Dual Roles of Hemidesmosomal Proteins in the Pancreatic Epithelium: the Phosphoinositide 3-Kinase Decides

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ORIGINAL ARTICLE Dual roles of hemidesmosomal proteins in the pancreatic epithelium: the phosphoinositide 3-kinase decides

S Laval1,2, H Laklai1,2,5, M Fanjul1,2,5, M Pucelle1, H Laurell1,2, A Billon-Gale´s1,2, S Le Guellec3, M-B Delisle3, A Sonnenberg4, C Susini1,2, S Pyronnet1,2 and C Bousquet1,2

Given the failure of chemo- and biotherapies to ﬁght advanced pancreatic cancer, one major challenge is to identify critical events that initiate invasion. One priming step in epithelia carcinogenesis is the disruption of epithelial cell anchorage to the basement membrane which can be provided by hemidesmosomes (HDs). However, the existence of HDs in pancreatic ductal epithelium and their role in carcinogenesis remain unexplored. HDs have been explored in normal and cancer pancreatic cells, and patient samples. Unique cancer cell models where HD assembly can be pharmacologically manipulated by somatostatin/sst2 signaling have been then used to investigate the role and molecular mechanisms of dynamic HD during pancreatic carcinogenesis. We surprisingly report the presence of mature type-1 HDs comprising the integrin a6b4 and bullous pemphigoid antigen BP180 in the human pancreatic ductal epithelium. Importantly, HDs are shown to disassemble during pancreatic carcinogenesis. HD breakdown requires phosphoinositide 3-kinase (PI3K)-dependent induction of the matrix-metalloprotease MMP-9, which cleaves BP180. Consequently, integrin a6b4 delocalizes to the cell-leading edges where it paradoxically promotes cell migration and invasion through S100A4 activation. As S100A4 in turn stimulates MMP-9 expression, a vicious cycle maintains BP180 cleavage. Inactivation of this PI3K-MMP- 9-S100A4 signaling loop conversely blocks BP180 cleavage, induces HD reassembly and inhibits cell invasion. We conclude that mature type-1 HDs are critical anchoring structures for the pancreatic ductal epithelium whose disruption, upon PI3K activation during carcinogenesis, provokes pancreatic cancer cell migration and invasion.

Oncogene (2014) 33, 1934–1944; doi:10.1038/onc.2013.146; published online 29 April 2013 Keywords: pancreatic ductal epithelium; hemidesmosome; phosphoinositide 3-kinase; cell migration and invasion; G protein- coupled somatostatin receptor

INTRODUCTION required, including during normal epithelial cell division, differ- Pancreatic ductal adenocarcinoma (PDAC) is a particularly entiation or migration. HD disruption facilitates not only cell challenging malignancy, given its usually advanced invasive stage de-adhesion from the BM but also the release of the integrin a6b4, at diagnosis and its rather limited treatment options.1 A better which is hijacked from its primary ‘cell-anchoring’ function in HDs 6 understanding of the molecular pathways involved in early PDAC to promote cell migration. Integrin a6b4 delocalization is at least invasion is clearly warranted. partially triggered by, and dependent on, growth factor- One initiating step in the process of invasion is the detachment stimulated phosphorylation of its b4 subunit, thereby facilitating 5,7,8 of epithelial cells from the basement membrane (BM).2 its dissociation from its hemidesmosomal partners. Under this Hemidesmosomes (HDs) are multiprotein complexes that phosphorylated form, the integrin a6b4 is clustered at the cell- provide stable adhesion of epithelial cells to the underlying BM. leading edges where it promotes motility by reorganizing 6 Mature type-1 HDs are composed of the integrin a6b4, plectin, the cytoskeletal actin, as well as proliferation and survival. bullous pemphigoid (BP) antigens BP180 (also called BPAG2 or Interestingly, the integrin a6b4 is overall overexpressed and type XVII collagen) and BP230 (BPAG1). Transmembrane integrin delocalized to the tumor-invasive fronts in invasive and metastatic a6b4 and BP180 bind to laminin-332 in the BM, and intracellular carcinomas, including breast, colon, squamous cell carcinoma and 9–12 HD stabilization occurs via their association with keratin PDAC. In squamous cell carcinoma, altered integrin a6b4 intermediate filaments through the two plakins, plectin and expression and localization have been shown to be associated BP230, thus creating a stable anchoring complex.3 These mature with HD breakdown, which represents a critical step toward type-1 HD structures have been described in (pseudo-) stratified invasion of cancer cells. Additionally, plectin is the first biomarker and certain complex epithelia, but only in the simple epithelium of to be identified for PDAC that allows the detection by imaging of mammary origin.4 Indeed, simple epithelium adhesiveness to the primary and metastatic PDAC lesions,13,14 but mechanisms and underlying BM is generally described to rather involve immature significance of its overexpression in pancreatic cancer cells is type-2 HDs that contain only integrin a6b4 and plectin.5 enigmatic. A recent report describes an interaction between HDs are highly dynamic structures that can quickly disassemble plectin and integrin b4 in pancreatic cancer cells that is disrupted under conditions in which (partial) detachment from the BM is upon RON receptor activation, facilitating cell migration.15

