Tj And Breast Cancer Metastasis

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4.1 Role of TJs in Breast Cancer Metastasis

Cancer metastasis proceeds by a series of steps, among which the capacity of cancer cells to invade surrounding normal tissues is of central importance in the dissemination of disease (113). The interaction between cancer cells and mesothelial cells lining the cavity is crucial for achieving the complex sequence of cancer cell dissemination into the body cavity. In the process of submesothelial invasion of cancer cells, TJs of mesothelial cells may function as a defence against the invasion of cancer cells, because the TJs are known to work as a barrier to the paracellular passage of cells and substances between epithelial or endothelial cells (113).

Metastasis is the primary cause of fatality in breast cancer patients. Although there are believed to be numerous events contributing to the process of metastasis, it is widely accepted that the loss of cell-cell adhesion in neoplastic epithelium is necessary for invasion of surrounding stromal elements and subsequent metastatic events (10). Regulation of vascular permeability is one of the most important functions of endothelial cells, and endothelial cells from different organ sites show different degrees of permeability (114). Tumour blood vessels are more permeable on macro-molecular diffusion than normal tissue vessels. However, the cause and mechanism of hyperpermeability of human vessels had not been clear (114). Although, tumour-cell-conditioned medium increases endothelial cell permeability irreversibly. A timely discussion of the organization of TJs in mammary gland biology can be found in (65).

4.2 Expression of TJ Proteins in Breast Cancer

Most cancers, including breast cancer, originate from epithelial tissues and are characterised by aberrant growth control, and loss of differentiation and tissue architecture. It is a fundamental property of cancer cells that their mutual adhesiveness is significantly weaker than that of normal cells.

Reduced cell-cell interaction allows cancer cells to disobey the social order, resulting in destruction of overall tissue architecture, the morphological hallmark of malignancy. Loss of contact inhibition, which reflects disorder in the signal transduction pathways that connect cell-cell interactions are typical of both early (loss of cell polarity and growth control) and late (invasion and metastasis) stages of tumour progression.

Recent studies have shown that several TJ components are, directly or indirectly, involved in breast cancer progression including ZO-1, ZO-2, claudin-7, claudin-1, and occludin.

4.2.1 Transmembrane protein expression in breast cancer

Claudin-1 (SEMP-1) is normally expressed in mammary gland-derived epithelial cells, but is absent in most human breast cancer cell lines. Claudin-1 expression was not detectable in subconfluent MDA-MB-435 and MDA-MB-361 breast cancer cells (9). Neither of these cell lines express occludin protein, and MDA-MB-435 do not express ZO-1 protein. Claudin-1 retroviral transduced breast cancer cells showed expression of Claudin-1 at the usual cell-cell contact sites, suggesting that other proteins may be able to target claudin-1 to the TJ in the absence of occludin and ZO-1. Moreover, paracellular permeability was reduced in these transduced cells. The authors suggest that Claudin-1 gene transfer may be in itself enough to exert TJ-mediated gate function in metastatic breast cancer cells even in the absence of other TJ associated proteins such as occludin.

Claudin-1 expression has been observed in human mammary epithelial cells, but was observed to be at low or undetectable levels in a number of breast tumours and breast cancer cell lines (11). This points to a possible tumour suppressor function. Kramer et al. found that in sporadic and hereditary breast cancer, there were no genetic changes, implying that regulatory or epigenetic factors may be involved in the downregulation of the claudin-1 gene during breast cancer development.

Loss of claudin-7 correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast (10). The expression of claudin-7 is lost in both pre-neoplastic and invasive ductal carcinoma of the breast occurring predominately in high grade lesions. Expression is also frequently lost in LCIS correlating with the increased cellular discohesion observed in LCIS. Additionally, the majority of IDC cases displaying a low claudin-7 expression have a positive lymph node status. Such findings suggest that the loss of claudin-7 may aid in tumour cell dissemination and augment metastatic potential. Moreover, silencing of claudin-7 expression correlated with promoter hypermethylation in 3/3 breast cancer cell lines but not in invasive ductal carcinomas (0/5). In addition, HGF treatment results in disassociation of MCF-7 and T47D cells in culture, and a loss of claudin-7 expression within 24 hours.

