Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial–mesenchymal transition of pancreatic cancer

Carcinogenesis, Jun 2013

Protein kinase Cα (PKCα) is highly expressed in pancreatic cancer. However, the effects of PKCα on Snail and claudin-1, which play crucial roles in epithelial cell polarity during epithelial–mesenchymal transition (EMT), remain unclear. In this study, we investigated the mechanisms of regulation of Snail and claudin-1 via a PKCα signal pathway during EMT in pancreatic cancer cells and in normal human pancreatic duct epithelial cells (HPDEs). By immunostaining, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1. Treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. The PKCα inhibitor prevented upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment and under hypoxia in PANC-1 cells. The effects of the PKCα inhibitor were in part regulated via an extracellular signal-regulated kinase (ERK) signaling pathway. The PKCα inhibitor also prevented downregulation of the barrier function and fence function during EMT in well-differentiated pancreatic cancer cell line HPAC. In normal HPDEs, the PKCα inhibitor transcriptionally induced not only claudin-1 but also claudin-4, -7 and occludin without a change of Snail. Treatment with the PKCα inhibitor in normal HPDEs prevented downregulation of claudin-1 and occludin by TGF-β1 treatment and enhanced upregulation of claudin-1, -4, -7 and occludin under hypoxia. These findings suggest that PKCα regulates claudin-1 via Snail- and mitogen-activated protein kinase/ERK-dependent pathways during EMT in pancreatic cancer. Thus, PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells.

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Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial–mesenchymal transition of pancreatic cancer

Advance Access publication February Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial-mesenchymal transition of pancreatic cancer Daisuke Kyuno 0 1 Takashi Kojima 0 Hiroshi Yamaguchi 1 Tatsuya Ito 1 Yasutoshi Kimura 1 Masafumi Imamura 1 Akira Takasawa 0 Masaki Murata 0 Satoshi Tanaka 0 Koichi Hirata 1 Norimasa Sawada 0 0 Department of Pathology, Sapporo Medical University School of Medicine , Sapporo 060-8556 , Japan 1 Department of Surgery Protein kinase Cα (PKCα) is highly expressed in pancreatic cancer. However, the effects of PKCα on Snail and claudin-1, which play crucial roles in epithelial cell polarity during epithelial-mesenchymal transition (EMT), remain unclear. In this study, we investigated the mechanisms of regulation of Snail and claudin-1 via a PKCα signal pathway during EMT in pancreatic cancer cells and in normal human pancreatic duct epithelial cells (HPDEs). By immunostaining, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1. Treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. The PKCα inhibitor prevented upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment and under hypoxia in PANC-1 cells. The effects of the PKCα inhibitor were in part regulated via an extracellular signal-regulated kinase (ERK) signaling pathway. The PKCα inhibitor also prevented downregulation of the barrier function and fence function during EMT in well-differentiated pancreatic cancer cell line HPAC. In normal HPDEs, the PKCα inhibitor transcriptionally induced not only claudin-1 but also claudin-4, -7 and occludin without a change of Snail. Treatment with the PKCα inhibitor in normal HPDEs prevented downregulation of claudin-1 and occludin by TGF-β1 treatment and enhanced upregulation of claudin-1, -4, -7 and occludin under hypoxia. These findings suggest that PKCα regulates claudin-1 via Snail- and mitogen-activated protein kinase/ERK-dependent pathways during EMT in pancreatic cancer. Thus, PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells. Introduction Pancreatic cancer, which has a strong invasive capacity with frequent metastasis and recurrence, is known to be one of the most malignant human diseases and its death rate has not decreased over the past few decades ( 1 ). Thus, there is an urgent need to develop novel diagnostic and therapeutic strategies to reduce the mortality of pancreatic cancer patients. Protein kinase C (PKC) belongs to the family of serine-threonine kinases and regulates various cellular functions, including adhesion, secretion, proliferation, differentiation and apoptosis ( 2 ). At least 12 Abbreviations: EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HPDEs, human pancreatic duct epithelial cells; hTERT, telomerase reverse transcriptase; JNK, c-Jun N-terminal kinase; mRNA, messenger RNA; NF-κB, nuclear factor-kappaB; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; pMAPK, phospho-MAPK; pPKCα, phospho-PKCα; siRNA, small interference RNA; TER, transepithelial electrical resistance; TGF-β1, transforming growth factor-β1; TPA, 12-O-tetradecanoylphorbol 