Gene Summary

Gene:CLDN4; claudin 4
Summary:The protein encoded by this intronless gene belongs to the claudin family. Claudins are integral membrane proteins that are components of the epithelial cell tight junctions, which regulate movement of solutes and ions through the paracellular space. This protein is a high-affinity receptor for Clostridium perfringens enterotoxin (CPE) and may play a role in internal organ development and function during pre- and postnatal life. This gene is deleted in Williams-Beuren syndrome, a neurodevelopmental disorder affecting multiple systems. [provided by RefSeq, Sep 2013]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Source:NCBIAccessed: 15 March, 2017


What does this gene/protein do?
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Pathways:What pathways are this gene/protein implicaed in?
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Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 15 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Tight Junctions
  • Adenocarcinoma
  • Cell Proliferation
  • Messenger RNA
  • Chromosome 7
  • Promoter Regions
  • Stomach Cancer
  • Ovarian Cancer
  • Zonula Occludens-1 Protein
  • Neoplastic Cell Transformation
  • Western Blotting
  • Immunohistochemistry
  • Pancreatic Cancer
  • Triple Negative Breast Cancer
  • Up-Regulation
  • Sp1 Transcription Factor
  • Survival Rate
  • Biomarkers, Tumor
  • DNA Methylation
  • Enterotoxins
  • Claudin-1
  • Breast Cancer
  • Claudin-3
  • Membrane Proteins
  • Uterine Cancer
  • Claudins
  • Gene Expression Profiling
  • Cancer Gene Expression Regulation
  • Clostridium perfringens
  • Down-Regulation
  • siRNA
  • Squamous Cell Carcinoma
  • Immunoenzyme Techniques
  • Cystadenocarcinoma, Serous
  • Neoplasm Invasiveness
  • Claudin-4
  • Signal Transduction
  • Cell Movement
  • Oligonucleotide Array Sequence Analysis
Tag cloud generated 15 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: CLDN4 (cancer-related)

Wahdan-Alaswad R, Harrell JC, Fan Z, et al.
Metformin attenuates transforming growth factor beta (TGF-β) mediated oncogenesis in mesenchymal stem-like/claudin-low triple negative breast cancer.
Cell Cycle. 2016; 15(8):1046-59 [PubMed] Free Access to Full Article Related Publications
Mesenchymal stem-like/claudin-low (MSL/CL) breast cancers are highly aggressive, express low cell-cell adhesion cluster containing claudins (CLDN3/CLDN4/CLDN7) with enrichment of epithelial-to-mesenchymal transition (EMT), immunomodulatory, and transforming growth factor-β (TGF-β) genes. We examined the biological, molecular and prognostic impact of TGF-β upregulation and/or inhibition using in vivo and in vitro methods. Using publically available breast cancer gene expression databases, we show that upregulation and enrichment of a TGF-β gene signature is most frequent in MSL/CL breast cancers and is associated with a worse outcome. Using several MSL/CL breast cancer cell lines, we show that TGF-β elicits significant increases in cellular proliferation, migration, invasion, and motility, whereas these effects can be abrogated by a specific inhibitor against TGF-β receptor I and the anti-diabetic agent metformin, alone or in combination. Prior reports from our lab show that TNBC is exquisitely sensitive to metformin treatment. Mechanistically, metformin blocks endogenous activation of Smad2 and Smad3 and dampens TGF-β-mediated activation of Smad2, Smad3, and ID1 both at the transcriptional and translational level. We report the use of ID1 and ID3 as clinical surrogate markers, where high expression of these TGF-β target genes was correlated to poor prognosis in claudin-low patients. Given TGF-β's role in tumorigenesis and immunomodulation, blockade of this pathway using direct kinase inhibitors or more broadly acting inhibitors may dampen or abolish pro-carcinogenic and metastatic signaling in patients with MCL/CL TNBC. Metformin therapy (with or without other agents) may be a heretofore unrecognized approach to reduce the oncogenic activities associated with TGF-β mediated oncogenesis.

Madaras L, Balint N, Gyorffy B, et al.
BRCA Mutation-Related and Claudin-Low Breast Cancer: Blood Relatives or Stepsisters.
Pathobiology. 2016; 83(1):1-12 [PubMed] Related Publications
BACKGROUND: BRCA mutation-associated (BRCAmut) breast cancer represents a heterogeneous group displaying certain molecular features. Claudin-low breast cancers (CLBC) overlap with characteristics of BRCAmut tumors; therefore, we have investigated whether these are identical subtypes.
METHODS: Using public gene expression data, CLDN, CDH1, 9-cell line claudin-low predictor (9CLCLP) and PAM50 expression was evaluated in BRCAmut and BRCA wild-type (BRCAwt) breast cancer cases focusing on their possible overlap with the CLBC subtype. A separate formalin-fixed, paraffin-embedded (FFPE) cohort of 22 BRCAmut and 19 BRCAwt tumor tissues was used for immunohistochemical examination of AR, CD24, CD44, CK5/6, claudin-1, -3, -4 and -7, E-cadherin, EGFR, estrogen receptor (ER), EZH2, HER2, Ki67, p53, progesterone receptor (PgR) and vimentin expression.
RESULTS: In the data sets, CLDN1 (ROC = 0.785, p < 0.001), CDH1 (ROC = 0.785, p < 0.001), CLDN7 (ROC = 0.723, p < 0.001), CLDN3 (ROC = 0.696, p = 0.020) and CLDN4 (ROC = 0.685, p = 0.027) were expressed at higher level in BRCAmut than BRCAwt tumor tissue. The PAM50 subtype differed from the assigned immunohistochemistry (IHC)-based subtype in 30%. Based on accessible 9CLCLP predictor genes, BRCAmut breast cancer does not display the claudin-low phenotype. Utilizing FFPE samples, claudins were evidently expressed in both BRCAmut and BRCAwt cases. However, at the protein level, only claudin-3 expression was higher in BRCAmut tumors, while claudin-1, -4 and -7 and E-cadherin expression was lower compared to BRCAwt cases. A CD24low/CD44high phenotype was found in BRCAmut tumors upon comparison with BRCAwt cases (p < 0.001 and p = 0.001, respectively).
CONCLUSIONS: There is a prominent correlation between the genes under focus herein and BRCA mutation status. BRCAmut tumors bear stem cell characteristics displaying a distinct cell adhesion molecule profile characterized by high expression of CDH1 and CLDN4 according to public gene expression data set analysis, and higher claudin-3 expression as detected by IHC; thus, BRCAmut breast carcinomas are not identical with the previously identified claudin-low subtype of breast cancer.

