Proteomics reveals a therapeutic vulnerability via the combined blockade of APE1 and autophagy in lung cancer A549 cells

BMC Cancer. 2020 Jul 8;20(1):634. doi: 10.1186/s12885-020-07111-w.

Abstract

Background: Drug resistance is a major cause of therapeutic failure that is often associated with elevated autophagy and apurinic/apyrimidinic endonuclease 1 (APE1) expression. Herein, we investigated the role of APE1 and autophagy in A549 cells treated with cisplatin.

Methods: SILAC proteomics was applied to obtain a panoramic view of cisplatin treatment in KRASG12S-mutant A549 cells. Quantity analysis of cellular apoptosis and autophagy was based on flow cytometry. Western blotting was used to examine the expression levels of apoptosis- and autophagy-related proteins, as well as those of APE1. Knockdown of APE1 was achieved by RNA interference. Immunoprecipitation was further employed to reveal the molecular interaction of APE1, p53, and LC3 when A549 cells were exposed to cisplatin.

Results: SILAC proteomics revealed that 72 canonical pathways, including base excision repair (BER) and autophagy signalling pathways, were regulated after cisplatin treatment in A549 cells. Cisplatin markedly induced autophagy and apoptosis in A549 cells, accompanied by remarkable APE1 increase. Suppression of autophagy enhanced the inhibition effect of cisplatin on cell growth, proliferation, and colony formation; however, APE1 inhibition enhanced the expression of LC3-I/II, suggesting that APE1 and autophagy are compensatory for cell survival to evade the anticancer action of cisplatin. Immunoprecipitation results revealed the triple complex of APE1-p53-LC3 in response to cisplatin plus CQ in A549 cells. Dual inhibition of APE1 and autophagy significantly enhanced cisplatin-induced apoptosis, which eventually overcame drug resistance in cisplatin-resistant A549 cells.

Conclusions: Dual inhibition of APE1 and autophagy greatly enhances apoptosis in parental KRASG12S-mutant A549 cells and cisplatin-resistant A549 cells via regulation of APE1-p53-LC3 complex assembly, providing therapeutic vulnerability to overcome cisplatin resistance in the context of KRASG12S-mutant lung cancer.

Keywords: APE1; Apoptosis; Autophagy; Chemotherapy; Cisplatin; Non-small cell lung cancer.

MeSH terms

  • A549 Cells
  • Antineoplastic Combined Chemotherapy Protocols / pharmacology*
  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use
  • Apoptosis / drug effects
  • Apoptosis / genetics
  • Autophagy / drug effects*
  • Autophagy / genetics
  • Cell Proliferation / drug effects
  • Cell Proliferation / genetics
  • Chloroquine / pharmacology
  • Chloroquine / therapeutic use
  • Cisplatin / pharmacology
  • Cisplatin / therapeutic use
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / genetics*
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / metabolism
  • Drug Resistance, Neoplasm / drug effects
  • Drug Resistance, Neoplasm / genetics
  • Gene Knockdown Techniques
  • Humans
  • Lung Neoplasms / drug therapy*
  • Lung Neoplasms / pathology
  • Microtubule-Associated Proteins / metabolism
  • Proteomics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • Chloroquine
  • APEX1 protein, human
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • Cisplatin