Joseph Andrews

beta-arrestins regulate interleukin-8-induced CXCR1 internalization.

The functional role of neutrophils during acute inflammatory responses is regulated by two high affinity interleukin-8 receptors (CXCR1 and CXCR2) that are rapidly desensitized and internalized upon binding their cognate chemokine ligands. The efficient re-expression of CXCR1 on the surface of neutrophils following agonist-induced internalization suggests that CXCR1 surface receptor turnover may involve regulatory pathways and intracellular factors similar to those regulating β2-adrenergic receptor internalization and re-expression. To examine the internalization pathway utilized by ligand-activated CXCR1, a CXCR1-GFP construct was transiently expressed in two different cell lines, HEK 293 and RBL-2H3 cells. While interleukin-8 stimulation promoted CXCR1 sequestration in RBL-2H3 cells, receptor internalization in HEK 293 cells required co-expression of G protein-coupled receptor kinase 2 and β-arrestin proteins. The importance of β-arrestins in CXCR1 internalization was confirmed by the ability of a dominant negative β-arrestin 1-V53D mutant to block internalization of CXCR1 in RBL-2H3 cells. A role for dynamin was also demonstrated by the lack of CXCR1 internalization in dynamin I-K44A dominant negative mutant-transfected RBL-2H3 cells. Agonist-promoted co-localization of transferrin and CXCR1-GFP in endosomes of RBL-2H3 cells confirmed that receptor internalization occurs via clathrin-coated vesicles. Our data provides a direct link between agonist-induced internalization of CXCR1 and a requirement for G protein-coupled receptor kinase 2, β-arrestins, and dynamin during this process.

Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays

IntroductionBreast cancer metastasis is a complex, multi-step biological process. Genetic mutations along with epigenetic alterations in the form of DNA methylation patterns and histone modifications contribute to metastasis-related gene expression changes and genomic instability. So far, these epigenetic contributions to breast cancer metastasis have not been well characterized, and there is only a limited understanding of the functional mechanisms affected by such epigenetic alterations. Furthermore, no genome-wide assessments have been undertaken to identify altered DNA methylation patterns in the context of metastasis and their effects on specific functional pathways or gene networks.MethodsWe have used a human gene promoter tiling microarray platform to analyze a cell line model of metastasis to lymph nodes composed of a poorly metastatic MDA-MB-468GFP human breast adenocarcinoma cell line and its highly metastatic variant (468LN). Gene networks and pathways associated with metastasis were identified, and target genes associated with epithelial–mesenchymal transition were validated with respect to DNA methylation effects on gene expression.ResultsWe integrated data from the tiling microarrays with targets identified by Ingenuity Pathways Analysis software and observed epigenetic variations in genes implicated in epithelial–mesenchymal transition and with tumor cell migration. We identified widespread genomic hypermethylation and hypomethylation events in these cells and we confirmed functional associations between methylation status and expression of the CDH1, CST6, EGFR, SNAI2 and ZEB2 genes by quantitative real-time PCR. Our data also suggest that the complex genomic reorganization present in cancer cells may be superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes.ConclusionThis is the first whole-genome approach to identify genome-wide and gene-specific epigenetic alterations, and the functional consequences of these changes, in the context of breast cancer metastasis to lymph nodes. This approach allows the development of epigenetic signatures of metastasis to be used concurrently with genomic signatures to improve mapping of the evolving molecular landscape of metastasis and to permit translational approaches to target epigenetically regulated molecular pathways related to metastatic progression.

Genome-wide H3K9 Histone Acetylation Profiles Are Altered in Benzopyrene-treated MCF7 Breast Cancer Cells

