High α-actinin-1 expression in basal-like breast cancer cells destabilizes E-cadherin based adhesions.

<p>(A) Western blotting analysis of MDA-MB-231 cells expressing either GFP (Control) or GFP-tagged E-cadherin (+ E-cadherin) in combination with siRNA mediated downregulation using non-targeting (siNT) or α-actinin-1 (siA1) oligos, as indicated. Dotted lines indicate removal of intervening lanes. (B) Merged immunofluorescence images of phalloidin (F-actin, green) stained MDA-MB-231 cells expressing GFP (Control) or GFP-E-cadherin (+ E-cadherin). GFP-signal is pseudo-colored to red, and Hoechst visualizes nuclei. The arrowhead points to punctate E-cadherin and arrows point to subcortical actin fibers. Scale bar, 10 μm (C) A representative example of a change from punctate to linear E-cadherin following α-actinin-1 downregulation (siA1) in MDA-MB-231 cells re-expressing E-cadherin. (D) Phalloidin (F-actin, green) and Hoechst (blue) co-stained HCC1937 cells following siRNA-mediated downregulation using non-targeting (siNT), α-actinin-1 (siA1) or α-actinin-4 (siA4) oligos, as indicated. Arrows show F-actin reorganization. Scale bar, 10 μm. (E) Zoom-in immunofluorescence images of E-cadherin or merged F-actin/E-cadherin following downregulation of control (siNT), α-actinin-1 (siA1) or α-actinin-4 (siA4), as indicated. (F) Immunofluorescence image of a wild-type HCC1937 cell stained for α-actinin-1 (purple) and merged image of α-actinin-1 (purple) and F-actin (green) to demonstrate the localization of endogenous α-actinin-1 on radial (white arrowheads) and arc-like (yellow arrows) actin fibers at E-cadherin based adhesion (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196986#pone.0196986.g005" target="_blank">Fig 5</a> schematic presentation). (G) Western blotting analysis with the indicated antibodies following downregulation of control (siNT), α-actinin-1 (siA1) or α-actinin-4 (siA4) in HCC1937 cells.</p

α-actinin-1 expression is increased in human breast cancer.

<p>(A) Comparison of <i>ACTN1</i> mRNA in healthy (grey boxes) and cancerous tissues (red boxes) as indicated. Box plots show median expression as a line, with 25 and 75 percentiles as lower and upper boxes with whiskers and outlier points extending to cover remaining data [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196986#pone.0196986.ref029" target="_blank">29</a>]. The x-axis indicates the number of patients. (B) Representative immunohistochemistry images of a healthy and two breast cancer sections stained for α-actinin-1 (A1-341 ab). The black arrowhead in healthy section indicates a luminal cell, and a black arrow indicates a myoepithelial cell. The blue arrow in the same image shows a stromal fibroblast positive for α-actinin-1. Scale bar, 50 μm. (C) D-HSCORE score [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196986#pone.0196986.ref033" target="_blank">33</a>] to compare α-actinin-1 staining signal of healthy (n = 19) and breast cancer tissues (n = 46) stained in (B). ***P<0.001 by student’s t-test. (D) Human breast cancer tissue lysate array immunoblotted for α-actinin-1. Each vertical line of spots represents triplicates of cancer (ca) and matched healthy (contr) tissues from a patient (n = 55). The columns represent the expression ratio of α-actinin-1 between cancer and adjacent healthy tissue of each patient based on the average spot intensity of triplicates. The red columns indicate ≥1.5-fold increase in α-actinin-1 levels.</p

Schematic presentation of destabilized E-cadherin-based adhesions and associated actin fibers following increased α-actinin-1 expression.

<p>Schematic presentation of destabilized E-cadherin-based adhesions and associated actin fibers following increased α-actinin-1 expression.</p

α-actinin-1 expression is higher in basal-like breast cancer cells and is associated with poor survival.

<p>(A) Comparison of α-actinin-1 mRNA (<i>ACTN1</i> log abundance) between luminal (n = 25) and basal-like (n = 26) breast cancer cell lines. ***<i>P</i><0.001 by Student’s <i>t</i>-test. (B) Western blotting analysis of four luminal and five basal-like breast cancer cell line lysates using α-actinin-1, ER-α, α-actinin-4 or β-actin antibodies as indicated. (C-F) Kaplan-Meier survival analysis showing relapse-free survival (Survival probability) based on α-actinin-1 (<i>ACTN1</i>) expression in ER+ (C), ER- (D), luminal A (E) or basal-like (F) breast cancer subtypes. Curves were generated using KM blotter (<a href="http://kmplot.com/breast/" target="_blank">http://kmplot.com/breast/</a>). Patients with high (red) or low (black) <i>ACTN1</i> expression were split based on the median value calculated across the entire dataset to generate two groups of equal size. Numbers of patients at risk at specific time points are indicated below each diagram. n indicates number of patients. Hazard ratios (HRs) and log-rank P-values are depicted for each survival analysis, P-values of <0.05 were considered to be statistically significant.</p