Increased alpha-actinin-1 destabilizes E-cadherin-based adhesions and associates with poor prognosis in basal-like breast cancer

The controlled formation and stabilization of E-cadherin-based adhesions is vital for epithelial integrity. This requires co-operation between the E-cadherin-based adhesions and the associated actin cytoskeleton. In cancer, this co-operation often fails, predisposing cells to migration through molecular mechanisms that have only been partially characterized. Here, we demonstrate that the actin filament cross-linker alpha-actinin-1 is frequently increased in human breast cancer. In mammary epithelial cells, the increased alpha-actinin-1 levels promote cell migration and induce disorganized acini-like structures in Matrigel. This is accompanied by a major reorganization of the actin cytoskeleton and the associated E-cadherin-based adhesions. Increased expression of alpha-actinin-1 is particularly noted in basal-like breast cancer cell lines, and in breast cancer patients it associates with poor prognosis in basal-like subtypes. Downregulation of alpha-actinin-1 in E-cadherin expressing basal-like breast cancer cells demonstrate that alpha-actinin-1-assembled actin fibers destabilize E-cadherin-based adhesions. Taken together, these results indicate that increased alpha-actinin-1 expression destabilizes E-cadherin-based adhesions, which is likely to promote the migratory potential of breast cancer cells. Furthermore, our results identify alpha-actinin-1 as a candidate prognostic biomarker in basal-like breast cancer.Peer reviewe

SUMOylation of AMPK alpha 1 by PIAS4 specifically regulates mTORC1 signalling

AMP-activated protein kinase (AMPK) inhibits several anabolic pathways such as fatty acid and protein synthesis, and identification of AMPK substrate specificity would be useful to understand its role in particular cellular processes and develop strategies to modulate AMPK activity in a substrate-specific manner. Here we show that SUMOylation of Z attenuates AMPK activation specifically towards mTORC1 signalling. SUMOylation is also important for rapid inactivation of AMPK, to allow prompt restoration of mTORC1 signalling. PIAS4 and its SUMO E3 ligase activity are specifically required for the AMPK alpha 1 SUMOylation and the inhibition of AMPK alpha 1 activity towards mTORC1 signalling. The activity of a SUMOylation-deficient AMPK alpha 1a mutant is higher than the wild type towards mTORC1 signalling when reconstituted in AMPKa-deficient cells. PIAS4 depletion reduced growth of breast cancer cells, specifically when combined with direct AMPK activator A769662, suggesting that inhibiting AMPK alpha 1 SUMOylation can be explored to modulate AMPK activation and thereby suppress cancer cell growth.Peer reviewe

Cyclin G-associated kinase promotes microtubule outgrowth from chromosomes during spindle assembly

Peer reviewe

Assembly of non-contractile dorsal stress fibers requires α-actinin-1 and Rac1 in migrating and spreading cells

Cell migration and spreading is driven by actin polymerization and actin stress fibers. Actin stress fibers are considered to contain aactinin crosslinkers and nonmuscle myosin II motors. Although several actin stress fiber subtypes have been identified in migrating and spreading cells, the degree of molecular diversity of their composition and the signaling pathways regulating fiber subtypes remain largely uncharacterized. In the present study we identify that dorsal stress fiber assembly requires α-actinin-1. Loss of dorsal stress fibers in α-actinin-1-depleted cells results in defective maturation of leading edge focal adhesions. This is accompanied by a delay in early cell spreading and slower cell migration without noticeable alterations in myosin light chain phosphorylation. In agreement with the unaltered myosin II activity, dorsal stress fiber trunks lack myosin II and are resistant to myosin II ATPase inhibition. Furthermore, the non-contractility of dorsal stress fibers is supported by the finding that Rac1 induces dorsal stress fiber assembly whereas contractile ventral stress fibers are induced by RhoA. Loss of dorsal stress fibers either by depleting α-actinin-1 or Rac1 results in a β-actin accumulation at the leading edge in migrating and spreading cells. These findings molecularly specify dorsal stress fibers from other actin stress fiber subtypes. Furthermore, we propose that non-contractile dorsal stress fibers promote cell migration and early cell spreading through Rac1-induced actin polymerization

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

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

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