1INSERM UMR 1037, Laboratoire d’excellence Toulouse Cancer (labex TOUCAN), Equipe labelliseé Ligue Nationale Contre le Cancer (LNCC), Centre de Recherche en Cance´rologie de Toulouse (CRCT), Toulouse, France; 2Universite´ Toulouse III Paul Sabatier, Toulouse, France; 3Services d’Anatomie et Cytologie Pathologique of Hoˆpital Rangueil, Toulouse, France and 4Department of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. Correspondence: Dr C Bousquet, INSERM UMR 1037, Centre de Recherche en Cance´rologie de Toulouse (CRCT), 1 avenue Jean Poulhe`s, BP84225, cedex4, Toulouse 31432, France. E-mail: [email protected] 5These authors contributed equally to this work. Received 27 July 2012; revised 14 February 2013; accepted 11 March 2013; published online 29 April 2013 Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1935 This literature suggests the critical role of hemidesmosomal and in BxPC-3 cancer cells where this BP120 form predominates proteins in PDAC. However, their role in the normal pancreatic (Figure 1e, T1–5 and BxPC-3, respectively). These results describe for ductal epithelium and during carcinogenesis remained unknown. the first time the presence of mature type-1 HDs containing BP180 We have previously demonstrated that the expression of the G in the simple pancreatic ductal epithelium. These anchoring protein-coupled sst2 receptor is lost in 90% of human PDAC. Upon structures are destroyed in pancreatic cancer specimen, perhaps re-expression in pancreatic cancer cells, sst2 presents oncosup- via BP180 cleavage, suggesting a role in PDAC cell invasiveness. pressive activities through inhibition of tumor growth, angiogen- esis and metastatic progression.16–18 Using several sst2-expressing cancer cell models including pancreatic, we report that sst2 Activation of the somatostatin sst2 receptor: a pharmacological inhibits cell migration and invasion by forcing the reassembly of way to reassemble HDs HDs. Importantly, we present the first description in the simple We have previously demonstrated that expressing the somatos- pancreatic ductal epithelium of the presence of mature type-1 tatin receptor sst2 in BxPC-3 cancer cells reduces their tumor- HDs, which are disrupted during pancreatic carcinogenesis but igenicity.17 HD status has therefore been compared in mock- restored upon sst2 re-expression. This represents the first (BxPC-3/mock) versus sst2- (BxPC-3/sst2) transfected cells by pharmacological peptide/receptor signal described to induce the confocal microscopy. In parental BxPC-3/mock cells, the integrin reassembly of these anchoring structures. Using sst2-expressing subunits b4 and a6 (Supplementary Figure S1A) colocalize at the cell models, molecular mechanisms for HD turnover in pancreatic cell-leading edges (arrows) (Supplementary Figure S1A) in a close cancer cells are identified. They involve regulation of the master contact with the focal complex-associated protein paxillin oncogenic phosphoinositide 3-kinase (PI3K) pathway. (Figure 2a), whereas plectin is diffusely distributed in the cytoplasm (Supplementary Figure S1B). By contrast, patch-like structures typical of HD and comprising the colocalized integrin RESULTS subunits a6 and b4 with BP180 and plectin (Figure 2a, Mature type-1 HDs containing BP180 are present in human Supplementary Figures S1C and S1D, arrowheads) are observed pancreatic ducts, but disassemble in PDAC in association with at the basal cell–substrate contacts of BxPC-3/sst2 cells, indicating BP180 cleavage that restoration of sst2 signaling rescues the formation of mature While the b4 subunit of the integrin a6b4 is overexpressed and type-1 HDs similar to those observed in non-transformed HPDE delocalized in human PDAC,10 its function in the normal pancreas cells (Figure 1b). Accordingly, co-immunoprecipitation experi- is unknown. The presence of HDs has therefore been explored in ments using an anti-integrin b4 antibody confirm the presence of human PDAC and normal adjacent tissue samples, as well as in BP180 and of integrin a6 in a complex with b4 in BxPC-3/sst2, human pancreatic ductal epithelium (HPDE19) normal and cancer whereas only integrin a6, but not BP180, is co-immunoprecipi- (BxPC-3, CFPAC-1) cells (Figure 1). When visualized by immuno- tated with b4 in BxPC-3/mock cells (Figure 2b). We have previously histochemistry in normal pancreatic ducts, both integrin b4and demonstrated that activation of the sst2 receptor relies on an BP180 proteins concentrate at the basal layer of the ductal autocrine loop whereby transfection of sst2 in BxPC-3 cells, which epithelium, in contact with the BM (Figure 1a, N, arrowheads). In do not express endogenously this receptor, induces the expres- PDAC samples however, both proteins delocalize from these cell– sion and secretion of the sst2 ligand, somatostatin, which in turn substrate contact sites: analyses of 60 cases of human PDAC continuously activates its receptor.17 Interestingly, knocking down (Table 1) indicate that the integrin b4 and BP180 are highly somatostatin expression using a specific small interfering RNA expressed in 65 and 80% of PDAC, where they are frequently (siRNA) (siSom), which blunts this autocrine loop,18 abrogates delocalized in 95 and 83% (delocalization in 425% of cells), HD assembly in BxPC-3/sst2 cells, and induces integrin b4 respectively, at cell-to-cell lateral contacts of cancer cells present in delocalization to the cell-leading edges (Supplementary Figure ‘ductal glands’ (Figure 1a, T, arrows), and, for integrin b4, at the S1B). Inversely, challenging BxPC-3/sst2 cells (previously transmembrane-migrating structures of isolated invasive cancer cells fected with siSom) with a specific sst2 agonist, BIM23197, rescues (Figure 1a, T, insert). Interestingly, BP180 delocalization is correlated HDs, indicating that HD reassembly is dependent on somatostatin with the tumor grade (r2 ¼ 0.68), and 17% of PDAC grade 1 still (Supplementary Figure S1E). To directly visualize HDs in vivo, present a basal localization of BP180 versus 5% for integrin b4, transmission electron microscopy has been performed on tumors indicating that integrin b4 delocalizes early during the pancreatic resulting from the subcutaneous xenograft of BxPC-3/mock or cancerogenesis as already reported.10 Nevertheless, when BP180 is BxPC-3/sst2 cells in nude mice.17 The images reveal the presence delocalized, integrin b4 is also delocalized (r2 ¼ 0.66). In cellulo, of mature type-1 HDs in slow-growing sst2-expressing tumor presence of mature type-1 HDs is attested by the co-expression of xenografts (Figure 2c, arrowheads), but not in fast-growing mock both integrin a6, b4 and BP180 in patch-like structures (typical of BxPC-3 tumors (Supplementary Figure S1F). HD), visualized at the basal layer of normal HPDE cells by confocal We have then investigated whether reassembly of mature microscopy, and where integrin a6 and BP180 colocalize type-1 HDs upon sst2 activation can be generalized to other sst2- (Figure 1b, HPDE, arrowheads point to HDs). By contrast, both expressing cells. The HaCaT keratinocytes are known to present HDs integrin b4 and BP180 are delocalized to the cell-leading edges and express sst2 (Supplementary Figure S2A). Epidermal growth (b4) and to cell–cell contacts (b4 and BP180) of cancer BxPC-3 and factor (EGF) treatment is known to disassemble HDs in keratinocytes, CFPAC-1 cells (Figure 1b, arrows). In BxPC-3 cells, integrins a6 as visualized by the delocalization of both the integrin b4 to the cell- and b4, but not BP180, colocalize in lamellipodia (Supplementary leading edges (migrating structures) and of BP180 to cell-to-cell Figure S1A, arrows). The integrin b4 co-immunoprecipitates with contacts (Supplementary Figure S2B, arrows). Cell co-treatment with the other hemidesmosomal components of mature type-1 HD, that BIM23197 (sst2 agonist) reverses these EGF-induced delocalizations, is, BP180, BP230, plectin and integrin a6 in normal HPDE, but not in thereby forcing the reassembly of HDs, as visualized by the presence cancer BxPC-3 cells where only the integrins a6andb4 are in of the integrin b4 and BP180 in patch-like structures (Supplementary complex (Figure 1c). Transmission electron microscopy confirms Figure S2B, arrowheads). Similarly, treatment of the oropharyngeal the presence of mature type-1 HDs at the extracellular matrix (ECM) squamous cell carcinoma HSC-3 cells, which express the sst2 contacts in ductal cells (Figure 1d, arrows, normal human receptor (Supplementary Figure S2A) and where the integrin b4is pancreas). The full-length hemidesmosomal BP180 protein appears present at the cell-leading edges (Supplementary Figure S2C, predominantly expressed in human normal pancreatic tissue and arrows), with BIM23197 restores HD assembly (Supplementary HPDE cell samples (Figure 1e, N1,2,3 and HPDE, respectively). Figure S2C, arrowheads). These results demonstrate that sst2 Surprisingly, BP180 is cleaved into a 120-kDa form (BP120) in PDAC controls HD assembly in epithelial cells of different origins.

