Original Article | Published:

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

Oncogene volume 33, pages 23632374 (01 May 2014) | Download Citation

Abstract

Desmoglein 3 (Dsg3), the pemphigus 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.

Introduction

Desmoglein 3 (Dsg3), the pemphigus vulgaris antigen, is one of seven transmembrane desmosomal cadherins.1, 2 Although a large body of evidence suggests that Dsg3 has a paramount role in cell–cell adhesion,1 an in vitro study showed that it mediates weak homophilic cell–cell adhesion.3 There are two pools of Dsg3 protein existing in epithelial cells, non-junctional (Triton soluble) and junctional (Triton insoluble); the latter is associated with the desmosomes.4, 5, 6, 7, 8 Our previous studies show that non-junctional Dsg3 is involved in E-cadherin signaling via Src and Rac1/Cdc42, suggesting cross talk between the desmosomal and classical cadherins in epithelial cells.8, 9, 10

Alterations in the expression of desmosomal components could contribute to cancer progression similar to E-cadherin;11, 12, 13 however, the correlation between the expression levels of desmosomal components and the state of tumor development has not been fully established. Recent studies have shown upregulation of Dsg3 in squamous cell carcinoma (SCC) in various tissues, particularly in the head and neck and lungs, which is correlated with T stage, N stage and overall stage.14, 15, 16, 17, 18 Moreover, DSG3 messenger RNA and protein have been identified as the best discriminatory (with 100% accuracy) biomarker among 40 potential candidate genes for accurate intraoperative staging of cervical sentinel lymph nodes in the head and neck SCC.18, 19, 20 These findings suggest strongly a pro-metastatic role for Dsg3 in tumor cell biology; however, the underlying molecular mechanism remains poorly understood.

Ezrin is a member of the Ezrin/Radixin/Moesin (ERM) proteins that act as plasma membrane-actin linkers and are enriched in microvilli, ruffles, filopodia, uropods and cell adhesion sites. The ERM proteins exist in two forms, active and inactive. The inactive proteins predominantly localize in the cytosol21, 22 and are present in a closed conformation. Recruitment of these proteins to the plasma membrane leads to the release of intramolecular interactions, exposing the binding site for actin at their C-terminus and for plasma membrane proteins such as CD44,23, 24 or adaptor proteins such as EBP5025 at the N-terminus. Phosphatidylinositol 4,5-bisphosphate binding and phosphorylation of Ezrin on Thr567 are involved in this activation process.26, 27, 28 Protein kinase C (PKC) and Rho kinase (ROCK) are responsible for Ezrin-Thr567 phosphorylation29, 30, 31, 32, 33, 34, 35, 36 and also activation of the transcriptional factor activator protein 1 (AP-1) downstream that regulates the expression of many genes, including those encoding for Ezrin and CD44, which are necessary for implementation of the invasion program.37 Ezrin participates in diverse cellular functions including cell adhesion, polarization and morphogenesis,21, 38, 39 and more importantly has been identified as a potent regulator of tumor cell invasion and metastasis.32, 33, 35, 40, 41, 42, 43 A positive correlation between Ezrin expression and cervical lymph node metastasis and clinical staging has been found in various tumors.43, 44, 45, 46, 47 In vitro RNA interference studies in cancer cell lines have demonstrated that knocking down Ezrin expression suppresses cell growth, invasion, tumor progression and metastasis,48, 49, 50 a phenotype that overlaps strikingly with that of Dsg3 silencing.14

In this study, we investigated the potential link between Dsg3 signaling and Ezrin activation, and describe a novel phosphorylation mechanism of the ERM proteins, primarily Ezrin-Thr567 that is regulated by Dsg3 in a PKC-dependent manner in A431 cells. We showed that non-junctional Dsg3 forms a complex with and is capable of activating Ezrin, which regulates actin-based cell shape change and migratory behavior. We confirmed that this pathway can be abrogated by inhibition of various signaling molecules including PKCs. Finally, we demonstrated a strong correlation between AP-1 activity and Dsg3 expression levels in cells. Collectively, our study uncovers a novel signaling role of Dsg3 that acts as a cell surface activator of AP-1 and the PKC/Ezrin pathway that promotes cancer cell migration and invasion.

Results

Dsg3 induces morphological change and promotes membrane protrusion and cell spreading

To establish the role of Dsg3 in adhesion-induced membrane morphogenesis,51 we analyzed A431-hDsg3.myc cells10 and its derived clones with high or low Dsg3 levels and also an oral SCC line, SqCC/Y113 with transduced hDsg3.myc (Sq-D3). Recently, we have shown that Dsg3 functions as an upstream regulator of Rac1/Cdc42 in the regulation of actin organization and dynamics.9 In this study, we performed immunostaining for Ezrin and analyzed membrane coverage with Ezrin-positive protrusions. We showed that cells with high Dsg3 levels exhibited exaggerated membrane protrusions and cell spreading (Figure 1a and Supplementary Figure 1). Consistently, the transient transfection of hDsg3.myc construct in 293T cells resulted in striking membrane branching and micro-spikes compared with green fluorescent protein control cells (Supplementary Figure 1b). To demonstrate the specificity of the phenotype, Dsg3 in both A431-V and -D3 were depleted by RNA interference, and subsequent immunostaining showed that cells with Dsg3 depletion displayed remarkable collapse of the membrane protrusions and a concomitant inhibition of E-cadherin junction assembly (Figures 1b and c). These findings are consistent with our hypothesis that Dsg3 is involved, at least in part, in the regulation of actin organization.9 To address whether Dsg3 overexpression enhances cell–cell adhesion, we performed the hanging drop assay (Supplementary Figure 2) and consistently showed that overexpression failed to enhance cell–cell adhesion, as we reported previously.10

Figure 1
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 >50% of cell periphery in the field (experiment n>3, 100 cells were analyzed from each group and are presented as mean±s.d., *P<0.05, ***P<0.001). (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 μm.

