Invading Basement Membrane Matrix Is Sufficient for MDA-MB-231 Breast Cancer Cells to Develop a Stable In Vivo Metastatic Phenotype

1 - ArticleIntroduction: The poor efficacy of various anti-cancer treatments against metastatic cells has focused attention on the role of tumor microenvironment in cancer progression. To understand the contribution of the extracellular matrix (ECM) environment to this phenomenon, we isolated ECM surrogate invading cell populations from MDA-MB-231 breast cancer cells and studied their genotype and malignant phenotype. Methods: We isolated invasive subpopulations (INV) from non invasive populations (REF) using a 2D-Matrigel assay, a surrogate of basal membrane passage. INV and REF populations were investigated by microarray assay and for their capacities to adhere, invade and transmigrate in vitro, and to form metastases in nude mice. Results: REF and INV subpopulations were stable in culture and present different transcriptome profiles. INV cells were characterized by reduced expression of cell adhesion and cell-cell junction genes (44% of down regulated genes) and by a gain in expression of anti-apoptotic and pro-angiogenic gene sets. In line with this observation, in vitro INV cells showed reduced adhesion and increased motility through endothelial monolayers and fibronectin. When injected into the circulation, INV cells induced metastases formation, and reduced injected mice survival by up to 80% as compared to REF cells. In nude mice, INV xenografts grew rapidly inducing vessel formation and displaying resistance to apoptosis. Conclusion: Our findings reveal that the in vitro ECM microenvironment per se was sufficient to select for tumor cells with a stable metastatic phenotype in vivo characterized by loss of adhesion molecules expression and induction of proangiogenic and survival factors

Regulation of CDKN2B expression by interaction of Arnt with Miz-1 - a basis for functional integration between the HIF and Myc gene regulatory pathways

BACKGROUND: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. METHODS: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student’s t-test and ANOVA. RESULTS: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. CONCLUSIONS: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis

GR protein immunofluorescence is shown as mean Gray values ±SEM for female and male mice in the different genotype and treatment groups.

<p>Gray values monitored in each brain region (DG, CA1 and CA3) from unstressed (No) and stressed (0h, 30min and 2h recovery) mice were compared and significance determined by One-way ANOVA with Tukey’s adjustment for multiple comparisons. Statistical analyses were performed separately for female and male groups.</p

Epac1 and Epac2 mRNA levels are increased in wild type female mice following restraint stress.

<p>Female (<b>A</b>) and male (<b>B</b>) wt mice were kept at standard housing conditions (-) or exposed to 30min of restraint stress (+). The hippocampus was dissected out after the mice had been culled immediately after the stressor (0h), or after recovery from the stress for 30min or 2h. qPCR analyses were performed to determine the mRNA levels of Epac1 (black bars) and Epac2 (white bars). The qPCR values were normalized to the expression of the housekeeping genes Sdha and Ppib, and are shown as average of relative fold change ±SEM of three independent experiments performed in triplicates. Epac1 and Epac2 expression, and males and females, were analyzed separately. One-way ANOVA with Tukey’s adjustment for multiple comparisons was used for statistical analysis. *p≤0.05, **p≤0.01, ***p≤0.001. n = 7–11 mice per group. F-statistics (F(DFn, DFd)) Female Epac1: F(3, 40) = 4.217, p = 0.0111, Female Epac2: F(3, 34) = 7.649, p = 0.0005, Male Epac1: F(3, 29) = 3.286, p = 0.0347 and Male Epac2: F(3, 30) = 0.05371, p = 0.9833.</p

GR translocation is delayed in female Epac1/2^-/- mice.

