Desmoglein 1 Regulates Invadopodia by Suppressing EGFR/Erk Signaling in an Erbin-Dependent Manner Alejandra Valenzuela-Iglesias1, Hope E

Published OnlineFirst January 17, 2019; DOI: 10.1158/1541-7786.MCR-18-0048

Signal Transduction Molecular Cancer Research Desmoglein 1 Regulates Invadopodia by Suppressing EGFR/Erk Signaling in an Erbin-Dependent Manner Alejandra Valenzuela-Iglesias1, Hope E. Burks1, Christopher R. Arnette1, Amulya Yalamanchili1, Oxana Nekrasova1, Lisa M. Godsel1,2, and Kathleen J. Green1,2,3

Abstract

Loss of the desmosomal cell–cell adhesion molecule, sion by decreasing the number of invadopodia and matrix Desmoglein 1 (Dsg1), has been reported as an indicator degradation. Moreover, Dsg1 requires Erbin to downregu- of poor prognosis in head and neck squamous cell carci- late EGFR/Erk signaling and to fully suppress invadopodia nomas (HNSCC) overexpressing epidermal growth factor formation. Our ﬁndings indicate a novel role for Dsg1 in receptor (EGFR). It has been well established that EGFR the regulation of invadopodia signaling and provide poten- signaling promotes the formation of invadopodia, actin- tial new targets for development of therapies to prevent based protrusions formed by cancer cells to facilitate inva- invadopodia formation and therefore cancer invasion and sion and metastasis, by activating pathways leading to actin metastasis. polymerization and ultimately matrix degradation. We previously showed that Dsg1 downregulates EGFR/Erk signal- Implications: Our work exposes a new pathway by which a ing by interacting with the ErbB2-binding protein Erbin desmosomal cadherin called Dsg1, which is lost early in head (ErbB2 Interacting Protein) to promote keratinocyte differ- and neck cancer progression, suppresses cancer cell invado- entiation. Here, we provide evidence that restoring Dsg1 podia formation by scaffolding ErbB2 Interacting Protein and expression in cells derived from HNSCC suppresses inva- consequent attenuation of EGF/Erk signaling.

In invasive cancer cells, activation of EGFR and its downstream Introduction effectors results in the formation of actin-based protrusions Squamous cell carcinoma comprises more than 90% of known as invadopodia (7). These structures contain an actin-rich cancers of the head and neck (HNSCC) and represents the sixth core and actin-regulatory molecules (e.g., cortactin, Tks5, coﬁlin, most common cancer worldwide (1, 2). Although there have been Arp2/3, N-WASP, MT1-MMP, among others; ref. 8) that facilitate advances in treating HNSCC patients, the overall survival rate degradation of the basement membrane and extracellular matrix remains low (1). Overexpression of epidermal growth factor by the targeted delivery and secretion of matrix metalloprotei- receptor (EGFR) occurs in more than 90% of HNSCC patients nases (MMP) to sites of invasion. The ability of human cancer cells and correlates with enhanced invasion and nodal metastasis (3, 4). to form invadopodia has been correlated with their invasiveness, Targeted therapy against EGFR is considered one of the most both in vitro and in vivo (9–11), and represents a mechanism by promising molecular therapies for HNSCC; however, single treat- which cancer cells enter into the bloodstream and disseminate to ment with agents directed against EGFR provides only a modest distant organs (12, 13). In previous studies, HNSCC cells have beneﬁt due in part to development of drug resistance. Mutations proven to be an excellent model for assessing the role of the EGFR in the EGFR gene, altered expression and/or activity of EGFR pathway and the actin-regulatory machinery in invadopodium effectors, or activation of alternate signaling pathways are impli- dynamics. It has been shown that inhibition of EGFR and down- cated in acquisition of therapeutic resistance (5, 6). stream effectors such as Src and Erk1/2 reduces the number of invadopodia and matrix degradation by suppressing invadopodia signaling and/or phosphorylation of cortactin (12, 14–16). 1 Department of Pathology, Northwestern University Feinberg School of Another critical step in tumor cell invasion and metastasis is 2 Medicine, Chicago, Illinois. Department of Dermatology, Northwestern modulation of intercellular adhesion between cells in the primary University Feinberg School of Medicine, Chicago, Illinois. 3Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago and tumor (17). The importance of classic cadherins and associated Evanston, IL. intercellular adherens junction components in tumor progression is widely appreciated (18, 19). Although less well understood, a Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). role for desmosomal cadherins and associated desmosome components has more recently emerged (20–26). Desmosomes are H.E. Burks and C.R. Arnette contributed equally to this article. intercellular junctions that mediate strong cell–cell adhesion in Corresponding Author: Kathleen J. Green, Northwestern University, 303 E tissues that suffer large amounts of mechanical strain, such as the Chicago Ave., Chicago, IL 60611. Phone: 312-503-5300; Fax: 312-503-8240 or epidermis and myocardium (26, 27). They are composed of three 312-503-8249; E-mail: [email protected] main protein families: the desmosomal cadherins (desmogleins doi: 10.1158/1541-7786.MCR-18-0048 and desmocollins) and their associated armadillo family 2019 American Association for Cancer Research. proteins (plakoglobin and plakophilins), which in turn are linked

