α-Actinin-4 Enhances Colorectal Cancer Cell Invasion by Suppressing Focal Adhesion Maturation

α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion

Structural Insight into TNIK Inhibition

TRAF2- and NCK-interacting kinase (TNIK) has emerged as a promising therapeutic target for colorectal cancer because of its essential role in regulating the Wnt/β-catenin signaling pathway. Colorectal cancers contain many mutations in the Wnt/β-catenin signaling pathway genes upstream of TNIK, such as the adenomatous polyposis coli (APC) tumor suppressor gene. TNIK is a regulatory component of the transcriptional complex composed of β-catenin and T-cell factor 4 (TCF4). Inhibition of TNIK is expected to block the aberrant Wnt/β-catenin signaling caused by colorectal cancer mutations. Here we present structural insights into TNIK inhibitors targeting the ATP-binding site. We will discuss the effects of the binding of different chemical scaffolds of nanomolar inhibitors on the structure and function of TNIK

α-Actinin-4 Enhances Colorectal Cancer Cell Invasion by Suppressing Focal Adhesion Maturation

<div><p>α-Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance cancer cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the rapid turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion.</p></div

Effect of ACTN1 and ACTN4 on DLD-1 cell morphology.

<p><b>(A)</b> ACTN1 and ACTN4 expression was silenced by siGENOME SMARTpool siRNA in DLD-1 cells. Efficient knockdown was confirmed by western blotting using ACTN isoform-specific antibodies. The arrowhead indicates endogenous ACTN1. The asterisk indicates a non-specific crossreacting band. <b>(B)</b> Cells transfected with the indicated siRNAs were stained for vinculin (VCL) and F-actin and observed by confocal microscopy. The arrows and arrowheads indicate focal adhesions and stress fibers, respectively. Scale bar = 10 μm. <b>(C)</b> The percentages of siRNA-treated cells forming stress fibers are graphed. Data represent the mean ± SD of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01. <b>(D)</b> The focal adhesion numbers per cell of siRNA-treated cells were graphed. Data represent the mean ± SD of 71 cells for control, 73 cells for si-ACTN1 cells, and 55 cells for si-ACTN4 cells. *<i>P</i> < 0.05. <b>(E)</b> Transient expression of ACTN1-GFP and ACTN4-GFP in DLD-1 cells was confirmed by western blotting using ACTN isoform-specific antibodies. <b>(F)</b> GFP, ACTN1-GFP, and ACTN4-GFP were expressed in DLD-1 cells, which were then stained for VCL and F-actin and observed by confocal microscopy. The arrows and arrowheads indicate focal adhesions and stress fibers, respectively. Scale bar = 10 μm. <b>(G)</b> The percentages of transfected cells forming stress fibers are graphed. Data represent the mean ± SD of three independent experiments. Tfx indicates transfection. **<i>P</i> < 0.01. <b>(H)</b> The focal adhesion numbers per cell of cells expressing GFP, ACTN1-GFP, and ACTN4-GFP were graphed. Data represent the mean ± SD of 130 cells for GFP, 52 cells for ACTN1-GFP, and 73 cells for ACTN4-GFP. *<i>P</i> < 0.05.</p

ZYX binding to ACTN1 and ACTN4.

<p><b>(A)</b> Pull-down assay showing that a putative ACTN-binding site, GST-ZYX-N (aa 1–51), binds specifically to His-ACTN1, but not to His-ACTN4. Bound His-ACTN1 or His-ACTN4 was analyzed by western blotting using anti-His-tag antibody. Equal amounts of input proteins were confirmed by SDS-PAGE followed by Coomassie blue staining (CBB). <b>(B)</b> FLAG-ZYX-full (aa 1–572) or FLAG-ZYX-C (aa 52–572) bound to anti-FLAG M2 beads were mixed either with His-ACTN1 or His-ACTN4 proteins, or without ACTN proteins (denoted by “-”). Bound His-ACTN1 or His-ACTN4 was analyzed by western blotting using anti-pan-actinin antibody. Note that the faint bands detected in lanes 1 and 4 of the pull-down blot represent non-specific background signals of pan-actinin antibody. Equal amounts of ZYX proteins were confirmed by western blotting using anti-FLAG antibody.</p

Invasion of DLD-1 cells expressing GFP, ACTN1-GFP, and ACTN4-GFP.

<p><b>(A)</b> Stable expression of GFP, ACTN1-GFP, and ACTN4-GFP in DLD-1 cells was examined by western blotting using the antibodies indicated to the left of each blot. <b>(B)</b> The invasion assay was performed using GFP-, ACTN1-GFP-, or ACTN4-GFP-expressing stable cell lines. Cells were seeded into Matrigel invasion chambers and allowed to invade into the Matrigel for 26 h. Then, the invaded cells were fixed and imaged by examining the GFP signals on the undersurface of the chamber using fluorescence confocal microscopy. Scale bar = 100 μm. <b>(C)</b> The GFP-positive cells shown in (B) were quantified. At least six microscopic fields obtained from duplicated chambers were used for quantification in each experiment. The results represent the mean ± SD of three independent experiments. *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Turnover rate of focal adhesions in mCherry-, ACTN1-mCherry-, or ACTN4-mCherry-overexpressing DLD-1 cells.

<p><b>(A)</b> mCherry, ACTN1-mCherry, or ACTN4-mCherry were co-expressed with GFP-VCL and VCL time lapse image was observed by total internal reflection fluorescence (TIRF) microscopy to trace the assembly and disassembly of focal adhesions. Upper panels show representative kymographs of GFP-VCL fluorescence along 5-μm long lines drawn in regions of extending lamellipodia. Fluorescence intensity in the upper panel was plotted against time and shown as a graph to the bottom of respective kymographs. The turnover phases of focal adhesion assembly, stability, and disassembly are presented as red dashed lines in the mCherry vector graph. <b>(B–D)</b> Duration of each turnover phase exemplified in the left graph in A was analyzed and quantified from 51 adhesions for mCherry-expressing cells, 57 adhesions for ACTN1-mCherry-expressing cells, and 64 adhesions for ACTN4-mCherry-expressing cells. Data are presented as box and whisker plots with boxes representing 25th–75th percentile range and whiskers representing 10th–90th percentile range. n.s., not significant, **<i>P</i> < 0.01.</p