Dsg3 promotes cell migration and invasion

Previously, we have shown that overexpression of Dsg3 enhanced cell migration by a wound scratch assay.10 To complement this observation, we analyzed both A431 and SqCC/Y1 stably overexpressing Dsg3 under various conditions. Transwell migration assays indicated that all Dsg3-overexpressing cells exhibited greater cell motility, especially the A431-C2- and -C7-cloned cells (Figure 2a) while knockdown of Dsg3 had no effect in this migration assay. Consistent with this result, a transwell invasion assay showed that Dsg3 overexpression significantly promoted cell invasion compared with control in SqCC/Y1 cells (Figure 2b) and furthermore, we demonstrated that the enhanced migratory activity in Sq-D3 cells could be recapitulated in an organotypic cell invasion assay52 (Figure 2c) that showed a significantly greater number of cell clusters invading into the Matrigel:collagen matrix compared with control cells (Figures 2c–e). Collectively, these data suggest that Dsg3 promotes cancer cell migration and invasion.

Figure 2
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=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., *P<0.05, **P<0.01, ***P<0.001). (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=13, mean±s.d., *P<0.05, ***P<0.001). (e) Cell invasion index80 indicated a significant increase in Sq-D3 cells compared with control cells (mean±s.e.m.,***P<0.001). Scale bars, 50 μm.

Dsg3 associates with Ezrin at the plasma membrane

Our preliminary observation of the Dsg3/Ezrin colocalization10 led us to hypothesize that Dsg3 may have a role in regulating cell morphology and migratory behavior through a mechanism involving Ezrin. To address this question, we, first of all, analyzed protein–protein interaction by the proximity ligation assay (PLA) and demonstrated a marked increase of PLA signals that were generated by close proximity of Dsg3/Ezrin in Dsg3-overexpressing cells compared with control (Figure 3a). In line with this, the fluorescence resonance energy transfer (FRET) analysis of A431-D3 cells showed positive FRET (14.6% of regions of interest) predominantly localized to the membrane projections (Figure 3b). Furthermore, the association between Dsg3/Ezrin was verified by co-immunoprecipitation assay, demonstrating reproducibly that both endogenous and ectopic Dsg3 formed a 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, we analyzed the association in Triton-soluble and -insoluble (NP-40 soluble) fractions by immunofluorescence and co-immunoprecipitation. For immunofluorescence, cells were treated with or without Triton buffer before fixation and immunostaining. Cells treated with Triton showed significant reduction of phospho-ERM and its colocalization with Dsg3, particularly at the cell borders (Supplementary Figure 3a and b). In support, co-immunoprecipitation demonstrated that the association predominantly existed in Triton-soluble fractions in all cell lines (Supplementary Figure 3c). Together, these results suggest that non-junctional Dsg3 forms a complex with Ezrin outside of desmosomes. In order to search for other potential-binding partners, mass spectrometry was applied. A construct of the cytoplasmic tail of Dsg3 tagged with Halo at the N-terminus was cloned into pFN21A (Promega, Southampton, UK) and the plasmid or Halo vector control (Vect) was transfected into A431 parental cells. The lysates were subjected to Halo tag purification and the resulting complex was analyzed using one-dimensional SDS–polyacrylamide gel electrophoresis proteomics approach. Proteins were separated using SDS–polyacrylamide gel electrophoresis and bands were digested by trypsin before further analyzed by liquid chromatography-tandem mass spectrometry (Supplementary Figure 4). Actin was identified as the major component present in the test sample but not in the empty vector (Supplementary Figure 4d).

Figure 3
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 (+) and negative (−) controls (mean±s.d., **P<0.01). 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=16 and Positive: n=13, mean±s.d., ***P<0.001). Scale bars, 10 μm in (a), 20 μm in (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 μm.

Image analyses showed that changes in Dsg3 levels directly affected its colocalization with Ezrin (also phosphorylated form; Supplementary Figures 5 and 6) indicating an association between 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 pronounced (Supplementary Figure 7). Quantitation of immunofluorescence intensity of Ezrin at the plasma membrane showed approximately a twofold increase in A431-D3 compared with control cells (immunofluorescence intensity/pixel in D3 vs Vect is 66.0±15.6 vs 36.7±16.1), implying that Dsg3 overexpression enhances Ezrin activity. Consistent with this notion, triple staining for Dsg3/pERM/F-actin showed a positive correlation and significantly enhanced colocalization of the three proteins at the cell borders in D3 compared with Vect cells, particularly in those stratified cells located in the center of the colonies (Figure 4a arrows). As Ezrin activation is associated with its interaction with CD44 and F-actin at the plasma membrane,23, 24 we evaluated the colocalization of Ezrin/F-actin or CD44/F-actinin A431-D3 with or without Dsg3 knockdown. As shown in Figures 4a and b, a significant reduction of their colocalization was seen in RNA interference-treated cells compared with controls.

Figure 4
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., ***P<0.001). (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. (*P<0.05, ***P<0.001). Scale bars, 20 μm.