13-acetate. different isozymes of PKC are known and can be subdivided into three classes (classic or conventional, novel and atypical isozymes) according to their responsiveness to activators ( 3,4 ). Levels of PKCα, PKCβ1, PKCδ and PKCι are higher in pancreatic cancer, whereas that of PKCε is higher in normal tissue ( 5,6 ). In addition, PKCα is thought to be one of the biomarkers for diagnosis of cancers ( 7 ). In pancreatic cancer, tumorigenicity is directly related to PKCα expression as demonstrated by decreased survival when it is overexpressed ( 8 ). The increased level of PKCα is also associated with pancreatic cancer cell proliferation ( 9 ). PKCα is also one of the regulators and therapeutic targets in cancer ( 10 ). Epithelial–mesenchymal transition (EMT) is closely related to carcinoma progression and acts as a major driver of morphogenesis and tumor progression ( 11 ). The activation of PKC is involved in EMT. The PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) induces EMT in human prostate cancer cells and pancreatic cancer cell line HPAC ( 12,13 ). Expression of PKCα and PKCδ closely contributes to EMT in colon cancer cells ( 14,15 ). Snail, which is a transcription repressor that plays a central role in EMT, directly binds to E-boxes of the promoters of claudin/occludin genes, resulting in repression of their promoter activities and loss of epithelial cell polarity ( 16 ). In several human cancers, including pancreatic cancer, some tight junction protein claudins are abnormally regulated and therefore promising molecular targets for diagnosis and therapy ( 17,18 ). Tight junctions are the most apical components of intercellular junctional complexes in epithelial and endothelial cells. They separate the apical and basolateral cell surface domains, maintaining cell polarity (termed the ‘fence’ function), and selectively control solute and water flow through the paracellular space (termed the ‘barrier’ function) ( 19–22 ). It is thought that loss of tight junction function in part leads to invasion and metastasis of cancer cells ( 23 ). In particular, claudin-1, which is expressed in various types of epithelial cells, plays an important role in epithelial cell polarity, cancer invasion and metastasis ( 24–28 ). Tight junction proteins are regulated by various cytokines and growth factors via distinct signal transduction pathways including PKC ( 29,30 ). We previously found that, in pancreatic cancer cell line HPAC, tricellulin localized at tricellular tight junctions was in part regulated via PKCδ and PKCε pathways (31), and the expression of claudin-18 and localization of claudin-4 and occludin were in part regulated via a PKCα pathway ( 13,32,33 ). Furthermore, in normal human pancreatic duct epithelial cells (HPDEs), some tight junction proteins are regulated via PKCα and PKCδ ( 34 ). However, little is known about how PKCα regulates claudin-1 in pancreatic cancer cells and normal HPDEs. In this study, overexpression of PKCα and downregulation of claudin-1 were observed in poorly differentiated human pancreatic cancer tissues and pancreatic cancer cell lines. Treatment with the PKCα inhibitor Gö6976 prevents upregulation of Snail and downregulation of claudin-1 during EMT induced by transforming growth factor-β1 (TGF-β1) treatment or under hypoxia in a pancreatic cancer cell line ( 28 ). Our findings suggest that PKCα inhibitors may be potential therapeutic agents against human pancreatic cancer cells. Materials and methods Reagents and inhibitors Rabbit polyclonal anti-occludin, anti-claudin-1, anti-claudin-4, anti-claudin-7, anti-Snail and mouse monoclonal anti-occludin (OC-3F10) antibodies were obtained from Zymed Laboratories (San Francisco, CA). A rabbit polyclonal anti-actin antibody and a nuclear factor-kappaB (NF-κB) inhibitor (IMD0354) were purchased from Sigma–Aldrich, (St Louis, MO). Rabbit polyclonal anti-Snail, anti-phospho-PKCα (pPKCα) and PKCα antibodies were obtained from Cell Signaling (Beverly, MA). Alexa 488 (green)-conjugated anti-rabbit IgG and Alexa594 (red)-conjugated anti-mouse IgG antibodies were purchased from Molecular Probes (Eugene, OR). Inhibitors of panPKC (GF109203X), PKCα (Gö6976), mitogen-activated protein kinase (MAPK) (U0126), p38 MAPK (SB203580), phosphatidylinositol 3-kinase (PI3K) (LY294002) and c-Jun N-terminal kinase (JNK) (SP600125) were purchased from CalbiochemNovabiochem Corporation (San Diego, CA). TGF-β1 was purchased from PeproTech EC (London, UK). Immunohistochemical analysis Immunohistochemical analysis was performed to evaluate the expression and distribution of PKCα and claudin-1 in normal pancreatic tissues, well-differentiated and poorly differentiated pancreatic cancer tissues. Deparaffinized tissue sections were immersed in 10 mM Tris–1 mM ethylenediaminetetraacetic acid buffer (pH 9.0) for staining of PKCα or 10 mM