The paxillin-plectin-EPLIN complex promotes apical elimination of RasV12-transformed cells by modulating HDAC6-regulated tubulin acetylation

Recent studies have revealed that newly emerging RasV12-transformed cells are often apically extruded from the epithelial layer. During this cancer preventive process, cytoskeletal proteins plectin and Epithelial Protein Lost In Neoplasm (EPLIN) are accumulated in RasV12 cells that are surrounded by normal cells, which positively regulate the apical elimination of transformed cells. However, the downstream regulators of the plectin-EPLIN complex remain to be identified. In this study, we have found that paxillin binds to EPLIN specifically in the mix culture of normal and RasV12-transformed cells. In addition, paxillin is accumulated in RasV12 cells surrounded by normal cells. Paxillin, plectin and EPLIN mutually influence their non-cell-autonomous accumulation, and paxillin plays a crucial role in apical extrusion of RasV12 cells. We also demonstrate that in RasV12 cells surrounded by normal cells, acetylated tubulin is accumulated. Furthermore, acetylation of tubulin is promoted by paxillin that suppresses the activity of histone deacetylase (HDAC) 6. Collectively, these results indicate that in concert with plectin and EPLIN, paxillin positively regulates apical extrusion of RasV12-transformed cells by promoting microtubule acetylation. This study shed light on the unexplored events occurring at the initial stage of carcinogenesis and would potentially lead to a novel type of cancer preventive medicine.

At the initial stage of carcinogenesis, an oncogenic mutation occurs in single cells within the epithelium. Recent studies have revealed that the newly emerging transformed cells and the surrounding normal epithelial cells often compete with each other for survival [1][2][3][4][5][6][7][8][9][10] . This phenomenon is called cell competition; the loser cells are eliminated from epithelial tissues, while the winner cells proliferate and fill the vacant spaces. By using Madin-Darby canine kidney (MDCK) epithelial cells stably expressing RasV12 in a tetracycline-inducible manner, we have demonstrated that when Ras-transformed cells appear within the epithelial monolayer, the transformed cells are extruded into the apical lumen of the epithelium in a cell death-independent fashion, a process called apical extrusion 11 . Together with other studies, it has become evident that normal epithelial cells can recognize and actively eliminate the neighbouring transformed cells from epithelial tissues via cell competition. This cancer preventive mechanism is termed Epithelial Defense Against Cancer (EDAC) 12,13 . In the cell competition between normal and RasV12-transformed epithelial cells, the presence of normal cells profoundly influences various cellular processes and signalling pathways in the neighbouring transformed cells, which positively regulate their apical extrusion. In the previous studies, we have reported that cytoskeletal proteins plectin and Epithelial Protein Lost In Neoplasm (EPLIN) are accumulated in RasV12 cells when they are surrounded by normal cells 14,15 . The plectin-EPLIN complex then induces α-tubulin polymerization, leading to the accumulation of microtubule filaments. This process plays a crucial role in the apical extrusion of RasV12 cells, however the molecular mechanism of how plectin and EPLIN regulate the organization of microtubules remains unknown.
The structure and physical property of microtubule filaments are dynamically regulated by various mechanisms including acetylation of α-tubulin K40 16,17 . In addition, acetylation of tubulin can also influence a variety of cellular processes including vesicle transport, signalling pathways and cell migration 18,19 . Acetylation of tubulin is catalysed by α-tubulin acetyltransferase (αTAT) 1 20,21 , while deacetylation is mediated by histone deacetylase (HDAC) 6 22,23 and sirtuin (SIRT) 2 24 . The activity of HDAC6 can be regulated by multiple mechanisms such as suppression by paxillin 25 . Paxillin is one of the key adaptor proteins in the integrin-based focal adhesion complex 26 . But, additionally, paxillin localizes in the cytosol and can play other cellular functions 25 .
