| Breast cancer is one of the most common malignant tumors and the first killer of women in the world. Breast cancer metastasis is a hallmark of tumor and the principal cause of cancer-related mortality.Meanwhile, patients with advanced breast cancer always suffered pain from cancer metastasis, and thus the quality of their life is seriously affected. Therefore, understand the mechanism of breast tumorigenesis, tumor progression and metastasis is necessary and will bring a novel maneuver of treatment for breast cancer.Extracellular nucleotides such as UDP, UTP and ATP are accepted as the substrates of RNA and DNA synthesis, and it is reported that extracellular nucleotides exert a series of physiological functions through several kinds of P2Y receptors in many tissues and cells, including translating cell signaling, participating in immune response, and regulating cell proliferation, differation and apototics. P2Y6as a GPCR have essential roles in physiological and pathological processes, including cancer growth and metastasis. It is generally believed that nucleotides, released by apoptotic cells act as a "find-me"and "eat me"signal, coupled to P2Y receptors to promote phagocytic clearance. A great of studies have shown that GPCRs and the GPCR ligands express aberrantly in cancer. However, the function and mechanism of P2Y6receptor in tumor metastasis has not been clarified.This study aims to find the relationship between P2Y6and breast cancer progression. Finally, it will facilitate the development of new potential targeted drugs for clinical application and be useful as diagnostic or prognostic targets of human breast cancers.After the comparison of the expression of P2Y6in primary tumor and adjacent non-malignant tissues from breast cancer patients with different aggressive levels by immunohistochemistry, we find that P2Y6is closely related to the breast cancer tumorgenesis and progression:The expression of P2Y6can only be found in aggressive breast cancer samples, and hard to be detected in the control samples taken from adjacent non-malignant tissues or the normal breast tissues. Furthermore, we used Real-time PCR to confirm our result in normal breast cell and breast cancer cell lines, the result support our finding, P2Y6expressed more in breast cancer cells, and has low expression in normal cell (MCF-10A).In order to understand the function of P2Y6receptor in breast cancer, we use different concentration of UDP and MRS2578to treate the breast cancer cells, and found UDP promote cells migration and invasion, and this kind of promotion could be inhibited by MRS2578. In order to investigate the influence of P2Y6on breast cancer metastasis in vivo, firefly luciferase plasmid transfected MDA-MB-231were injected into the left ventricle to set up the in vivo breast cancer metastasis mouse model. We also find UDP promote breast cancer metastasis and MRS2578suppress breast cancer metastasis. So we conclude that P2Y6receptor could promote breast cancer metastasis in vitro and in vivo. And this kind of promotion is inhibited by MRS2578.To explore the underlying mechanism of P2Y6receptor in promoting metastasis, RT-PCR, Zymography, Immunofluorescence, Pull-down, Western blotting and Luciferase assay were used to analysis the signaling involved in tumor metastasis. Firstly we found that the activity of MMP-9and the ratio of protrusion formation were significantly increased by UDP and suppressed by MRS2578, but the activity of MMP-2which also play a very important role in tumor metastasis were little affected. Meanwhile, the phosphorylation of MAPKs, IκBα and the activation of GTPases which involved in protrusion formation and tumor metastasis were dramatically influenced by P2Y6receptor in MDA-MB-231cells. We implicate that the activation of AP-1and NF-κB were also influenced by P2Y6receptor.Taken together, our results indicated that the expression of human P2Y6increased in breast cancer tissues or cells. P2Y6receptor could be acting as a novel receptor promotes breast cancer metastasis by increasing the activation of MMP-9through AP-1and NF-κB associated signaling pathway and inducing RhoGTPases-dependented protrusion formation at the leading edge. |
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