sodium citrate (pH 6.0) for staining of claudin-1 and boiled for antigen retrieval by microwave (95°C, 30 min). Endogenous peroxidase activity was blocked using 3% hydrogen peroxidase for 10 min. The sections were incubated with rabbit polyclonal PKCα and claudin-1 antibodies (1:100 dilution) overnight at 4°C. The sections were incubated with a Dako REALTM EnVisionTM/HRP, Rabbit/Mouse (Dako REALTM EnVisionTM Detection System; Dako, code K5007) for 1 h at room temperature. After washing with phosphate-buffered saline, the labeled secondary antibody was visualized by adding Dako REALTM Substrate Buffer (Dako REALTM EnVisionTM Detection System; Dako, code K5007) containing Dako REALTM DAB + Chromogen (Dako REALTM EnVisionTM Detection System: Dako, code K5007). The sections were counterstained with hematoxylin. Cultures of cell lines and treatment Human pancreatic cancer cell lines PANC-1, HPAF-II, BXPC-3 and HPAC were purchased from American Type Culture Collection (Manassas, VA). PANC-1 and HPAC cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich) supplemented with 10% dialyzed fetal bovine serum (FBS; Invitrogen, Carlsbad, CA). HPAF-II cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% FBS and supplemented with 0.1 mM non-essential amino acids (Sigma–Aldrich) and 1 mM sodium pyruvate (Sigma–Aldrich). BXPC-3 cells were maintained in RPMI-1640 (Sigma–Aldrich) supplemented with 10% FBS. The media for all cell lines contained 100 U/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin-B. All cells were plated on 60 mm culture dishes (Corning Glass Works, Corning, NY) that were coated with rat tail collagen (500 µg of dried tendon/ ml in 0.1% acetic acid) and incubated in a humidified 5% CO2 incubator at 37°C. PANC-1 and HPAC cells were treated with 0.01–2 µg/ml Gö6976 for 24 h or 100 ng/ml TGF-β1 for 24 and 48 h. PANC-1 cells were pretreated with 20 µM U0126, 10  µM SB203580, 10  µM LY294002, 10  µM GF109203X, 10  µM SP600125 and 0.1  µM IMD-0354 for 30 min before treatment with 1  µg/ml Gö6976 for 24 h and were pretreated with 1 µg/ml Gö6976 for 30 min before treatment with 100 ng/ml TGF-β1 for 24 h. The PANC-1 cells were incubated in a 2% CO2:2% O2 incubator balanced with nitrogen with or without 1 μg/ml Gö6976 for 24 h. HPAC cells were pretreated with 1 µg/ml Gö6976 for 30 min before treatment with 100 ng/ml TGF-β1 for 24 h. For RNA interference studies, small interference RNA (siRNA) duplexes targeting the messenger RNA (mRNA) sequences of human Snail were purchased from Invitrogen. The sequences were as follows: siRNA-1 of Snail (sense 5′-CCUCGCUGCCAAUGCUCAUCUGGGA-3′), siRNA-2 of Snail (sense 5′-AGGCCAAGGAUCUCCAGGCUCGAAA-3′), siRNA of PKCα (sense 5′-CCGAGUGAAACUCACGGACUUCAAU-3′) and siRNA of PKCβ (sense 5′-GAGACCGGAUGAAACUGACCGAUUU-3′). A scrambled siRNA sequence (BLOCK-iT Alexa Fluor fluorescent; Invitrogen) was employed as control siRNA. One day before transfection, the PANC-1 cells were plated in medium without antibiotics such that they would be half confluent at the time of transfection. The cells were transfected with 100 nM siRNAs using Lipofectamine RNAiMAX (Invitrogen) as a carrier according to the manufacturer’s instructions. Isolation and culture of HPDEs Human pancreatic tissues were obtained from patients with pancreatic or biliary tract diseases who underwent pancreatic resection in the Sapporo Medical University hospital. Informed consent was obtained from all patients, and the study was approved by the ethics committee of Sapporo Medical University. The procedures for primary culture of HPDEs were as reported previously ( 31,32,34 ). Some primary cultured HPDEs were transfected with the catalytic component of telomerase, the human catalytic subunit of the telomerase reverse transcriptase (hTERT) gene as described previously ( 31,32,34 ). The hTERTHPDEs were cultured in serum-free Bronchial Epithelial Cell Medium kit (Lonza Walkersville, Walkersville, MD) and incubated in a humidified, 5% CO2:95% air incubator at 37°C. In this experiment, second and third passaged cells were used. The hTERT-HPDEs were treated with 1 and 2  µg/ml Gö6976 for 24 h or 20 ng/ml TGF-β1 for 24 and 48 h. Some cells were pretreated with 20  µM U0126, 10  µM SB203580, 10  µM LY294002, 10  µM GF109203X, 10  µM SP600125 and 0.1  µM IMD-0354 for 30 min before treatment with 1  µg/ml Gö6976 for 24 h and some cells were pretreated with 1  µg/ml Gö6976 for 30 min before treatment with 20 ng/ml TGF-β1 for 24 h. The hTERT-HPDEs were incubated in a 2% CO2:2% O2 incubator balanced with nitrogen with or without 1 μg/ml Gö6976 for 24 h. Western blot analysis Western blot analysis was performed as described previously ( 31,32,34 ). The membranes were incubated with polyclonal anti-claudin-1, anti-claudin-4, anti-claudin-7, anti-occludin, anti-Snail, anti-pPKCα, anti-PKCα, anti-phospho-extracellular signal-regulated