Current toxicogenomic approaches generate transcriptional profiles that can identify functional gene expression signatures of environmental toxicants. However, the intricate processes governing transcription are overlaid with a complex set of molecular instructions involving epigenetic modifications. These commands regulate both gene expression and chromatin organization through coordinated sets of histone modifications and heritable DNA methylation patterns. Although the effects of specific environmental toxicants on gene expression are the subject of much study, the epigenetic effects of such compounds are poorly understood. Here we have used human promoter tiling arrays along with chromatin immunoprecipitation to identify changes in histone acetylation profiles because of chemical exposure. Chromatin from cells exposed to the polyaromatic hydrocarbon benzo(a)pyrene was immunoprecipitated with antibodies against acetylated histones. Affymetrix promoter tiling microarrays were probed to generate epigenomic profiles of hypo- and hyperacetylated chromatin localized to gene promoter regions. Statistical analyses, data mining, and expression studies revealed that treated cells possessed differentially acetylated gene promoter regions and gene-specific expression changes. This chromatin immunoprecipitation-on-chip approach permits genome-wide profiling of histone acetylation patterns that can identify chromatin-related signatures of environmental toxicants and potentially determine the molecular pathways these changes target. This approach also has potential applications for profiling histone modifications and DNA methylation changes during embryonic development, in cancer biology, and in the development and assessment of cancer therapeutics.

Multi-Platform Whole-Genome Microarray Analyses Refine the Epigenetic Signature of Breast Cancer Metastasis with Gene Expression and Copy Number

Background We have previously identified genome-wide DNA methylation changes in a cell line model of breast cancer metastasis. These complex epigenetic changes that we observed, along with concurrent karyotype analyses, have led us to hypothesize that complex genomic alterations in cancer cells (deletions, translocations and ploidy) are superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes observed in breast cancer metastasis. Methodology/Principal Findings We undertook simultaneous high-resolution, whole-genome analyses of MDA-MB-468GFP and MDA-MB-468GFP-LN human breast cancer cell lines (an isogenic, paired lymphatic metastasis cell line model) using Affymetrix gene expression (U133), promoter (1.0R), and SNP/CNV (SNP 6.0) microarray platforms to correlate data from gene expression, epigenetic (DNA methylation), and combination copy number variant/single nucleotide polymorphism microarrays. Using Partek Software and Ingenuity Pathway Analysis we integrated datasets from these three platforms and detected multiple hypomethylation and hypermethylation events. Many of these epigenetic alterations correlated with gene expression changes. In addition, gene dosage events correlated with the karyotypic differences observed between the cell lines and were reflected in specific promoter methylation patterns. Gene subsets were identified that correlated hyper (and hypo) methylation with the loss (or gain) of gene expression and in parallel, with gene dosage losses and gains, respectively. Individual gene targets from these subsets were also validated for their methylation, expression and copy number status, and susceptible gene pathways were identified that may indicate how selective advantage drives the processes of tumourigenesis and metastasis. Conclusions/Significance Our approach allows more precisely profiling of functionally relevant epigenetic signatures that are associated with cancer progression and metastasis.

Gene signatures of breast cancer progression and metastasis.

Breast cancer is a heterogeneous disease. Patient outcome varies significantly, depending on prognostic features of patients and their tumors, including patient age, menopausal status, tumor size and histology, nodal status, and so on. Response to treatment also depends on a series of predictive factors, such as hormone receptor and HER2 status. Current treatment guidelines use these features to determine treatment. However, these guidelines are imperfect, and do not always predict response to treatment or survival. Evolving technologies are permitting increasingly large amounts of molecular data to be obtained from tumors, which may enable more personalized treatment decisions to be made. The challenge is to learn what information leads to improved prognostic accuracy and treatment outcome for individual patients.

Human 21T breast epithelial cell lines mimic breast cancer progression in vivo and in vitro and show stage-specific gene expression patterns

Early breast cancer progression involves advancement through specific morphological stages including atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and invasive mammary carcinoma (IMC), although not necessarily always in a linear fashion. Observational studies have examined genetic, epigenetic and gene expression differences in breast tissues representing these stages of progression, but model systems which would allow for experimental testing of specific factors influencing transition through these stages are scarce. The 21T series cell lines, all originally derived from the same patient with metastatic breast cancer, have been proposed to represent a mammary tumor progression series. We report here that three of the 21T cell lines indeed mimic specific stages of human breast cancer progression (21PT-derived cells, ADH; 21NT-derived cells, DCIS; 21MT-1 cells, IMC) when grown in the mammary fat pad of nude mice, albeit after a year. To develop a more rapid, readily manipulatable in vitro assay for examining the biological differences between these cell lines, we have used a 3D Matrigel system. When the three cell lines were grown in 3D Matrigel, they showed characteristic morphologies, in which quantifiable aspects of stage-specific in vivo behaviors (ie, differences in acinar structure formation, cell polarization, colony morphology, cell proliferation, cell invasion) were recapitulated in a reproducible fashion. Gene expression profiling revealed a characteristic pattern for each of the three cell lines. Interestingly, Wnt pathway alterations are particularly predominant in the early transition from 21PTci (ADH) to 21NTci (DCIS), whereas alterations in expression of genes associated with control of cell motility and invasion phenomena are more prominent in the later transition of 21NTci (DCIS) to 21MT-1 (IMC). This system thus reveals potential therapeutic targets and will provide a means of testing the influences of identified genes on transitions between these stages of pre-malignant to malignant growth.

