Dysregulation of Claudin-7 Leads to Loss of E-Cadherin Expression and the Increased Invasion of Esophageal Squamous Cell Carcinoma Cells
2007; Elsevier BV; Volume: 170; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.060343
ISSN1525-2191
AutoresMercedes Lioni, Patricia Brafford, Claudia D. Andl, Anil K. Rustgi, Wafik S. El‐Deiry, Meenhard Herlyn, Keiran S.M. Smalley,
Tópico(s)Cancer Cells and Metastasis
ResumoThe claudins constitute a 24-member family of proteins that are critical for the function and formation of tight junctions. Here, we examine the expression of claudin-7 in squamous cell carcinoma (SCC) of the esophagus and its possible role in tumor progression. In the normal esophagus, expression of claudin-7 was confined to the cell membrane of differentiated keratinocytes. However, in the tumor samples, claudin-7 expression is often lost or localized to the cytoplasm. Assaying esophageal SCC lines revealed variable expression of claudin-7, with some lacking expression completely. Knockdown of claudin-7 in SCC cell lines using a small interfering RNA approach led to decreased E-cadherin expression, increased cell growth, and enhanced invasion into a three-dimensional matrix. The opposite was observed when claudin-7 was overexpressed in esophageal SCC cells lacking both claudin-7 and E-cadherin. In this context, the claudin-7-overexpressing cells became more adhesive and less invasive associated with increased E-cadherin expression. In summary, we demonstrate that claudin-7 is mislocalized during the malignant transformation of esophageal keratinocytes. We also demonstrate a critical role for claudin-7 expression in the regulation of E-cadherin in these cells, suggesting this may be one mechanism for the loss of epithelial architecture and invasion observed in esophageal SCC. The claudins constitute a 24-member family of proteins that are critical for the function and formation of tight junctions. Here, we examine the expression of claudin-7 in squamous cell carcinoma (SCC) of the esophagus and its possible role in tumor progression. In the normal esophagus, expression of claudin-7 was confined to the cell membrane of differentiated keratinocytes. However, in the tumor samples, claudin-7 expression is often lost or localized to the cytoplasm. Assaying esophageal SCC lines revealed variable expression of claudin-7, with some lacking expression completely. Knockdown of claudin-7 in SCC cell lines using a small interfering RNA approach led to decreased E-cadherin expression, increased cell growth, and enhanced invasion into a three-dimensional matrix. The opposite was observed when claudin-7 was overexpressed in esophageal SCC cells lacking both claudin-7 and E-cadherin. In this context, the claudin-7-overexpressing cells became more adhesive and less invasive associated with increased E-cadherin expression. In summary, we demonstrate that claudin-7 is mislocalized during the malignant transformation of esophageal keratinocytes. We also demonstrate a critical role for claudin-7 expression in the regulation of E-cadherin in these cells, suggesting this may be one mechanism for the loss of epithelial architecture and invasion observed in esophageal SCC. The development of cancer is often viewed as a disruption of normal homeostatic balance. Under physiological conditions, epithelial cells exist as tightly regulated, organized cellular sheets. Under these circumstances, growth and motility are regulated closely by a network of soluble growth factors as well as intercellular communication via cell-cell adhesion, cell-matrix adhesion, and gap junctional communication.1Haass NK Smalley KS Herlyn M The role of altered cell-cell communication in melanoma progression.J Mol Histol. 