The roles of tight junction protein cingulin in human endometrioid endometrial cancer.

The bicellular tight junction molecule cingulin (CGN) binds to microtubules in centrosomes. Furthermore, CGN contributes to the tricellular tight junction (tTJ) proteins lipolysis-stimulated lipoprotein receptor (LSR) and tricellulin (TRIC). CGN as well as LSR decreased during the malignancy of endometrioid endometrial cancer (EEC). Although tTJ protein LSR is involved in the malignancy of some cancers, including EEC, the role of CGN is unknown. In this study, we investigated the roles of CGN with tTJ proteins in human EEC cells by using the CGN-overexpressing EEC cell line Sawano. In 2D cultures, CGN was colocalized with LSR and TRIC at tTJ or at γ-tubulin-positive centrosomes. In immunoprecipitation with CGN antibodies, CGN directly bound to LSR, TRIC, and β-tubulin. Knockdown of CGN by the siRNA decreased the epithelial barrier and enhanced cell proliferation, migration and invasion, as well as knockdown of LSR. In the Sawano cells cocultured with normal human endometrial stromal cells, knockdown of CGN decreased expression of LSR and TRIC via MAPK and AMPK pathways. In 2.5D cultures, knockdown of CGN induced the formation of abnormal cysts and increased the permeability of FD-4 to the lumen. In 2D and 2.5D cultures, treatment with β-estradiol with or without EGF or TGF-β decreased CGN expression and the epithelial permeability barrier and enhanced cell migration, and pretreatment with EW7197+AG1478, U0126 or an anti-IL-6 antibody prevented this. In conclusion, CGN, with tTJ proteins might suppress the malignancy of human EEC and its complex proteins are sensitive to estrogen and growth factors derived from stromal cells.

Endometrial cancer is the most common female genital malignancy in industrialized countries, and both morbidity and mortality rates are on the rise.Citation1,Citation2 The number of endometrial cancer cases and deaths in Japan have increased 1.7 and 1.4 fold, respectively, over the past 10 years.Citation3 Therefore, it is necessary to elucidate the pathophysiology of endometrial cancer in order to develop new treatment methods for this disease. Tight junctions are composed of membrane proteins, such as claudin (CLDN), cingulin (CGN), occludin (OCLN), and junctional adhesion molecule (JAM), and scaffold proteins such as the Zonula occludens family that bind to these membrane proteins.Citation4 Tight junction molecules regulate gene expression, cell proliferation, and migration of epithelial cells, and their abnormalities are involved in the malignant transformation of various tumors.Citation5,Citation6 The overexpression, mislocalization and loss of CLDNs contribute to the malignancy of endometrial carcinoma (EC) cells.Citation7 Overexpression of the leaky type tight junction molecule CLDN-2 promotes tumorigenesis of some cancer cells.Citation8–10 In human endometrial cancer, CLDN-2 is elevated in type I endometrioid endometrial carcinoma (EEC), while overexpression of CLDN-1 is observed in type II seropapillary endometrial carcinoma.Citation11,Citation12 Knockdown of CLDN-2 in endometrioid endometrial carcinoma results in increased epithelial barrier function and decreased cell migration and proliferation.Citation13 When the bicellular tight junction molecule CGN is phosphorylated by AMP-activated protein kinase (AMPK), the CGN conformation is changed and it binds to actin filaments and microtubules to regulate the epithelial barrier functions.Citation14 Depletion of CGN expression increases CLDN-2 expression and cell proliferation via RhoA- and c-Jun NH2-terminal kinase (JNK)-dependent pathways.Citation15 Tricellular tight junctions (tTJs) are intercellular junctions that seal the intercellular space in the tricellular junction where the apexes of the three epithelial cells come together.Citation16 The tricellular tight junction protein lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a constituent molecule of the tricellular junction. LSR expression decreases with malignant transformation in EEC, and downregulation of LSR decreases the epithelial barrier and increases cell migration, invasion, and proliferation of EEC cells.Citation17,Citation18 Although CGN may contribute to LSR and TRIC, the relationship is yet unknown in EEC cells. Epithelial integrity and barrier function are maintained during cytokinesis in vertebrate epithelial tissues.Citation19,Citation20 At the midbody and centrosome during cytokinesis, the tricellular tight junction molecules LSR and tricellulin, bicellular tight junction molecules CGN, OCLN, CLDN-7, and ZO-1 and the epithelial polarized related molecules PAR3 and apoptosis-stimulating of p53 protein 2 (ASPP2) are detected in EEC cells.Citation21 The major risk factors for the malignancy of endometrial cancer are estrogen and the growth factors Epidermal growth factor (EGF) and Transforming growth factor-β (TGF-β), which promote epithelial-mesenchymal transition (EMT) via various signaling pathways.Citation22–24 Circulating tumor cells (CTCs) in peripheral blood escape the tumor after the epithelial-mesenchymal transition.Citation25 CTCs are detected in high-risk endometrial cancer.Citation26 CTC counts and CLDN-4 expression are independent predictors of poor prognosis in breast cancer patients.Citation27 In the present study, CGN expression decreased during the malignancy of EEC, while CGN was also detected in CTCs. In EEC cell line Sawano, CGN was colocalized with LSR and TRIC at the membranes of tTJ or at γ-tubulin-positive centrosomes, and it directly bound to LSR, TRIC, and β-tubulin. Knockdown of CGN decreased the epithelial permeability barrier and enhanced cell proliferation, migration, and invasion as well as knockdown of LSR, and it decreased expression of LSR and TRIC via MAPK and AMPK pathways. Treatment with β-estradiol with or without EGF or TGF-β decreased CGN expression and the epithelial permeability barrier and enhanced cell migration. CGN with tTJ proteins might suppress the malignancy of human EEC.