Hatta M, Naganuma K, Kato K, Yamazaki J
3-Deazaneplanocin A suppresses aggressive phenotype-related gene expression in an oral squamous cell carcinoma cell line.
Biochem Biophys Res Commun. 2015 Dec 4-11; 468(1-2):269-73 [PubMed] Related Publications
In tumor tissues, alterations of gene expression caused by aberrant epigenetic modifications confer phenotypic diversity on malignant cells. Although 3-deazaneplanocin A (DZNep) has been shown to reactivate tumor suppressor genes in several cancer cells, it remains unclear whether DZNep attenuates the malignant phenotypes of oral squamous cell carcinoma (OSCC) cells. In this study, we investigated the effect of DZNep on the expression of genes related to aggressive phenotypes, such as epithelial-mesenchymal transition, in OSCC cells. We found that DZNep reduced the cellular levels of polycomb group proteins (EZH2, SUZ12, BMI1, and RING1A) and the associated trimethylation of Lys27 on histone H3 and monoubiquitination of Lys119 on histone H2A in the poorly differentiated OSCC cell line SAS. Immunocytochemical staining demonstrated that DZNep induced the reorganization of filamentous actin and the membrane localization of E-cadherin associated with cell-cell adhesions. We also found an inhibitory effect of DZNep on cell proliferation using a WST assay. Finally, quantitative RT-PCR analysis demonstrated that genes involved in the aggressive phenotypes (TWIST2, EGFR, ACTA2, TGFB1, WNT5B, and APLIN) were down-regulated, whereas epithelial phenotype genes (CDH1, CLDN4, IVL, and TGM1) were up-regulated in SAS cells treated with DZNep. Collectively, our findings suggest that DZNep reverses the aggressive characteristics of OSCC cells through the dynamic regulation of epithelial plasticity via the reprogramming of gene expression patterns.

DE Vicente JC, Fernández-Valle Á, Vivanco-Allende B, et al.
The prognostic role of claudins -1 and -4 in oral squamous cell carcinoma.
Anticancer Res. 2015; 35(5):2949-59 [PubMed] Related Publications
Claudin dysregulation has been described in various tumor types; however, its clinical relevance is poorly understood. We present a study in which we assessed the expression of claudin 1 (CLDN1) and CLDN4 in oral squamous cell carcinoma (OSCC), as well as their prognostic relevance. Immunohistochemical analysis of CLDN1 and CLDN4 expression was carried out on tissue sections from 65 OSCCs. The presence of CLDN1 in the invasive front of tumor islands was associated with neck node metastasis, and the expression of CLDN4 was associated with higher histological grade, and tumor recurrence. Membranous staining for CLDN4 in tumor cells, and weak intensity of CLDN4 immunoexpression were predictive for poorer survival. In a multivariate analysis for disease recurrence, CLDN1 immunostaining was statistically significant. Specifically, CDLN1 expression in the tumor invasive front was associated with tumor recurrence. Our results indicate that CLDN4 expression is correlated with poor prognosis, and CLDN1 expression may be an indicator of recurrence of OSCC.

Yang Y, Cheon S, Jung MK, et al.
Interleukin-18 enhances breast cancer cell migration via down-regulation of claudin-12 and induction of the p38 MAPK pathway.
Biochem Biophys Res Commun. 2015; 459(3):379-86 [PubMed] Related Publications
Interleukin-18 (IL-18) was recently reported to have a pro-tumor effect in various cancers. Increased IL-18 levels in the serum of cancer patients correlated with malignancy, and IL-18 acts a crucial factor for cell migration in gastric cancer and melanoma. Claudins, which are the most important tight junction proteins, are also linked with cancer progression and metastasis. However, the relationship between claudins and IL-18 is not well-understood. Here, we show that the migratory ability of MCF-7 cells was reduced when endogenous IL-18 expression was inhibited with IL-18 siRNA. Moreover, exogenous IL-18 enhanced breast cancer cell migration and suppressed the expression of the tight junction proteins claudin-1, claudin-3, claudin-4, and claudin-12 in MCF-7 cells. Knockdown of claudin-3, claudin-4, and claudin-12, but not claudin-1, increased breast cancer migration with maximal effects observed in claudin-12 siRNA-transfected cells. To investigate whether the mitogen-activated protein kinase (MAPK) signaling pathway is involved in IL-18-induced cell migration and claudin-12 expression, cells were pretreated with SB203580 (an inhibitor of p38 MAPK) or PD98059 (an inhibitor of ERK1/2) prior to the addition of IL-18. Although pretreatment of MCF-7 cells with SB203580 blocked both the enhanced cell migration and the decreased claudin-12 expression, PD98059 only blocked cell migration and did not affect claudin-12 expression. In addition, exogenous IL-18 induced rapid phosphorylation of p38 MAPK. These results suggest that IL-18 is an important factor inducing breast cancer cell migration through down-regulation of claudin-12 and activation of the p38 MAPK pathway.