kinase (ERK1/2), anti-ERK1/2 and antiactin antibodies (1:1000) for 1 h at room temperature. The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Dako A/S, Copenhagen, Denmark) at room temperature for 1 h. The immunoreactive bands were detected using an enhanced chemiluminescence western blotting analysis system (GE Healthcare, Little Chalfont, UK). RNA isolation and real-time PCR analysis Total RNA was extracted and purified using TRIzol (Invitrogen). One microgram of total RNA was reverse transcribed into complementary DNA using a mixture of oligo (deoxythymidine) and superscript II reverse transcriptase according to the manufacturer’s recommendations (Invitrogen). Real-time PCR detection was performed using a TaqMan Gene Expression Assay kit with a StepOnePlusTM real-time PCR system (Applied Biosystems, Foster City, CA). The amount of 18S ribosomal RNA (Hs99999901) mRNA in each sample was used to standardize the quantity of the following mRNAs: claudin-1 (Hs00221623), claudin-4 (Hs00533616), claudin-7 (Hs00154575), occludin (Hs00170162) and Snail1 (Hs00195591). The relative mRNA expression levels between the control and treated samples were calculated by the difference of the threshold cycle (comparative CT [∆C T] method) and presented as the average of triplicate experiments with a 95% confidence interval. Immunocytochemistry The cells were grown on 35 mm glass-base dishes (Iwaki, Chiba, Japan) coated with rat tail collagen. They were fixed with cold acetone and ethanol (1:1) at 20°C for 10 min. After rinsing in phosphate-buffered saline, the sections and cells were incubated with polyclonal anti-claudin-1 and monoclonal anti-occludin antibodies (1:100) at room temperature for 1 h and then with Alexa Fluor 488 (green)-conjugated anti-rabbit IgG (1:200) and Alexa Fluor 594 (red)-conjugated anti-mouse IgG (1:200) at room temperature for 1 h. 4′,6-diamidino-2-phenylindole (Sigma–Aldrich) was used for counterstaining of nuclei in the cells. The specimens were examined using an epifluorescence microscope (Olympus, Tokyo, Japan). Measurement of transepithelial electrical resistance The cells were cultured to confluence on inner chambers of 12mm Transwell 0.4 µm pore-size filters (Corning Life Science). Transepithelial electrical resistance (TER) was measured using an EVOM voltmeter with an ENDOHM-12 (World Precision Instruments, Sarasota, FL) on a heating plate (Fine, Tokyo, Japan) adjusted to 37°C. The values are expressed in standard units of ohms per square centimeter and presented as the mean ± SD of triplicate experiments. For calculation, the resistance of blank filters was subtracted from that of filters covered with cells. Diffusion of BODIPY-sphingomyelin For measurement of the tight junctional fence function, we used diffusion of BODIPY-sphingomyelin with some modification ( 28 ). The samples were analyzed by confocal laser scanning microscopy (LSM510; Carl Zeiss, Jena, Germany). All pictures shown were generated within the first 5min of analysis. Data analysis Signals were quantified using Scion Image Beta 4.02 Win (Scion, Frederick, MD). Each set of results shown is representative of at least three separate experiments. Results are given as means ± standard error of the mean. Differences between groups were tested by analysis of variance followed by a post hoc test and an unpaired two-tailed Student’s t-test. Results Expression and distribution of PKCα and claudin-1 in normal pancreatic ducts, well- and poorly differentiated pancreatic duct carcinomas In this study, we examined the expression and distribution of PKCα and claudin-1 in normal pancreatic ducts, well- and poorly differentiated pancreatic duct carcinomas (Figure  1). PKCα was detected in cytoplasm of both pancreatic duct carcinomas and the expression in poorly differentiated pancreatic duct carcinoma was stronger than in well-differentiated pancreatic duct carcinoma, whereas in normal pancreatic ducts, PKCα was not detected. Claudin-1 was localized at the cell membranes of normal pancreatic ducts and well-differentiated pancreatic carcinoma, whereas in poorly differentiated pancreatic carcinoma, it was weakly detected in cytoplasm. Expression patterns of PKCα, Snail, claudin-1, -4, -7 and occludin in hTERT-HPDEs and human pancreatic cancer cell lines To study the relationship between activation of PKCα and expression of tight junction proteins in human pancreatic cancer cells and normal pancreatic duct epithelial cells, we first investigated the expression patterns of pPKCα, panPKCα and claudin-1 in hTERT-HPDEs, which are models of normal pancreatic duct epithelial cells ( 34 ), and human pancreatic cancer cell lines HPAF-II and HPAC, which are models of well- or moderately differentiated pancreatic cancers and BXPC-3 and PANC-1, which are models of poorly differentiated pancreatic cancers ( 35 ). pPKCα and panPKCα were detected in