Research IndicatorsGraph generated 16 March 2017 using data from PubMed using criteria.
Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic. Tag cloud generated 16 March, 2017 using data from PubMed, MeSH and CancerIndex
Specific Cancers (4)
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).
OMIM, Johns Hopkin University
Referenced article focusing on the relationship between phenotype and genotype.
International Cancer Genome Consortium.
Summary of gene and mutations by cancer type from ICGC
Cancer Genome Anatomy Project, NCI
COSMIC, Sanger Institute
Somatic mutation information and related details
GEO Profiles, NCBI
Search the gene expression profiles from curated DataSets in the Gene Expression Omnibus (GEO) repository.
Latest Publications: OCLN (cancer-related)
Martin TA, Jordan N, Davies EL, Jiang WGMetastasis to Bone in Human Cancer Is Associated with Loss of Occludin Expression.
Anticancer Res. 2016; 36(3):1287-93 [PubMed
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BACKGROUND: Occludin is an integral membrane protein localised at tight junctions (TJ). There is no consensus regarding its paramount role in TJ. In previous work we showed that occludin is aberrantly expressed in both human breast tissues and cancer cell lines. This study demonstrates a link to bone metastasis in human cancer.
MATERIALS AND METHODS: Primary breast tumours (n=124) and matched normal tissues (n=30) were processed for quantitative polymerase chain reaction (QPCR) analysis. A hammerhead ribozyme was constructed to create occludin knockdown cell lines, MDA-MB-231(ΔOcc) and PC-3(ΔOcc). The effect of human bone matrix extract (BME) was investigated using cell growth and electric cell impedance sensing (ECIS) technology to measure changes in attachment/migration. Trans-epithelial resistance (TER) was measured for assessing changes in TJ function. Cells used were MDA-MB-231, PC-3, CORL-23, SKMES-1 and A-549 human cancer cell lines.
RESULTS: Tumours from patients with bone metastasis had significantly lower occludin expression compared to those remaining alive/well (60.7±21 vs. 331±98, respectively, p=0.008). This was striking in ductal carcinomas, where patients alive/well had significantly higher occludin expression compared to those with bone metastasis (391±12.5 vs. 67.9±28, respectively, p=0.0014). ECIS demonstrated that MDA-MB-231(ΔOcc) showed reduced attachment to 5% BME compared to controls (84% vs. 100%) that prevented closure of wounded cell layers. Moreover, these cells had reduced growth on BME. In addition, BME changed the TER of a number of human cell lines and was able to effect changes in the growth of MDA-MB-241 and PC-3 cells, with greater effect on knockdown cells.
CONCLUSION: This is the first study to demonstrate that occludin expression has a clear relationship with bone metastasis in human cancer. The discrepancy between this and the in vitro data indicating a reduction in migration/growth rate of occludin knockdown indicates that loss of occludin leads to complex changes in human cancer cell phenotype.
Zhang X, Yu X, Jiang G, et al.Cytosolic TMEM88 promotes invasion and metastasis in lung cancer cells by binding DVLS.
Cancer Res. 2015; 75(21):4527-37 [PubMed
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Transmembrane protein 88 (TMEM88) is a transmembrane protein that plays a crucial role in regulating human stem cell differentiation and embryonic development. However, its expression and clinicopathologic significance in human neoplasms is unclear. In this study, the expression and subcellular localizations of TMEM88 were assessed in 214 cases of non-small cell lung cancer (NSCLC). Notably, TMEM88 was highly expressed in the cytosol of ∼60% NSCLC specimens examined. Higher expression of cytosolic TMEM88 in NSCLC correlated significantly with poor differentiation, high TNM stage, lymph node metastasis, and inferior survival. In NSCLC cells displaying membrane-localized TMEM88, we observed an inhibition of canonical Wnt signaling due to interactions of TMEM88 with the Wnt pathway factor Dishevelled (DVLS). In contrast, NSCLC cells with cytosol-localized TMEM88 lacked effects on Wnt signaling. Cytosolic interactions of TMEM88 and DVLS increased the expression of phosphorylated, active forms of p38, GSK3β (Thr390), and Snail, thereby reducing the expression of the tight junction-associated proteins ZO-1 and occludin, effects associated with enhanced invasive and metastatic cell characters. Importantly, attenuating the expression of cytosolic TMEM88 reduced metastatic prowess in xenograft models. Overall, our findings show how mislocalization of TMEM88 to the cytosol in NSCLC cells ablates its Wnt pathway regulatory properties, thereby promoting invasion and metastasis by activating the p38-GSK3β-Snail signaling pathway.
Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.
Blood-tumor barrier (BTB) limits the delivery of chemotherapeutic agent to brain tumor tissues. Long non-coding RNAs (lncRNAs) have been shown to play critical regulatory roles in various biologic processes of tumors. However, the role of lncRNAs in BTB permeability is unclear. LncRNA TUG1 (taurine upregulated gene 1) was highly expressed in glioma vascular endothelial cells from glioma tissues. It also upregulated in glioma co-cultured endothelial cells (GEC) from BTB model in vitro. Knockdown of TUG1 increased BTB permeability, and meanwhile down-regulated the expression of the tight junction proteins ZO-1, occludin, and claudin-5. Both bioinformatics and luciferase reporter assays demonstrated that TUG1 influenced BTB permeability via binding to miR-144. Furthermore, Knockdown of TUG1 also down-regulated Heat shock transcription factor 2 (HSF2), a transcription factor of the heat shock transcription factor family, which was defined as a direct and functional downstream target of miR-144. HSF2 up-regulated the promoter activities and interacted with the promoters of ZO-1, occludin, and claudin-5 in GECs. In conclusion, our results indicate that knockdown of TUG1 increased BTB permeability via binding to miR-144 and then reducing EC tight junction protein expression by targeting HSF2. Thus, TUG1 may represent a useful future therapeutic target for enhancing BTB permeability.
