Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication
Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication
复制标题
癌症相关成纤维细胞通过连接蛋白43形成的单向间隙连接细胞间通讯加速非小细胞肺癌的恶性进展
DOI：
10.1159/000495232
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发表时间：
2018-01-01
影响因子：
--
通讯作者：
Yang, Jie
中科院分区：
文献类型：
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作者：
Luo, Min;Luo, Yanmei;Yang, Jie
背景/目的：缝隙连接由连接蛋白组装而成，可直接连接相邻细胞的细胞质，实现相邻细胞间的缝隙连接通讯(GJIC)和代谢偶联。在这里，我们研究了连接蛋白43(Cx43)及其衍生的GJIC在非小细胞肺癌(NSCLC)细胞和癌症相关成纤维细胞(CAF)之间相互作用中的作用。方法：CAF与NSCLC细胞直接接触共培养，用流式细胞仪进行分离。用比色法、Western blotting和酶联免疫吸附试验(ELISA)检测分离的CAF对葡萄糖的摄取、乳酸的产生以及PKM-2和LDH-A的表达和活性。同时，采用免疫印迹法、创伤宽度法和Transwell法检测E-钙粘蛋白和N-钙粘蛋白的表达以及分选的NSCLC细胞的迁移和侵袭能力。分离的NSCLC细胞丙酮酸、乙酰辅酶A和柠檬酸水平、ATP水平以及LDH-B和-KG活性分别用比色法或荧光法测定。用降落伞试验和免疫荧光法检测细胞间GJIC功能和连接蛋白的亚细胞定位。免疫组织化学方法检测NSCLC组织中-SMA、Cx43和LDH-B的表达水平。结果：Cx43聚集在细胞膜上，有利于CAF向NSCLC细胞组装不对称的单向GJIC。CAF经历了更多的有氧糖酵解，并促进了NSCLC细胞上皮-间充质的转变、迁移和侵袭。相反，在CAF刺激下，NSCLC细胞经历了氧化磷酸化增强，ATP生成增加，从而激活了PI3K/Akt和MAPK/ERK通路。CAF与NSCLC细胞之间的代谢偶联受Cx43形成的单向GJIC的严格控制。SMA、Cx43和LDH-B高三表达患者的总生存期和无复发生存期比单独过表达或高双表达患者短。结论：Cx43形成的单向GJIC在CAF和NSCLC细胞之间密切的代谢协作中发挥关键作用，从而支持NSCLC的恶性进展。
Background/Aims: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). Methods: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and -KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a Parachute assay and immunofluorescence. Levels of -SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. Results: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of -SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. Conclusion: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.