Infiltrating ductal carcinomas of the breast are believed to arise from the terminal mammary duct. During the process of tumorigenesis and development, mammary epithelial cells acquire mesenchymal characteristics (epithelial/mesenchyme transition, EMT), with altered cell polarity and adhesion, which contributes to the dissemination of tumor cells through blood vessels and lymphatics from the original site [18
]. Current reports suggest that tight junction protein expression or locations of specific tight junction-associated expression are altered before the initiation of EMT [19
Tight junctions are located at the extreme apical region of the junction complex in epithelial and endothelial cells, where they can seal intracellular gaps, maintain cell polarity, adhesion, and permeability as well as regulate cell proliferation and differentiation [1
Tight junctions mainly consist of CLDNs, occludin, junctional adhesion molecules. However, CLDNs are major constituents and determine the barrier properties of tight junctions [18
]. CLDN6, one of 24 members of the CLDN family, was first identified by searching for sequences in genomic databases [5
]. CLDN6 is similar to CLDN1, 7, 8, 10, 14, 15, 17, and 19 [20
], as it four transmembrane domains, and two extracellular loops [21
]. CLDN6 is located on chromosome 16 p13.3 and encodes a 23 kDa membrane protein of 219 amino acids [5
]. Recent reports show that CLDN6 plays important roles in cells under both physiological and pathological conditions. For example, CLDN6 can regulate adipogenesis and fat deposition [22
], it is required for normal blastocyst formation [23
], and its expression occurs in early development, with particular attention to definitive endoderm derivatives [24
]. CLDN6 is expressed very early in epidermal morphogenesis [1
], and it is important for maintaining permeability barriers and transepithelial resistance in epidermal cells [11
The current study herein has shown that CLDN6 was silenced in esophageal squamous cell carcinoma [25
]. In our previous work, we have identified CLDN6 as a potential mammary cancer suppressor gene, which may contribute to the mammary cancer resistant phenotype observed in Copenhagen rats, and CLDN6 expression was undetectable or at low levels in human and rat mammary cancer cell lines [12
In addition, we demonstrated that MCF-7 cells transfected with CLDN6 grow slower, show an increased rate of cell death, have reduced 2-D and 3-D colony-forming ability, and decreased invasiveness and cell migration [13
]. These results suggest that CLDN6 could have beneficial effects for inhibiting carcinogenesis and the malignant progression of certain types of breast cancers. Our further studies found that 17β-estradiol upregulates CLDN6 mRNA and protein expression in MCF-7 cells [15
]. However, the regulatory mechanisms involved in the process of 17β-estradiol induced CLDN6 expression are not fully understood.
A significant association exists between CLDN1, 3, and 4, and ERs in breast cancers [16
]. However, to our knowledge, no information is available about CLDN6. So, we examined CLDN6 and ERs expression in the tissues of 80 patients with breast cancers using immunohistochemistry, where the results showed a significant association between CLDN6 and ERα expression. In 50 ERα-positive tumors, 25 (50%) stained positive for CLDN6, but only eight of 30 ERα-negative tumors (27%) were positive for CLDN6 (). But no significant association was observed between CLDN6 and ERβ expression (). Previous reports have shown that ERα-negative tumors represent a more invasive phenotype and a poorer prognosis compared with ERα-positive breast cancers [26
]. The decreased expression of CLDN6 protein in the ERα-negative breast cancers was coincident with our hypothesis that CLDN6 acts as an anti-oncogene, and its expression might be regulated by estrogen via ERα.
A recent report shows that 17β-estradiol has biphasic effects on occludin expression [17
]. In our previous study, we found that 17β-estradiol significantly upregulated CLDN6 expression with a maximal effect at 5×10-9
mol/L for 24 hours and that the effect of 17β-estradiol could be blocked by the ERs antagonist ICI [15
]. To investigate which ER subset was involved in the process of 17β-estradiol induced CLDN6 expression, we examined the expression of ERs in MCF-7 cells. Similar to an earlier report, MCF-7 cells expressed ERα, but not ERβ () [17
]. The ERα agonist PPT and the ERs antagonist ICI were employed in the present study, and the results revealed that CLDN6 mRNA and protein expression could be induced by PPT (), and that the induction could be blocked by ICI (). We detected the location of CLDN6 protein in MCF-7 cells using immunofluorescent staining, but only the MCF-7 cells treated with 17β-estradiol or (and) PPT showed CLDN6 expression located at the plasma membranes ().
Estrogen has a wide array of cell and tissue-specific effects. The biological effects of estrogen are mediated by ERα and ERβ [27
], which are members of a large superfamily of nuclear receptors regulating the expression of target genes via two pathways. The first is a genomic pathway, and the second is non-genomic pathway The latter is mediated by membrane-associated ERs and occurs so rapidly that the effects of estrogen do not depend on RNA and protein synthesis activation [28
]. In the classical genomic pathway, estrogen binds to nuclear receptors, the receptors dimerize and bind to specific response elements known as estrogen response elements (EREs) located in target gene promoter [28
]. However, a number of studies have shown that ERs can regulate the expression of several genes without binding directly to DNA, but through modulating the function of other transcription factors by protein-protein interactions in the nucleus [29
]. The interaction of ERs with the activator protein 1 (AP-1) transcription factor complex is an example of such an ERE-independent genomic actions [30
]. This mode of activation regulates the expression of specific downstream genes and may require hours to days in a single cell type [29
]. According to our previous works, 17β-estradiol could upregulate CLDN6 mRNA and protein expression in MCF-7 cells after 24 hours [15
]. In this study, PPT induced CLDN6 mRNA and protein expression at 24 hours (), indicating that estrogen could modify the expression of CLDN6 mRNA and protein through a genomic pathway mediated by ERα.
Our study provides the first direct evidence that CLDN6 expression is associated with ERα in breast cancer tissues, and that PPT could induces CLDN6 mRNA and protein expression in MCF-7 cells. A further study showed that this effect could be blocked by the ER antagonist, ICI. We concluded that estrogen could induce CLDN6 expression via an ERα pathway in breast cancer tissues and MCF-7 cells.