Zhao L, Wang P, Liu Y, et al.miR-34c regulates the permeability of blood-tumor barrier via MAZ-mediated expression changes of ZO-1, occludin, and claudin-5.
J Cell Physiol. 2015; 230(3):716-31 [PubMed
] Related Publications
The purposes of this study were to investigate the potential roles of miR-34c in regulating blood-tumor barrier (BTB) functions and its possible molecular mechanisms. The over-expression of miR-34c significantly impaired the integrity and increased the permeability of BTB, which were detected in an in vitro BTB model by transendothelial electric resistance and horseradish peroxidase flux assays, respectively. Meanwhile, real-time quantitative PCR (qRT-PCR), Western blot and immunofluorescence assays successively demonstrated downregulation of ZO-1, occludin, and claudin-5 and miR-34c silencing uncovered the opposite results. Dual-luciferase reporter assays results revealed myc-associated zinc-finger protein (MAZ) is a target gene of miR-34c. Besides, mRNA and protein expressions of MAZ were reversely regulated by miR-34c. The down-expression of MAZ significantly impaired the integrity and increased the permeability of BTB as well as downregulated the expressions of ZO-1, occludin, and claudin-5. And chromatin immunoprecipitation verified that MAZ interacted with "GGGCGGG," "CCCTCCC," and "GGGAGGG" DNA sequence of ZO-1, occludin, and claudin-5 promoter, respectively. The over-expression or silencing of either miR-34c or MAZ was performed simultaneously to further explore their functional relations, and results elucidated that miR-34c and MAZ displayed reverse regulatory effects on the integrity and permeability of BTB as well as the expressions of ZO-1, occludin, and claudin-5. In conclusion, our present study indicated that miR-34c regulated the permeability of BTB via MAZ-mediated expression changes of ZO-1, occludin, and claudin-5.
Karagiannis GS, Schaeffer DF, Cho CK, et al.Collective migration of cancer-associated fibroblasts is enhanced by overexpression of tight junction-associated proteins claudin-11 and occludin.
Mol Oncol. 2014; 8(2):178-95 [PubMed
] Related Publications
It has been suggested that cancer-associated fibroblasts (CAFs) positioned at the desmoplastic areas of various types of cancer are capable of executing a migratory program, characterized by accelerated motility and collective configuration. Since CAFs are reprogrammed derivatives of normal progenitors, including quiescent fibroblasts, we hypothesized that such migratory program could be context-dependent, thus being regulated by specific paracrine signals from the adjacent cancer population. Using the traditional scratch assay setup, we showed that only specific colon cancer cell lines (i.e. HT29) were able to induce collective CAF migration. By performing quantitative proteomics (SILAC), we identified a 2.7-fold increase of claudin-11, a member of the tight junction apparatus, in CAFs that exerted such collectivity in their migratory pattern. Further proteomic investigations of cancer cell line secretomes revealed a specific signature, involving TGF-β, as potential mediator of this effect. Normal colonic fibroblasts stimulated with TGF-β exerted myofibroblastic differentiation, occludin (OCLN) and claudin-11 (CLDN11) overexpression and cohort formation. Subsequently, inhibition of TGF-β attenuated all the previous effects. Immunohistochemistry of the universal tight junction marker occludin in a cohort of 30 colorectal adenocarcinoma patients defined a CAF subpopulation expressing tight junctions. Overall, these data suggest that cancer cells may induce CLDN11 overexpression and subsequent collective migration of peritumoral CAFs via TGF-β secretion.
The basic helix-loop-helix transcription factor AP4/TFAP4/AP-4 is encoded by a c-MYC target gene and displays up-regulation concomitantly with c-MYC in colorectal cancer (CRC) and numerous other tumor types. Here a genome-wide characterization of AP4 DNA binding and mRNA expression was performed using a combination of microarray, genome-wide chromatin immunoprecipitation, next-generation sequencing, and bioinformatic analyses. Thereby, hundreds of induced and repressed AP4 target genes were identified. Besides many genes involved in the control of proliferation, the AP4 target genes included markers of stemness (LGR5 and CD44) and epithelial-mesenchymal transition (EMT) such as SNAIL, E-cadherin/CDH1, OCLN, VIM, FN1, and the Claudins 1, 4, and 7. Accordingly, activation of AP4 induced EMT and enhanced migration and invasion of CRC cells. Conversely, down-regulation of AP4 resulted in mesenchymal-epithelial transition and inhibited migration and invasion. In addition, AP4 induction was required for EMT, migration, and invasion caused by ectopic expression of c-MYC. Inhibition of AP4 in CRC cells resulted in decreased lung metastasis in mice. Elevated AP4 expression in primary CRC significantly correlated with liver metastasis and poor patient survival. These findings imply AP4 as a new regulator of EMT that contributes to metastatic processes in CRC and presumably other carcinomas.
Lee NPThe blood-biliary barrier, tight junctions and human liver diseases.