Taken together, our findings demonstrate that non-junctional Dsg3 forms a complex with Ezrin at the plasma membrane and regulates its activity that is required for membrane projections and cell migration/invasion.31, 53

Dsg3 activates the ERM proteins, particularly Ezrin via the phosphorylation at Ezrin-Thr567

The phosphorylation of Ezrin-Thr567 leads to its activation, which has been implicated in cell invasion and metastasis.32, 35, 54, 55, 56, 57 To show that Ezrin was activated in response to Dsg3 overexpression, we analyzed the phosphorylation of ERM proteins including Ezrin-Thr567. Using a rabbit antibody (pERM) specific for the conserved C-terminal phospho-threonine of ERM proteins, we observed a significant increase in pERM levels in Dsg3-overexpressing cells compared with Vect or C11 that 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 moesin in A431–C7 using an Ezrin- or moesin-specific small interfering RNA. Cell lysates were analyzed by western blotting for pERM. In the control the upper band—Ezrin and radixin (81 kDa) appeared to be almost double that of the lower (77 kDa) phospho-moesin (Figure 5b). The RNA interference-mediated Ezrin knockdown caused a significant reduction in the upper band, suggesting that Ezrin is the predominant phosphorylated isoform in A431 cells (Figure 5b), as reported previously.58 Thus, it is reasonable to conclude that the elevated pERM in A431-D3 and C7 (Figure 5a) was due to increased phospho-Ezrin rather than phospho-radixin or -moesin. To further validate this finding, we performed PLA using an antibody pair for Ezrin/pERM and showed a significant increase of PLA signals in D3 compared with the control, indicating the enhanced pERM indeed was phospho-Ezrin-Thr567 (Figure 5c). Ezrin activation was also reported to induce shape changes including cell rounding in addition to the membrane projections in A431 cells,21 and such a rounded cell phenotype coupled with pronounced membrane protrusions was also observable in A431-D3 compared with control cells or cells with low levels of Dsg3 in the same sample (Figure 5d). These results further support the notion that Dsg3 regulates Ezrin activity through phosphorylation at Thr567.

Figure 5
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 n>6, mean±s.e.m., *P<0.05). (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 μg 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., ***P<0.001). (c) PLA assay using antibody pair for pERM/Ezrin showed significantly increased PLA signal in D3 cells compared with Vect cells (mean±s.d., *P<0.05). (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 μm.

Dsg3-induced phosphorylation of Ezrin-Thr567 can be abrogated by inhibitors of PKC as well as various signaling molecules

To investigate whether Ezrin activation by Dsg3 was PKC-dependent, a dose–response experiment with the broad spectrum PKC inhibitor, bisindolylmakeimide I59 was performed. Although a significant inhibition of pERM was seen in all cell lines in a dose-dependent manner, the increased levels of pERM in Dsg3-overexpressing cells was abrogated effectively only at higher concentrations (>10 μM) comparable to that in control cells (only 5 μM; red dotted box in Figure 6a). This result implied that the increased pERM in Dsg3-overexpressing cells was PKC-dependent. Consistent with this observation, a time course study with bisindolylmakeimide I showed inhibition of pERM to a lesser degree in D3 and C7 compared with vector and C11 controls (Figure 6b).

Figure 6
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 μM) for 4 h that showed a dose-dependent inhibition of pERM levels in all cell lines. Note that higher concentrations (<20 μM) 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 (<10 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-Rö 31-7549: 2 μM for 5 h or 50 μM for 45 min, PCK-BIM: 5 μM for 4 h, Src-PP2: 10 μM for 5 h, RhoA-C3: 2 μg for 5 h, ROCK-Y-27652: 10 μM for 4 h, Rac1-NSC23766: 30 μM for 5 h and p38-SB202190: 20 μM 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.).

Ezrin-Thr567 is also known as a substrate of ROCK,35, 36 and PKC-mediated ROCK activation increases actomyosin contractility and cell rounding,60 the phenotype also observed in our study (Figure 5d). Thus, a dose-dependent experiment with the ROCK inhibitor Y-27632 was performed and similar inhibition of pERM was observed in Dsg3-overexpressing cells. To further explore what other potential signaling molecules are likely involved in the Ezrin-Thr567 phosphorylation in our system, we tested various inhibitors including Rö-31-7549 for conventional PKC,61 PP2 for Src,10 C3 for RhoA, Y-27632 for ROCK,62 NSC23766 for Rac163 and SB202190 for p38,64 and found inhibition at different degrees for most inhibitors except for PP2 (Figure 6c). As conventional PKCs are Ca2+-dependent, whereas novel and atypical isoforms do not require Ca2+ for their activation, we wanted to determine which group of PKCs was responsible for the ERM phosphorylation in our system. We performed a dose-dependent experiment with three PKC inhibitors bisindolylmakeimide I (broad spectrum), Rö-31-7549 and Gö6976 (conventional), and showed significant inhibition of pERM by all inhibitors tested in a dose-dependent manner (Supplementary Figure 8a), suggesting both conventional and nonconventional PKCs are involved in ERM phosphorylation. In addition, we conducted an experiment with these inhibitors in conjunction with a calcium switch and observed that in Ca2+-free conditions, the serum-induced pERM could be abrogated effectively by bisindolylmakeimide I or Rö-31-7549 but to a lesser extent by Gö6976 (Supplementary Figure 8b). Addition of calcium in the absence of inhibitors caused an increase of pERM compared with dimethyl sulfoxide control in calcium-free conditions indicative of calcium-induced activation of PKCs. Calcium addition also enhanced the expression of Dsg3, as we reported previously.8 We observed that the calcium-induced pERM could only be partially abrogated by PKC inhibition (Supplementary Figure 8b, plus Ca2+) and this incomplete inhibition suggests that multiple mechanisms (as shown in Figure 6c), either mediated by calcium or Dsg3, are involved in ERM phosphorylation.

