T-cadherin loss promotes experimental metastasis of squamous cell carcinoma

European Journal of Cancer (2013) 49, 2048– 2058

Available at www.sciencedirect.com

journal homepage: www.ejcancer.info

T-cadherin loss promotes experimental metastasis of squamous cell carcinoma Maria Philippova a,f, Dennis Pfaff a,f, Emmanouil Kyriakakis a, Stanislaw A. Buechner b, Giandomenica Iezzi c, Giulio C. Spagnoli c, Andreas W. Schoenenberger d, Paul Erne e, Therese J. Resink a,⇑ a

Department of Biomedicine, Laboratory for Signal Transduction, Basel University Hospital, CH 4031 Basel, Switzerland Blumenrain 20, CH 4051 Basel, Switzerland c Institute for Surgical Research and Hospital Management, University of Basel, CH 4051 Basel, Switzerland d Division of Geriatrics, Department of General Internal Medicine, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland e Division of Cardiology, Kantonsspital Luzern, CH 6000 Luzern, Switzerland b

Available online 29 January 2013

KEYWORDS Squamous cell carcinoma T-cadherin Experimental metastasis Endothelial cells Adhesion Transendothelial migration

T-cadherin is gaining recognition as a determinant for the development of incipient invasive squamous cell carcinoma (SCC). However, effects of T-cadherin expression on the metastatic potential of SCC have not been studied. Here, using a murine model of experimental metastasis following tail vein injection of A431 SCC cells we report that loss of T-cadherin increased both the incidence and rate of appearance of lung metastases. T-cadherin-silenced SCC metastases were highly disordered with evidence of single cell dissemination away from main foci whereas SCC metastases overexpressing T-cadherin developed as compact, tightly organised sheets. SCC cell adhesion to vascular endothelial cells (EC) in culture was increased for T-cadherin-silenced SCC and decreased for T-cadherin-overexpressing SCC. Confocal microscopy showed that T-cadherin-silenced SCC adherent on EC display an elongated morphology with long thin extensions and a high degree of intercalation within the EC monolayer, whereas SCC overexpressing T-cadherin formed poorly-spread multicellular aggregates that remain on the outer surface of the EC monolayer. T-cadherin-deficient SCC or human keratinocyte cells exhibited increased transendothelial migration in vitro which could be attenuated in the presence of EGFR inhibitor gefitinib. Our data suggest that loss of T-cadherin can increase metastatic potential and aggressiveness of SCC, possibly due to facilitating arrest and extravasation through the vascular wall and/or more efficient establishment of metastases in the new microenvironment. Ó 2013 Elsevier Ltd. All rights reserved.

Abstract

⇑ Corresponding author: Tel.: +41 61 265 2422; fax: +41 61 265 2350. f

E-mail address: [email protected] (T.J. Resink). Contributed equally to the study.

0959-8049/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejca.2012.12.026

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1. Introduction

2.2. Viral vector transduction

Cutaneous squamous cell carcinoma (SCC) accounts for 20% of cutaneous malignancies and unlike other non-melanoma cancers such as basal cell carcinoma is characterised by risk of distant metastasis.1,2 Mechanisms underlying malignisation and metastatic spreading of SCC remain poorly understood. Increased invasiveness and metastatic potential of SCC has been associated with altered expression/function of a variety of cell surface receptors. T-cadherin (encoded by Cdh13), an atypical member of the cadherin superfamily of intercellular adhesion molecules, has been shown to participate in (patho)physiological processes such as axon guidance, angiogenesis and cancerogenesis.3,4 Accumulating data supports T-cadherin as a determinant of cutaneous SCC progression. In healthy skin T-cadherin expression is largely restricted to the basal keratinocyte layer.5,6 In cutaneous SCC regional loss of T-cadherin expression is associated with histological features of potentially more malignant and invasive tumours.7,8 Gain- and loss-of-function studies in vitro using normal keratinocyte and SCC cell lines demonstrate that T-cadherin can regulate growth9,10 and invasion.7,10,11 To date only one study, performed by our laboratory and using a murine xenograft model with subcutaneous injection of A431 SCC cells, has addressed effects of T-cadherin on SCC progression in vivo.10 Xenografts composed of T-cadherin-deficient SCC exhibited increased growth and intra-tumoural proliferative activity compared to controls, confirming stimulatory effects of T-cadherin-deficiency on proliferation in vitro.10 Paradoxically, increased expansion of xenografts composed of T-cadherin-overexpressing SCC also occurred, but via mechanisms involving enhanced intra-tumoural angio/lymphangiogenesis through increased VEGF expression by T-cadherin-overexpressing SCC. To determine effects of T-cadherin-overexpression/ silencing on metastatic potential of SCC in vivo the present study used a murine model of experimental metastasis whereby tumour cells injected into the tail vein may extravasate from peripheral blood vessels and establish metastases in tissues of arrest, primarily lung.12 We demonstrate that T-cadherin-silencing in SCC promotes experimental metastasis in vivo and facilitates adhesion to and transmigration across endothelial monolayers in vitro.