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to plakin proteins (desmoplakin; refs. 28, 29). Misregulation hepatitis B and C testing is routinely performed. Keratinocyte of desmosomal cadherins or desmosomal armadillo family purity is assessed by immunostaining for epidermal keratinocyte– proteins has been associated with cell invasion and metastasis specific markers, such as keratins K1/10 and K5/14. Mycoplasma in different types of cancer (17, 20–22, 24, 25, 30–35). Moreover, is routinely performed for all lines using the Lonza MycoAlert desmosome loss can occur even before the disappearance of mycoplasma detection Kit and/or by real-time PCR (IDEXX E-cadherin, consistent with this step being an important early BioResearch). event in the process of epithelial–mesenchymal transition (EMT) contributing to cancer progression (20, 30, 36). Antibodies Desmoglein 1 (Dsg1) is a desmosomal cadherin that is first The following primary antibodies were used: the cortactin expressed as cells transit out of the basal proliferating layer of antibody (ab33333) was from Abcam. The GAPDH (sc- stratified epithelial tissues and become more strongly concentrat- 365062) antibody was from Santa Cruz Biotechnology. The Tks5 ed in the superficial epithelial layers of the epidermis and oral (sc-30122) was from Santa Cruz Biotechnology. A second Tks5 cavity (26, 27). Although Dsg10s roles in maintaining tissue antibody from EMD Millipore (MABT336) was used after sc- integrity have been well-established, Dsg1 is also now known to 30122 became unavailable. The Dsg1 (AF944) antibody was from be a key regulator of signaling pathways to modulate the balance R&D Systems. The Erbin (22438-1-AP) antibody was from Pro- of proliferation and differentiation. Through its cytoplasmic tail, teintech. The EGFR (4267), pEGFR Y1068 (2234), and pErk1/2 Dsg1 inhibits both EGFR and the Erk/MAPK pathways (37, 38). By (4370) antibodies were from Cell Signaling Technology. The interacting with the ErbB2-binding protein Erbin (ErbB2 Inter- Erk1/2 (V114A) antibody was from Promega. The MT1-MMP acting Protein), Dsg1 inhibits the formation of Ras–Raf com- (AB8345) and MMP-2 (AB809) antibodies were from Chemicon plexes mediated by Shoc2, leading to Erk1/2 signaling down- International. The ADAM 10 (CSA-835) antibody was from regulation, which induces keratinocyte differentiation (37). In Stressgen Biotechnologies. The M2 Flag (F1804), Flag (F7425), addition, Dsg1 is downregulated in different types of cancer that and GAPDH (G9545) antibodies were from Sigma-Aldrich. The frequently overexpress EGFR, such as HNSCC. Reduced Dsg1 in Dsg2 (610121) antibody was from Progen. The Dsg3 (MABT335) these tumors correlates with a poorly differentiated phenotype antibody was from Millipore. The E-cadherin (HECD1) antibody and highly invasive carcinoma with low survival rates (39, 40). was a gift from M. Takeichi and O. Abe, RIKEN Center for Here, we demonstrate a role for Dsg1 in suppressing EGF-depen- Developmental Biology, Kobe, Japan. The plakoglobin (1407) dent invadopodia formation and function, and show that Dsg10s antibody was generated by Aves Laboratories. The K5 and K1 ability to efficiently inhibit HNSCC cell invasion depends on its antibodies were a gift from J. Segre, National Human Genome associated protein Erbin. Research Institute. Actin was visualized using phalloidin from Thermo Fisher (A22287). Western blot analysis included use of peroxidase-conjugated Materials and Methods anti-mouse, -rabbit, and -chicken secondary antibodies pur- Cell culture and drugs chased from Kirkegaard & Perry Laboratories. AlexaFluor 405/ Human-derived squamous cells, carcinoma Cal33 cells, and 488/568/647–conjugated donkey anti-mouse, -goat, and –rabbit UMSCC1 squamous carcinoma cells were cultured in DMEM/ secondary antibodies (Invitrogen) were used in immunofluores- F-12 media supplemented with 10% FBS and 1% penicillin/ cence studies. streptomycin, and were used within five passages. For EGF stimulation experiments, Cal33, UMSCC1 cells and/or spheroids were DNA constructs serum starved in 0.5% FBS and 0.8% BSA in DMEM F-12 media LZRS-Dsg1 full-length Flag Tag, LZRS-mCherry, LZRS-Dsg1- for 16 hours before stimulation with 50 ng/mL EGF. For experi- ICS, LZRS-dPg, and LZRS-909 constructs were generated as pre- ments using inhibitors, Cal33 cells were serum starved for 16 viously described (37, 38, 41, 42). fi hours before treatment with DMSO (Thermo Fisher Scienti c), Retroviral infections m 5 mol/L EGFR inhibitor, AG1478 (Selleck Chemicals), or The phoenix packaging cell line (provided by G. Nolan, Stan- m 5 mol/L Erk inhibitor, U0126 (Cell Signaling Technology). ford University, Stanford, CA) was cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and Cell lines authentication transfected with LZRS constructs (4 mg/mL) using Lipofectamine. Cell lines obtained from the following sources were all sub- The next day, cells were reseeded into selection media with 1 mg/ fi jected to short tandem repeat pro ling to detect both contami- mL puromycin. When cells reached 70% confluency, they were fi nation and misidenti cation, including intra- and interspecies switched to DMEM without puromycin and incubated at 32C for contamination by IDEXX BioResearch: Cal33 (Sharon Stack, 24 hours. Viral supernatant was collected and concentrated using University of Notre Dame); SCC25 (Jennifer Grandis, University Amicon Ultra-15 Centrifugal Filter Units (Millipore). Cal33 cells of Pittsburgh); SCC9 (J. Rheinwald, Harvard Medical School, were infected with virus and 8 mg/mL polybrene for 60 to 90 Boston, MA); SCC22B and UMSCC1 (Thomas Carey, University minutes at 32C. Next, cells were washed with PBS and returned to of Michigan); Cal27 cells were purchased from the ATCC (CRL- growth at 37C in complete DMEM/F-12 media. 2095). With one exception, lines had scores of 80% to 100%, indicating the samples are consistent with the cell line of origin. siRNA treatment and transfection The SCC22B cell line had a score below 80% and due to uncer- Cal33 and/or UMSCC1 cells at approximately 30% to 50% tainty about its identity was not used beyond Supplementary Fig. confluency were transfected with siRNA oligonucleotides at a final S1 in this study. Primary normal human epidermal keratinocyte concentration of 20 nmol/L via DharmaFECT. siRNA directed isolates (NHEK) are obtained through the Northwestern Univer- toward Erbin (Invitrogen Stealth siRNA: 50-CCACACTGTTGTAT- sity Skin Disease Research Core, where mycoplasma, HIV-1, and GATCAACCATT-30), Dsg2 (Dharmacon ON-TARGETplus

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SMARTpool, L-011645-00-0005: 50-CAAUAUACCUGUAGUA- Statistical analysis GAA-30,50-GAGAGGAUCUGUCCAAGAA-30,50-GAGAGGA- Statistical analysis was performed using Excel or GraphPad UCUGUCCAAGAA-30,50-CCUUAGAGCUACGCAUUAA-30, Prism software. P values for two groups were calculated using two- 50-CCAGUGUUCUACCUAAAUA-30), and scramble/nontarget- tailed unpaired Student t tests. P values for three or more groups ing siRNA (Dharmacon, D-001206-14-20). were calculated using one-way ANOVA. P values for multiple dependent variables were calculated using two-way ANOVA. Each Invadopodial matrix degradation assay group includes measurements from three independent experi- The invadopodial matrix degradation assay was performed ments. P values less than 0.05 were considered statistically sig- as previously described with minor modifications (9, 11, 43). nificant. Error bars represent the SEM. All graphs are displayed as Briefly, 75,000 cells were plated on 488-labeled gelatin over- mean SEM. night. The next day, cells were fixed in 3.7% paraformaldehyde for 20 minutes, permeabilized in 0.1% Triton-X-100 for 30 minutes, and stained with antibodies recognizing cortactin Results or actin, Tks5, and Dsg1. At least ten different fields were Dsg1 expression suppresses invadopodia formation and matrix acquired per condition, and each independent experiment was degradation in HNSCC cells performed 3 times. Invadopodia were identified as Tks5 and In light of our previous findings that Dsg1 suppresses EGFR/Erk cortactin-positive punctate structures. Invadopodia were man- signaling in keratinocytes (37, 38), we hypothesized that Dsg1 ually counted from images and reported as invadopodia per may suppress HNSCC tumor invasion through its ability to cell. Degradation area was determined by thresholding images interfere with EGF-dependent formation of invadopodia. To and determining the number of dark pixels within punctate establish a model system for the study, we first evaluated a panel regions of degradation using the Analyze Particles command in of oral SCC lines, including Cal33, Cal27, SCC25, SCC22B, SCC9, ImageJ. The total degradation area in pixels was divided by and UMSCC1, for expression of junction proteins, EMT markers, the total number of cells sampled or by the average total cell and ability to degrade gelatin. All cell lines tested had lost area (in pixels) of the sampled cells to arrive at mean degra- expression of Dsg1 (Supplementary Fig. S1). Cal27, SCC25, dation area/cell or /cell area. The data were normalized to SCC22B, and SCC9 cells were excluded from the study as they control (mCherry) by dividing each area of degradation by the had already progressed to express EMT markers such as N-cad- mean degradation area of mCherry, setting the mean area of herin, vimentin, or both. Cal33 and UMSCC1 retained expression mCherry-expressing cells to a value of 1. of junctional proteins such as E-cadherin without having begun to express EMT markers. Therefore, we selected these lines for further 0 Spheroid invasion assay evaluation of Dsg1 s role in invadopodia formation and function. To generate spheroids, cells were plated at low density (0.5 Although Cal33 cells have lost expression of Dsg1, they still 104 cells/mL) into ultralow attachment 96-well round-bottom express the desmoglein isoforms, Dsg2 and Dsg3 (Supplementary plates as described (44). After 4 days of incubation, the spheroids Fig. S2), and the classic cadherin adhesion machinery, including were embedded in 100 mL of 5 mg/mL rat-tail collagen I (Corning E-cadherin and associated catenins (Fig. 1A). This selective loss of Inc.). The collagen solution was prepared according to the man- Dsg1 suggests that Cal33 calls have progressed to a more tumor- ufacturer's protocol (Alternate Gelation Procedure). EGF was igenic phenotype, but still retain their baseline adhesion machin- added into each well as indicated. For the image analysis of ery. Toward addressing whether exogenous Dsg1 can suppress spheroids, both the core and the distal margin of the invasion invadopodia formation, Cal33 cells were transduced with LZRS zone were manually outlined based on phase-contrast images Dsg1 full-length Flag-tag (Dsg1FL). mCherry was used as a con- using ImageJ. Then, the area of the core was subtracted to obtain trol. Western blot analysis of whole-cell lysates confirmed the the area of invasion. efficacy of the transduction (Fig. 1A). Next, Cal33-mCherry and Cal33-Dsg1FL cells were assayed for Microscope imaging invadopodium formation. Cells were plated on 488-gelatin over- For invadopodial matrix degradation assays, imaging was night, then fixed and stained for Tks5 and cortactin as invado- performed on a wide-field microscope (Upright Leica, model podia markers. Dsg1FL cells were also stained for Dsg1, and only DMR) fitted with a 40X (NA 1.0) oil immersion objective. Images those cells expressing Dsg1 at cell–cell interfaces were counted for were captured with an Orca 100 CCD camera (model C4742-95; the assay. Invadopodia were identified as Tks5 and cortactin- Hammamatsu) and MetaMorph version 7.7.0 imaging software positive punctate structures and reported as invadopodia per cell. (Universal Imaging Corp.). For 3D invasion assays, spheroids Imaging analysis showed that upon Dsg1 expression, the number were imaged using a phase-contrast inverted microscope (Carl of invadopodia decreased by 75% (Fig. 1B and C). It should be Zeiss, Axiovert 40 CFL) fitted with 10X objective. Images were noted that whereas the basal keratin K5 is present regardless of analyzed using ImageJ software (NIH version 1.6.0). Dsg1 expression status, exogenous expression of Dsg1 in Cal33 cells does not induce the expression of differentiation markers Western blot analysis such as K1 (Fig. 1A). This finding is consistent with the idea that 0 For analysis of protein expression levels, whole-cell lysates Dsg1 s ability to interfere with invadopodia formation is not an were obtained by using urea-SDS buffer or RIPA buffer (for indirect effect of promoting differentiation. phosphoprotein analysis). Samples were run on 7.5% SDS- Because the primary function of invadopodia is to degrade PAGE gels and transferred to nitrocellulose membranes to be basement membranes through secretion of MMPs, we analyzed probed with appropriate primary and secondary antibodies. the ability of the cells to degrade the underlying gelatin. Degra- Densitometric measurements of scanned immunoblots were dation area was calculated as the total area covered by degradation performed using ImageJ. holes/cell in thresholded images using the Analyze Particles

