Mitochondrial ROS drive resistance to chemotherapy and immune-killing 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. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo Vγ9Vδ2 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-1α (HIF-1α), CCAAT/Enhancer Binding Protein-β (C/EBP-β) isoforms LAP and LIP, ABCB1, ABCC1 and ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS, depolarization, O2 consumption were monitored by spectrophotometer and electronic sensors. The role of ROS/HIF-1α/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 O2 consumption, 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 splicing of C/EBP-β 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 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. 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-1α/LAP cascade. Conclusions 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.

FLVCR1a Controls Cellular Cholesterol Levels through the Regulation of Heme Biosynthesis and Tricarboxylic Acid Cycle Flux in Endothelial Cells

“…Mitochondria were extracted as reported in [24]. Cells were lysed in 0.5 ml mitochondria lysis buffer (50 mM Tris-HCl, 100 mM KCl, 5 mM Mg Cl 2 , 1.8 mM ATP, and 1 mM EDTA at pH7.2), supplemented with protease inhibitor cocktail III (Sigma Aldrich), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 250 mM NaF.…”

New Dual P-Glycoprotein (P-gp) and Human Carbonic Anhydrase XII (hCA XII) Inhibitors as Multidrug Resistance (MDR) Reversers in Cancer Cells

“…This feature makes the compounds particularly promising as chemosensitizer agents in the most aggressive and drug-resistant tumors that coexpress both P-gp and hCA XII. This is the case of tumors rich in stem cells, which are often responsible for tumor relapse, metastization, and generation of drug-resistance clones, − or hypoxic tumors that are the most invasive and resistant to the conventional therapies in use, − where the transcription factor HIF-1α transcriptionally upregulates P-gp and hCA XII …”

Fighting Multidrug Resistance with Ruthenium–Cyclopentadienyl Compounds: Unveiling the Mechanism of P-gp Inhibition

“…15 The main reason for chemoresistance to cisplatin in the three cell lines analyzed is the presence of ABC transporters, as previously mentioned. 26 Notably, compounds 2 and 3 inhibited the activity of P-gp and MRP1 (Figure 7A,B), with a potency superimposable to that of RT150 and RT151 in the same cell lines. 15 Overall, these results are clear evidence of the importance that the ABC transporters surely have on the mechanism of action for these compounds, in particular for RT150 and RT151, which are only active against cells overexpressing MRP1 and P-gp.…”

“…42 Three boxes (R-site, M-site, and H-site) were used in our protocol, each encompassing the residues of the three substrate-binding sites introduced in Ferreira et al 43 (Figure 8 and Table S6 of the SI). Each docking box was created using a spacing of 1.0 Å, the Autodock Vina v1.2 standard, with sizes (20,30,30), (24,30,28), and (26,28,32) and centered at (−10.466,−5.677,−27.858), (−3.294,−1.928,− 49.003), and (3.456,7.964,−33.394), for the R, M, and H-site, respectively. The maximum number of binding modes and the search exhaustiveness were set to 20.…”