基于三维基质筛选模型靶向ACAD11促进乳腺癌分化诱导的机制研究

三阴乳腺癌的显著特征是分化差、恶性程度高、预后差，分化诱导治疗策略具有潜在的应用价值，但是其去分化的机制仍不清楚。前期研究中申请人建立了肿瘤细胞三维培养模型，发现组蛋白甲基化调控鼻咽癌细胞分化的机制（Nat Commun，2014）。最近申请人运用基于CRISPR/Cas9的文库筛选技术，在三维基质模型中鉴定了脂代谢相关基因ACAD11是调控乳腺癌细胞去分化的关键分子并分别在细胞和动物模型中初步验证其调控分化的作用。本课题拟在三维细胞模型中通过RNA干扰和基因表达回复实验，结合转录组、质谱分析等，探讨ACAD11影响乳腺癌细胞分化的信号通路和下游靶分子及代谢物。进一步，在三维PDX模型中探讨靶向ACAD11及其通路的抑制剂对乳腺癌细胞的分化诱导效果，最后，通过免疫组化和质谱等检测ACAD11及其靶基因与代谢物在乳腺癌病人标本中的水平并探讨其临床意义，为三阴乳腺癌的治疗提供新靶点。

Triple-negative breast cancers (TNBCs) generally are undifferentiated and more aggressive with poor prognosis, suggesting the differentiation therapy maybe a promising treatment strategy for TNBCs. However, the reason for the differentiation block is unclear. In the previous study, we have established a three-dimensional culture model of tumor cells and found histone methylation are involved in regulation of nasopharyngeal carcinoma cells (Nat Commun, 2014). Recently, we conducted a CRISPR/Cas9 library screening in the three-dimensional matrix model, and identified the lipid metabolism-related gene ACAD11, which played key role in suppressing differentiation of TNBC cells. The function of ACAD11 in regulating dedifferentiation was further confirmed by in vitro and in vivo models. Next, to explore the molecular mechanism of ACAD11 suppressed differentiation, we will perform RNA interference and gene expression recovery experiments, combine with RNA-seq and mass spectrometry analysis to find ACAD11 signaling pathways, its downstream molecular targets and metabolites. Further, we will build three-dimensional PDX models, and investigate the effects of inhibitors targeting ACAD11 pathway or metabolites on inducing differentiation of TNBC cells. Finally, we will detect the levels of ACAD11 pathway and metabolites by immunohistochemistry or mass spectrometry assays, then evaluate their clinical significance in TNBC patient specimens. This study will provide new therapeutic targets or potential strategies for the treatment of TNBCs.