缝隙连接在肿瘤相关成纤维细胞反向Warburg效应调控非小细胞肺癌恶性进展中的作用及机制研究

Carcinoma-associated fibroblasts (CAFs) undergo "reverse Warburg effect" (aerobic glycolysis), which is closely associated with cancer progression. The gap junction (GJ) between cancer cells and the surrounding cells can promote tumor invasion and metastasis. Our previous study found that GJ between cancer cells was involved in the regulation of NSCLC progress.In addition, we found that the existence of one-way GJ function between co-cultured A549 cells and CAFs. Enhanced functionality of GJ by RA (a widely accepted GJ enhancer) can increase the proliferation, invasion and Akt phosphorylation levels of A549 cells. Moreover, the "reverse Warburg effect"of CAFs and proliferation and invasion of A549 cells were both increased in A549 cells/ CAFs co-cultures. Specifically, inhibition of "reverse Warburg effect" of CAFs by DCA (specific aerobic glycolysis inhibitor) significantly reduced proliferation and invasion of A549 cells, and this effect could be obviously reversed by combination treatment of DCA with GJ enhancer RA. Thus, we propose that "reverse Warburg effect" of CAFs might activate PI3K/Akt/mTOR signaling pathway in NSCLC via GJ, thereby promoting malignant progression of NSCLC. Based on these findings, the present study will clarify the role of GJ in the malignant progression of NSCLC regulated by "reverse Warburg effect" of CAFs and the underlying mechanisms in cells, animals and specimens of patients. Our study will provide the basis for the new therapeutic strategies targeting NSCLC tumor microenvironment.

肿瘤相关成纤维细胞（CAFs）存在反向Warburg效应（有氧糖酵解）并与肿瘤进展密切相关。肿瘤细胞与周围细胞间的缝隙连接（GJ）可促进肿瘤侵袭转移。我们前期研究发现，肿瘤细胞间GJ参与调控NSCLC进展。此外，共培养A549细胞与CAFs间存在单向性GJ功能，增强GJ功能可促进A549细胞增殖、侵袭及磷酸化Akt水平；共培养可增强CAFs反向Warburg效应，并促进A549细胞增殖和侵袭；抑制CAFs反向Warburg效应可减少A549细胞增殖及侵袭，此时增强GJ功能可明显逆转此效应。由此我们提出新的科学假设：CAFs反向Warburg效应可能通过GJ激活NSCLC的PI3K/Akt/mTOR信号通路，从而促进NSCLC恶性进展。本课题将通过细胞、动物及临床标本明确GJ在CAFs反向Warburg效应调控NSCLC恶性进展中的作用及机制，为靶向肿瘤微环境的NSCLC治疗策略提供新依据。

肿瘤相关成纤维细胞（cancer-associated fibroblasts,CAFs）是肿瘤微环境中最主要的基质细胞，CAFs发生反向 Warburg 效应（有氧糖酵解）并与肿瘤进展相关。肿瘤细胞与CAFs细胞均表达连接蛋白（connexin,Cx）,且相邻两种细胞间能形成缝隙连接通讯（gap junctional intercellular communication，GJIC），促进肿瘤侵袭转移。但CAFs与非小细胞肺癌（non-small lung cancer, NSCLC）是否存在GJIC以及GJIC是否参与CAFs与NSCLC间代谢共生尚未阐明。我们发现细胞间存在单向GJIC并证实这种GJIC由Cx43组成的。本研究探讨Cx43 及其构成的GJIC 介导CAFs与NSCLC细胞间代谢偶联和调控侵袭转移的作用。采用GJ抑制剂18α-GA或者干扰Cx43阻断CAFs-NSCLC间的单向GJ可抑制共培养后NSCLC的侵袭迁移，减弱氧化磷酸化（oxidative phosphorylation，OXPHOS）代谢；反之，采用GJ增强剂RA或者过表达Cx43能促进共培养后NSCLC的侵袭转移，增强OXPHOS代谢。此外，同时抑制单向GJIC和CAFs上的糖酵解可进一步抑制共培养后NSCLC细胞的侵袭迁移，减弱OXPHOS代谢，减少P-AKT,P-ERK的表达；反之，可进一步促进共培养后NSCLC细胞的侵袭迁移，增强OXPHOS代谢，增加 P-AKT,P-ERK的表达。用糖酵解激活剂1，6二磷酸果糖（FBP）预处理CAFs，也无法恢复GJIC缺失的共培养NSCLC细胞侵袭迁移能力的减弱。用糖酵解抑制剂2-去氧-D-葡萄糖（2-DG）预处理CAFs后，可以部分逆转GJIC增强的共培养NSCLC细胞侵袭迁移能力增加。外加ATP处理，可显著增加NSCLC细胞的PI3K/Akt、MAPK/ERK信号通路的激活,促进NSCLC的侵袭转移，还可部分逆转LY294002和（或）U0126引起的NSCLC细胞PI3K/Akt和 MAPK/ERK信号通路抑制以及NSCLC细胞的侵袭转移能力的下降。以上表明发生有氧糖酵解的CAFs通过Cx43介导的单向GJIC增强NSCLC的氧化磷酸化，其主要产物ATP能诱导NSCLC细胞的PI3K/Akt 和MAPK/ERK信号通路激活和侵袭转移