& 2014 Macmillan Publishers Limited Oncogene (2014) 1934 – 1944 Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1936

Figure 1. Mature type-1 HD-like structures containing BP180 are present in human pancreatic ducts, but disassemble in PDAC in association with BP180 cleavage. (a) Immunohistochemistry using an anti-b4 or anti-BP180 antibody in normal (N) or tumoral (T) paraffin-embedded pancreatic section (representative of 12 or 60 different normal pancreatic or PDAC samples, respectively). (b) Immunofluorescence confocal microscopy analyses of integrins a6, b4 or BP180 in pancreatic normal HPDE, and in cancer BxPC-3 and CFPAC-1 cells. Colocalization of integrin a6 and BP180 is shown (merge). Arrowheads point to hemidesmosomes, arrows to migration structures at cell-leading edges (one field representative of n ¼ 5 independent analyses). Inserts represent higher magnification views of hemidesmosomes. (c) Serial immunoblottings using anti-BP180, -BP230, -plectin, -integrin a6, and then integrin b4 (loading control) antibodies using HPDE or BxPC-3 cell extracts, following integrin b4-immunoprecipitation, or using total protein extracts (no immunoprecipitation (ip): input ¼ 10% of extracts used for ip) (representative of n ¼ 3 independent experiments). (d) TEM of normal human pancreas (one field representative of n ¼ 3 independent analyses). (e) Immunoblotting using anti-BP180 and anti-pan-cytokeratin (CK) or anti-b-tubulin (loading controls) antibodies of proteins from pancreatic normal HPDE and cancer BxPC-3 cells, and from normal (N1,2,3) and tumoral (T1,2,3,4,5) pancreatic samples.

Oncogene (2014) 1934 – 1944 & 2014 Macmillan Publishers Limited Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1937 Table 1. Frequency of normal (basal, at cell-to-basement membrane contacts)/abnormal (lateral sides of the cells) localization of integrin b4 and BP180 together with the level of expression of MMP-9

Integrin b4 BP180 MMP-9

Expression Score S Tumor grade Expression Score S Tumor grade Expression Score S Tumor grade (% of (localization) (% of cases (% of (localization) (% of cases for (% of (% of cells x (% of cases for cases) for each cases) each score) cases) intensity) each score) score)

S % of cases 1 2 3 S % of cases 1 2 3 S % of 123 cases

Normal 100 0 100% N/A 100 0 100% N/A 0 N/A N/A pancreas Interlobular Interlobular ducts ducts PDAC 65 0 0 N/A 80 0 15 100 0 0 100 1 21 82 9 9 samples 1550500 12 86140 24445838 2 37 21 36 43 2 50 4 65 31 3 30 6 41 53 3 58 9 50 41 3 33 0 25 75 4 5 0 34 66 Abbreviation: N/A, not applicable. Analysis of integrin b4 and BP180 delocalization was performed by attributing a score (S: 0–3) as follow: 0, no delocalization; 1, delocalized in o25% of cells; 2, delocalized in 25–50% of cells; 3, delocalized in 450% of cells. Analysis of MMP-9 expression was performed by attributing a score (S: 1–4), considering the % of expressing cells and the level of expression. For each score, the % of samples in each tumor grade (1–3) is given.

