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

doi:10.21203/rs.3.rs-1503033/v1

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

https://doi.org/10.21203/rs.3.rs-1503033/v1

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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.

intermittent hypoxia

chemo-immuno-resistance

ABC transporters

CCAAT/Enhancer Binding Protein-β

mitochondrial ROS

non-small cell lung cancer

Rapidly growing solid tumors often generate hypoxic regions within their bulk, characterized by a partial O₂ pressure (pO₂) around 1-1.3%. The activation of neo-angiogenesis partially compensates the drop in pO₂, but the poorly organized architecture and the easy collapse caused by tumor growth and extracellular matrix deposition do not allow a good compensation of hypoxia [1]. The alternation between the vessels formation and their collapse generates repeated cycles of hypoxia and normoxia [2], a condition that resembles the hypoxia/reoxygenation observed in cardiovascular diseases.

Intermittent hypoxia contributes to generate tumor niches more resistant to chemotherapy, by multiple mechanisms. Hypoxic conditions stabilize the hypoxia induced factor α subunit (HIF-α) that is subtracted to the O₂-dependent hydroxylation by prolyl hydroxylase domain (PHD) enzymes and subsequent degradation [3]. α subunit of HIF is free to associate the O₂-independent β subunit, forming an active transcription factor that controls more than 200 genes, promoting cell proliferation, epithelial mesenchymal transition, invasion, metabolic adaptation to the O₂-deprived environment and chemoresistance [1]. For instance, HIF-1α activates anti-apoptotic proteins of Bcl-2 family and survivins, destabilizes TP53, up-regulates DNA repair genes such as DNA-PKs, Ku80 and Ku70 [4]. As a result, HIF-1α reduces the efficacy of chemotherapeutic drugs that damage DNA, as cisplatin and gemcitabine [4,5]. In parallel, the increased anaerobic metabolism of glucose, favored by the up-regulation of glucose transporter 1 and glycolytic enzymes, determines intratumor acidosis that neutralizes weak bases chemotherapeutic drugs, such anthracyclines, favoring their sequestration within lysosomes [6]. The reduction of oxidative phosphorylation (OXPHOS)-based metabolism and the increased mitophagy associated with hypoxia, which allows the recovery of ATP and oxide-reductive cofactors, further protect cancer cells from chemotherapy-induced damages [7]. Furthermore, HIF-1α is a transcriptional inducer of ATP Binding Cassette (ABC) transporter genes, such as ABC transporter B1/P-glycoprotein (ABCB1/Pgp) [8] and ABC transporter C1/multidrug resistance related protein 1 (ABCC1/MRP1) [9] that actively efflux several chemotherapeutic drugs and determine a multidrug resistant phenotype [10].

Hypoxia also generates endoplasmic reticulum (ER) stress that can produce cell death or survival, favoring or counteracting chemotherapy effects [11]. In neurons, intermittent hypoxia has been shown to activate the CCAAT/Enhancer Binding Protein (C/EBP) [12], which is of paramount importance in tumor resistance to chemotherapy. Indeed, by alternative splicing, C/EBP-β generates the full-length LAP isoform that prevents apoptosis and up-regulates ABCB1, or the C-terminal LIP isoform that has opposite effects [13].

The ratio between LIP/LAP [14,15]upon ER stress also affects the immuno-resistance of cancer cells, by pleiotropic mechanisms. First, ABCB1 impairs the immuno-sensitizing functions of calreticulin, an ER-residing protein that is exposed on plasma membrane in case of ER stress or chemotherapy and favors the phagocytosis of cancer cells by dendritic cells [16]. Second, LIP is a transcriptional activator of calreticulin, while LAP is a calreticulin repressor [14]. Furthermore, ER stress regulates the levels of ABC transporter A1 (ABCA1) [17], physiologically involved in cholesterol efflux, but also in the anti-tumor activity of T-cells. We previously identified ABCA1 as the efflux transporter of isopentenyl pyrophosphate (IPP), an isoprenoid metabolite of the cholesterol synthetic pathway [18]. IPP is the endogenous activator of Vγ9Vδ2 T-cells [19], a lymphocytic subset with strong killing properties against tumor cells [20] and considered the immune-infiltrating population with highest positive prognostic value [21]. To close the molecular circuitry linking chemo- and immuno-resistance, in 3D cultures, characterized by constitutively activation of HIF-1α and chemoresistance cells [16], ABCB1 and ABCC1 are up-regulated, while ABCA1 is down-regulated [15]. This phenotype makes cells resistant to chemotherapy and immune-killing.