Adv Exp Med Biol. 2012; 763:171-85 [PubMed
] Related Publications
Tight junction (TJ) composes of an intriguing class of cell junction molecules, for which these molecules share similar organizations and structure features among different organs. Fourtypes of transmembrane molecules namely occludins, claudins, junctional adhesion molecules and coxsackievirus and adenovirus receptors act as core units and each link directly and indirectly with a panel of peripheral molecules and underlying cytoskeletons to constitute the functional protein complexes at TJs. Individual TJ complex alone or in co-operation with other complexes via cross-talk mediated by peripheral molecules activate signaling pathways pertinent to various physiological and pathological processes in livers. In human livers, TJs are located at two regions in association with hepatocytes and cholangiocytes and perform major roles in controlling bile flow and metabolism. Apart from this physiological function, the other functions of TJs relating to liver diseases of hepatitis and liver cancer are gradually uncovered. The understanding of how TJs are involved in these clinical conditions hint for the development of new treatments at the molecular level.
Tight junction (TJ) proteins are involved in a number of cellular functions, including paracellular barrier formation, cell polarization, differentiation, and proliferation. Altered expression of TJ proteins was reported in various epithelial tumors. Here, we used tissue samples of human cutaneous squamous cell carcinoma (SCC), its precursor tumors, as well as sun-exposed and non-sun-exposed skin as a model system to investigate TJ protein alteration at various stages of tumorigenesis. We identified that a broader localization of zonula occludens protein (ZO)-1 and claudin-4 (Cldn-4) as well as downregulation of Cldn-1 in deeper epidermal layers is a frequent event in all the tumor entities as well as in sun-exposed skin, suggesting that these changes result from chronic UV irradiation. In contrast, SCC could be distinguished from the precursor tumors and sun-exposed skin by a frequent complete loss of occludin (Ocln). To elucidate the impact of down-regulation of Ocln, we performed Ocln siRNA experiments in human keratinocytes and uncovered that Ocln downregulation results in decreased epithelial cell-cell adhesion and reduced susceptibility to apoptosis induction by UVB or TNF-related apoptosis-inducing ligand (TRAIL), cellular characteristics for tumorigenesis. Furthermore, an influence on epidermal differentiation was observed, while there was no change of E-cadherin and vimentin, markers for epithelial-mesenchymal transition. Ocln knock-down altered Ca(2+)-homeostasis which may contribute to alterations of cell-cell adhesion and differentiation. As downregulation of Ocln is also seen in SCC derived from other tissues, as well as in other carcinomas, we suggest this as a common principle in tumor pathogenesis, which may be used as a target for therapeutic intervention.
Saintigny P, Peng S, Zhang L, et al.Global evaluation of Eph receptors and ephrins in lung adenocarcinomas identifies EphA4 as an inhibitor of cell migration and invasion.
Mol Cancer Ther. 2012; 11(9):2021-32 [PubMed
] Free Access to Full Article Related Publications
The Eph family of receptors is the largest family of receptor tyrosine kinases, but it remains poorly studied in lung cancer. We aimed to systematically explore the human Eph receptors and their ligands, the ephrins, in lung adenocarcinoma. The prognostic impact of Eph receptor and ephrin gene expression was analyzed using 2 independent cohorts of lung adenocarcinoma. Gene expression profiles in lung adenocarcinoma compared with normal adjacent lung were studied in 3 independent cohorts and in cell lines. Gene expression profiles were validated with quantitative polymerase chain reaction (qPCR) and Western blotting in cell lines. Functional studies to assess the role of Eph receptor A4 (EphA4) were carried out in vitro. The biological effects of EphA4 in lung cancer cell lines were assayed following overexpression and knockdown. Of the 11 Eph receptors and 8 ephrins analyzed, only EphA4 and ephrin A1 gene expression were consistently associated with an improved outcome in patients with lung adenocarcinoma. Expression levels of EphA4 by microarray correlated well with expression levels measured by qPCR and Western blotting. EphA4 overexpression reduced cell migration and invasion but did not affect cell cycle, apoptosis, or drug sensitivity. Surprisingly, EphA4 was expressed at higher levels in cancer compared with non-cancer tissues and cell lines. EphA4 gene expression is associated with an improved outcome in patients with resected lung adenocarcinoma, possibly by affecting cancer cell migration and invasion.
The thyroid transcription factor 1 gene (TTF-1 or NKX2-1) is essential to lung development; however, it is also a critical factor in lung cancer. TTF-1 is amplified in lung cancers, suggesting that it is a gain-of-function lung oncogene. Conversely, TTF-1 counters epithelial to mesenchymal transition in cell-based studies and inhibits progression of primary lung adenocarcinomas to metastases in an animal model of lung adenocarcinomas. The unifying theory regarding TTF-1 is that it exhibits both pro-oncogenic and anti-metastatic function depending on the cellular context. Occludin is the first discovered constituent of the epithelial tight junction; in recent years, a functional role of occludin as a tumor suppressor has begun to emerge. Here, we demonstrate that TTF-1 transactivated the expression of the epithelial tight junction molecules occludin (OCLN) and claudin-1 (CLDN1). We show that transcriptional activation occurred through a direct interaction of TTF-1 with the OCLN and CLDN1 promoters. Furthermore, in cells that lack TTF-1, exogenous TTF-1 expression dampened the inhibitory effect of TGF-β on occludin and claudin-1 content. Using cells derived from a genetically engineered mouse model of lung adenocarcinomas, we observed that silenced TTF-1 expression down-regulated occludin, which we supported with additional siRNA experiments. Finally, TTF-1 knockdown conferred human lung cancer cells resistance to anoikis, and expression of occludin restored cellular sensitivity to anoikis. Overexpression of occludin impeded migration and induced anoikis in lung carcinoma cells. Collectively, these data suggest that TTF-1 transcriptionally regulates occludin, which represents another avenue of TTF-1-mediated metastasis suppression.
Liu K, Hearne K, Mrsny A, et al.A strategy to improve selectivity and targeting to epithelial-derived cancer cells.