HD breakdown stimulates pancreatic cancer cell migration and (Supplementary Figure S3C). Interestingly, BxPC-3/mock tumors invasion display invasive fronts of migrating cells into the CAM, whereas We then hypothesized that HD breakdown during pancreatic BxPC-3/sst2 tumors remain small and localized (Figure 3d). carcinogenesis is critical for acquisition of cell migratory and Importantly, siBP180-transfected BxPC-3/sst2 cells invade more invasive properties. We took advantage of our HD-inducible system efficiently the CAM than siCTR-transfected cells, as attested by a by sst2 to explore this hypothesis. Scaning electron microscopy deeper penetration into the CAM (Figure 3e). Silencing BP180 in reveals drastic phenotypic changes induced upon sst2 expression in BxPC-3/sst2 cells also results in enlarged CAM tumors (28.9±9.1 3 BxPC-3 and HaCaT cells (Supplementary Figures S3A and S4A, versus 2.8±1.9 mm in siCTR-transfected tumors) (Supplementary respectively). Membrane protrusions, also called dorsal ruffles, Figure S3C), although it does not affect BxPC-3/sst2 cell prolife- rippling on the surface of BxPC-3/mock and EGF-treated HaCaT cell ration in the CAM in vivo assay (Figure 3d, Ki67 labeling), nor cell membrane, and giving rise to filopodia and lamellipodia are survival or proliferation in vitro (Supplementary Figure S3D). indicative of a migratory activity. This is also attested by the Interestingly, upon BP180 silencing in BxPC-3/sst2 cells, increased distribution of F-actin displaying a cortical pattern at the ECM deposition is observed in the corresponding CAM tumors lamellipodia spreading of BxPC-3/mock cells (Supplementary (asterisks, Figure 3e), explaining why these tumors are enlarged as Figure S3B, arrow). Membrane protrusions are absent from sst2- compared with controls (siCTR-transfected). This is consistent with expressing BxPC-3/sst2 cells (Supplementary Figure S3B) where an increased secretion of laminin-332 by siBP180-, as compared F-actin organizes into a polygonal actin network characteristic of with siCTR-, transfected BxPC-3/sst2 cells (Figure 3f). These results non-motile cells. Similarly, EGF þ BIM23197-treated HaCaT cells demonstrate that the somatostatin/sst2 signaling induces HD flatten onto the dish displaying extensive cell–cell contacts exactly assembly in different epithelial cells, including pancreatic cancer as HaCaT cells left untreated (Supplementary Figure S4A). cells and keratinocytes; HD assembly consequently results in the Quantitatively, migratory properties of BxPC-3/sst2 cells are inhibition of cell migratory and/or invasive properties. drastically reduced as compared with those of BxPC-3/mock cells (Figure 3a), and BIM23197 significantly decreases EGF pro-migratory Molecular mechanisms underlying HDs breakdown in pancreatic action in HaCaT cells (Supplementary Figure S4B, lower panel). cancer cells To investigate whether the decrease in cell migration elicited by Involvement of BP180 cleavage (into BP120, Figure 1e) in HD sst2 relies on HD reassembly, we forced HD disassembly by breakdown has then been explored. BP180 cleavage into BP120 transfecting a siRNA targeting BP180 (siBP180) in BxPC-3/sst2 or in has been described to occur extracellularly, releasing the BP120 HaCaT cells. BP180 silencing in BxPC-3/sst2 cells induces the fragment in the ECM. HD reassembly induced by sst2 expression breakdown of HDs as attested by the disappearance of b4-stained in BxPC-3 cells correlates with the accumulation of BP180 in cell patch-like structures (Figure 3b) and migration, as well as invasion extracts (Figure 4a, Cells), and with the decrease of BP120 are enhanced to levels similar to those observed in BxPC-3/mock expression in cell-conditioned media (Figure 4a, CM), whilst cells (Figures 3a and c). By contrast, abrogating BP180 expression BP180 mRNA expression is not affected by sst2 (Supplementary in BxPC-3/mock cells does not affect cell migration or invasion Figure S5A). We have previously demonstrated that sst2 inhibits (not shown). Upon BP180 silencing in HaCaT cells (Supplementary the PI3K in BxPC-3 cells.17 Inhibiting PI3K (LY294002) in BxPC-3/ Figure S4B, upper panel), BIM23197 no longer prevents HD mock cells decreases BP180 cleavage into BP120 (Figure 4b), and disassembly provoked by EGF (Supplementary Figure S4C), and mimicks sst2 effect by restoring HD assembly: integrin b4 and consequently no longer prevents EGF-stimulated migration BP180 are relocalized to typical patch-like HD type-1 structures (Supplementary Figure S4B, lower panel). where BP180 and integrin a6 colocalize (Figures 4c and d, HD involvement in pancreatic cancer cell invasiveness was then arrowheads), and BP180 co-immunoprecipitates with the integrin explored by xenografting BxPC-3 cells in vivo on the chick a6b4 (Figure 2b). Restoration of HD assembly upon PI3K inhibition chorioallantoic membrane (CAM). As previously shown,18 sst2 has been confirmed in the other human pancreatic cancer CFPAC- expression significantly reduces tumor growth (6.6±1.9 mm3 1 cell line (Figure 4e). By contrast to sst2wt, expression in BxPC-3 for BxPC-3/sst2 versus 30.1±8.3 mm3 for BxPC-3/mock cells) cells of a mutant sst2 protein (sst2Y71F), which is inefficient to

& 2014 Macmillan Publishers Limited Oncogene (2014) 1934 – 1944 Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1938

Figure 2. sst2 induces type-1 HD assembly in pancreatic cancer cells. (a) Co-staining using anti-b4 and anti-paxillin antibodies in BxPC-3/mock cells, or anti-BP180 and anti-a6 antibodies in BxPC-3/sst2 cells, analyzed by confocal imaging (one field representative of n ¼ 5 independent analyses). Arrowheads and arrows are as in Figure 1b. Insert in merge image represents a higher magnification view of hemidesmosomes. (b) Serial immunoblottings using anti-BP180, -integrin a6, and then -integrin b4 (loading control) antibodies using BxPC-3/mock or BxPC-3/ sst2 cell extracts, or BxPC-3/mock cells treated for 24 h with LY294002, following integrin b4-immunoprecipitation, or using total protein extracts (no immunoprecipitation (ip): input ¼ 10% of extracts used for ip) (representative of n ¼ 3 independent experiments). (c) TEM images of tumors obtained from the subcutaneous xenograft of BxPC-3/sst2 cells showing typical tripartite electron-dense type-1 HDs at the cell– substrate contact sites. D are desmosomes (one field representative of n ¼ 3 independent analyses).