Ethics statement The protocol for human study was reviewed and approved by the ethics committee of the Sapporo Medical University School of Medicine. Written informed consent was obtained from each patient who participated in the investigation. All experiments were carried out in accordance with the approved guidelines and with the Declaration of Helsinki. Reagents and antibodies Taxol (paclitaxel) was obtained from Sigma-Aldrich (33069-62-4, St. Louis, MO, USA). Recombinant human EGF (AF-100-15) and TGF-β (100–21) were obtained from Peprotech (Rock Hill, NJ, USA). β-estradiol was obtained from Tokyo Chemical Industry Co., Ltd. (50-28-2, Tokyo, Japan). TGF-β receptor type 1 inhibitor (EW-7197, 1352608-82-2) and EGF receptor inhibitor (AG-1478, 153436-53-4) were obtained from Cayman Chemical (Ann Arbor, MI, USA). Mitogen-activated protein kinase (MAPKK) inhibitor (U0126) was obtained from the Calbiochem-Novabiochem Corporation (109511-58-2, San Diego, CA, USA). Rabbit polyclonal anti-cingulin (CGN) was obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA). Mouse monoclonal CGN (G6) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit polyclonal anti-lipolysis-stimulated lipoprotein receptor (LSR) antibodies were from Novus Biologicals (Littleton, CO, USA). Rabbit polyclonal anti-actin and anti-γ-tubulin antibodies and mouse monoclonal anti-acetylated tubulin (T7451) were obtained from Sigma-Aldrich (St. Louis, Mo., USA). Rabbit polyclonal anti-tricellulin (TRIC) was obtained from Zymed Laboratories (San Francisco, CA, USA). Rabbit polyclonal anti-phosphorylated MAPK (pMAPK) and AMPK (pAMPK) antibodies were from Cell Signaling Technology (Danvers, MA, USA). Alexa 488 (green)-conjugated anti-rabbit IgG and Alexa 594 (red)-conjugated anti-mouse IgG antibodies were purchased from Molecular Probes, Inc. (Eugene, OR). The ECL Western blotting system was obtained from GE Healthcare UK, Ltd. (Buckinghamshire, UK). Immunohistochemical analysis Human endometriosis tissues and human endometrial carcinoma tissues were obtained from 6 patients with endometriosis and 15 patients with endometrial adenocarcinoma (G1: 7, G2: 4, and G3: 4) who underwent hysterectomy at Sapporo Medical University Hospital. Written informed consent was obtained from all patients. The study was approved by the ethics committee of Sapporo Medical University. The diagnosis and grades of the tumors were determined according to the guidelines of the WHO classification. The diagnoses of endometriosis and endometrial adenocarcinomas were established by both gynecologists and pathologists by hematoxylin and eosin-stained slides. All endometrial adenocarcinoma was the classic endometrial type I. In G1, the gland-like structures were clear observed in the whole area of the endometrial adenocarcinoma tissues, while in G2, the gland-like structures were decreased less than 50%. In G3, the gland-like structures were almost never seen. Human endometriosis and endometrial cancer tissues were embedded in paraffin after fixation with 10% formalin in PBS. Briefly, 5-μm-thick sections were dewaxed in xylene, rehydrated in ethanol, and heated with Vision BioSystems Bond Max using ER2 solution (Leica) in an autoclave for antigen retrieval. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide in methanol for 10 min. The tissue sections were then washed twice with Tris-buffered saline (TBS) and preblocked with Block Ace for 1 h. After washing with TBS, the sections were incubated with a mouse monoclonal anti-CGN (1:100) antibody for 1 h. The sections were then washed three times in TBS and incubated with Vision BioSystems Bond Polymer Refine Detection kit DS9800. After three washes in TBS, a diamino-benzidine tetrahydrochloride working solution was applied. Finally, the sections were counterstained with hematoxylin. Cell line culture and treatment The human endometrioid endometrial cancer cell line Sawano (RCB1152) was purchased from RIKEN Bio-Resource Center (Tsukuba, Japan). The cells were maintained with MEM (Sigma-Aldrich) supplemented with 10% dialyzed fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). The medium contained 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml amphotericin-B. Sawano cells were plated on 35- and 60-mm culture dishes, which were coated with rat tail collagen (500 μg dried tendon/ml in 0.1% acetic acid) and incubated in a humidified 5% CO2 incubator at 37°C. Some cells were treated with 100 ng/ml Taxol, 100 ng/ml IL-6, 100 ng/ml β-estradiol 100 ng/ml TGF-β1 and/or 100 ng/ml EGF after pretreatment with or without the IL-6 antibody in 10 μM U0126, 10 μM EW-7197 and/or 10 μM AG-1478 for 24 h. Dimensional (2.5D) matrigel