J Control Release. 2012; 164(2):205-12 [PubMed
] Related Publications
Examination of genomic and proteomic changes associated with ras-driven epithelial to mesenchymal transformation (EMT) of polarized epithelial cells has led to an improved understanding of surface-expressed structures and alterations in components involved in intracellular trafficking events that are altered as normal cells become cancerous. We have previously identified a mechanism involved in the establishment of tight junction (TJ) cell-cell contacts orchestrated by the protein occludin (Ocln) and its ability to reverse EMT events. Previous studies have suggested an increased functional expression of a cell-surface import system for small peptides, hPepT1, in several types of cancer cells. We now describe two approaches to identify agents capable of re-activating Ocln expression which could be modified into selective substrates of hPepT1. A screen for agents to re-activate suppressed occludin gene (OCLN) expression resulting from Ras/Raf/MEK/ERK pathway activation led to the identification of several small molecules. Using phage panning we have also identified several short peptide sequences that bind to the E-box used by the suppressor protein Slug to block OCLN expression. Thus, the current studies have identified several molecules and a roadmap to generate additional agents that could be examined for their ability to selectively enter cancer cells via hPepT1. We believe this strategy could result in reduced off-target drug distribution and thus greater functional targeting could be achieved for epithelial-derived cancers to prime them for the actions of established chemotherapeutic agents.
Speisky D, Duces A, Bièche I, et al.Molecular profiling of pancreatic neuroendocrine tumors in sporadic and Von Hippel-Lindau patients.
Clin Cancer Res. 2012; 18(10):2838-49 [PubMed
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PURPOSE: Von Hippel-Lindau (VHL) disease is an inherited syndrome caused by germline mutations in the VHL tumor suppressor gene, predisposing to a variety of neoplasms including pancreatic neuroendocrine tumors (PanNET). In VHL disease, PanNET probably progress according to a specific pathway of carcinogenesis. Our aim was to characterize by molecular quantitative analysis a panel of molecules implicated in the VHL pathway and in tumor progression in the PanNET of patients with VHL.
EXPERIMENTAL DESIGN: The expression of 52 genes was studied by quantitative reverse transcriptase PCR in 18 patients with VHL operated on for PanNET and compared with 16 non-VHL PanNET. The VHL and non-VHL tumors were matched according to their size and cell proliferation. For some genes, we looked for differences in the protein expression in VHL PanNET (n = 31), microadenomas (n = 22), and non-VHL PanNET (n = 16), included in tissue microarray blocks.
RESULTS: Nineteen (36%) genes were significantly upregulated and three (6%) downregulated in VHL PanNET. The upregulated genes were related to (i) hypoxia-inducible factor (HIF) molecules (CA9, HIF2A, and GLUT1), (ii) angiogenesis (CDH5, VEGFR1, EDNRA, ANGPT2, CD34, VEGFR2, VEGFA, and ANGPT1), (iii) the processes of epithelial-mesenchymal transition (VIM) and/or metastasis (LAMA4 and CXCR4), (iv) growth factors and receptors (PDGFB, IRS1, and ERBB1), or (v) cell cycle (CCND1 and CDKN2A). The downregulated genes were related to (i) EMT (OCLN) and (ii) signaling pathways (RPS6KB1 and GADD45B).
CONCLUSION: This study shows that the progression of PanNET in patients with VHL tumors follows a specific pathway and supports that targeting molecules specifically involved may be of therapeutic importance.
Kojima T, Takasawa A, Kyuno D, et al.Downregulation of tight junction-associated MARVEL protein marvelD3 during epithelial-mesenchymal transition in human pancreatic cancer cells.
Exp Cell Res. 2011; 317(16):2288-98 [PubMed
] Related Publications
The novel tight junction protein marvelD3 contains a conserved MARVEL (MAL and related proteins for vesicle trafficking and membrane link) domain like occludin and tricellulin. However, little is yet known about the detailed role and regulation of marvelD3 in normal epithelial cells and cancer cells, including pancreatic cancer. In the present study, we investigated marvelD3 expression in well and poorly differentiated human pancreatic cancer cell lines and normal pancreatic duct epithelial cells in which the hTERT gene was introduced into human pancreatic duct epithelial cells in primary culture, and the changes of marvelD3 during Snail-induced epithelial-mesenchymal transition (EMT) under hypoxia, TGF-β treatment and knockdown of FOXA2 in well differentiated pancreatic cancer HPAC cells. MarvelD3 was transcriptionally downregulated in poorly differentiated pancreatic cancer cells and during Snail-induced EMT of pancreatic cancer cells in which Snail was highly expressed and the fence function downregulated, whereas it was maintained in well differentiated human pancreatic cancer cells and normal pancreatic duct epithelial cells. Depletion of marvelD3 by siRNAs in HPAC cells resulted in downregulation of barrier functions indicated as a decrease in transepithelial electric resistance and an increase of permeability to fluorescent dextran tracers, whereas it did not affect fence function of tight junctions. In conclusion, marvelD3 is transcriptionally downregulated in Snail-induced EMT during the progression for the pancreatic cancer.
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.