Lentiviral-mediated generation of stably transduced A431 and HaCAT with respect to T-cadherin-overexpression (Tcad+) or T-cadherin-deficiency using T-cadherintargeted shRNA (shTcad), and empty vector (E) or nontarget shRNA (shC) as respective controls, has been detailed7. A431 transductants were co-transduced with lentiviral pGreenfire1-CMV reporter vector co-expressing firefly luciferase and GFP (gift from Dr. Jeroen Geurts, Department of Biomedicine, Basel University). T-cadherin-overexpression/silencing was confirmed by immunoblotting (Supplementary data). GFP-expression levels were measured using a CyAN ADP flow cytometer and Summit v4.3 software (DakoCytomation, Fort Collins, CO, USA). Luciferase expression levels were measured using Dual Luciferase Reporter Assay Kit (Promega, Du¨bendorf, Switzerland). Adenoviral-mediated overexpression of T-cadherin in EC was performed as described.13

2. Materials and methods 2.1. Cells Sources and culture conditions (Supplementary data) for human microvascular endothelial cell (EC) line (HMEC-1), primary human umbilical vein EC (HUVEC), human lymphatic EC line (hTERT-HDLEC), A431 (epidermoid carcinoma of skin; ATTC, CRL-1555) and HaCAT (normal human keratinocytes) cells have been detailed.7,10,13

2.3. Animal experiments and non-invasive imaging Animal experimental protocols were approved by the Kantonal veterinary ethics committee. Animals were handled according to Swiss veterinary laws. Cell suspensions (1.5  106 cells/100 ll PBS), prepared ex tempora, pre-warmed and filtered (40 lm cell-strainer), were inoculated through the tail vein of 6–8-week-old NOD/ SCID mice. Tumour development in vivo was monitored using the LB983 NightOWL II imaging system (Berthold Technologies GmbH, Bad Wildbad, Germany) as described14 with minor modifications (Supplementary data). The time-point for sacrifice was based on a combination of two criteria: when total luminescence intensity of the tumour area peak reached 106 counts and/or when mice showed signs of pain and distress. 2.4. Histological and immunofluorescence analyses of mouse tissue Tissues harvested post-sacrifice were macroscopically examined for presence of tumour nodules before processing for microscopic analyses. Cryosections (8 lm) were analysed for H&E staining, fluorescence microscopy detection of GFP-positive foci and immunofluorescence staining for T-cadherin and epidermal growth factor receptor (EGFR). Supplementary data details tissue processing procedures, staining protocols and antibody sources. 2.5. Analysis of SCC adhesion, morphology and transmigration Protocols for adhesion assay and analysis of A341 morphology in co-culture with endothelial cells (EC) are detailed in Supplementary data. Transmigration of A431 and HaCAT across EC monolayers was evaluated as described15 with modifications (Supplementary data).

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2.6. Statistical analysis All in vitro experiments were performed on at least three separate occasions. Unless otherwise stated all results are given as mean ± S.D. Differences were determined using 1-way repeated measures ANOVA with Tukey’s multiple comparison using GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA). In vivo experiments were performed twice, using 6 and 10 mice for every transductant on the first and second occasions, respectively. Mice survival was evaluated by Kaplan–Meier method using GraphPad Prism 5.0 software. Incidence of tumours was determined using Kaplan–Meier failure function. Incidence rates between groups were compared using incidence rate ratios (IRR) with 95% confidence intervals. To test for equality of failure functions a log-rank test was used. Data were analysed using Stata 11.2 (StataCorp LP, College Station, TX, USA). A p value of <0.05 was considered significant.