all pancreatic cancer cell lines and were strongly expressed in PANC-1 cells, whereas in hTERT-HPDEs, they were not detected (Figure 1B). Snail, an EMT marker, was highly expressed only in PANC-1 cells (Figure 1B). Claudin-1, -4 and occludin were detected in all cell types, but at a low level in PANC-1 cells compared with other cancer cells, whereas claudin-7 was not detected in PANC-1 cells (Figure 1C). In the poorly differentiated pancreatic cancer cell line PANC-1, PKCα and Snail were upregulated and claudin-1 was downregulated. PKCα inhibitor downregulates Snail and upregulates claudin-1 and occludin in PANC-1 cells To investigate the effects of the PKCα inhibitor on the expression of Snail and tight junction proteins in the poorly differentiated pancreatic cancer cell line PANC-1, the cells were treated with 0.01–2 μg/ml PKCα inhibitor Gö6976 for 24 h. In western blots, a significant decrease of Snail and a significant increase of claudin-1 and occludin were observed after treatment with Gö6976 in a dose-dependent manner, whereas no changes of claudin-4 and -7 were observed compared with the control (Figure 2A; Supplementary Figure  1, available at Carcinogenesis Online). In realtime PCR, a significant decrease of Snail mRNA and a significant increase of claudin-1 mRNA were induced by treatment with Gö6976, whereas no change of occludin mRNA was observed (Figure  2B). In immunocytochemistry, claudin-1 and occludin were strongly observed at the membranes after treatment with Gö6976 compared with the control (Supplementary Figure 2, available at Carcinogenesis Online). PKCα inhibitor upregulates claudin-1, -4, -7 and occludin and increases TER values in hTERT-HPDEs To investigate the effects of the PKCα inhibitor on tight junction proteins in normal pancreatic duct epithelial cells, hTERT-HPDEs were treated with 1 and 2 μg/ml Gö6976 for 24 h. In western blots, claudin-1,-4,-7 and occludin were significantly increased by treatment with Gö6976 (Figure  2C; Supplementary Figure  1, available at Carcinogenesis Online). In real-time PCR, claudin-1,-4,-7 and occludin mRNAs were significantly increased by treatment with Gö6976 (Figure 2D). In immunocytochemistry, claudin-1 and occludin were detected at the membranes after treatment with Gö6976 (Supplementary Figure 2, available at Carcinogenesis Online). Furthermore, to investigate whether the PKCα inhibitor affected barrier function in normal pancreatic duct epithelial cells, hTERT-HPDEs were treated with 1 μg/ml Gö6976 for 24 h and then TER was measured. TER values were significantly increased from 2 h after treatment with Gö6976 (Figure 2E). PKCα inhibitor induces activation of MAPK/ERK in both PANC-1 cells and hTERT-HPDEs PKCα functions as a potent activator of c-Raf-1 and turns on the MAPK/ERK cascade ( 36 ). Thus, we investigated the effect of Gö6976 on MAPK/ERK activation in pancreatic cancer cells and normal pancreatic duct epithelial cells. Western blots revealed that Gö6976 induced phosphorylation of MAPK of both PANC-1 cells and hTERT-HPDEs in a dose-dependent manner (Figure  3A and B; Supplementary Figure 3, available at Carcinogenesis Online). MAPK/ERK inhibitor prevents upregulation of claudin-1 and occludin by treatment with PKCα inhibitor in PANC-1 cells, but not in hTERT-HPDEs To investigate whether MAPK/ERK activation affected changes of claudin-1, occludin and Snail induced by treatment with Gö6976, PANC-1 cells and hTERT-HPDEs were pretreated with 20 μM MAPK inhibitor U0126 before treatment with 1  μg/ml Gö6976. In western blots, U0126 prevented upregulation of phospho-MAPK (pMAPK) after treatment with Gö6976 in both PANC-1 cells and hTERTHPDEs (Figure  3C and D; Supplementary Figure  3, available at Carcinogenesis Online). In PANC-1 cells, U0126 prevented upregulation of claudin-1 and occludin after treatment with Gö6976, whereas downregulation of Snail was decreased (Figure  3C; Supplementary Figure  3, available at Carcinogenesis Online). In hTERT-HPDEs, upregulation of occludin after treatment with Gö6976 was enhanced by treatment with U0126, whereas upregulation of claudin-1 was not affected by the treatment (Figure 3D; Supplementary Figure 3, available at Carcinogenesis Online). Upregulation of claudin-1 by treatment with PKCα inhibitor is involved in distinct signaling pathways in PANC-1 cells and in hTERT-HPDEs As shown in Figure 3, MAPK inhibitor U0126 prevented upregulation of claudin-1 after treatment with Gö6976 in PANC-1 cells but not in hTERT-HPDEs. To investigate which signaling pathways were associated with induction of claudin-1 by Gö6976 in PANC-1 cells and hTERT-HPDEs, the cells were pretreated with U0126, p38 MAPK inhibitor SB203580, PI3K inhibitor LY294002, panPKC inhibitor GF109203X, JNK inhibitor SP600125 and NF-κB inhibitor IMD0354 before treatment with Gö6976. In western blots, induction of claudin-1 after treatment