Patterns of MMP and TIMP RNA Levels Seen During T3 Induced Metamorphosis in <i>X. laevis</i> Intestine and Tail as Derived From RT-PCR Data.

<p>Levels of RNA found during induced metamorphosis of the intestine and tail demonstrated that MT1-MMP and TIMP-2 shared similar RNA expression patterns, dipping after 1 day and elevating thereafter. MMP-2 and MT3-MMP also shared similar RNA expression patterns, elevating after 1 day in both the intestine and tail. TIMP-3 expression patterns were not similar between the intestine and tail, nor were they similar to the four other genes. Changes in RNA levels are not quantitative, but instead illustrate patterns of expression.</p

Representative RT-PCR Results of Similar MT1-MMP and TIMP-2 Levels of RNA During Natural and T3 Induced Metamorphosis.

<p>PCR products were agarose gel fractionated, stained, and photographed. MT1-MMP and TIMP-2 transcription patterns mimic each other at all stages and conditions. In the intestine, MT1-MMP and TIMP-2 transcripts are poorly detected at stage 56 prior to metamorphosis. Levels of both increase during stages 58, 61 and 63 and then drop to pre-metamorphic levels at stage 65. Conversely, in the tail, transcripts are detectable at stage 56, rise to stage 61, and then begin to drop at stage 63. T3 treatment induces a drop in levels after one day, but an increase after three days for both genes. EF1α RNA levels are shown for all comparable stages and treatments.</p

Patterns of MMP and TIMP RNA Levels Seen during <i>X. laevis</i> Intestine Metamorphosis Derived From RT-PCR Data.

<p>Levels of RNA found during intestine metamorphosis demonstrated that MT1-MMP, TIMP-2 and TIMP-3 shared similar RNA expression patterns, with peaks present at stages 58 and63. MT3-MMP and MMP-2 RNA expression patterns in the intestine did mirror each other, but were not similar to the three other genes. Changes in RNA levels are not quantitative, but instead illustrate patterns of expression.</p

Patterns of MMP and TIMP RNA Levels Seen During <i>X. laevis</i> Tail Metamorphosis Derived From RT-PCR Data.

<p>Levels of RNA found during tail metamorphosis demonstrated that MT1-MMP, TIMP-2 and MMP-2 shared similar RNA expression patterns, peaking at stage 61 and staying elevated at stage 63. MT3-MMP and TIMP-3 expression patterns in the tail were not similar to the three other genes, nor to each other. Changes in RNA levels are not quantitative, but instead illustrate patterns of expression.</p

Remodeling of the Methylation Landscape in Breast Cancer Metastasis

<div><p>The development of breast cancer metastasis is accompanied by dynamic transcriptome changes and dramatic alterations in nuclear and chromatin structure. The basis of these changes is incompletely understood. The DNA methylome of primary breast cancers contribute to transcriptomic heterogeneity and different metastatic behavior. Therefore we sought to characterize methylome remodeling during regional metastasis. We profiled the DNA methylome and transcriptome of 44 matched primary breast tumors and regional metastases. Striking subtype-specific patterns of metastasis-associated methylome remodeling were observed, which reflected the molecular heterogeneity of breast cancers. These divergent changes occurred primarily in CpG island (CGI)-poor areas. Regions of methylome reorganization shared by the subtypes were also observed, and we were able to identify a metastasis-specific methylation signature that was present across the breast cancer subclasses. These alterations also occurred outside of CGIs and promoters, including sequences flanking CGIs and intergenic sequences. Integrated analysis of methylation and gene expression identified genes whose expression correlated with metastasis-specific methylation. Together, these findings significantly enhance our understanding of the epigenetic reorganization that occurs during regional breast cancer metastasis across the major breast cancer subtypes and reveal the nature of methylome remodeling during this process.</p></div

Methylome remodeling in metastasis across molecular subtypes of breast cancer.

<p><b>A</b>. Hierarchical clustering of top 1000 differentially methylated loci (defined by SAM). Primary tumor or metastasis is noted along the top. PAM50 subtype classification is labeled. Color scale indicates normalized β-value. <b>B</b>. Box plots of metastasis-specific methylation change across top 25% differentially methylated probes common to all molecular subtypes. Y-axis, mean beta-value change. The median, 1 standard deviation (box plot), and 10–90 percentile (whiskers) are indicated in the graph. <b>C</b>. Frequency of differentially hypermethylated and hypomethylated loci as a function of relationship to CGIs (top panel), functional location (middle panel) and location within core promoter (bottom panel). <b>D</b>. Gene set enrichment analysis (GSEA) plot. GSEA was performed with PRC2 target list from Lee et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103896#pone.0103896-Lee1" target="_blank">[37]</a> The graph on the bottom represents the ranked, ordered, non-redundant list of genes (by SAM). Genes on the far left (red) correlated the most with metastases, and genes on the far right (blue) correlated the most primary samples. The vertical black lines indicate the position of each of the genes of the studied gene set in the ordered, non-redundant data set. The green curve corresponds to the ES (enrichment score) curve, which is the running sum of the weighted enrichment score obtained from GSEA software.</p

Chromosome characterization of subtype-specific methylation change in metastasis.

