Mitochondrial ROS drive resistance to chemotherapy and immune-killing in hypoxic non-small cell lung cancer

Background: Solid tumors subjected to intermittent hypoxia are characterized by resistance to chemotherapy and immune-killing by effector T-Iymphocytes, particularly tumor-infiltrating Vy9V delta 2 T-lymphocytes. The molecular circuitries determining this double resistance are not known.Methods: We analyzed a panel of 28 human non-small cell lung cancer (NSCLC) lines, using an in vitro system simulating continuous and intermittent hypoxia. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo V gamma 9V delta 2 T-lymphocyte expansion and immune-killing by flow cytometry. Targeted transcriptomics identified efflux transporters and nuclear factors involved in this chemo-immuno-resistance. The molecular mechanism linking Hypoxia-inducible factor-1 alpha (HIF-1 alpha), CCAAT/Enhancer Binding Protein-beta (C/EBP-beta) isoforms LAP and LIP, ABCB1, ABCC1 and ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS, depolarization, O-2 consumption were monitored by spectrophotometer and electronic sensors. The role of ROS/HIF-1 alpha/LAP axis was validated in knocked-out or overexpressing cells, and in humanized (Hu-CD34(+)NSG) mice bearing LAP-overexpressing tumors. The clinical meaning of LAP was assessed in 60 NSCLC patients prospectively enrolled, treated with chemotherapy.Results: By up-regulating ABCB1 and ABCC1, and down-regulating ABCA1, intermittent hypoxia induced a stronger chemo-immuno-resistance than continuous hypoxia in NSCLC cells. Intermittent hypoxia impaired the electron transport chain and reduced O-2 consumption, increasing mitochondrial ROS that favor the stabilization of C/EBP-beta mRNA mediated by HIF-1 alpha. HIF-1 alpha/C/EBP-beta mRNA binding increases the splicing of C/EBP-beta toward the production of LAP isoform that transcriptionally induces ABCB1 and ABCC1, promoting the efflux of cisplatin and docetaxel. LAP also decreases ABCA1, limiting the efflux of isopentenyl pyrophosphate, i.e. the endogenous activator of V gamma 9V delta 2 T-cells, and reducing the immune-killing. In NSCLC patients subjected to cisplatin-based chemotherapy, C/EBP-beta LAP was abundant in hypoxic tumors and was associated with lower response to treatment and survival. LAP-overexpressing tumors in Hu-CD34(+)NSG mice recapitulated the patients' chemo-immuno-resistant phenotype. Interestingly, the ROS scavenger mitoquinol chemo-immuno-sensitized immuno-xenografts, by disrupting the ROS/HIF-1 alpha/LAP cascade.Conclusions: The impairment of mitochondrial metabolism induced by intermittent hypoxia increases the ROS-dependent stabilization of HIF-1 alpha/LAP complex in NSCLC, producing chemo-immuno-resistance. Clinically used mitochondrial ROS scavengers may counteract such double resistance. Moreover, we suggest C/EBP-beta LAP as a new predictive and prognostic factor in NSCLC patients.