culture Thirty-five-mm culture glass-coated dishes were coated with 100% Matrigel (15 μl/dish) at 4°C and incubated at 37°C for 30 min. Sawano cells (5 × 104 cells/well) were plated in MEM supplemented with 10% dialyzed fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.). After 48 h of plating, 10 spheroids were examined. RNA interference and transfection siRNA duplex oligonucleotides against siRNA of CGN-A (sense: 5’-CCCACCAUGCUGCAGUUCAA AUCAA-3’; antisense: 5’-UUGAU-UUGAACUGCAGCAUGGUGGG-3’), CGN-B (sense: 5’-CCUCUGUGAGGAGGAAGGUUAGUUU-3’; antisense: 5’-AAACUAACCUUCCUCCUCACAGAGG-3’), and LSR (forward sense 5’-CCCACGCAACCCAUCGUCAUCUGGA-3’, reverse sense 5’-UCCAGAUGACGAUGGGUUGCGUGGG-3’) were synthesized by Thermo Fisher Scientific (Waltham, MA). A scrambled siRNA sequence (BLOCK-iT Alexa Fluor Fluorescent, Invitrogen) was employed as control siRNA. At 24 h after plating, Sawano cells were transfected with 100 nM siRNAs of CGN and LSR, using LipofectamineTM RNAiMAX Reagent (Invitrogen). Immunocytochemical staining Cells cultured in 35-mm glass-coated wells (Iwaki, Chiba, Japan) were fixed with cold acetone and ethanol (1:1) at −20°C for 10 min. After rinsing in PBS, the cells were incubated with anti-CGN (1:100), anti-LSR (1:100), and anti-γ-tubulin (1:100) antibodies overnight at 4°C. Alexa Fluor 488 (green)-conjugated anti-rabbit IgG and Alexa Fluor 594 (red)-conjugated anti-mouse IgG (Invitrogen) were used as secondary antibodies. The specimens were examined using an epifluorescence microscope (Olympus, Tokyo, Japan) and a confocal laser scanning microscope (LSM5; Carl Zeiss, Jena, Germany). Western blot analysis The cultured cells were scraped from 60 mm dishes containing 400 μl of buffer (1 mM NaHCOCitation3 and 2 mM phenylmethylsulfonyl fluoride) collected in microcentrifuge tubes, and then sonicated for 10 s. The protein concentrations of the samples were determined using a BCA protein assay regent kit (Pierce Chemical Co.; Rockford, IL, USA). Aliquots of 15 μl of protein/lane for each sample were separated by electrophoresis in 5–20% SDS polyacrylamide gels (Wako, Osaka, Japan), and electrophoretically transferred to a nitrocellulose membrane (Immobilon; Millipore Co.; Bedford, UK). The membrane was saturated with blocking buffer (25 mM Tris, pH 8.0, 125 mM NaCl, 0.1% Tween 20, and 4% skim milk) for over 30 min at room temperature and incubated with anti- CGN (1:1000), anti-LSR (1:1000), anti-TRIC (1:1000), anti-β-tubulin (1:2000), anti-Ac-tubulin (1:2000), anti-pMAPK (1:1000), anti-pAMPK (1:1000) and anti-actin (1:1000) antibodies at room temperature for over 1 h. Then, it was incubated with HRP-conjugated anti-mouse and anti-rabbit IgG antibodies at room temperature for 1 h. The immunoreactive bands were detected using an ECL Western blot system. Coimmunopreciptitation The dishes were washed with PBS twice and 300 μl of NP-40 lysis buffer (50 mM Tris – HCl, 2% NP-40, 0.25 mM Na-deoxycholate, 150 mM NaCl, 2 mM EGTA, 0.1 mM Na3VO4, 10 mM NaF, 2 mM PMSF) was added to the 60-mm dishes. The cells were scraped off, collected in microcentrifuge tubes and then sonicated for 10 s. Cell lysates were incubated with protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology, Uppsala, Sweden) for 1 h at 4°C and then clarified by centrifugation at 15,000 g for 10 min. The supernatants were incubated with the polyclonal anti-CGN antibody bound to protein A-Sepharose CL-4B overnight at 4°C. After incubation, immunoprecipitates were washed extensively with the same lysis buffer and subjected to Western blot analysis with anti-LSR, anti-TRIC and anti-β-tubulin antibodies. Matrigel invasion assay For the invasion assay, we used Matrigel (Becton Dickinson Labware, Bedford, MA) and Cell Culture Insert (pore size 8 μm; Becton Dickinson Labware). Sawano cells were plated onto the upper chamber coated with Matrigel and the lower chamber of the Transwell was filled with human fibroblast conditioned medium containing 10 nM EGF as an adhesive substrate. Then the cells were incubated for 36 h, after which the upper chamber was fixed with 100% methanol for 10 min and stained with Giemsa for 20 min. The areas of invading cells were measured using a microscope imaging system (Olympus, Tokyo, Japan). Migration assay After the Sawano cells were plated onto the 35 mm dishes, they were cultured to confluence. The cell layer was wounded by using a plastic pipette tip (P200) and then measured the length of the wound by using a microscope imaging system (Olympus, Tokyo, Japan). Cell cycle assay Sawano cells cultured in 35 mm dishes were collected with 0.05％ Trypsin-EDTA and washed once with PBS. After that, the cells were