Differentiation and injury-repair signals modulate the interaction of E2F and pRB proteins with novel target genes in keratinocytes.

E2F transcription factors are central to epidermal morphogenesis and regeneration afterinjury. The precise nature of E2F target genes involved in epidermal formation and repair has yetto be determined. Identification of these genes is essential to understand how E2F proteinsregulate fundamental aspects of epidermal homeostasis and transformation. We have conducted agenome-wide screen using CpG island microarray analysis to identify novel promoters bound byE2F3 and E2F5 in human keratinocytes. We further characterized several of these genes, anddetermined that multiple E2F and retinoblastoma (pRb) family proteins associate with them inexponentially proliferating cells. We also assessed the effect on E2F and pRb binding to thosegenes in response to differentiation induced by bone morphogenetic protein-6 (BMP-6), or toactivation of repair mechanisms induced by transforming growth factor-β (TGF-β). Thesestudies demonstrate promoter- and cytokine-specific changes in binding profiles of E2F and/orpRb family proteins. For example, E2F1, 3, 4 and p107 were recruited to the N-myc promoter incells treated with BMP-6, whereas E2F1, 3, 4, 5, p107 and p130 were bound to this promoter inthe presence of TGF-β. Functionally, these different interactions resulted in transcriptionalrepression by BMP-6 and TGF-β of the N-myc gene, via mechanisms that involved E2F bindingto the promoter and association with pRb-family proteins. Thus, multiple combinations of E2Fand pRb family proteins may associate with and transcriptionally regulate a given target promoterin response to differentiation and injury-repair stimuli in epidermal keratinocytes.

The impact of blunted beta-adrenergic responsiveness on growth regulatory pathways in hypertension

The effects of vasodilator hormones acting through receptors linked to adenylyl cyclase are impaired in the hypertensive state. This has been ascribed to impaired receptor-G protein coupling. However, these receptors also act via effectors not linked to adenylyl cyclase activation. These “alternate” mechanisms may be especially important in growth regulation and might be unaffected (or enhanced) with G protein-coupled receptor-G protein uncoupling. Therefore, we assessed the effects of β-adrenergic activation on 1) regulation of phosphatidylinositol 3-kinase (PI3 kinase) and extracellular signal-regulated kinase (ERK) activation—two tyrosine kinase-dependent enzymes linked to cell growth—and 2) microarray analysis in vascular smooth muscle cells from spontaneously hypertensive rats (SHR). Isoproterenol-stimulated phosphorylation of ERK1/2 was impaired in SHR. The effect of forskolin was unaltered. In contrast, both vasopressin and angiotensin 2-mediated stimulation of ERK activation was enhanced in SHR. In addition, β-adrenergic-mediated inhibition of PI3 kinase activity was attenuated in SHR (whereas the effect of forskolin remained intact). In microarray studies, the effect of isoproterenol to regulate transcription was significantly impaired in SHR (as was the effect of forskolin). Together, these data support the hypothesis that the blunted vasodilator effects of hormones linked to adenylyl cyclase activation are an index of a more generalized impairment in modulating growth regulatory pathways. Furthermore, this study supports the hypothesis that the blunting of β-adrenergic responses relating to increased G protein-coupled receptor kinase 2 expression reflects a “generalized uncoupling” of β-adrenergic-mediated responses and do not support the concept of “enhanced coupling” of “alternate” pathways of β-adrenergic growth regulatory pathways in the hypertensive state.

Genome-Wide Analysis in Human Colorectal Cancer Cells Reveals Ischemia-Mediated Expression of Motility Genes via DNA Hypomethylation

DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumours, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel data suggests that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes.