Oncogene (2014) 33, 2363–2374 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc

ORIGINAL ARTICLE Desmoglein 3 promotes cancer cell migration and invasion by regulating activator protein 1 and protein kinase C-dependent-Ezrin activation

L Brown1, A Waseem1, IN Cruz2, J Szary1, E Gunic1, T Mannan1, M Unadkat1, M Yang2, F Valderrama3,EAO0Toole4 and H Wan1

Desmoglein 3 (Dsg3), the vulgaris antigen, has recently been shown to be upregulated in squamous cell carcinoma (SCC) and has been identified as a good tumor-specific marker for clinical staging of cervical sentinel lymph nodes in head and neck SCC. However, little is known about its biological function in cancer. The actin-binding protein Ezrin and the activator protein 1 (AP-1) transcription factor are implicated in cancer progression and metastasis. Here, we report that Dsg3 regulates the activity of c-Jun/AP-1 as well as protein kinase C (PKC)-mediated phosphorylation of Ezrin-Thr567, which contributes to the accelerated motility of cancer cells. Ectopic expression of Dsg3 in cancer cell lines caused enhanced phosphorylation at Ezrin-Thr567 with concomitant augmented membrane protrusions, cell spreading and invasive phenotype. We showed that Dsg3 formed a complex with Ezrin at the plasma membrane that was required for its proper function of interacting with F-actin and CD44 as Dsg3 knockdown impaired these associations. The increased Ezrin phosphorylation in Dsg3-overexpressing cells could be abrogated substantially by various pharmacological inhibitors for Ser/Thr kinases, including PKC and Rho kinase that are known to activate Ezrin. Furthermore, a marked increase in c-Jun S63 phosphorylation, among others, was found in Dsg3-overexpressing cells and the activation of c-Jun/AP-1 was further supported by a luciferase reporter assay. Taken together, our study identifies a novel Dsg3- mediated c-Jun/AP-1 regulatory mechanism and PKC-dependent Ezrin phosphorylation that could be responsible for Dsg3- associated cancer metastasis.

Oncogene (2014) 33, 2363–2374; doi:10.1038/onc.2013.186; published online 10 June 2013 Keywords: desmoglein; ERM proteins; AP-1 transcription factor; cell migration and invasion; keratinocytes

INTRODUCTION of cervical sentinel lymph nodes in the head and neck SCC.18–20 Desmoglein 3 (Dsg3), the antigen, is one of These findings suggest strongly a pro-metastatic role for Dsg3 in seven transmembrane desmosomal .1,2 Although a large tumor cell biology; however, the underlying molecular mechanism body of evidence suggests that Dsg3 has a paramount role in cell– remains poorly understood. ,1 an in vitro study showed that it mediates weak Ezrin is a member of the Ezrin/Radixin/Moesin (ERM) proteins homophilic cell–cell adhesion.3 There are two pools of Dsg3 that act as plasma membrane-actin linkers and are enriched in protein existing in epithelial cells, non-junctional (Triton soluble) microvilli, ruffles, filopodia, uropods and cell adhesion sites. The and junctional (Triton insoluble); the latter is associated with the ERM proteins exist in two forms, active and inactive. The inactive .4–8 Our previous studies show that non-junctional proteins predominantly localize in the cytosol21,22 and are present Dsg3 is involved in E- signaling via Src and Rac1/Cdc42, in a closed conformation. Recruitment of these proteins to the suggesting cross talk between the desmosomal and classical plasma membrane leads to the release of intramolecular cadherins in epithelial cells.8–10 interactions, exposing the binding site for actin at their Alterations in the expression of desmosomal components could C-terminus and for plasma membrane proteins such as contribute to cancer progression similar to E-cadherin;11–13 CD44,23,24 or adaptor proteins such as EBP5025 at the however, the correlation between the expression levels of N-terminus. Phosphatidylinositol 4,5-bisphosphate binding and desmosomal components and the state of tumor development phosphorylation of Ezrin on Thr567 are involved in this activation has not been fully established. Recent studies have shown process.26–28 Protein kinase C (PKC) and Rho kinase (ROCK) are upregulation of Dsg3 in squamous cell carcinoma (SCC) in responsible for Ezrin-Thr567 phosphorylation29–36 and also various tissues, particularly in the head and neck and lungs, activation of the transcriptional factor activator protein 1 (AP-1) which is correlated with T stage, N stage and overall stage.14–18 downstream that regulates the expression of many genes, Moreover, DSG3 messenger RNA and protein have been identified including those encoding for Ezrin and CD44, which are as the best discriminatory (with 100% accuracy) biomarker among necessary for implementation of the invasion program.37 Ezrin 40 potential candidate genes for accurate intraoperative staging participates in diverse cellular functions including cell adhesion,

1Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Centre for Clinical and Diagnostic Oral Sciences, Institute of Dentistry, London, UK; 2Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, London, UK; 3Division of Biomedical Sciences, St George’s, University of London, Cranmer Terrace, London, UK and 4Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Centre for Cutaneous Research, Blizard Institute, London, UK. Correspondence: Dr H Wan, Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Centre for Clinical and Diagnostic Oral Sciences, Institute of Dentistry, Blizard Building, Whitechapel, London E1 2AT, UK. E-mail:[email protected] Received 8 May 2012; revised 12 February 2013; accepted 4 April 2013; published online 10 June 2013 Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2364 polarization and morphogenesis,21,38,39 and more importantly has significantly greater number of cell clusters invading been identified as a potent regulator of tumor cell invasion and into the Matrigel:collagen matrix compared with control cells metastasis.32,33,35,40–43 A positive correlation between Ezrin (Figures 2c–e). Collectively, these data suggest that Dsg3 expression and cervical lymph node metastasis and clinical promotes cancer cell migration and invasion. staging has been found in various tumors.43–47 In vitro RNA interference studies in cancer cell lines have demonstrated that knocking down Ezrin expression suppresses cell growth, invasion, Dsg3 associates with Ezrin at the plasma membrane tumor progression and metastasis,48–50 a phenotype that overlaps Our preliminary observation of the Dsg3/Ezrin colocalization10 led strikingly with that of Dsg3 silencing.14 us to hypothesize that Dsg3 may have a role in regulating cell In this study, we investigated the potential link between Dsg3 morphology and migratory behavior through a mechanism signaling and Ezrin activation, and describe a novel phosphoryla- involving Ezrin. To address this question, we, first of all, analyzed tion mechanism of the ERM proteins, primarily Ezrin-Thr567 that is protein–protein interaction by the proximity ligation assay (PLA) regulated by Dsg3 in a PKC-dependent manner in A431 cells. We and demonstrated a marked increase of PLA signals that were showed that non-junctional Dsg3 forms a complex with and is generated by close proximity of Dsg3/Ezrin in Dsg3-overexpres- capable of activating Ezrin, which regulates actin-based cell shape sing cells compared with control (Figure 3a). In line with this, the change and migratory behavior. We confirmed that this pathway fluorescence resonance energy transfer (FRET) analysis of A431-D3 can be abrogated by inhibition of various signaling molecules cells showed positive FRET (B14.6% of regions of interest) including PKCs. Finally, we demonstrated a strong correlation predominantly localized to the membrane projections between AP-1 activity and Dsg3 expression levels in cells. (Figure 3b). Furthermore, the association between Dsg3/Ezrin Collectively, our study uncovers a novel signaling role of Dsg3 was verified by co-immunoprecipitation assay, demonstrating that acts as a cell surface activator of AP-1 and the PKC/Ezrin reproducibly that both endogenous and ectopic Dsg3 formed a pathway that promotes cancer cell migration and invasion. complex with Ezrin in A431 cells in a Dsg3 dose-dependent manner (Figures 3c and d). To determine whether the association exists in the detergent soluble pool, that is, non-junctional Dsg3, RESULTS we analyzed the association in Triton-soluble and -insoluble Dsg3 induces morphological change and promotes membrane (NP-40 soluble) fractions by immunofluorescence and co-immu- protrusion and cell spreading noprecipitation. For immunofluorescence, cells were treated with To establish the role of Dsg3 in adhesion-induced membrane or without Triton buffer before fixation and immunostaining. Cells morphogenesis,51 we analyzed A431-hDsg3.myc cells10 and its treated with Triton showed significant reduction of phospho-ERM derived clones with high or low Dsg3 levels and also an oral SCC and its colocalization with Dsg3, particularly at the cell borders line, SqCC/Y113 with transduced hDsg3.myc (Sq-D3). Recently, we (Supplementary Figure 3a and b). In support, co-immunoprecipi- have shown that Dsg3 functions as an upstream regulator of tation demonstrated that the association predominantly existed in Rac1/Cdc42 in the regulation of actin organization and dynamics.9 Triton-soluble fractions in all cell lines (Supplementary Figure 3c). In this study, we performed immunostaining for Ezrin and analyzed Together, these results suggest that non-junctional Dsg3 forms a membrane coverage with Ezrin-positive protrusions. We showed complex with Ezrin outside of desmosomes. In order to search for that cells with high Dsg3 levels exhibited exaggerated membrane other potential-binding partners, mass spectrometry was applied. protrusions and cell spreading (Figure 1a and Supplementary A construct of the cytoplasmic tail of Dsg3 tagged with Halo at the Figure 1). Consistently, the transient transfection of hDsg3.myc N-terminus was cloned into pFN21A (Promega, Southampton, UK) construct in 293T cells resulted in striking membrane branching and the plasmid or Halo vector control (Vect) was transfected into and micro-spikes compared with green fluorescent protein control A431 parental cells. The lysates were subjected to Halo tag cells (Supplementary Figure 1b). To demonstrate the specificity of purification and the resulting complex was analyzed using one- the phenotype, Dsg3 in both A431-V and -D3 were depleted by dimensional SDS–polyacrylamide gel electrophoresis proteomics RNA interference, and subsequent immunostaining showed that approach. Proteins were separated using SDS–polyacrylamide gel cells with Dsg3 depletion displayed remarkable collapse of the electrophoresis and bands were digested by trypsin before further membrane protrusions and a concomitant inhibition of E-cadherin analyzed by liquid chromatography-tandem mass spectrometry junction assembly (Figures 1b and c). These findings are consistent (Supplementary Figure 4). Actin was identified as the major with our hypothesis that Dsg3 is involved, at least in part, in the component present in the test sample but not in the empty vector regulation of actin organization.9 To address whether Dsg3 (Supplementary Figure 4d). overexpression enhances cell–cell adhesion, we performed the Image analyses showed that changes in Dsg3 levels directly hanging drop assay (Supplementary Figure 2) and consistently affected its colocalization with Ezrin (also phosphorylated form; showed that overexpression failed to enhance cell–cell adhesion, Supplementary Figures 5 and 6) indicating an association between as we reported previously.10 them. Detailed analysis of confocal image stacks revealed that the interaction was predominantly located at the basolateral domain of the plasma membrane where membrane protrusions were Dsg3 promotes cell migration and invasion pronounced (Supplementary Figure 7). Quantitation of immuno- Previously, we have shown that overexpression of Dsg3 enhanced fluorescence intensity of Ezrin at the plasma membrane showed cell migration by a wound scratch assay.10 To complement this approximately a twofold increase in A431-D3 compared with observation, we analyzed both A431 and SqCC/Y1 stably control cells (immunofluorescence intensity/pixel in D3 vs Vect is overexpressing Dsg3 under various conditions. Transwell 66.0±15.6 vs 36.7±16.1), implying that Dsg3 overexpression migration assays indicated that all Dsg3-overexpressing cells enhances Ezrin activity. Consistent with this notion, triple staining exhibited greater cell motility, especially the A431-C2- and -C7- for Dsg3/pERM/F-actin showed a positive correlation and sig- cloned cells (Figure 2a) while knockdown of Dsg3 had no effect in nificantly enhanced colocalization of the three proteins at the cell this migration assay. Consistent with this result, a transwell borders in D3 compared with Vect cells, particularly in those invasion assay showed that Dsg3 overexpression significantly stratified cells located in the center of the colonies (Figure 4a promoted cell invasion compared with control in SqCC/Y1 cells arrows). As Ezrin activation is associated with its interaction with (Figure 2b) and furthermore, we demonstrated that the enhanced CD44 and F-actin at the plasma membrane,23,24 we evaluated the migratory activity in Sq-D3 cells could be recapitulated in an colocalization of Ezrin/F-actin or CD44/F-actinin A431-D3 with or organotypic cell invasion assay52 (Figure 2c) that showed a without Dsg3 knockdown. As shown in Figures 4a and b,

Oncogene (2014) 2363 – 2374 & 2014 Macmillan Publishers Limited Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2365

Figure 1. Overexpression of Dsg3 induces filopodia formations in A431 cells. (a) Fluorescent confocal images of Vect, polyclonal (D3) and monoclonal (C2, C7) cells labeled with mouse Ezrin antibody. Pronounced membrane protrusions were particularly seen in Dsg3- overexpressing cells. The bar chart on the right shows quantitation of the percentage of cells with membrane projections covering 450% of cell periphery in the field (experiment n43, B100 cells were analyzed from each group and are presented as mean±s.d., *Po0.05, ***Po0.001). (b) The enhanced membrane projections in A431-D3 cells ((i) epi-fluorescent microscopic image, inset: confocal image) were largely inhibited by Dsg3 knockdown (KD; arrows in (ii) and (iii)). Note that the collapse of membrane projections was coupled with disruption of E-cadherin adhesion in knock-down cells compared with control. The enlarged images of boxed areas in (ii) are displayed on the right. Scram, scrambled small interfering RNA control. (c) Western blotting of Dsg3 KD in A431-Vect and -D3 cells. Scale bars, 20 mm. a significant reduction of their colocalization was seen in RNA expression, we analyzed the phosphorylation of ERM proteins interference-treated cells compared with controls. including Ezrin-Thr567. Using a rabbit antibody (pERM) Taken together, our findings demonstrate that non-junctional specific for the conserved C-terminal phospho-threonine of Dsg3 forms a complex with Ezrin at the plasma membrane and ERM proteins, we observed a significant increase in pERM levels regulates its activity that is required for membrane projections in Dsg3-overexpressing cells compared with Vect or C11 that and cell migration/invasion.31,53 expressed low Dsg3 levels (Figure 5a). No change was seen for total ERM or Ezrin. To delineate the increased pERM was mainly due to activation of Ezrin-Thr567, we knocked down Ezrin or Dsg3 activates the ERM proteins, particularly Ezrin via the moesin in A431–C7 using an Ezrin- or moesin-specific small phosphorylation at Ezrin-Thr567 interfering RNA. Cell lysates were analyzed by western blotting The phosphorylation of Ezrin-Thr567 leads to its activation, which for pERM. In the control the upper band—Ezrin and radixin has been implicated in cell invasion and metastasis.32,35,54–57 (B81 kDa) appeared to be almost double that of the lower To show that Ezrin was activated in response to Dsg3 over- (77 kDa) phospho-moesin (Figure 5b). The RNA interference-

& 2014 Macmillan Publishers Limited Oncogene (2014) 2363 – 2374 Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2366

Figure 2. Dsg3 promotes cell migration and invasion. (a) Transwell cell migration assay of A431 (including polyclonal and monoclonal cells) and SqCC/Y1 cells showed that overexpression of Dsg3 enhanced cell migration compared with Vect cells. HaCaT (HaC) normal keratinocytes served as a negative control in this experiment. Data of A431 were pooled from three independent experiments (n ¼ 9, mean±s.e.m.). (b) Transwell cell invasion assays of SqCC/Y1 cells showed that overexpression of Dsg3 (both polyclonal and monoclonal cells) promoted cell invasion compared with control cells (mean±s.d., *Po0.05, **Po0.01, ***Po0.001). (c) Hematoxylin and eosin staining of 2-week-organotypic culture of SqCC/Y1 cells shows augmented cell invasion in SqCC/Y1 with overexpression of Dsg3 (Sq-D3; red arrow in Sq-D3). The middle panels are the representative binary images and the epithelial compartment is highlighted in red that enable better visualization of cell invasion. The lower panels are immunohistochemistry for myc-tag staining demonstrating positive staining of myc-tag in hDsg3.myc- transducing cells, but only weak nucleus staining (endogenous c-Myc) in control. (d) Quantitation of cell invasion from two independent experiments and the invaded epithelial cell clusters scored for the parameters are shown (n ¼ 13, mean±s.d., *Po0.05, ***Po0.001). (e) Cell invasion index80 indicated a significant increase in Sq-D3 cells compared with control cells (mean±s.e.m.,***Po0.001). Scale bars, 50 mm.

mediated Ezrin knockdown caused a significant reduction in the enhanced pERM indeed was phospho-Ezrin-Thr567 (Figure 5c). upper band, suggesting that Ezrin is the predominant phos- Ezrin activation was also reported to induce shape changes phorylated isoform in A431 cells (Figure 5b), as reported including cell rounding in addition to the membrane projections previously.58 Thus, it is reasonable to conclude that the in A431 cells,21 and such a rounded cell phenotype coupled with elevated pERM in A431-D3 and C7 (Figure 5a) was due to pronounced membrane protrusions was also observable in A431- increased phospho-Ezrin rather than phospho-radixin or -moesin. D3 compared with control cells or cells with low levels of Dsg3 in To further validate this finding, we performed PLA using an the same sample (Figure 5d). These results further support the antibody pair for Ezrin/pERM and showed a significant increase of notion that Dsg3 regulates Ezrin activity through phosphoryla- PLA signals in D3 compared with the control, indicating the tion at Thr567.

Oncogene (2014) 2363 – 2374 & 2014 Macmillan Publishers Limited Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2367

Figure 3. Association of Dsg3 with Ezrin. (a) In situ PLA assay in A431 cells showed increased signals of Dsg3/Ezrin interaction (red dots) in cells with overexpression of Dsg3 (polyclone:D3; monoclones: C2, C7 and C11) compared with Vect or positive ( þ ) and negative ( À ) controls (mean±s.d., **Po0.01). Here, the negative control was Dsg3/myc-tag in A431-Vect cells and the positive was Dsg3/myc-tag in A431-D3 cells. (b) Confocal analysis of FRET by acceptor photobleaching: the representative images are shown and the enlarged region 1 (Reg-1) for each channel is displayed at the bottom panels. The bleach of acceptor was 61.24% (average of Reg-1 and -2) and the enhanced fluorescent intensity in region 1 and 2 compared with internal and photo-damage regions is shown below the images. Among 89 regions comprising both membrane projections and intercellular junctions, 13 displayed positive FRET efficiency (14.6%) above thresholding of 5%. The FRET efficiency was calculated by a formula (Materials and methods), and is shown at the bottom of (b; Negative: n ¼ 16 and Positive: n ¼ 13, mean±s.d., ***Po0.001). Scale bars, 10 mmin(a), 20 mmin(b). (c, d) Co-immunoprecipitation (co-IP) in both A431-Vect and -D3 cells. The representatives from at least six independent co-IPs shown in (c) and from at least two independent co-IPs in both polyclonal and monoclonal cell lines are shown in (d), demonstrating that the association exists for both endogenous and exogenous Dsg3 and is in a Dsg3 dose- dependent manner. Scale bars, 10 mm.

Dsg3-induced phosphorylation of Ezrin-Thr567 can be abrogated dependent manner, the increased levels of pERM in Dsg3- by inhibitors of PKC as well as various signaling molecules overexpressing cells was abrogated effectively only at higher To investigate whether Ezrin activation by Dsg3 was PKC- concentrations (410 mM) comparable to that in control cells (only dependent, a dose–response experiment with the broad spectrum 5 mM; red dotted box in Figure 6a). This result implied that the PKC inhibitor, bisindolylmakeimide I59 was performed. Although a increased pERM in Dsg3-overexpressing cells was PKC-dependent. significant inhibition of pERM was seen in all cell lines in a dose- Consistent with this observation, a time course study with

& 2014 Macmillan Publishers Limited Oncogene (2014) 2363 – 2374 Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2368

Figure 4. Dsg3 is required for the localization and activation of Ezrin at the plasma membrane in A431 cells. (a) Confocal images of A431-Vect and -C7 with high levels of Dsg3 showed strong colocalization between Dsg3 (purple), the peripheral pERM (red) and cortical F-actin bundles (green), and its correlation with cell contraction and stratification. Arrows indicate the central stratified rounded cells in the colony that express high levels of Dsg3 coupled with strong cortical actin bundles and the pERM expression. These changes however appeared to be missing in A431-Vect cells in which Dsg3 expression remained low and little pERM signal was seen. The quantitation for each channel is presented on the right (mean±s.e.m., ***Po0.001). (b) Colocalization of the indicated proteins in A431 cells with or without Dsg3 knockdown (KD). Confocal images were obtained in six arbitrary fields from each coverslip. Percentage of colocalization of the indicated proteins was determine by ImageJ (highlighted in white) and is presented as mean±s.d. (*Po0.05, ***Po0.001). Scale bars, 20 mm.

bisindolylmakeimide I showed inhibition of pERM to a lesser system. We performed a dose-dependent experiment with three degree in D3 and C7 compared with vector and C11 controls PKC inhibitors bisindolylmakeimide I (broad spectrum), (Figure 6b). Ro¨-31-7549 and Go¨6976 (conventional), and showed significant Ezrin-Thr567 is also known as a substrate of ROCK,35,36 and inhibition of pERM by all inhibitors tested in a dose-dependent PKC-mediated ROCK activation increases actomyosin contractility manner (Supplementary Figure 8a), suggesting both conventional and cell rounding,60 the phenotype also observed in our study and nonconventional PKCs are involved in ERM phosphorylation. (Figure 5d). Thus, a dose-dependent experiment with the ROCK In addition, we conducted an experiment with these inhibitors in inhibitor Y-27632 was performed and similar inhibition of pERM conjunction with a calcium switch and observed that in Ca2 þ -free was observed in Dsg3-overexpressing cells. To further explore conditions, the serum-induced pERM could be abrogated effec- what other potential signaling molecules are likely involved in the tively by bisindolylmakeimide I or Ro¨-31-7549 but to a lesser Ezrin-Thr567 phosphorylation in our system, we tested various extent by Go¨6976 (Supplementary Figure 8b). Addition of calcium inhibitors including Ro¨-31-7549 for conventional PKC,61 PP2 for in the absence of inhibitors caused an increase of pERM compared Src,10 C3 for RhoA, Y-27632 for ROCK,62 NSC23766 for Rac163 and with dimethyl sulfoxide control in calcium-free conditions SB202190 for p38,64 and found inhibition at different degrees for indicative of calcium-induced activation of PKCs. Calcium addition most inhibitors except for PP2 (Figure 6c). As conventional PKCs also enhanced the expression of Dsg3, as we reported previously.8 are Ca2 þ -dependent, whereas novel and atypical isoforms do not We observed that the calcium-induced pERM could only be require Ca2 þ for their activation, we wanted to determine which partially abrogated by PKC inhibition (Supplementary Figure 8b, group of PKCs was responsible for the ERM phosphorylation in our plus Ca2 þ ) and this incomplete inhibition suggests that multiple

Oncogene (2014) 2363 – 2374 & 2014 Macmillan Publishers Limited Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2369

Figure 5. Dsg3 activates the ERM proteins including Ezrin at the Thr567 residue. (a) Western blotting analysis with the indicated antibodies showed an enhanced phosphorylation of pERM in A431 cells with high Dsg3 expression levels. The representative blots are shown on the left and bar charts on the right (experiment n46, mean±s.e.m., *Po0.05). (b) C7 cells transfected with either the scrambled control or Ezrin- or moesin-specific small interfering RNA for 2 days before protein extraction followed by western blotting. Total protein samples (10 mg per lane) were resolved in 3–8% SDS–polyacrylamide gel electrophoresis that showed a good separation of Ezrin/radixin (81 kD) and moesin (77 kD). There was a good knocking down for Ezrin but less effect for moesin, and the bar chart summarizes the data of four independent blots (mean±s.e.m., ***Po0.001). (c) PLA assay using antibody pair for pERM/Ezrin showed significantly increased PLA signal in D3 cells compared with Vect cells (mean±s.d., *Po0.05). (d) Confocal images of A431 cells showed rounded cells coupled with pronounced membrane protrusions (enlarged on the left) in cells with high Dsg3 levels when compared with control cells or cells with low Dsg3 levels in the same sample. Scale bar, 20 mm. mechanisms (as shown in Figure 6c), either mediated by calcium with Vect. In addition to the Src family as we have previously or Dsg3, are involved in ERM phosphorylation. reported,8,10 increased phosphorylation was also seen for c-Jun S63 (2.6-fold), PLCg-1-Y783 (1.6-fold) and p70 S6 Kinase-T229 (1.8-fold; Figure 7a). C-Jun is part of the AP-1 transcription factor, a Dsg3 enhances the phosphorylation of c-Jun and activates the proto-oncogene that has been implicated in the regulation of a transcriptional activity of AP-1 variety of cellular responses including cell migration and To explore the involvement of other kinases, we performed a oncogenesis. The phosphorylation of S63 at the N-terminal phospho-kinase profiler array study and observed that a number transactivating domain of c-Jun is known to be mediated by Jun of kinases were increased in Dsg3-overexpressing cells compared kinase,65 suggesting the Jun kinase pathway is activated by Dsg3.

& 2014 Macmillan Publishers Limited Oncogene (2014) 2363 – 2374 Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2370

Figure 6. Dsg3-induced Ezrin-Thr567 phosphorylation could be abrogated by various inhibitors including those for PKCs. (a) Western blotting of total lysates of A431 lines treated with the broad spectrum PKC inhibitor, bisindolylmakeimide I (BIM), at various concentrations (0.5, 5, 10, 20 and 40 mM) for 4 h that showed a dose-dependent inhibition of pERM levels in all cell lines. Note that higher concentrations (o20 mM) of the inhibitor were required to abrogate the enhanced pERM in Dsg3-overexpressing cells (D3 and C7) compared with Vect and C11 cells that were treated at lower concentrations (o10 uM). In addition, all Dsg3-transducted cells appeared to be more sensitive than control cells to BIM. The results are representative of at least three independent experiments. (b) Quantitation of the western blots of a time course experiment in which four cell lines were stimulated in normal growth medium for 3 h after serum starvation, before treatment with BIM for 0.5 and 1 h. Again, less inhibition was seen in Dsg3-overexpressing cell lines. (c) The phosphorylation of the ERM proteins can be abrogated by the inhibitors for various signaling molecules in addition to PKCs. Cells were serum starved for 1 day before being treated with the inhibitors as follows: PKC-Ro¨ 31-7549: 2 mM for5hor50mM for 45 min, PCK-BIM: 5 mM for 4 h, Src-PP2: 10 mM for 5 h, RhoA-C3: 2 mg for 5 h, ROCK-Y-27652: 10 mM for 4 h, Rac1-NSC23766: 30 mM for 5 h and p38-SB202190: 20 mM for 4 h. Lysates were extracted and subsequently analyzed by western blotting. Except for Src inhibitor PP2, almost all the inhibitors showed inhibition of pERM to different degrees. The results are representative of three independent experiments, and shown below are the quantitation data (mean±s.d.).

To confirm that the AP-1 activity is indeed induced by Dsg3, we abrogated by various inhibitors and observed partial inhibition performed a luciferase reporter assay in A431 parental cells with with inhibitors of PKC, ROCK and p38. transient co-transfection of an AP-1 reporter construct together with various concentrations of the pBABE-hDsg3.myc construct and observed a strong dose-dependent change in AP-1 activity in DISCUSSION relation to Dsg3 expression levels with B14-fold increase in cells This study identifies the desmosomal cadherin Dsg3 as an with the highest amount of Dsg3 complementary DNA (Figure 7b). upstream cell surface activator for AP-1 and the PKC/Ezrin Furthermore, this finding was validated by loss-of-function pathway in the control of cell motility and invasion in cancer analysis showing a more than twofold reduction in luciferase (Figure 8). Dsg3 and Ezrin have been independently implicated in activity in Dsg3 knockdown cells compared with those treated cancer progression and metastasis.32,33,35,40–43 This study provides with scrambled control small interfering RNA (Figure 7c). Finally, direct evidence of cross talk between Dsg3 and Ezrin at the we tested whether the enhanced luciferase activity can be plasma membrane that is responsible, at least in part, for

Oncogene (2014) 2363 – 2374 & 2014 Macmillan Publishers Limited Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2371

Figure 7. Overexpression of Dsg3 enhances the c-Jun S63 phosphorylation and activates the AP-1 transcriptional activity. (a) Human phosphokinase array data (part) presented as the log 2 ratio of fold increase in A431-D3 against Vect cells. Significant increase in the phosphorylation of c-Jun S63 was observed among other kinases. (b) Dsg3 enhanced the AP-1 activity assessed by the luciferase assay. AP-1 reporter or Vect plasmid was transfected into A431 parental cells together with various concentrations of pBABE-hDsg3.myc topped up to 1 mg plasmid(s) with pBABE-GFP. A clear dose-dependent increase of luciferase activity was consistently seen in cells with overexpression of Dsg3 (experiment n43, **Po0.01, ***Po0.001). (c) Dsg3 silencing in A431 reduced the luciferase activity more than twofold compared with scrambled small interfering RNA-treated cells (**Po0.01).

cadherins via Src activation.8,10 However, to enable tumor cells to invade into the neighboring tissues, additional properties that involve multigenic processes and coordinated rearrangements of the actin are required. AP-1 is known as a proto- oncogene and a critical regulator of a complex gene expression program that defines the invasive phenotype.65 Ezrin is one of its functional effectors and has a key role in remodeling actin dynamics and cell adhesion that associates with invasion. AP-1 comprises heterodimers of Fos and Jun or homodimers of Jun that regulate gene expression by binding to a consensus DNA motif in the promoter region of target genes. Sustained activation of AP-1 is required for transformation by many oncogenes. It is well known that the growth factor signaling pathway is an upstream regulator for sustained activation of AP-1.66 In this study, we report that the cell adhesion protein Dsg3 also functions as an upstream activator of AP-1 and is capable of activating one of its Figure 8. A model illustrating that Dsg3 acts as an upstream target effectors, Ezrin,37 at the plasma membrane to promote cell regulator for the Ezrin phosphorylation at Thr567 (activation) shape change and migratory capacity. through the PKC/AP-1regulation pathway that is required for Dsg3 (pemphigus vulgaris antigen) has recently been impli- promoting membrane projections, cell spreading and invasive motility in tumor cells. cated in tumor metastasis and progression of SCC in multiple organs, especially the head and neck and lungs,14–16,18–20 suggesting it has a pivotal role in cancer cell biology. In support, our gain-of-function studies demonstrated that, rather than promoting cell–cell adhesion, overexpression of this gene results promoting cell migration and invasion in Dsg3-associated SCC. We in compromised intercellular adhesion and a reduction in the demonstrated that Dsg3 is capable of forming a complex with expression of classical and desmosomal cadherins.8,10 In this Ezrin at the plasma membrane and regulates its phosphorylation study, we describe an additional mechanism by which Dsg3 at the Thr567 residue, the activity required for proper interaction promotes cell invasive capability by activating AP-1- and the with F-actin and CD44 that is a prerequisite for cell migration and PKC-dependent Ezrin-Thr567 pathway that are likely to be invasion. We also showed that the Dsg3-induced Ezrin-Thr567 responsible for tumor metastasis in SCC. Evidence that supports phosphorylation can be abrogated by various inhibitors including this conclusion are as follows: (1) overexpression of Dsg3 enhances those for PKC and ROCK, indicating the likelihood that both membrane protrusions, cell spreading and rounding that are kinases are partially regulated by Dsg3. Furthermore, we the necessary prerequisites for cell migration/invasion; (2) discovered that Dsg3 also regulates the activity of AP-1, via overexpression of Dsg3 accelerates cell migration/invasion in phosphorylation of c-Jun S63, a transcription factor that has a both A431 and SqCC/Y1 cancer cell lines; (3) Dsg3 forms a pivotal role in tumor metastasis. complex with and activates Ezrin at the Thr567 residue that is Cell invasion is one of the hallmarks of cancer. Our previous dependent upon several kinases including PKC; (4) modulation of studies showed that overexpression of human Dsg3 in cancer cell Dsg3 levels directly influenced the activity of Ezrin that directly lines causes dissolution of both classical and desmosomal associates with its interaction with F-actin and CD44 at the plasma

& 2014 Macmillan Publishers Limited Oncogene (2014) 2363 – 2374 Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2372 membrane; and finally, (5) Dsg3 functions as an upstream activator from Ezrin to Rho GTPases and ROCK was not ruled out here.60 of AP-1that could be abrogated by PKC inhibition. Evidence indicating that Rac1 is coupled with Ezrin activation in The positive role of Ezrin in cancer progression and metastasis the control of E-cadherin-mediated assembly is has been shown by many studies in the context of cortical documented in the literature.79 In summary, our findings suggest cytoskeleton organization, filopodia extension, cell spreading, that Dsg3 might serve as a master cell surface regulator for a proliferation, survival and motility.32,67,68 Ezrin activation is variety of signal transduction pathways that influence the activity dynamically regulated in a spatiotemporal manner during of Ezrin, one of the effector proteins of these signaling cascades. metastatic progression.33 Given that upregulation of Dsg3 and In conclusion, this study provides the first evidence of the Ezrin has been independently found in lymph node metastasis of desmosomal cadherin Dsg3 that functions as a key membrane the head and neck cancer,18,33,40–42 our study suggests it is likely activator for AP-1 and Ezrin activation that are associated with that the Dsg3/PKC/Ezrin-Thr567 pathway is activated at the various cellular responses such as membrane dynamics, cell leading edge or invasive front, in lymph node metastasis of the spreading, migration and invasion. This pathway may involve head and neck cancer.18–20 several signaling molecules including PKCs that are regulated by A number of transmembrane proteins including the hyaluronan Dsg3.8–10 These findings will advance our understanding of the receptor, CD44, have been characterized as ERM-binding pro- role of Dsg3 in tumor progression and invasion, and likely facilitate teins.24,69 This study has identified Dsg3 as a novel membrane development of new therapeutic strategies for the head and neck protein that associates with Ezrin and is able to regulate its and lung cancers. activation. We showed that both endogenous and ectopic Dsg3 form a complex with Ezrin at the plasma membrane where membrane projections are pronounced. Dsg3 knockdown not MATERIALS AND METHODS only affected its association with Ezrin but also impaired Cell culture and reagents E-cadherin adhesion and the interaction between Ezrin and A431 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) CD44/F-actin, indicating the functional significance of such a plus 10% fetal calf serum. Human oral SCC cell line SqCC/Y1 was cultured complex. Although the actual nature of the binding between Dsg3 in EpiLife (Invitrogen, Life Technologies Ltd, Paisley, UK) or keratinocyte and Ezrin remains to be elucidated, our mass spectrometric growth medium.13 Primary human oral fibroblasts were cultured in DMEM analysis suggests it could be indirect that warrants further plus 10% donor calf serum and cells with passage number o10 were used. investigation. Collectively, the findings from this study and our A431 clones with transduction of hDsg3.myc10 were obtained using the 10 previous reports8–10 support the view that non-junctional Dsg3 limited dilution assay. The protocol, as described previously, was used for has a signaling role in epithelial cells, acting as a membrane the generation of stable SqCC/Y1 lines with transduction of human Dsg3 as receptor in regulating a diverse array of cellular events that control well as the matched Vects. Antibodies and small interfering RNA sequences are listed in Supplementary Tables 1 and 2. actin-based cell–cell adhesion, cell shape determination and migratory behavior. It is possible that cells use Dsg3 to recruit Ezrin to the basolateral domain to form membrane projections, Immunofluorescence and confocal microscopy such as filopodia and lamellipodia, in order to facilitate contacts Immunofluorescence was performed as described previously.10 The with neighboring cells that is a prerequisite for junction formation coverslips were examined with a Zeiss Meta 510LSM/510LSM laser and cell polarization in normal keratinocytes.51 However, this scanning confocal (Carl Zeiss, Cambridge, UK) or Leica DM5000 epi- process is somehow hijacked in transformed tumor cells where fluorescence microscope (Leica Microsystems (UK) Ltd. Milton Keynes, UK). the stability and organization of intercellular junctions are drastically altered. Evidence also suggesting the signaling roles Transwell cell migration and invasion assays of Dsg3 comes from many in vitro studies based on pemphigus vulgaris (PV-IgG).70–72 Nevertheless, no report has yet shown its The assays were conducted with 24-well transwell inserts, which contain a permeable membrane with 8 mm pore size (VWR International Ltd, West association with Ezrin, although the link between actin and 5 73 Sussex, UK). Cells (1 Â 10 ) in basal medium (alpha-DMEM or DMEM:Ham’s pemphigus acantholysis is just beginning to be appreciated. F12 at 3:1) plus 1% bovine serum albumin were placed on the upper layer Serine/threonine kinases such as ROCK, PKC, p38 and LOK have of an insert, which was placed into a 24-well plate containing 500 ml alpha- been identified as the ERM kinases for phosphorylating the minimum essential medium plus 10% fetal calf serum for A431 cells and C-terminal threonine residue in a different cellular context.32,74–77 keratinocyte growth medium for SqCC/Y1, which acted as a chemoat- Among them, PKC family isoforms seem to be ubiquitously tractant. The plate was kept in an incubator for 2–3 days to allow cells to involved in all cell types. PKC isoforms such as a, g and i are migrate into the bottom chamber. The total cell number migrating to the capable of phosphorylating Ezrin-Thr567 at the plasma bottom chambers was determined using the CASY cell counter (Roche membrane, particularly the membranous protrusions that Innovatis AG, Reutlingen, Germany). For the cell invasion, Matrigel diluted 33 1:3 in DMEM was loaded on the upper layer of the inserts and allowed to control cell motility and agents that block this phosphorylation gel uniformly before addition of the cell suspension. also abrogate PKC a-dependent migration.32 Dsg3 is a calcium- dependent protein and becomes stabilized (increase in expression levels) upon calcium switching8 that also activates PKC Organotypic culture 80 (conventional). Our results with the PKC inhibitors that showed The Matrigel:collagen organotypic raft culture is described elsewhere. significant inhibition of pERM in Dsg3-overexpressing cells indicate it is likely that the Dsg3-mediated Ezrin-Thr567 Histology, immunohistochemistry and quantitative analysis phosphorylation involves PKCs, particularly calcium-dependent Raft cultures were collected after 14 days and fixed in Trumps (100 mM conventional isoforms. There are seven PKC isoforms, that is, a, bII, NaH2PO4, 67.5 mM NaOH, 4% formaldehyde, 1% glutaraldehyde). Speci- d, Z, e, l, z, which were shown to be expressed in A431 cells.78 mens were subjected to routine procedures for histology. Five-micrometer To delineate which isoform(s) is responsible for Dsg3-mediated sections were stained with hematoxylin and eosin or for keratin with LP34, followed by Dako Cytomation EnVision þ dual Link System Peroxidase. The Ezrin activation merits further investigation. In addition, our 0 studies (this and the study by Tsang et al.9) also support the peroxidase was visualized using 3,3 -Diaminobenzidine (DAB) substrate (DAKO, Dako UK Ltd, Cambridgeshire, UK). Quantitation of cell invasion is general notion that other kinases such as ROCK, p38 MAPK, Rac1 described elsewhere.80 Images were acquired in a MBF stereology system and RhoA may also have a role in Dsg3-mediated Ezrin activation, (MBF Bioscience, Williston, VT, USA) and analyzed by ImageJ (Wayne as our phospho-kinase array and the inhibitor analyses showed a Rasband, NIH, Bethesda, MD, USA). Parameters such as particle number, wide range of signaling pathways that had been activated in total area and average size of particles, and invasion depth were analyzed. Dsg3-overexpressing cells (Figures 6c and 7a). Positive feedback The ‘invasion index’ was calculated.80