added to 1 ml of ice cold 70°C ethanol and incubated for at least 3 h at − 20°C. The cells were then washed once with PBS and with 200 µL of Muse Cell Cycle reagent (Merck Millipore, MA, USA) before being incubated for 30 min at room temperature in the dark. We used a Muse®Cell Analyzer to measure the cell cycle according to the manufacturer’s instructions. Measurement of transepithelial electrical resistance (TEER) Sawano cells were cultured to confluence in the inner chambers of 12-mm transwell inserts with 0.4-μm pore-size filters (Corning Life Sciences). TEER was measured using an EVOM voltameter with an ENDOHM-12 (World Precision Instruments, Sarasota, FL). The values were expressed in standard units of ohms per square centimeter and presented as the mean ± S.D. For calculation, the resistance of blank filters was subtracted from that of filters covered with cells. Fluorescein isothiocyanate (FITC) permeability assay To assess barrier permeability, function, the permeability of fluorescein isothiocyanate (FITC)-dextran (FD-4, MW 4.0 kDa) from the outside into the spheroid lumen was examined by using 2.5D Matrigel cultures on 35-mm glass-coated dishes. The 2.5D Matrigel-cultured cells were incubated in the medium with 1% FD-4 at 37°C for 2 h. In all experiments ten spheroids were photographed and measured using a confocal laser scanning microscope with imaging software (LSM5 PASCAL; Carl Zeiss, Jena, Germany). Data analysis Each set of results shown is representative of at least three separate experiments. Results are given as means ± SEM. Differences between groups were tested by ANOVA followed by a post hoc test and an unpaired two-tailed Student’s t test.

Expression and localization of CGN in endometriosis and endometrial carcinoma To investigate the distribution and expression of CGN during the carcinogenesis of human endometrial cancer, immunohistchemical staining for CGN was performed using paraffin-embedded sections of endometriosis and endometrial cancer tissues. In endometriosis (EM), endometrial carcinoma G1 and G2, CGN were observed in the membranes of gland-like structures (). In endometrial carcinoma G3, the expression of CGN decreased with the loss of gland-like structures (). Furthermore, CGN was also found to be expressed in some circulating tumor cells (CTCs) that appeared to be intravascular cells in the invasive area (). Expression and localization of CGN in Sawano cells In the controlled Sawano cells in 2D culture, CGN was localized at the bicellular membranes, and LSR and TRIC were localized at the tricellular membranes (). Furthermore, CGN was colocalized with LSR and TRIC at the tricellular membranes (). In coimmunoprecipitation assays, the immunoprecipitates by using anti-CGN antibody detected LSR, TRIC, and β-tubulin (). To investigate whether the tight junction molecules CGN, LSR, and TRIC localized in the centrosomes during cytokinesis, Sawano cells were treated with 100 ng/ml Taxol and subjected to immunocytochemistry for CGN, LSR, TRIC, and γ-tubulin. All three junction molecules were localized in the centrosomes during the cytokinesis induced by Taxol (). Knockdown of CGN affects the epithelial barrier and the cell cycle in Sawano cells To investigate whether CGN suppression affected the epithelial barrier and the cell cycle, Sawano cells were transfected with two siRNAs of CGN with or without Taxol-treatment. Knockdown of CGN with or without Taxol-treatment did not affect expression and distribution of LSR and TRIC (). Knockdown of CGN decreased the TEER values indicating the epithelial barrier (). In the cell cycle, a decrease in the number of cells in the G0/G1 phase and an increase in the number of cells in the S phase were observed by knockdown of CGN (). Knockdown of CGN promoted cell proliferation in Sawano cells. Knockdown of CGN induces cell invasion and migration in Sawano cells We investigated whether CGN suppression affected cell invasion and migration in Sawano cells. Knockdown of CGN by the siRNA induced cell invasion and migration, as well as the changes by knockdown of LSR (). Knockdown of CGN affects the spheroid shape and induces the epithelial permeability barriers in 2.5D cultures of Sawano cells It is known that CGN expression affects lumen formation of ductal epithelial cells.Citation28 We investigated whether CGN suppression affected the formation of spheroids and induced the epithelial permeability barriers in 2.5D cultures of Sawano cells. Knockdown of CGN by the siRNA in part induced the formation of abnormal spheroids and the hyperpermeability of FD-4 into the lumina of cells in 2.5D cultures (). At the membranes of the lumina in 2.5D cultures, knockdown