• 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • br reversed the increased ECM adhesion of ADD overexpressed


    reversed the increased ECM adhesion of ADD1-overexpressed H1299 Belinostat (PXD101) (Suppl. Fig. B,C). Unfortunately, a long-time (~16 h) leptomycin treatment, which was required for examining cell migration, produced uninterpretable results by markedly inhibiting transfilter migration of control cells (data not shown). These results suggest that at least some effects of ADD1-overexpression of the adhesion and migration of NSCLC cells could be mediated by the non-nuclear pool of ADD1.
    3.5. Src inhibition reverses the increased adhesion and inhibited migration of ADD1-overexpressing lung cancer cells
    Next, we sought to elucidate if activation of Src is responsible for the increased ECM adhesion and attenuated motility of ADD1-over-expressing NSCLC cells by using siRNA-mediated downregulation of Src expression (Fig. 7A). Depletion of Src decreased paxillin phosphoryla-tion and inhibited the formation of non-retractable tails in ADD1-overexpressing H1299 cells (Fig. 7A, B). Consistently, Src knockdown reversed the enhanced ECM adhesion of ADD1-overexpressing cells without affecting adhesion of control cells (Fig. 7C, D). Interestingly, downregulation of Src had a complex effect on H1299 cell motility by decreasing transfilter migration of control cells, but increasing migra-tion of ADD1-overexpressing cells (Fig. 7E, F). This most likely reflects
    Fig. 4. Overexpression of ADD1 but not ADD3, inhibits lung cancer cell motility. FLAG-tagged ADD1 or ADD3 were stably expressed in H1299 lung cancer cells using a lentiviral expression vector. (A) Immunoblotting analysis shows the levels of ADD1 and ADD3 proteins in the generated cell lines. (B) Quantification of the planar migration of the control, ADD1 or ADD3-overexpressing H1299 cell monolayers after 12 h of wound healing. (C, D) Representative images and quantitative analysis of the DAPI-labeled control, ADD1, or ADD3-overexpressing H1299 cells after 12 h transfilter migration in the Boyden chamber. (E, F) Representative images and quantitative analysis of the DAPI-labeled control, ADD1, or ADD3-overexpressing H1299 cells after 24 h invasion into Matrigel. Data are presented as mean ± SE (n = 3); *p < 0.05; **p < 0.005, as com-pared to the control group.
    multiple roles of Src in the regulation of cell motility that include ad-hesion-dependent and independent mechanisms [56,57]. Together, our data demonstrate that ADD1 overexpression results in Src activation that increases ECM adhesion and impedes motility of NSCLC cells.
    3.6. Upregulation of cadherin-11 mediates the increased motility of adducin-depleted lung cancer cells
    In the final series of experiments, we investigated the mechanisms that mediate ECM adhesion-independent acceleration of motility of adducin-depleted NSCLC cells. Since adducins were previously im-plicated in the regulation of cadherin-based intercellular junctions and epithelial differentiation [31,33,58,59] we asked if the loss of adducins could trigger the phenotypic alterations of NSCLC cells. Immunoblot-ting analysis was used to examine the expression of major epithelial and mesenchymal cadherins in control and adducin-depleted H1573 cells. While the levels of epithelial-type E- and P-cadherins were not affected by ADD1 and ADD3 knockout, expression of mesenchymal-type cad-herin-11 was dramatically (up to 31-fold) upregulated in ADD1-defi-cient and in less extent (6–8 fold) in ADD3 knockout cells (Fig. 8A,B). 
    Consistently, cadherin-11 protein expression was downregulated in ADD1-overexpressing H1299 cells (data not shown). To examine the functional roles of cadherin-11 overexpression in accelerated motility of adducin-depleted H1573 cells, cadherin-11 functions were inhibited by two different approaches. One approach involved siRNA-mediated knockdown of cadherin-11 (Fig. 8C, D), while the other approach used an anti-cadherin-11 inhibitory antibody (Fig. 8E, F). Both approaches produced consistent results by selectively attenuating transfilter mi-gration of ADD1-deficient H1573 cells, while having little effects on the motility of control cells (Fig. 8C–F). Similar results were obtained when cadherin-11 was inhibited in ADD3-deficient H1573 cells (Suppl. Fig. 9). Together, our results demonstrate that upregulation of cad-herin-11 expression plays a major role in the accelerated motility of adducin-deficient NSCLC cells.
    Fig. 5. Overexpression of ADD1 increases extracellular matrix adhesion and promotes focal adhesion assembly in lung cancer cells. (A, B) Representative images and quantification of 30 min adhesion to the collagen I matrix by the control and ADD1-overexpressing H1299 cells. (C, D) Representative immunofluorescence images and quantitative analysis of phosphorylated