Loss of tight junction barrier function and its role in cancer metastasis

As the most apical structure between epithelial and endothelial cells, tight junctions (TJ) are well known as functioning as a control for the paracellular diffusion of ions and certain molecules. It has however, become increasingly apparent that the TJ has a vital role in maintaining cell to cell integrity and that the loss of cohesion of the structure can lead to invasion and thus metastasis of cancer cells. This article will present data showing how modulation of expression of TJ molecules results in key changes in TJ barrier function leading to the successful metastasis of a number of different cancer types.     

Claudin-16 reduces the aggressive behavior of human breast cancer cells

Claudin-16 (Paracellin-1) is a transmembrane tight junction (TJ) protein originally described as having a critical role in the re-absorption of magnesium and calcium in the kidney. This study examined expression of Claudin-16 in human breast cells and tissues to identify a possible link between expression and aggressiveness in cells and between Claudin-16 levels and patient prognosis. Insertion of the Claudin-16 gene into MDA-MB-231 human breast cancer cells resulted in cells that were significantly less motile and invasive in behavior, with increased adhesion to matrix. These cells also exhibited significantly increased TJ functionality and "tighter" colony morphology. Moreover, growth rates were reduced in both in vitro and in vivo assays (P < 0.002). Frozen sections from breast cancer primary tumors (matched tumor 124 and background 33) were immuno-stained. RNA was reverse transcribed and analyzed by Q-PCR (standardized using beta-actin, normalized with cytokeratin-19 levels). Levels of expression of Claudin-16 were significantly decreased in node positive tumors compared to negative (P = 0.016). Expression was significantly lower in patients with node positive tumors (P = 0.016) and in those who had died from breast cancer or had general poor prognosis (P < 0.015). Immunohistochemical staining showed decreased expression of Claudin-16 in tumor sections (P < 0.00001). In conclusion, forced expression of Claudin-16 in breast cancer cells resulted in a less aggressive phenotype and reduced in vivo tumor volume. Claudin-16 expression was reduced in human breast cancer, particularly in patients with aggressive tumors and high mortality. This suggests that Claudin-16 plays a role beyond that of an initial metastasis repressor in this cancer type.     

Soluble epithin/PRSS14 secreted from cancer cells contains active angiogenic potential

Epithin (PRSS14/matriptase/ST14), a type II membrane protein, is involved in progression of epithelial cancers and metastasis as well as in the normal epidermal barrier function. When activated, it translocates into the cell-cell contacts and sheds into media. In order to understand the specific mechanism during tumor progression, we tested the angiogenic potential of secreted form of epithin. Epithin produced from the cancer cells shed more in hypoxia and induced motility of endothelial cells. Epithin enhanced the migration and invasion of mouse and bovine endothelial cells without cell proliferation. Furthermore, soluble epithin induced endothelial differentiation in the assay of the human endothelial microvessel-like tube formation and in that of the chicken chorioallantoic membrane. The knock-down of epithin in the 427 thymoma cell line abolished the protease activity of secreted epithin fraction, reduced the invasion of endothelial cells through matrigel, and tube formation activity. Only specific antibodies abolished the migration of endothelial cell and the vessel morphogenesis, suggesting that epithin specifically functions in these systems. Therefore, we propose that the secreted epithin in the hypoxic cancer microenvironment plays a role as a proangiogenic factor, and can be modulated with specific antibodies.     