3. Results 3.1. T-cadherin-silencing promotes experimental metastasis of SCC Prior to animal experimentation A431 transductants were controlled for efficiency of T-cadherin-overexpression/-silencing and expression-level equivalence of reporter genes GFP and luciferase (Supplementary Fig. S1). Distribution and growth of metastases in tissues following intravenous injection in NOD/SCID mice was monitored by in vivo chemiluminescence imaging. First monitoring 24 h post-injection (p.i.) showed that in 8 of the 64 mice injected cells were partly retained in the venous system of the tail (“tail trapping” phenomenon16) where they grew rapidly and formed large tumour nodules. These animals were excluded from the analysis. In every other animal, and as expected17, luciferase-positive cells were trapped within the pulmonary circulation (example in Fig. 1A); comparable luciferase signal intensities attested to equivalent inoculation of the different transductants (Supplementary Fig. S2A). Within 7-d p.i. luminescence was below relevant detection limits (example in Fig. 1A), confirming clearance of the majority of tumour cells from lungs and consistent with observations that <1% of injected cells survive to form lung colonies.16 Reappearance of luciferase signal first began after around 3-week post-injection (example in Fig. 1A). Metastases located primarily in lungs and back area (subsequently histologically identified as adrenal glands) and occasionally in brain and hindquarter (bone) (Fig. 1A and B). Lung tissue is recognised as “the organ of first encounter” for tumour cells injected into the venous circulation12,17 and therefore of primary interest for the current investigation of experimental metastasis. Compared with mice injected with T-cadherin-overexpressing

(Tcad+) or control transduced (shC, E) A431 the mice injected with T-cadherin-silenced A431 (shTcad) exhibited an accelerated rate of appearance and higher incidence of lung metastases (Fig. 1C). For shTcad group versus shC, E and Tcad+ groups incidence rate was 6.06%/week and 2.28%/week, respectively, which corresponded to an IRR of 2.65 (95%CI 0.89–7.75, p value (log-rank) 0.031). Survival was concomitantly significantly reduced in the shTcad group compared with shC, E or Tcad+ groups (Fig. 1D). There were no significant differences between any of the groups with respect to rate of appearance or incidence of metastases to other organs. 3.2. Macroscopic and immunofluorescence analyses of lung tissue metastases Presence of A431 tumour nodules in lung tissue was verified by macroscopic observation (Fig. 2A), H&Estaining (Fig. 2B), immunofluorescence staining for EGFR which is highly expressed in A431 (Fig. 2C) and fluorescence microscopy to detect GFP expressed by A431 (Fig. 2C and D). Staining for T-cadherin confirmed persistence of T-cadherin-overexpression or silencing during the time of the experiment (Fig. 2E). T-cadherin expression profoundly affected tumour structure/morphology. shTcad-tumours were disorganised, irregularly-shaped and dispersed whereas Tcad+ tumours were compact (Fig. 3A). Cells within shTcadtumours were loosely distributed and many solitary cells scattered away from main foci into surrounding lung tissue, while Tcad+-tumours developed as organised sheets of tightly interconnected cells (Fig. 3B). Structures of tumours formed by control transductants were intermediate and without marked evidence of cell scattering (Fig. 3A and B). 3.3. T-cadherin expression on SCC modulates adhesion to the vascular endothelium Increased experimental metastatic rates in vivo for T-cadherin-deficient A431 imply increased extravasation. Stable attachment of tumour cells to the endothelium is an initial step in the extravasation process.18–20 To assess effects of T-cadherin on heterotypic cell interactions we assayed in vitro adhesion of A431 transductants onto monolayers of macrovascular (HUVEC), microvascular (HMEC-1) and lymphatic (hTERTHDLEC) EC. T-cadherin-overexpression decreased while T-cadherin-silencing increased adhesion onto HUVEC (Fig. 4A) and HMEC-1 (Fig. 4B). The transductants did not differ with respect to adhesion onto hTERT-HDLEC (Fig. 4C). T-cadherin protein expression profiles for HUVEC, HMEC-1 and hTERTHDLEC were comparable (Fig. 4D). T-cadherin is expressed both on the apical surface of the endothelium and on circulating tumour cells and could affect tumour cell retention in the capillaries via