We showed previously that cruciferous vegetable constituent benzyl isothiocyanate (BITC) inhibits growth of cultured and xenografted human breast cancer cells and suppresses mammary cancer development in a transgenic mouse model. We now show, for the first time, that BITC inhibits epithelial-mesenchymal transition (EMT) in human breast cancer cells. Exposure of estrogen-independent MDA-MB-231 and estrogen-responsive MCF-7 human breast cancer cell lines and a pancreatic cancer cell line (PL-45) to BITC resulted in upregulation of epithelial markers (e.g., E-cadherin and/or occludin) with a concomitant decrease in protein levels of mesenchymal markers, including vimentin, fibronectin, snail, and/or c-Met. The BITC-mediated induction of E-cadherin protein was accompanied by an increase in its transcription, whereas BITC-treated MDA-MB-231 cells exhibited suppression of vimentin, snail, and slug mRNA levels. Experimental EMT induced by exposure to TGFβ and TNFα or Rb knockdown in a spontaneously immortalized nontumorigenic human mammary epithelial cell line (MCF-10A) was also partially reversed by BITC treatment. The TGFβ-/TNFα-induced migration of MCF-10A cells was inhibited in the presence of BITC, which was partially attenuated by RNA interference of E-cadherin. Inhibition of MDA-MB-231 xenograft growth in vivo in female athymic mice by BITC administration was associated with an increase in protein level of E-cadherin and suppression of vimentin and fibronectin protein expression. In conclusion, this study reports a novel anticancer effect of BITC involving inhibition of EMT, a process triggered during progression of cancer to invasive state.
Entry of hepatitis C virus (HCV) into hepatocytes is a multi-step process that involves a number of different host cell factors. Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes. Physiologically, SR-BI is the major receptor for high-density lipoproteins (HDL) in the liver, where its expression is primarily controlled at the post-transcriptional level by its interaction with the scaffold protein PDZK1. However, the importance of interaction with PDZK1 to the involvement of SR-BI in HCV entry is unclear. Here we demonstrate that stable shRNA-knockdown of PDZK1 expression in human hepatoma cells significantly reduces their susceptibility to HCV infection, and that this effect can be reversed by overexpression of full length PDZK1 but not the first PDZ domain of PDZK1 alone. Furthermore, we found that overexpression of a green fluorescent protein chimera of the cytoplasmic carboxy-terminus of SR-BI (amino acids 479-509) in Huh-7 cells resulted in its interaction with PDZK1 and a reduced susceptibility to HCV infection. In contrast a similar chimera lacking the final amino acid of SR-BI (amino acids 479-508) failed to interact with PDZK1 and did not inhibit HCV infection. Taken together these results indicate an indirect involvement of PDZK1 in HCV entry via its ability to interact with SR-BI and enhance its activity as an HCV entry factor.
Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of OCLN in normal human liver and observed variable expression of alternative splice variants, including two known forms (WT-OCLN and OCLN-ex4del) and six novel forms (OCLN-ex7ext, OCLN-ex3pdel, OCLN-ex3del, OCLN-ex3-4del, OCLN-ex3p-9pdel, and OCLN-ex3p-7pdel). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in in vitro infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of OCLN splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of OCLN might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of OCLN expression and susceptibility to HCV infection remain to be elucidated.
Ordóñez-Morán P, Alvarez-Díaz S, Valle N, et al.The effects of 1,25-dihydroxyvitamin D3 on colon cancer cells depend on RhoA-ROCK-p38MAPK-MSK signaling.
J Steroid Biochem Mol Biol. 2010; 121(1-2):355-61 [PubMed
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Many studies support a protective action of vitamin D against colon cancer. 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts wide gene regulatory effects in human colon cancer cells. We previously reported that 1,25(OH)2D3 increases cytosolic Ca2+ concentration and transiently activates RhoA and its effector the Rho-associated coiled-kinase (ROCK), and later p38MAPK-MSK. We found that the inhibition of ROCK signaling by Y27632 or that of MSK by Ro318220 prevent the formation of epithelioid islands of SW480-ADH cells by 1,25(OH)2D3 and disrupts the adhesive phenotype of HT29 cells. ROCK and MSK inhibition also abrogates the induction of 1,25(OH)2D3 24-hydroxylase (CYP24), E-cadherin, and vinculin and the repression of cyclin D1 by 1,25(OH)2D3. Moreover, 1,25(OH)2D3 does not promote the localization of the tight junction protein occludin at the plasma membrane in cells expressing a dominant negative RhoA (N19-RhoA). In addition, 1,25(OH)2D3 specifically increases the level of the cysteine protease-inhibitor cystatin D, whereas that of cystatin SN is unaffected. The increase of cystatin D protein caused by 1,25(OH)2D3 is abrogated in N19-RhoA cells. Thus, activation of the RhoA-ROCK-p38MAPK-MSK signaling pathway is essential for the regulation of the phenotype and of the CST5/cystatin D candidate tumor suppressor and other target genes by 1,25(OH)2D3 in colon cancer cells.
Benedicto I, Molina-Jiménez F, Barreiro O, et al.Hepatitis C virus envelope components alter localization of hepatocyte tight junction-associated proteins and promote occludin retention in the endoplasmic reticulum.
Hepatology. 2008; 48(4):1044-53 [PubMed
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UNLABELLED: Hepatocyte tight junctions (TJ) play key roles in characteristic liver functions, including bile formation and secretion. Infection by hepatitis C virus (HCV) may cause alterations of the liver architecture and disruption of the bile duct, which ultimately can lead to cholestasis. Herein, we employed the HCV replicon system to analyze the effect of HCV on TJ organization. TJ-associated proteins occludin, claudin-1, and Zonula Occludens protein-1 (ZO-1) disappeared from their normal localization at the border of adjacent cells in Huh7 clones harboring genomic but not subgenomic replicons expressing only the nonstructural proteins. Furthermore, cells containing genomic replicons showed a cytoplasmic accumulation of occludin in the endoplasmic reticulum (ER). TJ-associated function, measured as FITC-dextran paracellular permeability, of genomic replicon-containing cells, was also altered. Interestingly, clearance of the HCV replicon by interferon-alpha (IFN-alpha) treatment and by short hairpin RNA (shRNA) significantly restored the localization of TJ-associated proteins. Transient expression of all HCV structural proteins, but not core protein alone, altered the localization of TJ-associated proteins in Huh7 cells and in clones with subgenomic replicons. Confocal analysis showed that accumulation of occludin in the ER partially co-localized with HCV envelope glycoprotein E2. E2/occludin association was further confirmed by co-immunoprecipitation and pull-down assays. Additionally, using a cell culture model of HCV infection, we observed the cytoplasmic dot-like accumulation of occludin in infected Huh7 cells.
