Correlation of the NDRG2 protein expression with HER2-receptor expression.

<p>Correlation of the NDRG2 protein expression with HER2-receptor expression.</p

NDRG2 expression loss leads to reduced cell proliferation and migration in basal type HCC1806 cells.

<p><b>(A)</b> NDRG2 knockdown in basal-type HCC1806 cells. <i>Upper graph</i>: <i>NDRG2</i> mRNA expression loss after 48 h, 96 h and 144 h RNA interference treatment. <i>Vertical lines</i>: standard deviation of three independent analyses. House-keeping gene <i>GAPDH</i> was used for normalization. <i>Lower graph</i>: Representative western blot illustrates the NDRG2 protein expression loss after 48 h, 96 h and 144 h RNA interference treatment. β-Actin served as loading control. <b>(B</b> to <b>C)</b> Cell proliferation due to transient NDRG2 knockdown: <b>(B)</b> Cell proliferation rate is decreased due to NDRG2 expression loss (green line; median proliferation rate of NDRG2-specific siRNA #4, #6 as well as a combination of #4 and #6) compared to HCC1806 cells treated with control siRNA (red line). <i>Vertical lines</i>: <i>standard error of mean</i> (SEM) of triplicates <b>(C)</b> Box plot represents averages of triplicate experiments. (<b>D</b> to <b>F</b>) Cell migration was analyzed by performing a scratch wound healing assay. <b>(D)</b> Representative images of the wound size are shown for HCC1806-siRNA control and HCC1806-NDRG2 siRNA for 0 h, 30 h and 48 h is shown. <b>(E)</b> Mean migration rate of a control cell set (HCC1806-siRNA control) and independent HCC1806-NDRG2 siRNA cells over 48 h is shown. Cell-free area on day 0 was set as 100% and used for standardization. Δ_{FC 30 hours}: differences of cell-free areas after 30 h. <b>(F)</b> Detailed comparison of wound closure after 30 h.</p

Abundant <i>NDRG2</i> Expression Is Associated with Aggressiveness and Unfavorable Patients’ Outcome in Basal-Like Breast Cancer

<div><p><i>NDRG2</i>, a member of the N-myc downstream-regulated gene family, is thought to be a putative tumor suppressor gene with promising clinical impact in breast cancer. Since breast cancer comprises heterogeneous intrinsic subtypes with distinct clinical outcomes we investigated the pivotal role of NDRG2 in basal-type breast cancers. Based on subtype classified tumor (n = 45) and adjacent normal tissues (n = 17) we examined <i>NDRG2</i> mRNA expression and CpG-hypermethylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA). In addition, NDRG2 protein expression was evaluated immunohistochemically using a tissue micro array (TMA, n = 211). <i>In vitro</i>, we investigated phenotypic effects caused by NDRG2 silencing in the basal A-like HCC1806 as well as NDRG2 over-expression in basal A-like BT20 compared to luminal-type MCF7 breast cancer cells. Our tissue collections demonstrated an overall low <i>NDRG2</i> mRNA expression in breast cancer subtypes compared to normal breast tissue in line with an increased CpG-hypermethylation in breast cancer tissue. Independent TCGA data sets verified a significant (P<0.001) expression loss of <i>NDRG2</i> in breast tumors. Of interest, basal-like tumors more frequently retained abundant <i>NDRG2</i> expression concordant with a lower CpG-hypermethylation. Unexpectedly, basal-like breast cancer revealed an association of <i>NDRG2</i> expression with unfavorable patients’ outcome. In line with this observation, <i>in vitro</i> experiments demonstrated reduced proliferation and migration rates (~20%) in HCC1806 cells following <i>NDRG2</i> silencing. In contrast, NDRG2 over-expressing luminal-type MCF7 cells demonstrated a 26% decreased proliferation rate. Until now, this is the first study investigating the putative role of NDRG2 in depth in basal-type breast cancer. Our data indicate that the described putative tumor suppressive function of NDRG2 may be confined to luminal- and basal B-type breast cancers.</p></div

<i>NDRG2</i> revealed a divergent expression and methylation pattern in basal- compared to luminal-type breast cancer.

