Integrinαvβ3/MAPK/ERK通路活化GLI1在胃癌腹腔转移多细胞团簇干性调节中的作用及机制研究

Tumor multicellular aggregates (MCAs) is an independent structure unit，and is widespread concerned about its biological function. The exfoliated gastric cancer cells are consisted of mainly MCAs, which are the actual “seed” cells during the process of peritoneal metastasis for gastric cancer. In previous study we found MCAs are aggregated together by Integrin alpha v beta 3, and MCAs are acquired tumor stem-like characteristics, however the underlying mechanism of sustaining stemness for MCAs is still unclear. Based on recent literature and the results of previous studies, we postulate that crosstalk occurred between Integrinαvβ3/MAPK and Hedgehog pathways in MCAs, GLI1 is activated and the stemness is contained by Integrinαvβ3/MAPK pathway in a noncanonical way. This project intends to revolve around abdominal gastric MCAs, confirms Integrinαvβ3/MAPK phosphorylation of GLI1 by direct serine/threonine, activate GLI1, starting downstream associated nuclear transcription factors expression, thereby maintaining the MCAs cancer stem cells by using molecular biology, in vitro kinase activity assays, and mass spectrometry analysis. In vivo experiment further verifies these results. Thereby reveal the mechanism of abdominal gastric MCAs stemness maintenance, thus offer a novel theoretical basis for the prevention and treatment of peritoneal metastasis.

肿瘤多细胞团簇（MCAs）是肿瘤转移中的独立结构单元，生物学功能备受关注。腹腔脱落胃癌细胞通常以MCAs形式存在，是胃癌腹腔转移真正“种子”细胞，前期研究发现胃癌腹腔转移MCAs主要以Integrinαvβ3介导粘附聚集而成，具有肿瘤干样细胞特性，但干性维持机制不清。本项目基于前期研究结果，推测胃癌腹腔转移MCAs中Integrinαvβ3/MAPK与Hedgehog-GLI1通路发生crosstalk，Integrinαvβ3/MAPK以非经典途径活化GLI1维持MCAs肿瘤干细胞特性。本课题拟以胃癌MCAs为研究对象，应用分子生物学、体外激酶活性测定、质谱分析等方法证实Integrinαvβ3/MAPK通路磷酸化GLI1丝/苏氨酸，活化GLI1，启动干细胞核转录因子表达，维持MCAs干样特性。从而深入揭示胃癌腹腔转移MCAs干性调节分子机制，为胃癌腹腔转移防治提供新的理论依据。

脱落癌细胞是胃癌腹腔转移种子细胞，具有干样特性，机制不清，推测可能通过Integrinαvβ3/MAPK/ERK途径激活GLI1维持其干性，本项目研究成果如下：1.成功分离、鉴定胃癌腹腔脱落MCAs；2.证实MCAs自我更新能力增强，肿瘤干细胞相关蛋白表达增强，MCAs具有更强的增殖、耐药能力和致瘤性； 3.Western-blot结果显示，胃癌腹腔脱落MCAs中Integrin αvβ3, p-ERK1/2, GLI1蛋白水平均明显增高。4.分别用Integrin αvβ3抑制剂、Integrin αvβ3刺激配体、空白对照处理BGC823 MCAs、SGC7901 MCAs，GLI1蛋白在Integrin αvβ3抑制剂组下调，在Integrin αvβ3刺激配体组上调，GLI1蛋白与Integrin αvβ3活性变化呈正相关；5.分别用Integrin αvβ3刺激配体或与ERK抑制剂联合处理MCAs，蛋白免疫印迹检测GLI1蛋白水平，证实Integrin αvβ3通过ERK1/2途径调节GLI1表达；6.证实胃癌MCAs主要通过非经典Integrin αvβ3/ERK1/2通路调节GLI1；7.构建GLI1荧光素酶报告基因载体，证实ERK1/2直接调控GLI1转录；8. 抑制Integrin αvβ3、ERK1/2、GLI1，胃癌MCAs增殖能力、自我更新能力、干性相关分子表达下降，胃癌MCAs致瘤能力下降；9.证实shGLI1#1组胃癌MCAs干样特性下降；10.构建裸鼠胃癌腹腔转移模型，证实Integrin β3、GLI1降低组裸鼠腹腔致瘤能力减弱，胃癌腹腔转移灶Integrin αvβ3、 p-ERK1/2、GLI1表达高于原发灶。综上研究证实Integrin αvβ3介导胃癌MCAs细胞间黏附，形成独立功能单元，通过ERK1/2途径激活GLI1，启动下游相关靶基因转录，以维持胃癌MCAs干样表型