Dsg3 enhances the phosphorylation of c-Jun and activates the transcriptional activity of AP-1

To explore the involvement of other kinases, we performed a phospho-kinase profiler array study and observed that a number of kinases were increased in Dsg3-overexpressing cells compared with Vect. In addition to the Src family as we have previously reported,8, 10 increased phosphorylation was also seen for c-Jun S63 (2.6-fold), PLCγ-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 proto-oncogene that has been implicated in the regulation of a variety of cellular responses including cell migration and oncogenesis. The phosphorylation of S63 at the N-terminal transactivating domain of c-Jun is known to be mediated by Jun kinase,65 suggesting the Jun kinase pathway is activated by Dsg3. To confirm that the AP-1 activity is indeed induced by Dsg3, we performed a luciferase reporter assay in A431 parental cells with 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 relation to Dsg3 expression levels with 14-fold increase in cells with the highest amount of Dsg3 complementary DNA (Figure 7b). Furthermore, this finding was validated by loss-of-function analysis showing a more than twofold reduction in luciferase activity in Dsg3 knockdown cells compared with those treated with scrambled control small interfering RNA (Figure 7c). Finally, we tested whether the enhanced luciferase activity can be abrogated by various inhibitors and observed partial inhibition with inhibitors of PKC, ROCK and p38.

Figure 7
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 μg plasmid(s) with pBABE-GFP. A clear dose-dependent increase of luciferase activity was consistently seen in cells with overexpression of Dsg3 (experiment n>3, **P<0.01, ***P<0.001). (c) Dsg3 silencing in A431 reduced the luciferase activity more than twofold compared with scrambled small interfering RNA-treated cells (**P<0.01).

Discussion

This study identifies the desmosomal cadherin Dsg3 as an upstream cell surface activator for AP-1 and the PKC/Ezrin pathway in the control of cell motility and invasion in cancer (Figure 8). Dsg3 and Ezrin have been independently implicated in cancer progression and metastasis.32, 33, 35, 40, 41, 42, 43 This study provides direct evidence of cross talk between Dsg3 and Ezrin at the plasma membrane that is responsible, at least in part, for promoting cell migration and invasion in Dsg3-associated SCC. We demonstrated that Dsg3 is capable of forming a complex with Ezrin at the plasma membrane and regulates its phosphorylation at the Thr567 residue, the activity required for proper interaction with F-actin and CD44 that is a prerequisite for cell migration and invasion. We also showed that the Dsg3-induced Ezrin-Thr567 phosphorylation can be abrogated by various inhibitors including those for PKC and ROCK, indicating the likelihood that both kinases are partially regulated by Dsg3. Furthermore, we discovered that Dsg3 also regulates the activity of AP-1, via phosphorylation of c-Jun S63, a transcription factor that has a pivotal role in tumor metastasis.

Figure 8
Figure 8

A model illustrating that Dsg3 acts as an upstream regulator for the Ezrin phosphorylation at Thr567 (activation) through the PKC/AP-1regulation pathway that is required for promoting membrane projections, cell spreading and invasive motility in tumor cells.

Cell invasion is one of the hallmarks of cancer. Our previous studies showed that overexpression of human Dsg3 in cancer cell lines causes dissolution of both classical and desmosomal 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 cytoskeleton 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 target effectors, Ezrin,37 at the plasma membrane to promote cell shape change and migratory capacity.

Dsg3 (pemphigus vulgaris antigen) has recently been implicated in tumor metastasis and progression of SCC in multiple organs, especially the head and neck and lungs,14, 15, 16, 18, 19, 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 in compromised intercellular adhesion and a reduction in the expression of classical and desmosomal cadherins.8, 10 In this study, we describe an additional mechanism by which Dsg3 promotes cell invasive capability by activating AP-1- and the PKC-dependent Ezrin-Thr567 pathway that are likely to be responsible for tumor metastasis in SCC. Evidence that supports this conclusion are as follows: (1) overexpression of Dsg3 enhances membrane protrusions, cell spreading and rounding that are the necessary prerequisites for cell migration/invasion; (2) overexpression of Dsg3 accelerates cell migration/invasion in both A431 and SqCC/Y1 cancer cell lines; (3) Dsg3 forms a complex with and activates Ezrin at the Thr567 residue that is dependent upon several kinases including PKC; (4) modulation of Dsg3 levels directly influenced the activity of Ezrin that directly associates with its interaction with F-actin and CD44 at the plasma membrane; and finally, (5) Dsg3 functions as an upstream activator of AP-1that could be abrogated by PKC inhibition.

The positive role of Ezrin in cancer progression and metastasis has been shown by many studies in the context of cortical cytoskeleton organization, filopodia extension, cell spreading, proliferation, survival and motility.32, 67, 68 Ezrin activation is dynamically regulated in a spatiotemporal manner during metastatic progression.33 Given that upregulation of Dsg3 and Ezrin has been independently found in lymph node metastasis of the head and neck cancer,18, 33, 40, 41, 42 our study suggests it is likely that the Dsg3/PKC/Ezrin-Thr567 pathway is activated at the leading edge or invasive front, in lymph node metastasis of the head and neck cancer.18, 19, 20