Tokes et al. (115) compared levels of protein and mRNA expression of three members of the claudin family in malignant breast tumours and benign lesions. Altogether, 56 sections from 52 surgically resected breast specimens were analysed for claudin-1, claudin-3, and claudin-4 expression by immunohistochemistry and mRNA was also analysed using real-time PCR. Claudins were rarely observed exclusively at TJ structures. Claudin-1 was present in the membrane of normal duct cells and in some of the cell membranes from ductal carcinoma in situ, and was frequently observed in eight out of nine areas of apocrine metaplasia, whereas invasive tumours were negative for claudin-1 or it was present in a scattered distribution among such tumour cells (in 36/39 malignant tumours). Claudin-3 was present in 49 of the 56 sections and calsuin-4 was present in all 56 tissue sections. However, claudin-4 was highly positive in normal epithelial cells and was decreased or absent in 17 out of 21 ductal carcinoma grade 1, in special types of breast carcinoma (mucinous, papillary, tubular) and in areas of apocrine metaplasia. Claudin-1 mRNA was downregulated by 12-fold in the tumour group. Claudin-3 and claudin-4 mRNA exhibited no difference in expression between invasive tumours and surrounding tissue. The significant loss of claudin-1 protein in breast cancer cells suggests that this protein may play a role in invasion and metastasis. The loss of claudin-4 expression in areas of apocrine metaplasia and in the majority of grade 1 invasive carcinomas also suggests a particular role for this protein in mammary glandular cell differentiation and carcinogenesis.

Soini (116), evaluated the expression of claudin-2, claudin-3, claudin-4, and claudin-5 in 20 cases of Paget's disease (13 mammary and 7 extra-mammary cases), and compared the results with those of other neoplastic skin lesions, including actinic keratoses, basal cell carcinomas, and malignant melanomas. To compare claudin expression in Paget's disease and breast neoplasia, it was also studied in a large set of breast carcinomas. Membrane-bound claudin-3 and -4 expression was seen in all cases of Paget's disease, whereas claudin-5 was seen in 50% of cases and claudin-2 was seen in 32% of cases. In contrast, claudin-3, claudin-4, and claudin-5 were not seen in the other skin lesions, and claudin 2 was seen in most of them, suggesting an inverse expression of these claudins between Paget's disease and epidermal and nevocytic lesions. Claudin expression in breast carcinomas was claudin-2 in 52%, claudin-3 in 93%, claudin-4 in 92%, and claudin-5 in 47%. Claudins-2 and claudin-5 were found more often in ductal carcinomas than in lobular carcinomas. Expression of claudins were frequently associated with each other. They were not associated with estrogen or progesterone receptor status or with tumour grade. No significant differences were found between claudin expression in Paget's disease and breast carcinomas. The results demonstrate that claudins could be useful in diagnosing Paget's disease and in differentiating these lesions from other epidermal lesions, such as actinic keratoses, basal cell carcinomas, and nevocytic lesions. The lack of difference in claudin expression between Paget's disease and breast tumours suggests that changes in the phenotype of claudin-2, claudin-3, claudin-4, and claudin-5 are not necessary for epidermal invasion.

Claudin-16 (Paracellin-1), Ponsin, ZO-2, AF6, Vinculin, and Nectin are reduced with poor prognosis of patients with breast cancer, however JAM-2 does not show differences in expression (117). The levels of transcripts of claudin-16 and vinculin were significantly lower in patients that had poor prognosis (with metastasis, recurrence, or mortality), compared with those that remained healthy after a median follow-up of 72.2 months Immunohistochemistry confirmed these results, as there is decreased levels in staining for claudin-16 and AF6. In normal tissue, staining was confined to the intercellular regions whereas in the tumour tissues the staining was diffuse and cytosolic. The conclusion was that low levels of TJ molecules claudin-16 and vinculin in breast cancer are associated with poor prognosis in patients, underscoring the idea that regulation of TJs could be of fundamental importance in the prevention of metastasis of breast cancer cells.

