Objective: To investigate the mechanism of metformin for regulating tumor-stromal cell cross-talk in breast cancer.
Methods: Tumor associated fibroblasts (CAFs) co-cultured with breast cancer cells were treated with metformin, and the changes in expressions of hypoxia-inducible factor-1α (HIF-1α), p-AMPK, stroma-derived factor-1 (SDF-1) and interleukin-8 (IL-8) in the CAFs were detected using ELISA, RT-qPCR or Western blotting; Transwell assay was used to evaluate the invasiveness of the tumor cells and its changes following treatment with exogenous SDF-1, IL-8 and TGF-β1. The effects of HIF-1α shRNA or overexpression plasmid, AMPK shRNA, and treatment with OG (a proline hydroxylase inhibitor) or 2-OXO (a proline hydroxylase activator) were examined on p-AMPK, HIF-1α, SDF-1 and IL-8 expressions and invasiveness of the CAFs.
Results: Metformin treatment significantly increased the expression levels of p-AMPK, SDF-1 and IL-8 (P<0.05) and decreased HIF-1α expression (P<0.05) without affecting AMPK expression level (P>0.05) in the CAFs. The invasion ability of metformintreated breast cancer cells was significantly decreased (P<0.05). Exogenous SDF-1 and IL-8, HIF-1α overexpression, and OGinduced upregulation of HIF-1α all significantly attenuated the inhibitory effects of metformin on breast cancer cell invasion (P<0.05) and HIF-1α, SDF-1 and IL-8 expressions in CAFs (P<0.05). Transfection with HIF-1α shRNA or treatment with 2-OXO significantly decreased the invasiveness of breast cancer cells (P<0.05). P-AMPK knockdown significantly suppressed the inhibitory effect of metformin on HIF-1α expression in CAFs and on invasion of breast cancer cells (P<0.05). Treatment with TGF-β1 partially decreased the inhibitory effect of metformin on HIF-1α expression in CAFs and invasiveness of the breast cancer cells (P<0.05).
Conclusion: Metformin suppresses HIF-1α expression in CAFs to block tumor-stromal cross talk in breast cancer.
目的: 探讨二甲双胍（Met）对乳腺癌肿瘤-间质细胞交互作用的影响及机制。
方法: 将肿瘤相关成纤维细胞（CAFs）与乳腺癌细胞共培养，运用二甲双胍进行干预，分为对照组和Met干预组，ELISA及RT-qPCR检测Met对CAFs中HIF-1α、p-AMPK、基质衍生因子-1（SDF-1）和白细胞介素-8（IL-8）等因子的表达变化以及Transwell检测肿瘤细胞侵袭能力的变化。运用外源性SDF-1、IL-8干预后，Transwell检测肿瘤细胞侵袭能力的变化。运用缺氧诱导因子-1α（HIF-1α）shRNA或过表达质粒调节CAFs-HIF-1α的表达，以及AMPK-shRNA抑制AMPK的表达，并运用OG和2-OXO调节脯氨酸羟化酶的表达，及运用外源性TGF-β1干预后，Western blot及RT-qPCR检测CAFs中p-AMPK、HIF-1α、SDF-1、IL-8的表达，Transwell检测肿瘤细胞侵袭能力的变化。
结果: 相较于对照组，Met干预组中CAFs的p-AMPK、SDF-1和IL-8的表达水平升高（P<0.05），HIF-1α表达水平下降（P<0.05），AMPK的表达水平差异无统计学意义（P>0.05），Met组中乳腺癌细胞侵袭能力下降（P<0.05）。外源性SDF-1、IL-8干预可降低Met对乳腺癌细胞侵袭的抑制作用，增加乳腺癌细胞的侵袭能力（P<0.05）。过表达HIF-1α及运用脯氨酸羟化酶抑制剂OG提高HIF-1α的表达后，可降低Met对CAFs中HIF-1α、SDF-1及IL-8表达的抑制作用，并可降低Met对乳腺癌细胞侵袭的抑制作用（P<0.05）；运用HIF-1α-shRNA及运用脯氨酸羟化酶激活剂2-OXO抑制HIF-1α的表达后，降低乳腺癌细胞的侵袭能力（P<0.05）；运用AMPK-shRNA抑制p-AMPK的表达后，可降低Met对CAFs中HIF-1α表达的抑制作用，并可降低Met对乳腺癌细胞侵袭的抑制作用（P<0.05）；加入外源性TGF-β1后，可部分降低Met对CAFs中HIF-1α表达的抑制作用，并可部分降低Met对乳腺癌细胞侵袭的抑制作用（P<0.05）。
结论: Met通过抑制CAFs-HIF-1α的表达进而发挥阻断乳腺癌细胞-间质细胞交互作用。
Keywords: cancer-associated fibroblasts; hypoxiainducible factor-1α; metformin; phospho-AMPK; tumor microenvironment.