A number of transmembrane proteins including the hyaluronan receptor, CD44, have been characterized as ERM-binding proteins.24, 69 This study has identified Dsg3 as a novel membrane protein that associates with Ezrin and is able to regulate its 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 only affected its association with Ezrin but also impaired E-cadherin adhesion and the interaction between Ezrin and CD44/F-actin, indicating the functional significance of such a complex. Although the actual nature of the binding between Dsg3 and Ezrin remains to be elucidated, our mass spectrometric analysis suggests it could be indirect that warrants further investigation. Collectively, the findings from this study and our previous reports8, 9, 10 support the view that non-junctional Dsg3 has a signaling role in epithelial cells, acting as a membrane receptor in regulating a diverse array of cellular events that control 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, such as filopodia and lamellipodia, in order to facilitate contacts with neighboring cells that is a prerequisite for junction formation and cell polarization in normal keratinocytes.51 However, this process is somehow hijacked in transformed tumor cells where the stability and organization of intercellular junctions are drastically altered. Evidence also suggesting the signaling roles of Dsg3 comes from many in vitro studies based on pemphigus vulgaris (PV-IgG).70, 71, 72 Nevertheless, no report has yet shown its association with Ezrin, although the link between actin and pemphigus acantholysis is just beginning to be appreciated.73

Serine/threonine kinases such as ROCK, PKC, p38 and LOK have been identified as the ERM kinases for phosphorylating the C-terminal threonine residue in a different cellular context.32, 74, 75, 76, 77 Among them, PKC family isoforms seem to be ubiquitously involved in all cell types. PKC isoforms such as α, γ and ι are capable of phosphorylating Ezrin-Thr567 at the plasma membrane, particularly the membranous protrusions that control cell motility33 and agents that block this phosphorylation also abrogate PKC α-dependent migration.32 Dsg3 is a calcium-dependent protein and becomes stabilized (increase in expression levels) upon calcium switching8 that also activates PKC (conventional). Our results with the PKC inhibitors that showed significant inhibition of pERM in Dsg3-overexpressing cells indicate it is likely that the Dsg3-mediated Ezrin-Thr567 phosphorylation involves PKCs, particularly calcium-dependent conventional isoforms. There are seven PKC isoforms, that is, α, βII, δ, η, ɛ, λ, ζ, which were shown to be expressed in A431 cells.78 To delineate which isoform(s) is responsible for Dsg3-mediated Ezrin activation merits further investigation. In addition, our studies (this and the study by Tsang et al.9) also support the general notion that other kinases such as ROCK, p38 MAPK, Rac1 and RhoA may also have a role in Dsg3-mediated Ezrin activation, as our phospho-kinase array and the inhibitor analyses showed a wide range of signaling pathways that had been activated in Dsg3-overexpressing cells (Figures 6c and 7a). Positive feedback from Ezrin to Rho GTPases and ROCK was not ruled out here.60 Evidence indicating that Rac1 is coupled with Ezrin activation in the control of E-cadherin-mediated adherens junction assembly is documented in the literature.79 In summary, our findings suggest that Dsg3 might serve as a master cell surface regulator for a variety of signal transduction pathways that influence the activity of Ezrin, one of the effector proteins of these signaling cascades.

In conclusion, this study provides the first evidence of the desmosomal cadherin Dsg3 that functions as a key membrane activator for AP-1 and Ezrin activation that are associated with various cellular responses such as membrane dynamics, cell spreading, migration and invasion. This pathway may involve several signaling molecules including PKCs that are regulated by Dsg3.8, 9, 10 These findings will advance our understanding of the role of Dsg3 in tumor progression and invasion, and likely facilitate development of new therapeutic strategies for the head and neck and lung cancers.

Materials and methods

Cell culture and reagents

A431 cells were maintained in Dulbecco’s modified Eagle medium (DMEM) plus 10% fetal calf serum. Human oral SCC cell line SqCC/Y1 was cultured in EpiLife (Invitrogen, Life Technologies Ltd, Paisley, UK) or keratinocyte growth medium.13 Primary human oral fibroblasts were cultured in DMEM plus 10% donor calf serum and cells with passage number <10 were used. A431 clones with transduction of hDsg3.myc10 were obtained using the limited dilution assay. The protocol, as described previously,10 was used for the generation of stable SqCC/Y1 lines with transduction of human Dsg3 as well as the matched Vects. Antibodies and small interfering RNA sequences are listed in Supplementary Tables 1 and 2.

Immunofluorescence and confocal microscopy

Immunofluorescence was performed as described previously.10 The coverslips were examined with a Zeiss Meta 510LSM/510LSM laser scanning confocal (Carl Zeiss, Cambridge, UK) or Leica DM5000 epi-fluorescence microscope (Leica Microsystems (UK) Ltd. Milton Keynes, UK).

Transwell cell migration and invasion assays

The assays were conducted with 24-well transwell inserts, which contain a permeable membrane with 8 μm pore size (VWR International Ltd, West Sussex, UK). Cells (1 × 105) in basal medium (alpha-DMEM or DMEM:Ham’s F12 at 3:1) plus 1% bovine serum albumin were placed on the upper layer of an insert, which was placed into a 24-well plate containing 500 μl alpha-minimum essential medium plus 10% fetal calf serum for A431 cells and keratinocyte growth medium for SqCC/Y1, which acted as a chemoattractant. The plate was kept in an incubator for 2–3 days to allow cells to migrate into the bottom chamber. The total cell number migrating to the bottom chambers was determined using the CASY cell counter (Roche Innovatis AG, Reutlingen, Germany). For the cell invasion, Matrigel diluted 1:3 in DMEM was loaded on the upper layer of the inserts and allowed to gel uniformly before addition of the cell suspension.