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A C *** 40 Cal33 mCherry NHEK Cal33 Dsg1FL Cal33 165 KDa Dsg1 30 165 KDa Flag Dsg1 staining in B: 135 KDa E-cad 20

75 KDa Pg Number of

60 KDa K5 invadopodia/cell 10 58 KDa K1

35 KDa GAPDH 0

B mCherry Dsg1FL E **** 5

mCherry 4

Tks5 Cortactin 3

1 Degradation area/cell (norm)

Dsg1FL 0 Tks5 Cortactin mCherry Dsg1FL D F *** 5

3 mCherry Gelatin 2

Degradation area/cell area (norm) 0

Dsg1FL Dsg1 Gelatin mCherry

Figure 1. Dsg1 regulates invadopodia formation and function. A, Western blot of whole-cell lysates from Cal33, Cal33-mCherry, Cal33-Dsg1FL, and NHEK cells probed for Dsg1, Flag, E-cadherin (E-cad), Plakoglobin (Pg), Keratin 5 (K5), Keratin 1 (K1), and GAPDH (NHEK was used for comparison). B, Cal33- mCherry and Cal33-Dsg1FL cells stained for invadopodia markers, Tks5 and cortactin. Inset shows magnified image of invadopodia in red box. Smaller image shows Dsg1 staining in Dsg1FL cells. C, Quantification of the number of invadopodia per cell. n > 400 invadopodia from >100 cells; three independent experiments. Unpaired Student t test with Welch correction. , P ¼ 0.0005. Error bars indicate SEM. D, Cal33-mCherry and Cal33- Dsg1FL cells after overnight plating on 488-labeled gelatin. E, Quantification of invadopodial matrix degradation area per cell normalized to control. Normalization of data was done by dividing each area of degradation by the mean degradation area of mCherry-expressing cells. n > 100 cells; three independent experiments. Unpaired Student t test with Welch correction. , P < 0.0001. Error bars indicate SEM. F, Quantification of degradation area per cell area normalized to mCherry-expressing cells. n > 100 cells; three independent experiments. Unpaired Student t test with Welch correction. , P ¼ 0.0003. Error bars indicate SEM. Scale bars, 10 mm.