Rho‐associated kinases in tumorigenesis: re‐considering ROCK inhibition for cancer therapy

The Rho‐associated (ROCK) serine/threonine kinases have emerged as central regulators of the actomyosin cytoskeleton, their main purpose being to promote contractile force generation. Aided by the discovery of effective inhibitors such as Y27632, their roles in cancer have been extensively explored with particular attention focused on motility, invasion and metastasis. Recent studies have revealed a surprisingly diverse range of functions of ROCK. These insights could change the way ROCK inhibitors might be used in cancer therapy to include the targeting of stromal rather than tumour cells, the concomitant blocking of ROCK and proteasome activity in K‐Ras‐driven lung cancers and the combination of ROCK with tyrosine kinase inhibitors for treating haematological malignancies such as chronic myeloid leukaemia. Despite initial optimism for therapeutic efficacy of ROCK inhibition for cancer treatment, no compounds have progressed into standard therapy so far. However, by carefully defining the key cancer types and expanding the appreciation of ROCK's role in cancer beyond being a cell‐autonomous promoter of tumour cell invasion and metastasis, the early promise of ROCK inhibitors for cancer therapy might still be realized.     

C-Jun N-Terminal kinase mediates disassembly of apical junctions in model intestinal epithelia

Dynamic remodeling of intercellular junctions is a critical determinant of epithelial barrier function in both physiological and pathophysiological states. While the disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ) has been well-described in response to pathogens and other external stressors, the role of stress-related signaling in TJ/AJ regulation remains poorly understood. The aim of this study was to define the role of stress-activated c-Jun N-terminal kinase (JNK) in disruption of intercellular junctions in model intestinal epithelia. We show that rapid AJ/TJ disassembly triggered by extracellular calcium depletion of T84 and SK-CO15 cell monolayers was accompanied by activation (phosphorylation) of JNK, and prevented by pharmacological inhibitors of JNK. The opposite process, TJ/AJ reassembly, was accelerated by JNK inhibition and suppressed by the JNK activator anisomycin. JNK1 but not JNK2 was found to colocalize with intercellular junctions, and siRNA-mediated down-regulation of JNK1 attenuated the TJ/AJ disruption caused by calcium depletion. JNK inhibition also blocked formation of characteristic contractile F-actin rings in calcium-depleted epithelial cells, suggesting that JNK regulates junctions by remodeling the actin cytoskeleton. In this role JNK acts downstream of the actin-reorganizing Rho-dependent kinase (ROCK), since ROCK inhibition abrogated JNK phosphorylation and TJ/AJ disassembly after calcium depletion. Furthermore, JNK acts upstream of F-actin-membrane linker proteins of the ERM (ezrin-radixin-moesin) family, but in a complex relationship yet to be fully elucidated. Taken together, our findings suggest a novel role for JNK in the signaling pathway that links ROCK and F-actin remodeling during disassembly of epithelial junctions.     

The Expression of the Nectin Complex in Human Breast Cancer and the Role of Nectin-3 in the Control of Tight Junctions during Metastasis

Introduction

Nectins are a family of integral protein molecules involved in the formation of functioning Adherens and Tight Junctions (TJ). Aberrant expression is associated with cancer progression but little is known how this effects changes in cell behaviour. This study aimed to ascertain the distribution of Nectins-1 to -4 in human breast cancer and the effect on junctional integrity of Nectin-3 modulation in human endothelial and breast cancer cells.

Methods

A human breast tissue cohort was processed for Q-PCR and immunohistochemistry for analysis of Nectin-1/-2/-3/-4. Nectin-3 over-expression was induced in the human breast cancer cell line MDA-MB-231 and the human endothelial cell line HECV. Functional testing was carried out to ascertain changes in cell behaviour.

Results

Q-PCR revealed a distinct reduction in node positive tumours and in patients with poor outcome. There was increased expression of Nectin-1/-2 in patients with metastatic disease, Nectin-3/-4 was reduced. IHC revealed that Nectin-3 expression showed clear changes in distribution between normal and cancerous cells. Nectin-3 over-expression in MDA-MB-231 cells showed reduced invasion and migration even when treated with HGF. Changes in barrier function resulted in MDAN3 cells showing less change in resistance after 2h treatment with HGF (p<0.001). Nectin-3 transformed endothelial cells were significantly more adhesive, irrespective of treatment with HGF (p<0.05) and had reduced growth. Barrier function revealed that transformed HECV cells had significantly tighter junctions that wildtype cells when treated with HGF (p<0.0001). HGF-induced changes in permeability were also reduced. Overexpression of Nectin-3 produced endothelial cells with significantly reduced ability to form tubules (p<0.0001). Immunoprecipitation studies discovered hitherto novel associations for Nectin-3. Moreover, HGF appeared to exert an effect on Nectin-3 via tyrosine and threonine phosphorylation.