inhibit PI3K,17 neither affects BP180/BP120 ratio (Supplementary the extracellular domain of BP180, thereby inducing HD disas- Figure S5B) nor restores HD assembly (Supplementary Figure S5C). sembly. Whereas MMP-9 is not expressed in normal human HD assembly in BxPC-3/sst2 cells is also associated with the pancreatic ductal cells, its expression is observed in 100% of a PI3K-independent accumulation of the other hemidesmo- panel of 60 cases of human PDAC (Figure 5a). The calculated score somal component BP230 mRNA and protein (Supplementary of MMP-9 expression (both in cancer and stromal cells) is Figure S5A and Supplementary Figure S5B) of the HD-linked positively correlated with the delocalization of BP180 from cytokeratins 5 and 14 (Supplementary Figure S5D and E), and with cell–substrate contact sites (r2 ¼ 0.84). Treatment of BxPC-3/mock a marked densification of the cytokeratin network (Supplementary cells with a recombinant, active MMP-9 (rMMP-9) is sufficient to Figure S5D, transmission electron microscopy). induce HD breakdown whose reassembly had been stimulated As we demonstrate that sst2 decreases the expression of the by the PI3K inhibitor LY294002 (Figure 5b, arrows point to matrix-metalloprotease MMP-9 (Figure 4a, CM), which is well migration structures). Similarly, rMMP-9 treatment of BxPC-3/sst2 expressed and positively regulated by the PI3K pathway in cells is sufficient to promote HD breakdown (Figure 5c). BxPC-3/mock cells (treatment with LY294002 decreases its siRNA-mediated silencing of MMP-9 in BxPC-3/mock cells pre- expression: Figure 4b, CM), we hypothesized that MMP-9 cleaves cludes BP180 cleavage into BP120 (Figure 5d, CM and Cells),

Oncogene (2014) 1934 – 1944 & 2014 Macmillan Publishers Limited Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1939

Figure 3. HD breakdown stimulates pancreatic cancer cell migration and invasion. (a, c) Quantification of migration (a), or invasion (c) of BxPC- 3/mock versus BxPC-3/sst2, and of siCTR- versus siBP180-transfected BxPC-3/sst2 cells. Results are the average (±s.e.m.) of three independent experiments, normalized to values obtained for BxPC-3/mock cells or for siCTR-transfected BxPC-3/sst2 cells, respectively. (***Po0.005; **Po0.01; *Po0.05). (b) Immunoblotting using an anti-BP180 or anti-CK18 (loading control) antibody of protein extracts from siCTR- or siBP180-transfected BxPC-3/sst2 cells (upper panels) (representative of n ¼ 3 independent experiments). Immunofluorescence confocal imaging of b4 subunit in siCTR- or siBP180-transfected BxPC-3/sst2 cells (lower panels) (one representative field of n ¼ 5 independent analyses). Arrowheads and arrows are as in Figure 1b. (d, e) Hematoxylin-eosin (HE) staining offrozen sections of tumor obtained after xenografting onto the CAM of BxPC-3/mock or BxPC-3/sst2 cells (d), or siCTR- or si-BP180-transfected BxPC-3/sst2 cells (e) (one representative field of n ¼ 3 independent experiments, 10 eggs per condition). Samples in (e) have also been labeled with a Ki67 antibody. (f) Immunoblottings using an anti-laminin-332 or MMP-2 (loading control) antibody of conditioned media (CM) from siCTR- or si-BP180-transfected BxPC-3/sst2 cells (representative of n ¼ 3 independent experiments). whereas BxPC-3/sst2 cell treatment with rMMP-9 stimulates the return of integrin a6b4 to HD in BxPC-3-sst2 cells is conversely cleavage of BP180 into BP120 (Figure 5e, CM and Cells). MMP-9 accompanied by tyrosine dephosphorylation of the b4 subunit knockdown in BxPC-3/mock cells (siRNA) is, however, not (Figure 6a, upper panels). Similarly, somatostatin-analog treatment sufficient to prime HD reassembly (Supplementary Figure S6A). prevents EGF-induced b4 phosphorylation in HaCaT cells Here, the role of MMP-9 in BP180 cleavage is also confirmed in the (Supplementary Figure S2D). sst2-induced dephosphorylation of squamous cell carcinoma HSC-3 cells (Supplementary Figure S6B), b4 is associated with a robust decreased expression of S100A4 which also express a cleaved BP120 form. These results provide protein (Figure 6a, lower panels) and mRNA (Figure 6b) that the first demonstration that PI3K inhibition (using its specific depends on PI3K inhibition, as the sst2Y71F mutant has no effect inhibitor LY294002 or via somatostatin/sst2 signaling) is necessary (Supplementary Figure S5B, lower panels). Consistently, inhibiting and sufficient to prime HD reassembly in pancreatic cancer cells. PI3K (LY294002) in BxPC-3 cells also abrogates S100A4 expression Conversely, expressing an active form of the PI3K effector MMP-9 (Figure 6c). S100A4 expression (and consequent pro-invasive induces their breakdown, even so its inhibition appears necessary function) has been shown to be transcriptionally regulated by the (to inhibit BP180 cleavage) but not sufficient to restore HD. integrin a6b4 only when the b4 subunit is phosphorylated.21 Silencing BP180 in BxPC-3/sst2 cells, or treatment of BxPC-3/sst2 cells with recombinant MMP-9, similarly results in HD breakdown Switching on HD assembly prevents pancreatic cancer cell (because of the absence of BP180 expression or BP180 cleavage, invasiveness by having an impact on the integrin a6b4 respectively) and in integrin a6b4 delocalization to invasive fronts pro-invasive signaling pathway (Figures 3b and 5c, respectively). In these conditions, both integrin When delocalized to invasive fronts, the b4 subunit of the integrin b4 phosphorylation and S100A4 expression are increased a6b4 is phosphorylated on tyrosine residues, thereby initiating (Figures 6d and e). These results indicate that sst2-induced HD survival and invasive signaling pathways.20 We show here that the reassembly in pancreatic cancer cells is associated with integrin

& 2014 Macmillan Publishers Limited Oncogene (2014) 1934 – 1944 Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1940

