ISL1介导的肝癌干细胞与非癌干细胞转换在转移中的作用和分子机制

It is hypothesized that a rare subset of cancer cells, often operationally referred to as cancer stem cells (CSCs) or tumor-initiating cells, are responsible for sustaining tumor growth and metastasis. The acquirement and maintenance of cancer stem cell traits often requires epithelial-mesenchymal transition (EMT), while the differentiation of CSCs results in epithelial-mesenchymal transition (MET), hence the stem cell properties of cancer cells are closely linked to EMT. Although it is still debating, it is generally accepted that tumor cell metastasis requires at least two phenotypic transition processes: an EMT-mediated de-differentiation triggering invasion and dissemination, and a subsequent “re-differentiation” achieved through MET which is crucial for metastatic colonization. However, how the conversion is dynamically regulated and how the transition contributes to tumor metastasis remain elusive. We have previously identified a subpopulation of liver CSCs that expressing the isoform 5 of α2δ1, which is encoded by CACNA2D1, a composing subunit of a voltage-gated calcium channel. The α2δ1 positive liver CSCs, which are of mesenchymal traits, are invasive and cause metastases when they were orthotopically transplanted in livers of NOD/SCID mice. Furthermore, we found the expression levels of islet1 (ISL1), a transcriptional factor of LIM/homeodomain family, determine the conversion between α2δ1 positive liver CSCs and non-CSCs, EMT and MET. Interestingly, ISL1 can activate the expression of ZEB1/ZEB2 through the interaction with NPM1. These findings lead to the proposal that ISL1 is a master molecule that determines the conversion between liver α2δ1+ CSCs and non-CSCs, EMT-MET, which subsequently regulates the metastasis of hepatocellular carcinoma. In this project, we are going to investigate the roles and the underlying mechanisms of ISL1 in the regulation of the conversion between liver α2δ1+ CSCs and non-CSCs, EMT and MET, and how non-CSCs and MET contribute to hepatocellular carcinoma metastasis. Moreover, the mechanisms that determine the levels of ISL1 will also be determined. Hopefully, we can uncover new roles and mechanisms of ISL1 in the conversion between liver α2δ1 positive CSCs and non-CSCs, EMT and MET, which regulate hepatocellular carcinoma metastasis. This study would contribute to the understanding of the metastasis of hepatocellular carcinoma at both the cellular and molecular levels and advance the development of prognostic and therapeutic strategies.

肿瘤干细胞被认为是引起肿瘤复发转移的起源细胞，其干性的获得与分化常伴随上皮-间质转化（EMT）和间质-上皮转化（MET）,然而决定这些转化的分子机制及其如何影响肿瘤的转移尚存大量未知。我们发现α2δ1+阳性肝癌干细胞具有间质细胞特性，具高侵袭性，能引起转移；进一步发现转录因子LIM家族胰岛因子1（Islet1, ISL1）的表达水平决定了EMT和MET、干性和非干性之间的的转换，从而在肝癌转移中发挥作用，ISL1转录活性可能受NPM1等分子影响。本课题拟对ISL1在不同表型转换中的作用及作用的方式、作用的分子机制，不同的表型尤其是发生MET的非肿瘤干细胞对肝癌转移的影响和作用的分子机制，以及决定细胞内ISL1表达水平的调控机制进行系统深入研究，以期揭示ISL1在肝癌干细胞-非癌干细胞、EMT-MET转换中的作用和分子机制及对肝癌转移的影响，为肝癌转移预警和和治疗分子靶点提供线索。

肿瘤干细胞被认为是引起肿瘤复发转移的起源细胞，其干性的获得与分化常伴随上皮-间质转化（epithelial-mesenchymal transition，EMT）和间质-上皮转化（mesenchymal-epithelial transition，MET）,然而决定这些转化的分子机制及其如何影响肿瘤的转移尚存大量未知。本项目基于前期发现电压门控钙通道α2δ1亚基阳性肝癌干细胞具有间质细胞特性，具高侵袭性，能引起转移，对转录因子LIM家族成员胰岛因子1（Islet1, ISL1）在肝细胞癌干细胞与非干细胞、上皮-间质不同表型转换中的作用及作用的方式、作用的分子机制以及决定细胞内ISL1表达水平的调控机制进行系统深入研究。研究结果表明ISL1在 α2δ1 阳性肝癌干细胞干性获得与维持、EMT 和 MET 转换中发挥重要作用，ISL1可与超增强子元件BRD4结合直接调控电压门控钙离子通道α2δ1亚基（由基因CACNA2D1编码）等基因的表达。明确了α2δ1还是非小细胞癌和胰腺癌的干细胞标志物和治疗靶点。α2δ1介导的钙离子内流可以通过CaMKII调控去乙酰化酶SIRT4的表达，SIRT4进一步可以引起MCCC2第269位点赖氨酸的去乙酰化，去乙酰化的MCCC2-K269与MCCC形成的异源二聚体能力增强，乙酰辅酶A的生成量增加，促使H3K27乙酰化水平升高，使与干性调控相关的信号通路活化，促进了肝癌干细胞自我更新和成瘤能力的增加。此外课题还揭示了α2δ1 阳性肝癌干细胞中IP3R2介导的内质网钙的释放是钙振荡产生的原因之一，阻断该分子可以抑制钙振荡和癌干细胞的自我更新，是一潜在治疗靶点。靶向α2δ1的人源化抗体1B50-1在肝癌荷瘤小鼠模型中具有核素显像作用，亦可通过降低EGFR的表达克服肝细胞的伦伐替尼耐药。通过解人源化抗体1B50-1结合抗原α2δ1复合物分辨率达3.1A°的冷冻电镜结构，明确了1B50-1抗体与α2δ1结合的关键氨基酸位点，并发现1B50-1可干扰α2δ1与离子通道α1亚基的结合，从而降低钙离子的内流和抑制干性。这些发现揭示了ISL1在肝癌干细胞-非癌干细胞、EMT-MET转换中的作用和机制及对肝癌转移的影响，为肝癌预后判断和治疗分子靶点、抗体药物奠定了理论和实验依据。