Organotypic culture

The Matrigel:collagen organotypic raft culture is described elsewhere.80

Histology, immunohistochemistry and quantitative analysis

Raft cultures were collected after 14 days and fixed in Trumps (100 mM NaH2PO4, 67.5 mM NaOH, 4% formaldehyde, 1% glutaraldehyde). Specimens were subjected to routine procedures for histology. Five-micrometer sections were stained with hematoxylin and eosin or for keratin with LP34, followed by Dako Cytomation EnVision+dual Link System Peroxidase. The peroxidase was visualized using 3,3′-Diaminobenzidine (DAB) substrate (DAKO, Dako UK Ltd, Cambridgeshire, UK). Quantitation of cell invasion is described elsewhere.80 Images were acquired in a MBF stereology system (MBF Bioscience, Williston, VT, USA) and analyzed by ImageJ (Wayne Rasband, NIH, Bethesda, MD, USA). Parameters such as particle number, total area and average size of particles, and invasion depth were analyzed. The ‘invasion index’ was calculated.80

A Duolink in situ PLA for protein–protein interactions

The PLA was conducted following the protocol described in the Duolink PLA kit (Cambridge Bioscience Limited, Cambridge, UK).

FRET analysis

FRET efficiency was measured using acceptor photobleaching as described previously.10 Eighty-nine regions of both junctional and free edges were analyzed and FRET efficiency was calculated, and the FRET efficiency >5% was considered to be positive.10

Co-immunoprecipitation, protein fractionation and western blotting

The details of the procedures are described previously.8, 10

Human phosphokinase profiler array

A431-Vect and -D3 cell lines seeded at equal density in 10-cm culture dishes were extracted and the phosphokinase array was carried out using 500 μg of total protein in each sample, according to the protocol in the kit (Catalog number: ARY003, R&D Proteome Profiler Array, R&D Systems Europe Ltd., Abingdon, UK).

Luciferase assay

An AP-1 reporter construct was made using the pGL4.26 vector (Promega) containing a minimal eukaryotic promoter. An oligo containing six repeats of the consensus AP-1-binding motif (TGAC[G]TCA) was cloned into KpnI and BglII sites of pGL4.26.AP-1 promoter. A431 parental cells seeded in 6-well plate were co-transfected with equal amount (1 μg) of AP-1 reporter plasmid or empty pGL Vect along with pBABE-hDsg3.myc10 at various concentrations using Fugene HD reagent (Promega). The total amount of pBABE plasmid was topped up by pBABE-GFP to 1 μg in each transfection. Cells were extracted after 2 days or were subjected to serum starvation for 1 day before treatment with various inhibitors. Luciferase assays with cell lysates were performed within 3 days of transfection using the Luciferase Assay System (Promega). Luciferase activities were normalized against protein concentration determined by Bio-Rad DC protein assay (Bio-Rad Laboratories Ltd., Hertfordshire, UK).

References

  1. 1.

    , , . Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion. Cell 1991; 67: 869–877.

  2. 2.

    , , , . Pemphigus vulgaris antigen is a desmosomal desmoglein. Dermatology 1994; 189(Suppl 1): 24–26.

  3. 3.

    , , , , . Extracellular domain of pemphigus vulgaris antigen (desmoglein 3) mediates weak homophilic adhesion. J Invest Dermatol 1994; 103: 609–615.

  4. 4.

    , , , , , et al. Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies. J Biol Chem 2006; 281: 7623–7634.

  5. 5.

    , , , , , . Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism. J Biol Chem 2008; 283: 18303–18313.

  6. 6.

    , , , , , et al. P120-catenin is a novel desmoglein 3 interacting partner: identification of the p120-catenin association site of desmoglein 3. Exp Cell Res 2008; 314: 1683–1692.

  7. 7.

    , , , , , et al. Pemphigus vulgaris identifies plakoglobin as key suppressor of c-Myc in the skin. EMBO J 2006; 25: 3298–3309.

  8. 8.

    , , , , , et al. Non-junctional human desmoglein 3 acts as an upstream regulator of Src in E-cadherin adhesion, a pathway possibly involved in the pathogenesis of pemphigus vulgaris. J Pathol 2012; 227: 81–93.

  9. 9.

    , , , , , et al. Desmoglein 3 acting as an upstream regulator of Rho GTPases, Rac-1/Cdc42 in the regulation of actin organisation and dynamics. Exp Cell Res 2012; 318: 2269–2283.

  10. 10.

    , , , , , et al. Desmoglein 3, via an interaction with E-cadherin, is associated with activation of Src. PLoS One 2010; 5: e14211.

  11. 11.

    , . Desmosomes: a role in cancer? Br J Cancer 2007; 96: 1783–1787.

  12. 12.

    , , , , , et al. Immunohistochemical study of desmosomes in oral squamous cell carcinoma: correlation with cytokeratin and E-cadherin staining, and with tumour behaviour. J Pathol 1998; 184: 369–381.

  13. 13.

    , , , , , . A molecular study of desmosomes identifies a desmoglein isoform switch in head and neck squamous cell carcinoma. J Oral Pathol Med 2011; 40: 67–76.

  14. 14.

    , , , , , et al. DSG3 is overexpressed in head neck cancer and is a potential molecular target for inhibition of oncogenesis. Oncogene 2007; 26: 467–476.

  15. 15.

    , , , , , et al. Desmoglein 3 is overexpressed in inverted papilloma and squamous cell carcinoma of sinonasal cavity. Laryngoscope 2010; 120: 26–29.

  16. 16.

    , , , , , et al. The role of desmoglein-3 in the diagnosis of squamous cell carcinoma of the lung. Am J Pathol 2009; 174: 1629–1637.

  17. 17.

    , , , , , et al. DSG3 as a biomarker for the ultrasensitive detection of occult lymph node metastasis in oral cancer using nanostructured immunoarrays. Oral Oncol 2012; 49: 93–101.

  18. 18.

    , , , , , et al. Molecular staging of cervical lymph nodes in squamous cell carcinoma of the head and neck. Cancer Res 2005; 65: 2147–2156.

