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.
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。.
Keywords: Breast neoplasms; CDK9; Drug resistance; Programmed death-1; Tyro3.