Ma F, Ding X, Fan Y, et al.
A CLDN1-negative phenotype predicts poor prognosis in triple-negative breast cancer.
PLoS One. 2014; 9(11):e112765 [PubMed] Free Access to Full Article Related Publications
INTRODUCTION: Triple-negative breast cancer (TNBC) is a heterogeneous disease with no definitive prognostic markers. As a major component of tight junctions, claudins (CLDNs) presumably play an important role in carcinogenesis and progression of breast cancer. This study was aimed at determining the relationship between the expression of CLDNs and the clinical outcomes of TNBCs.
MATERIALS AND METHODS: The surgical specimens of primary breast tumors from a consecutive cohort of 173 TNBC patients were retrospectively collected. The membranous expression of CLDN1, CLDN2, CLDN4, and CLDN7 was measured by immunohistochemistry. Then, the associations between CLDN expression, clinicopathological features, and clinical outcomes were assessed.
RESULTS: Positive CLDN1, CLDN2, CLDN4, and CLDN7 membrane expression was detected in 44.5%, 54.9%, 76.9%, and 73.4% of the cohort specimens, respectively. A lack of CLDN1 expression was related to only lymph node metastasis (P = 0.014). The rate of CLDN4-positive tumors was significantly increased in tumors of a higher grade (P = 0.003). Importantly, negative CLDN1 expression was associated with worse relapse-free survival (RFS) in both lymph node positive (LN+) and negative (LN-) cases (both P<0.001). Similarly it was also associated with shorter overall survival (OS)(P = 0.003 in LN+ cases; P = 0.018 in LN- cases). In the LN+ subgroup, CLDN2-negative cases had a significantly higher risk of recurrence (P = 0.008). Multivariate analysis revealed that negative CLDN1 expression was an independent prognostic factor for high risk of both recurrence and death (HR 5.529, 95% CI 2.664-11.475, P<0.001; HR 3.459, 95% CI 1.555-7.696, P = 0.002). However, neither CLDN4 nor CLDN7 expression was associated with survival.
CONCLUSION: In TNBC, the CLDN1-negative phenotype predicts a high risk of recurrence and death. The absence of CLDN1 expression is strongly suggested to be an independent adverse prognostic factor in this heterogeneous subtype of breast cancer.

Al Sawah E, Marchion DC, Xiong Y, et al.
The Chinese herb polyphyllin D sensitizes ovarian cancer cells to cisplatin-induced growth arrest.
J Cancer Res Clin Oncol. 2015; 141(2):237-42 [PubMed] Related Publications
PURPOSE: We evaluated the effects of polyphyllin D (PD), a natural compound with anti-neoplastic activity and a major component of the Chinese herb Paris polyphylla, on ovarian cancer (OVCA) cell line proliferation and platinum sensitivity.
METHODS: A panel of 20 OVCA cell lines was subjected to PD treatment, MTS proliferation assays, and determination of IC50. Pre-treatment, baseline genome-wide Affymetrix expression analysis was performed on each cell line, and Pearson's correlation was performed to identify genes associated with OVCA PD sensitivity. Twelve cell lines were treated with PD with and without cisplatin, and the effects of PD on cisplatin IC50 were quantified. Genes associated with OVCA PD sensitivity were evaluated for associations with survival in a publically available clinico-genomic dataset of 218 patients with OVCA.
RESULTS: Our results showed that PD exhibited anti-proliferative effects against all OVCA cell lines tested, with IC50 values ranging from 0.2 to 1.4 μm. Furthermore, in all cell lines, PD treatment significantly decreased cisplatin IC50 (mean IC50 reduction of 2.1 µm; P < 0.02). Pearson's correlation test identified 25 probe sets, representing 18 unique genes to be associated with PD sensitivity (FDR = 0). We found that one of these genes was associated with overall survival in women with OVCA: CLDN4 (P = 0.014).
CONCLUSION: Our findings highlight the value of PD as a natural product with anti-cancer properties, which may also enhance the activity of existing therapeutic agents.

Liszka L
Ductal adenocarcinoma of the pancreas usually retained SMAD4 and p53 protein status as well as expression of epithelial-to-mesenchymal transition markers and cell cycle regulators at the stage of liver metastasis.
Pol J Pathol. 2014; 65(2):100-12 [PubMed] Related Publications
There are limited data on the biology of metastatic pancreatic ductal adenocarcinoma (PDAC). The aim of the present study was to compare the expression of immunohistochemical markers that may be involved in the development of metastatic disease in primary PDAC and in synchronous liver metastatic tissues. Thirty-two stains (corresponding to proteins encoded by 31 genes: SMAD4, TP53, ACTA2, CDH1, CDKN1A, CLDN1, CLDN4, CLDN7, CTNNB1, EGFR, ERBB2, FN1, KRT19, MAPK1/MAPK3, MAPK14, MKI67, MMP2, MMP9, MUC1 (3 antibodies), MUC5AC, MUC6, MTOR, MYC, NES, PTGS2, RPS6, RPS6KB1, TGFB1, TGFBR1, VIM) were evaluated using tissue microarray of 26 pairs of primary PDACs and their liver metastases. There were no significant differences in expression levels of examined proteins between primary and secondary lesions. In particular, metastatic PDAC retained the primary tumour's SMAD4 protein status in all and p53 protein status in all but one case. This surprising homogeneity also involved expression levels of markers of epithelial-to-mesenchymal transition as well as cell cycle regulators studied. In conclusion, the biological profiles of primary PDACs and their liver metastases seemed to be similar. Molecular alterations of PDAC related to a set of immunohistochemical markers examined in the present study were already present at the stage of localized disease.

Kraiklang R, Pairojkul C, Khuntikeo N, et al.
A novel predictive equation for potential diagnosis of cholangiocarcinoma.
PLoS One. 2014; 9(2):e89337 [PubMed] Free Access to Full Article Related Publications
Cholangiocarcinoma (CCA) is the second most common-primary liver cancer. The difficulties in diagnosis limit successful treatment of CCA. At present, histological investigation is the standard diagnosis for CCA. However, there are some poor-defined tumor tissues which cannot be definitively diagnosed by general histopathology. As molecular signatures can define molecular phenotypes related to diagnosis, prognosis, or treatment outcome, and CCA is the second most common cancer found after hepatocellularcarcinoma (HCC), the aim of this study was to develop a predictive model which differentiates CCA from HCC and normal liver tissues. An in-house PCR array containing 176 putative CCA marker genes was tested with the training set tissues of 20 CCA and 10 HCC cases. The molecular signature of CCA revealed the prominent expression of genes involved in cell adhesion and cell movement, whereas HCC showed elevated expression of genes related to cell proliferation/differentiation and metabolisms. A total of 69 genes differentially expressed in CCA and HCC were optimized statistically to formulate a diagnostic equation which distinguished CCA cases from HCC cases. Finally, a four-gene diagnostic equation (CLDN4, HOXB7, TMSB4 and TTR) was formulated and then successfully validated using real-time PCR in an independent testing set of 68 CCA samples and 77 non-CCA controls. Discrimination analysis showed that a combination of these genes could be used as a diagnostic marker for CCA with better diagnostic parameters with high sensitivity and specificity than using a single gene marker or the usual serum markers (CA19-9 and CEA). This new combination marker may help physicians to identify CCA in liver tissues when the histopathology is uncertain.