Martin and Jiang (118) investigated the expression of occludin in human breast cancer tissues and cell lines. Tissues and breast cancer cell lines were amplified for functional regions of occludin. 6/6 tumour tissues showed truncated and/or variant signals for N-terminal and first trans-membrane loop of occludin; 4/6 tumour tissues did not express the C-terminal region of Occludin. Paired background tissues showed similar expression profiles. None of the breast tissues showed methylation of the occludin promotor. Of the 10 human breast cancer cell lines, 3 did not express the N-terminal, 6 expressed 2 or more variants; 3 did expressed a truncated message for the first trans-membrane loop, 5 expressed the correct message, MDA-MB-231 cells did not express this region; the C-terminal region was expressed correctly in 3 cell lines, 4 expressed variants, and 4 were missing this region. Overall, only 3/10 breast cancer cell lines expressed full length occludin; interestingly, these were of the more invasive phenotype. Methylation of the promotor was not observed. Western blotting also demonstrated variants of occludin when probed with 3 antibodies specific for the N-terminus, first membrane loop and C-terminus. These variants did not fit the expected occludin signals for changes in phosphorylation status of the protein. Immuno-staining showed similarly disparate levels of expression, with more invasive cell lines showing reduced cell junction location. This study showed for the first time that occludin is differentially expressed in breast tumour tissues and in human breast cancer cell lines. The changes in occludin message indicate that variants are expressed in tumour tissue. The loss of or truncation of the N-terminus indicates reduced assembly of TJ structure and reduced maintenance of barrier function. Loss of C-terminal expression suggests reduced intracellular trafficking of occludin to the basolateral membrane and binding to ZO-1, resulting in reduced TJ anchoring, assembly, and cell-cell adhesion. This has clear repercussions as to the importance of occludin in maintaining TJ integrity in breast tissues. Such inappropriate expression may play a part in breast cancer development.

4.2.2 Peripheral Plaque protein expression in breast cancer

ZO-2 can be expressed in two isoforms, ZO-2A and ZO-2C, in normal epithelia. ZO-2A is absent in pancreatic adenocarcinoma of the ductal type, with none of the common mechanisms of gene inactivation responsible (13). Analysis of the ZO-2 promotors (PA and PC) showed that lack of expression of ZO-A in neoplastic pancreatic cells is caused by inactivation of the downstream promoter PA, probably due to structural or functional alterations in the regulatory elements localised outside the analysed promoter region as hypermethylation was not a convincing reason in early cancers. However, methylation of PA is responsible for the inactivation of the suppressed promoter at the late stages of tumour development (111). ZO-2 was found to be deregulated in breast adeno-carcinoma, but not in colon or prostate adenocarcinoma, both of which are considered to be of acinar rather than ductal type.

MAGUKs may play a vital role in cellular functions preventing tumori-genesis as indicated by neoplastic phenotypes in Drosophila; Normal breast tissues have shown the expected intense staining at cell-cell junctions; however, ZO-1 staining is found to be reduced or lost in 69% of breast cancers analysed using immunohistochemistry (12). Normal tissue showed intense staining for ZO-1 at the position of the epithelial TJs, but this was lost or reduced in 69% of breast cancers analysed. In infiltrating ductal carcinomas there was a reduction in staining in 42% of well-differentiated, in 83% of moderately differentiated and in 93% of poorly differentiated tumours. ZO-1 was positively correlated with tumour differentiation, and more specifically with the glandular differentiation of tumours. The ZO-1 gene tjp-1 was mapped relative to other markers flanking the gene. There was a loss of heterozygosity in 23% of informative tumours. Loss of a tjp-1-linked marker suggests that genetic loss may, in some cases, be responsible for a reduction in ZO-1 in breast cancer.

In 18 breast cancer cell lines, the most poorly differentiated, fibroblastic cell lines were ZO-1 negative, and were highly invasive (119).