2004; 35: 309-318Crossref PubMed Scopus (127) Google Scholar Epithelial sheet architecture is maintained through the coordinated actions of tight junctions, adherens junction, and desmosomes. Of these, adherens junction proteins, such as E-cadherin, and desmosomes are primarily responsible for the adhesion between adjacent cells, whereas tight junctions regulate permeability and the paracellular passage of water, ions, and macromolecules through the epithelial sheet.2Tsukita S Furuse M Pores in the wall: claudins constitute tight junction strands containing aqueous pores.J Cell Biol. 2000; 149: 13-16Crossref PubMed Scopus (405) Google Scholar, 3Morin PJ Claudin proteins in human cancer: promising new targets for diagnosis and therapy.Cancer Res. 2005; 65: 9603-9606Crossref PubMed Scopus (450) Google Scholar The tight junction family comprises three main classes of protein: claudins, occludins, and junctional adhesion molecules. The claudins and occludins constitute the functional unit responsible for the tight sealing of the cells in the epithelial sheet, whereas the tight-junction proteins, such as zonula occludens (ZO) protein-1, are responsible for linking the claudins and occludins to the actin cytoskeleton.4Smalley KS Brafford P Haass NK Brandner JM Brown E Herlyn M Up-regulated expression of zonula occludens protein-1 in human melanoma associates with N-cadherin and contributes to invasion and adhesion.Am J Pathol. 2005; 166: 1541-1554Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 5Stevenson BR Anderson JM Goodenough DA Mooseker MS Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance.J Cell Biol. 1988; 107: 2401-2408Crossref PubMed Scopus (196) Google Scholar, 6Anderson JM Stevenson BR Jesaitis LA Goodenough DA Mooseker MS Characterization of ZO-1, a protein component of the tight junction from mouse liver and Madin-Darby canine kidney cells.J Cell Biol. 1988; 106: 1141-1149Crossref PubMed Scopus (285) Google Scholar The claudins constitute a family of 24 distinct transmembrane proteins that are composed of four transmembrane domains and two extracellular loops, which are involved in the homophilic and heterophilic interactions with other adjacent claudins.7Peacock RE Keen TJ Inglehearn CF Analysis of a human gene homologous to rat ventral prostate.1 protein.Genomics. 1997; 46: 443-449Crossref PubMed Scopus (17) Google Scholar, 8Itoh M Furuse M Morita K Kubota K Saitou M Tsukita S Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.J Cell Biol. 1999; 147: 1351-1363Crossref PubMed Scopus (906) Google Scholar, 9Morita K Furuse M Fujimoto K Tsukita S Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands.Proc Natl Acad Sci USA. 1999; 96: 511-516Crossref PubMed Scopus (981) Google Scholar Claudins exhibit distinct patterns of expression that are tissue-specific.10Turksen K Troy TC Barriers built on claudins.J Cell Sci. 2004; 117: 2435-2447Crossref PubMed Scopus (345) Google Scholar Most cells express multiple claudin isoforms that interact in a homotypic and heterotypic manner to regulate junctional permeability and confer the selectivity and strength of the tight junctions. During oncogenic transformation, tumor cells typically lose tight junction function, leading to derangement of tissue architecture and loss of cell polarity. The loss of tight junction permeability leads to impairment of epithelial integrity, allowing the free flow of nutrients and growth factors to the nascent tumor. Claudin expression has been shown to be either deregulated or lost in cancer.3Morin PJ Claudin proteins in human cancer: promising new targets for diagnosis and therapy.Cancer Res. 2005; 65: 9603-9606Crossref PubMed Scopus (450) Google Scholar Expression of claudin-7 is lost in both head and neck cancer11Al Moustafa AE Alaoui-Jamali MA Batist G Hernandez-Perez M Serruya C Alpert L Black MJ Sladek R Foulkes WD Identification of genes associated with head and neck carcinogenesis by cDNA microarray comparison between matched primary normal epithelial and squamous carcinoma cells.Oncogene. 2002; 21: 2634-2640Crossref PubMed Scopus (189) Google Scholar and invasive breast cancer.12Kominsky SL Argani P Korz D Evron E Raman V Garrett E Rein A Sauter G Kallioniemi OP Sukumar S Loss of the tight junction protein claudin-7 correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast.Oncogene. 2003; 22: 2021-2033Crossref PubMed Scopus (379) Google Scholar Likewise, claudin-1 expression is down-regulated in colon cancer, leading to increased tumor growth and metastasis.13Resnick MB Konkin T Routhier J Sabo E Pricolo VE Claudin-1 is a strong prognostic indicator in stage II colonic cancer: a tissue microarray study.Mod Pathol. 