FAT1通过调控Wnt/β-catenin信号通路影响食管鳞癌细胞干性的机制研究

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor in China. Cancer stem cell (CSC) plays an important role in the recurrence and metastasis of cancers, the prognosis of ESCC is poor. However, there are few studies on esophageal squamous cell carcinoma stem cell(ESCC-CSC). Previously we performed whole-genome sequencing of ESCC tumors and adjacent normal tissue, and found FAT1 was one of significant mutant genes. Previous experiments showed that FAT1 inhibits the migration and invasion of ESCC cell via prevention the progress of epithelial-mesenchymal transition(EMT), and FAT1 had a lower expression in CSCs than that in the bulk cells. In FAT1 knockdown ESCC cells, the results of RNA sequencing and tentative experiments showed that the altering genes were enriched in the Wnt/β-catenin signaling pathway and stem cell markers had a significant increase. These results suggest FAT1 inhibits the stemness of ESCC cell through Wnt/β-catenin signaling pathway. This proposal aims to validate the effect of FAT1 on the stemness of ESCC, ESCC cell lines were used for knockdown experiment while ESCC stem cell lines were used for over-expression experiment, and then cell phenotype and stemness were detected. The mechanism of FAT1 was illuminated by co-immunoprecipitation, western blot, immunofluorescence, dual-luciferase reporter assay and flight mass spectrometry analysis of protein. In addition, we will collect clinical cases of ESCC and analyse the correlation of the expression levels of FAT1 and clinical pathology, stem cell markers, Wnt/β-catenin signaling protein. The study will contribute to understanding the mechanisms that recurrence and metastasis of ESCC and may provide the clinical strategies for ESCC-CSC treatment.

食管鳞癌是我国常见恶性肿瘤，术后易复发转移，预后极差，肿瘤干细胞可能是其重要原因，但目前针对食管鳞癌肿瘤干细胞的研究很少。课题组对食管鳞癌基因组测序发现FAT1存在显著失活突变，可抑制上皮间质转化发挥抑癌作用，且FAT1在食管鳞癌肿瘤干细胞表达显著降低。转录组测序和初步实验表明FAT1敲低后差异基因主要富集在Wnt/β-catenin信号通路，且干细胞标志物显著增加，提示FAT1可能通过调控该通路抑制肿瘤细胞干性。本项目拟在食管鳞癌细胞敲低FAT1，在食管鳞癌干细胞过表达FAT1后检测细胞表型和干性变化；采用免疫共沉淀、免疫荧光、双荧光素酶实验、蛋白质飞行质谱等明确FAT1作用机制；收集临床病例，研究FAT1在食管鳞癌组织中表达的意义及其与干细胞标志物和Wnt/β-catenin信号通路蛋白相关性。本研究将有助于阐明食管鳞癌转移复发机制和为筛选针对食管鳞癌干细胞的特定靶向药物提供理论基础。

在世界范围内，食管癌发病率位居第七，死亡率居于第六。食管癌病理类型主要为食管鳞癌（ESCC）和食管腺癌（EAC），而我国食管鳞癌占大多数。化疗是治疗ESCC的主要方法之一。然而，有效率低至40%，复发率高达80%，5年生存率仍低于30%。原发性或获得性耐药是ESCC化疗失败和复发的原因，但ESCC中导致耐药性的机制目前尚未明了。我们之前研究发现FAT非典型钙黏蛋白1（FAT1）可抑制 ESCC上皮-间质转化（EMT）。EMT使癌细胞具有肿瘤干细胞（CSC）样特征，在多种癌症的耐药性发展中起着关键作用。本研究我们对FAT1调节ESCC细胞干性和耐药性进行了探讨。我们发现FAT1在ESCC干细胞球中下调，并与干细胞相关标记物ALDH1A1和KLF4呈负相关。FAT1敲低后ESCC细胞成球能力、抗顺铂能力和药物外排能力增强，耐药相关基因ABCC3表达上调。另外，我们发现FAT1敲低后可诱导β-catenin向细胞核移位，并增强其转录活性。ChIP结果显示β-catenin在ABCC3启动子区域富集。同时，β-catenin促进ABCC3的转录和表达。总之，我们发现FAT1敲低后可通过Wnt/β-catenin信号通路增强ESCC细胞的干性和ABCC3相关的顺铂耐药性。靶向FAT1及其下游基因ABCC3可能是克服ESCC化疗耐药性的一种有前景的治疗策略。