In this study, we have found that paxillin is a vital regulator of apical extrusion of RasV12-transformed cells by linking the plectin-EPLIN complex and acetylation of microtubules. (a) Co-immunoprecipitation of EPLIN with paxillin. MM, normal MDCK cells cultured alone; MR, 1:1 mix culture of normal MDCK and MDCK-pTR GFP-RasV12 cells; RR, MDCK-pTR GFP-RasV12 cells cultured alone. (b) Immunofluorescence images of paxillin. MDCK-pTR GFP-RasV12 cells were mixed with normal MDCK cells or cultured alone on collagen gels. Cells were fixed after 16 h incubation with tetracycline and stained with anti-paxillin antibody (grey) and Hoechst (blue). Scale bar, 10 μM. (c) Quantification of the fluorescence intensity of paxillin. Data are mean ± SD from three independent experiments. *P < 0.05, n.s., not significant; n ≧ 30 cells for each experimental condition. Values are expressed as a ratio relative to MDCK cells. Note that accumulation of paxillin is also observed in the 1:1 mixed culture by immunofluorescence, but rather moderately. In the 1:1 mixed culture condition, both normal and transformed cells are included, and certain fractions of transformed cells do not directly interact with normal cells. Therefore, non-cellautonomous changes in transformed cells are diluted by the presence of normal cells and transformed cells that do not interact with normal cells, and thus often difficult to be detected biochemically in the 1:1 mixed culture condition.

Results
Paxillin plays a crucial role in apical elimination of RasV12-transformed cells. EPLIN and plectin are accumulated in RasV12-transformed cells surrounded by normal cells and play a vital role in apical extrusion of the transformed cells 14,15 . In a previous study, EPLIN was shown to interact with paxillin 27 . We thus examined the interaction between EPLIN and paxillin in our in vitro cell competition model system 11 . Paxillin was co-immunoprecipitated with EPLIN, and the interaction was enhanced under the mix culture condition of normal and RasV12 cells (Fig. 1a). In addition, by immunofluorescence, we demonstrated that paxillin was accumulated and partially co-localized with EPLIN in RasV12 cells that were surrounded by normal cells, but not in RasV12 cells cultured alone (Figs 1b,c, 2a and 3a).
To examine the functional role of paxillin, we have established RasV12-transformed cells stably expressing paxillin-shRNA1 or -shRNA2 ( Supplementary Fig. S1a). Knockdown of paxillin substantially diminished the accumulation of EPLIN in RasV12 cells that were surrounded by normal cells (Fig. 2a S1b). In addition, paxillin-knockdown also suppressed accumulation of plectin in RasV12 cells surrounded by normal cells (Fig. 2c,d and Supplementary Fig. S1c). Conversely, knockdown of EPLIN (Fig. 3a,b) 15 or plectin (Fig. 3c,d) 14 significantly suppressed accumulation of paxillin. When RasV12 cells were cultured alone, knockdown of paxillin or EPLIN did not affect expression of the other proteins ( Supplementary Fig. S1d,e). Collectively, these results indicate that paxillin, plectin and EPLIN mutually influence their non-cell-autonomous accumulation in RasV12 cells. We next examined whether knockdown of paxillin affects the fate of RasV12-transformed cells upon cell competition with the surrounding normal cells. Knockdown of paxillin strongly suppressed apical extrusion of RasV12 cells, and most of paxillin-knockdown RasV12 cells remained within the epithelium (Fig. 4a,b), indicating that paxillin is a crucial regulator for the elimination of the transformed cells.

Acetylation of tubulin is enhanced in RasV12-transformed cells surrounded by normal cells.
In a previous study, we have reported that α-tubulin, a major component of microtubules, accumulates at the apical domain of RasV12-transformed cells that are surrounded by normal cells 14 . Plectin and EPLIN regulate the accumulation of α-tubulin, but the molecular linkage between the plectin-EPLIN complex and tubulin accumulation remains unclear. The organization of microtubule filaments is often regulated by tubulin acetylation 16,17 . We thus examined acetylation of α-tubulin by immunofluorescence. In RasV12-transformed cells, accumulation of acetylated tubulin was mainly observed in the apical region, which overlapped with that of tubulin (Fig. 5a). Moreover, acetylation of α-tubulin was substantially elevated when RasV12 cells were surrounded by normal cells, compared with that in RasV12 cells cultured alone (Fig. 5a,b), indicating the non-cell-autonomous upregulation of tubulin acetylation in RasV12 cells. Acetylation of α-tubulin can be regulated by deacetylases: HDAC6 and SIRT2 14 . We then examined the effect of the inhibitor for HDAC6 (tubacin) or SIRT2 (AGK2). Acetylation of α-tubulin in RasV12 cells was strongly enhanced by tubacin, but not by AGK2 (Fig. 5c). Furthermore, treatment of tubacin, but not AGK2, significantly promoted apical extrusion of RasV12 cells (Fig. 5d), suggesting that acetylation of tubulin in RasV12 cells may be regulated by HDAC6, which plays a positive role in the elimination of transformed cells from epithelia.

Paxillin regulates tubulin acetylation thereby promoting apical extrusion of RasV12-transformed cells.
A previous study demonstrated that paxillin positively regulates acetylation of α-tubulin by suppressing HDAC6 25 . We found that paxillin was partially colocalised with acetylated α-tubulin in RasV12 cells that were surrounded by normal cells (Fig. 6a). In addition, knockdown of paxillin profoundly diminished the accumulation of acetylated tubulin (Fig. 6b), demonstrating that paxillin is a crucial upstream regulator of tubulin acetylation upon cell competition between normal and RasV12 cells. Knockdown of EPLIN or plectin also significantly suppressed the accumulation of acetylated tubulin ( Supplementary Fig. S2a,b). Furthermore, tubacin restored accumulation of acetylated tubulin in paxillin-knockdown cells (Fig. 7a,b), suggesting that paxillin regulates tubulin acetylation by suppressing HDAC6. Moreover, tubacin partially rescued the inhibitory effect of paxillin-knockdown on the apical extrusion of RasV12 cells (Fig. 7c). Collectively, these data indicate that HDAC6-regulated tubulin acetylation is one of the downstream effectors of the paxillin-plectin-EPLIN complex in the apical elimination of transformed cells (Fig. 7d).

Discussion
In this study, we have demonstrated that paxillin is a novel regulator for the elimination of RasV12-transformed cells from the epithelium. Paxillin regulates the accumulation of other regulators plectin and EPLIN, and vice versa. In addition, paxillin, in concert with plectin and EPLIN, induces acetylation of α-tubulin, leading to reorganization of microtubule filaments, at least partly, via HDAC6 (Fig. 7d). Plectin and paxillin bind to microtubules and/or HDAC6; thus the paxillin-plectin-EPLIN complex could act as a scaffolding-platform that efficiently induces the HDAC6-mediated acetylation of tubulin. However, suppression of the HDAC6 activity only partially rescued the inhibitory effect of paxillin-knockdown on the apical extrusion of RasV12 cells, suggesting that other molecules may also function downstream of the paxillin-plectin-EPLIN complex.
It still remains unclear how the accumulation of the paxillin-plectin-EPLIN complex is regulated and how tubulin acetylation positively regulates apical extrusion of transformed cells. We have previously reported that at the boundary between normal and transformed epithelial cells, various non-cell autonomous changes occur in both cells. For example, Rab5-mediated endocytosis is enhanced in RasV12 cells when they are surrounded by normal cells 28 . In addition, Warburg effect-like metabolic changes, increased glycolysis and decreased mitochondrial activity, occur in RasV12 cells neighbouring normal cells 29 . Furthermore, cytoskeletal proteins filamin and vimentin accumulate in normal cells at the interface with transformed cells, providing physical forces for apical extrusion 12 . In future studies, it needs to be elucidated whether and how these processes function upstream or downstream of the paxillin-plectin-EPLIN complex.
Apical extrusion of transformed cells can be observed in vivo as well, and the extruded transformed cells disappear from the tissues 29 , implying that apical extrusion is a cancer preventive phenomenon. Therefore, the molecules governing this process could be potential therapeutic targets for cancer preventive medicine. Expression of HDAC6 is upregulated in various cancer cells 30,31 . In addition, inhibition of the HDAC6 activity can suppress tumourigenesis and diminish tumor cell migration 32,33 . Thus, HDAC6 currently attracts substantial attention as one of the potential drug targets for cancer treatment [34][35][36] . Our data suggest that HDAC6 inhibitor could facilitate the eradication of potentially precancerous cells at the initial stage of carcinogenesis, implying that HDAC6 inhibitor can be applied not only to cancer treatment, but also cancer prevention. Further understanding of cytoskeletal organization machineries in apical extrusion would represent an interesting challenge for biological fields and cancer preventive medicine.