Abd El-Ghani SF, Kasem RF, Ghallab NA, Shaker OG
Detection of claudin-4 in salivary gland neoplasms (a study utilizing RT-PCR and immunohistochemistry).
J Oral Pathol Med. 2013; 42(10):781-7 [PubMed] Related Publications
BACKGROUND: Claudins are transmembrane proteins of tight junctions emerging as targets for diagnosis, prediction of prognosis, disease recurrence, and metastasis. Our goal was to evaluate expression of claudin-4 in the most common benign and malignant salivary gland neoplasms.
METHODS: Claudin-4 gene levels and protein expression were determined by real-time polymerase chain reaction (PCR), and immunohistochemistry in a total of 30 specimens containing normal salivary tissue, pleomorphic adenoma, Warthin's tumor, mucoepidermoid carcinoma, and adenoid cystic carcinoma.
RESULTS: We identified down-regulation of claudin-4 gene levels and protein expression from normal control to benign salivary gland neoplasms and reached their lowest values in the malignant salivary gland neoplasms.
CONCLUSIONS: Low claudin-4 expression could be considered as a sign of increasing cellular disorientation and invasion of salivary gland tumors.

Siar CH, Abbas SA
Claudin expression and tight junction protein localization in the lining epithelium of the keratocystic odontogenic tumors, dentigerous cysts, and radicular cysts.
Oral Surg Oral Med Oral Pathol Oral Radiol. 2013; 115(5):652-9 [PubMed] Related Publications
OBJECTIVE: The aim of this study was to evaluate the expression and localization of tight junction proteins (TJPs) or claudins in the keratocystic odontogenic tumor (KCOT) and to correlate with its biological behavior.
STUDY DESIGN: Five claudins (-1, -3, -4, -5, and 7) were examined immunohistochemically in 25 KCOTs and compared with 10 dentigerous cysts (DCs) and 10 radicular cysts (RCs).
RESULTS: Marked claudin-3 loss of expression in KCOT basal layer (n=24/25; 96%) compared with DCs (n=1/10; 10%) and RCs (n=5/10; 50%) (P<.05) suggests that claudin-3 downregulation may indicate altered or loss of basal cell polarity and impaired barrier function of KCOT lining epithelium and this might contribute indirectly to its biological behavior. In contrast, claudins-1, -4, -5, and -7 distribution patterns were less distinctive in all three entities, suggesting that these TJP molecules probably play limited roles in influencing their different growth potentials.
CONCLUSION: Present findings suggest that differential claudin expressions in the lining epithelium of KCOTs, DCs, and RCs probably reflect their neoplastic or nonneoplastic nature.

Hashimi SM, Yu S, Alqurashi N, et al.
Immunotoxin-mediated targeting of claudin-4 inhibits the proliferation of cancer cells.
Int J Oncol. 2013; 42(6):1911-8 [PubMed] Related Publications
Immunotoxins are engineered chimeric proteins that consist of a fragment of a toxin fused to a modified antibody or growth factor capable of targeting specific cells. Furthermore, these proteins can be targeted to receptors that are commonly overexpressed on cancer cells. The majority of immunotoxins function by binding to cells, translocating into the cytosol and inhibiting protein synthesis. In this study, the expression of claudin‑4 (CLDN4) in various cancer cells was analysed as a potential target for immunotoxins. To target CLDN4-expressing cancer cells, the c-terminal CLDN4‑binding domain of Clostridium perfringens enterotoxin (CPE) was fused to the Pseudomonas aeruginosa exotoxin A (ETA) domain to create an immunotoxin (CPE‑ETA'). Subsequently, the capacity of such an immunotoxin in suppressing the proliferation of CLDN4-positive cancer cells was investigated. We report that head and neck squamous carcinoma cells (HN5) have an elevated CLDN4 expression compared to the other cell lines tested. Our findings further demonstrate that CPE‑ETA' is highly potent against MCF-7 breast [50% inhibitory concentration (IC50) 9.8 ng/ml] and HN5 head/neck (IC50 8.8 ng/ml) cancer cell lines, while it has no cytotoxic effects on HeLa cells (CLDN4‑negative). The immunotoxin was subsequently expressed in the tumour colonising oncolytic strain, Clostridium ghonii. Most importantly, the strictly anaerobic Clostridium ghonii was able to overexpress and secrete a functional CPE‑ETA' fusion protein. Our findings open the possibility of the targeted delivery of the immunotoxin locally to tumour sites at a high concentration using strictly anaerobic Clostridium ghonii for the treatment of CLDN4-positive cancer cells.

Nordfors K, Haapasalo J, Sallinen PK, et al.
Expression of claudins relates to tumour aggressivity, location and recurrence in ependymomas.
Histol Histopathol. 2013; 28(9):1137-46 [PubMed] Related Publications
The aim of our study was to assess the nature and importance of claudin expression in grade I-III ependymomas. The expression of claudins 2-5, 7, 10, TWIST, and ZEB1 were investigated in a series of 61 ependymomas using immunohistochemistry. All the claudins were expressed in ependymomas, except for CLDN4. CLDN5 positive tumours were associated with higher grade (p=0.049), whereas CLDN10 was lower in higher grade tumours (p=0.039). CLDN5 and CLDN3 were overexpressed in ependymomas of cerebral location (p=0.036, p=0.007, respectively). CLDN5 positive tumours showed more nuclear atypia, endothelial proliferation, mitosis, and hypercellularity (p=0.007, p=0.018, p=0.041, p=0.010, respectively). CLDN5 positivity correlated to higher proliferation (p=0.015). CLDN7 was more often positive in primary tumours (p=0.041). Positive ZEB1 expression was associated with CLDN2 negativity (p=0.031). TWIST-negative tumours were more often also CLDN5 and 10 negative (p=0.013, p=0.017, respectively). CLDN5 was related to more aggressive tumours compared to CLDN2 and 10, which tended to display a better degree of differentiation and a better prognosis. CLDN2 and CLDN5 were expressed commonly in ependymomas, while the parental ependymal cells in the central nervous system were usually negative. Evidently, claudins influence growth and differentiation in ependymomas.

