title: Repression of the autophagic response sensitises lung cancer cells to radiation and chemotherapy.
abstract: The cellular autophagic response to radiation is complex. Various cells and tissues respond differentially to radiation, depending on both the dose of exposure and the time post irradiation. In the current study, we determined the autophagosomal and lysosomal response to radiation in lung cancer cell lines by evaluating the expression of the associated proteins, as well as the effect of relevant gene silencing in radio and chemosensitisation. Furthermore, tumour sensitisation was evaluated in in vivo autophagic gene silencing model after irradiation.
  
  
  
  A549 and H1299 cell lines were utilised as in vitro cancer models. Both cell lines were transfected with various small-interfering RNAs, silencing auto-lysosomal genes, and irradiated with 4 Gy. Cell growth response was evaluated with AlamarBlue assay. Western blot and confocal microscopy were utilised for the characterisation of the auto-lysosomal flux. Also, the H1299 cell line was stable transfected with small-hairpin RNA of the MAP1LC3A gene, and the tumour radiosensitisation in Athymic Nude-Foxn1(nu) was evaluated.
  
  
  
  Following exposure to 4 Gy of radiation, A549 cells exhibited a significant induction of the autophagic flux, which was not supported by transcriptional activation of auto-lysosomal genes (LC3A, LC3B, p62, TFEB and LAMP2a), resulting in aggresome accumulation. Recovery of transcriptional activity and autophagy efficacy occurred 7 days post irradiation. Alternatively, H1299 cells, a relatively radio-resistant cell line, sharply responded with an early (at 2 days) transcriptional activation of auto-lysosomal genes that sustained an effective autophagosomal flux, resulting in adequate aggresome clearance. Subsequently, we tested the silencing of four genes (LC3A, LC3B, TFEB and LAMP2a), confirming a significant radiosensitisation and chemosensitisation to various chemotherapeutic agents, including cisplatin and taxanes. In mouse xenografts, exposure to radiation significantly reduced tumour growth (P<0.001), which was exacerbated among shLC3A-H1299 transfected tumours.
  
  
  
  The ability of lung cancer cells to survive after irradiation at 4 Gy depends on their ability to sustain a functional autophagic flux. Abrogation of such ability results in increased radiosensitivity and susceptibility to various chemotherapy agents. Selective inhibitors of cancer cell autophagic function may prove important for the eradication of lung cancer.
authors.affiliation: Department of Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis 68100, Greece.
authors.forename: Ilias V
authors.initials: IV
authors.lastname: Karagounis
authors.affiliation: Department of Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis 68100, Greece.
authors.forename: Dimitra
authors.initials: D
authors.lastname: Kalamida
authors.affiliation: Department of Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis 68100, Greece.
authors.forename: Achilleas
authors.initials: A
authors.lastname: Mitrakas
authors.affiliation: Department of Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis 68100, Greece.
authors.forename: Stamatia
authors.initials: S
authors.lastname: Pouliliou
authors.affiliation: Department of Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis 68100, Greece.
authors.forename: Maria V
authors.initials: MV
authors.lastname: Liousia
authors.affiliation: Department of Pathology, Democritus University of Thrace, Alexandroupolis 68100, Greece.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Giatromanolaki
authors.affiliation: Department of Radiotherapy/Oncology, Democritus University of Thrace, Alexandroupolis 68100, Greece.
authors.forename: Michael I
authors.initials: MI
authors.lastname: Koukourakis
mesh_terms: D000818:Animals; D001343:Autophagy; D045744:Cell Line, Tumor; D020868:Gene Silencing; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000473:pathology* / Q000532:radiotherapy; D051379:Mice; D008819:Mice, Nude; D011836:Radiation Tolerance; D023041:Xenograft Model Antitumor Assays