Martin et al. (16) investigated the expression of Zonula Occludens (ZO) proteins ZO-1, ZO-2, and ZO-3, and MUPP-1 in patients with primary breast cancer (Figure 4). Breast cancer primary tumours. Standar dised transcript levels of ZO-1 and MUPP-1 were significantly lower in patients with metastatic disease compared with those remaining disease-free (median follow-up 72.2 months). Immunohistochemistry confirmed these results, with decreased levels in ZO-1 staining. For both ZO-1 and ZO-3, staining was confined to the intercellular regions in normal tissue, whereas in tumour tissues staining was diffuse and cytosolic. Q-PCR revealed a reduction in the levels of ZO-1 and MUPP-1 in patients with disease recurrence. Prognostic indicators of breast cancer were also inversely correlated with ZO-1 expression. It was concluded that low levels of TJ plaque molecules, such as ZO-1 and MUPP-1, in breast cancer are associated with poor patients prognosis.

Figure 4. Panel shows the differential expression of peripheral/plaque proteins in representative sections from patients with breast cancer. (A) Immunohistochemical staining (x100) of ZO-1, ZO-2, and ZO-3 in human breast cancer tissues. Clear staining is shown in normal tissue (left), reduced staining for ZO-1 and ZO-3 shown in the right. (B) Western blotting of paired normal and tumour tissues and densimetric analysis. Total levels of ZO-1 and ZO-3 were seen in tumours (8/10). (C) Comparison of grade and RNA transcript level of plaque proteins. All four were reduced with increasing tumour grade. (D) Comparison of histology of primary tumours and RNA transcript level of plaque proteins. ZO-1 and ZO-2 were increased in lobular carcinoma compared to other types. ZO-3 was significantly reduced. MUPP-1 was significantly reduced in ductal carcinoma.

Figure 4. Panel shows the differential expression of peripheral/plaque proteins in representative sections from patients with breast cancer. (A) Immunohistochemical staining (x100) of ZO-1, ZO-2, and ZO-3 in human breast cancer tissues. Clear staining is shown in normal tissue (left), reduced staining for ZO-1 and ZO-3 shown in the right. (B) Western blotting of paired normal and tumour tissues and densimetric analysis. Total levels of ZO-1 and ZO-3 were seen in tumours (8/10). (C) Comparison of grade and RNA transcript level of plaque proteins. All four were reduced with increasing tumour grade. (D) Comparison of histology of primary tumours and RNA transcript level of plaque proteins. ZO-1 and ZO-2 were increased in lobular carcinoma compared to other types. ZO-3 was significantly reduced. MUPP-1 was significantly reduced in ductal carcinoma.

ZO-1 can upregulate HER-2/neu expression in vitro by sequestering a repressor of the Her-2/neu gene promoter (14). ZO-1 was examined in a series of breast cancers: one group contained those invasive cancers scoring for HER-2/neu status (negative (12), 2+ (13) and 3+ (10)) and we re analysed by IHC: ZO-1 expression did not correlate with HER-2/neu expression in breast carcinomas, and so other causes of HER-2/neu overexpression should be sought. Interestingly, the authors report that ZO-1 IHC stained DCIS were positive for ZO-1 in 18/20 cases, with 4/18 negative for ZO-1 in the invasive tumour.

Interestingly, the nectin family has been little studied as regards TJs in cancer, being originally described as molecules involved in adherens junctions only. Recently however, it has become apparent the nectins are also involved in recruitment and maintenance of proteins within the TJ. Studies have shown that nectin-3 expression showed clear changes in distribution between normal and cancerous cells (120). However, there was little difference in overall expression when analysed by Q-PCR. Breast cancer cell lines screened showed aberrant expression for nectin-3. nectin-3 transformed cells showed retarded invasion, even when treated with HGF. Invasion was significantly different between these cells and the wild type and when treated with HGF. Moreover, these transformed cells were significantly less motile and more resistance to HGF-induced reduced TJ functionality. As anticipated, breast cancer cells with endogenous nectin-3 knocked out using ribozyme technology showed both increased invasiveness and motility. The staining pattern in human breast cancer tissues indicated that the distribution of the molecule is more crucial than the level of expression. The introduction of nectin-3 into human breast cancer cells results in breast cancer cells with reduced invasive phenotype and increased TJ function; conversely, breast cancer cells with nectin-3 knockout showed increased invasion and motility. This, together with the reported aberrant expression of other nectins in human cancer, indicates that nectin-3 may be a key component in the formation of cell-cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells (120).