<p>Paraffin-embedded brain sections were prepared from female (A-C, G) and male (D-F) wt, Epac1^-/-, Epac2^-/- and Epac1/2^-/- mice kept at standard housing conditions (-) or exposed to 30min restraint stress (+). The hippocampus was dissected out after the mice had been culled immediately after the stressor (0h), or after recovery from the stress for 30min or 2h. Paraffin sections were subjected to IHC using a GR-specific antibody, and GR immunofluorescence was quantified using Image J software. Data are presented as average ±SEM gray values recorded from the DG, CA1, and CA3 regions as indicated. Two-way ANOVA with Dunnett’s adjustment for multiple comparisons was used to determine differences between genotypes in each treatment group in each region. *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001 wt mice compared to Epac1^-/-, Epac2^-/- and Epac1/2^-/- mice (same timepoint). n = 3 mice per group, and for each mouse, 3 sections of the hippocampus where quantified for GR staining. F-statistics (F(DFn, DFd)) Female DG; Interaction: F(9, 128) = 35.68, p<0.0001, Female CA1; Interaction: F(9, 128) = 68.28, p<0.0001, Female CA3; Interaction: F(9, 128) = 26.84, p<0.0001, Male DG; Interaction: F(9, 131) = 2.861, p = 0.0041, Male CA1; Interaction: F(9, 131) = 4.433, p<0.0001, Male CA3; Interaction: F(9, 131) = 2.754, p = 0.0056. G) Representative GR IHC staining of the CA1 region of wt and Epac1/2^-/- female mice. Paraffin-embedded coronal brain sections (15μm) were stained with a GR-specific antibody and visualized under a 60X objective of the Nikon Te 2000-e microscope with a TRITC fluorescent light filter.</p

Serum corticosterone levels are disturbed immediately after restraint stress in the absence of Epac.

<p>Female <b>(A)</b> and male <b>(B)</b> wt, Epac1^-/-, Epac2^-/- and Epac1/2^-/- mice were kept at standard housing conditions (-) or exposed to 30min restraint stress (+). Mice exposed to stress were culled either immediately after the stressor (0h), or after recovery from the stress for 30min or 2h, and trunk blood collected. Serum corticosterone levels were determined by ELISA. The corticosterone levels are shown as average ±SD. Two-way ANOVA with Tukey’s adjustment for multiple comparisons was used for statistical analysis. *p≤0.05,**p≤0.01, ***p<0.001 and ****p<0.0001. n = 7–9 mice per group. F-statistics (F(DFn, DFd)) for the female group: Interaction F(9,102) = 3.713, p = 0.0005 and male group: Interaction F(9,108) = 1.759, p = 0.0846.</p

Deletion of exchange proteins directly activated by cAMP (Epac) causes defects in hippocampal signaling in female mice

<div><p>Previous studies demonstrate essential roles for the exchange proteins directly activated by cAMP 1 and 2 (Epac1 and Epac2; here collectively referred to as Epac) in the brain. In the hippocampus, Epac contributes to the control of neuronal growth and differentiation and has been implicated in memory and learning as well as in anxiety and depression. In the present study we address the hypothesis that Epac affects hippocampal cellular responses to acute restraint stress. Stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis, and glucocorticoid receptor (GR) signaling is essential for proper feedback regulation of the stress response, both in the brain and along the HPA axis. In the hippocampus, GR expression is regulated by cAMP and the brain enriched micro RNA miR-124. Epac has been associated with miR-124 expression in hippocampal neurons, but not in regulation of GR. We report that hippocampal expression of Epac1 and Epac2 increased in response to acute stress in female wild type mice. In female mice genetically deleted for Epac, nuclear translocation of GR in response to restraint stress was significantly delayed, and moreover, miR-124 expression was decreased in these mice. Male mice lacking Epac also showed abnormalities in miR-124 expression, but the phenotype was less profound than in females. Serum corticosterone levels were slightly altered immediately after stress in both male and female mice deleted for Epac. The presented data indicate that Epac1 and Epac2 are involved in controlling cellular responses to acute stress in the mouse hippocampus and provide novel insights into the underlying transcriptional and signaling networks. Interestingly, we observe sex specific differences when Epac is deleted. As the incidence and prevalence of stress-related diseases are higher in women than in men, the Epac knockout models might serve as genetic tools to further elucidate the cellular mechanisms underlying differences between male and female with regard to regulation of stress.</p></div