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command in ImageJ. Expression of Dsg1FL resulted in a decrease mation, we tested several previously published Dsg1 mutants in in the degradation area per cell (Fig. 1D and E) and degradation Cal33 cells (37, 38, 41, 42). These include Dsg1-dPg, a triple-point area per cell area (Fig. 1F) by 68% and 54%, respectively. This is mutant of the catenin-like region, which interferes with plako- most likely due to a decrease in the number of invadopodia, rather globin binding; Dsg1-909 mutant that lacks the last 140 aa on than a decrease in MMP secretion, because quantification of the Dsg1 cytoplasmic tail; and Dsg1-ICS mutant that lacks the PL, degradation area per invadopodium showed no differences RUD, and TD domains, and is unable to bind Erbin (Dsg1- between control and Dsg1FL cells (Supplementary Fig. S3). In ICS; Fig. 3A and B). We previously showed that through its addition, the expression levels of MT1-MMP, ADAM10, and association with Erbin, Dsg1 suppresses Erk1/2 activation down- MMP-2, the major proteinases secreted by invadopodia, remained stream of EGFR by inhibiting the Ras-Raf scaffolds mediated by unchanged upon Dsg1 expression (Supplementary Fig. S4), sug- Shoc2 (37). gesting that invadopodia formed in both types of cells are equally Next, we assayed all the mutants for invadopodia formation active. after overnight plating on fluorescent gelatin and found that the number of invadopodia formed by Cal33 Dsg1-dPg and Dsg1- EGFR stimulation does not increase the number of invadopodia 909 were suppressed to the same level as Cal33-Dsg1FL cells. or invasion of Cal33-Dsg1–expressing cells Conversely, cells expressing the Dsg1-ICS mutant failed to Invadopodia formation is a multistep process that starts with suppress invadopodia, exhibiting a similar number as control the formation of a precursor, which is a structure constituted by all cells (Fig. 3C and D). Consistent with these results, quantification the proteins that form the invadopodium core, but has not yet of the degradation area per cell decreased compared with control acquired the capacity to degrade the extracellular matrix (ECM). and showed no differences between Dsg1-FL, Dsg1-dPg, and Subsequent precursor maturation then results in full proteolytic Dsg1-909. In contrast, the Dsg1-ICS mutant failed to suppress activity (8). It is well known that invadopodia can form through degradation (Fig. 3E and F). activation of EGFR by EGF or other stimuli (e.g., TGF-b or PDGF) Collectively, these results show that the Dsg1 domain known to in different types of cancer cells (7, 45). As we previously showed be required for Erbin binding is necessary to regulate invadopodia that Dsg1 attenuates EGFR activity and downstream MAPK sig- formation and function. naling in an adhesion-independent manner (37, 38), we tested whether Dsg1 regulates the early stages of invadopodium precur- Efficient suppression of invadopodia by Dsg1 is sor formation. Erbin-dependent To test this hypothesis, Cal33-mCherry and Cal33-Dsg1FL cells Because the Dsg1-ICS mutant lacks several domains on the were serum starved overnight and stimulated with 50 ng/mL EGF Dsg1 tail that could be affecting other unknown binding partners, for 0 minute (untreated) and 3 minutes to synchronously induce to more directly address Dsg10s dependence on Erbin to suppress precursor formation. Whereas the number of invadopodia invadopodia, we next silenced Erbin in Cal33 cells in the presence increased 4.5-fold in response to EGF treatment in mCherry cells, and absence of Dsg1 expression (Fig. 4A). Introducing Dsg1 along Dsg1FL-expressing cells failed to exhibit an increase in the number with a control siRNA resulted in a 73% reduction in the number of of invadopodia precursors upon EGF stimulation (Fig. 2A and B). invadopodia, similar to that shown above. However, in cells To determine whether Dsg1 is also capable of suppressing expressing Dsg1 in the presence of Erbin knockdown, the reduc- invadopodia in a more physiologically relevant 3D environment, tion in the number of invadopodia was only 37% when compared we carried out invasion assays using spheroids in which tumor with control cells, suggesting that Dsg1 depends at least in part on cells are organized into a 3D structure mimicking a tumor micro- Erbin for its invadopodia-suppressing function (Fig. 4B and C). region. Spheroids are embedded into rat-tail collagen 1, where Importantly, Erbin knockdown alone did not affect the number of MMP activity is required to invade out of the tumor mass into the invadopodia compared with control cells, suggesting that its role surrounding matrix (46, 47). At 0 hour (no stimulation), there are in regulating invadopodia formation requires the presence of no obvious differences in size and morphology between the Dsg1 (Fig. 4B and C). Similar results were obtained when ana- spheroids from Cal33-mCherry and Cal33-Dsg1FL cells lyzing matrix degradation per cell. Dsg1 in the presence of control (Fig. 2C). However, after 24 hours of EGF stimulation, Dsg1FL- siRNA suppresses matrix degradation, but its ability to do so is expressing spheroids fail to invade into the collagen, showing a impaired upon Erbin depletion. Depletion of Erbin alone, how- reduction in the invasion area by 80% when compared with ever, did not have any effect on matrix degradation compared with spheroids from control cells (Fig. 2C and D). Overall, these results control (Fig. 4D and E). These observations are consistent with the suggest that Dsg1 expression inhibits the early stages of EGF- idea that Erbin acts in a complex with Dsg1 to inhibit invadopodia induced invadopodium precursor formation leading to a decrease formation and matrix degradation. in the invasion capacity. We next examined whether Erbin was required for Dsg1- dependent inhibition of invadopodia following EGF stimulation. The Erbin-binding region on Dsg1 is required to suppress Dsg1FL-expressing cells did not increase invadopodia formation invadopodia formation and function in response to EGF stimulation. Erbin depletion in Dsg1- In addition to the shared intracellular cadherin-typical expressing cells exhibited a 1.7-fold increase in invadopodia sequence (ICS) required for Pg binding, the cytoplasmic tail of formation upon EGF stimulation compared with the unstimu- the Dsg isoforms contains unique C-terminal domains not found lated control (Supplementary Fig. S5; P ¼ 0.078 trending toward in other desmosomal or classical cadherins. These include an significance). intracellular proline-rich linker (PL), a variable number of repeat- In order to demonstrate the impact of the Dsg1–Erbin complex ing unit domains (RUD), and a glycine-rich desmoglein-specific on invadopodia formation is not cell type specific, we expressed terminal domain (TD; refs. 29, 48; Fig. 3A). To address which Dsg1 in the HNSCC line UMSCC1. Both invadopodia formation Dsg1 domains are important for suppressing invadopodia for- and matrix degradation were also reduced in this cell type in the

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A Tks5 Cortactin

B n.s. 25 **** ****

mCherry 20 n.s.

nim 0 nim 15

5 Dsg1FL Number of invadopodia/cell 0

Dsg1FL mCherry Dsg1FL mCherry 0 min 3 min 50 ng/mL EGF

FGE mCherry

Lm/gn 05 ni 05 Lm/gn Dsg1 staining in A: 3 min 0 min 50 ng/mL EGF

m 3 m Dsg1FL Dsg1FL

CD0 h 24 h 8 *

6 mCherry 4

2 Area of invasion/spheroid 0 Dsg1FL

mCherry Dsg1FL

Figure 2. Dsg1 suppresses EGF-induced invadopodium precursors and 3D invasion. A, Cal33-mCherry and Cal33-Dsg1FL cells stained for invadopodia markers Tks5 (red) and cortactin (green) at 0 minute (untreated) and 3 minutes after 50 ng/mL EGF stimulation. Inset shows magnified image of invadopodia in red box. B, Quantification of the number of invadopodia per cell at 0 minute (untreated) and 3 minutes after EGF stimulation. n > 500 invadopodia from >250 cells; three independent experiments. Two-way ANOVA with Sidak multiple comparison test. , P < 0.0001. Error bars, SEM. C, Phase-contrast images of invasion of Cal33-mCherry and Cal33-Dsg1FL spheroids embedded in rat-tail collagen type I (5 mg/mL) at 0 hour (untreated) and 24 hours after EGF stimulation. D, Quantification of area of invasion per spheroid normalized to control at 24 hours after EGF stimulation. n ¼ 30 spheroids; three independent experiments. Unpaired Student test with Welch correction. , P ¼ 0.034. Error bars, SEM. Scale bars, 10 mm.

presence of Dsg1 (72% and 82% respectively compared with ultimately matrix degradation (15, 45). Because we previously mCherry controls), in an Erbin-dependent fashion (Supplemen- showed that Dsg1–Erbin interaction attenuates EGFR/Erk signal- tary Fig. S6). ing (37, 38), we measured their activation levels in Cal33 cells It has been reported that activation of EGFR and Erk is required expressing Dsg1 in the presence and absence of Erbin. Western to initiate the pathways that lead to actin polymerization and blot analysis showed that in control cells, Erbin depletion has no

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Dsg1 staining in C: A Cytoplasmic domains Dsg1FL Dsg1-dPg (aa 570-1049) B

1 1049 Extracellular domain TM IA ICS PL RUD I RUD II RUD III TD

Flag Dsg1FL Cal33 mCherry Cal33 Dsg1FL Cal33 Dsg1-dPg Cal33 Dsg1-909 Cal33 Dsg1-ICS AAA Dsg1-909 Dsg1-ICS Flag Dsg1-dPg 165 KDa Flag Flag Dsg1-909 35 KDa GAPDH Flag Dsg1-ICS

C Tks5 Cortactin E mCherry/Dsg1 Gelatin

D 40 mCherry mCherry 30 **** n.s. **** 20 **** Dsg1FL Dsg1FL 10 Number of invadopodia/cell 0