4.3 Regulation of TJs in Breast Cancer

Application of all transretinoic acid correlates well with paracellular barrier function of endothelial cells, significantly reducing the rate by which tumour cells transmigrate across the endothelial cell monolayers (8, 113). Such experiments (6) suggest that TJs of vascular endothelium in vivo function as a barrier between blood and tissues against metastatic cancer cells. Dexamethasone induces "normal-like" differentiated property of TJ formation, and suppresses growth of the rat Con8 mammary epithelial tumour cell line (121).

An early paper looked at metastatic, weakly metastatic, and parent clones spontaneously developed from rat mammary carcinoma (7). EMs were used to look at TJ formation. When highly and weakly metastatic clones were co-cultured with normal fibroblasts, TJ structures were observed only in the weakly metastatic clones. Ultrastructural differences were related to the proliferation and detachment of tumour cells from the primary site in the initial stage of tumour metastasis (7).

In Con8 rat mammary epithelial tumour cells, dexamethasone stimulates TER, promotes remodelling of apical junctions, and downregulates the level of fascin, an actin-bundling protein that can bind beta-catenin. It has been shown that TGFa ablates dexamethsone-induced remodelling of the apical junction and stimulation of TER (122). This response was polarised in that basolateral but not apical exposure reversed fascin production and TJ formation. The authors propose the regulation of fascin protein levels as a target of cross-talk between the Ras-dependent growth factor signalling and dexamethasone (glucocorticoid) signalling pathways that control TJ dynamics in mammary epithelial tumour cells.

Hyperactivation of the insulin-like growth factor I receptor (IGF-IR) contributes to primary breast cancer development, but its role in metastasis is unclear. IGF-IR overexpression markedly stimulates aggregation in E-cadherin positive MCF-7 cells, but not in E-cadherin negative MDA 231 cells (123). IGF-IR-dependent cell-cell adhesion of MCF-7 cells coincided with the upregulation of ZI-1. ZO-1 expression (mRNA and protein) was induced by IGF-I and was blocked in MCF-7 cells with a tyrosine kinase-defective IGF-IR mutant. ZO-1 associates with the E-cadherin complex (immunoprecipitation) and IGF-IR. High levels of ZO-1 coincide with increased IGF-IR/alpha-catenin/ZO-1-binding and improved ZO-1/actin association, whereas downregulation of ZO-1 by anti-ZO-1 RNA inhibited IGF-IR-dependent cell-cell adhesion. The results are suggestive of a mechanism by which activated IGF-IR regulated E-cadherin-mediated cell-cell adhesion by over-expression of ZO-1 and the resulting stronger connections between the E-cadherin complex and the actin cytoskeleton. IGF-IR may thus provide an anti-metastatic effect in E-cadherin positive breast cancer cells.

It has been demonstrated that involvement of the Ras-MEK-ERK pathway is likely not involved in the dysregulated TJ formation in breast tumour cells and indicates that elevated activity of Ras might not be of general importance for the disruption of TJ structures in breast tumours

(124). Constitutive activation of Ras of Ras-mediated signalling pathways is one of the initial steps during tumorogenesis that promotes neoplastic transformation.