Shang X, Lin X, Alvarez E, et al.
Tight junction proteins claudin-3 and claudin-4 control tumor growth and metastases.
Neoplasia. 2012; 14(10):974-85 [PubMed] Free Access to Full Article Related Publications
The extent of tight junction (TJ) formation is one of many factors that regulate motility, invasion, and metastasis. Claudins are required for the formation and maintenance of TJs. Claudin-3 (CLDN3) and claudin-4 (CLDN4) are highly expressed in the majority of ovarian cancers. We report here that CLDN3 and CLDN4 each serve to constrain the growth of human 2008 cancer xenografts and limit metastatic potential. Knockdown of CLDN3 increased in vivo growth rate by 2.3-fold and knockdown of CLDN4 by 3.7-fold in the absence of significant change in in vitro growth rate. Both types of tumors exhibited increase in birth rate as measured by Ki67 staining and decrease in death rate as reflected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Knockdown of either claudin did not alter expression of other TJ protein but did reduce TJ formation as measured by transepithelial resistance and paracellular flux of dextran, enhance migration and invasion in in vitro assays, and increase lung colonization following intravenous injection. Knockdown of CLDN3 and CLDN4 increased total lung metastatic burden by 1.7-fold and 2.4-fold, respectively. Loss of either CLDN3 or CLDN4 resulted in down-regulation of E-cadherin mRNA and protein, increased inhibitory phosphorylation of glycogen synthase kinase-3β (GSK-3β), and activation of β-catenin pathway signaling as evidenced by increases in nuclear β-catenin, the dephosphorylated form of the protein, and transcriptional activity of β-catenin/T-cell factor (TCF). We conclude that both CLDN3 and CLDN4 mediate interactions with other cells in vivo that restrain growth and metastatic potential by sustaining expression of E-cadherin and limiting β-catenin signaling.

Xiao-Yu P, Yan J, Cui-Ping F, et al.
Altered claudin-4 expression in progesterone-treated endometrial adenocarcinoma cell line Ishikawa.
Int J Gynecol Cancer. 2012; 22(9):1585-90 [PubMed] Related Publications
OBJECTIVE: To detect the expression change of claudin-4 in Ishikawa endometrial adenocarcinoma cell line in response to progesterone. To determine whether claudin-4 is involved in the anticancer effect of progesterone.
METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the 50% inhibitory concentration (IC50) of megestrol acetate (MA) in treating Ishikawa cells. After the Ishikawa cells were treated with MA at IC50, cell apoptosis was examined by flow cytometry and transmission electron microscopy. The messenger RNA and protein expression levels of claudin-4 were further quantified by real-time polymerase chain reaction and Western blot. The localization of claudin-4 was examined by immunofluorescent staining.
RESULTS: The IC50 of MA on Ishikawa cells was 15 mg/L incubated for 72 hours. Apoptosis percentage was elevated from 0.07% ± 0.02% to 3.93% ± 0.81% after MA treatment. The expression of claudin-4 at both protein and messenger RNA levels was significantly decreased after the treatment of MA (P < 0.05). The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus.
CONCLUSION: Megestrol acetate can inhibit the growth of Ishikawa cells. It may work through decreasing claudin-4 expression and cell apoptosis. The localization change of claudin-4 may also be involved in the anticancer effect of progesterone.

Shang X, Lin X, Manorek G, Howell SB
Claudin-3 and claudin-4 regulate sensitivity to cisplatin by controlling expression of the copper and cisplatin influx transporter CTR1.
Mol Pharmacol. 2013; 83(1):85-94 [PubMed] Related Publications
Claudin-3 (CLDN3) and claudin-4 (CLDN4) are the major structural molecules that form tight junctions (TJs) between epithelial cells. We found that knockdown of the expression of either CLDN3 or CLDN4 produced marked changes in the phenotype of ovarian cancer cells, including an increase in resistance to cisplatin (cDDP). The effect of CLND3 and CLDN4 on cDDP cytotoxicity, cDDP cellular accumulation, and DNA adduct formation was compared in the CLDN3- and CLDN4-expressing parental human ovarian carcinoma 2008 cells and CLDN3 and CLDN4 knockdown sublines (CLDN3KD and CLDN4KD, respectively). Knockdown of CLDN3 or CLDN4 rendered human ovarian carcinoma 2008 cells resistant to cDDP in both in vitro culture and in vivo xenograft model. The net accumulation of platinum (Pt) and the Pt-DNA adduct levels were reduced in CLDN3KD and CLDN4KD cells. The endogenous mRNA levels of copper influx transporter CTR1 were found to be significantly reduced in the knockdown cells, and exogenous expression of CTR1 restored their sensitivity to cDDP. Reexpression of an shRNAi-resistant CLDN3 or CLDN4 up-regulated CTR1 levels, reversed the cDDP resistance, and enhanced TJ formation in the knockdown cells. Baseline copper (Cu) level, Cu uptake, and Cu cytotoxicity were also reduced in CLDN3KD and CLDN4KD cells. Cu-dependent tyrosinase activity was also markedly reduced in both types of CLDN knockdown cells when incubated with the substrate l-DOPA. These results indicate that CLDN3 and CLDN4 affect sensitivity of the ovarian cancer cells to the cytotoxic effect of cDDP by regulating expression of the Cu transporter CTR1.

