Apoptosis signal-regulating kinase 1 is associated with the effect of claudin-6 in breast cancer
© Guo et al.; licensee BioMed Central Ltd. 2012
Received: 12 July 2012
Accepted: 20 August 2012
Published: 27 August 2012
Previous studies have demonstrated that claudin-6 functions as a cancer suppressor in human MCF-7 breast cancer cells. The growth inhibitory effect could be attributed to inhibition of cell proliferation and induction of apoptosis. The purpose of the current study was to examine the involvement of apoptosis signal-regulating kinase 1 (ASK1) in the anticancer effect of claudin-6.
Immunohistochemical analysis was performed to evaluate the ASK1 protein expression and the correlation between ASK1, claudin-6 and clinicopathological features in 85 samples of breast invasive ductal carcinomas (IDC). Western blotting and RT-PCR was carried out to examine the expression of ASK1 and claudin-6 in MCF-7 cell clones transfected with claudin-6.
Immunohistochemical analysis showed that ASK1 expression was significantly related with that of claudin-6 in breast invasive ductal carcinomas (P < 0.05). In addition, a positive correlation between ASK1 and C-erb B 2 protein expression was identified (P < 0.05). Western blotting and RT-PCR consistently revealed that the level of ASK1 protein and mRNA was upregulated in MCF-7 cell clones transfected with claudin-6.
Our data suggests, for the first time, that the ASK1 signal may play a positive role in the inhibitory effect of claudin-6 in breast cancer.
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Breast cancer is one of the most frequent and deadly cancers in women . Emerging evidence points toward a pivotal role of tight junction (TJ) in mediating tumorigenic growth of breast cancer [2, 3]. TJs are junctional complexes which mediate cell-to-cell adhesion in epithelial and endothelial cellular sheets , and which affect cell polarity and tight junction formation .
Claudins (CLDNs) constitute a family of integral membrane proteins and have been identified as prominent structural components of TJ strands [5, 6]. The CLDNs which include 27 members at least , encode 20-27 kDa proteins with four transmembrane domains and two extracellular loops . The expression of CLDNs is often different in various types of human tumor [9, 10]. Many studies have demonstrated that claudins may participate in several signal transduction pathways [11–15]. For instance, inhibition of c-jun NH2-terinal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) selectively modulates the expression of claudin-4, -8 and −9 to enhance TJ barrier function in mammary epithelial cells , And p38 MAPK activity is involved in the epithelial barrier dysfunction in which claudin-7 protein plays a major role . It is well known that apoptosis signal-regulating kinase 1 (ASK1) phosphorylates and actives both p38 and JNK pathway . ASK1 is a member of the MAPKKK family and functions as a promoting apoptosis gene in response to common pro-apoptosis stresses . However, there is little knowledge about the relationship of ASK1 and claudins, especially claudin-6.
In our previous study, we found that claudin-6 is preferentially expressed in mammary epithelial cells and functions as a potential breast cancer suppressor gene , which is supported by the follow-up study of Osanai . Recently, we have discovered that the low level expression of claudin-6 gene contributed to malignant progression of breast cancer . A previous study has described that breast cancer tissues also expressed lower levels of ASK1 compared with normal mammary tissues . Therefore, the purpose of the current study is to discover the relationship between ASK1 and claudin-6 in breast cancer and to explore the pathways involves the activation of ASK1.
Patients and tissue samples
Clinicopathological features and the expression of ASK1 in 85 IDC samples
ASK1 expression (n)
I ~ II
Lymph node metastasis
Human breast cancer cell line MCF-7 cell clones expressing an vector pcDNA3.1 (+) or claudin-6 were cultured as previously described .
Primers used for PCR
Sequence of primers
Size of PCR(bp)
Denaturation temperature (°C)
Western blotting analysis
The Western blotting analyses were performed as described previously . Primary antibodies included ASK1 (rabbit polyclonal antibody, 1:1000, Bioworlde Technology), claudin-6 (rabbit polyclonal antibody, 1:1000, Bioworlde Technology) and β-actin (mouse polyclonal antibody, 1:1000, Santa Cruz). Secondary antibodies for the detection: anti-rabbit IgG (1:2000, Proteintech Group) and anti-mouse IgG (1:2000, Proteintech Group).
Immunohistochemical staining was carried out as described in our previouspublications . The primary antibodies against ASK1 (rabbit polyclonal antibody, 1:200, Bioworlde Technology) and claudin-6 (rabbit polyclonal antibody, 1:200, Bioworlde Technology) were used. The negative controls were handled in the same way except PBS instead of primary antibody.
