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

Introduction Breast 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. Methods We 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. Results We 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. Conclusion This 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

TMEM43 Mutation p.S358L Alters Intercalated Disc Protein Expression and Reduces Conduction Velocity in Arrhythmogenic Right Ventricular Cardiomyopathy

<div><p>Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.</p></div

Expression and distribution of TMEM43 in non-transfected HL-1 cells and cells transfected with GFP-tagged wild-type or p.S358L TMEM43.

<p><b>A</b>. Western blot analysis demonstrating expression of endogenous and exogenous (transfected) TMEM43, with GAPDH as loading control. Rabbit polyclonal TMEM43 antibody specifically detected GFP-TMEM43 and native TMEM43, indicated by the presence of bands at 70 kDa and 43 kDa respectively, in transfected cells and only 43 kDa native TMEM43 in non-transfected HL-1 cells (control). The 70 kDa band indicates the GFP-tagged wild-type (TMEM43-WT) or mutant (TMEM43-S358L) TMEM43 <b>B</b>. Immunofluoresence staining with TMEM43 antibody showed cytoplasmic as well as nuclear envelope distribution of endogenous TMEM43 in non-transfected HL-1 control cells. TMEM43 was detected using anti-TMEM43 antibody and Cy3-labeled anti-rabbit IgG. <b>C</b>. Immunofluorescence staining of TMEM43-WT or TMEM43-S358L cells. Expressed wild-type or mutant TMEM43 were observed by GFP fluorescence (left column); GFP-TMEM43 showed more cytoplasmic distribution. TMEM43 was detected in the cytoplasm of both the stably expressing wild-type (top row) and mutant (bottom row) cell lines. Nuclei are visualized with DAPI (blue, right column). GFP (green, left column) fluorescent, Cy3 (red, middle column) fluorescent and merged images (right column) are shown. Co-localization of signals due to GFP and antibody binding (yellow, right column).</p

Expression of IC disc proteins in HL-1 cells transfected with wild-type and mutant TMEM43.

<p><b>A</b> and <b>B</b>. The stably transfected cells were lysed and Western blot was performed for ZO-1, α-catenin, N-cadherin, β-catenin, JUP, Cx43 and GAPDH. The expression of α-catenin, N-cadherin, β-catenin and JUP were similar in non-transfected HL-1 (control) and stably-transfected cells (TMEM43-WT and TMEM43-S358L). <b>A</b>. The level of ZO-1 was significantly decreased in TMEM43-S358L cells compared to TMEM43-WT and control. <b>B</b>. P2 and P0 represent the phosphorylated and non-phosphorylated forms of Cx43. The level of the non-phosphorylated Cx43 isoform (P0) was increased in mutant cells compared to the control and TMEM43-WT cells. <b>C</b> and <b>D</b>. Densitometry analysis of ZO-1/GAPDH and phosphorylated Cx43 P2/P0. * denotes p<0.05. The densitometry results showed the combined analysis of three separate western blots and the western blot images are representative of three independent cell culture experiments.</p

TMEM43 distribution and association with ZO-1 and JUP in the intercalated disc of normal murine myocardium.

<p><b>A</b>. Electron micrograph of a double immunogold labeled section of the perinuclear region of a cardiac myocyte that has been labeled with TMEM43. Particles were located diffusely throughout the nucleus (N), on the nuclear membrane (arrows) and on components of the sarcotubular network (arrowheads). <b>B</b>. Sarcoplasm of a cardiac myocyte. Note the labeling on the sarcoplasmic reticulum (arrowheads) on vesicular components of the sarcotubular network (asterisk). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109128#pone-0109128-g004" target="_blank">Figure 4C</a> represents co-localization of TMEM43 (10 nm gold particles) with ZO-1 (15 nm gold particles). Arrows indicate two adherens junctions that contain both ZO-1 and TMEM43. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109128#pone-0109128-g004" target="_blank">Figure 4D</a> represents co-localization of TMEM43 (10 nm gold particles) with JUP (15 nm gold particles) in desmosomes and adherens junctions (arrows) of the intercalated disc. All bars equal 0.5 µm.</p

Effects of TMEM43 on conduction velocity and sodium channel expression.

<p><b>A</b>. Electrograms recorded from adjacent MEA electrodes in HL-1 cell cultures. All cells beat spontaneously. Control and TMEM43-WT cells beat with a regular, synchronous rhythm. TMEM43-S358L cells beat with an irregular, comparatively slowed rhythm. <b>B</b>. Average velocity illustrating a decrease in conduction velocity of the TMEM43-S358L cells (1.2 cm/s) compared to control cells (2.5 cm/s) and TMEM43-WT cells (2.1 cm/s) (n = 7 control, n = 6 TMEM43-WT and n = 6 TMEM43-S358L, * p<0.01). All results are representative of six independent culture experiments. <b>C</b>. Western blot demonstrating expression of Na_V1.5 in control, TMEM43-WT and TMEM43-S358L cells. GAPDH was used as a loading control. Rabbit polyclonal Na_V1.5 antibody specifically detected sodium ion channel (Na_V1.5) expression in all three cell types. Neither wild-type nor mutant TMEM43 altered the Na_V1.5 expression. All results are representative of three independent Western blot experiments.</p

Transmission Electron Micrograph of transfected HL-1 cells labeled for TMEM43 with immunogold.

<p><b>A</b> and <b>B</b>. Single immunogold labeling experiments used 15 nm gold particles to label GFP. <b>A</b>. Immunogold-labeled TMEM43-WT cells. The label (large dot) is associated with the endoplasmic reticulum (arrows), nuclear envelope, and the nucleus (N). <b>B</b>. Immunogold-labeled TMEM43-S358L cells. Immunogold particles (large dot) are confined to bundles of cytoplasmic microfilaments (arrows) and what vacuolated endosomal structures (asterisk). The endoplasmic reticulum or nuclear envelope did not show TMEM43 expression. <b>C</b> and <b>D</b>. Double immunogold labeling experiments used 15 nm gold particles to label GFP and 5 nm gold particles to label TMEM43. <b>C</b>. Cytoplasm of a wild-type cell that has been labeled with both TMEM43 (small dot) and GFP (large dot) antibodies. Note the co-localization on the endoplasmic reticulum (arrows). <b>D</b>. Mutant protein labeled with antibody specific for TMEM43 (small dot) or the GFP Tag (large dot) were found in a large vacuolated structure (asterisk) as well as discrete clusters in the cytoplasm (arrows) of stably transfected cells. All bars equal 0.5 µm. <b>E</b>. Quantitative analysis of the distance between large gold particles (GFP-15 nm) and small gold particles (TMEM43-5 nm) in TMEM43-WT (open column) and TMEM43-S358L (filled column) cells. Data are expressed mean ± SD from three independent experiments.</p