Ricardo S, Gerhard R, Cameselle-Teijeiro JF, et al.
Claudin expression in breast cancer: high or low, what to expect?
Histol Histopathol. 2012; 27(10):1283-95 [PubMed] Related Publications
The evaluation of claudins (CLDNs) expression pattern in tumours can be important to understand breast carcinogenesis. The study of CLDNs became more appealing since it was found that CLDN3 and CLDN4 are putative therapeutic targets for Clostridium perfrigens enterotoxin (CPE), as well as for monoclonal antibody-based therapy. Moreover, the recently characterized CLDN-low molecular subgroup of breast tumours increased the interest in these molecules. Based on these facts, our aim was to explore the pattern of expression of CLDNs among a large series of invasive breast carcinomas. We also analysed the correlation between the combinatorial expression of CLDN3/CLDN4 and classical prognostic factors and biological markers. In addition, we also compared the characteristics of tumours with low expression of CLDN3, CLDN4 and CLDN7, assessed by immunohistochemistry (IHC), and the ones from CLDN-low subgroup of tumours previously defined by genomic assays. The combinatorial analysis of the expression of CLDN3/CLDN4 showed a significant association between high CLDN3/CLDN4 levels and triple-negative tumours, as well as with worse patient outcome. This combined analysis may provide useful information for breast carcinomas, since these two CLDN members are putative therapeutic targets. Comparing tumours with low expression of CLDN3, CLDN4 and CLDN7 with tumours previously referred to as CLDN-low by genomic assays, we demonstrated that the single IHC evaluation of these three specific CLDNs is insufficient to identify the CLDN-low molecular subtype of breast tumours. The analysis of several other molecular markers, such as EMT (epithelial-to-mesenchymal transition) and CSC (cancer stem cell) markers should probably be added to improve the identification of this subgroup of tumours by IHC, which probably are enriched in carcinomas with metaplastic differentiation.

No JH, Kim K, Park KH, Kim YB
Cell-free DNA level as a prognostic biomarker for epithelial ovarian cancer.
Anticancer Res. 2012; 32(8):3467-71 [PubMed] Related Publications
BACKGROUND: The use of cell-free DNA (cfDNA) as a non-invasive biomarker has been evaluated in many types of cancer. This study investigated the prognostic significance of cfDNA level for ovarian cancer.
MATERIALS AND METHODS: Preoperative sera of 36 patients with ovarian cancer and of 16 with benign tumors were analyzed using commercially available copy number assay kits to measure the cfDNA level of genes including beta-2-microglobulin (B2M), member RAS oncogene family (RAB25), claudin 4 (CLDN4) and ATP-binding cassette subfamily F member 2 (ABCF2). Cox regression analysis was used to calculate hazard ratios (HR) and 95% confidence intervals (CI).
RESULTS: cfDNA level of these genes had no association with other prognostic factors of ovarian cancer. In particular, in patients with advanced stage disease, a low RAB25 level was an independent prognostic factor for disease-free survival (HR=18.2, 95% CI=2.0-170.0) and overall survival (HR=33.6, 95% CI=1.8-634.8).
CONCLUSION: Our findings suggest that the preoperative serum cfDNA level of RAB25 could be a useful biomarker predicting survival outcomes in patients with advanced ovarian cancer.

Kojima T, Kyuno D, Sawada N
Targeting claudin-4 in human pancreatic cancer.
Expert Opin Ther Targets. 2012; 16(9):881-7 [PubMed] Related Publications
INTRODUCTION: Pancreatic cancer is one of the most malignant human diseases and there is an urgent need to develop novel diagnostic and therapeutic strategies. Claudin-4, overexpressed in pancreatic cancer and its precursor lesions, is a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE and monoclonal antibodies against claudin-4 are useful as novel therapeutic tools for pancreatic cancer.
AREAS COVERED: This review describes and discusses the studies targeting claudin-4 in normal human pancreatic duct epithelial (HPDE) cells and cancer cells.
EXPERT OPINION: Claudin-4 is in part regulated via a PKCα signal transduction pathway in pancreatic cancer cell lines. PKCα inhibitors may represent potential therapeutic agents against human pancreatic cancer cells by the use of CPE cytotoxicity via claudin-4. The COOH-terminal half fragment of CPE (C-CPE) enhances the effectiveness of clinically relevant chemotherapies and can be used as a carrier for drugs and other bacterial toxins to claudin-4-positive cancer cells. hTERT-HPDE cells, in which the human telomerase reverse transcriptase (hTERT) gene is introduced into normal HPDE cells, may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer.

Lee KW, Lee NK, Kim JH, et al.
Twist1 causes the transcriptional repression of claudin-4 with prognostic significance in esophageal cancer.
Biochem Biophys Res Commun. 2012; 423(3):454-60 [PubMed] Related Publications
Twist1 is a transcription factor that is involved in epithelial-mesenchymal transition by suppressing intercellular adhesion. In this study, we aimed to determine the role of Twist1 in the regulation of claudin-4 expression and investigate its specific mechanisms and clinical implications using human esophageal carcinoma cell lines and tissues. As a result, up-regulation of Twist1 decreased both gene and protein expression levels of endogenous claudin-4 and the suppression was mediated by direct binding of Twist1 to the canonical E-box in the promoter region of claudin-4. In addition, there was a significant inverse correlation of claudin-4 with Twist1 in esophageal cancer tissues. High Twist1 and low claudin-4 expression was associated with the poorest prognosis and was more highly correlated with adverse outcome than any other subgroup with statistical significance (p=0.001). Our results indicate that Twist1 induces the repression of claudin-4 expression during the epithelial-mesenchymal transition in esophageal carcinoma.

Park HS, Kim GY, Choi IW, et al.
Inhibition of matrix metalloproteinase activities and tightening of tight junctions by diallyl disulfide in AGS human gastric carcinoma cells.
J Food Sci. 2011; 76(4):T105-11 [PubMed] Related Publications
The effect of diallyl disulfide (DADS), a major component of an oil-soluble allyl sulfide garlic (Allium sativum) derivative, on the correlation between anti-invasive activity and tightening of tight junctions (TJs) was investigated in human gastric adenocarcinoma AGS cells. Our data indicated that the inhibitory effects of DADS on cell motility and invasiveness were found to be associated with increased tightness of the TJs, which was demonstrated by an increase in transepithelial electrical resistance. Activities of matrix metalloprotease (MMP)-2 and -9 in AGS cells were dose-dependently inhibited by treatment with DADS, and this was also correlated with a decrease in expression of their mRNA and proteins; however, tissue inhibitor of metalloproteinase (TIMP)-1 and -2 mRNA levels and proteins were increased. Additionally, immunoblotting results indicated that DADS repressed the levels of claudin proteins (claudin-2, -3, and -4), major components of TJs that play key roles in control and selectivity of paracellular transport. Although further studies are needed, these results suggest that DADS treatment may inhibit tumor cell motility and invasion and, therefore, act as a dietary source to decrease the risk of cancer metastasis.

