C/EBP-β LAP regulates chemo-immuno-resistance in hypoxic non-small cell lung cancer

Abstract Background: Solid tumors subjected to intermittent hypoxia are characterized by resistance to chemotherapy and immune-killing by effector T-lymphocytes, particularly tumor-infiltrating Vγ9Vδ2 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 to. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo Vγ9Vδ2 T-lymphocyte expansion and immune-killing by flow cytometry. Targeted transcriptomic identified efflux transporters and nuclear factors involved in chemo-immuno-resistance. The molecular mechanism linking Hypoxia-inducible factor-1α (HIF-1α), CCAAT/Enhancer Binding Protein-β (C/EBP-β) isoforms LAP and LIP, and ABCB1, ABCC1, ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS and depolarization were measured spectrofluorimetrically. The role of ROS/HIF-1α/LAP axis was validated in HIF-1α/LAP knocked-out/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, increasing mitochondrial ROS that favor the stabilization of C/EBP-β mRNA mediated by HIF-1α. HIF-1α/C/EBP-β mRNA binding increases 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 Vγ9Vδ2 T-cells, and reducing the immune-killing. In NSCLC patients subjected to cisplatin-based chemotherapy, C/EBP-β LAP was abundant in hypoxic tumors and was associated with lower response to treatment and survival. Accordingly, in Hu-CD34+NSG mice, LAP-overexpressing tumors were more chemo-immuno-resistant than parental tumors. Interestingly, the ROS scavenger mitoquinol re-induced chemo-immuno-sensitization, by disrupting the ROS/HIF-1α/LAP cascade. Conclusion: The impairment of mitochondrial metabolism induced by intermittent hypoxia increases the ROS-dependent stabilization of HIF-1α/LAP complex in NSCLC, producing chemo-immuno-resistance. Clinically used mitochondrial ROS scavengers may counteract such double resistance. Moreover, we suggest C/EBP-β LAP as a new predictive and prognostic factor in NSCLC patients.