of CGN deceased CGN expression but did not affect LSR expression (). Knockdown of CGN affected lumen formation and epithelial permeability barriers in 2.5D cultures of Sawano cells. Coculture with stromal cells enhances the effects caused by knockdown of CGN in 2.5D cultures of Sawano cells To investigate the effects of stromal cells on expression of CGN, LSR, and TRIC, knockdown of CGN by the siRNA was performed in 2D cultures of Sawano cells cocultured with the stromal cells (). Western blotting showed that knockdown of CGN by the siRNAs decreased expression of CGN, LSR, and TRIC and increased expression of phosphorylated MAPK and phosphorylated AMPK (). Knockdown of CGN promoted the activation of MAPK and AMPK. β-estradiol with or without EGF or TGF-β promoted cell migration and epithelial permeability barriers in 2D and 2.5D cultures of Sawano cells To investigate whether β-estradiol and the growth factors EGF and TGF-β affect the cell migration, invasion and epithelial permeability barriers via CGN expression, the cells in 2D and 2.5D cultures were pretreated with the inhibitors EW7197 (ALK5 inhibitor), AG1478 (EGFR inhibitor) and U0126 (MAPK inhibitor) at 10 μM before treatment with β-estradiol with or without EGF and TGF-β at 100 ng/ml. Treatment with β-estradiol with or without EGF and TGF-β promoted cell migration and increased the TEER value, and pretreatement with EW7197, AG1478 and U0126 prevented the changes induced by the treatment with β-estradiol with or without EGF and TGF-β (). Western blotting showed that treatment with β-estradiol with or without EGF and TGF-β decreased CGN expression and the pretreatment with EW7197 and AG1478 prevented the downregulation (). Pretreatment with EW7197, AG1478 and U0126 decreased phosphorylated MAPK expression (). Treatment with β-estradiol with EGF and TGF-β promoted epithelial permeability and decreased LSR expression at membranes and the pretreatment with EW7197, AG1478 and U0126 prevented these changes in 2.5D cultures (). Anti-IL-6 antibody prevented an increase of epithelial permeability by β-estradiol in 2.5D cultures of Sawano cells Treatment with IL-6 as well as β-estradiol promoted epithelial permeability and decreased LSR expression at the membranes in 2.5D cultures (). Pretreatment with the anti-IL-6 antibody prevented the changes caused by treatment with IL-6 or β-estradiol in 2.5D cultures ().

In the present study, CGN expression decreased during the malignancy of EEC, while CGN was detected in CTCs. In EEC cell line Sawano, CGN was colocalized with LSR and TRIC at the membranes of tTJ and at γ-tubulin-positive centrosomes during cytokinesis, and it directly bound to LSR, TRIC, and β-tubulin. Knockdown of CGN decreased epithelial permeability barriers and enhanced cell proliferation, migration, and invasion via dependent or independent expression of LSR and TRIC by MAPK and AMPK pathways. Treatment with β-estradiol with or without EGF or TGF-β decreased CGN expression and epithelial permeability barriers and enhanced cell migration. CGN expression with tTJ proteins may suppress the malignancy of human EEC. In EM and EEC G1, LSR is highly expressed, and it is localized not only in the subapical region but also throughout the lateral region.Citation29 In EEC G2 and G3 LSR expression is reduced during the malignancy.Citation29 In EM, EEC G1 and EEC G2, CGN were observed in the subapical region. In EEC G3, the expression of CGN decreased with the loss of gland-like structures. CGN expression and LSR decreased during the malignancy of EEC. CGN interacts with TJ proteins ZO-1, ZO-2, ZO-3, and AF6.Citation30 In EEC cell line Sawano, CGN was colocalized with LSR and TRIC at the membranes of tTJ and it directly bound to LSR, TRIC, and β-tubulin. LSR, tricellulin, CGN, OCLN, CLDN-7, ZO-1, PAR3, and ASPP2 are detected at γ-tubulin-positive centrosomes during cytokinesis of Sawano cells.Citation21 Although the roles of the tight junction proteins concentrated at the centrosomes are not well known, it is possible that CGN may play an important role in cell division by interacting with these molecules. CGN also binds to both actin filaments and microtubules (MTs).Citation14 Furthermore, a planar apical network of noncentrosomal MTs is associated with TJs including CGN, and the CGN organizes the apical MT network formation.Citation14 It is thought that the network formation in the presence of CGN indicates epithelial differentiation. In the present study, since CGN directly bound to MTs, CGN expression might have suppressed dedifferentiation of ECC. Depletion of CGN expression increases CLDN-2 expression and cell proliferation via RhoA- and