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Fig. 1. T-cadherin-silencing in A431 cells promotes experimental metastasis in a mouse model of SCC. NOD/SCID mice were injected intravenously with Tcad+, shTcad or control E and shC A431 transductants co-expressing GFP and firefly luciferase. Non-invasive in vivo luciferase imaging was performed using the NightOWL II system after injection of D-luciferin. (A) Representative images illustrating trapping of tumour cells in the lungs (24 h p.i.), clearance from the lungs (7 d p.i.) and the typical anatomical locations of metastatic foci appearing during the course of the experiment are shown. Images are displayed as pseudocolour images of peak bioluminescence, with variations in colour representing light intensity at a given location. The colour bars indicate relative signal intensity; red represents the most intense light emission, while violet corresponds to the weakest signal. (B) Incidence and tissue distribution of metastases. (C) Rate of appearance of lung metastases. Values in parentheses indicate numbers of mice with lung tumours/total number of mice for each group. For the shTcad group data points beyond 8 weeks remain unchanged since all shTcad mice were sacrificed within this period. (D) Survival rates among the different animal groups. Beyond the 8weeks p.i. period within which all shTcad mice had been sacrificed no further lung tumours appeared in the other groups, and therefore 11-weeks p.i. was selected as the experimental end-point. # Indicates significant difference in survival between shTcad and each other group (# < 0.05, ## < 0.01).

homophilic interactions. To investigate this issue we examined whether adenoviral vector-driven overexpression of T-cadherin in EC (Fig. 4H) influenced tumour cell adhesion. T-cadherin-overexpression in EC did not augment tumour cell adhesion (Fig. 4E–G) indicating that T-cadherin-dependent homophilic adhesive interactions21 are not essential for stabilising heterotypic interactions. The alternative scenario of T-cadherindependent deadhesive negative guidance22,23 also seems unlikely since adhesion was not worsened on a “substratum” of T-cadherin-overexpressing EC (Fig. 4E–G). 3.4. T-cadherin expression on SCC cells modulates their EC-adherent phenotype We applied confocal microscopy to examine how T-cadherin expression in A431 affects their morphology/

phenotype when plated onto HUVEC monolayers. Tcad+ remained predominantly rounded and formed poorly-spread multicellular aggregates, whereas shTcad were elongated with thin long protrusions extending among EC (Fig. 5A). Control transductants displayed an intermediate degree of spreading. 3D-reconstruction showed that shTcad were largely embedded within the endothelial monolayer and formed long plasmic podia intercalated with EC whereas Tcad+ rather stayed on the outer surface (Fig. 5B and C). 3.5. Downregulation of T-cadherin facilitates transendothelial migration We also examined in vitro transmigration of A431 and HaCAT across monolayers of HUVEC, HMEC-1 and hTERT-HDLEC. T-cadherin-silencing increased

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C EGFR

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Fig. 2. Histological analysis of lung tissue of mice injected with SCC cells. The presence of tumour nodules in lung tissue was verified by (A) macroscopic observation, (B) H&E-staining, (C) immunofluorescence staining for EGFR (red) with respective control for non-specific staining of tumour-free lung tissue, and (C and D) fluorescence microscopy to detect GFP-fluorescence (green) from injected cells. (E) Immunofluoresence staining for T-cadherin confirmed that differential levels of T-cadherin expression (red) in cell transductants was retained during the experiment. n.i., control staining with non-immune antibodies as substitution for primary anti-T-cadherin IgG. (C–E) Nuclei were counterstained with Hoechst 33342 (blue). Representative images are shown. Bars = 1 cm (A), 500 lm (B–D) and 100 lm (E).

transmigration of A431 (Fig. 6A) and HaCAT (Fig. 6B). We have previously demonstrated that T-cadherin negatively regulates EGFR activity in A431 cells.11 In order to assess the impact of EGFR in T-cadherin-dependent effects on SCC cell transendothelial migration a second series of experiments included EGFR tyrosine kinase inhibitor gefitinib. Gefitinib significantly attenuated the stimulation of A431 transmigration by T-cadherin silencing (Fig. 6C), supporting that the effects of T-cadherin loss on SCC transmigration depend on EGFR activation. We performed control experiments to measure proliferation of A431 cells transductants cultured without or with inclusion of gefitinib. Within the time frame of the transmigration experiments (18 h) cell numbers were equivalently increased in all transductants and inhibitory effects of gefitinib on proliferation were not detectable (data not shown).

