Effect Of JQ1 On The Expression Of Fra1 Gene In Tumor - Associated Macrophages On The Growth And Metastasis Of Mouse Breast Cancer

Objective:Breast cancer is the most common invasive cancer in women. It affects about 12% women worldwide. Recruited by various cytokines and chemokines, TAMs in tumor microenvironment are derived from circulating monocytes or resident tissue macrophages, and lead an important role in tumor invasion, migration, angiogenesis, immune resistance activities. FOS related antigen-1(Fral) is an important downstream transcription factor of oncogenes activation, and involves in many biological processes including transcriptional and posttranscriptional regulation. In tumor microenvironment Fral is highly expressed in TAMs, which leads macrophages to M2 phenotype. JQ1, a cell-permeable small molecule inhibitor that binds competitively to acetyl-lysine recognition motifs, or bromodomains 4 (BRD4). Previous work shows that Fral expression can be inhibited by JQ1, and hence inhibited clone formation of breast cancer cell, and decreased ALDH+ cancer stem cells. In this study we investigated influence of Fral inhibited TAMs on breast tumor cell; and in vivo, we also explore therapeutic effect of breast tumor in site and lung metastasis in mice, which are treated by Fral inhibitor JQ1.Method:First we detect Fral expression in macrophages and tumor cells in vitro, and use Western blot to detect Fral protein expression inhibited by JQ1 and M1(NOS2)/M2(Argl) marker in TAMs; Cell Counting Kit-8 (CCK8) was performed to detect JQ1 toxicity on mouse macrophages (RAW 264.7). Reverse transcription & real-time polymerase chain reaction (RT-qPCR) was used to check the expression of M1(IL-1b, Nos2, MCP-1, TNF-a) and M2 (Arg1, Fizz, Fral, Yml) markers in macrophages. and flow cytometry was used to detect M2 cell proportion in macrophages after treated by JQ1; Transwell test was performed to confirm 4T1 migration affected byJQl treated RAW264.7. In vivo,4T1 tumor cells transplanted mice were used to test therapeutic effect of JQ1 and combined therapy with Taxol.Results:1.Fral protein is widely expressed in RAW264.7, and in THP1, U937,4T1, MDA-MB-231,Hut78.2.JQ1 can inhibit Fral in cell RAW264.7.3.10nM is the appropriate concentration that can both inhibit Fral and have low toxicity to RAW264.74.M1 macrophage associated marker molecules(IL-1b、Nos2、Mcp1、TNF-α) are down-regulated in TAMs, and JQ1 can increase these molecules’ expression.5.M2 macrophage associated marker molecules(Arg1、Fizz1、Fra1、Ym1) are up-regulated in TAMs, and JQ1 can decrease these molecules’expression.6.JQ1 can up-regulated Nos2 and down-regulate Argl in TAMs.7.IL-4 can enhance CD206+ cells proportion in macrophage, while JQ1 can rescue(down-regulate) the proportion back to normal.8.Fra-1 inhibition in macrophages can decrease migration of 4T1 cells.9.JQ1, combined with Taxol, inhibits tumor growth and lung metastasis in 4T1 breast cancer model.Conclusions:Macrophages in which the expression of Fra1 was down regulated by JQ1 can suppress the M2 phenotype of macrophages, and decrease 4T1 tumor cell migration, and futher inhibits tumor growth and lung metastasis in mouse breast cancer model.