<p><b>(A)</b> Box plot analysis (based on our own subtype classified tissue collective) illustrates a decreased <i>NDRG2</i> mRNA expression in breast cancer compared with normal breast tissue. An increased <i>NDRG2</i> mRNA expression (median expression level: 0.334) in triple negative tumors is observed comparing to HER2-enriched (median expression level: 0.275) and luminal-type (median expression level: 0.229) carcinomas. <b>(B)</b> Heatmap of <i>NDRG2</i> expression is shown. <i>Red</i>: high-, <i>black</i>: mean-, <i>green</i>: low-expression. <i>Left panel</i>: breast cancer subtypes. <i>Middle panel</i>: <i>NDRG2</i> mRNA expression. <i>Right panel</i>: sample type (<i>dark grey</i>: primary tumor; <i>light grey</i>: solid normal tissues). Breast tumor samples are stratified by subtypes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159073#pone.0159073.ref003" target="_blank">3</a>]. <b>(C)</b> Box plot demonstrating a significant loss of <i>NDRG2</i> mRNA expression in luminal-type and HER2-enriched tumors with respect to basal-type cancer specimens. <b>(D)</b> Statistical association of <i>NDRG2</i> mRNA expression and <i>NDRG2</i> hypermethylation in breast cancer. Pearson correlation coefficient: r = -0.5478, P<0.001. <b>(E)</b> Box plot analysis (based on our own subtype classified tissue collective) showing significant increased CpG-hypermethylation of the <i>NDRG2</i> promoter in luminal-type and HER2-enriched tumors compared to normal tissue samples. Horizontal lines: grouped medians. Boxes: 25–75% quartiles. Vertical lines: range, peak, and minimum. ***P<0.001, **P<0.01, ns: not significant.</p

Abundant <i>NDRG2</i> mRNA expression in invasive ductal carcinoma is associated with unfavorable patients’ recurrence score.

<p><b>(A)</b> Heatmap of <i>NDRG2</i> expression and breast cancer 21-gene recurrence score is shown. <i>Red</i>: high-, <i>black</i>: mean-, <i>green</i>: low-expression respectively score values. <i>Left panel</i>: breast cancer histological subtypes (<i>light orange</i>: invasive ductal carcinoma (IDC); <i>dark orange</i>: invasive lobular carcinoma (ILC); <i>light grey</i>: solid normal tissues). <i>Middle panel</i>: <i>NDRG2</i> mRNA expression. <i>Right panel</i>: 21-gene signature score values. <b>(B</b> to <b>C)</b> Statistical association of <i>NDRG2</i> mRNA expression and 21-gene recurrence score in <b>(B)</b> IDC samples (Pearson correlation coefficient: r = 0.2274, P<0.0001) and <b>(C)</b> ILC cases (Pearson correlation coefficient: r = -0.310, P<0.0001).</p

Clinicopathological parameters in relation to <i>NDRG2</i> mRNA expression of the TCGA data portal.

<p>Clinicopathological parameters in relation to <i>NDRG2</i> mRNA expression of the TCGA data portal.</p

Loss of NDRG2 protein expression in human breast cancer.

<p><b>(A)</b> Strong NDRG2 expression in epithelial cells of normal breast tissue (<b>a</b> and <b>b</b>). Moderate to low NDRG2 immunoreactivity in luminal-type (<b>c</b> and <b>d</b>) and triple-negative (<b>e</b> and <b>f</b>) breast carcinoma. <b>(B)</b> Box plot analysis showing significant loss of NDRG2 expression in tumor tissue. <b>(C</b> to <b>D)</b> Box plot analysis illustrating pronounced loss of NDRG2 expression in luminal-type and HER2-enriched tumors (P<0.001) compared to triple negative cancers (P = 0.025) <b>(C)</b> and in line with a significant higher median NDRG2 staining intensity in triple negative cancer <b>(D)</b>. Horizontal lines: group medians. Boxes: 25–75% quartiles. Vertical lines: range, peak and minimum. ***P<0.001. *P<0.05.</p