Dsg1FL mCherry Dsg1-dPGDsg1-909Dsg1-ICS Dsg1-dPg F 8

6 Dsg1-909 Dsg1-909 4 n.s. 2 **** **** ***

Degradation area/cell (norm) 0 Dsg1-ICS Dsg1-ICS Dsg1-dPg

sg1-dPGsg1-909 sg1-ICS mCherryDsg1FLD D D

Figure 3. Erbin-binding domain of Dsg1 is required to regulate invadopodia. A, Schematic of Dsg1 constructs. B, Western blot of whole-cell lysates from Cal33-mCherry, -Dsg1FL, -Dsg1-dPg, -Dsg1-909, and -Dsg1-ICS probed for Flag and GAPDH. C, Cal33-mCherry, -Dsg1FL, -Dsg1-dPg, -Dsg1-909, and -Dsg1-ICS cells stained for invadopodia markers, Tks5 (red) and cortactin (green). Inset shows magnified image of invadopodia in red box. Smaller images show Dsg1 staining of cells expressing Dsg1 constructs. D, Quantification of the number of invadopodia per cell. n > 1,600 invadopodia from n > 300 cells; three independent experiments. One-way ANOVA with Dunn post hoc test. , P < 0.0001. Error bars, SEM. E, Cal33-mCherry, -Dsg1FL, -Dsg1-dPg, -Dsg1-909, and -Dsg1-ICS cells after overnight plating on 488-labeled gelatin. F, Quantification of invadopodial matrix degradation area per cell normalized to control. n > 300 cells; three independent experiments. One-way ANOVA with Dunn post hoc test. , P ¼ 0.0002 and , P < 0.0001. Error bars, SEM. Scale bars, 10 mm. effect on the phosphorylation state of EGFR and Erk1/2 (Fig. 5A). cells in the absence and presence of Dsg1 expression. Next, to In Dsg1FL cells treated with control siRNA, activation of EGFR and initiate invadopodia formation, cells were cultured in complete Erk1/2 was suppressed as previously reported for normal epider- media for 6 hours and treated with the vehicle DMSO as a mal keratinocytes (37, 38). However, Dsg1FL was unable to control, EGFR inhibitor (5 mmol/L AG1478), or Erk inhibitor suppress EGFR and Erk1/2 in cells that were also transfected with (5 mmol/L U0126). Erbin RNAi (Fig. 5A). Together, these results suggest that Dsg1 Consistent with previous experiments, Dsg1FL-ctrl siRNA cells requires Erbin to efficiently downregulate EGFR/Erk1/2 signaling treated with DMSO suppressed matrix degradation by 67% when in Cal33 HNSCC cells, comparable with normal epidermal compared with control cells (Fig. 5B and C). Erbin depletion keratinocytes. reduced Dsg10s effectiveness; under these conditions matrix deg- We next took a pharmacologic approach to address the extent radation was suppressed by 37% when compared with control to which the observed difference in matrix degradation between cells (Fig. 5B and C). Erbin knockdown in control cells did not Dsg1-expressing cells and Erbin-deficient Dsg1-expressing cells show any effect (Fig. 5B and C). EGFR or Erk inhibitors reduced is EGFR or Erk1/2-dependent. We first silenced Erbin in Cal33 matrix degradation in mCherry (control) cells 60% compared

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ABTks5 Cortactin DmCherry/Dsg1 Gelatin ctrl si mCherry ctrl si mCherry Erb si Dsg1FL Erb si Dsg1FL

180 KDa Erbin

165 KDa Flag mCherry ctrl si mCherry ctrl si 35 KDa GAPDH

C **** 40 n.s. mCherry Erb si 30 mCherry Erb si *

10 ctrl si L Number of invadopodia/cell Dsg1FL ctrl si Dsg1FL 0 Dsg1F

Dsg1FL ctrl si mCherry ctrlmCherry si Erb si Dsg1FL Erb si L Erb si L Erb si E **** L 4 n.s. Dsg1F

** Dsg1F

3 Dsg1 staining in B: 2 Dsg1FL ctrl si Dsg1FL Erb si

Degradation area/cell (norm) 0

mCherry ctrlmCherry si ErbDsg1FL si ctrlDsg1FL si Erb si

Figure 4. Dsg1 mediates invadopodia formation and function through Erbin. A, Cal33-mCherry and Cal33-Dsg1 FL cells were transfected with control or Erbin siRNA, and whole-cells lysates were immunoblotted after 72 hours of transfection with Erbin, Flag, and GAPDH antibodies. B, Cal33-mCherry and Cal33-Dsg1FL cells treated with control or Erbin siRNA and stained for invadopodia markers, Tks5 (red), and cortactin (green). Inset shows magnified image of invadopodia in red box. Smaller images show Dsg1 staining in Dsg1-expressing cells. C, Quantification of the number of invadopodia per cell. n > 800 invadopodia from n > 200 cells; three independent experiments. One-way ANOVA with Dunn multiple comparisons test. , P ¼ 0.0319 and , P < 0.0001. Error bars, SEM. D, Cal33-mCherry and Cal33-Dsg1FL cells treated with control or Erbin siRNA and plated on 488-labeled gelatin overnight.E, Quantification of invadopodial matrix degradation per cell normalized to control. n > 200 cells; three independent experiments. One-way ANOVA with Dunn multiple comparison test. , P < 0.01 and , P < 0.0001. Error bars, SEM. Scale bars, 10 mm.

with DMSO-treated cells, as expected from previous reports (15, radation when reintroduced into cells that have lost Dsg1 expres- 49). EGFR or Erk inhibitors reduced matrix degradation in Dsg1- sion. These data also reveal an Erbin-independent component of Erbin siRNA cells and mCherry cells with and without Erbin, to Dsg1 invadopodia inhibition in addition to the EGFR/Erk-depen- levels similar to those observed in Dsg1FL-ctl siRNA-treated cells dent component in the Dsg1 siErbin cells. (Fig. 5B and C). No further reduction in matrix degradation To determine whether loss of Dsg1 is associated with progres- occurred in Dsg1FL-expressing cells treated with the inhibitors. sion of HNSCC in vivo, we used The Cancer Genome Atlas (TCGA) This is consistent with the idea that Dsg1, in the presence of Erbin, database (50) to compare Dsg1 mRNA expression levels in tumors is just as powerful as the pharmacologic inhibitors of EGFR/Erk to with histologic grades 1–4. Signiﬁcant reductions in Dsg1 mRNA suppress these downstream effectors and associated matrix deg- occur at each stage from 1 to 3 (Supplementary Fig. S7A).

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A C 1.5 mCh Ctrl si mCh Erb si Ctrl si Dsg1FL Erb si Dsg1FL mCh Ctrl si mCh Erb si Dsg1FL Erb si Dsg1FL Dsg1FL Ctrl si Dsg1FL 175 KDa EGFR 44 KDa Erk 1.0 175 KDa 44 KDa pErk ## pEGFR (Y1068) #### 1.0 1.1 0.6 1.4 1.0 1.4 0.6 1.3 35 KDa GAPDH 35 KDa GAPDH 0.5 **** **** **** **** ** mCherry ctrl si ** mCherry Erb si 165 KDa Dsg1 180 KDa Erbin Dsg1FL ctrl si

35 KDa GAPDH 35 KDa GAPDH Degradation area/cell (norm) 0.0 Dsg1FL Erb si

mol/L mol/L DMSO µ µ U0126 5 AG1478 5

mCherrymCherry Dsg1 Dsg1 B mCherry ctrl si mCherry Erb si Dsg1FL ctrl si Dsg1FL Erb si DMSO 5 µ mol/L AG1478 5 µ mol/LU0126

Figure 5. Dsg1 attenuates invadopodia signaling in an Erbin-dependent manner. A, Cal33-mCherry and Cal33-Dsg1FL cells were transfected with control or Erbin siRNA, and whole-cells lysates were immunoblotted after 72 hours of transfection with EGFR, pEGFR (Y1068), Erk, pErk (p44/42), Erbin, Flag, and GAPDH antibodies. For quantification of phosphorylated proteins, each band was normalized to the amount of total protein. Measurements represent an average of three independent experiments. B, Cal33-mCherry and Cal33-Dsg1FL cells treated with control or Erbin siRNA plus vehicle (DMSO), EGFR (5 mmol/L AG1478), and Erk (5 mmol/L U0126) inhibitors and plated on 488-labeled gelatin for 6 hours. Smaller images show mCherry IF and Dsg1 staining. Scale bars, 10 mm. C, Quantification of invadopodial matrix degradation per cell. n > 700 cells; three independent experiments. Two-way ANOVA with Dunn post hoc test. Analysis within groups reveals that only DMSO treatment shows significant decreases in Dsg1FL ctrl si and Dsg1FL Erb si as compared with mCherry ctrl si. ##, P ¼ 0.032 and ####, P < 0.0001. Analysis across groups reveals significant decreases in degradation area/cell following AG1478 and U0126 drug treatment under all expression conditions except Dsg1FL ctrl si. , P ¼ 0.0073 and , P < 0.0001. Error bars, SEM.

