| Breast cancer, as we all known, severely endangers women’s health in the global world and its morbidity rank highest among other cancers in female of developed country. Unfortunately, in recent years, its morbidity is gradually raised in developing country. Treatment against breast cancer is usually operation, radiotherapy and chemotherapy. These therapies fail to limit the metastasis though they usually suppress the primary lesion effectively.Following development of research on cancer metastasis, certain signal molecules are up regulated in tumor microenvironment induced by cell injury by radiotherapy or chemotherapy which promote metastasis of cancer cells. As was mentioned above, this mechanism maybe one of reason that promote tumor metastasis after treatment. However, the study of this field is still limited.Many studies suggest that extracellular nucleotides play an important role in cells as a signal of cell injury. When cells infected by virus, met DNA damage or stimulated by environment pressure, nucleotide will be released from intracellular to extracellular and then recognized by P2 receptors, then the cell physiological function is changed. In addition, P2 receptors act an important role in proliferation, differentiation and migration of cancer. Only few study reported P2Y6 signal in tumor migration mediated by UDP. However the effect of P2Y6 in repairing DNA damage and immunologic function in innate immunity suggested the important role of P2Y6 in cell injury signaling. Earlier research in our laboratory found that UDP induced P2Y6 signal pathway activation and promote the breast cancer cell migration. This process is similar to the up regulating of tumor cancer metastasis after cell injury. Thus, we infer that there is closely releationship between extracellular UDP, receptor P2Y6 and the process of tumor metastasis after cell injury. To figure out the detail mechanism, we first analysised the alteration of P2Y6 in different tumor through GEO, EGA and TCGA datebase and then find that amplification ratio of P2Y6 in breast canceris is highest. Second, western blotting in various breast cancer cell lines showed that P2Y6 expression different from each others in cell line. Then tissue microarray obtained from clinical cases of breast cancer was analyzed by immunohistochemical staining. The results showed that P2Y6 expression rise in most tumor tissue and is associated with tumor stage and prognosis. These results show P2Y6 expression is likely to releated with breast cancer development and occurrence.Next, we used doxorubcicn to establish breast cancer cell injury model and confirm it by FACS. In this research, we found that concentration of UDP increased in supernatant of injuried breast cancer cells by fluorescence polarization assay. Subsequently, in order to confirm the pathway of UDP release, we used different channel inhibitor:CBX (Pannexin inhibitor), FFA (Connexin inhibitor) and NEM (exocytosis inhibitor). Results showed that CBX and FFA can inhibit UDP releasing. Further study showed that pannexin channel is opened in damaged cells by FACS. This can be concluded that UDP maybe released through pannexin and connexin channels in injured breast cancer cells. After then, transwell assay shows that supernatant of injured breast cancer cell can improve breast cancer cell migration and this process can be inhibited by P2Y6 specific inhibitor, MRS2578 and pannexin inhibitor, CBX. Then we confirmed that the injured cells mediated breast cancer cells metastasis is mainly throug UDP and P2Y6. To confirm the function of P2Y6 in breast cancer mouse model, we generated P2Y6 knockout mice by Crispr/Cas9 technology, and mating with MMTV-PyMt mice (a mouse model for breast cancer spontaneous metastasis) to obtain P2Y6 knockout MMTV-PyMt mice. Our research enrich the study on the function of extracellular nucleotide UDP during the tumor development and progression, contribute to the study of breast cancer migration mechanism and theoretical basis of drug development. |
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