2005; 18: 511-518Crossref PubMed Scopus (201) Google Scholar The importance of claudin loss in cancer is demonstrated by the fact that claudin-4 re-expression reduces the invasion of pancreatic cancer cells14Michl P Barth C Buchholz M Lerch MM Rolke M Holzmann KH Menke A Fensterer H Giehl K Lohr M Leder G Iwamura T Adler G Gress TM Claudin-4 expression decreases invasiveness and metastatic potential of pancreatic cancer.Cancer Res. 2003; 63: 6265-6271PubMed Google Scholar and that claudin-1 re-expression leads to apoptosis of breast cancer cells in a three-dimensional spheroid model.15Hoevel T Macek R Swisshelm K Kubbies M Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids.Int J Cancer. 2004; 108: 374-383Crossref PubMed Scopus (103) Google Scholar Conversely, certain tumor types are characterized by increased claudin expression, with overexpression of claudin-3 and -4 expression being reported in ovarian, breast, prostate, and pancreatic cancer.16Rangel LB Agarwal R D'Souza T Pizer ES Alo PL Lancaster WD Gregoire L Schwartz DR Cho KR Morin PJ Tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer but not in ovarian cystadenomas.Clin Cancer Res. 2003; 9: 2567-2575PubMed Google Scholar, 17Gress TM Muller-Pillasch F Geng M Zimmerhackl F Zehetner G Friess H Buchler M Adler G Lehrach H A pancreatic cancer-specific expression profile.Oncogene. 1996; 13: 1819-1830PubMed Google Scholar, 18Kominsky SL Vali M Korz D Gabig TG Weitzman SA Argani P Sukumar S Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4.Am J Pathol. 2004; 164: 1627-1633Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 19Long H Crean CD Lee WH Cummings OW Gabig TG Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in prostate cancer epithelium.Cancer Res. 2001; 61: 7878-7881PubMed Google Scholar Again, like down-regulation of claudin expression, the increased expression of claudins in cancer cells is also linked to increased invasiveness, through the recruitment of matrix metalloproteinases.20Agarwal R D'Souza T Morin PJ Claudin-3 and claudin-4 expression in ovarian epithelial cells enhances invasion and is associated with increased matrix metalloproteinase-2 activity.Cancer Res. 2005; 65: 7378-7385Crossref PubMed Scopus (304) Google Scholar To date, little is known about the expression or biological roles of the claudins in the normal human esophagus or esophageal squamous cell carcinoma (SCC). Herein, we demonstrate for the first time the expression of claudin-4 and -7 in the cell membranes of differentiated keratinocytes in the normal human esophagus. However, in esophageal SCC, claudin expression is mislocalized to the cytoplasm, with expression being lost in more advanced tumors. Genetic approaches were used to investigate the role of claudin-7 in SCC. We demonstrate that knockdown of claudin-7 leads to reduced E-cadherin expression, impairment of homotypic adhesion, and increased cell invasion. Conversely, re-expression of claudin-7 in claudin-7-deficient SCC lines results in increased E-cadherin expression and suppression of invasion. This study is the first demonstration that claudin-7 expression regulates E-cadherin expression and invasion in esophageal SCC and suggests that this may be an early event in the development of these tumors. Primary human esophageal keratinocytes EPC2 and the immortalized EPC2 derivatives EPC2-hTERT have been described previously.21Andl CD Mizushima T Nakagawa H Oyama K Harada H Chruma K Herlyn M Rustgi AK Epidermal growth factor receptor mediates increased cell proliferation, migration, and aggregation in esophageal keratinocytes in vitro and in vivo.J Biol Chem. 2003; 278: 1824-1830Crossref PubMed Scopus (220) Google Scholar, 22Harada H Nakagawa H Oyama K Takaoka M Andl CD Jacobmeier B von Werder A Enders GH Opitz OG Rustgi AK Telomerase induces immortalization of human esophageal keratinocytes without p16INK4a inactivation.Mol Cancer Res. 2003; 1: 729-738PubMed Google Scholar Cells were grown at 37°C under 5% CO2 in serum-free medium (keratinocyte-SFM) supplemented with 50 μg/ml bovine pituitary extract and 1 ng/ml epidermal growth factor (Invitrogen, Carlsbad, CA). TE cell lines (TE1, -2, -3, -8, -11, -12) are available commercially and through the National Institutes of Health/National Institute of Diabetes Digestive and Kidney Diseases Center for Molecular Studies in the Digestive and Liver Diseases’ Cell Culture Core Facility. Cells were cultured in Dulbecco's modified Eagle's medium (Mediatech, Herndon, VA) containing 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. Most of the tumor samples were obtained from resected primary lesions of esophageal cancer. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. Rabbit polyclonal claudin-7 and ZO-1 and mouse monoclonal claudin-4 primary antibodies were from Zymed (San Francisco, CA). Matrix metalloproteinase (MMP)-2 and -9 antibodies were from Cell Signaling Technology (Beverly, MA). Mouse anti-E-cadherin was from BD Pharmingen, Franklin Lakes, NJ. Mouse anti-β-actin antibody was from Sigma-Aldrich (St. Louis, MO). Alexa488 anti-rabbit secondary antibody was from Molecular Probes (Eugene, OR). Texas Red-conjugated anti-mouse secondary antibody was from Vector Laboratories (Burlingame, CA). Total human RNA from EPC2, EPC1a, TE1, TE8, TE11, and TE12 was isolated from each sample using the RNeasy kit (Qiagen, Valencia, CA) with on-column DNase digestion. Ten μg of total RNA was processed according to the standard protocol recommend by Affymetrix for use on their U133A arrays. Arrays were scanned and data generated using Affymetrix ArraySuite 5.0. The data were further analyzed using GeneSpring and GenMapp. The protein expression of claudin-1, -4, and -7 in esophageal tissues was assessed by immunohistochemical staining using a tissue microarray created at the Morphology Core from the Center for Molecular Studies in Digestive and Liver Diseases at the University of Pennsylvania. In addition AccuMax array A128(I) (Accurate Chemical and Scientific Corp., Westbury, NY) containing 40 samples (two replicates each) of formalin-fixed esophagus squamous cancer tissues and four normal esophageal epithelium were stained and scored for claudin-7 and E-cadherin expression. Scoring of claudin-7 and E-cadherin immunostaining was based on semiquantitative evaluation of stain intensity from 0 to 2. Marginal or no staining of less than 5% of the cells was graded as 0 (negative), mild to moderate stain of 5 to 50% of cells was graded as 1, and moderate to intense staining of more than 50% of the cells was classified as grade 2. Slides were scored for staining intensity by the Pathology Department of the Fox Chase Cancer Center (Philadelphia, PA), by two independent observers in a blinded manner. The adenoviral vector E-cad/Ad5 carrying the gene for E-cadherin protein has been described.23Hsu MY Meier FE Nesbit M Hsu JY Van Belle P Elder DE Herlyn M E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.Am J Pathol. 2000; 156: 1515-1525Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar The control adenoviral vector GFP has already been described24de Martin R Raidl M Hofer E Binder BR Adenovirus-mediated expression of green fluorescent protein.Gene Ther. 1997; 4: 493-495Crossref PubMed Scopus (59) Google Scholar was obtained from Dr. James Wilson (University of Pennsylvania Vector Core). Three clones of TE8 cells were stably transduced using ViraPower lentiviral expression system containing the gene for claudin-7 (CLDN7a-c). Control GFP lentivirus was raised in our laboratory. TE8 cells were transduced in the presence of 6 μg/ml polybrene. Forty-eight hours after transduction cells were selected in the presence of 10 μg/ml blasticidin for 14 days. Western blotting for claudin-7 and GFP were performed as described above. Claudin-7 expression was knocked down using Dharmacon SMARTpool RNAi claudin-7. TE1 cells were plated in six-well dishes at 50 to 60% confluence and transfected with 200 pmol of duplex RNA plus 4 μl of Lipofectamine 2000 (Life Technologies, Inc., Carlsbad, CA) following the manufacturer's protocol and as described.25Elbashir SM Harborth J Weber K Tuschl T Analysis of gene function in somatic mammalian cells using small interfering RNAs.Methods. 