JNK-dependent pathways.Citation15 In human endometrial cancer, CLDN-2 is elevated in type I endometrioid endometrial carcinoma (EEC), while overexpression of CLDN-1 is observed in type II seropapillary endometrial carcinoma.Citation11,Citation12 Knockdown of CLDN-2 in Sawano cells results in increased epithelial barrier function and decreased cell migration and proliferation.Citation14 Downregulation of LSR also decreases the epithelial barrier and increases proliferation, migration, and invasion of Sawano cells.Citation17 In the present study, knockdown of CGN decreased the epithelial barrier and enhanced cell proliferation, migration, and invasion in 2D cultures of Sawano cells. In 2.5D cultures, knockdown of CGN induced the formation of abnormal cysts and increased the epithelial permeability. In the Sawano cells cocultured with normal human endometrial stromal cells, knockdown of CGN decreased expression of LSR and TRIC via MAPK and AMPK pathways. It is known that CGN phosphorylated by AMPK, binds to actin filaments and microtubules to regulate the epithelial barrier function.Citation14 In human lung adenocarcinoma cell line A549 cells, the knockdown of CGN increased bicellular TJ protein CLDN-2 via MAPK and AMPK pathways and induced cell migration.Citation31 CGN expression with tTJ proteins may suppress the malignancy of human EEC. CGN expression in endometrial epithelial cells may be affected by stromal cells. Endometrial cancer is known to be a hormone-dependent tumor, and estrogen closely contributes to its development and growth.Citation32 Unopposed estrogen conditions, such as late menopause, childbearing, obesity, and single-drug estrogen replacement therapy, are known risk factors for endometrial cancer.Citation32 It is known that there are many signaling pathways via estrogen receptors (ER).Citation32 Estrogen binding to cell surface ER activates the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway, which may play an important role in estrogen-induced endometrial cancer growth.Citation33 The proliferative effects of estrogen on the uterus are also known to involve growth factors.Citation34 EGF binds to epidermal growth factor receptors (EGFRs) on the cell surface and is involved in the regulation of cell proliferation. EGF controls the cell cycle and cell proliferation through activation of the ERK/MAPK signaling pathway.Citation35 TGF-β acts on various cells to regulate proliferation and differentiation. TGF-β is known to be involved in tumor cell invasion and metastasis by inducing angiogenesis and immunosuppression.Citation34 It has also been reported that overexpression of TGF-β in endometrial stromal cells activates the ERK/MAPK signaling pathway.Citation36 The growth factors TGF-β and EGF affect the epithelial permeability barrier, cell migration, and expression of bicellular and tricellular tight junction molecules in 2D and 2.5D cultures of Sawano cells.Citation37 EW7197 (a TGF-β receptor inhibitor), AG1478 (an EGFR inhibitor), and SP600125 (a JNK inhibitor) prevent the changes induced by TGF-β and EGF in these cultures of Sawano cells.Citation37 In the present study, treatment with β-estradiol with or without EGF or TGF-β decreased CGN expression and the epithelial permeability barrier and enhanced cell migration in 2D and 2.5D cultures of Sawano cells. Their changes were prevented by the inhibitors EW7197, AG1478, U0126 (a MAPK inhibitor) and an IL-6 antibody. Treatment with combined EW7197 and AG1478 induced CGN expression in the membranes of human salivary duct adenocarcinoma cell line A253 cells.Citation38 IL-6 is upregulated in endometriosis and endometrioid adenocarcinoma.Citation39 In conclusion, there are abnormalities of tight junction expression and functions, and cell migration in human endometriosis and endometrial carcinoma.Citation17 Depletion of CGN in EEC cells decreases the epithelial permeability barrier and expression of tTJ proteins and increased cell migration and proliferation, which are involved in malignant transformation of endometrial cancer. In a protein-protein interaction map, CGN can be seen to directly interact with LSR and TRIC (MARVELD2) (supplemental Figure S1). CGN, with tTJ proteins, might suppress the malignancy of human EEC and its complex proteins are sensitive to estrogen and the growth factors derived from stromal cells. Authors contributions AK, Takayuki K, TS, and Takashi K conceived and designed the study. AK, KS, TO, SN, DI, and Takumi K performed the experiments and verified data quality. MM, SN, and DI performed the statistical analysis of obtained data. AK and Takashi K wrote the paper and submitted it to the evaluation of the whole consortium. All authors have read and agreed to the published version of the manuscript. Ethics statement The protocol for the human study was reviewed and approved by the ethics committee of the Sapporo Medical University School of Medicine. All experiments were carried out in accordance with the approved guidelines and the Declaration of Helsinki. Supplemental material Supplemental Material Download Zip (1.2 MB)Supplemental MaterialDisclosure statement No potential conflict of interest was reported by the author(s). supplementary material Supplemental data for this article can be accessed online at https://doi.org/10.1080/21688370.2024.2361976. 