Tyro3和CDK9与乳腺癌抗程序性死亡蛋白1治疗耐药的关系

Tyro3 and CDK9 as biomarkers for drug resistance to breast cancer anti-PD-1 therapies

目的探讨乳腺癌抗程序性死亡蛋白1(PD-1)治疗耐药的机制和克服抗PD-1治疗耐药的途径。方法建立人三阴性乳腺癌耐药细胞株BT-549R5和小鼠乳腺癌耐药细胞株4T1R3,应用全基因测序分析筛选候选耐药关联分子。采用Western blot检测4T1R3细胞中受体酪氨酸激酶TAM(Tyro3、Axl和MerTK)的表达。通过T细胞杀伤实验观察下调CDK9对T细胞杀伤BT-549R5细胞的影响,通过小鼠成瘤实验观察下调Tyro3和CDK9对乳腺癌移植瘤抗PD-1治疗疗效的影响。结果成功建立了抗PD-1治疗耐药的乳腺癌细胞株和动物模型。TAM家族Tyro3、Axl和MerTK在4T1R3细胞中高表达。全基因组测序显示,Tyro3和CDK9在BT-549R5细胞中高表达。T细胞杀伤实验显示,在效靶比分别为1∶1、5∶1和10∶1的情况下,BT-549R5细胞组细胞存活率均高于BT-549细胞组(均P<0.05),但CDK9下调组BT-549R5细胞的存活率下降迅速。小鼠成瘤实验显示,在抗PD-1治疗的情况下,与4T1细胞组相比,4T1R3细胞组的移植瘤生长迅速,第20天时,肿瘤体积大于4T1细胞组(P<0.05);但CDK9敲降4T1R3细胞组和Tyro3敲降4T1R3细胞组的肿瘤生长速度与4T1细胞组相近,第20天时与4T1R3细胞组肿瘤体积差异均有统计学意义(均P<0.05)。结论Tyro3和CDK9与乳腺癌抗PD-1治疗耐药有关,抑制Tyro3和CDK9的表达可逆转乳腺癌的耐药。

Objective PD-1/PD-L1 immune checkpoint treatment is effective for some triple-negative breast cancer populations with PD-L1 expression,but the response rate is still not satisfactory.This study aims to explore the mechanism of drug resistance to breast cancer anti-PD-1 therapies and the strategies for overcoming the resistance to PD-1therapies.Methods By constructing a human triple-negative breast cancer drug-resistant cell line called BT-549R5 and a mouse breast cancer drug-resistant cell line called 4T1R3,and applying the whole-gene shRNA library screening,candidate drug resistance-associated molecules were obtained and verified by cytological experiments.The expression of Tyro3,Axl and MerTK of the TAM family in the 4T1R3 group was tested using the Western blot method.The down-regulation of CDK9 on the effect of T cells killing the BT-549R5 cells was observed through T cell killing tests,while the down-regulation of Tyro3 and CDK9 on the effect of anti-PD-1 therapies for transplanted breast tumors was observed in mouse tumor formation experiments.Results The cell lines and animal models of breast cancer resistant to PD-1 treatment were successfully constructed.Tyro3,Axl and MerTK were highly expressed in 4T1R3 cells.Whole genome sequencing showed that Tyro3 and CDK9 were highly expressed in BT-549R5 cells.T cell killing experiment showed that the survival rate of BT-549R5 cells in the CDK9 down-regulated group and the control group decreased gradually with the increase of T cells,but the survival rate of BT-549R5 cells in the CDK9 down-regulated group decreased rapidly.Tumor formation experiment in mice showed that under anti-PD-1 treatment,the transplanted tumor in the 4T1R3 cell group grew rapidly compared with the 4T1 cell group(P<0.05),and the tumor volume of the 4T1R3 group was larger than that of the 4T1 group on Day 20.Nevertheless,the tumor growth rates in the CDK9-knockdown 4T1R3 cell group and the Tyro3-knockdown 4T1R3 cell group were similar to that of the 4T1 cell group,and the tumor volumes at day 20 were signiference lower than that of 4T1R3 cell group(P<0.05).Conclusions Tyro3 and CDK9 are associated with the drug resistance to anti-PD-1 therapies for breast cancer.Inhibiting the expression of Tyro3 and CDK9 can reverse the drug resistance to breast cancer treatment.