Although Dsg1 in stage 4 tumors was not further reduced com- elevated in expression from stages 1–3 (ref. 50; Supplementary pared with stage 3, a relatively low number of samples was in the Fig. S7A). To determine whether Dsg2 has an impact on invado- database for stage 4. podia formation, we carried out Dsg2 knockdown experiments in Interestingly, Dsg2, which was previously shown to be elevated Cal33 cells, which express this basal desmoglein endogenously. in malignant skin carcinoma (51) and to predispose transgenic Notably, knockdown of Dsg2 suppressed both invadopodia animals in which it is misexpressed to SCC development (52), was formation and degradation in these cells, indicating it has a

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reciprocal role to Dsg1 in regulating invadopodia (Supplemen- In our study, a Dsg1 mutant with a 95% decreased ability to tary Fig. S7B–S7E). binding to plakoglobin was still able to suppress invadopodia formation and function. Thus, it seems likely that the remaining Dsg domains are sufficient to exert control over cell growth or Discussion migration mediated by plakoglobin in this setting. These con- EGFR is overexpressed in >90% of HNSCC patients, and its siderations also suggest that our previous demonstration that expression correlates with enhanced invasion and metasta- plakoglobin suppresses keratinocyte migration through Src- and sis (3, 4). We previously showed that Dsg1 dampens EGFR/Erk FN-dependent mechanisms cannot explain Dsg10s control over signaling (37, 38), and downstream effectors of EGFR are known HNSCC cell invasion (54, 55). to promote formation of invadopodia protrusions to facilitate We previously showed that Dsg1 interacts with the Erbin to cancer cell invasion and metastasis (7, 45).Therefore, we asked suppress MAPK/Erk signaling and support normal keratinocyte whether Dsg1 serves as an invasion suppressor through its ability differentiation (37). Erbin localizes at the basolateral membrane to regulate EGFR signaling. We demonstrate for the first time that to regulate cell junctions and polarity in epithelial cells (56, 57). expression of Dsg1 in oral cancer cells is sufficient to suppress However, the role of Erbin during cancer progression varies invadopodia formation and invasion in 2D and 3D assays. The depending on context. It has been reported that Erbin can act as potential in vivo relevance of this finding is supported by analysis a tumor suppressor by inhibiting TGFb and Erk1/2 signaling of TCGA data from staged HNSCC tumors, showing progressive (58–60). In HeLa cells, Erbin depletion causes an increase in cell loss of Dsg1 mRNA expression with increased histologic grade proliferation and migration (61). Conversely, Erbin can also (ref. 50; Supplementary Fig. S7A). This analysis supports previous promote tumorigenesis and tumor growth in colorectal cancer smaller studies associating Dsg1 loss with increased metastasis cells by interacting with c-Cbl and preventing ubiquitination of and poor patient outcome in HNSCC (39, 40). EGFR (62). One prominent feature of most cancer cells is that they fail to In our study, we showed that Erbin knockdown in the absence differentiate normally (53); however, exogenous expression of of Dsg1 has no effect on the number of invadopodia and matrix Dsg1 in HNSCC cells does not induce the expression of differ- degradation. However, Erbin knockdown impairs Dsg10s ability entiation markers, such as K1, suggesting that the mechanism by to efficiently suppress invadopodia and matrix degradation to the which Dsg1 is suppressing invasion is independent from the same level as Dsg1FL-expressing cells transfected with control differentiation process. siRNA. The same phenotype is observed when a Dsg1 mutant that Dsg10s role in suppressing EGFR is in contrast to that of the cannot bind to Erbin is expressed (Dsg1-ICS). Furthermore, desmosomal cadherin, Dsg2, which promotes growth factor invadopodia signaling is downregulated when Dsg1 and Erbin signaling pathways when overexpressed in the basal layer of are both present. When Erbin is depleted, Dsg1 by itself can no mouse epidermis (52) and increases cell growth and migration longer efficiently suppress EGFR/Erk signaling or invadopodia- in an EGFR-dependent manner in vitro (21, 22). Here, we show that Dsg2 acts in a reciprocal fashion to Dsg1 by promoting invadopodia formation and matrix degradation in HNSCC cells (Supplementary Fig. S7B–S7D). In addition, analysis of TCGA data shows that Dsg2 expression increases, rather than decreases, with HNSCC histologic grade (ref. 50; Supplementary Fig. S7A). EGF Dsg20s role in promoting EGFR signaling may be more broadly important in cancer, as it was also observed that Dsg2 downregulation inhibited EGFR signaling and cell proliferation through downstream Erk activation in colon cancer cells (23). These data are consistent with the idea that Dsg2 promotes, Dg1 whereas Dsg1 inhibits, growth factor signaling to maintain a balance of proliferation and differentiation in complex tissues. Erbin EGFR In addition, the work suggests that reintroducing Dsg1 into HNSCC cells is sufficient to overcome any proinvasive signaling that may be occurring through endogenous Dsg2 in HNSCC. Similarly, the presence of Dsg1 may also counter signaling Erk 1/2 through Dsg3, a basal keratinocyte desmoglein that promotes cell migration and invasion by controlling cortical regulators of the actin cytoskeleton and is upregulated in SCC (24, 36). Desmoglein armadillo proteins including plakoglobin and Invadopodia plakophilins (1–3) also have an impact on cancer progression. Like Dsg1, plakophilin 1 is downregulated in cancer (30, 31), and it has been shown that its expression suppresses cell migration and invasion in HNSCC cells (30). Conversely, plakophilin 2 and 3 promote neoplastic progression in different types of cancers where their expression is frequently upregulated (32, 34, 35). The Figure 6. expression of all three plakophilins in Cal33 cells suggests that Model: Dsg1 through its interaction with Erbin downregulates invadopodia Dsg1, alone or together with plakophilin 1, is enough to suppress signaling by dampening EGFR/Erk activation, which ultimately leads to a any potential proinvasive effects exerted by plakophilin 2 and 3. decrease in invadopodia formation and matrix degradation.

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mediated matrix degradation. Addition of pharmacologic inhi- Acquisition of data (provided animals, acquired and managed patients, bitors of EGFR or Erk1/2 reduces levels of degradation in Erbin- provided facilities, etc.): A. Valenzuela-Iglesias, H.E. Burks, C.R. Arnette, depleted cells to that observed in Dsg1 cells expressing Erbin. A. Yalamanchili, L.M. Godsel Analysis and interpretation of data (e.g., statistical analysis, biostatistics, These data indicate the importance of Erbin in suppressing these computational analysis): A. Valenzuela-Iglesias, H.E. Burks, C.R. Arnette, downstream effectors of invadopodia and matrix degradation. A. Yalamanchili, L.M. Godsel, K.J. Green Collectively, our data suggest that reintroducing Dsg1 into Writing, review, and/or revision of the manuscript: A. Valenzuela-Iglesias, HNSCC cells that have lost Dsg1 expression inhibits invadopodia C.R. Arnette, L.M. Godsel, K.J. Green formation and function in an Erbin-dependent manner by atten- Administrative, technical, or material support (i.e., reporting or organizing uating EGFR/Erk signaling (Fig. 6). Because Erbin alone is not data, constructing databases): A. Valenzuela-Iglesias, C.R. Arnette, K.J. Green Study supervision: K.J. Green sufﬁcient to mediate these effects, these data are consistent with the idea that Dsg1 positions Erbin in proximity to the EGFR/Erk1/ Acknowledgments 2 machinery to dampen invadopodia-promoting signals and/or The authors would like to thank Jennifer L. Koetsier for optimization of to promote invadopodia-inhibitory signaling. experiments, Bethany Perez-White for her helpful advice on the spheroid In addition to the Erbin-dependent ability to suppress EGFR/ invasion assay, and all the members of the Green laboratory for insightful Erk1/2-mediated matrix degradation in the presence of Dsg1, discussions during development of this project. Dsg1 exhibited an Erbin-independent component to its suppres- Gifts were provided by Sharon Stack (Cal33 cell line), Jennifer Grandis sive ability (e.g. Fig. 5B and C). In this case, suppression occurs (SCC25 cell line), J. Rheinwald (SCC9 cell line), Thomas Carey (SCC22B and UMSCC1 cell lines), M. Takeichi and O. Abe (E-cadherin antibody), Julie Segre through a mechanism independent of EGFR/Erk status, possibly (K5 and K1 antibodies), and G. Nolan (Phoenix package cell line). NHEK cells through other binding partners of the Dsg1 downstream of the were obtained from the Keratinocyte Core of the Northwestern University Skin conserved ICS domain (Fig. 3A). These data provide new insight Disease Research Center. into how Dsg1 may function as an invasion suppressor in This work was supported by NIH grants R01 CA122151, R01 AR041836, HNSCC, and open up possible new targets for development of and R37 AR043380 with partial support from the J.L. Mayberry Endowment to strategies for interfering with head and neck cancer progression. K.J. Green. A. Valenzuela-Iglesias was supported by a Postdoctoral Fellowship Grant by the Consejo Nacional de Ciencia y Tecnología, CONACYT, Mexico.

