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.