Clostridium perfringens enterotoxin (CPE), induces cytolysis very rapidly through binding to its receptors, the TJ proteins claudin-3 and -4

(125). In primary human breast cancers (21) claudin-3 and claudin-4

were both detected and compared to normal mammary epithelium, were over-expressed in approximately 62% - 26%, respectively. Treatment of breast cancer cell lines with CPE resulted in a dose-dependent cytolysis exclusively in cells expressing claudin-3 and claudin-4. In vivo models demonstrated a significant reduction on volume with accompanying necrosis after CPE treatment. Necrotic reaction was also observed in fre sh resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast cancers underwent rapid and complete cytolysis within 1 hour. Thus, expression of claudin-3 and claudin-4 sensitises breast carcinomas to CPE-mediated cytolysis and emphasises the potential of CPE in breast cancer therapy.

Although most malignant tumours are epithelia-derived carcinomas, methods for specific and effective delivery of anti-tumour agents to carcinomas have not been developed. Recent reports indicate that epithelia overexpress claudin-3 and claudin-4, which are integral membrane proteins of epithelial TJs. This suggests that claudins can be targeted for tumour therapy, but there is not currently a method for delivering drugs to claudin-expressing cells. It was evaluated whether a potent claudin-4-binding C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) would allow targeting to claudin-4-expressing cells (126). C-CPE was fused to the protein synthesis inhibitory factor (PSIF), which lacks the cell-binding domain of Pseudomonas exotoxin. This fusion protein, C-CPE-PSIF, was cytotoxic to MCF-7 human breast cancer cells, which express endogenous claudin-4, but it was not toxic to mouse fibroblast L cells, which lack endogenous claudin-4. The cytotoxicity of C-CPE-PSIF was attenuated by pretreating the MCF-7 cells with C-CPE but not bovine serum albumin. Also, deletion of the claudin-4-binding region of C-CPE reduced the cytotoxicity of C-CPE-PSIF. Finally, it was found that C-CPE-PSIF is toxic to L cells expressing claudin-4 but not to normal L cells or cells expressing claudin-1, claudin-2, or claudin-5. These results indicate that use of the C-CPE peptide may provide a novel way to target drugs to claudin-expressing cells (126).

HGF, a cytokine secreted by stromal cells, is capable of modulating expression and function of TJ molecules in human breast cancer cell lines (127). HGF decreases transepithelial resistance and increases para-cellular permeability of human breast cancer cell lines, MDA-MB-231 and MCF-7. Q-PCR shows that HGF modulates the levels of several TJ molecule (occludin, claudin-1 and claudin-5, JAM-1 and JAM-2) mRNA transcripts in MDA-MB-231 and MCF-7 cells. Western blotting and immunohistochemistry also showed modulation of expression of the TJ molecule, occludin. It is suggested that HGF disrupts TJ function in human breast cancer cells by effecting changes in the expression of TJ molecules at both the mRNA and protein levels. The conclusion was that regulation of TJs could be of fundamental importance in the prevention of metastasis of breast cancer cells (Figure 5).

Figure 5. Effect of HGF on expression of TJ molecules in human breast cancer cells. (A) Reduction of transepithelial resistance by HGF. Changes effected by HGF could be inhibited by NK4, the HGF antagonist. (B) Modulation of TJ molecules by HGF as assessed using Quantitative-PCR. Results are expressed as transcript copy/50 ng/RNA. (C) Western blots of TJ proteins after HGF treatment. (D) HGF and the increased phosphorylation status of ZO-1 in human breast cancer cell lines. This suggests deactivation of ZO-1 by HGF. (E) Immunostaining of human breast cancer cell lines treated with HGF for 1 hour. Cells were stained with ZO-1 or Occludin. MCF-7 cells, but not MDA-MB-231 cells showed typical TJ pattern staining for ZO-1 and Occludin at 0 hour. HGF reduced staining of both by 1 hour. Both cell lines show increased cytosolic staining and relocation of Occludin and ZO-1 to ruffled membrane areas.