CONCLUSION: We propose that HCV structural proteins, most likely those of the viral envelope, promote alterations of TJ-associated proteins, which may provide new insights for HCV-related pathogenesis.
Chao YC, Pan SH, Yang SC, et al.Claudin-1 is a metastasis suppressor and correlates with clinical outcome in lung adenocarcinoma.
Am J Respir Crit Care Med. 2009; 179(2):123-33 [PubMed
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RATIONALE: Claudin (CLDN)-1, a key component of tight junction complexes, was down-regulated in human lung adenocarcinomas.
OBJECTIVES: To investigate the clinical significance of CLDN1 expression in patients with lung adenocarcinoma and its role in cancer invasion and metastasis.
METHODS: We examined the CLDN1 mRNA expression in tumor specimens from 64 patients with lung adenocarcinoma and protein expression by immunohistochemistry in an independent cohort of 67 patients with lung adenocarcinoma. CLDN1 functions in cancer cell migration, invasion, and metastatic colonization were studied by overexpression and knockdown of CLDN1. Affymetrix microarrays were performed to identify gene expression changes associated with CLDN1 overexpression.
MEASUREMENTS AND MAIN RESULTS: We found that low-CLDN1 mRNA expression had shorter overall survival (P = 0.027, log-rank test) in 64 patients with lung adenocarcinoma, and we confirmed by immunohistochemistry that low CLDN1 expression had shorter overall survival (P = 0.024, log-rank test) in an independent cohort of 67 patients with lung adenocarcinoma. Overexpression of CLDN1 inhibited cancer cell dissociation in time-lapse imaging of wound healing, and suppressed cancer cell migration, invasion, and metastasis. Knockdown CLDN1 expression increased cancer cell invasive and metastatic abilities. Affymetrix microarrays identified a panel of genes altered by CLDN1 overexpression. CLDN1 increased expressions of cancer invasion/metastasis suppressors (e.g., connective tissue growth factor [CTGF], thrombospondin 1 [THBS1], deleted in liver cancer 1 [DLC1], occludin [OCLN], zona occludens 1 [ZO-1]) and suppressed expressions of invasion/metastasis enhancers (e.g., secreted phosphoprotein 1 [SPP1], cut-like homeobox 1 [CUTL1], transforming growth factor alpha [TGF-alpha], solute carrier family 2 [faciliated glucose transporter] member 3 [SLC2A3], placental growth factor [PGF]), supporting a role for CLDN1 as an invasion and metastasis suppressor.
CONCLUSIONS: CLDN1 is a cancer invasion/metastasis suppressor. CLDN1 is also a useful prognostic predictor and potential drug treatment target for patients with lung adenocarcinoma.
Kuang SQ, Tong WG, Yang H, et al.Genome-wide identification of aberrantly methylated promoter associated CpG islands in acute lymphocytic leukemia.
Leukemia. 2008; 22(8):1529-38 [PubMed
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We performed a genome-wide analysis of promoter associated CpG island methylation using methylated CpG island amplification (MCA) coupled to representational differential analysis (RDA) or a DNA promoter microarray in acute lymphoblastic leukemia (ALL). We identified 65 potential targets of methylation with the MCA/RDA approach, and 404 with the MCA/array. Thirty-six (77%) of the genes identified by MCA/RDA were shared by the MCA/array approach. Chromosomal location of these genes was evenly distributed in all autosomes. Functionally, 303 of these genes clustered in 18 molecular pathways. Of the 36 shared genes, 31 were validated and 26 were confirmed as being hypermethylated in leukemia cell lines. Expression analysis of eight of these genes was epigenetically modulated by hypomethylating agents and/or HDAC inhibitors in leukemia cell lines. Subsequently, DNA methylation of 15 of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) was validated in primary ALL samples. Patients with methylation of multiple CpG islands had a worse overall survival. This is the largest published list of potential methylation target genes in human leukemia offering the possibility of performing rational unbiased methylation studies in ALL.
Gu JM, Lim SO, Park YM, Jung GA novel splice variant of occludin deleted in exon 9 and its role in cell apoptosis and invasion.
FEBS J. 2008; 275(12):3145-56 [PubMed
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The tight junction protein occludin participates in cell adhesion and migration and has been shown to possess antitumorigenic properties; however, the exact mechanism underlying these effects is poorly understood. In liver cell lines, we identified an occludin splice variant deleted in exon 9 (Occ(DeltaE9)). Furthermore, comparison analysis of wild-type occludin (Occ(WT)) and Occ(DeltaE9) revealed that exon 9 played important roles in the induction of mitochondria-mediated apoptosis and the inhibition of invasion, along with the downregulation of matrix metalloproteinase expression. In addition, by using the calcium indicator X-rhod-1, and the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate, we found that Occ(WT) but not Occ(DeltaE9) increased calcium release from the endoplasmic reticulum. In conclusion, our results showed that occludin mediates apoptosis and invasion by elevating the cytoplasmic calcium concentration and that exon 9 of occludin is an important region that mediates these effects.