Conclusions

Nectin-3 may be a key component in the formation of cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells.     

Enhanced tight junction function in human breast cancer cells by antioxidant, selenium and polyunsaturated lipid

Paracellular permeability (PCP) is governed by tight junctions (TJs) in epithelial cells, acting as cell-cell adhesion structures, the aberration of which is known to be linked to the dissociation and metastasis of breast cancer cells. This study hypothesized that the function of TJs in human breast cancer cells can be augmented by gamma linolenic acid (GLA), selenium (Se), and iodine (I) in the presence of 17-beta-estradiol, as these molecules are known to increase TJ functions in endothelial cells, using assays of trans-epithelial resistance (TER), PCP, immunofluorescence, and in vitro invasion and motility models. GLA, I, and Se individually increased TER of MDA-MB-231 and MCF-7 human breast cancer cells. The combination of all three agents also had a significant increase in TER. Addition of GLA/Se/I reduced PCP of both breast cancer cell lines. GLA/Se/I reversed the effect of 17-beta-estradiol (reduced TER, increased PCP). Immunofluorescence revealed that after treatment with Se/I/GLA over 24 h, there was increasing relocation to breast cancer cell-cell junctions of occludin and ZO-1 in MCF-7 cells. Moreover, treatment with GLA/Se/I, alone or in combination, significantly reduced in vitro invasion of MDA-MB-231 cells through an endothelial cell barrier (P < 0.0001) and reduced 17-beta-estradiol induced breast cancer cell motility (P < 0.0001). Our previous work has demonstrated that GLA, I, and Se alone, or in combination are able to strengthen the function of TJs in human endothelial cells; this has now proved to be true of human breast cancer cells. This combination also completely reversed the effect of 17-beta-estradiol in these cells.     

Mechanism of IFN-gamma-induced endocytosis of tight junction proteins: myosin II-dependent vacuolarization of the apical plasma membrane

Disruption of epithelial barrier by proinflammatory cytokines such as IFN-gamma represents a major pathophysiological consequence of intestinal inflammation. We have previously shown that IFN-gamma increases paracellular permeability in model T84 epithelial cells by inducing endocytosis of tight junction (TJ) proteins occludin, JAM-A, and claudin-1. The present study was designed to dissect mechanisms of IFN-gamma-induced endocytosis of epithelial TJ proteins. IFN-gamma treatment of T84 cells resulted in internalization of TJ proteins into large actin-coated vacuoles that originated from the apical plasma membrane and resembled the vacuolar apical compartment (VAC) previously observed in epithelial cells that lose cell polarity. The IFN-gamma dependent formation of VACs required ATPase activity of a myosin II motor but was not dependent on rapid turnover of F-actin. In addition, activated myosin II was observed to colocalize with VACs after IFN-gamma exposure. Pharmacological analyses revealed that formation of VACs and endocytosis of TJ proteins was mediated by Rho-associated kinase (ROCK) but not myosin light chain kinase (MLCK). Furthermore, IFN-gamma treatment resulted in activation of Rho GTPase and induced expressional up-regulation of ROCK. These results, for the first time, suggest that IFN-gamma induces endocytosis of epithelial TJ proteins via RhoA/ROCK-mediated, myosin II-dependent formation of VACs.     

