br altered in the kidneys
altered in the kidneys of mice with either individual ADD3 knockout or a dual loss of ADD2 and ADD3 . Hence, it is reasonable to suggest that ADD1 in addition to canonical heterooligomerization with ADD3 or ADD2, is capable of forming homooligomers and functioning in-dependently from other adducin isoforms. Further studies are needed to compare the functional activities of homooligomers and hetero-oligomers of adducins.
4.3. High expression of ADD1 suppresses lung cancer cell motility by strengthening ECM adhesion
We found that ADD1 suppressed lung epithelial cell and NSCLC cell motility via two diﬀerent mechanisms, depending on the cellular level of this protein. High ADD1 expression inhibited cell migration by in-creasing the avidity of ECM adhesion, while moderate ADD1 expression attenuated cell migration by downregulating expression of the promi-gratory cadherin-11 (Figs. 5 & 8). Both mechanisms are novel and have not been previously associated with functional activities of adducins. The observed diﬀerent modes of ADD1 actions are not surprising, given its known roles as the membrane skeleton component and the actin-binding protein involving in multiple interactions [22,23]. The out-comes of such interactions are likely to be an assembly of diﬀerent cytoskeletal complexes, which abundancy and functions depend on the cellular level of ADD1.
Our data suggest that high ADD1 expression creates a strong ECM adhesion associated with the increased FA signaling events, such as Src activation (Figs. 5 & 7). Such enhanced FA are likely to be resistant to disassembly, thereby impeding detachment of the trailing cellular edge and attenuating cell motility (Fig. 6; Suppl. Movies 1 & 2). Similar formation of large integrin-based FA was previously reported in rat renal epithelial Etoposide after expression of an ADD1 point mutant asso-ciated with human hypertension . While we did not investigate the precise mechanisms that underline the increased Src activation and the enhanced FA assembly in ADD1-overexpressing cells, these events are likely to be related to the adducin-dependent reorganization of the actomyosin cytoskeleton. Indeed, ADD1-overexpressing NSCLC cells were characterized by the peripheral protrusions that contained pro-minent actomyosin bundles (Fig. 6). These findings are in agreement to
Fig. 7. Downregulation of Src expression reverses the increased adhesion and atte-nuated motility of ADD1-overexpressing lung cancer cells. Control and ADD1-over-expressing H1299 cells were transfected with either Src or non-targeted siRNAs and were analyzed on day 3 post-transfection.
(A) Immunoblotting analysis shows the ex-pression of Src and p-paxillin in the control and ADD1-overexpressing cells transfected with either non-targeting or Src-specific siRNAs. (B) Immunolabeling analysis of NM IIB in ADD1-overexpressing lung cancer cells transfected with either control or Src-specific siRNAs. Arrows show long NM IIB-rich protrusions in ADD1-overexpressing cells that disappear after Src depletion. (C, D) Representative images and quantifica-tion of collagen I matrix adhesion of the control and ADD1-overexpressing H1299 cells transfected with either control or Src-specific siRNAs. (E, F) Representative images and quantification analysis of transfilter migration of control and ADD1-overexpressing H1299 cells transfected with either control or Src-specific siRNAs. Data are presented as mean ± SE (n = 3); **p < 0.005.
previous studies showing that adducins promoted actin filament as-sembly in intestinal and renal epithelial cells [31,63] and that loss of ADD1 increased F-actin dynamics in the neuronal growth cone . Furthermore, the described eﬀects of ADD1 on the actin cytoskeleton in live cells are consistent with the known ability of adducins to bundle and cap actin filaments in cell-free systems [18–21]. Therefore, a functional outcome of the ADD1 overexpression could be creation of stable F-actin bundles. On the basal side of the cells, these F-actin bundles promote FA assembly and increase cell ECM attachment [54,55,64]. It is unclear why the formation of peripheral F-actin bun-dles requires the high level of ADD1 expression. One possibility is that some optimal actin-ADD1 stoichiometry should be achieved, while another possibility is that ADD1 competes with other highly-expressed actin-bundling and capping proteins for binding to actin filaments.
4.4. Adducins are novel regulators of cadherin-11 expression in lung cancer cells
One of the most surprising findings of this study is that adducins modulate NSCLC cell motility by regulating expression of cadherin-11. Indeed, loss of ADD1 and ADD3 resulted in marked increase of cad-herin-11 expression in epithelial-type H1573 cells, which played a causal role in the accelerated motility of adducin-depleted cells (Fig. 8, Suppl. Fig. 9). Furthermore, ADD1 overexpression downregulated cadherin-11 expression in mesenchymal H1299 cells (data not shown), which could synergize with the increased ECM adhesion in attenuating motility of these cells. Cadherin-11 is a member of the cadherin family of adhesion proteins and is known to be essential for embryonic de-velopment and tumorigenesis . Cadherin-11 is highly expressed in