Oncogene (2014) 2363 – 2374 & 2014 Macmillan Publishers Limited Dsg3 promotes cell migration via AP-1 and ezrin L Brown et al 2373 A Duolink in situ PLA for protein–protein interactions 8 Tsang SM, Brown L, Lin K, Liu L, Piper K, O’Toole EA et al. Non-junctional human The PLA was conducted following the protocol described in the Duolink desmoglein 3 acts as an upstream regulator of Src in E-cadherin adhesion, a PLA kit (Cambridge Bioscience Limited, Cambridge, UK). pathway possibly involved in the pathogenesis of pemphigus vulgaris. J Pathol 2012; 227: 81–93. 9 Tsang SM, Brown L, Gadmor H, Gammon L, Fortune F, Wheeler A et al. FRET analysis Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 FRET efficiency was measured using acceptor photobleaching as described in the regulation of actin organisation and dynamics. Exp Cell Res 2012; 318: 10 previously. Eighty-nine regions of both junctional and free edges were 2269–2283. analyzed and FRET efficiency was calculated, and the FRET efficiency 45% 10 Tsang SM, Liu L, Teh MT, Wheeler A, Grose R, Hart IR et al. Desmoglein 3, via an 10 was considered to be positive. interaction with E-cadherin, is associated with activation of Src. PLoS One 2010; 5: e14211. Co-immunoprecipitation, protein fractionation and western 11 Chidgey M, Dawson C. Desmosomes: a role in cancer? Br J Cancer 2007; 96: blotting 1783–1787. 8,10 12 Shinohara M, Hiraki A, Ikebe T, Nakamura S, Kurahara S, Shirasuna K et al. The details of the procedures are described previously. Immunohistochemical study of desmosomes in oral squamous cell carcinoma: correlation with cytokeratin and E-cadherin staining, and with tumour behaviour. Human phosphokinase profiler array J Pathol 1998; 184: 369–381. 13 Teh MT, Parkinson EK, Thurlow JK, Liu F, Fortune F, Wan H. A molecular study of A431-Vect and -D3 cell lines seeded at equal density in 10-cm culture desmosomes identifies a desmoglein isoform switch in head and neck squamous dishes were extracted and the phosphokinase array was carried out using cell carcinoma. J Oral Pathol Med 2011; 40:67–76. 500 mg of total protein in each sample, according to the protocol in the kit 14 Chen YJ, Chang JT, Lee L, Wang HM, Liao CT, Chiu CC et al. DSG3 is overexpressed (Catalog number: ARY003, R&D Proteome Profiler Array, R&D Systems in head neck cancer and is a potential molecular target for inhibition of onco- Europe Ltd., Abingdon, UK). genesis. Oncogene 2007; 26: 467–476. 15 Huang CC, Lee TJ, Chang PH, Lee YS, Chuang CC, Jhang YJ et al. Desmoglein 3 is Luciferase assay overexpressed in inverted papilloma and squamous cell carcinoma of sinonasal An AP-1 reporter construct was made using the pGL4.26 vector (Promega) cavity. Laryngoscope 2010; 120: 26–29. containing a minimal eukaryotic promoter. 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