丝氨酸生物合成途径介导肺腺癌细胞EGFR-TKIs靶向治疗适应性耐药

Serine Biosynthesis Pathway Mediated the Adaptive Resistance to EGFR-TKIs in Lung Adenocarcinoma

目的:研究丝氨酸生物合成途径(SSP)在肺腺癌使用表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)治疗后引起的适应性耐药中发挥的作用,探究早期适应性耐药机制以寻找抗耐药靶标。方法:使用EGFR-TKIs药物短时刺激肺腺癌细胞系后,利用Western blotting和qRT-PCR技术检测丝氨酸生物合成途径中关键酶的蛋白及m RNA水平变化,同时利用LC-MS检测细胞内丝氨酸生物合成途径产物及相关代谢产物变化情况。通过CCK8法检测敲低关键酶对细胞增殖的影响。体内实验进行肺腺癌细胞裸鼠皮下移植瘤注射,采用剂量爬坡法构建体内适应性耐药模型,检测肿瘤组织中关键酶表达情况。结果:1.细胞内丝氨酸生物合成途径关键酶PHGDH、PSAT1、PSPH的蛋白表达水平在不同药物作用时间和浓度下有不同程度上调,且m RNA水平也上调了20-50%左右(P<0.05);2.HCC827细胞中SSP及下游代谢通路产物如P-Serine、Serine、Glycine、AMP等均有显著性上调(P<0.01);3.敲低关键酶PSAT1及PSPH后可抑制细肺腺癌细胞HCCC827及PC9的增殖,与对照组相比最高抑制率可达60%左右(P<0.01);4.体内诱导PC9细胞适应性耐erlotinib后,肿瘤组织中的PHGDH及PSAT1表达均有明显上调。结论:丝氨酸生物合成途径介导了肺腺癌EGFR-TKIs靶向治疗的适应性耐药,其关键酶有望作为抗耐药靶标进行联合治疗,从而提高EGFR-TKIs靶向药物的早期疗效并最终克服耐药性的产生。

Objective: To investigate the role of serine biosynthesis pathway in adaptive resistance to EGFR-TKIs in lung adenocarcinoma and to explore the adaptive resistance mechanism in initial therapy, thus nominating the novel targets of overcoming drug resistance. Methods: The protein expression of serine biosynthesis pathway key enzymes in lung adenocarcinoma cells was measured by Western blotting and the m RNA levels of them was detected by qRT-PCR after the short-time treatment with EGFR-TKIs, meanwhile,the metabolites of serine biosynthesis pathway and the relevant metabolites in lung adenocarcinoma cells were detected by LC-MS. The effect of cell proliferation was measured by using CCK8 assay after the knockdown of key enzymes. And the adaptive resistance model in vivo was constructed to detect the protein levels of the key enzymes in tumors by using murine subcutaneous xenografts which were treated with dosage escalation of erlotinib. Results: 1. The protein levels of serine biosynthesis pathway key enzymes including PHGDH,PSAT1, PSPH in lung adenocarcinoma cells were all significantly up-regulated under different drug concentration and treatment time,and their m RNA levels also increased about 20 %-50 %(P<0.05);2. The metabolites of serine biosynthesis pathway and downstream pathways such as P-Serine, Serine, Glycine and AMP were up-regulated significantly in HCC827 cells(P<0.01);3. The depletion of key enzymes PSAT1 and PSPH suppressed the proliferation of lung adenocarcinoma cells and the highest inhibition rate compared to control can be up to about 60 %(P<0.01);4.After the PC9 xenografts adaptively resistant to erlotinib in vivo, the protein levels of PHGDH and PSAT1 in tumors were both up-regulated. Conclusions: Serine biosynthesis pathway mediated the adaptive resistance to EGFR-TKIs in lung adenocarcinoma, and its key enzymes could be the promising targets for combination strategy to enhance the response of EGFR-TKIs and eventually overcome the occurrence of drug resistance.