4. Discussion This study is the first to have examined in vivo outcomes of T-cadherin overexpression and silencing on metastatic potential of SCC, and also provides the

first evidence that T-cadherin expression levels on SCC influence their ability to interact with EC. Collectively our data support that T-cadherin loss favours extravasation and formation of metastases in distant tissues. The metastatic process comprises a cascade of events including dissemination of subpopulations of highly metastatic cancer cells from the primary tumour, degradation of the extracellular matrix, intravasation into the lumen of blood or lymphatic vessels, survival in the circulatory system, lodging/adhesion to the vascular endothelium at distant sites, extravasation into the tissue and finally growth and formation of secondary tumour nodules.24 A number of animal models allow mimicry of various stages in vivo.12,25 The model of spontaneous metastasis from implanted primary xenografts covers the whole spectrum of the metastatic process, but has certain limitations. In particular, rapid growth of the primary tumour together with constraints on permitted study time-frames may restrict the ability to detect development of distant metastases. This limitation applied to our previous xenograft study10 and we therefore chose the model of experimental metastasis for this study. While injecting tumour cells directly into the

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A

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Fig. 3. T-cadherin expression level influences metastatic spreading and tumour tissue structure in the lungs of mice injected with SCC cells. Fluorescence microscopy (A) and confocal microscopy (B) of lung metastases from animals inoculated with E, Tcad+ or shTcad cells. Nuclei were counterstained with Hoechst 33342. Representative images are shown. Note the greater number of microscopically visible GFP-positive-tumour foci in shTcad-innoculated animals (A), as well as the marked distinction between the loose and disseminated structure for shTcad foci and the wellorganised sheets of tightly interconnected cells in Tcad+ foci (B). Arrows indicate direction of tumour front spreading from the foci. Bars = 500 lm (A, top panels), 200 lm (A, bottom panels) and 50 lm (B).

systemic circulation bypasses the early stages of the metastatic process (dissemination and intravasation), it is possible to monitor extravasation and homing at distant sites which is believed to be one of the critical and ratelimiting events in metastatic colonisation. We detected metastases in several tissues. As expected a major metastatic site was lung tissue which contains the first and largest capillary bed encountered by tumour cells after their exit from larger vessels of the venous system.12,17 A second major lodging site was the adrenal gland. Previous studies demonstrated that a small proportion of tumour cells escape entrapment within the pulmonary circulation by either reducing their size due to “pinching off” large parts of cytoplasm, or bypassing capillaries and travelling through arteriovenous shunts.12 For cells spreading via systemic arterial circulation, adrenals are the preferred site supporting metastatic growth.17 Escape of tumour cells to arterial

circulation probably also underlies formation of occasional brain and bone metastases, although bone tumours in the hindquarters might be also caused by diversion of injected cells into venous plexuses en route to lungs.16 Increased incidence and rate of appearance of experimental metastases/tumours in mice injected with shTcad cells clearly indicates that T-cadherin loss increases metastatic potential and aggressiveness of SCC, likely due to stimulation of extravasation through the vascular wall and/or more efficient tumour establishment in the new microenvironment. There are many open questions regarding mechanisms of tumour cell extravasation. While initial trapping of tumour cells occurs due to mechanical retention in capillaries, specific adhesive interactions with the endothelium contribute to further tumour cell arrest.18–20 Several cell surface receptors potentially mediate initial heterotypic contact between trapped tumour cells and

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Fig. 4. T-cadherin-silencing in SCC cells promotes adhesion to vascular endothelial monolayers in vitro. Tcad+, shTcad or control E and shC A431 transductants co-expressing GFP were plated onto monolayers of macrovascular HUVEC (A and E), microvascular HMEC-1 (B and F) or lymphatic hTERT-HDLEC (C and G). (A–D) Wild type EC. (E–H) EC transduced with control adenoviral vector (Adv-E) (E) or T-cadherin expressing adenoviral vector (Adv-Tcad+). T-cadherin levels in wild type (D) or transduced (H) EC were determined by immunoblotting, with bactin used as loading control. For quantification of A431 adhesion co-cultures were rinsed and lysed after 1 h, and GFP-fluorescence from A431 was measured using a fluorescence ELISA reader (histograms). In any given experimental set the average absolute value of adhesion for the E group was taken as baseline and used to normalise the adhesion rates for all four groups (E, Tcad+, shC and shTcad). * < 0.05, ** < 0.01, *** < 0.001.