Figure 4. Role of PI3K-dependent cleavage of BP180 in HD breakdown. (a, b) Immunoblotting using an anti-BP180, CK18 (loading control), BP120, MMP-9 or MMP-2 (loading control) antibody of cell extracts (Cells) or conditioned media (CM) from BxPC-3/mock and BxPC-3/sst2 cells (a), or from BxPC-3/mock cells treated for 24 h with DMSO or 25 mM LY294002 (b) (representative of n ¼ 3 independent experiments). (c–e) Immunofluorescence confocal imaging on BxPC-3/mock (c, d), or CPAC-1 (e) cells treated with DMSO or LY294002 as in (b), using the anti-b4 and anti-BP180 antibodies (c, e), or co-stained with the anti-BP180 and anti-a6 antibodies (d) (one representative field of n ¼ 5 independent analyses). Arrowheads and arrows are as in Figure 1b. Inserts represent higher magnification views of hemidesmosomes.

b4 relocalization to HDs, where it is dephosphorylated and is BP180. We, therefore, hypothesized that type-1 HDs have a critical unable to induce S100A4 expression. Rescuing S100A4 expression role in anchoring the pancreatic ductal epithelium to the in BxPC-3/sst2 cells (Figure 6f, upper panel) reverses sst2- underlying BM; their disruption during carcinogenesis probably inhibitory action on both MMP-9 expression (Figure 6f, lower involving BP180 cleavage may, however, support cell migration panel) and in vitro cell invasion (Figure 6g). These results indicate and invasion. that S100A4 itself stimulates MMP-9 expression, as also observed Accordingly, our data demonstrate that the reassembly of in siBP180-transfected cells where both S100A4 and MMP-9 type-1 HDs in pancreatic cancer cells inhibits cancer cell expression are increased (Figure 6d, lower panels). By inhibiting migration and invasion. Interestingly, this rescue can be achieved PI3K, sst2 abrogates the MMP-9/S100A4/MMP-9 vicious cycle that through inhibition of PI3K activity or triggered by activation promotes in pancreatic cancer cells BP180 cleavage, HD disrup- of the somatostatin/sst2 receptor system that inhibits PI3K.17 tion, and finally cell migration and invasion (Figure 7). Conversely, in PI3K-inhibited cancer cells, disrupting type-1 HDs is sufficient to stimulate invasion. Our data unravel an original PI3K-dependent scenario in pancreatic cancer cells, whereby DISCUSSION high PI3K activity induces the MMP-9-dependent cleavage of Apart from stratified epithelia, mature type-1 HDs had only been BP180, which is sufficient to induce type-1 HD breakdown observed in the normal mammary simple epithelium, however and the subsequent release from HDs of integrin b4 that they are lost in breast cancer cells.4 Our results describe for the delocalizes to the lateral membrane sides of cancer cells. first time the presence of mature type-1 HDs (containing BP180) in It may also explain why plectin is delocalized in pancreatic the simple exocrine pancreatic ductal epithelium. In pancreatic cancer cells.14 Therefore, localized at the cell-leading edges, cancer cells, however, we show that type-1 HDs are absent integrin b4 is tyrosine-phosphorylated and stimulates ex- despite the overexpression of integrin b4 and plectin. pression of the pro-invasive protein S100A4. Strikingly, we show Delocalization of integrin b4 and BP180 is frequently observed that, in turn, S100A4 induces MMP-9 expression, thereby in human PDAC and occurs from ductal cell—ECM contacts to maintaining a positive feedback loop on MMP-9 activation and lateral sides of the cell. Interestingly, we observed that HD loss subsequent BP180 cleavage. By inhibiting PI3K, sst2 breaks this during pancreatic carcinogenesis is associated with a cleavage of vicious cycle.

Oncogene (2014) 1934 – 1944 & 2014 Macmillan Publishers Limited Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1941

Figure 5. Role of MMP-9-dependent cleavage of BP180 in HD breakdown. Immunohistochemistry using an anti-MMP-9 antibody in normal (N) or tumoral (T) paraffin-embedded pancreatic section (representative of 12 or 60 different normal pancreatic or PDAC samples, respectively). (b, c) b4 immunofluorescence confocal imaging on BxPC-3/mock (b), or BxPC-3/sst2 (c) cells treated with DMSO, LY294002 or with a combination of 25 mM LY294002 and 100 ng/ml of recombinant MMP-9 (rMMP-9) (b), or with rMMP-9 (c) (one representative field of n ¼ 5 independent analyses). Arrowheads and arrows are as in Figure 1b. (d, e) Immunoblotting using an anti-BP180, CK18 (loading control), BP120, MMP-9 or MMP-2 (loading control) antibody of siCTR- or siMMP-9-transfected BxPC-3/mock cell extracts and conditioned media (d), or of rMMP-9-treated BxPC-3/sst2 cell extracts and conditioned media (e) (representative of n ¼ 3 independent experiments). Note that the anti- MMP-9 antibody recognizes the endogenous (e) form of MMP-9 in BxPC-3/mock cells, whereas it recognizes the recombinant (r) form of faster mobility in SDS–PAGE than endogenous, in BxPC-3/sst2 cells (which do not express the endogenously form).