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- Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA A Cancer J Clin. 2018;68(6):1–17. doi:10.3322/caac.21492. - Katanoda K, Hori M, Matsuda T, Shibata A, Nishino Y, Hattori M, Soda M, Ioka A, Sobue T, Nishimoto H. An updated report on the trends in cancer incidence and mortality in Japan, 1958–2013. Japanese J Clin Oncol. 2015;45(4):390–401. doi:10.1093/jjco/hyv002. - Hori M, Matsuda T, Shibata A, Katanoda K, Sobue T, Nishimoto H; Cancer Statistics. Cancer information service, national cancer center, japan (monitoring of cancer incidence in japan (MCIJ), cancer incidence and incidence rates in japan in 2009: a study of 32 population-based cancer registries for the monitoring of cancer incidence in Japan (MCIJ) project. Jpn J Clin Oncol. 2015;45(9):884–891. doi:10.1093/jjco/hyv088. - Wibbe N, Ebnet K. Cell Adhesion at the tight junctions: new aspects and new functions. Cells. 2023;12(23):2701. doi:10.3390/cells12232701. - Matter K, Balda MS. Signalling to and from tight junctions. Nat Rev Mol Cell Biol. 2003;4(3):225–237. doi:10.1038/nrm1055. - Balda MS, Matter K. Tight junctions and the regulation of gene expression. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2009;1788(4):761–767. doi:10.1016/j.bbamem.2008.11.024. - Capoferri D, Bignotti E, Ravaggi A, Mitola S, Romani C. Finding the junction between claudins and endometrial carcinoma. Biochim Biophys Acta Rev Cancer. 2023;1878(6):189019. doi:10.1016/j.bbcan.2023.189019. - Ikari A, Sato T, Watanabe R, Yamazaki Y, Sugatani J. Increase in claudin-2 expression by an EGFR/MEK/ERK/c-Fos pathway in lung adenocarcinoma A549 cells. Biochim Biophys Acta. 2012;1823(6):1110–1118. doi:10.1016/j.bbamcr.2012.04.005. - Kinugasa T, Huo Q, Higashi D, Shibaguchi H, Kuroki M, Tanaka T, Futami K, Yamashita Y, Hachimine K, Maekawa S, et al. Selective up-regulation of claudin-1 and claudin-2 in colorectal cancer. Anticancer Res. 2007;27(6A):3729–3734. - Xin S, Huixin C, Benchang S, Aiping B, Jinhui W, Xiaoyan L, Yu WB, Minhu C. Expression of Cdx2 and claudin-2 in the multistage tissue of gastric carcinogenesis. Oncology. 2007;73(5–6):357–365. doi:10.1159/000135351. - Sobel G, Németh J, Kiss A, Lotz G, Szabó I, Udvarhelyi N, Schaff Z, Páska C. Claudin 1 differentiates endometrioid and serous papillary endometrial adenocarcinoma. Gynecol Oncol. 2006;103(2):591–598. doi:10.1016/j.ygyno.2006.04.005. - Szabó I, Kiss A, Schaff Z, Sobel G. Claudins as diagnostic and prognostic markers in gynecological cancer. Histol Histopathol. 2009;24(12):1607–1615. doi:10.14670/HH-24.1607. - Okada H, Konno T, Kohno T, Shimada H, Saito K, Satohisa S, Saito T, Kojima T. Possibility of targeting claudin-2 in therapy for human endometrioid endometrial carcinoma. Reprod Sci. 2020;27(11):2092–2103. doi:10.1007/s43032-020-00230-6. - Yano T, Matsui T, Tamura A, Uji M, Tsukita S. The association of microtubules with tight junctions is promoted by cingulin phosphorylation by AMPK. J Cell Bio. 2013;203(4):605–614. doi:10.1083/jcb.201304194. - Guillemot L, Citi S. Cingulin regulates claudin-2 expression and cell proliferation through the small GTPase RhoA. MboC. 2006;17(8):3569–3577. doi:10.1091/mbc.e06-02-0122. - Furuse M, Oda Y, Higashi T, Iwamoto N, Masuda S. Lipolysis-stimulated lipoprotein receptor: a novel membrane protein of tricellular tight junctions. Ann N. Y. Acad Sci. 2012;1257(1):54–58. doi:10.1111/j.1749-6632.2012.06486.x. - Shimada H, Kohno T, Konno T, Okada T, Saito K, Shindo Y, Kikuchi S, Tsujiwaki M, Ogawa M, Matsuura M, et al. The roles of tricellular tight junction protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in endometriosis and endometrioid-endometrial carcinoma. Cancers Basel. 2021;13(24):6341. doi:10.3390/cancers13246341. - Saito K, Konno T, Kohno T, Shimada H, Matsuura M, Okada T, Kura A, Ishii D, Kondoh M, Saito T, et al. LSR antibody promotes apoptosis and disrupts epithelial barriers via signal pathways in endometrial cancer. Tissue Barriers. 