br p paxillin Tyr labeling in the control
(p) paxillin (Tyr118) labeling in the control and ADD1-overexpressing H1299 cells. Arrows indicate small peripherally located focal adhesions in the control cells. Arrowheads point at enlarged focal adhe-sions in ADD1-overexpressing cells. (E) Immunoblotting analysis of expression and phosphorylation of major focal adhesion proteins in the control and ADD1-over-expressing H1299 cells. Data are presented as mean ± SE (n = 3); *p < 0.05; **p < 0.005, as compared to the control group.
4.1. Altered expression and localization of adducins in NSCLC and other cancers
Adducins are scaﬀolding proteins, best known for their roles in regulating the organization and dynamics of the plasma membrane-associated cytoskeleton [22,23]. They are dysregulated in diﬀerent types of solid tumors, although functional roles of adducins in tumor-igenesis remain poorly characterized . The present study identifies adducins as potent inhibitors of NSCLC cell motility and reveals non-canonical mechanisms of their actions involving the regulation of ECM adhesion signaling and cell-cell adhesion proteins. Importantly, our data suggest that functional activities of adducins are impaired in NSCLC cells. First, mRNA expression of ADD1 and ADD3 was sig-nificantly decreased in lung adenocarcinoma patient samples as
compared to normal human lung tissue (Fig. 1A). Second, the level of ADD3 protein was dramatically decreased in mesenchymal NSCLC cells, the most aggressive and metastatic type of lung cancer (Fig. 1B, C; Suppl. Fig. 1). Finally, ADD1 was translocated from the cell cortex into cytoplasmic and nuclear compartments of mesenchymal-type NSCLC cells (Fig. 1B, C; Suppl. Fig. 1). Such mislocalization of ADD1 could be due to phosphorylation by oncogenic kinases that weaken its interac-tions with cortical Mocetinostat (MGCD0103, MG0103) filaments and spectrin oligomers, although this suggestion has not been proved experimentally. Since deletion of ADD1 and ADD3 potently promoted migration and invasion of NSCLC and non-transformed lung epithelial cells (Figs. 2 & 3; Suppl. Figs. 2 & 3), it is reasonable to suggest that adducin dysfunctions due to either ex-pressional downregulation or mislocalization contribute to the in-creased motility of mesenchymal NSCLC cells. Importantly, previous studies demonstrated that loss of adducins blocked the formation of the lateral plasma membrane domain and impaired assembly of
Fig. 6. ADD1 overexpression induces the formation of long actomyosin-rich protrusions. A dual immunofluorescence analysis of F-actin (red) and non-muscle myosin IIB (NM IIB, green) in the control and ADD1-overexpressing H1299 cells. Arrows show long protrusions enriched in F-actin and NMIIB induced by ADD1 expression.
intercellular junctions in normal epithelial and cancer cells [29–31,33]. These findings highlight another possible consequence of adducin dysfunctions in lung cancer, which is destabilization of intercellular contacts. Collectively, our present and published data suggest that the net eﬀect of the impaired functional activity of adducins would be in-creased metastatic dissemination of NSCLC cells. This suggestion could be extended to other types of cancers characterized by the diminished expression and altered localization of adducins. Indeed, loss of ADD3 was reported in human glioma patients [60,61], whereas ADD1 was downregulated in ovarian cancer cell lines as compared to normal human ovarian surface epithelium . Additional evidence were provided by animal cancer model studies where decreased expression and mislocalization of ADD1 and ADD3 were found in rat renal carci-noma [28,46] and alternative splicing of ADD3 was associated with highly metastatic murine breast tumor . While these clinical and experimental evidence suggest that adducins could act as a general suppressor of tumor metastasis, our study provides the first mechanistic insights that may explain the antimetastatic activity of these proteins.
4.2. ADD1 is a potent suppressor of lung cancer cell motility
Our findings primarily implicate ADD1 in the regulation of lung cancer cell motility, while providing suggestive evidence about anti-migratory activity of ADD3. Indeed, knockout of either ADD1 or ADD3 that increased migration and invasion of normal lung epithelial and NSCLC cell, resulted in simultaneous down-regulation of both adducin isoforms (Figs. 2 & 3; Suppl. Figs. 2 & 3). On the other hand, ADD1 but not ADD3 overexpression inhibited the motility of mesenchymal NSCLC cells (Fig. 4). It is generally believed that ADD1 and ADD3 stabilize each other by forming heterodimers or heterotetramers [22,23] and our present and previous results with genetic depletion of diﬀerent ad-ducins support this postulate . However, under certain conditions, ADD1 appears to be stable in the absence of other adducin isoforms. For example, mesenchymal NSCLC cells demonstrated high expression of ADD1 along with the very low level of ADD3 and undetectable ADD2 protein (Fig. 1; Suppl. Fig. 1 and data not shown). Overexpression of exogenous ADD1 in these cells just marginally increased ADD3 level (Fig. 4A). Furthermore, expression of the ADD1 protein was minimally