with Gö6976 was inhibited by U0126, SB203580, LY294002 and GF109203X in PANC-1 cells (Figure 3E; Supplementary Figure  3, available at Carcinogenesis Online), whereas in hTERT-HPDEs, induction of claudin-1 after treatment with Gö6976 was inhibited by SB203580, LY294002, GF109203X, SP600125 and IMD-0354 (Figure 3F; Supplementary Figure 3, available at Carcinogenesis Online). TGF-β1 upregulates Snail and downregulates claudin-1, -4 and occludin together with activation of pPKCα and pMAPK/ERK in PANC-1 cells To investigate changes of Snail and claudins/occludin caused by PKCα and MAPK/ERK during EMT induced by TGF-β in PANC-1 cells, the cells were treated with 100 ng/ml TGF-β1 for 24 and 48 h. PANC-1 cells acquired a spindle cell morphology from 24 h after treatment with TGF-β1 (Supplementary Figure  4, available at Carcinogenesis Online). In western blots, claudin-1,-4 and occludin were decreased and Snail was increased at 24 and 48 h after treatment with TGF-β1, whereas no change of claudin-7 was observed (Figure  4A; Supplementary Figure  5, available at Carcinogenesis Online). Furthermore, TGF-β1 enhanced phosphorylation of PKCα and MAPK/ERK at 24 and 48 h, respectively (Figure  4A; Supplementary Figure 5, available at Carcinogenesis Online). TGF-β1 downregulates claudin-1, -7 and occludin and upregulates claudin-4 in hTERT-HPDEs To investigate changes of claudins/occludin, Snail, PKCα and MAPK induced by treatment with TGF-β1 in hTERT-HPDEs, the cells were pretreated with 4% FBS for 24 h and then treated with 20 ng/ml TGFβ1 for 24 and 48 h. In phase-contrast images, hTERT-HPDEs also acquired spindle cell morphology from 24 h after treatment with TGFβ1 (Supplementary Figure 4, available at Carcinogenesis Online). In western blots, claudin-1 and occludin were decreased and claudin-4 was increased at 24 and 48 h after treatment with TGF-β1, whereas no changes of claudin-7 and pMAPK/ERK were observed and Snail and PKCα were not detected (Figure  4B; Supplementary Figure  5, available at Carcinogenesis Online). Claudin-1 in PANC-1 cells is in part regulated via the Snail gene To investigate whether claudin-1 in PANC-1 cells was directly regulated via the Snail gene, knockdown of Snail using siRNAs was performed and the cells were treated with or without 100 ng/ ml TGF-β1 for 48 h. In the control cells, claudin-1 was significantly increased by knockdown of Snail (Figure 4C; Supplementary Figure  5, available at Carcinogenesis Online). In the cells treated with TGF-β1, regardless of the fact that an increase of TGF-β1induced Snail was prevented by the siRNAs, the downregulation of claudin-1 by TGF-β1 was not completely reversed (Figure 4D). These findings indicated that claudin-1 in PANC-1 cells during EMT induced by TGF-β, was in part regulated via the Snail gene, though also by other factors. PKCα inhibitor prevents upregulation of Snail and downregulation of claudin-1 by TGF-β in PANC-1 cells To investigate whether the PKCα inhibitor affected changes of Snail, claudins/occludin and pMAPK/ERK induced by TGF-β in PANC-1 cells, the cells were pretreated with Gö6976 before treatment with 100 ng/ml TGF-β1. Western blots showed that Gö6976 prevented the increase of Snail and decrease of claudin-1 after treatment with TGF-β1, but it did not affect the changes of claudin-4, -7 and occludin (Figure 5A; Supplementary Figure 6, available at Carcinogenesis Online). Gö6976 enhanced the upregulation of pMAPK/ERK caused by treatment with TGF-β1 (Figure  5A; Supplementary Figure  6, available at Carcinogenesis Online). PKCα inhibitor prevents downregulation of claudin-1 and occludin and enhanced upregulation of claudin-4 by TGF-β in hTERT-HPDEs hTERT-HPDEs were pretreated with 4% FBS for 24 h and then treated with Gö6976 before treatment with 20 ng/ml TGF-β1. Gö6976 prevented decreases of claudin-1 and occludin after treatment with TGF-β1 and enhanced the increase of claudin-4, whereas it did not affect claudin-7 or pMAPK/ERK (Figure  5B; Supplementary Figure  6, available at Carcinogenesis Online). After treatment with TGF-β1, PKCα was not detected with or without Gö6976 (Figure 5B; Supplementary Figure 6, available at Carcinogenesis Online). PKCα inhibitor prevents upregulation of Snail and pPKCα and downregulation of claudin-1 and pMAPK/ERK under hypoxia in PANC-1 cells To investigate whether the PKCα inhibitor affected changes of Snail, claudin-1, occludin, pPKCα and pMAPK/ERK caused by hypoxia in PANC-1 cells, the cells were incubated in a 2% CO2:2% O2 incubator balanced with nitrogen for 24 h with or without Gö6976. In western blots, in PANC-1 cells under hypoxia, increases of Snail and pPKCα and decreases of claudin-1 and pMAPK/ERK were observed compared with the control (Figure  5C; Supplementary Figure  6, available at Carcinogenesis Online). Gö6976 prevented these increases of Snail and pPKCα and decreases of