Neesse A, Griesmann H, Gress TM, Michl P
Claudin-4 as therapeutic target in cancer.
Arch Biochem Biophys. 2012; 524(1):64-70 [PubMed] Related Publications
BACKGROUND: Intercellular junctional complexes such as adherens junctions and tight junctions are critical regulators of cellular polarity, paracellular permeability and metabolic and structural integrity of cellular networks. Abundant expression analysis data have yielded insights into the complex pattern of differentially expressed cell-adhesion proteins in epithelial cancers and provide a useful platform for functional, preclinical and clinical evaluation of novel targets.
SCOPE OF REVIEW: This review will focus on the role of claudin-4, an integral constituent of tight junctions, in the pathophysiology of epithelial malignancies with particular focus pancreatic cancer, and its potential applicability for prognostic, diagnostic and therapeutic approaches.
MAJOR CONCLUSIONS: Claudin-4 expression is widely dysregulated in epithelial malignancies and in a number of premalignant precursor lesions. Although the functional implications are only starting to unravel, claudin-4 seems to play an important role in tumour cell invasion and metastasis, and its dual role as receptor of Clostridium perfringens enterotoxin (CPE) opens exciting avenues for molecular targeted approaches.
GENERAL SIGNIFICANCE: Claudin-4 constitutes a promising molecular marker for prognosis, diagnosis and therapy of epithelial malignancies.

Riski M, Santala M, Soini Y, Talvensaari-Mattila A
Claudins 1, 3M, 3S, 4, 5 and 7 in vulvar neoplasms compared with vulvar squamous cell carcinoma.
Tumour Biol. 2012; 33(2):537-42 [PubMed] Related Publications
The purpose of this study was to evaluate the expression of claudins 1, 3M (membrane-bound), 3S (cytoplasmic), 4, 5 and 7 in vulvar epithelial neoplasia (VIN I-III) and to compare those with invasive vulvar squamous cell carcinoma. Paraffin tissue sections from 73 vulvar neoplasms (12 VIN I, 12 VIN II-III and 49 vulvar carcinomas) were studied by immunohistochemistry for the expression of claudins 1, 3M, 3S, 4, 5 and 7. Claudin 1 stained strongly in all groups, whereas claudin 3M, 3S and 4 immunostaining were moderate in all groups. Claudin 7 stained strongly in all groups. Claudin 3M expression was higher in VIN I compared to carcinoma, while no difference was found between VIN I and VIN II-III or between VIN II-III and carcinoma. Claudin 1 and claudin 3S expressions also showed the same decreasing tendency from VIN towards vulvar carcinoma. Claudin 5 showed only weak staining in VIN I and VIN II-III, and positive expression was also low in the carcinoma group. Expressions of claudins 1, 3M, 3S, 4 and 7 were found in VIN and vulvar carcinoma. Changes in claudin 1 and claudin 3 expression during progression from VIN to vulvar carcinoma suggests a connection with claudin expression and differentiation of vulvar squamous cells. Claudin 5 does not seem to be important in VIN or vulvar carcinoma.

Walther W, Petkov S, Kuvardina ON, et al.
Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors.
Gene Ther. 2012; 19(5):494-503 [PubMed] Related Publications
Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

Lang JY, Hsu JL, Meric-Bernstam F, et al.
BikDD eliminates breast cancer initiating cells and synergizes with lapatinib for breast cancer treatment.
Cancer Cell. 2011; 20(3):341-56 [PubMed] Free Access to Full Article Related Publications
Breast cancer initiating cells (BCICs), which can fully recapitulate the tumor origin and are often resistant to chemo- and radiotherapy, are currently considered as a major obstacle for breast cancer treatment. Here, we show that BIKDD, a constitutively active mutant form of proapoptotic gene, BIK, effectively induces apoptosis of breast cancer cells and synergizes with lapatinib. Most importantly, BikDD significantly reduces BCICs through co-antagonism of its binding partners Bcl-2, Bcl-xL, and Mcl-1, suggesting a potential therapeutic strategy targeting BCICs. Furthermore, we developed a cancer-specific targeting approach for breast cancer that selectively expresses BikDD in breast cancer cells including BCICs, and demonstrated its potent antitumor activity and synergism with lapatinib in vitro and in vivo.

Kwon MJ, Kim SH, Jeong HM, et al.
Claudin-4 overexpression is associated with epigenetic derepression in gastric carcinoma.
Lab Invest. 2011; 91(11):1652-67 [PubMed] Related Publications
The tight junction (TJ) protein claudin-4 is aberrantly upregulated in gastric cancer, but its clinical significance and the molecular mechanisms underlying claudin-4 overexpression in gastric cancer remain unclear. Here, we investigated its roles and epigenetic mechanisms regulating CLDN4 expression in gastric cancer. We show that increased membranous expression of claudin-4 in gastric carcinoma is associated with better patient prognosis, whereas cytoplasmic claudin-4 expression did not show a significant association with prognosis. Consistent with the correlation of increased membranous claudin-4 with favorable clinicopathological factors, claudin-4 overexpression inhibited the migration and invasion of gastric cancer cells; in contrast, it did not affect cell growth. Claudin-4 expression also increased the barrier function of TJs. Claudin-4 upregulation was strongly correlated with DNA hypomethylation in both gastric tissues and gastric cancer cells. Moreover, CLDN4 expression was repressed in normal gastric tissues in association with bivalent histone modifications, and loss of repressive histone methylations and gain of active histone modifications were associated with CLDN4 overexpression in gastric cancer cells. Interestingly, CLDN4 repression could be markedly derepressed by combined treatments that simultaneously target both histone modifications and DNA demethylation in CLDN4-hypermethylated cells, whereas concomitant changes in histone methylations and acetylations are required for CLDN4 induction in CLDN4-repressed cells with low DNA methylation. Taken together, this study reveals that membranous claudin-4 expression is associated with gastric cancer progression and that it is an independent positive prognosis marker in gastric carcinoma. Furthermore, our findings suggest that epigenetic derepression may be a possible mechanism underlying CLDN4 overexpression in gastric cancer and that claudin-4 may have potential as a promising target for the treatment of gastric cancer.