TNF-alpha induced NFκB signaling and p65 (RelA) overexpression repress Cldn5 promoter in mouse brain endothelial cells

Inflammatory cytokine TNFα enhances permeability of brain capillaries constituting blood brain barrier (BBB). In the monoculture endothelial models of BBB TNFα alters tight junction (TJ) structure and protein content. Claudin-5 (Cldn5) is a key TJ protein whose expression in the brain endothelial cells is critical to the function of BBB. TNFα reduces Cldn5 promoter activity and mRNA expression in mouse brain derived endothelial cells but the regulatory elements and signaling mechanism involved are not defined. Here we report that TNFα acts through NFκB signaling and requires a conserved promoter region for the down-regulation of Cldn5 expression. Overexpression of the NFκB subunit p65 (RelA) alone repressed Cldn5 promoter activity in mouse brain endothelial cells. We observed partial loss of Cldn5 protein expression after prolonged TNFα treatment in primary endothelial culture isolated from C56BL/6 mice brain. Taken together, our results confirm and extend previous observations of TNFα induced down-regulation of Cldn5 expression in mouse brain endothelial cells.     

Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression

Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.     

Numb modulates the paracellular permeability of intestinal epithelial cells through regulating apical junctional complex assembly and myosin light chain phosphorylation

Numb is highly expressed throughout the crypt-villus axis of intestinal mucosa and functions as cell fate determinant and integrator of cell-to-cell adhesion. Increased paracellular permeability of intestinal epithelial cells is associated with the epithelial barrier dysfunction of inflammatory bowel diseases (IBDs). The apical junctional complex (AJC) assembly and myosin light chain (MLC) phosphorylation regulate adherens junctions (AJ) and tight junctions (TJ). We determined whether and how Numb modulate the paracellular permeability of intestinal epithelial cells. Caco-2 intestinal epithelial cells and their Numb-interfered counterparts were used in the study for physiological, morphological and biological analyses. Numb, expressed in intestinal epithelial cells and located at the plasma membrane of Caco-2 cells in a basolateral to apical distribution, increased in the intestinal epithelial cells with the formation of the intestinal epithelial barrier. Numb expression decreased and accumulated in the cytoplasm of intestinal epithelial cells in a DSS-induced colitis mouse model. Numb co-localized with E-cadherin, ZO-1 and Par3 at the plasma membrane and interacted with E-cadherin and Par3. Knockdown of Numb in Caco-2 cells altered the F-actin structure during the Ca²⁺ switch assay, enhanced TNFα-/INF-γ-induced intestinal epithelial barrier dysfunction and TJ destruction, and increased the Claudin-2 protein level. Immunofluorescence experiments revealed that NMIIA and F-actin co-localized at the cell surface of Caco-2 cells. Numb knockdown in Caco-2 cells increased F-actin contraction and the abundance of phosphorylated MLC. Numb modulated the intestinal epithelial barrier in a Notch signaling-independent manner. These findings suggest that Numb modulates the paracellular permeability by affecting AJC assembly and MLC phosphorylation.     

Tumor autocrine motility factor is an angiogenic factor hat stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine that primarily stimulates tumor cell motility via receptor-mediated signaling pathways and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for anti-angiogenic treatment.     

Claudin-5 expression in the vasculature of the developing chick embryo

The claudin family of proteins are integral components of tight junctions and are responsible for determining the ion specificity and permeability of paracellular transport within epithelial and endothelial cell layers. Studies in human, mouse, Xenopus, and zebrafish have shown that only a limited number of claudins are expressed in endothelial cells. Here, we report the expression pattern of Claudin-5 during chick development. Between HH stage 4 and 6 Claudin-5 expression was observed exclusively in extraembryonic tissue. Claudin-5 expression was not observed in the embryo until HH stage 8, coincident with the onset of embryonic vascularization. Claudin-5 expression was maintained in the developing vasculature in the embryonic and extraembryonic tissue throughout organogenesis (HH stage 19–35), including the vasculature of the ectoderm and of organs derived from the mesoderm and endoderm lineages. These data describe a conserved expression pattern for Claudin-5 in the endothelial tight junction barrier and is the first report of the onset of Claudin-5 expression in a vertebrate embryo.     