In the present work we investigated how the response to chemotherapy and immune-killing by Vγ9Vδ2 T-cells are affected by hypoxia. We focused on non-small cell lung cancer (NSCLC), the first tumor for number of deaths worldwide, characterized by resistance to chemotherapy or immunotherapy in up to 30% patients [22]. By reproducing in vitro the intermittent hypoxia that often characterizes solid tumors, we found that the mitochondrial reactive oxygen species (mtROS) produced by intermittent hypoxia promotes the interaction between HIF-1α and C/EBP-β mRNA, favoring the generation of LAP isoform. LAP induced chemo-immuno-resistance, and was as a negative prognostic factor in NSCLC patients. On the other hand, mtROS scavengers emerged as potent chemo-immuno-sensitizer agents in hypoxic tumors.

Chemicals. Fetal bovine serum (FBS) and culture medium were from Invitrogen Life Technologies (Carlsbad, CA). Plasticware for cell cultures was from Falcon (Becton Dickinson, Franklin Lakes, NJ). The protein content was assessed with the BCA kit from Sigma Aldrich. (St. Louis, MO). Cis-diammineplatinum (II) dichloride, docetaxel, elesclomol were purchased by Sigma Aldrich. [10-(2,5-dihydroxy-3,4-dimethoxy-6,ethylphenyl)decyl]triphenyl-phosphonium, monomethanesulfonate (mitoquinol or mitoQ) was from Cayman Chemical (Ann Arbor, MI).

Cells. Human NSCLC cells (NCI-H1650, NCI-H1385, NCI-H460, NCI-H522, NCI-H661, NCI-H2126, NCI-H23, NCI-H1703, NCI-H1435, NCI-H596, NCI-H2286, NCI-H1437, NCI-H1651, NCI-H2085, NCI-H2342, NCI-H2073, NCI-H1793, NCI-H2170, NCI-H1299, NCI-H2066, NCI-H2347, NCI-H1734, NCI-H1563, NCI-H441, NCI-H1975, A549, Calu-3, NCI-H2228) were purchased from ATCC (Manassas, VA). Cells were maintained in the respective medium supplemented with 10% v/v FBS, 1% v/v penicillin-streptomycin, 1% v/v L-glutamine, in a Heracell incubator (ThermoFisher, Waltham, MA) with different pO₂. The experimental conditions were set as it followed: 24h at 20% O₂ (normoxia), 24h at 1% O₂ (hypoxia), 12 h at 1% O₂followed by12h at 20% O₂ (hypoxia/normoxia), 12h at 1% O₂followed by12h at 20% O₂ and 12h at 1% O₂(hypoxia/normoxia/hypoxia or intermittent hypoxia).

Cell viability. Cells were incubated 48h with increasing concentrations (from 1×10^-9 to 1×10^-5M) of cisplatin or docetaxel. Cell viability was measured by the ATPlite Luminescence Assay System (PerkinElmer, Waltham, MA), as per manufacturer’s instructions, using a Synergy HT Multi-Detection Microplate Reader (Bio-Tek Instruments, Winooski, VT) to measure the relative luminescence units (RLUs). The RLUs of untreated cells were considered as 100% viability; the results were expressed as a percentage of viable cells versus untreated cells. The IC₅₀ was calculated applying the [inhibitor] vs. normalized dose-response equation (GraphPad Prism 9 software, https://www.graphpad.com/).