Figure 5. Effect of HGF on expression of TJ molecules in human breast cancer cells. (A) Reduction of transepithelial resistance by HGF. Changes effected by HGF could be inhibited by NK4, the HGF antagonist. (B) Modulation of TJ molecules by HGF as assessed using Quantitative-PCR. Results are expressed as transcript copy/50 ng/RNA. (C) Western blots of TJ proteins after HGF treatment. (D) HGF and the increased phosphorylation status of ZO-1 in human breast cancer cell lines. This suggests deactivation of ZO-1 by HGF. (E) Immunostaining of human breast cancer cell lines treated with HGF for 1 hour. Cells were stained with ZO-1 or Occludin. MCF-7 cells, but not MDA-MB-231 cells showed typical TJ pattern staining for ZO-1 and Occludin at 0 hour. HGF reduced staining of both by 1 hour. Both cell lines show increased cytosolic staining and relocation of Occludin and ZO-1 to ruffled membrane areas.

Ye et al. (128) sought to determine the role of oestrogen in the regulation of TJs and expression of molecules making TJs in endothelial cells. Human endothelial cell, HECV, which express ER-beta but not ER-alpha was used. 17beta estradiol induced a concentration- and time-dependent biphasic effect on TJ. At 10(-9) and 10(-6) M, it decreased the level of occludin and increased in paracellular permeability of HECV cells, but at 10(-12) M it decreased in paracellular permeability and increased the level of occludin. The transendothelial electrical resistance (TER), however, was reduced by 17beta estradiol at lower concentrations (as low as 10(-12) M). Furthermore, the time-dependent biphasic effect was observed over a period of 4 days, with the first reduction of TER seen within 15 minutes and the second drop occurring 48 hours after 17beta estradiol treatment. It was further revealed that protein and mRNA levels of occludin, but not claudin-1 and -5, and ZO-1, were reduced by 17beta estradiol, in line with changes of TER. This study shows that 17beta estradiol can induce concentration- and time-related biphasic effects on TJ functions expression of occludin in endothelial cells and that this perturbation of TJ functions may have implications in the etiology of mastalgia and the vascular spread of breast cancer.

Glucocorticoid hormones stimulate adherens and TJ formation in Con8 mammary epithelial tumour cells through a multi-step process in which the membrane organisation of structural apical junction proteins and TJ sealing is controlled by specific signal transduction components. Dexa-methasone stimulation of apical junction formation requires downregula-tion of the small GTPase RhoA. Rnd3/RhoE, a GTPase-deficient Rho family member and RhoA antagonist was defined as a key regulator of apical junction dynamics. Exogenously expressed Rnd3/RhoE co-localised with actin at the cell periphery and induced the localisation of the adherens junction protein p-catenin and the TJ protein ZO-1 to sites of cell-cell contact, and led to the formation of highly sealed TJs. Treatment with glucocorticoids was not required to achieve complete apical junction remodeling. Consistent with Rnd3/RhoE acting as an antagonist of RhoA, expression of Rnd3/RhoE rescued the disruptive effects of constitutively active RhoA on apical junction organisation. Therse results demonstrate a new role for the Rho family member Rnd3/RhoE in regulating the assembly of the apical junction complex and TJ sealing (129).

Transforming growth factor beta (TGF-beta) facilitates metastasis during the advanced stages of cancer. Smad6, Smad7, and c-Ski block signaling by the TGF-beta superfamily proteins through different modes of action. Expression of Smad7 in JygMC(A) cells was associated with increased expression of major components of adherens and TJs, including E-cadherin, decreased expression of N-cadherin, and decreases in the migratory and invasive abilities of the JygMC(A) cells. Smad7 inhibits metastasis, possibly by regulating cell-cell adhesion. Systemic expression of Smad7 may be a novel strategy for the prevention of metastasis of advanced cancers (130).

Snail and Slug bind to the E-box motifs present in the human Claudin-1 promoter (131). Moreover, an inverse correlation in the levels of Claudin-1 and Slug transcripts were observed in breast cancer cell lines. E-box elements in the Claudin-1 promoter were found to play a critical negative regulatory role in breast cancer cell lines that expressed low levels of Claudin-1 transcript. Significantly, in invasive human breast tumours, high levels of Snail and Slug correlated with low levels of Claudin-1 expression. Taken together, these results support the hypothesis that Claudin-1 is a direct downstream target gene of Snail family factors in epithelial cells.

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