Hahn-Strömberg V, Edvardsson H, Bodin L, Franzén LDisturbed expression of E-cadherin, beta-catenin and tight junction proteins in colon carcinoma is unrelated to growth pattern and genetic polymorphisms.
APMIS. 2008; 116(4):253-62 [PubMed
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Adhesion proteins are responsible for the structural integrity of epithelial tissue and in tumors this integrity is often lost, resulting in a disorganization of the tissue. In the present study the complexity of the invasive front of colon carcinomas was correlated with cell adhesion protein expression and with polymorphisms in their genes. A complexity index was constructed from 32 colon carcinomas using computer-assisted morphometry estimating fractal dimension and tumor cell clusters followed by tree analysis. Immunohistochemical staining of beta-catenin, E-cadherin, occludin and claudin 2 was used for assessment of protein expression. Genetic screening of tissue from the tumor invasion front with laser microdissection was performed using SSCP and DNA sequencing. Adhesion protein distribution was significantly disturbed in most carcinomas. A single mutation in the gene of beta-catenin was found but there was no correlation between protein expression and genetic polymorphism. Nor was there any correlation between the complexity of the invasive border and protein distribution or genetic alterations. The results indicate that the complexity of colon carcinoma invasion is not dependent on genetic derangements in the genes of adhesion proteins or the protein distribution. Rather, aberrations in the function of other proteins related to the adhesive proteins could be responsible.
Orbán E, Szabó E, Lotz G, et al.Different expression of occludin and ZO-1 in primary and metastatic liver tumors.
Pathol Oncol Res. 2008; 14(3):299-306 [PubMed
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Tight junction (TJ) components were found to be correlated with carcinogenesis and tumor development. TJs are composed of three main integral membrane proteins; occludin, claudins and JAMs. Alteration of the TJ protein expression may play an important role in the process of cell dissociation, which is among the first steps of tumor invasion and metastasis. Reduced expression of ZO-1 has been reported to be associated with invasion of several tumors. The aim of the present study was to detect differences between occludin and ZO-1 expression in normal liver samples, HCCs and colorectal liver metastases. Expression of occludin and ZO-1 was analysed in 25 surgically removed human hepatocellular carcinomas (HCC) and 25 human colorectal liver metastases. Gene expression levels were measured by real-time RT PCR, protein expression was determined by immunohistochemistry, comparing tumors with the surrounding nontumorous parenchyma and with seven normal liver samples. Occludin and ZO-1 mRNAs showed significant downregulation in HCCs in comparison with normal liver and were also downregulated in the metastases when compared with normal liver. Occludin and ZO-1 proteins were weakly expressed on hepatocytes in normal liver, while strong expression was found on bile canaliculi. In HCCs occludin and ZO-1 did not show immunopositivity on tumor cells, while colorectal metastatic tumors revealed high levels of these molecules. HCCs and metastases are characterized by markedly different protein expression pattern of occludin and ZO-1, which phenomenon might be attributed to the different histogenesis of these tumors.
Osanai M, Murata M, Nishikiori N, et al.Epigenetic silencing of occludin promotes tumorigenic and metastatic properties of cancer cells via modulations of unique sets of apoptosis-associated genes.
Cancer Res. 2006; 66(18):9125-33 [PubMed
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Occludin is the first identified integral protein for the tight junction (TJ), and its long COOH-terminal domain is considered to have functions in receiving and transmitting cell survival signals. Loss of TJ-associated molecules, such as occludin, has been correlated with tumor progression in carcinogenesis; however, the precise molecular mechanisms explaining its loss of expression and whether occludin expression has any effects on cancer phenotypes remain to be clarified. Here, we show that forced expression of occludin in cancer cells exhibits enhanced sensitivity to differently acting apoptogenic factors, and thus inhibits the tumorigenicity of transformed cells, via modulation of unique sets of apoptosis-associated genes. In addition, studies using deletion mutants of occludin constructs show that 44 amino acids at the COOH-terminal end play a critical role in modifying the cellular phenotypes. Interestingly, occludin decreases cellular invasiveness and motility, thereby abrogating metastatic potencies of cancer cells. We also found that occludin expression is silenced by CpG island hypermethylation on its promoter region. Synergy with a demethylator and histone deacetylase inhibitor or retinoids that stimulate retinoic acid receptor alpha induces endogenous occludin, which is sufficient for apoptotic sensitization. Our results show the functional diversity of occludin and suggest that methylator phenotype of occludin provides enhanced tumorigenic, invasive, and metastatic properties of cancer cells, identifying occludin as a likely candidate for a tumor-suppressor gene in certain types of cancer.
AIM: To establish the DNA methylation patterns of the promoter CpG islands of 14 "drug-resistance" genes in hepatocellular carcinoma (HCC).
METHODS: The methylation specific polymerase chain reaction in conjunction with sequencing verification was used to establish the methylation patterns of the 14 genes in the liver tissues of four healthy liver donors, as well as tumor and the paired non-cancerous tissues of 30 HCC patients.
RESULTS: While 11 genes (ATP-binding cassette, sub-family G (WHITE), member 2(ABCG2), activating transcription factor (ATF2), beta-2-microglobulin (B2M), deoxycytidine kinase (DCK), occludin (OCLN), v-raf-1 murine leukemia viral oncogene homolog (RAF1), ralA binding protein 1 (RALBP1), splicing factor (45 kD) (SPF45), S-phase kinase-associated protein 2 (p45) (SKP2), tumor protein p53 (Li-Fraumeni syndrome) (TP53) and topoisomerase (DNA) II beta (TOP2B)) maintained the unmethylated patterns, three genes displayed to various extents the hypermethylation state in tumor tissues in comparison with the normal counterparts. The catalase (CAT) was hypermethylated in tumor and the neighboring non-cancerous tissue of one case (3.3%). Both glutathione S-transferase pi (GSTpi) (80%, 24/30 in tumor and 56.7%, 17/30 in the paired non-cancerous tissues) and cystic fibrosis transmembrane conductance regulator, ATP-binding cassette (sub-family C, member 7) (CFTR) (77%, 23/30 in tumor and 50%, 15/30 in the paired non-cancerous tissues) genes were prevalently hypermethylated in HCC as well as their neighboring non-cancerous tissues. No significant difference in the hypermethylation occurrence was observed between the HCC and its neighboring non-cancerous tissues.
