蛋白酶ACADS调控酸性微环境介导自噬在结直肠癌肝转移中的机制研究

Liver metastasis is the most pivotal factor of survival in colorectal cancer patients, and the internal mechanism remains to be elucidated. We used two-dimensional electrophoresis and mass spectrometry-based proteomics approach to study the differentially expressed proteins in tumor and adjacent normal tissue samples from 5 pathologically confirmed colorectal liver metastasis patients, and identified that acetyl coenzyme A dehydrogenase (ACADS) is highly expressed in colorectal tumor tissue with liver metastasis. Knockdown and overexpression of ACADS could affect metastasis. Further research on mechanism show that ACADS promotes cancer cells to form acidic microenvironment via Na+/H+ ions metabolic regulation, and then cancer cells take part in autophagy on the feedback of microenvironment, resulting in metastasis get stronger. On the above results, we want to conduct in vivo and in vitro experiments to study the molecular mechanisms of ACADS regulating autophagy and promoting cancer cell metastasis, clear molecular target of ACADS regulating MAPK signaling pathway, describe downstream molecular pathways of ACADS, and then validate molecular regulation through the large sample validation of human tissue and analyze its clinical significance. At present, ACADS study in malignant tumors is still rare, the mechanism on colorectal liver metastasis has not been reported, and we expect to achieve a high level of research results on the basis of preliminary work, and find potential new targets for targeted therapy of colorectal liver metastasis.

肝转移是影响结直肠癌患者预后的最主要原因，其内在机制有待阐明。申请人前期利用双向电泳结合质谱手段筛查肠癌肝转移相关蛋白，发现乙酰辅酶A脱氢酶（ACADS）在伴肝转移的肠癌组织中表达显著上调；敲减和过表达肠癌细胞中的ACADS证实其具有促进转移的表型。进一步研究其作用机制表明：ACADS先经由Na+/H+离子代谢调控促进肠癌细胞形成酸性微环境，然后微环境又反馈肠癌细胞发生自噬效应，致使肿瘤获得更强的转移能力。本项目拟从细胞、动物水平结合体内和体外实验深入探讨ACADS调节自噬以及促进肠癌细胞转移的分子机制，明确ACADS调控AMPK信号通路的分子靶点，描绘出ACADS下游的分子途径，并在组织水平通过大样本验证分子间调控作用和分析其临床意义。目前国内外对ACADS在恶性肿瘤中的研究尚处于起步阶段，我们在大量前期工作基础上深入探讨其调节自噬影响转移的分子机制，并为临床靶向治疗提供潜在的新靶点。

肝转移是影响结直肠癌患者预后的最主要原因，其内在机制有待阐明。申请人前期利用双向电泳结合质谱手段筛查肠癌肝转移相关蛋白，发现乙酰辅酶A脱氢酶（ACADS）在伴肝转移的肠癌组织中表达显著上调；敲减和过表达肠癌细胞中的ACADS证实其具有促进转移的表型。进一步研究其作用机制表明ACADS先经由Na+/H+离子代谢调控促进肠癌细胞形成酸性微环境，然后微环境又反馈肠癌细胞发生自噬效应，致使肿瘤获得更强的转移能力。随后在本项目的资助下，首先应用组织切片免疫组化检测ACADS在153例结直肠癌患者肠癌原发灶中的表达情况，发现ACADS阳性表达仅和异时性肝转移明显相关，ACADS阳性患者的总体生存和无疾病生存明显短于ACADS阴性患者，而且ACADS阳性是结直肠癌患者不良预后的独立预测因素。随后在裸鼠体内模型中验证发现过表达ACADS，裸鼠皮下成瘤能力增强，而敲减ACADS，裸鼠皮下成瘤能力则降低，而使用脾包膜下注射肠癌细胞构建肠癌肝转移模型，发现敲减ACADS，裸鼠肝转移成瘤能力也减弱。然后进一步探究分子机制，发现结直肠癌细胞系SW620中ACADS和pMKK6存在同时性表达，敲减ACADS可以调控pMKK6和pMAPK蛋白表达。因此，本研究从细胞、动物水平结合体内和体外实验深入探讨ACADS调节自噬以及促进肠癌细胞转移的分子机制，明确ACADS调控MAPK信号通路，描绘出ACADS下游的分子途径，并在组织水平通过大样本验证和分析其临床意义。后续还要深入研究具体的精细调控机制等。期待进一步明确相关机制，从而有望为临床靶向治疗提供潜在的新靶点。