Abstract: Background and purpose: Increasing evidences have shown the key roles of chemokines in the formation and metastasis of cancers. In this study, we investigated the molecular mechanisms of liver metastasis from breast cancer and the effects of ulinastatin on liver metastasis from breast cancer mediated by CCL17/CCL22–CCR4 pathway. Methods: A mouse xenograft model and corresponding control were established by subcutaneously inoculating 4T1 breast cancer cells into mouse mammary fat pad. Fifteen days later, the mice were sacrificed, and the breast tumors were weighed. Immunohistochemistry was performed to detect CCR4 protein expression in breast tumors and CCL22 and CCL17 protein expressions in liver metastasis. The CCR4 gene was found to be inhibited by lentiviral transduction in 4T1 breast cancer cells. Western blot was used to examine the inhibitory effect. The same method was used to induce breast tumorigenesis and to examine tumor growth. Three different concentrations of ulinastatin were used to treat 4T1 tumor-bearing mice. Fifteen days later, immunohistochemistry was used to assess the expression of CCR4 in breast tumors and the expression of CCL22 and CCL17 in liver tissues. Western blot and real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) were used to detect the expression of TGF-β, microRNA-34a and microRNA-31. We also analyzed the correlation between TGF-β and microRNA-34a, microRNA-31, CCL22, and CCL17. Results: Our results showed that CCR4 was highly expressed in mouse breast tumors. CCL22 and CCL17 were highly expressed in the liver metastasis. CCR4 expression was silenced in the 4T1 breast cancer cells and the in vivo growth of breast cancer xenograft tumor was inhibited. Ulinastatin significantly inhibited CCR4, TGF-β, CCL22, microRNA-31 and CCL17, but upregulated microRNA-34a. Conclusion: These results showed that ulinastatin can inhibit liver metastasis of breast cancer. The specific mechanism may involve ulinastatin acting on TGF-β-microRNA-34a-CCL22 and microRNA-31-TGF-β-CCL17 axes to inhibit CCL17/CCL22-CCR4 signaling pathway in liver tissues.

Key words: Breast cancer, Liver metastasis, Uinastatin, CCR4, CCL22, CCL17