Disclosure of Potential Conflicts of Interest The costs of publication of this article were defrayed in part by the payment of No potential conflicts of interest were disclosed. page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Authors' Contributions Conception and design: A. Valenzuela-Iglesias, O. Nekrasova, K.J. Green Received January 17, 2018; revised December 7, 2018; accepted January 8, Development of methodology: A. Valenzuela-Iglesias, O. Nekrasova 2019; published first January 17, 2019.

References 1. Marur S, Forastiere AA. Head and neck squamous cell carcinoma: update on 12. Eddy RJ, Weidmann MD, Sharma VP, Condeelis JS. Tumor cell invado- epidemiology, diagnosis, and treatment. Mayo Clin Proc 2016;91:386–96. podia: invasive protrusions that orchestrate metastasis. Trends Cell Biol 2. Curry JM, Sprandio J, Cognetti D, Luginbuhl A, Bar-ad V, Pribitkin E, et al. 2017;8:595–607. Tumor microenvironment in head and neck squamous cell carcinoma. 13. Bravo-Cordero JJ, Hodgson L, Condeelis J. Directed cell invasion and Semin Oncol 2014;41:217–34. migration during metastasis. Curr Opin Cell Biol 2012;24:277–83. 3. Echarri JM, Lopez-Martin A, Hitt R. Targeted therapy in locally advanced 14. Hayes KE, Walk EL, Ammer AG, Kelley LC, Martin KH, Weed S. Ableson and recurrent/metastatic head and neck squamous cell carcinoma (LA-R/M kinases negatively regulate invadopodia function and invasion in head and HNSCC). Cancers 2016;8:1–22. neck squamous cell carcinoma by inhibiting an HB-EGF autocrine loop. 4. Sacco AG, Worden FP. Molecularly targeted therapy for the treatment of Oncogene 2013;32:4766–77. head and neck cancer: a review of the ErbB family inhibitors. OncoTargets 15. Ayala I, Baldassarre M, Giacchetti G, Caldieri G, Tete S, Luini A, et al. Ther 2016;9:1927–43. Multiple regulatory inputs converge on cortactin to control invadopodia 5. Hartmann S, Bhola NE, Grandis JR. HGF / met signaling in head and neck biogenesis and extracellular matrix degradation. J Cell Sci 2008;121: cancer: impact on the tumor microenvironment. Clin Cancer Res 2016;22: 369–78. 4005–13. 16. Hwang YS, Park KK, Chung WY. Invadopodia formation in oral squamous 6. Boeckx C, Baay M, Wouters A, Specenier P, Vermorken JB, Peeters M, et al. cell carcinoma: the role of epidermal growth factor receptor signaling. Anti-epidermal growth factor receptor therapy in head and neck squamous Arch Oral Biol 2012;57:335–43. cell carcinoma: focus on potential molecular mechanisms of drug resis- 17. Huber O, Petersen I. 150th anniversary series: desmosomes and the hall- tance. Oncologist 2013;18:850–64. marks of cancer. Cell Commun Adhes 2015;22:15–28. 7. Beaty BT, Condeelis J. Digging a little deeper: the stages of invadopodium 18. Rodriguez FJ, Lewis-Tuffin LJ, Anastasiadis PZ. E-cadherin's dark side: formation and maturation. Eur J Cell Biol 2014;93:438–44. possible role in tumor progression. Biochim Biophys Acta 2012;1826: 8. Oser M, Eddy R, Condeelis J. Actin-based motile processes in tumor cell 23–31. invasion. In Carlier MF, editor. Actin-based motility. Dordrecht: Springer; 19. Rajwar YC, Jain N, Bhatia G, Sikka N, Garg B, Walia E. Expression and 2010. p. 125–64. significance of cadherins and its subtypes in development and progression 9. Valenzuela-Iglesias A, Sharma VP, Beaty BT, Ding Z., Gutierrez-Millan LE, of oral cancers: a review. J Clin Diagnostic Res 2015;9:5–7. Roy P, et al. Profilin1 regulates invadopodium maturation in human breast 20. Dusek RL, Attardi LD. Desmosomes: new perpetrators in tumour suppres- cancer cells. Eur J Cell Biol 2015;94:78–89. sion. Nat Rev Cancer 2011;11:317–23. 10. Beaty BT, Sharma VP, Bravo-Cordero JJ, Simpson MA, Eddy RJ, Koleske AJ, 21. Overmiller AM, Pierluissi JA, Wermuth PJ, Sauma S, Martinez-Outschoorn et al. b1 integrin regulates Arg to promote invadopodial maturation and U, Tuluc M, et al. Desmoglein 2 modulates extracellular vesicle release from matrix degradation. Mol Biol Cell 2013;24:1661–75. squamous cell carcinoma keratinocytes. FASEB 2017;31:3412–24. 11. Beaty BT, Wang Y, Bravo-Cordero JJ, Sharma VP, Miskolci V, Hodgson L, 22. Overmiller AM, McGuinn KP, Roberts BJ, Cooper F, Brennan-Crispi DM, et al. Talin regulates moesin-NHE-1 recruitment to invadopodia and Deguchi T, et al. c-Src / Cav1-dependent activation of the EGFR by Dsg2. promotes mammary tumor metastasis. J Cell Biol 2014;205:737–51. Oncotarget 2016;7:37536–55.

www.aacrjournals.org Mol Cancer Res; 2019 OF11

Valenzuela-Iglesias et al.

