Construction of crizotinib resistant models with CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutations to explore resistance mechanism and treatment strategy

Cell Signal. 2023 Jan:101:110497. doi: 10.1016/j.cellsig.2022.110497. Epub 2022 Oct 17.

Abstract

Targeted therapy is an essential treatment for non-small cell lung cancer (NSCLC) that is always associated with the drug resistance. c-ros oncogene 1 (ROS1) gene point mutation is one of the leading factors causing drug resistance. However, the point mutation cell models of crizotinib are challenging to obtain, causing few reports on the drug resistance mechanism and the treatment strategy. We constructed CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant plasmids by fusion PCR technology and transfected them into A549 cells. Western blot and MTT assay proved that the drug-resistant cell lines were successfully transfected. The transwell assay confirmed that the mutant cells' motor abilities were significantly increased compared with the wild-type group. In addition, focal adhesion kinase (FAK) was significantly increased in mutant cells. Moreover, crizotinib resistance occurred in the mutant cells through the activation of FAK / phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) pathway. Next, crizotinib was combined with defactinib, a FAK inhibitor, to further explore its therapeutic effect. The results showed that the combination could significantly inhibit the proliferation, invasion and migration of mutant cells. In conclusion, we proved that CD74-ROS1 D2033N and CD74-ROS1 S1986F point mutant NSCLC cells were resistant to crizotinib through the activation of FAK/PI3K/AKT signaling pathway, and inhibiting FAK/PI3K/AKT signaling pathway activation by defactinib could overcome drug resistance in mutant cells.

Keywords: Crizotinib; Defactinib; FAK; NSCLC; ROS1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carcinoma, Non-Small-Cell Lung* / drug therapy
  • Carcinoma, Non-Small-Cell Lung* / genetics
  • Cell Line, Tumor
  • Crizotinib / pharmacology
  • Crizotinib / therapeutic use
  • Drug Resistance, Neoplasm / genetics
  • Humans
  • Lung Neoplasms* / drug therapy
  • Lung Neoplasms* / genetics
  • Oncogenes
  • Phosphatidylinositol 3-Kinases / genetics
  • Point Mutation
  • Protein Kinase Inhibitors / pharmacology
  • Protein Kinase Inhibitors / therapeutic use
  • Protein-Tyrosine Kinases / genetics
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins c-akt / genetics
  • Pyrazoles / pharmacology
  • Pyridines / pharmacology
  • Pyridines / therapeutic use

Substances

  • Crizotinib
  • Protein-Tyrosine Kinases
  • Proto-Oncogene Proteins c-akt
  • defactinib
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins
  • Pyrazoles
  • Pyridines
  • Protein Kinase Inhibitors
  • ROS1 protein, human