Disulfiram通过miR-30/SOX4/WNT7B调控EMT和肿瘤干细胞抑制乳腺癌转移的机制

Downregulation of miR-30 might be associated with epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) in breast cancer. However, the exact role of miR-30 in breast cancer is not yet fully understood. In addition, how to use small molecular compounds to upregulate the expression of miR-30 for treatment of human cancer remains unknown. Our previous data have revealed that small molecular compound disulfiram can inhibit TGF-β induced EMT and stem-like features in breast cancer via ERK/NF-κB/Snail pathway. The results of miRNA expression profiling and real time RT-PCR demonstrated that disulfiram can affect the expression of miR-30; TargetScan and miRanda both predict that SOX4 and WNT7B are the direct targets of miR-30, and both of them were involved in EMT program. We therefore hypothesize that the inhibitory effects of disulfiram on EMT and CSC may result from its upregulation of miR-30 and interference of TGF-β and Wnt pathway by targeting miR-30/SOX4/WNT7B. To test the hypothesis, we will first determine the effect of disulfiram on miR-30 expression and the role of miR-30 in breast cancer EMT and CSC in vitro and in vivo. Second, we will determine whether SOX4 and WNT7B are miR-30 target genes. Third, we will investigate how miR-30 regulate TGF-β and Wnt pathway by targeting SOX4 and WNT7B to understand the mechanisms by which miR-30 suppress tumor EMT and stem-like properties. Regulation of miRNA expression by small molecular compounds may be a novel strategy for anticancer therapy.

miR-30下调可能与乳腺癌EMT和CSC相关，但问题在于①miR-30的作用机制尚未完全明确，②如何利用小分子化合物来上调miR-30表达，发挥其抗肿瘤作用，目前尚无相关报道。前期研究结果显示：①小分子化合物双硫仑可通过抑制NFκB通路抑制乳腺癌EMT和干样特性；②miRNA芯片及RT-PCR结果揭示双硫仑可上调miR-30表达；③预测SOX4和WNT7B是miR-30新靶标，且二者均与EMT相关。基于此，我们推测：双硫仑上调miR-30的表达，通过调控miR-30/SOX4/WNT7B，干扰TGF-β和Wnt通路，抑制EMT和CSC，降低乳腺癌转移风险。本课题拟通过乳腺癌体内外模型，探讨新靶标SOX4和WNT7B在miR-30对EMT和CSC调控中的作用及相互关联，阐明双硫仑通过上调miR-30而逆转EMT和CSC的新机制。利用小分子化合物调控miRNA将为乳腺癌精准治疗提供新策略。

上皮间质转化（EMT）和肿瘤干细胞（CSC）在乳腺癌转移中起着重要作用。据报道，微小RNA中miR-30家族和转录因子早期生长因子（EGR）家族对EMT和CSC起着重要作用，但是其潜在的作用机制尚不明确。近年来有多项研究报道小分子化合物双硫仑（disulfiram, DSF）具有抗肿瘤效应，在不同的肿瘤模型中表现出许多抗癌特性，包括抑制肿瘤细胞增殖、促进细胞凋亡、抑制侵袭迁移能力等。在我们的研究中，我们通过体内和体外实验证实了miR-30a对乳腺癌EMT和CSC表型具有抑制作用，并证实了DSF通过miR-30a/SOX4双负反馈环调控乳腺癌EMT和CSC表型进而抑制乳腺癌的转移。同时我们确定了LRP6是miR-30e的靶基因，证实了EGR1/miR-30e/LRP6轴对三阴乳腺癌的侵袭转移具有抑制作用。我们进一步探索了DSF对转录因子EGR1的调控以及对肿瘤免疫微环境的调控，证实DSF可以上调EGR1表达，并通过NFκB/p65/CD47信号通路调节EMT及TAM的极化；DSF上调肿瘤细胞表面PD-L1的表达，联合PD-1单抗可提高其抗肿瘤疗效。本研究结果从多角度探讨了DSF抑制乳腺癌转移的机制，并探索性的研究了DSF对免疫检查点分子及免疫微环境的调控作用，将为DSF的抗肿瘤机制提供了重要的新的理论依据，为推动DSF在临床上“老药新用”提供坚实的理论基础。