A Non-genetic Mechanism Involving the Integrin b4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer. A Non-genetic Mechanism Involving the Integrin b4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer

Tumor heterogeneity and cisplatin resistance are major causes of tumor relapse and poor survival. Here, we show that in lung cancer, interaction between paxillin (PXN) and integrin b4 (ITGB4), components of the focal adhesion (FA) complex, contributes to cisplatin resistance. Knocking down PXN and ITGB4 attenuated cell growth and improved cisplatin sensitivity, both in 2D and 3D cultures. PXN and ITGB4 independently regulated expression of several genes. In addition, they also regulated expression of common genes including USP1 and VDAC1 that are required for maintaining genomic stability and mitochondrial function, respectively. Mathematical modelling suggested that bistability could lead to stochastic phenotypic switching between cisplatin-sensitive and resistant states in these cells. Consistently, purified subpopulations of sensitive and resistant cells recreated the mixed parental population when cultured separately. Altogether, these data point to an unexpected role of the FA complex in cisplatin resistance, and highlight a novel non-genetic mechanism. Overall design: Knockdown of ITGB4 (Cat #: SR302473C) at the mRNA level was executed using siRNAs purchased from OriGene Technologies (Rockville, MD, USA). Knockdown of PXN was achieved by siRNA purchased from Life Technologies Corporation (Cat #: 4392421). JetPRIME transfection reagent (Polyplus Transfection, Illkirch, France) was used to transfect the siRNAs according to the manufacturer’s protocol. Cells were seeded in 6-well plates (200,000 cells/well) and allowed to adhere overnight. Next day, 10 nM siRNA was transfected with 4 μl jetPRIME reagent in complete growth medium for each well. Cell growth medium was changed the next day and expression was detected 72 h post-transfection by qPCR and immunoblot. The RNA extracted at 72hr of transfection was sent to the core for RNA sequencing. The experiments were repeated two times with SiRNA ITGB4 or SiRNA PXN or both SiRNA ITGB4 and PXN.

肿瘤异质性与顺铂耐药是肿瘤复发及不良预后的核心诱因。本研究证实，在肺癌中，黏着斑（focal adhesion, FA）复合物组分桩蛋白（paxillin, PXN）与整合素β4（integrin b4, ITGB4）之间的相互作用，可介导顺铂耐药。在二维与三维培养体系中，同时敲低PXN与ITGB4可削弱细胞增殖能力，并增强顺铂敏感性。PXN与ITGB4可独立调控多种基因的表达；此外，二者还共同调控包括USP1与VDAC1在内的共有基因，这两种基因分别对维持基因组稳定性与线粒体功能不可或缺。数学建模结果显示，双稳态可诱导这类细胞在顺铂敏感与耐药表型间发生随机表型转换。与之一致的是，分离纯化的敏感与耐药细胞亚群单独培养时，可重现混合亲本群体的表型特征。综上，本研究揭示了黏着斑复合物在顺铂耐药中此前未被认知的作用，并阐明了一种全新的非遗传耐药机制。

总体实验设计：采用购自美国马里兰州罗克维尔市OriGene Technologies公司的小干扰RNA（siRNA，货号：SR302473C）在mRNA水平敲低ITGB4；通过Life Technologies Corporation公司提供的siRNA（货号：4392421）实现PXN的敲低。依照制造商说明书，使用JetPRIME转染试剂（法国Illkirch市Polyplus Transfection公司出品）完成siRNA转染。将细胞以2×10^5个/孔的密度接种于6孔板中，过夜贴壁。次日，每孔使用4 μl JetPRIME试剂与10 nM siRNA在完全生长培养基中进行转染。转染次日更换细胞生长培养基，于转染后72小时通过实时定量PCR（qPCR）与免疫印迹（immunoblot）检测基因表达水平。于转染72小时提取RNA，送至测序中心进行RNA测序。本实验针对ITGB4 siRNA、PXN siRNA或二者联合转染组，均重复开展两次。