细胞命运决定因子DACH1抑制肝癌干细胞样特性的分子机制研究

Hepatocellular carcinoma (HCC) is the second-leading cause of cancer-related death. The poor outcome of patients with HCC is attributed to recurrence of the disease after curative treatment and the resistance of HCC cells to conventional chemotherapy, which are associated with stem cell-like characteristics of HCC cells. A deep understanding of the underlying mechanisms will be of considerable value to the progress of treatment and prognosis prediction of patients with HCC..Cell fate determination factor human dachshund homolog 1 (DACH1) is a tumor suppressor gene. We have reported that DACH1 can inhibit c-myc expression in HCC. The loss of DACH1 expression is associated with poor differentiation and chemoresistance of HCC cells, which are tied to the stem cell-like properties of cancer cells. Therefore, it is hinted that DACH1 may serve as a tumor suppressor gene via inhibiting stem cell-like properties of HCC cells. It was also shown that re-expression of DACH1 suppresses the activity of Wnt/β-catenin signaling, which has been implicated in the stemness maintenance of cancer cells. However, mechanisms of how DACH1 inhibiting stem cell-like properties has not been clarified. Interestingly, it was reported that the binding of FOXM1 and β-catenin was the key of persistent activation of β-catenin. Therefore, it is postulated that DACH1 may suppress the stem cell-like characteristics of HCC cells by inhibiting the Wnt signaling via reducing the binding of FOXM1 and β-catenin. Decreasing transcription or nuclear translocation, or interacting with β-catenin may be involved in the repression of β-catenin by DACH1. .To test the hypothesis, the effect of ectopic expression or silencing of DACH1 on the stem cell-like phenotypes of HCC cells is detected using sphere formation assay, flow cytometry and in vivo tumorigenicity etc. It will be investigated how DACH1 regulates the activation of β-catenin by molecular biological technology, laser scanning confocal microscopy, co-immunoprecipitation and chromatin immunoprecipitation..The main theme of the proposed research is tantamount to elucidate the role of DACH1 in inhibiting stem cell-like characteristics of HCC cells and the underlying mechanisms thereof. This study may demonstrate a better understanding of the role of DACH1 in inhibiting HCC, and favor overcoming recurrence and chemoresistance of HCC by novel therapy based on DACH1 protein.

肿瘤复发和耐药是肝癌患者预后差的主要原因，肿瘤干细胞样特性与此密切相关，但调控机制不清。我们的前期研究提示细胞命运决定因子DACH1可能抑制肝癌的干细胞样特性，并发现它能下调与干性紧密相关的Wnt通路的活性，但分子机理不清。文献报道FOXM1与β-catenin的结合是β-catenin活化的关键因素。我们推测肝癌中DACH1可能通过抑制基因转录，或抑制蛋白核转位，或竞争结合等途径抑制FOXM1和β-catenin的相互作用，进而抑制Wnt通路以及肝癌干细胞样特性。本项目拟干扰和上调细胞DACH1的表达，应用流式细胞术、成瘤实验及球体形成实验等研究DACH1对肝癌干性相关表型的影响，应用激光扫描共聚焦，免疫共沉淀以及染色质免疫共沉淀等方法探讨DACH1抑制肝癌Wnt通路的机制。本项目将阐明DACH1调控肝癌干细胞样特性的机制，为发展基于DACH1的肝癌复发及化疗耐药的防治手段提供理论依据。

肝细胞肝癌（HCC）是常见的恶性肿瘤，其恶性程度高，进展快，手术切除后远处转移和复发率高，对常规化疗均不敏感，而肿瘤干细胞样特性与此相关。细胞命运决定因子DACH1是视网膜决定基因网络家族的关键基因，为正常组织发育所必需，通过直接与特定的DNA序列结合或与其他转录因子相互作用而调控靶基因的表达。现有研究提示，DACH1可能成为新的肿瘤抑制基因。我们的前期研究提示DACH1可能抑制肝癌的干细胞样特性。本研究旨在评估DACH1对肝癌干细胞样特性，包括耐药性等的影响，并探讨其调控机制。在本项目中，我们分析了公共基因表达数据库，构建了稳定转染的细胞系和动物模型，采用蛋白免疫印迹、激光共聚焦、细胞增殖实验和球体形成实验等方法探讨了DACH1对肝癌干细胞样特性等的抑制作用。通过上述研究，初步阐明DACH1破坏了FOXM1-β-catenin的相互作用，通过双重途径抑制了FOXM1介导的β-catenin细胞核内积累，进而抑制Wnt/β-catenin通路的活性，抑制肝癌细胞的干细胞样特性。我们发现肝癌中DACH1表达普遍下调，与HCC发生、索拉非尼的耐药性以及肿瘤复发呈负相关。过表达DACH1可以提高肝癌细胞对索拉非尼的敏感性。功能研究表明，DACH1可以抑制肝癌细胞的自我更新、对多柔比星和顺铂的耐药性、去分化状态以及在裸鼠体内的肿瘤形成能力。体内和体外实验均证实DACH1能够抑制干性基因的表达。机制研究表明，DACH1能够促进β-catenin的降解并抑制其核内积累，同时伴随着FOXM1的表达水平的下降和核内积累的减少。对细胞、肿瘤组织、临床和公共数据库的分析均表明，DACH1与FOXM1的表达呈负相关。进一步研究表明，DACH1通过抑制EGFR的表达，下调ERK的磷酸化，减少FOXM1的核转位，同时在细胞核内与FOXM1竞争性地结合β-catenin，相应地FOXM1和β-catenin的核内累积减少，C-myc、Nanog等干性基因的表达明显下降。提示DACH1依赖的调节通路将能在肿瘤形成中限制Wnt/β-catenin通路的活性程度和作用时间，而诱导DACH1的表达可能有助于发展更有效的肝癌治疗手段。本研究有利于我们更好地理解肝癌复发和耐药的机制，完善肝癌的分子分型和靶向药物的开发，优化诊治和随访体系，最终达到改善患者预后的目的。