Lu Xiao, Zhang Jiao, Li Yan-Qi, Liu Quan-Xing, Zhou Dong, Deng Xu-Feng, Qiu Yuan, Chen Qian, Li Man-Yuan, Liu Xiao-Qing, Dai Ji-Gang, Zheng Hong
Department of Thoracic Surgery, Xinqiao Hospital, Army Medical University, Chongqing, China.
Department of General Surgery, Xinqiao Hospital, Army Medical University, Chongqing, China.
Front Cell Dev Biol. 2022 Feb 3;10:830046. doi: 10.3389/fcell.2022.830046. eCollection 2022.
Almost all lung adenocarcinoma (LUAD) patients with EGFR mutant will develop resistance to EGFR-TKIs, which limit the long-term clinical application of these agents. Accumulating evidence shows one of the main reasons for resistance to EGFR-TKIs is induction of autophagy in tumor cells. Our previous study found that circumsporozoite protein (CSP) in can suppress autophagy in host hepatocytes. However, it is unknown whether CSP-mediated inhibition of autophagy could improve the anti-tumor effect of EGFR-TKIs. We constructed A549 and H1975 cell lines with stable overexpression of CSP (OE-CSP cells). CCK-8, Lactate Dehydrogenase (LDH), flow cytometry, and colony analysis were performed to observe the effect of CSP overexpression on cell viability, apoptosis rate, and colony formation ratio. The sensitizing effect of CSP on gefitinib was evaluated using a subcutaneous tumor model in nude mice and immunohistochemical assay. The role of CSP in regulation of autophagy was investigated by laser confocal microscopy assay and western blotting. A transcriptome sequencing assay and real-time polymerase chain reaction were used to determine the levels of mRNA for autophagy-related proteins. Cycloheximide (CHX), MG132, TAK-243, and immunoprecipitation assays were used to detect and confirm proteasomal degradation of LC3B. OE-CSP A549 and H1975 cells were more sensitive to gefitinib, demonstrating significant amounts of apoptosis and decreased viability. In the OE-CSP group, autophagy was significantly inhibited, and there was a decrease in LC3B protein after exposure to gefitinib. Cell viability and colony formed ability were recovered when OE-CSP cells were exposed to rapamycin. In nude mice with xenografts of LUAD cells, inhibition of autophagy by CSP resulted in suppression of cell growth, and more marked apoptosis during exposure to gefitinib. CSP promoted ubiquitin-proteasome degradation of LC3B, leading to inhibition of autophagy in LUAD cells after treatment with gefitinib. When LUAD cells were treated with ubiquitin activating enzyme inhibitor TAK-243, cell viability, apoptosis, and growth were comparable between the OE-CSP group and a control group both and . CSP can inhibit gefitinib-induced autophagy via proteasomal degradation of LC3B, which suggests that CSP could be used as an autophagy inhibitor to sensitize EGFR-TKIs.
几乎所有携带表皮生长因子受体（EGFR）突变的肺腺癌（LUAD）患者都会对EGFR酪氨酸激酶抑制剂（EGFR-TKIs）产生耐药性，这限制了这些药物的长期临床应用。越来越多的证据表明，对EGFR-TKIs产生耐药性的主要原因之一是肿瘤细胞中自噬的诱导。我们之前的研究发现，[具体内容缺失]中的环子孢子蛋白（CSP）可以抑制宿主肝细胞中的自噬。然而，CSP介导的自噬抑制是否能提高EGFR-TKIs的抗肿瘤效果尚不清楚。我们构建了稳定过表达CSP的A549和H1975细胞系（OE-CSP细胞）。进行细胞计数试剂盒-8（CCK-8）、乳酸脱氢酶（LDH）、流式细胞术和集落分析，以观察CSP过表达对细胞活力、凋亡率和集落形成率的影响。使用裸鼠皮下肿瘤模型和免疫组织化学分析评估CSP对吉非替尼的增敏作用。通过激光共聚焦显微镜分析和蛋白质印迹法研究CSP在自噬调节中的作用。使用转录组测序分析和实时聚合酶链反应来确定自噬相关蛋白的mRNA水平。使用放线菌酮（CHX）、MG132、TAK-243和免疫沉淀分析来检测和确认LC3B的蛋白酶体降解。OE-CSP A549和H1975细胞对吉非替尼更敏感，表现出大量细胞凋亡和活力下降。在OE-CSP组中，自噬被显著抑制，暴露于吉非替尼后LC3B蛋白减少。当OE-CSP细胞暴露于雷帕霉素时，细胞活力和集落形成能力恢复。在携带LUAD细胞异种移植的裸鼠中，CSP对自噬的抑制导致细胞生长受到抑制，并且在暴露于吉非替尼期间凋亡更明显。CSP促进了LC3B的泛素-蛋白酶体降解，导致吉非替尼处理后的LUAD细胞中自噬受到抑制。当用泛素激活酶抑制剂TAK-243处理LUAD细胞时，OE-CSP组和对照组之间的细胞活力、凋亡和生长情况相当。CSP可以通过LC3B的蛋白酶体降解抑制吉非替尼诱导的自噬，这表明CSP可以用作自噬抑制剂来增敏EGFR-TKIs。