BP180 proteolytic cleavage into BP120 has been described to 1 PDAC, where BP180 is not yet delocalized. Paradoxically, our occur within the extracellular non-collagenous 16A domain, results indicate that MMP-9 is already expressed (mostly score 1) in adjacent to the cell membrane.22 It results in type-1 HD these low-grade tumors; but it has been reported that, although breakdown by destabilizing the interactions of BP180 with expressed, MMP-9 is not activated yet,27 explaining why BP180 is integrin a6b4 and BP230.22 The molecular mechanisms not delocalized. These data are of critical importance because they responsible for BP180 cleavage into a BP120 form have not been unravel the critical role in pancreatic cancers cells of PI3K in documented in carcinomas. This cleavage has been attributed to a regulating hemidesmosomal protein expression/phosphorylation, neutrophil-derived MMP-9 in the autoimmune BP skin-blistering and subsequently type-1 HD turnover. disease23 or to ADAMs in normal primary keratinocytes.24 MMP-9 is Unraveling that somatostatin/sst2 signaling acts at the cell/ECM a critical protease involved in pancreatic cancer cell invasiveness.25 interface to promote HD reassembly is original because it is the Interestingly, MMP-9 has recently been shown to also cleave the first identification of an inducible peptide/receptor system able to integrin b4 ectodomain, leading to epithelial corneal erosions in regulate positively the dynamics of type-1 HD assembly. As similar mice through HD disassembly.26 Although we did not observe observations apply in epithelial cells of different origins (pancreas, such integrin b4 cleavage in pancreatic cancer cells, we cannot oropharynx and skin), we suggest that the hormone somatostatin, exclude that it concerns a fraction of the protein. Nevertheless, this through activation of sst2, is generally involved in the main- reinforces the critical role of MMP-9 in type-1 HD breakdown tenance of epithelia integrity. This is consistent with sst2 loss of during pancreatic carcinogenesis, although acting through BP180 expression during pancreatic carcinogenesis,18,28 and with sst2 cleavage. Cleavage of other substrates than BP180 outside HDs is oncosuppressive properties upon re-expression in pancreatic also probably involved in MMP-9 pro-invasive activity on cancer cells.17,18 sst2-inhibitory role on pancreatic cancer cell pancreatic cancer cells. migration and invasion is here ascribed to its capability to rescue Our data indicate that in contrast to PI3K inhibition (using the type-1 HD assembly. PI3K inhibitor LY294002, or through the expression of sst2), which is In summary, we here unravel the existence of type-1 HDs in sufficient to restore type-1 HD assembly in pancreatic cancer cells, normal human pancreatic ducts, which are disrupted in PDAC. MMP-9 silencing, although precluding BP180 cleavage, is not. It Furthermore, sst2-expressing cells have served as a unique tool to suggests that other event(s) under the control of the PI3K pathway identify the molecular mechanisms involved in HD turnover, and are necessary. In PI3K-inhibited pancreatic cancer cells, our to provide mechanistic insights into the early acquisition of an hypothesis is that dephosphorylation of the integrin b4constitutes invasive behavior by the pancreatic cancer cells (Figure 7). the second event that is, in addition to inhibition of BP180 cleavage, necessary for HD assembly. Consistently, dephospho- phorylation of the integrin b4 is also observed upon expression of MATERIALS AND METHODS sst2. Integrin b4 dephosphorylation likely facilitates its interaction with plectin, a first step-forming labile type-2 HDs; then BP180 and Cells and reagents BP230 are recruited to this pre-formed complex to form mature Human pancreatic cancer BxPC-3 cells were maintained in DMEM (1 g/l type-1 HDs, as described in migrating keratinocytes.8 Accordingly, it glucose) and CFPAC-1 in Iscove’s Modified Dulbecco’s Medium, supplemented with 10% foetal calf serum. BxPC-3 cells have been stably transfected has been recently reported that integrin b4 phosphorylation on 71 18 8 with the human wild-type or mutated Y Fsst2cDNA, and BxPC-3/sst2 T1736 alters its association with plectin, and that RON-mediated with a S100A4-FLAG cDNA. Human normal pancreatic ductal HPDE cells PI3K activation in pancreatic cancer cells induces integrin b4 (HPV-16E6E7-immortalized, gift from Dr Tsao, University of Toronto) were 15 dissociation from plectin. This hypothesis is also consistent with cultured in keratinocyte-SFM supplemented with 5 ng/ml EGF and 50 g/ml our results demonstrating that integrin b4 delocalizes early in grade bovine pituitary extract (Life Technologies SAS, Saint Aubin, France).

& 2014 Macmillan Publishers Limited Oncogene (2014) 1934 – 1944 Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1942

Figure 6. Role for S100A4 in integrin a6b4 pro-invasive signaling. (a) Serial immunoblottings using anti-phosphotyrosine (Y-P) and then b4 (loading control) antibodies following b4-immunoprecipitation of BxPC-3/mock or BxPC-3/sst2 cell extracts (upper panels), or immunoblotting using an anti-S100A4 or b-tubulin (loading control) antibody of cell extracts from BxPC-3/mock and BxPC-3/sst2 cells (lower panels) (representative of n ¼ 3 independent experiments). (b) RT–qPCR quantiﬁcation of S100A4 mRNA in BxPC-3/mock and BxPC-3/sst2 cells, normalized to the value obtained for BxPC-3/mock cells. Results are the average (±s.e.m.) of three independent experiments. (**Po0.01). (c) Immunoblotting using an anti-S100A4 or anti-b-tubulin (loading control) antibody of cell extracts obtained from BxPC-3 cells treated for 30 min with DMSO or 25 mM of LY294002 (representative of n ¼ 3 independent experiments). (d, e) Immunoblotting using an anti-BP180, S100A4 or b-tubulin (loading control) antibody, or anti-Y-P and then anti-b4 (loading control) antibody, following b4-immunoprecipitation of siCTR- or siBP180-transfected (d), or of treated or not with rMMP-9 (e), BxPC-3/sst2 cell extracts. Immunoblotting using MMP-9 or MMP-2 (loading control) antibody is performed in the same conditions as above but on cell-conditioned media (representative of n ¼ 3 independent experiments). (f) Immunoblotting using an anti-S100A4 or b-tubulin (loading control) antibody of S100A4-transfected BxPC-3/sst2 cell extracts, or using an anti-MMP-9 or MMP-2 (loading control) antibody of S100A4-transfected BxPC-3/sst2 cell-conditioned media (representative of n ¼ 3 independent experiments). (g) Quantiﬁcation of in vitro invasion assays of BxPC-3/mock, BxPC-3/sst2 and mock- or S100A4-transfected BxPC-3/sst2 cells. Data are normalized to the value obtained for the respective mock cells, and are the average (±s.e.m.) of three independent experiments (***Po0.001).

