br Downregulation of ADD and ADD enhances
3.2. Downregulation of ADD1 and ADD3 enhances individual migration of NSCLC cells
To investigate the roles of adducins in the regulation of lung cancer cell motility, we deleted either ADD1 or ADD3 in epithelial-type H1573 and 16HBE14o Ko 143 by using CRISPR/Cas9-mediated gene editing. Cells were transfected with either non-targeted guide RNA or four dif-ferent single guide (sg) RNA oligonucleotides against each adducin isoform (ADD1 or ADD3), and stable knockout cell lines were obtained by puromycin treatment. This approach resulted in variable levels of adducin depletion (Figs. 2A, 3A, Suppl. Figs. 2 & 3). Two H1573 cell lines with the most eﬃcient knockout of ADD1 (transfected with sgRNAs 2 and 3) and two stable cell lines with deepest ADD3 depletion (created by sgRNAs 4 and 5) were used for subsequent functional ex-periments. It is noteworthy that loss of ADD1 markedly decreased ADD3 protein level, and vice versa (Figs. 2 & 3; Suppl. Figs. 2 & 3). This phenomenon was previously described in adducin-depleted colonic epithelial cells  and tissues of either ADD1 or ADD3 null mice [50,51] and could be due to stabilizing eﬀects of the ADD1/ADD3 hetero-oligomerization, protecting these proteins from proteolytic de-gradation. Therefore, the outcome of either ADD1 or ADD3 knockout in bronchial epithelial and lung cancer cells was a dual ADD1/ADD3 de-pletion.
The roles of adducins in regulating NSCLC cell motility were de-termined by examining three diﬀerent modes of cell migration: col-lective cell migration during scratch wound closure, chemotactic transfilter migration of individual cells in the Boyden chamber, and cell invasion into Matrigel. Loss of ADD1 in H1573 cells had no eﬀect on wound closure (Fig. 2B), but significantly accelerated transfilter mi-gration and Matrigel invasion of these cells (Fig. 2C–F). Likewise, ADD3 knockout accelerated individual migration and invasion of NSCLC cells without aﬀecting their wound closure (Fig. 3). Consistently, knockout of ADD1 and ADD3 accelerated transfilter migration, but not wound healing of non-tumorigenic 16HBE14o cells (Suppl. Figs. 2 & 3).
3.3. Overexpression of ADD1 inhibits migration and invasion of mesenchymal NSCLC cells
Next, a gain-of-function approach was used to collaborate the re-sults obtained with adducins knockout in lung epithelial cells. H1299 cells, a known mesenchymal-type NSCLC cell line was chosen for stable overexpression of either FLAG-tagged ADD1, ADD3, or a control plasmid. Immunoblotting analysis demonstrated successful over-expression of either adducin isoform (Fig. 4A). Interestingly, while ADD3 overexpression also caused an approximately 3-fold increase in ADD1 level, the level of endogenous ADD3 was just marginally (~1.5 fold) increased in ADD1-overexpressing H1299 cells (Fig. 4A). This suggests that other mechanisms besides decreased protein stability could be responsible for low ADD3 expression in mesenchymal NSCLC cells. ADD1 overexpression significantly inhibited wound closure (Fig. 4B), transfilter migration (Fig. 4C, D) and Matrigel invasion (Fig. 4E, F) of H1299 cells. By contrast, ADD3 overexpression did not
Fig. 1. Altered expression and localization of ad-ducins in lung cancer cells. (A) A comparative ana-lysis of ADD1 and ADD3 mRNA expression levels in normal (n = 59) and tumor (n = 457) tissues from patients with lung adenocarcinoma. Error bars in-dicate the median absolute deviations. (B) Immunoblotting analysis of adducins expression in epithelial and mesenchymal-type non-small cell lung cancer cells. (C) Immunofluorescence labeling of ADD1 and ADD3 in non-tumorigenic bronchial epi-thelial cells (16HBE14o), epithelial-type (H1573) and mesenchymal-type (H1299) lung cancer cells. Arrows point on plasma membrane localization of ADD1 and ADD3 in epithelial-type cells. Arrowheads indicate cytoplasmic and nuclear localization of ADD1 in mesenchymal-type lung cancer cells.
aﬀect any of the examined modes of NSCLC cell migration (Fig. 4). Since both ADD1 and ADD3 knockouts decreased ADD1 expression and ADD1, but not ADD3 overexpression markedly elevated ADD1 level, results of our experiments suggest that ADD1 played a major role in suppressing migration and invasion of NSCLC cells.
3.4. Overexpression of ADD1 increases ECM adhesion and stimulates focal adhesion assembly
The next series of experiments were designed to elucidate molecular mechanisms that underline the anti-migratory function of ADD1 in
NSCLC cells. Since ADD1 was previously implicated in the control of mitosis [30,45], we examined the possibility that adducins regulate cell migration indirectly, by altering cell proliferation. However, MTT assay of either ADD1- and ADD3-depleted, or ADD1-overexpressing cells and their appropriate controls did not show significant eﬀects of adducins on NSCLC cell proliferation (Suppl. Fig. 4). Adhesion to the ECM is known to be essential for individual and collective epithelial cell mi-gration. Therefore, we asked if this process is aﬀected by altered ADD1 expression. Overexpression of ADD1 caused an almost two-fold increase in H1299 cell adhesion to collagen I (Fig. 5A, B), and fibronectin (data not shown). By contrast, knockout of ADD1 and ADD3 did not change