CAFs分泌的PDGFC通过PI3K-mTOR信号通路促进乳腺癌细胞对阿霉素耐药

PDGFC secreted by CAFs promotes resistance of breast cancer cells to doxorubicin by PI3K-mTOR signalling pathway

目的探讨肿瘤相关成纤维细胞(cancer-associated fibroblasts,CAFs)来源的血小板衍生生长因子C(platelet derived growth factor C,PDGFC)是否促进乳腺癌细胞对阿霉素(doxorubicin,DOX)耐药并探讨其机制。方法原代提取CAFs和正常成纤维细胞(normal fibroblasts,NFs)并获取CAFs和NFs的条件培养基(conditional medium,CM),观察CAFs-CM是否影响乳腺癌细胞对DOX的敏感性;检测CAFs及其条件培养基中PDGFC的表达;Western blot检测乳腺癌细胞凋亡相关蛋白BAX/BCL2、耐药蛋白ABCG2、线粒体膜蛋白TOM20与COXⅣ及信号蛋白(p-)PI3K与(p-)mTOR的表达;DCFH-DA荧光染色、JC-1试剂盒分别检测乳腺癌细胞内ROS水平与线粒体膜电位。结果CAFs-CM减少乳腺癌细胞中DOX的含量,诱导对DOX耐药,同时乳腺癌细胞中BAX减少,Bcl2增加,ROS减少,线粒体膜电位升高及线粒体膜蛋白TOM20与COXⅣ表达增加。进一步研究发现,CAFs及CAFs-CM中PDGFC均高表达,重组人PDGFC产生了与CAFs-CM相似的促进乳腺癌细胞对DOX耐药的作用,PDGFRα的特异性抑制剂则显著抑制了CAF-CM诱导的耐药;进一步机制研究表明,CAFs分泌的PDGFC通过激活PI3K-mTOR信号通路诱导了乳腺癌细胞耐药。结论CAFs分泌的PDGFC通过PI3K-mTOR通路促进乳腺癌细胞对DOX耐药,这一发现为开发靶向CAFs的抗癌药物提供了新的思路。

Aim To explore whether platelet-derived growth factor C(PDGFC)derived from cancer-associated fibroblasts(CAFs)can promote resistance of breast cancer cells to doxorubicin(DOX)and the underlying mechanisms.Methods CAFs and normal fibroblasts(NFs)were extracted from freshly resected breast cancer tissue and adjacent normal breast tissue respectively.Conditioned medium(CM)from CAFs and NFs was collected and co-cultured with breast cancer cells.Cell proliferation and toxicity were assessed using a Cell Counting Kit-8(CCK-8).The expression of PDGFC in CAFs,NFs and corresponding CM was detected by Western blot and ELISA respectively.The influence of CAFs-CM on intracellular doxorubicin content in breast cancer cells was observed by fluorescence microscopy.The impact of CAFs-CM on apoptosis-related proteins BAX and BCL2 was predicted and valifated using the Starbase database and Western blot.The changes in ROS levels,mitochondrial membrane potential,and mitochondrial membrane proteins TOM20 and COXⅣin breast cancer cells were measured using DCFH-DA fluorescence staining,JC-1 assay,and Western blot.Results CAFs-CM decreased the intracellular doxorubicin content and inhibited the sensitivity of breast cancer cells to doxorubicin.Additionally,the expression of apoptosis protein BAX decreased while the anti-apoptotic protein BCL2 increased in breast cancer cells cultured with CAFs-CM.Furthermore,CAFs-CM led to decreased ROS levels and increased mitochondrial membrane potential in breast cancer cells accompanied with elevated expression of mitochondrial membrane proteins TOM20 and COXⅣ.Further study found that PDGEF was highly expressed in CAFs and CAFs-CM,recombinant human PDGFC produced resistance of breast cancer cells to DOX similar to CAFs-CM,and the specific inhibitors of PDGFRαsignificantly inhibited CAFs-CM.Further mechanistic studies revealed that PDGFC in CAFs-CM induced chemoresistance by activating PI3K-mTOR signaling pathway.Conclusion PDGFC secreted by CAFs promotes doxorubicin resistance in breast cancer cells through PI3K-mTOR signaling pathway,which provides a new perspective for the development of anticancer drugs targeting CAFs.