Inhibition of angiogenic factor- and tumour-induced angiogenesis by gamma linolenic acid.

Angiogenesis, the formation of new blood vessels, is an essential feature of malignant tumour development. Gamma linolenic acid (GLA), a n-6 polyunsaturated fatty acid (PUFA), inhibits the growth and metastasis of a variety of tumour cells, including breast, prostate, pancreatic cancer and hepatoma cells and also has anti-metastatic effects on endothelial cells. In the current study, we tested whether GLA inhibited angiogenesis induced by tumour cells. A rat aortic ring assay and in vitro tube formation of human vascular endothelial cells were used to determine angiogenesis (spontaneous, angiogenic factor- and tumour cells-induced). Inclusion of GLA in this 3-D matrix culture system significantly inhibited angiogenesis from aortic rings in a concentration-dependent manner. The results from tube formation of vascular endothelial cell further confirmed that GLA suppressed angiogenesis. Furthermore, in the cell motility assay (phagokinetic assay and endothelial wounding assay), a significant reduction of the motility of vascular endothelial cells by GLA was seen. It is concluded that gamma linolenic acid inhibits angiogenic factor and tumour-induced angiogenesis in vitro at least in part via its inhibitory effect on the motility of vascular endothelial cells.     

The C-terminal cytoplasmic tail of claudins 1 and 5 but not its PDZ-binding motif is required for apical localization at epithelial and endothelial tight junctions

Claudins are a family of tetraspan transmembrane proteins that represent the major constituents of epithelial and endothelial tight junctions (TJs). They form TJ strands representing the major barrier regulating paracellular transport of solutes and water. Intracellularly, claudins are connected via a C-terminal PDZ-binding motif with several TJ-associated proteins containing PDZ domains. Although these interactions can provide a link to the actin cytoskeleton, they appear to be dispensable for the TJ localization of claudins. To identify TJ-targeting elements in the C-terminal cytoplasmic domains of the claudins 1 and 5, we generated a series of C-terminal deletion mutants and analyzed their distribution in polarized epithelial (MDCK) and endothelial (HMEC-1) cells. TJ localization was revealed by establishing an in vivo cross-linking approach that stabilized claudin-TJ interactions. We show that residues located C-terminal to the last transmembrane domain are required for the proper targeting to apical TJ.s. While claudin derivatives lacking only the very C-terminal PDZ-binding motif continue to localize to TJs, mutants lacking the entire C-terminal juxtamembrane sequence do not associate with TJs and accumulate in intracellular structures. This indicates that crucial determinants for stable TJ incorporation of claudins reside in a cytoplasmic C-terminal sequence which up to now has not been implicated in specific protein-protein interactions.     

The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo

Angiogenesis consists of multistep pathways such as the degradation of the matrix, proliferation of the endothelial cells, motility of the endothelial cells, formation of the cord structure and network formation of microvessels. The small GTPase Rho participates in cell motility through actin fiber polymerization. The role of the small GTPase Rho signal transduction pathway in regulating angiogenesis, however, is still unknown. In this study, we investigated the role of the small GTPase Rho signal transduction pathway in angiogenesis in vitro and in vivo using the exoenzyme, Clostridium botulinum C3 transferase, which specifically suppresses Rho and a compound, Y-27632, which suppresses p160ROCK (Rho-associated coiled-coil containing protein kinase). In this paper, we showed that the small GTPase Rho-p160ROCK signal transduction pathway played an important role in angiogenesis both in vitro and in vivo. These results suggest that inhibition of the small GTPase Rho signal transduction pathway by the p160ROCK inhibitor could be a possible new strategy for angiogenic diseases.     

Tumor autocrine motility factor is an angiogenic factor that stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine which primarily stimulates tumor cell motility via receptor-mediated signaling pathways, and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for antiangiogenic treatment.