  19. 19.

    , , , , , et al. Pemphigus vulgaris antigen mRNA quantification for the staging of sentinel lymph nodes in head and neck cancer. Br J Cancer 2010; 102: 181–187.

  20. 20.

    , , , , , et al. Intraoperative qRT-PCR for detection of lymph node metastasis in head and neck cancer. Clin Cancer Res 2011; 17: 1858–1866.

  21. 21.

    . Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor. J Cell Biol 1989; 108: 921–930.

  22. 22.

    . Regulation of cortical structure by the ezrin-radixin-moesin protein family. Curr Opin Cell Biol 1999; 11: 109–116.

  23. 23.

    , , , , , . ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J Cell Biol 1994; 126: 391–401.

  24. 24.

    , . Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44. Curr Biol 1998; 8: 705–708.

  25. 25.

    , , . Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family. J Cell Biol 1997; 139: 169–179.

  26. 26.

    , , , , , et al. Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin. J Cell Biol 2004; 164: 653–659.

  27. 27.

    , , , . Rho-dependent and -independent activation mechanisms of ezrin/radixin/moesin proteins: an essential role for polyphosphoinositides in vivo. J Cell Sci 2002; 115: 2569–2580.

  28. 28.

    , , , , , et al. High turnover of ezrin T567 phosphorylation: conformation, activity, and cellular function. Am J Physiol Cell Physiol 2007; 293: C874–C884.

  29. 29.

    , , , , , et al. Protein kinase C regulates ezrin-radixin-moesin phosphorylation in canine osteosarcoma cells. Vet Comp Oncol 2011; 9: 207–218.

  30. 30.

    , . Actin microdomains on endothelial cells: association with CD44, ERM proteins, and signaling molecules during quiescence and wound healing. Histochem Cell Biol 2004; 121: 361–369.

  31. 31.

    , , , , . A novel PKC-regulated mechanism controls CD44 ezrin association and directional cell motility. Nat Cell Biol 2002; 4: 399–407.

  32. 32.

    , , , , , et al. Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility. EMBO J 2001; 20: 2723–2741.

  33. 33.

    , , , , , et al. The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC. Oncogene 2009; 28: 792–802.

  34. 34.

    , , , , , . Atypical protein kinase C (iota) activates ezrin in the apical domain of intestinal epithelial cells. J Cell Sci 2008; 121: 644–654.

  35. 35.

    , , , , , et al. Rho kinase phosphorylation promotes ezrin-mediated metastasis in hepatocellular carcinoma. Cancer Res 2011; 71: 1721–1729.

  36. 36.

    , , , , , et al. Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol 1998; 140: 647–657.

  37. 37.

    , , . Downregulated AP-1 activity is associated with inhibition of Protein-Kinase-C-dependent CD44 and ezrin localisation and upregulation of PKC theta in A431 cells. J Cell Sci 2002; 115: 2713–2724.

  38. 38.

    , , , . A possible mechanism for ezrin to establish epithelial cell polarity. Am J Physiol Cell Physiol 2010; 299: C431–C443.

  39. 39.

    , , , , . Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J Cell Biol 1997; 138: 423–434.

  40. 40.

    . Ezrin, a key component in tumor metastasis. Trends Mol Med 2004; 10: 201–204.

  41. 41.

    , , , , , et al. The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis. Nat Med 2004; 10: 182–186.

  42. 42.

    , , , , , et al. Ezrin promotes ovarian carcinoma cell invasion and its retained expression predicts poor prognosis in ovarian carcinoma. Int J Gynecol Pathol 2006; 25: 121–130.

  43. 43.

    , , , , , . Expression of ezrin is associated with invasion and dedifferentiation of hepatitis B related hepatocellular carcinoma. BMC Cancer 2009; 9: 233.

  44. 44.

    , , , , , et al. Intense cytoplasmic ezrin immunoreactivity predicts poor survival in colorectal cancer. Hum Pathol 2008; 39: 1737–1743.

  45. 45.

    , , , , , . Ezrin promotes invasion and metastasis of pancreatic cancer cells. J Transl Med 2010; 8: 61.

  46. 46.

    , , , . Expression of Ezrin and E-cadherin in nasopharyngeal carcinoma and its significance. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2010; 35: 969–975.

  47. 47.

    , , , , , et al. Expression of the membrane-cytoskeletal linker Ezrin in salivary gland adenoid cystic carcinoma. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 112: 96–104.

  48. 48.

    , , , , . Knockdown of ezrin via RNA interference suppresses Helicobacter pylori-enhanced invasion of gastric cancer cells. Cancer Biol Ther 2011; 11: 746–752.

  49. 49.

    , , , , , et al. Roles of ezrin in the growth and invasiveness of esophageal squamous carcinoma cells. Int J Cancer 2009; 124: 2549–2558.

  50. 50.

    , , , , , . RNAi-mediated silencing of ezrin gene reverses malignant behavior of human gastric cancer cell line SGC-7901. J Dig Dis 2009; 10: 258–264.

  51. 51.

    , , , . Directed actin polymerization is the driving force for epithelial cell-cell adhesion. Cell 2000; 100: 209–219.

  52. 52.

    , , , , , . Development of a quantitative method to analyse tumour cell invasion in organotypic culture. J Pathol 2005; 205: 468–475.

  53. 53.

    , . The ARP2/3 complex: an actin nucleator comes of age. Nat Rev Mol Cell Biol 2006; 7: 713–726.

  54. 54.