claudin-1 and pMAPK, whereas no change of occludin was observed under hypoxia with or without Gö6976 (Figure  5C; Supplementary Figure  6, available at Carcinogenesis Online). PKCα inhibitor enhances upregulation of claudin-1, -4, -7 and occludin under hypoxia in hTERT-HPDEs To investigate whether the PKCα inhibitor affected changes of Snail, claudin-1, -4, -7, occludin and pMAPK/ERK in hTERT-HPDEs caused by hypoxia, the cells were pretreated with 4% FBS for 24 h and were incubated in a 2% CO2:2% O2 incubator balanced with nitrogen for 24 h with or without Gö6976. In western blots, increases of claudin-1, -4, -7, occludin and pMAPK/ERK caused by hypoxia were observed (Figure  5D; Supplementary Figure  6, available at Carcinogenesis Online). Gö6976 enhanced increases of claudin-1, -4, -7 and occludin under hypoxia, whereas it did not affect upregulation of pMAPK/ERK (Figure  5D; Supplementary Figure  6, available at Carcinogenesis Online). Snail was not detected under hypoxia with or without Gö6976 (Figure 5D; Supplementary Figure 6, available at Carcinogenesis Online). PKCα inhibitor prevents downregulation of barrier function and fence function by treatment with TGF-β1 in HPAC cells To investigate whether the PKCα inhibitor affected the barrier and fence functions in pancreatic cancer cells, the well-differentiated pancreatic cancer cell line HPAC was used. It was pretreated with 100 ng/ ml TGF-β1 for 24 and 48 h with or without Gö6976. In western blots, upregulation of Snail and pPKCα and downregulation of claudin-1,-4, -7 and occludin were observed after treatment with TGF-β1 together with downregulation of the barrier and fence functions (Figure 6A, C and D; Supplementary Figure 7, available at Carcinogenesis Online). Gö6976 prevented upregulation of Snail and downregulation of claudin-1 after treatment with TGF-β1, whereas it did not affect changes of claudin-4, -7 and occludin (Figure  6B; Supplementary Figure  7, available at Carcinogenesis Online). These changes were similar to the changes in PANC-1 cells. Furthermore, Gö6976 prevented downregulation of the barrier and fence functions by TGF-β1 (Figure 6C and D). Discussion In this study, we demonstrated that PKCα inhibitor Gö6976 protected against downregulation of claudin-1 via Snail- and MAPK/ERKdependent pathways during EMT in human pancreatic cancer. The mechanisms involved in the regulation of claudin-1 by the inhibitor in pancreatic cancer were in part different from those in normal HPDEs. It is known that the PKC inhibitor Gö6976, which was used in this study, can inhibit not only α isoform of PKC but also β1 isoform of PKC. To clarify a role of PKCα in the regulation of claudins during EMT in pancreatic cancer, we performed using siRNA against PKCα or PKCβ. In PANC-1 cells, knockdown of PKCα by the siRNA induced claudin-1 and -4 and prevented downregulation of claudin-1 and -4 by treatment with TGF-β1, whereas the effects of knockdown of PKCβ by the siRNA were not observed (Supplementary Figure 8, available at Carcinogenesis Online). These results indicated that in this study, the effects of the PKC inhibitor Gö6976 were mainly caused via a specific PKCα pathway. PKCα is thought to be one of the biomarkers for diagnosis of cancer and regulators in cancer cells ( 7,10 ). In pancreatic cancer, cell proliferation and tumorigenicity are directly related to PKCα expression ( 8,9 ). On the other hand, PKC is associated with claudin-1 expression in melanoma cells ( 37 ). In HPAC cells, treatment with TPA modifies the activity of pPKCα and causes downregulation of claudin-1 and mislocalization of claudin-4 and occludin ( 13 ). In this study, in poorly differentiated human pancreatic cancer tissues and the pancreatic cancer cell line PANC-1, overexpression of PKCα and downregulation of claudin-1 were observed, whereas in normal HPDEs in vivo and in vitro, PKCα was not detected and claudin-1 was highly expressed. These findings suggest that, in pancreatic cancer, PKCα is a biomarker for diagnosis and may be a negative regulator against claudin-1 expression. The activation of PKC is involved in EMT. The PKC activator TPA induces EMT in human prostate cancer cells and HPAC cells ( 12,13 ). Expression of PKCα and PKCδ closely contributes to EMT in colon cancer cells ( 14,15 ). TGF-β1 induces PKCα in poorly differentiated pancreatic cancer cell line BXPC-3 ( 38 ). During EMT induced by treatment with TGF-β1 and under hypoxia, claudin-1 is directly regulated via the Snail gene ( 28 ). In this study, treatment with the PKCα inhibitor Gö6976 transcriptionally decreased Snail and increased claudin-1 in PANC-1 cells. Expression of claudin-1 in PANC-1 cells was increased by knockdown of Snail. Furthermore, the PKCα inhibitor prevented the upregulation of Snail and downregulation of claudin-1 in PANC-1 cells during EMT induced by treatment with TGF-β1 and