Role Of Chemokine XCL1 - XCR1 Biological Axis In Breast Cancer And Its Relationship With

Part I ERa takes part in the regulation of XCLl in XCR1 positive breast cancerPurpose:To study the crucial involvement of Estrogen receptors ER alpha (ERa) in the regulation of XCL1 in XCR1 positive breast cancer.Methods:We used Kaplan Meier Plotter software to predict the influence of XCL1 and its receptors XCR1 on diseases-free survival (DFS) of ERα- and ERα+ breast cancer patients. XCL1 and XCR1 expressions were detected in 9 kinds of human breast cancer cell lines (MDA-MB-231, MDA-MB-468, MDA-MB-231HM, MDA-MB-231BO, MCF-7, T47D, BCaP-37, ZR-75-30, SK-BR-3) and 1 kind of normal breast epithelial cell line (MCF-10A). The effects of XCL1 on the proliferation in vitro were tested by CCK8 with or without the co-culture of selective ER down regulator fulvestrant (ICI 182 780). ELISA was used to confirm the best reaction time of XCL1 in breast cancer cell MCF-7. The laser confocal microscopy analysis was used to detect the position of XCL1 and ERa. Immune co-precipitation experiment was used to detect the direct combination of XCL1 and ERα. Protein level of relative molecules in cell lines was examined by Western blot.Results:Our results indicated that XCL1 can prolong the DFS of ERα- breast cancer patients, and have no effect on DFS of ERα+ breast cancer patients. However, XCR1 have no apparent effect on the DFS of ERa" or ERα+ breast cancer patients. XCL1 expression is increased in MDA-MB-231 and ZR-75-30 compares with other cell lines and the normal breast epithelial cell line (MCF-10A). But XCR1 expression is higher in ERαa+ cell lines (MCF-7, T47D, BCaP-37, ZR-75-30) than that in ERα- cell lines (MDA-MB-231, MDA-MB-468). Immunohistochemical analysis of clinical specimens from the patients with breast cancer validate the correlation between XCR1 and ERa. MCF-7 had the biggest proliferative response to XCL1 at concentration of 200 ng/ml. Fulvestrant could decrease this growth effect through reducing the activity of MAPK signaling pathways. After adding the rhXCLl, total ERK and MEK protein had no obvious change, but pERK and pMEK were apparently up-regulated. However after the treatment with fulvestrant, pERK and pMEK expression were reduced. Some reports indicated that there may be the combining site between XCL1 and ERa. In order to conform this, we used laser confocal microscopy to detect the relation of their position, the result showed XCL1 and ERa were in the same position but could not combine together directly, suggesting there may exist the indirect binding protein between XCL1 and ERa.Conclusion:Our results suggest XCR1 expression is associated with ERa expression for high XCR1 expression in ERa positive human breast cancer cell lines and breast cancer specimens. XCL1 can promote cell proliferation in ERa positive human breast cancer cell line MCF-7 by activating MAPK signal transduction pathway. Fulvestrant which down-regulates the activation of ERa may decrease this growth effect. This suggests the crosstalk between the XCL1-XCR1 biological axis and estrogen receptor. XCL1 may be one of ERa downstream effect molecules. In addition, high XCL1 expression in human breast cancer cell lines MDA-MB-231HM indicates that XCL1 may play a key role in tumor metastasis.Part II The role and mechanism of XCR1 over-expression in the proliferation, invasion, metastasis and tumorigenesis in vivo of XCR1 negative breast cancer cell linesPurpose:This study is mainly to explore the role of XCR1 over-expression in breast cancer cell proliferation, invasion, metastasis, tumorigenesis in vivo and exam the relative mechanism.Methods:We established XCR1 over-expressing breast cancer cell line MDA-MB-231 (231/XCR1) in XCR1 low expression cell line MDA-MB-231 (231). The function of proliferation, invasion and metastasis were measured by CCK8, plate cloning formation and Transwell analysis respectively in 231/XCR1 and its parental cell line 231. mRNA level was examed by real-time PCR.5×106/100 u 1 cells were inoculated in mammary fat pad of BALB/c nude mice. There were 6 BALB/c nude mice in the experimental group and control group. Finally, we used Kaplan Meier Plotter software to predict the relationship between XCR1 and DFS in the patients in ERα+breast cancer with lymph node metastasis. Protein expression was analyzed by cell immunofluorescence and Western blot.Results:Real-time PCR, RT-PCR and Western blot results verified the establishment of XCRl over-expressing cell line 231/XCRl both in mRNA expression and protein level. Compared with control group, reduced cell proliferation and clone formation, increased cell migration and invasion were associated with XCRl over-expression (p<0.05). Western blot showed the proliferation inhibition effect may be related with reduced activation of MAPK and PI3K/AKT/mTOR signaling pathway, and the generation of p-P53 and LC3 to promote cell apoptosis. Immunohistochemistry of mice xenograft tumor also proved that p-P53 and autophagy protein LC3 are increased in 231/XCRl. At the same time, the β-catenin expression is decreased, thus promotes cell migration and invasion mediated by XCRl. In animal experiments, XCRl expression could reduce xenograft tumor size and tumorigenesis rate (50%). However lung metastasis in both experimental group and control group did not happen. Kaplan Meier Plotter software indicated XCRl expression influence the DFS in ERα+ breast cancer patients with lymph node metastasis, although it is not statistically significant, but still hints that XCR1 can be a poor prognosis factor of lymph node metastasis in ERα+ breast cancer patients.Conclusion:The growth of XCRl over-expressing human breast cancer cell line MDA-MB-231 in vitro was restrained and tumorigenesis in vivo was extenuated, its mechanism may involve in the inhibition of MAPK and PI3K/AKT/mTOR signalling pathway and increase of p-P53 and LC3 expression. However, over-expression of XCRl in human breast cancer cell line MDA-MB-231 in vitro can promote the migration and invasion by decreasing the protein level of β-catenin. Therefore, XCRl can affect the biological characteristics of breast cancer cells through complex signal transduction pathway.