Positive-staining exhibits brown staining, Claudin-6 was shown on the cell membrane and/or cytoplasm in breast cancer tissues , and ASK1 expressed on the breast cancer cytoplasm according to the manufacturer’s Instructions of ASK1 antibody. Immunostaining was observed under light microscopy with 400× magnification, and positive cells, negative cells and total cells of five different visual fields were numbered in each specimen. Scoring was performed as follows: negative (−), <10% positive tumor cells; positive (+), ≥10% positive tumor cells.
All computations were carried out using the software of SPSS version 19.0 for Windows (SPSS Inc, IL, USA). Chi-Square test was used to examine categorical data. Unpaired t-tests were performed to evaluated data of target mRNA and protein. The data are presented as means ± standard deviation (SD) from at least three independent experiments. P < 0.05 was considered statistically significant.
Association of ASK1 expression with the clinicopathological features of breast invasive ductal carcinomas
Correlation between the expression of ASK1 and claudin-6 in breast cancer tissues
The correlation between the expression of claudin-6 and ASK1 in breast invasive ductal carcinomas
Claudin-6 expression (n,%)
Correlation between the expression of claudin-6 and ASK1 in breast cancer cells
In the previous study, We found that the expression level of claudin-6 was lower in two human breast cancer cells (MCF-7 and BT474) and one breast cancer sample than that in normal breast tissues . In addition, we also discovered the growth, migration and invasion of MCF-7 cells were inhibited by overexpression of claudin-6 . One report has shown that the expression of ASK1 is lower in breast cancer tissues than that in normal tissues . As we all known, ASK1 is regulated in response to various cellular stresses, including cell survival, proliferation, differentiation, and so forth. Therefore, in the current study, we attempted to elucidate the relationship between the expression of claudin-6 and ASK1 using clinicopathological features and classical prognostic factors in breast pathology, including the expression of immunohistochemical markers of prognostic significance (ER, PR, C-erb B 2) (Table 1). To the best of our knowledge, this is the first study to demonstrate an association between the protein expression of claudin-6 and ASK1 in a large series of breast invasive ductal carcinomas and the breast cancer cells.
We have previously found that the expression of claudin-6 was negatively correlated with lymphatic metastasis of breast IDCs , but we did not found the correlation between ASK1 expression and the lymphatic metastasis (Table 1). This may be mainly due to that the ASK1 signal pathway is not the only pathway to be regulated by claudin-6. Besides that, we found the correlation of claudin-6 and ERα (p = 0.033), and also discovered that ERα regulated claudin-6 in MCF-7 cells . We failed to find the correlation between ASK1 and ER. And the reason high likely is the cross-talk among different signaling pathways, as we discussed in the case of failing to discover the correlation of ASK1 and lymphatic metastasis. However, we revealed the correlation between ASK1 and C-erb B 2 (Table 1). These results indicate the role of C-erb B 2 in ASK1 signal pathway. We next analyzed the relationship of C-erb B 2 and claudin-6, but we found no relationship between them (data not shown). Therefore, these data suggest that the inhibitory effect of claudin-6 in breast cancer mainly results from the regulation of ASK1.
Besides analysis of the breast cancer tissues, we also analyzed the correlation of ASK1 and claudin-6 mRNA and protein in breast cancer cell lines. We have found claudin-6 was a anti-cancer gene in claudins family , and the up-regulation of claudin-6 has important clinical implication, but details of the mechanism was not clear. C-jun NH2-terinal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) signal pathway played a positive role in the process of claudin-4, -8 and −9 enhancing TJ barrier function in mammary epithelial cells . ASK1 actives JNK and p38 pathway and induces apoptosis in various cells through mitochondria-dependent caspase activation [18, 26–28]. ASK1 activation depends on its binding proteins such as TNF receptor-associated factors2/6 , DAXX , TRADD , RIP1 , and FADD . And several cellular proteins, for example, thioredoxin , Hsp90  and 14-3-3  were also reported to interact with ASK1 and inhibit ASK1 activity. Here, we demonstrated that ASK1 was upregulated when claudin-6 gene was transfected into MCF-7 cells (Figure 3). Therefore, the present study indicates that ASK1 signal participates in the pro-apoptosis function of claudin-6.
As a conclusion, our study suggests that the ASK1 expression is low in breast cancer, and the levels of ASK1 mRNA and protein expression are correlated with that of claudin-6. We have identified a novel mechanism responsible for the pro-apoptosis function of claudin-6, and ASK1 may become a target for breast cancer treatments. However, we still need further study to clarify the detail of this mechanism.
This study was supported by National Natural Science Foundation of China (Code: 81172499) and Science and Technology Development Plan of the Office of Science and Technology Project in Jilin Province (Code: 20100731).
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