hypoxia. These findings suggested that, in pancreatic cancer cells, PKCα is activated by TGF-β1 and hypoxia induced EMT, and the PKCα inhibitor could prevent downregulation of claudin-1 by repressing Snail during EMT. PKC has been shown to induce both assembly and disassembly of tight junctions depending on the cell type and conditions of activation ( 39–41 ). It is also known to regulate epithelial barrier function via tight junctions ( 42,43 ). We previously reported that, in hTERTHPDEs, TPA enhanced expression of claudin-1, -4, -7 and -18, occludin, JAM-A, ZO-1, ZO-2 and tricellulin, and the expression of claudin-4 and -18 induced by TPA was inhibited by the PKCα inhibitor Gö6976 ( 32,34 ). In this study, using hTERT-HPDEs, treatment with the PKCα inhibitor transcriptionally induced expression of claudin-1, -4, -7 and occludin and enhanced the barrier function measured as the TER value. Furthermore, in hTERT-HPDEs, TGF-β1 caused downregulation of claudin-1 and occludin and upregulation of claudin-4. The PKCα inhibitor prevented the downregulation of claudin-1 and occludin and enhanced the upregulation of claudin-4. However, expression of Snail in hTERT-HPDE treated with the PKCα inhibitor and TGF-β1 was not detected. We have previously reported that treatment with TGF-β1 upregulates claudin-4 without a change of barrier function in normal human nasal epithelial cells ( 43 ). TGFβ1 transcriptionally upregulates claudin-4 and strengthens the intestinal barrier in a human intestinal cell line ( 44 ). TGF-β1 induces EMT in cultured human pancreatic duct cells ( 45 ). Our results indicated that the PKCα inhibitor upregulated tight junction proteins and the barrier function via a Snail-independent pathway in normal HPDEs. Furthermore, the PKCα inhibitor also prevented downregulation of claudin-1 via a Snail-independent pathway during EMT induced by treatment with TGF-β in normal HPDEs. Activated PKCα turns on the MAPK cascade via activation of downstream signaling ( 46 ). PKCα inhibition leads to increased ERK1/2 activity ( 47,48 ). The arrangement of the expression and distribution of claudin-1 are closely related to cell dissociation status in pancreatic cancer cells through the MAPK/ERK pathway (49). In this study, the PKCα inhibitor Gö6976 enhanced the phosphorylation of MAPK/ ERK in both PANC-1 cells and hTERT-HPDEs. In PANC-1 cells, the upregulation of claudin-1, but not the downregulation of Snail, by the PKCα inhibitor was prevented by the MAPK/ERK inhibitor U0126. In PANC-1 cells treated with TGF-β1, the downregulation of claudin-1 by TGF-β1 was not completely reversed via knockdown of Snail. In hTERT-HPDEs, the upregulation of claudin-1 by the PKCα inhibitor was prevented by treatment with inhibitors of p38MAPK, PI3K, panPKC, JNK and NF-κB, but not MAPK/ERK signaling pathways. These findings suggested that the signal transduction pathways of the PKCα inhibitor for the regulation of claudin-1 were different in pancreatic cancer cells and normal HPDEs, and in pancreatic cancer, claudin-1 was regulated via not only the Snail gene but also the MAPK/ERK signaling pathway. Loss of the fence function, which is important for maintenance of epithelial cell polarity, is closely associated with the expression of claudin-1 ( 24 ). The poorly differentiated pancreatic cancer cell line PANC-1 shows loss of the tight junctional barrier measured as TER and the fence function examined by diffusion of labeled BODIPYsphingomyelin ( 28 ). The well-differentiated pancreatic cancer cell line HPAC has high barrier and fence functions, and the fence function is downregulated by treatment with TGF-β1 and hypoxia ( 28 ). In this study, upregulation of Snail and downregulation of claudin-1 expression, barrier function and fence function were observed in HPAC cells treated with TGF-β1, and the PKCα inhibitor Gö6976 prevented all the changes induced by treatment with TGF-β1. These results suggest that the PKCα inhibitor may prevent downregulation of epithelial cell polarity during EMT in pancreatic cancer. In conclusion, the PKCα inhibitor strongly protected against downregulation of claudin-1 during EMT in pancreatic cancer. Furthermore, in normal HPDEs, the PKCα inhibitor induced some tight junction proteins, including claudin-1, and enhanced barrier function. Taken together, these results suggest that PKCα inhibitors may be potential therapeutic agents against the malignancy of human pancreatic cancer cells. 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Daisuke Kyuno, Takashi Kojima, Hiroshi Yamaguchi, Tatsuya Ito, Yasutoshi Kimura, Masafumi Imamura, Akira Takasawa, Masaki Murata, Satoshi Tanaka, Koichi Hirata, Norimasa Sawada. Protein kinase Cα inhibitor protects against downregulation of claudin-1 during epithelial–mesenchymal transition of pancreatic cancer, Carcinogenesis, 2013, 1232-1243, DOI: 10.1093/carcin/bgt057