Cell Culture. MDCK and MDCK-pTR GFP-RasV12 cells were cultured as previously described 11 .
To induce the expression of GFP-RasV12, the tetracycline-inducible MDCK-pTR GFP-RasV12 cell lines were treated with 2 μg ml −1 tetracycline (Sigma-Aldrich). For inhibitor treatment, the indicated inhibitors were simultaneously added together with tetracycline, and then cells were further cultured for 16 h or 24 h. For immunofluorescence, cells were seeded onto Type-I collagen-mounted coverslips as described below in the section of immunofluorescence.
Immunofluorescence. MDCK-pTR GFP-RasV12 cells were mixed with MDCK cells at a ratio of 1:50 and cultured on the collagen matrix as previously described 11 . For immunofluorescence analyses, the mixture of cells was incubated for 8-12 h until they formed a monolayer, followed by tetracycline treatment for 16 h. Cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.5% Triton X-100 in PBS, followed by blocking with 1% BSA in PBS. Primary or secondary antibodies were incubated for 2 h or 1 h, respectively at ambient temperature. Immunofluorescence images were acquired by the Olympus FV1200 system and the Olympus FV10-ASW software. For quantification of immunofluorescence intensity, 30 transformed cells were analysed for each experiment using the MetaMorph software (Molecular Devices). For analyses of apical extrusions, the samples were prepared as described above, except that cells were treated with tetracycline for 24 h (for Fig. 5d, apical extrusion was observed after 16 h of tetracycline). More than 95 cells were analysed for each experiment, and apically extruded cells were quantified.
Immunoprecipitation and western blotting. For immunoprecipitation, 0.6 × 10 7 MDCK cells and 0.6 × 10 7 MDCK-pTR GFP-RasV12 cells for mix culture or 1.2 × 10 7 MDCK or MDCK-pTR GFP-RasV12 cells for single culture were seeded in 14.5-cm dishes (two dishes for each experimental condition) (Greiner-Bio-One) and cultured at 37 °C for 6-8 h until a monolayer was formed. Tetracycline was then added to induce RasV12 expression. After 16 h culture with tetracycline, cells were washed with ice-cold PBS containing 1 mM Na 3 VO 4 and lysed for 30 min in NP-40 lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl and 1% NP-40) containing the following inhibitors: 1 mM Na 3 VO 4 , 0.1 mM Na 2 MoO 4 , 10 mM NaF, 5 μg ml −1 leupeptin, 1 mM phenylmethylsulfonyl fluoride and 7.2 trypsin inhibitor units of aprotinin. After centrifugation of the cell lysates at 21,500 g for 10 min, the supernatant was first pre-cleared with sepharose 4B (Sigma-Aldrich) at 4 °C for 30 min. This step was repeated three times. The pre-cleared cell lysates were then incubated with control IgG-conjugated Dynabeads protein G (Life Technologies) at 4 °C for 30 min and finally subjected to immunoprecipitation for 1 h with Dynabeads Protein G conjugated to rabbit anti-paxillin antibody (10 μg). Immunoprecipitated proteins were subjected to SDS-PAGE, followed by western blotting with the indicated antibodies. Western blotting data were acquired using ImageQuant TM LAS4010 (GE healthcare). To examine the efficiency of paxillin-knockdown, MDCK-pTR GFP-RasV12 cells stably expressing paxillin-shRNA were seeded onto 6-cm dishes (Greiner-Bio-One) at the density of 1 × 10 6 cells. After 24 h, the incubated cells were lysed with Triton X-100 lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl and 1% Triton X-100) containing protease inhibitors (5 μg ml −1 leupeptin, 1 mM phenylmethylsulfonyl fluoride and 7.2 trypsin inhibitor units of aprotinin) and directly boiled with SDS-PAGE sample buffer. Data Analyses. Two-tailed Student's t-tests were used to determine P-values for statistical analyses. Data Availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.