2002; 26: 199-213Crossref PubMed Scopus (1030) Google Scholar Using a six-transwell tray (Organogenesis, Canton, MA), 4.5 × 105 human skin fibroblast cells were mixed and seeded with a collagen matrix containing 1.68 mmol/L l-glutamine (Cellgro, Herndon, VA), 1× minimal essential medium with Earle's salts (EMEM), 10% fetal bovine serum, 0.15% sodium bicarbonate, 76.7% bovine collagen (PA-treated; Organogenesis). The collagen matrix was incubated with DMEM (JRH Biosciences, Lenexa, KS) containing 10% fetal bovine serum. After a 5-day incubation, wells and collagen matrix were washed with DMEM and Ham's F12 in a 3:1 ratio. Immortalized human esophageal epithelial cells (EPC2-hTERT) (5 × 105) were seeded onto the collagen matrix. Cells were fed for 2 days with epidermalization I medium, which contains DMEM/Ham's F-12 (3:1) supplemented with 4 mmol/L l-glutamine, 0.5 μg/ml hydrocortisone, 0.1 mmol/L O-phosphorylethanolamine, 20 pmol/L tri-iodothyronine, 0.18 mmol/L adenine, 1.88 mmol/L CaCl2, 4 pmol/L progesterone (Sigma); 10 μg/ml insulin, 10 μg/ml transferrin, 10 mmol/L ethanolamine, 10 ng/ml selenium (ITES); and 0.1% chelated newborn calf serum. For the following 2 days, cells were fed with epidermalization II medium, which is epidermalization I medium containing 0.1% unchelated newborn calf serum. Then cells were exposed to the air-liquid interface, cultured in epidermalization III medium for 8 days containing the same growth supplements as epidermalization II except with 2% newborn calf serum and no progesterone. Cells were fixed with 10% formaldehyde and embedded in paraffin. Activity of MMP-2 and MMP-9 in the culture medium of cells were assessed using gelatin zymography. TE8, ControlGFP, and CLDN7a-c cells were plated at equal density in 10-cm dishes and allowed to grow to 80% confluence. Cells were serum-starved for 24 hours, after which the cell-conditioned medium was collected. Equal amounts of proteins were then separated under nonreducing conditions on a 10% zymogram gel containing 0.1% gelatin (Invitrogen). After electrophoresis, the gel was incubated in zymogram renaturing buffer (Invitrogen) for 30 minutes at room temperature followed by a second incubation for 30 minutes at room temperature with zymogram developing buffer (Invitrogen). This was continued by overnight incubation at 37°C in the zymogram-developing buffer. Gels were then stained for 1 hour in GelCode blue stain reagent and destained for 1 hour in distilled water. EPC2-hTERT and TE1, TE2, and TE12 cells were seeded onto glass coverslips in six-well plates and incubated overnight. Cells were then fixed in 4% formaldehyde solution (Electron Microscopy Systems, Hatfield, PA) and permeabilized with Triton X-100 [0.2% (v/v)]. Samples were blocked in phosphate-buffered saline (PBS) containing 1% bovine serum albumin. Primary antibody incubations (claudin-4 and -7; Zymed) at 1:50 dilution were performed at 37°C in a humidified atmosphere for 1 hour. Coverslips were then washed three times in PBS before being incubated with secondary antibodies for 1 hour under similar conditions to the primary antibody (dilution factor of 1:250). Coverslips were then further washed in PBS and sterile water before being mounted with VectaShield with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and analyzed using immunofluorescence microscopy. For immunofluorescence detection of claudin-7 on the esophageal reconstructs, fixed paraffin-embedded tissue slides were deparaffinized with xylene two times for 10 minutes each, rehydrated with 100, 95, 75, and 50% ethanol for 2 minutes, and washed with PBS two times for 5 minutes. Antigen was retrieved by steaming the slides with 10 mmol/L citrate buffer for 20 minutes. Slides were thoroughly washed with distilled water and blocked for 30 minutes with 10% goat serum. Primary antibody was incubated overnight at 4°C. Slides were washed with PBS four times during 20 minutes, and secondary antibody was incubated at room temperature for 1 hour. After incubation, slides were washed with PBS four times during 20 minutes, treated with VectaShield, and then photographed under a Nikon E600 fluorescent microscope. Subconfluent cells were lysed in lysis buffer (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mmol/L ethylenediaminetetraacetic acid, and 2 mmol/L sodium orthovanadate) and a protease inhibitor mixture tablet (Roche Molecular Biochemicals, Indianapolis, IN). Protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL). The solution was subsequently solubilized in NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen) containing 50 mmol/L dithiothreitol. Total protein samples (20 μg) were separated on a 4 to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corp., Bedford, MA). The membrane was blocked in 5% nonfat milk in TBST (10 mmol/L Tris, 150 mmol/L NaCl, pH 8.0, and 0.1% Tween 20) for 1 hour at room temperature. Membranes were probed with primary antibody diluted 1:1000 in 5% TBST milk overnight at 4°C, washed three times in TBST, incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibody diluted 1:3000 in TBST for 1 hour at room temperature and then washed three times in TBST. The signal was visualized by an enhanced chemiluminescence solution (ECL Plus) and was exposed to film (Kodak, Rochester, NY). Esophageal cancer spheroids were prepared using the liquid overlay method.26Smalley KS Haass NK Brafford PA Lioni M Flaherty KT Herlyn M Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases.Mol Cancer Ther. 2006; 5: 1136-1144Crossref PubMed Scopus (381) Google Scholar Two hundred μl of TE cells (25,000 cells per ml) were added to a 96-well plate coated in 1.5% agar (Difco, Sparks, MD). Plates were left to incubate for 48 hours, by which time cells had organized into three-dimensional spheroids. Spheroids were then washed with PBS for 15 minutes. After removing the PBS, spheroids were treated with calcein-AM, ethidium bromide (Molecular Probes) for 1 hour at 37°C, according to the manufacturer's instructions. Pictures of the spheroids were taken using the Nikon-300 inverted fluorescence microscope. Cells (5 × 104)/chamber were used for each invasion assay. The upper parts of the Transwell (Corning Costar, Cambridge, MA) were coated with 70 μl of a bovine collagen matrix. Cells were plated onto the collagen-coated transwell in the presence of serum-free DMEM. In the lower chamber, 500 μl of 10% serum DMEM was added. The inserts were incubated for 4 days at 37°C. Cells that had invaded the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with DAPI. Ten random fields were counted per sample by light microscopy under a high-power field (×20). Data show the mean number of invading cells per field. Data show the mean of at least three independent experiments ± the SEM, unless stated otherwise. Statistically significant results were considered as P ≤ 0.05. Little is known about the expression patterns and function of the claudins either in the normal esophagus or in SCC of the esophagus. Preliminary microarray studies showed the up-regulation of claudin-1, -4, and -7 compared with control esophageal keratinocytes (Supplemental Figure 1 at http://ajp.amjpathol.org). To confirm these data, Western blotting was performed on protein extracts from seven esophageal SCC lines of various stages and primary esophageal keratinocytes. High expression of claudin-7 was observed in five of six SCC lines but not in the normal esophageal keratinocytes (Figure 1, A and B). A similar pattern of E-cadherin expression was seen compared with that of claudin-7 in the SCC lines (Figure 1B). By contrast, claudin-1 was expressed in the TE2, TE11, and TE12 cell lines only, and claudin-4 was only expressed in TE11 and TE12 (Figure 1, A and B). The normal esophageal keratinocyte line (EPC2) revealed no expression of any of the tested claudins. Similar results were observed with immunocytochemistry (Figure 1C). The distribution of claudin-7 in the TE1, TE2, and TE12 cell lines was primarily cytoplasmic and perinuclear. To expand on our initial observations, immunohistochemical staining for claudin-7 was performed on a tissue microarray containing samples from normal esophagus and SCC samples (Figure 2A; data summarized in Table 1 and Supplementary Tables 1 and 2 at http://ajp.amjpathol.org). The expression and distribution of the individual claudins in the normal squamous epithelia showed different