Recombinant MMP-9 and PI3K inhibitor LY294002 were from Calbio- (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A pool of four designed chem (Merck Millipore, Molsheim, France), EGF from R&D Systems (R&D siRNAs targeting each gene was first tested, and two specific siRNAs have Systems Europe, Abingdon, UK) and rat laminin-5 (332) from Millipore then been used separately in functional assays for each target gene to (Merck Millipore). knockdown (Supplementary Table 1). Results are presented for siRNA_1 Human normal pancreatic and PDAC protein extracts, and paraffin- but are also representative of siRNA_2, except for functional assays where embedded samples were from BioChain (CliniSciences s.a., Nanterre, results represent the mean of siRNA_1 and siRNA_2 effects. France), from US Biomax (Rockville, MD, USA) (tissue-microarrays PA207) and from the Pathology Department of Toulouse Hospital, Toulouse, France. This study was approved by the ethic committee of the Institution. Immunohistochemistry Paraffin-embedded normal and tumoral pancreas sections were incubated with primary antibodies (Supplementary Table 2), and then with peroxidase- RNA interference conjugated secondary antibodies (Dako, Carpinteria, CA, USA). AEC was Human siRNAs for BP180, somatostatin and MMP-9, non-targeting (control) applied as a chromogen. Sections were finally counterstained with Mayer siRNA and DharmaFECT-1 transfection reagent were from Dharmacon hemalun. Except when indicated, scales represent 10 mm.

Oncogene (2014) 1934 – 1944 & 2014 Macmillan Publishers Limited Roles of hemidesmosomal proteins in the pancreatic epithelium S Laval et al 1943

1 3 2

Normal Pancreatic Ductal Adenocarcinoma pancreatic duct PDAC

Lumen Lumen

HD-type I Migration structures Anchorage+++ HD breakdown 2 Migration+++ Epithelium integrity Invasion+++

sst2 activation Ple ctin 0 HD reassembly 3 P23 B

MMP-9 Somatostatin

CK S100A4 PI3K+++

Y-P sst2 RTK actin RTK plectin plectin plectin tors BP230 BP230 BP230 th fac Inhibition of PI3K w ro G 6 4 BP180 BP180 BP180 BP180 64 64 64 MMP-9

BP120 Figure 7. Scheme depicting molecular mechanisms underlying HD breakdown during PDAC carcinogenesis and HD restoration induced by sst2. (1) Mature type-1 hemidesmosomal structures comprising the integrin a6b4, plectin, BP230 and BP180, and providing cell anchorage to the basal lamina (laminin-332), are surprisingly present in the normal human pancreatic ducts, but disrupted in PDAC cells. (2) Indeed, high MMP-9 expression/ activity, stimulated by growth factor-induced PI3K activity in PDAC cells, results in BP180 cleavage, thereby conducting the HD breakdown. As consequences, migration and invasion are promoted at least through integrin a6b4 delocalization to migration structures where it is phosphorylated on tyrosine and stimulates expression of the pro-invasive S100A4 protein. S100A4 maintains itself a vicious cycle on BP180 cleavage because it increases MMP-9 expression. (3) Importantly, re-expressing/activating sst2 in PDAC cells blocks PI3K activity, thereby restoring HD assembly. This is achieved at least through inhibition of PI3K-MMP-9-dependent cleavage of BP180, thereby relocalizing integrin a6b4 to HD at the cell basal layer under a tyrosine-unphosphorylated form, where it does not anymore increase S100A4 expression. Consequently, PDAC cell migration and invasion are repressed.

Immunofluorescence HaCaT cells) to the lower chamber. BxPC-3 cells were allowed to migrate Cells grown on laminin-332 (0.5 mg/ml) were fixed with 4% para- for 20 h (migration), 72 h (invasion) and HaCaT for 8 h (migration). formaldehyde and permeabilized with 0.3% Triton X-100. Selected Cells that migrated to the lower side of the membrane were fixed with proteins were immunodetected using primary antibodies and then 4% paraformaldehyde and stained with crystal violet (0.2%) in 20% with fluorescent secondary antibodies (Supplementary Table 2). Cells were methanol. Quantification of the membrane surface area covered by visualized using a Zeiss LSM510 laser scanning confocal microscope migrated/invaded cells was performed with Morpho Expert software, (objective: Â 63/1,4 oil). To immunodetect hemidesmosomal components, Explora Nova (La Rochelle, France). images were obtained from horizontal sections (x–y plane) taken at cell-basement membrane contacts. Images are representative of at least CAM assay five different fields in at least three independent experiments. Scales Fertilized chicken eggs (Morizeau, France) were incubated at 37 1C and represent 10 mm. 80% humidified atmosphere. Cell xenografting was performed as previously described.18 Tumor volumes were estimated by the equation: V ¼ 4/3 r3, with r ¼ 1/2 O(d1 Â d2). Protein extraction and western blot Proteins were extracted in 0.4% SDS, 10 mM NaCl, 20 mM Tris–HCl, pH 7.5, Statistical analysis supplemented with 2 mM orthovanadate and a protease inhibitor cocktail (Roche S.A.S. Boulogne-Billancourt, France) for 20 min, sonicated and Statistical analyses were performed by comparing two by two independent boiled before adding Triton X-100 (final concentration 1%). Samples were conditions (with homogeneous variances) using an unpaired parametric then resolved by SDS–PAGE (7,5%), immunoblotted with primary t-test. All values are mean±s.e.m. antibodies (Supplementary Table 2) then with secondary HRP-conjugated antibodies (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and visualized by enhanced chemiluminescence (Pierce). CONFLICT OF INTEREST For assessing extracellular protein expression, cell media were The authors declare no conflict of interest. concentrated using 30 kDa centricons (Millipore, UFC803024) and sub- mitted to western blot analysis (Supplementary Table 2). ACKNOWLEDGEMENTS This work was supported by ARC (grant no. 5000, Association pour la Recherche Migration and in vitro invasion assays Contre le Cancer), LNCC (Ligue Nationale Contre le Cancer RAB09006BBA), the Migration and invasion assays were performed using transwell inserts Laboratoire d’Excellence Toulouse Cancer LABEX TOUCAN, the Canceropole Grand (8.0 mm pore size, BD Falcon, Becton Dickinson, Le Pont de Claix, France), Sud-Ouest (RMA04002BPA), the ANR (Agence Nationale pour la Recherche, project coated with Matrigel for invasion. Cells were seeded on the upper no. R06423BS) and the University Paul Sabatier of Toulouse (CR27 A01BQR-2007). SL chamber. Chemoattractant was added (serum for BxPC-3 and 10 nM EGF for was recipient of a fellowship from LNCC. HL was recipient of a fellowship from

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

Oncogene (2014) 1934 – 1944 & 2014 Macmillan Publishers Limited