Meng J, Mostaghel EA, Vakar-Lopez F, et al.
Testosterone regulates tight junction proteins and influences prostatic autoimmune responses.
Horm Cancer. 2011; 2(3):145-56 [PubMed] Free Access to Full Article Related Publications
Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry, and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an autoimmune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology.

Hess J, Thomas G, Braselmann H, et al.
Gain of chromosome band 7q11 in papillary thyroid carcinomas of young patients is associated with exposure to low-dose irradiation.
Proc Natl Acad Sci U S A. 2011; 108(23):9595-600 [PubMed] Free Access to Full Article Related Publications
The main consequence of the Chernobyl accident has been an increase in papillary thyroid carcinomas (PTCs) in those exposed to radioactive fallout as young children. Our aim was to identify genomic alterations that are associated with exposure to radiation. We used array comparative genomic hybridization to analyze a main (n = 52) and a validation cohort (n = 28) of PTC from patients aged <25 y at operation and matched for age at diagnosis and residency. Both cohorts consisted of patients exposed and not exposed to radioiodine fallout. The study showed association of a gain on chromosome 7 (7q11.22-11.23) with exposure (false discovery rate = 0.035). Thirty-nine percent of the exposed group showed the alteration; however, it was not found in a single case from the unexposed group. This was confirmed in the validation set. Because only a subgroup of cases in the exposed groups showed gain of 7q11.22-11.23, it is likely that different molecular subgroups and routes of radiation-induced carcinogenesis exist. The candidate gene CLIP2 was specifically overexpressed in the exposed cases. In addition, the expression of the genes PMS2L11, PMS2L3, and STAG3L3 correlated with gain of 7q11.22-11.23. An enrichment of Gene Ontology terms "DNA repair" (PMS2L3, PMS2L5), "response to DNA damage stimulus" (BAZ1B, PMS2L3, PMS2L5, RFC2), and "cell-cell adhesion" (CLDN3, CLDN4) was found. This study, using matched exposed and unexposed cohorts, provides insights into the radiation-related carcinogenesis of young-onset PTC and, with the exposure-specific gain of 7q11 and overexpression of the CLIP2 gene, radiation-specific molecular markers.

Yamaguchi H, Kojima T, Ito T, et al.
Effects of Clostridium perfringens enterotoxin via claudin-4 on normal human pancreatic duct epithelial cells and cancer cells.
Cell Mol Biol Lett. 2011; 16(3):385-97 [PubMed] Related Publications
The tight junction protein claudin-4 is frequently overexpressed in pancreatic cancer, and is also a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE are thought to be useful as a novel therapeutic tool for pancreatic cancer. However, the responses to CPE via claudin-4 remain unknown in normal human pancreatic duct epithelial (HPDE) cells. We introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture as a model of normal HPDE cells in vitro. hTERT-HPDE cells treated with or without 10% FBS and pancreatic cancer cell lines PANC-1, BXPC3, HPAF-II and HPAC were treated with CPE. In Western blotting, the expression of claudin-4 protein in hTERT-HPDE cells treated with 10% FBS was as high as it was in all of the pancreatic cancer cell lines. In hTERT-HPDE cells with or without 10% FBS, cytotoxicity was not observed at any concentration of CPE, whereas in all pancreatic cancer cell lines, CPE had a dose-dependent cytotoxic effect. In hTERT-HPDE cells with 10% FBS, claudin-4 was localized in the apical-most regions, where there are tight junction areas, in which in all pancreatic cancer cell lines claudin-4 was found not only in the apical-most regions but also at basolateral membranes. In hTERT-HPDE cells with 10% FBS after treatment with CPE, downregulation of barrier function and claudin-4 expression at the membranes was observed. In HPAC cells, the sensitivity to CPE was significantly decreased by knockdown of claudin-4 expression using siRNA compared to the control. These findings suggest that, in normal HPDE cells, the lack of toxicity of CPE was probably due to the localization of claudin-4, which is different from that of pancreatic cancer cells. hTERT-HPDE cells in this culture system may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer.

Georges R, Bergmann F, Hamdi H, et al.
Sequential biphasic changes in claudin1 and claudin4 expression are correlated to colorectal cancer progression and liver metastasis.
J Cell Mol Med. 2012; 16(2):260-72 [PubMed] Free Access to Full Article Related Publications
Terminal progression of colorectal cancer (CRC) culminates in liver metastasis. To identify genes that are involved in the metastatic phenotype, cDNA microarrays were used to analyse mRNA expression profiles of colorectal carcinoma (CC)531 rat colon adenocarcinoma cells for changes related to their homing into the liver. Briefly, CC531 cells were intraportally implanted into the liver of Wag-Rij rats and re-isolated after 3, 6, 9, 14 and 21 days. Compared to control CC531 cells, claudin1 and claudin4 were among the ≥8-fold initially down-regulated genes. The co-culture of tumour cells with isolated rat hepatocytes and Kupffer cells did not induce down-regulation of either claudin1 or 4. When the environment effective on circulating tumour cells was simulated by cell culture conditions favouring their adhesion, only claudin4 showed augmented expression. Knockdown of claudin1 and claudin4 mediated by small interfering RNA caused significantly increased migration and decreased clonogenic growth of tumour cells (P < 0.05), but had no effect on their proliferation. These experimental results were paralleled by increased claudin1 and claudin4 expression in human CRC samples in Union for International Cancer Control (UICC) stages I-III, as evaluated by real-time PCR. Increased claudin4 levels were correlated with significantly reduced overall survival (log-rank test, P= 0.018). Further, significantly (P < 0.05) reduced expression of claudin1 and claudin4 was observed in stage IV and liver metastasis by immunohistochemistry. In conclusion, sequential biphasic changes in claudin1 and claudin4 expression occur during the homing of rat CC531 CRC cells to the liver. This modulation is reflected by significant changes in claudin expression in human primary and metastatic CRC.

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