Laminin-332-beta1 integrin interactions negatively regulate invadopodia

Abstract

Adhesion of epithelial cells to basement membranes (BM) occurs through two major structures: actin-associated focal contacts and keratin-associated hemidesmosomes, both of which form on laminin-332 (Ln-332). In epithelial-derived cancer cells, additional actin-linked structures with putative adhesive properties, invadopodia, are frequently present and mediate BM degradation. A recent study proposed that BM invasion requires a proper combination of focal contacts and invadopodia for invading cells to gain traction through degraded BM, and suggested that these structures may compete for common molecular components such as Src kinase. In this study, we tested the role of the Ln-332 in regulating invadopodia in 804G rat bladder carcinoma cells, a cell line that secretes Ln-332 and forms all three types of adhesions. Expression of shRNA to Ln-332 gamma2 chain (gamma2-kd) led to increased numbers of invadopodia and enhanced extracellular matrix degradation. Replating gamma2-kd cells on Ln-332 or collagen-I fully recovered cell spreading and inhibition of invadopodia. Inhibition of alpha3 or beta1, but not alpha6 or beta4, phenocopied the effect of gamma2-kd, suggesting that alpha3beta1-mediated focal contacts, rather than alpha6beta4-mediated hemidesmosome pathways, intersect with invadopodia regulation. gamma2-kd cells exhibited alterations in focal contact-type structures and in activation of focal adhesion kinase (FAK) and Src kinase. Inhibition of FAK also increased invadopodia number, which was reversible with Src inhibition. These data are consistent with a model whereby actin-based adhesions can limit the availability of active Src that is capable of invadopodia initiation and identifies Ln-332-beta1 interactions as a potent upstream regulator that limits cell invasion.

Figure 1. Suppression of Ln-332 increases invadopodia number

(A) Representative overhead and Z-section slice (Z) confocal images of γ2-ctrl and γ2-kd cells cultured for 16 h on glass, fixed, and stained with antibodies against F-actin (blue), cortactin (green), and Ln-332 γ2 chain (red). Colocalization between F-actin and cortactin is turquoise in merged images. Scale bar = 10 µm and arrows indicate examples of invadopodia. (B) Box-and-whisker plot shows the number of invadopodia counted per cell. γ2-kd cells had significantly more invadopodia than control cells (N≥3; p<0.001). (C) Representative confocal images from in vitro matrix degradation assay. Cells were cultured for 16 h on cross-linked FITC-gelatin (green), fixed, and stained with antibodies against F-actin (red) and cortactin (blue) to identify invadopodia. Pink regions in merged images show colocalization between F-actin and cortactin; yellow shows colocalization between F-actin and FITC-gelatin; turquoise shows colocalization between F-actin and FITC-gelatin; and white shows colocalization of all three markers. Scale bar = 10 µm and arrows indicate examples of invadopodia. (D) Box-and-whisker plots show number of invadopodia per cell. Again, γ2-kd cells formed significantly more invadopodia per cell than control cells (N=3; p<0.001). (E) Matrix-degrading assays show γ2-kd degraded more ECM per cell than control cells (N=3; p<0.001).

Figure 2. Invadopodia number is increased by blocking integrin β1 but not β4

(A) Western blotting confirms that β4 was knocked down in ITGB4-kd cells by ~70%. (B) Representative images of vector-ctrl and β4-kd cells cultured on glass for 16 h, fixed, and stained with antibodies against TKS-5 (green) and F-actin (red). Yellow in merged images show colocalization of markers. Scale bar = 10 µm. (C) Box-and-whisker plots show number of invadopodia formed per cell. Knockdown of β4 had little effect on invadopodia formation compared to vector-ctrl cells (N=2; p=0.162). (D) Representative images of γ2-ctrl cells cultured on glass for 3 h, treated with Ha2/5 (integrin β1 blocking antibody; 10 µg/ml) or IgM (10 µg/ml) for 16 h, fixed, and stained with antibodies against cortactin (green) and F-actin (red). Yellow in merged images show colocalization of markers. (E) Blocking integrin β1 by Ha2/5 significantly increased number of invadopodia in γ2-ctrl cells (p<0.001). Scale bar is equal to 10 µm and arrows show podsome-like structure. (F) Plots show number of invadopodia formed per cell type plated on either Ln-332 (1 µg/ml), collagen I (Coll-I; 10 µg/ml), vitronectin (VN; 10 µg/ml), or Poly-D-lysine (Poly; 0.1mg/ml), or PBS. Addition of Ln-332 significantly reduced the formation of invadopodia on both cell types, and Coll-I significantly reduced invadopodia formation of γ2-kd cells (N≥3; P<0.001).

Figure 3. Ln-332 acts upstream of Src

(A) Box-and-whisker plots show number of invadopodia formed per cell in the presence of Src kinase inhibitor PP2, or DMSO for control, on glass (left) or on cross-linked FITC-collagen (right). (B) Representative images of SrcMEF cells plated on uncoated or Ln-332 (10 µg/ml) coated dishes, fixed, and stained with antibodies against Tks5 (green) and phalloidin (red). (C) Plot shows number of podosome structures (half ring) formed by SrcMEF cells. Ln-332 had some, but not a significant, impact on the number of ring structures formed by SrcMEF cells (N=1; p=0.057). (D) Representative western blotting of total Src, Src p416, and Src p527 is shown. Src p416 was decreased in γ2-kd cells by ~50% compared to γ2-ctrl, while total Src expression level and Src p527 was not altered.

Figure 4. Suppression of Ln-332 decreases pFAK Y397 and alters adhesion

(A) Representative western blot of pFAK Y397 and total FAK reveals that pFAK Y397 was decreased in γ2-kd cells compared to γ2-ctrl cells, but not in integrin β4-kd cells, while total FAK expression level was not altered. (B) Representative images of γ2-ctrl cells treated with PF573,228 (pFAK inhibitor; 5 µM) or DMSO for ~16 h, fixed, and stained with antibodies against cortactin (green), F-actin (red), and Hoechst (blue). Yellow in merged image shows colocalization between F-actin and cortactin. (C) Box-and-whisker plot shows that blocking FAK phosphorylation by PF573,228 increased the number of invadopodia formed by γ2-ctrl (N=3; p<0.001). (D) Representative F-actin (red), paxillin (left, green), and vinculin staining (right, green) are shown in images of γ2-ctrl and γ2-kd. (E) Quantification of the number of focal contacts per cell (left; N=2; p<0.001) and size of focal contacts (right; N=2; p=0.247) of γ2-ctrl and γ2-kd.