23. Kamekura R, Kolegraff KN, Nava P, Hilgarth RS, Feng M, Parkos CA, et al. desmosomal cadherin desmoglein 1 implications for exfoliative toxin- Loss of the desmosomal cadherin desmoglein-2 suppresses colon cancer mediated skin blistering. Am J Pathol 2010;177:2921–37. cell proliferation through EGFR signaling. Oncogene 2014;33:4531–6. 43. Sharma VP, Eddy R, Entenberg D, Kai M, Gertler FB, Condeelis J. Tks5 and 24. Brown L, Waseem A, Cruz IN, Szary J, Gunic E, Mannan T, et al. Desmoglein SHIP2 regulate invadopodium maturation, but not initiation, in breast 3 promotes cancer cell migration and invasion by regulating activator carcinoma cells. Curr Biol 2013;23:2079–89. protein 1 and protein kinase C-dependent-Ezrin activation. Oncogene 44. Vinci M, Box C, Eccles SA. Three-dimensional (3D) tumor spheroid 2014;33:2363–74. invasion. Assay J Vis Exp 2015;e52686. 25. Chidgey M, Dawson C. Desmosomes: a role in cancer? Br J Cancer 2007;96: 45. Yamaguchi H, Lorenz M, Kempiak S, Sarmiento C, Coniglio S, Symons M, 1783–7. et al. Molecular mechanisms of invadopodium formation: the role of the 26. Broussard JA, Getsios S, Green KJ. Desmosome regulation and signaling in N-WASP-Arp2/3 complex pathway and cofilin. J Cell Biol 2005;168: disease. Cell Tissue Res 2015;360:501–12. 441–52. 27. Johnson JL, Najor NA, Green KJ. Desmosomes: regulators of cellular 46. Tonisen€ F, Perrin L, Bayarmagnai B, van den Dries K, Cambi A, Grigorijevic signaling and adhesion in epidermal health and disease. Cold Spring Harb B. EP4 receptor promotes invadopodia and invasion in human breast Perspect Med 2014;4:a015297. cancer. Eur J Cell Biol 2017;96:218–26. 28. Kowalczyk A, Green K. Structure, function and regulation of desmosomes. 47. Sodek KL, Brown TJ, Ringuette MJ. Collagen I but not Matrigel matrices Prog Mol Biol Transl Sci 2013;116:95–118. provide an MMP-dependent barrier to ovarian cancer cell penetration. 29. Nekrasova O, Green K. Desmosome assembly and dynamics. Trends Cell BMC Cancer 2008;8:223. Biol 2013;23:537–46. 48. Getsios S, Huen AC, Green KJ. Working out the strength and flexibility of 30. Sobolik-Delmaire T, Katafiasz D, Keim SA, Mahoney MG, Wahl JK 3rd. desmosomes. Nat Rev 2004;5:271–81. Decreased Plakophilin-1 expression promotes increased motility in head 49. Williams KC, Coppolino MG. SNARE-dependent interaction of Src, EGFR and neck squamous cell carcinoma cells. Cell Commun Adhes 2007;14: and b 1 integrin regulates invadopodia formation and tumor cell invasion. 99–109. J Cell Sci 2014;127:1712–25. 31. Yang C, Fischer-Keso R, Schlechter T, Strobel P, Marx A, Hofmann I. 50. Grossman RL, Heath AP, Ferretti V, Varmus HE, Lowry DR, Kibbe WA, et al. Plakophilin 1-deficient cells upregulate SPOCK1: implications for prostate Toward a share vision for cancer genomic data. N Engl J Med 2016;375:12, cancer progression. Tumor Biol 2015;36:9567–77. 1109–12. 32. Arimoto KI, Burkart C, Yan M, Ran D, Weng S, Zhang DE. Plakophilin-2 51. Brennan D, Mahoney MG. Increased expression of Dsg2 in malignant skin promotes tumor development by enhancing ligand- dependent and -inde- carcinomas. Cell Adhesion Migr 2009;3:148–54. pendent epidermal growth factor receptor dimerization and activation. 52. Brennan D, Hu Y, Joubeh S, Choi YW, Whitaker-Menezes D, O'Brien T, et al. Mol Cell Biol 2014;34:3843–54. Suprabasal Dsg2 expression in transgenic mouse skin confers a hyperpro- 33. Khapare N, Kundu ST, Sehgal L, Sawant M, Priya R, Gosavi P, et al. liferative and apoptosis- resistant phenotype to keratinocytes. J Cell Sci Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 2007;120:758–71. dephosphorylation, which is required for transformation and metastasis. 53. Jogi€ A, Vaapil M, Johansson M, Pahlman S. Cancer cell differentiation PLoS One 2012;7:e38561. heterogeneity and aggressive behavior in solid tumors. Ups J Med Sci 2012; 34. Demirag GG, Sullu Y, Yucel I. Expression of plakophilins (PKP1, PKP2, 117:217–24. PKP3) in breast cancers. Med Oncol 2012;29:1518–22. 54. Todorovic V, Desai BV, Schroeder Patterson MJ, Amargo EV, Dubash AD, 35. Breuninger S, Reidenbach S, Sauer CG, Strobel P, Pfitzenmaier J, et al. Yin T, et al. Plakoglobin regulates cell motility through Rho- and fibro- Desmosomal plakophilins in the prostate and prostatic adenocarcinomas nectin-dependent Src signaling. J Cell Sci 2010;123:3576–86. implications for diagnosis and tumor progression. Am J Pathol 2010;176: 55. Yin T, Getsios S, Caldelari R, Kowalczyk AP, Muller EJ, Jones JCR, et al. 2509–19. Plakoglobin suppresses keratinocyte motility through both cell – cell 36. Brown L, Wan H. Desmoglein 3: a help or a hindrance in cancer progres- adhesion-dependent and -independent mechanisms. Proc Natl Acad sion. Cancers 2015;7:266–86. Sci U S A 2005;102:5420–5. 37. Harmon RM, Simpson CL, Johnson JL, Koetsier JL, Dubash AD, Najor NA, 56. Liu D, Shi M, Duan C, Chen H, Hu Y, Yang Z, et al. Downregulation of Erbin et al. Desmoglein-1/Erbin interaction suppresses Erk activation to support in Her2-overexpressing breast cancer cells promotes cell migration and epidermal differentiation. J Clin Invest 2013;123:1556–70. induces trastuzumab resistance. Mol Immunol 2013;56:104–12. 38. Getsios S, Simpson CL, Kojima SI, Harmon R, Sheu LJ, Dusek RL, et al. 57. Hu Y, Chen H, Duan C, Liu D, Qian L, Yang Z, et al. Deficiency of Erbin Desmoglein 1–dependent suppression of EGFR signaling promotes induces resistance of cervical cancer cells to anoikis in a STAT3-dependent epidermal differentiation and morphogenesis. J Cell Bio 2009;185: manner. Oncogenesis 2013;2:e52. 1243–58. 58. Dai P, Xiong WC, Mei L. Erbin inhibits RAF activation by disrupting the Sur- 39. Wong MP, Cheang M, Yorida E, Coldman A, Gilks CB, Hunstman D, 8-Ras-Raf complex . J Biol Chem 2006;281:927–33. et al. Loss of desmoglein 1 expression associated with worse prognosis 59. Dai F, Chang C, Lin X, Dai P, Mei L, Feng XH. Erbin inhibits transforming in head and neck squamous cell carcinoma patients. Pathology 2008; growth factor B signaling through a novel Smad-interacting domain. 40:611–6. Mol Cell Biol 2007;27:6183–94. 40. Tada H, Hatoko M, Tanaka A, Kuwahara M, Muramatsu T. Expression of 60. Huang YZ, Zang M, Xiong WC, Luo Z, Mei L. Erbin Suppresses the MAP desmoglein I and plakoglobin in skin carcinomas. J Cutan Pathol 2000;27: kinase pathway. J Biol Chem 2003;278:1108–14. 24–9. 61. Huang H, Song Y, Wu Y, Guo N, Ma Y, Qian L. Erbin loss promotes cancer 41. Nekrasova O, Harmon RM, Broussard JA, Koetsier JL, Godsel LM, Fitz GN, cell proliferation through feedback activation of Akt-Skp2-p27 signaling. et al. Desmosomal cadherin association with Tctex-1 and cortactin-Arp2/3 Biochem Biophys Res Commun 2015;463:370–6. drives perijunctional actin polymerization to promote basal cell delami- 62. Yao S, Zheng P, Wu H, Song LM, Ying XF, Xing C, et al. Erbin interacts with nation. Nat Commun 2018;9:1053. c-Cbl and promotes tumourigenesis and tumour growth in colorectal 42. Simpson CL, Kojima S, Cooper-Whitehair V, Getsios S, Green KJ. Plako- cancer by preventing c-Cbl-mediated ubiquitination and down-regulation globin rescues adhesive defects induced by ectodomain truncation of the of EGFR. J Path 2015;236:65–77.

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Alejandra Valenzuela-Iglesias, Hope E. Burks, Christopher R. Arnette, et al.

Mol Cancer Res Published OnlineFirst January 17, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-18-0048

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