patterns. Claudin-1 was expressed mainly in the basal layer of the epithelium and exclusively in the cytoplasm (Figure 2A) and disappeared toward the upper layers of the epithelium. Claudin-4 expression was detected throughout the epithelium (Figure 2A), particularly in the cytoplasm of cells in the basal layer. In contrast, the intermediate zone and the superficial layers showed strong staining in the membrane only. Claudin-7 was expressed in a very similar manner to claudin-4, with high levels of cytoplasmic staining in the suprabasal layer (Figure 2A) and strong membrane staining in combination with faint cytoplasmic staining throughout the rest of the epithelium. In the tumors, the expression of all three claudins was either cytoplasmic or absent (Figure 2A; and Supplementary Tables 1 and 2 at http://ajp.amjpathol.org). Indeed, most of the tumor samples analyzed (70%) did not express any claudin-7 (Table 1).Table 1Expression of Claudin-7 and E-Cadherin in Human Esophageal SCC SamplesPercentage of samples (n) expressing claudin-7 (immunostaining intensity)Percentage of samples (n) expressing E-cadherin (immunostaining intensity)DiagnosisNegative (0)12Negative (0)12Normal esophageal epithelium0% (0)25% (1)75% (3)0% (0)100% (4)0% (0)SCC70% (28)22.5% (9)7.5% (3)90% (36)10% (4)0% (0)Expression levels were scored by two independent observers on a scale of 0 to 2, where 2 indicates the highest level of expression (as described in Materials and Methods). Data show the pooled values from two tissue microarrays (raw data are available in Supplemental Tables 1 and 2 at http://ajp.amjpathol.org). Open table in a new tab Expression levels were scored by two independent observers on a scale of 0 to 2, where 2 indicates the highest level of expression (as described in Materials and Methods). Data show the pooled values from two tissue microarrays (raw data are available in Supplemental Tables 1 and 2 at http://ajp.amjpathol.org). Because the expression of claudins in keratinocytes of the normal esophagus was determined by differentiation state, we next investigated whether the cellular microenvironment was a critical determinant of claudin expression. Under standard cell culture conditions, esophageal keratinocytes expressed neither claudin-4 nor -7 (Figure 2B). However, when the same EPC2 human esophageal keratinocytes were grown in three-dimensional organotypic culture, which accurately recreates the tissue microenvironment, we observed claudin-4 and -7 staining at the cell membrane (Figure 2C). The localization of both claudin-4 and -7 was identical to that seen in the normal human esophagus (Figure 2A). The role of claudin-7 in SCC was investigated by knocking down the protein using an RNAi approach. Treatment of the TE1 cells with an RNAi to claudin-7 led to near complete (>95%) knockdown of claudin-7 expression after 48 hours (Figure 3A, left). In contrast, a control RNAi construct had no effect on claudin-7 expression (Figure 3A, right). Interestingly, knockdown of claudin-7 expression was accompanied by a marked decrease in E-cadherin expression (Figure 3A). Again, like claudin-7, the control RNAi had no effect on E-cadherin expression. To determine whether claudin-7 down-regulation influenced cell growth, RNAi-transfected TE1 cells were seeded into six-well plates, and cell counts were performed (Figure 3B). Although transfection of the cells with claudin-7 RNAi initially stimulated cell growth at days 2 and 3, the effect was no longer significant after prolonged periods of culture (Figure 3B). Because both claudin-7 and E-cadherin are important mediators of cell-cell adhesion, we investigated the role of claudin-7 in cell-cell adhesion by plating either control cells (untransfected or siControl) or claudin-7 RNAi cells (siRNACLDN7) onto 1.5% agar and observing them for 72 hours. After this time period, control cells were able to adhere to each other to form spheroid structures (Figure 3C). In contrast, the cells with the claudin-7 RNAi exhibited poor cell-cell adhesion and were positioned on top of the agar (Figure 3C). To exclude the possibility that the RNAi cells were undergoing apoptosis, samples were
Referência(s)