2023;11(3):2106113. doi:10.1080/21688370.2022.2106113. - Jinguji Y, Ishikawa H. Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine. Cell Struct Funct. 1992;17(1):27–37. doi:10.1247/csf.17.27. - Baker J, Garrod D. Epithelial cells retain junctions during mitosis. J Cell Sci. 1993;104(2):415–425. doi:10.1242/jcs.104.2.415. - Konno T, Kohno T, Kikuchi S, Shimada H, Satohisa S, Takano K, Saito T, Kojima T. Localization of tricellular tight junction molecule LSR at midbody and centrosome during cytokinesis in human epithelial cells. J Histochem Cytochem. 2020;68(1):59–72. doi:10.1369/0022155419886263. - Rodriguez AC, Blanchard Z, Maurer KA, Gertz J. (2019) Estrogen signaling in endometrial cancer: a key oncogenic pathway with several open questions. Horm Cancer. 2019;10(2–3):51–63. doi:10.1007/s12672-019-0358-9. - Chiu HC, Li CJ, Yiang GT, Tsai AP, Wu MY. Epithelial to mesenchymal transition and cell biology of molecular regulation in endometrial carcinogenesis. J Clin Med. 2019;8(4):439. doi:10.3390/jcm8040439. - Stancel GM, Gardner RM, Kirkland JL, Lin TH, Lingham RB, Loose-Mitchell DS, Mukku VR, Orengo CA, Verner G. Interactions between estrogen and EGF in uterine growth and function. Adv Exp Med Biol. 1987;230:99–118. doi:10.1007/978-1-4684-1297-0_6. - Li R, Gong Z, Liu Y, Zhao X, Guo SJNF. Detection of circulating tumor cells and single cell extraction technology: principle, effect and application prospect. Futures. 2021;5(3):032002. doi:10.1088/2399-1984/ac1325. - Bogani G, Liu MC, Dowdy SC, Cliby WA, Kerr SE, Kalli KR, Kipp BR, Halling KC, Campion MB, Mariani A. Detection of circulating tumor cells in high-risk endometrial cancer. Anticancer Res. 2015;35(2):683–687. - Chai J, Liu X, Hu X, Wang C. Correlation analysis of circulating tumor cells and Claudin-4 in breast cancer. Pathol Oncol Res. 2023;29:1611224. doi:10.3389/pore.2023.1611224. - Su Y, Long Y, Xie K. Cingulin family: Structure, function and clinical significance. Life Sci. 2024;341:122504. doi:10.1016/j.lfs.2024.122504. - Shimada H, Satohisa S, Kohno T, Takahashi S, Hatakeyama T, Konno T, Tsujiwaki M, Saito T, Kojima T. The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells. Oncotarget. 2016;7(19):27735–27752. doi:10.18632/oncotarget.8408. - Cordenonsi M, D’Atri F, Hammar E, Parry DA, Kendrick-Jones J, Shore D, Citi S. Cingulin contains globular and coiled- coil domains and interacts with ZO-1, ZO-2, ZO-3, and myosin. J Cell Bio. 1999;147(7):1569–1582. doi:10.1083/jcb.147.7.1569. - Ishii D, Shindo Y, Arai W, Konno T, Kohno T, Honda K, Miyajima M, Watanabe A, Kojima T. The roles and regulatory mechanisms of tight junction protein cingulin and transcription factor forkhead box protein o1 in human lung adenocarcinoma a549 cells and normal lung epithelial cells. Int J Mol Sci. 2024 Jan 24; 25(3):1411. doi:10.3390/ijms25031411. - Rodriguez AC, Blanchard Z, Maurer KA, Gertz J. Estrogen signaling in endometrial cancer: a key oncogenic pathway with several open question. Horm Cancer. 2019;10(2–3):51–63. doi:10.1007/s12672-019-0358-9. - Losel R, Wehling M. Nongenomic actions of steroid hormones. Nat Rev Mol Cell Biol. 2003;4(1):46–56. doi:10.1038/nrm1009. - De Wever O, Mareel M. Role of tissue stroma in cancer cell invasion. J Pathol. 2003;200(4):429–447. doi:10.1002/path.1398. - Kubota Y, O’Grady P, Saito H, Takekawa M. Oncogenic Ras abrogates MEK SUMOylation that suppresses the ERK pathway and cell transformation. Nat Cell Biol. 2011 [Epub 2011 Feb 20]. 13(3):282–291. doi:10.1038/ncb2169. - Liu Z, Yi L, Du M, Gong G, Zhu Y. Overexpression of TGF-β enhances the migration and invasive ability of ectopic endometrial cells via ERK/MAPK signaling pathway. Exp Ther Med. 2019 [Epub 2019 Apr 23]. 17:4457–4464. doi:10.3892/etm.2019.7522. - Konno T, Kohno T, Kikuchi S, Kura A, Saito K, Okada T, Shimada H, Yamazaki Y, Sugiyama T, Matsuura H, et al. The interplay between the epithelial permeability barrier, cell migration and mitochondrial metabolism of growth factors and their inhibitors in a human endometrial carcinoma cell line. Tissue Barriers. 2024 Jan 15; 2304443. doi:10.1080/21688370.2024.2304443. PMID: 38225862. - Nakano M, Ohwada K, Shindo Y, Konno T, Kohno T, Kikuchi S, Tsujiwaki M, Ishii D, Nishida S, Kakuki T, et al. Inhibition of HDAC and signal transduction pathways induces tight junctions and promotes differentiation in p63-Positive salivary duct adenocarcinoma. Cancers Basel. 2022 May 24; 14(11):2584. doi:10.3390/cancers14112584. - Slater M, Cooper M, Murphy CR. Human growth hormone and interleukin-6 are upregulated in endometriosis and endometrioid adenocarcinoma. Acta Histochemica. 2006 [Epub 2006 Mar 27]; 108(1):13–18. doi:10.1016/j.acthis.2006.01.004.