<p><b>A</b>. Chromosome view of smoothed averaged paired differential methylation between metastasis and primary tumors shown for luminal A (purple), luminal B (orange), basal-like (red) and Her2-enriched (green) subtypes along human chromosome 2. CpG islands (CGI) are shown in grey below. <b>B</b>. Methylation profile of a 20 Mb region is shown. Location of RNAseq transcripts for+and – strands is shown above. CGIs, lamin B1-associated domains (LAD), and peaks for H3K4-trimethylated (H3K4me3), H3K4-monomethylated (H3K4me1) and H3K9-acetylated chromatin marks from human mammary endothelial cells (HMEC) are shown (from <a href="http://www.genome.ucsc.edu/cgi-bin/hgTables" target="_blank">http://www.genome.ucsc.edu/cgi-bin/hgTables</a>). <b>C</b>. Volcano plots of differentially methylated sites between metastasis and primaries by subtype-specific ANOVA. β-value difference is shown on the x-axis, -log10 of FDR-corrected p-value is on the y-axis. β-values of top three loci from luminal A, luminal B and basal primaries and metastases are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103896#pone.0103896.s001" target="_blank">figure S1</a>.</p

Embryonic Morphogen Nodal Promotes Breast Cancer Growth and Progression

<div><p>Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. <em>In vitro</em>, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of <em>BAX</em> and <em>BCL2</em> gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (<100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.</p> </div

Nodal inhibition alters proliferation:apoptosis ratios in micrometastases.

<p>(A) GUSB staining of pulmonary micrometastases from MDA-MB-231 cells transfected with a Control shRNA (231+shControl) or a shRNA to Nodal (231+shNodal) in NOD/SCID/MPSVII mice 4 weeks post-intravenous injection (red and outlined with white dotted line). (B) Scatter plot representing the average number of micrometastases (<100 cells) per section of lung from NOD/SCID/MPSVII mice 4 weeks after injection with 231+shControl or 231+shNodal cells. The number of 231+shNodal micrometastases that formed after 4 weeks in NOD/SCID/MPSVII mice was not significantly reduced compared to the number of 231+shControl micrometastases (n≥5, p>0.05). Each point represents the average mean number of micrometastases per section per mouse. Black bars represent the median number of micrometastases per section per mouse. (C) Immunohistochemical analysis of Ki67 expression (brown) and TUNEL (brown) staining in pulmonary micrometastases from 231+shControl cells or 231+shNodal cells in NOD/SCID/MPSVII mice 4 weeks post-intravenous injection. Proliferation is indicated by Ki67 staining and apoptotic nuclei were detected with TUNEL. (D) Proliferation:apoptosis ratios in 4 week micrometastases were determined with immunohistochemical localization of Ki67 and TUNEL. At 4 weeks, lesions from 231+shControl cells had a positive proliferation ratio (1.57) whereas lesions from 231+shNodal cells had a negative proliferation:apoptosis ratio (0.74) (n≥3, p<0.05). Values represent mean average proliferation:apoptosis ratio in tumour lesions per mouse ± S.E.M.</p

Nodal knockdown decreases proliferation and increases apoptosis in aggressive MDA-MB-231 breast cancer cells.

<p>(A) Trypan Blue exclusion was used to count live cells daily to generate growth curves over 3 days, in response to altered Nodal expression. MDA-MB-231 cells transfected with a Nodal-targeted shRNA (231+shNodal) exhibited a significant decrease in proliferation over 3 days compared to cells transfected with a scrambled Control shRNA (231+shControl) (n = 3; p = 0.047). (B) Representative histogram of mean fluorescence intensity (mfi) in 231+shNodal and 231+shControl cells labelled with Cell Trace Violet (CTV) for 4 days after synchronization via serum starvation. There was a greater loss of CTV in 231+shControl compared to 231+shNodal cells (4801 and 6006, respectively) indicating that control cells proliferated more than their shNodal-treated counterparts. (C) Western blots demonstrating decreased phosphorylated histone H3 at 4 different sites, including Thr11, Ser10, Ser28 and Thr3 in 231+shNodal cells compared to 231+shContol cells. Total histone H3 and β-Actin are used as controls. (D) Representative images of TUNEL staining and corresponding quantification of percent TUNEL-positive cells in 231+shNodal and 231+shControl cells grown <i>in vitro</i>. TUNEL positive cells are delineated by arrows and nuclei are counterstained blue. Micron bars equal 25 µm. The percentage of TUNEL positive cells is significantly higher in 231+shNodal cells as compared to 231+shControl cells (n = 23; p = 0.001). (E) Western blot demonstrating that cleavage of caspase-9 is elevated in 231+shNodal compared to 231+shControl cells. Uncleaved caspase-9 and β-Actin are used as controls. (F) Real time RT-PCR analysis demonstrating that <i>BAX</i> mRNA expression is significantly higher in 231+shNodal cells compared to 231+shControl cells (n = 4, p = 0.029). (G) Real time RT-PCR analysis demonstrating that <i>BCL2</i> mRNA expression is significantly lower in 231+shNodal cells compared to 231+shControl cells (n = 4, p = 0.029). All bar graphs are presented as mean ± S.E.M. for replicate values. Asterisks indicate a significant difference as specified compared to controls.</p