Chu Jian, Hu Xing-Chi, Li Chang-Chun, Li Tang-Ya, Fan Hui-Wen, Jiang Guo-Qin
Department of Surgery, The Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China; Department of General Surgery, The First People's Hospital of Yancheng City, Yancheng 224000, Jiangsu Province, China.
Department of General Surgery, The First People's Hospital of Yancheng City, Yancheng 224000, Jiangsu Province, China.
Cell Signal. 2022 Apr;92:110242. doi: 10.1016/j.cellsig.2022.110242. Epub 2022 Jan 6.
To study the functions and underlying network of KLF14 in breast cancer invasion and tumor-associated macrophages (TAMs).
The expressions of gene or protein were assessed by qRT-PCR and western blot assays, respectively. Cell proliferation and invasion were investigated by colony formation, CCK-8 and transwell assays, respectively. Macrophage M2 polarization was identified by flow cytometry assay. The methylation level was tested by methylation Specific PCR (MSP). The molecular relationship between KLF14 and SOCS3 was validated by dual luciferase and ChIP assays. In vivo model was established to confirm effect of KLF14 on tumor growth and metastasis.
KLF14 was downregulated in breast cancer, and its level was modified by CpG-mediated methylation. Overexpression of KLF14 significantly inhibited the proliferation and invasion of breast cancer in vitro and in vivo. Moreover, KLF14-overexpressing breast cancer cells notably reduced M2 macrophages polarization and it related promoting factor of tumor microenvironment (EGF, TGFβ, MMP9 and VEGF). Mechanistically, KLF14 could positively activate SOCS3 transcription, then blocking the activation of RhoA/Rock/STAT3 signaling. Further rescue experiments identified that either SOCS3 silencing and activation of RhoA/Rock/STAT3 signaling dramatically restrained the regulatory roles of KLF14 overexpression in breast cancer invasion and M2 macrophages polarization.
Collectively, KLF14 suppressed breast cancer cell invasion and M2 macrophage polarization through modulating SOCS3/RhoA/Rock/STAT3 signaling, and these findings would provide a new potential target against breast cancer.
研究KLF14在乳腺癌侵袭及肿瘤相关巨噬细胞（TAM）中的作用及潜在网络。
分别通过qRT-PCR和蛋白质免疫印迹法检测基因或蛋白质的表达。分别通过集落形成实验、CCK-8实验和Transwell实验研究细胞增殖和侵袭。通过流式细胞术鉴定巨噬细胞M2极化。通过甲基化特异性PCR（MSP）检测甲基化水平。通过双荧光素酶实验和染色质免疫沉淀实验验证KLF14与SOCS3之间的分子关系。建立体内模型以确认KLF14对肿瘤生长和转移的影响。
KLF14在乳腺癌中表达下调，其水平受CpG介导的甲基化修饰。KLF14的过表达显著抑制乳腺癌在体外和体内的增殖和侵袭。此外，过表达KLF14的乳腺癌细胞显著降低M2巨噬细胞极化及其相关的肿瘤微环境促进因子（EGF、TGFβ、MMP9和VEGF）。机制上，KLF14可正向激活SOCS3转录，进而阻断RhoA/Rock/STAT3信号通路的激活。进一步的挽救实验表明，SOCS3沉默或RhoA/Rock/STAT3信号通路的激活均显著抑制KLF14过表达对乳腺癌侵袭和M2巨噬细胞极化的调节作用。
总体而言，KLF14通过调节SOCS3/RhoA/Rock/STAT3信号通路抑制乳腺癌细胞侵袭和M2巨噬细胞极化，这些发现将为乳腺癌提供一个新的潜在治疗靶点。