    , , , , , et al. N,N'-Dinitrosopiperazine-mediated ezrin phosphorylation via activating Rho kinase and protein kinase C involves in metastasis of nasopharyngeal carcinoma 6-10B cells. J Biol Chem 2011; 286: 36956–36967.

  55. 55.

    , , , . Expression of Ezrin and phosphorylated Ezrin (pEzrin) in pancreatic ductal adenocarcinoma. Cancer Invest 2010; 28: 242–247.

  56. 56.

    , , . Organizing the cell cortex: the role of ERM proteins. Nat Rev Mol Cell Biol 2010; 11: 276–287.

  57. 57.

    , , , , , . Androgen induction of prostate cancer cell invasion is mediated by ezrin. J Biol Chem 2006; 281: 29938–29948.

  58. 58.

    , . Heterotypic and homotypic associations between ezrin and moesin, two putative membrane-cytoskeletal linking proteins. Proc Natl Acad Sci USA 1993; 90: 10846–10850.

  59. 59.

    , , , , , . Protein kinase C alpha/beta inhibitor Go6976 promotes formation of cell junctions and inhibits invasion of urinary bladder carcinoma cells. Cancer Res 2004; 64: 5693–5701.

  60. 60.

    , , , , . Protein kinase C activation disrupts epithelial apical junctions via ROCK-II dependent stimulation of actomyosin contractility. BMC Cell Biol 2009; 10: 36.

  61. 61.

    , , , , , et al. Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability. Am J Respir Cell Mol Biol 2011; 45: 1185–1194.

  62. 62.

    , . ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nat Cell Biol 2002; 4: 408–415.

  63. 63.

    , , , , . Rational design and characterization of a Rac GTPase-specific small molecule inhibitor. Proc Natl Acad Sci USA 2004; 101: 7618–7623.

  64. 64.

    , , , , , et al. Desmosome signaling. Inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization. J Biol Chem 2005; 280: 23778–23784.

  65. 65.

    , , , . Transcription factors control invasion: AP-1 the first among equals. Oncogene 2007; 26: 1–10.

  66. 66.

    , , , , , et al. Regulation of a multigenic invasion programme by the transcription factor, AP-1: re-expression of a down-regulated gene, TSC-36, inhibits invasion. Oncogene 2000; 19: 5348–5358.

  67. 67.

    , , , . Co-operative effect of c-Src and ezrin in deregulation of cell-cell contacts and scattering of mammary carcinoma cells. J Cell Biochem 2004; 92: 16–28.

  68. 68.

    , , , , , et al. Initiation of malignancy by duodenal contents reflux and the role of ezrin in developing esophageal squamous cell carcinoma. Cancer Sci 2010; 101: 624–630.

  69. 69.

    , , , , , et al. Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol 1998; 140: 885–895.

  70. 70.

    , , , . Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation? J Invest Dermatol 2008; 128: 501–516.

  71. 71.

    , , , . Desmosomes and disease: pemphigus and bullous impetigo. Curr Opin Cell Biol 2004; 16: 536–543.

  72. 72.

    , , . A pathogenic autoantibody, pemphigus vulgaris-IgG, induces phosphorylation of desmoglein 3, and its dissociation from plakoglobin in cultured keratinocytes. Eur J Immunol 1999; 29: 2233–2240.

  73. 73.

    , , , , . Actin reorganization contributes to loss of cell adhesion in pemphigus vulgaris. Am J Physiol Cell Physiol 2010; 299: C606–C613.

  74. 74.

    , , . Amphetamine-induced ERM Proteins Phosphorylation Is through PKCbeta Activation in PC12 Cells. Korean J Physiol Pharmacol 2011; 15: 245–249.

  75. 75.

    , , , , . LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation. Proc Natl Acad Sci USA 2009; 106: 4707–4712.

  76. 76.

    , , , . Hypotonicity causes actin reorganization and recruitment of the actin-binding ERM protein moesin in membrane protrusions in collecting duct principal cells. Am J Physiol Cell Physiol 2007; 292: C1476–C1484.

  77. 77.

    , , , , , et al. Ezrin/radixin/moesin proteins are phosphorylated by TNF-alpha and modulate permeability increases in human pulmonary microvascular endothelial cells. J Immunol 2006; 176: 1218–1227.

  78. 78.

    , , , . Translocation of diacylglycerol kinase theta from cytosol to plasma membrane in response to activation of G protein-coupled receptors and protein kinase C. J Biol Chem 2005; 280: 9870–9878.

  79. 79.

    , , , , . Ezrin regulates E-cadherin-dependent adherens junction assembly through Rac1 activation. Mol Biol Cell 2003; 14: 2181–2191.

  80. 80.

    , , , , , et al. Psoriasin (S100A7) associates with integrin beta6 subunit and is required for alphavbeta6-dependent carcinoma cell invasion. Oncogene 2011; 30: 1422–1435.

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Acknowledgements

We thank Dr MT Teh, Professor K Parkinson, Dr SM Tsang, Dr MS Ikram and members of CDOS for helpful discussion and technical assistance. This work was supported by a British Skin Foundation-funded studentship awarded to HW and in part by the Institute of Dentistry as well as The Facial Surgery Research Foundation-Saving Faces.

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Affiliations

  1. Queen 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

    • L Brown
    • , A Waseem
    • , J Szary
    • , E Gunic
    • , T Mannan
    • , M Unadkat
    •  & H Wan
  2. Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, London, UK

    • I N Cruz
    •  & M Yang
  3. Division of Biomedical Sciences, St George’s, University of London, Cranmer Terrace, London, UK

    • F Valderrama
  4. Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Centre for Cutaneous Research, Blizard Institute, London, UK

    • E A O′Toole

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https://doi.org/10.1038/onc.2013.186

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