chemokines, the noncoding RNA Xist, and proteins involved in metabolic processes. Additionally, we found that Fbxl10 is recruited to the promoters of Ccl7, Xist, Crabp2, and RipK3. Promoter occupancy by Fbxl10 was accompanied by reduced levels of H3K4me3 but unchanged levels of H3K36me2. Furthermore, knockdown of Fbxl10 using small interfering RNA approaches showed inverse regulation of Fbxl10 target genes. In summary, our data reveal a regulatory role of Fbxl10 in cell morphology, chemokine expression, and the metabolic control of fibroblasts.

Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tfap2c in embryonic stem cells and primordial germ cell-like cells. We show that loss of Tfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation of maturation markers and induction of markers indicative for somatic differentiation, cell cycle, epigenetic remodeling and pluripotency. Chromatin-immunoprecipitation analyses demonstrated binding of TFAP2C to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of TFAP2C in primordial germ cells. Since Tfap2c deficient primordial germ cell-like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tfap2c develop with high incidence germ cell cancer resembling human pediatric germ cell tumors. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate that mice with a heterozygous deletion of the TFAP2C target gene Nanos3 are also prone to develop teratomas. These data highlight TFAP2C as a critical and dose-sensitive regulator of germ cell fate.

Inhibition of endocannabinoid-degrading enzyme fatty acid amide hydrolase increases atherosclerotic plaque vulnerability in mice

The role of endocannabinoids such as anandamide during atherogenesis remains largely unknown. Fatty acid amide hydrolase (FAAH) represents the key enzyme in anandamide degradation, and its inhibition is associated with subsequent higher levels of anandamide. Here, we tested whether selective inhibition of FAAH influences the progression of atherosclerosis in mice. Selective inhibition of FAAH using URB597 resulted in significantly increased plasma levels of anandamide compared to control, as assessed by mass spectrometry experiments in mice. Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high-fat, cholesterol-rich diet to induce atherosclerotic conditions. Simultaneously, mice received either the pharmacological FAAH inhibitor URB597 1mg/kg body weight (n=28) or vehicle (n=25) via intraperitoneal injection three times a week. After eight weeks, mice were sacrificed, and experiments were performed. Vascular superoxide generation did not differ between both groups, as measured by L012 assay. To determine whether selective inhibition of FAAH affects atherosclerotic plaque inflammation, immunohistochemical staining of the aortic root was performed. Atherosclerotic plaque formation, vascular macrophage accumulation, as well as vascular T cell infiltration did not differ between both groups. Interestingly, neutrophil cell accumulation was significantly increased in mice receiving URB597 compared to control. Vascular collagen structures in atherosclerotic plaques were significantly diminished in mice treated with URB597 compared to control, as assessed by picro-sirius-red staining. This was accompanied by an increased aortic expression of matrix metalloproteinase-9, as determined by quantitative RT-PCR and western blot analysis. Inhibition of fatty acid amide hydrolase does not influence plaque size but increases plaque vulnerability in mice.

Effect of chronic corticosterone application on depression‐like behavior in C57BL/6N and C57BL/6J mice

Many studies using genetic mouse models are performed with animals on either one of the two closely related genetic backgrounds, C57BL/6J or C57BL/6N. These strains differ only in a few genetic loci, but have some phenotypic differences that also affect behavior. In order to determine the effects of chronic stress hormone exposure, which is relevant for the pathogenesis of psychiatric disorders, we investigated here the behavioral manifestations of long‐term increase in corticosterone levels. Thus, male mice from both sub‐strains were subcutaneously implanted with corticosterone (20 mg) or placebo pellets that released the hormone for a period of 21 days and resulted in significantly elevated plasma corticosterone levels. Corticosterone significantly increased food intake in B6N, but not in B6J mice. At various time points after pellet implantation, we performed tests relevant to activity and emotional behaviors. B6J mice displayed a generally higher activity in the home cage and the open field. Corticosterone decreased the activity. In B6N mice, corticosterone also decreased sucrose preference, worsened the coat state and increased forced swim immobility, while it had no effect in the B6J strain. Altogether, these results indicate that B6N mice are more sensitive to some of the effects of chronic corticosterone treatment than B6J mice.

Exposure of mice to long-light: a new animal model to study depression.

Mood disorders are highly prevalent and often difficult to treat. One of the most important obstacles in research on depression is the limited availability of reliable and valid animal models. Here we demonstrate that the exposure of mice to artificial daylight for 22 h per day produces a spectrum of behavioral and endocrine symptoms reminiscent to those seen in animal models of depression. Oral administration of the antidepressant imipramine has strong impact on these symptoms. Our results indicate that long-light exposure of mice represents a simple new method to study depression.