EC of the blood or lymphatic vessels, among them avb3 and a4b1 integrins, CD24, Thomsen–Friedenreich glycoantigen and galectin-3 on tumour cells or ICAM-1, PECAM, VCAM-1, CD14, avb3 and galectin-3 on

EC.4,20,26,27 We considered whether surface glycoprotein T-cadherin might mediate heterotypic contact via direct homophilic interactions. However, experiments examining adhesion of A431 onto T-cadherin-overexpressing

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Fig. 5. T-cadherin modulates phenotype of SCC cells plated onto endothelial monolayers. Tcad+, shTcad or control E and shC A431 transductants co-expressing GFP (green) were plated onto monolayers of HUVEC pre-labelled with PKH-26 dye (red). After 4 h the co-cultures were fixed and counterstained with Hoechst 33342 to visualise nuclei (blue). Confocal microscopy was used to obtain 2D images of the co-cultures (A), 3D reconstruction of Z-stacks (B) and orthogonal projections (C) of the co-cultures. Note that T-cadherin-silenced cells (shTcad) exhibit elongated morphology with long plasmic podia and are integrated into the endothelial monolayer, while T-cadherin-overexpressing cells (Tcad+) remain on the outside of the EC monolayer. Bars = 50 lm.

EC indicate that adherence is not dependent on direct T-cadherin-mediated homophilic pro-adhesive21 or prodeadhesive/negative guidance22 interactions. Increased adhesion of T-cadherin-deficient A431 to EC may result from alterations in expression of other, as yet unidentified, adhesion molecules concomitant with the general cell phenotype changes caused by T-cadherin loss. Interestingly, T-cadherin-silencing did not affect adhesion to lymphatic EC. This further supports that SCC and EC express cell-type specific surface molecules other than

T-cadherin which mediate firm arrest of SCC to the vasculature, and also implies that in vivo T-cadherin-deficient SCC may metastasise through blood and lymphatic routes with different efficiency. T-cadherin-silencing also increased transendothelial migration. Confocal analysis of EC-A431 co-cultures showed that shTcad intercalated within endothelial monolayers more efficiently and exhibited an elongated phenotype with thin plasmic podia resembling those in 2D-cultures after EGF treatment11 or 3D-spheroids

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Fig. 6. T-cadherin-silencing promotes transmigration of SCC cells through endothelial monolayers in vitro. A431 (A and C) or HaCAT (B) transductants expressing Tcad+, shTcad or control E and shC vectors were plated onto HUVEC, HMEC-1 and hTERT-HDLEC monolayers in transwell chambers. (C) Transmigration of A431 cells through HUVEC monolayers was measured in the absence or in the presence of EGFR inhibitor gefitinib (1 lM). (A–C) Transendothelial migration was quantified after a 24 h period of co-culture using fluorescence microscopy for A431 (expressing GFP) or HaCAT (after staining for EGFR). In any given experimental set the average absolute value of transmigration for the E group was taken as baseline and used to normalise the transmigration rates for all four groups (E, Tcad+, shC and shTcad). n.s. not significant, ** < 0.01, *** < 0.001.