CONCLUSION: Hypermethylation of promoter CpG islands of both CFTR and GSTpi genes occurs prevalently in HCC, which may correlate with the low expression of these two genes at the mRNA level and has the profound etiological and clinical implications. It is likely to be specific to the early phase of HCC carcinogenesis.
Ara C, Devirgiliis LC, Massimi MInfluence of retinoic acid on adhesion complexes in human hepatoma cells: a clue to its antiproliferative effects.
Cell Commun Adhes. 2004 Jan-Feb; 11(1):13-23 [PubMed
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Retinoic acid exerts antiproliferative and differentiative effects in normal and transformed in vitro hepatocytes. In order to verify whether these effects are related to a modulation of adhesion molecules, we used Western blot analysis and immunofluorescence microscopy to investigate the E-cadherinl/beta-catenin complex, the main system of adherens junctions, and the occludin/ZO-1 complex present in the tight junctions in HepG2 cells cultured in the presence or absence of retinoic acid. Results showed that retinoic acid treatment increases the amount of beta-catenin bound to E-cadherin by decreasing its tyrosine-phosphorylation level. Similar results were obtained with the tight junction system, in which the amount of occludin/ZO-1 complex is increased by a similar mechanism that reduced the level of ZO-1 phosphorylation on tyrosine. Immunofluorescence images also confirm these results, showing the localization on the cell surface of both adhesion complexes. Their insertion into the plasma membrane could be suggestive of an optimal reassembly and function of adherens and tight junctions in hepatoma cells, indicating that retinoic acid, besides inhibiting cell proliferation, improves cell-cell adhesion, sustaining or inducing the expression of a more differentiated phenotype.
Tan X, Tamori Y, Egami H, et al.Analysis of invasion-metastasis mechanism in pancreatic cancer: involvement of tight junction transmembrane protein occludin and MEK/ERK signal transduction pathway in cancer cell dissociation.
Oncol Rep. 2004; 11(5):993-8 [PubMed
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Mitogen-activated protein kinase kinase 2 (MEK2) was detected as an invasion-metastasis related factor between highly invasive (PC-1.0) and weakly invasive (PC-1) pancreatic cancer cell lines in our previous study. On the other hand, tight junction (TJ) was found to be correlated with carcino-genesis and tumor development. In this study, the expressions and correlation of TJ transmembrane protein occludin and MEK/extracellular signal-regulated kinase (ERK) signaling pathway were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and human (AsPC-1 and CAPAN-2) pancreatic cancer cell lines were analyzed immunocytochemically with anti-occludin, phosphorylated MEK1/2 (p-MEK1/2), phosphorylated ERK1/2 (p-ERK1/2) antibodies. MEK1/2 inhibitor U0126 significantly induced the expression of occludin at the cell-cell junction and substantially suppressed the p-MEK1/2 and p-ERK1/2 expressions in PC-1.0 and AsPC-1 cells. In contrast, dissociation factor (DF) treatment obviously disrupted the occludin expressions at the sites of cell-cell junction and markedly induced the p-MEK1/2 and p-ERK1/2 expressions in PC-1 and CAPAN-2 cells. In addition, occludin expressions at cell-cell junction were restored and p-MEK1/2 and p-ERK1/2 expressions were suppressed by subsequent U0126-treatment in DF treated PC-1 and CAPAN-2 cells. Correspondingly, light microscopic images showed that DF induced the dissociation of cell island-like colonies in PC-1 and CAPAN-2 cells, and U0126-treatment induced cell aggregation in these pancreatic cancer cells. Occludin is involved in the cell dissociation in pancreatic cancer cells. Moreover, MEK/ERK signaling pathway probably regulates the cell dissociation status of pancreatic cancer through influencing the intracellular localization and expression of occludin.
Tobioka H, Isomura H, Kokai Y, et al.Occludin expression decreases with the progression of human endometrial carcinoma.
Hum Pathol. 2004; 35(2):159-64 [PubMed
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The tight junctions of the glandular epithelium are crucial for the maintenance of cell polarity, separating the plasma membrane into apical and basolateral domains. Thus abnormalities of the tight junctions may result in the structural disturbances of glandular epithelial neoplasia. In this study we introduced an anti-occludin monoclonal antibody for semiquantitative assay of the occludin expression in tissue sections of human normal and neoplastic endometrial epithelia using the Adobe Photoshop and NIH Image programs. Normal endometrial glands and samples of endometrial hyperplasia and endometrioid carcinoma grade 1 fully expressed occludin at the apical cell border. In endometrioid carcinomas grades 2 and 3, however, occludin disappeared in solid areas of the carcinomatous tissues. Occludin was also found at the apical borders of the cancer cells that formed glandular structures. Occludin expression decreased progressively in parallel with the increase in carcinoma grade, and the decreased occludin expression correlated with myometrial invasion and lymph node metastasis. These results suggest that the loss of tight junctions has a close relationship with structural atypia in the progression of human endometrial carcinomas and their malignant potential.