expanding in collagen gels.7 Such morphology is associated with invasive phenotypes in several cancer cell lines and mesenchymal stem cells28 and resembles epithelialto-mesenchymal (EMT)-like transformation exhibited by carcinoma cells invading nearby cell layers.12 Confocal analysis of the structure of lung tissue metastatic shTcad colonies also revealed a more invasive behaviour of shTcad-A431, namely chaotic dissemination from the metastatic core as single cells. This contrasts strikingly with the tightly organised structure of Tcad+-nodules. Thus, while increased adherence of T-cadherin-deficient SCC to vascular EC may facilitate initial stable tumour cell arrest in distant organ microvessels, the shift to a migratory, invasive and disseminating phenotype in T-cadherin-deficient SCC is likely the more important contributor to aggressive metastatic behaviour of SCC. The role for many “classical” cadherins in maintenance of epithelial integrity and polarity is well established. In most epithelial-derived tumours concomitant loss of E-cadherin and gain of N-cadherin is associated with EMT and acquisition of an aggressive invasive phenotype, and correlates inversely with tumour grade and mortality rates.29,30 Less is known about their role in later steps of the metastatic process. In inflammatory

breast cancer metastatic circulating carcinoma cells were found to express high E-cadherin levels, its precise role in regulation of extravasation and metastasis being unclear.31 N-cadherin was found necessary for transendothelial migration of melanoma32–34 and prostate35 cancer cells. N-cadherin upregulation during EMT induced by Bcl-2 overexpression was prerequisite for metastasis of SCC cells.36 Pro-metastatic actions of N-cadherin have been ascribed to increased tumour cell-EC adhesion due to N-cadherin-mediated homophilic binding and to activation of promigratory signalling pathways in tumour cells through mechanisms independent of N-cadherin adhesive functions.37,38 Mechanisms whereby T-cadherin loss in SCC increases metastatic potential are not known. Since ectopic alteration of T-cadherin expression in A431 or HaCAT is not accompanied by changes in epithelial (i.e. E-cadherin) or mesenchymal (e.g. N-cadherin, vimentin) marker levels7,11 mechanisms alternative to those typically associated with EMT likely contribute to enhanced transendothelial migration of T-cadherin-deficient cells. We recently reported that T-cadherin functions as a negative regulator of EGFR activity in A431 and that T-cadherin loss renders cells more responsive to

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EGF.10,11 Data herein demonstrating that gefitinib mitigated enhanced transendothelial migration of T-cadherin-deficient A431 support involvement of EGFR in mechanisms whereby loss of T-cadherin in SCC increased their metastatic potential. Interestingly, EGF can be produced by EC co-cultured with SCC.39 Therefore, local secretion of EGF from activated EC at the sites of tumour cell arrest in vivo might promote acquisition of a migratory phenotype by T-cadherin-silenced cells, their transmigration through the endothelial lining and growth within the invaded stroma. The present data, taken together with those obtained in the xenograft model10 carry interesting implications. Although T-cadherin upregulation in primary SCC tumours stimulates intra-tumoural angio/lymphangiogenesis10, the presence of neovessels in T-cadherin-positive tumours, while offering routes for tumour cell trafficking, likely favours tumour expansion rather than metastasis since T-cadherin-overexpressing SCC do not possess high metastatic activity. On the other hand, T-cadherin-silencing in SCC stimulates both primary tumour growth10 and metastatic potential. Therefore, T-cadherin loss in patients with SCC likely reflects an unfavourable prognosis with aggressive metastatic tumour behaviour. Conflict of interest statement None declared. Acknowledgements This work was supported by Krebsforschung Schweiz (Grant No. KFS 20447-08-2009), SHK Stiftung fu¨r Herz- und Kreislaufkrankheiten and Swiss Life Jubila¨ums Stiftung. These funding bodies had no role in the design, execution or interpretation of the study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.ejca.2012.12.026. References 1. Walsh JE, Lathers DM, Chi AC, et al. Mechanisms of tumor growth and metastasis in head and neck squamous cell carcinoma. Curr Treat Options Oncol 2007;8(3):227–38. 2. Martinez JC, Otley CC, Stasko T, et al. Defining the clinical course of metastatic skin cancer in organ transplant recipients: a multicenter collaborative study. Arch Dermatol 2003;139(3):301–6. 3. Philippova M, Joshi MB, Kyriakakis E, et al. A guide and guard: the many faces of T-cadherin. Cell Signal 2009;21(7):1035–44. 4. Resink TJ, Philippova M, Joshi MB, Kyriakakis E, Erne P. Cadherins and cardiovascular disease. Swiss Med Wkly 2009;139(9–10):122–34. 5. Buechner SA, Philippova M, Erne P, Mathys T, Resink TJ. High T-cadherin expression is a feature of basal cell carcinoma. Br J Dermatol 2009;161(1):199–202.

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