Flow cytometry. 1×10⁴ cells were washed in phosphate-saline buffer (PBS), pH 7.2, 0.5% bovine serum albumin and 2 mM EDTA, centrifuged at 300 × g for 10 min, incubated 20 min at room temperature in the dark with 250 µl of Inside Fix reagent (Inside Stain Kit, Miltenyi Biotec., Bergisch Gladbach, Germany), centrifuged at 300×g for 5 minutes, washed with 1 ml of Inside Perm (Inside Stain Kit), centrifuged at 300×g for 5 minutes, and incubated 30 minutes at room temperature with the following antibodies (all from Miltenyi): anti-CD243/ABCB1 antibody (PE-Vio® 770-conjugated); anti-MRP1/ABCC1 antibody (PE-conjugated); anti-ABCA1(DyLight 488-conjugated). Cells were washed with 1 ml of Inside Perm reagent, centrifuged at 300×g for 5 minutes and read using a Guava® easyCyte flow cytometer (Millipore, Billerica, MA), equipped with the InCyte software (Millipore).

PCR arrays and qRT-PCR. Total RNA was extracted and reverse-transcribed using the iScript^TM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA). The PCR arrays were performed on 1 μg cDNA, using customized Plus PCR Arrays (Bio-Rad Laboratories) pre-coated with primers for the ABC transporters or the transcription factors indicated in the Results section, as per manufacturer’s instructions. Data analysis was performed with the PrimePCR™ Analysis Software (Bio-Rad Laboratories). The qRT-PCR was performed with the IQ SYBR Green Supermix (Bio-Rad Laboratories). The primer sequences, designed using the qPrimerDepot database (http://primerdepot.nci.nih.gov/), were reported in the Supplemental Table S1. The relative quantification was performed by comparing each PCR product with the housekeeping PCR product S14, using the Bio-Rad Software Gene Expression Quantitation (Bio-Rad Laboratories).

Promoter and transcription factors analysis. The promoter sequences were identified from the Eukariotic Promoter Database (EPD; https://epd.epfl.ch//index.php), using as inputs: https://epd.epfl.ch/cgi-bin/get_doc?db=hgEpdNew&format=genome&entry=ABCB1_1 (RefSeq NM_001348945) for ABCB1, https://epd.epfl.ch/search_EPDnew.php?query=ABCC1&db=human (RefSeq NM_004996) for ABCC1, https://epd.epfl.ch/cgi-bin/get_doc?db=hgEpdNew&format=genome&entry=ABCA1_1 (RefSeq NM_080282) for ABCA1. The transcription factors predicted to bind ABCB1, ABCC1 and ABCA1 promoters (Supplemental Table S2) were identified using the TRANSFAC software v 8.3 (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) [23].

Immunoblotting. Cells were lysed in the MLB buffer (125mM Tris-HCl, 750mM NaCl, 1%v/v NP40, 10%v/v glycerol, 50mM MgCl₂, 5mM EDTA, pH 7.5, supplemented with the protease inhibitor cocktail set III (Sigma Aldrich), 25mM NaF, 1mM NaVO₄, 10mg/ml aprotinin), sonicated and centrifuged at 13000×g for 10 minutes at 4 °C. 50μg of proteins were subjected to immunoblotting and probed with the following antibodies: anti-HIF-1α (clone 54, BD Transduction Laboratories), C/EBP-β (clone C-19, directed against the common C-terminus of LIP and LAP, Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by a peroxidase-conjugated secondary antibody. Anti-actin (clone C-4, Sigma Aldrich) was used as a control of equal protein loading. The proteins were detected by enhanced chemiluminescence (Bio-Rad Laboratories). In co-immunoprecipitation assays,100μg of whole cell lysates were immuno-precipitated at 4°C overnight with the PureProteome Protein A/G Mix Magnetic Beads (Millipore) as per manufacturer’s instruction, in the presence of the anti-C/EBP-β antibody (C-19, diluted 1/50), then blotted with the anti-HIF-1α antibody as reported above.

RNA immunoprecipitation (RNA-IP). Total RNA was isolated using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Sigma Aldrich), as per manufacturer’s instructions. 2 µg RNA were immuno-precipitated for 3 h at 4°C using an anti-HIF-1α antibody (PA1-16601, Invitrogen Life Technologies). A blank was prepared incubating the samples without the antibody. The immunoprecipitated RNA was retro-transcribed using the iScript^TM cDNA Synthesis Kit (Bio-Rad Laboratories). RT-PCR was performed with the IQ SYBR Green Supermix (Bio-Rad Laboratories), using primers upstream and downstream the hypoxia-response elements (HREs) contained in C/EBP-β LAP and LIP, respectively (Supplemental Table S1). The expression in normoxic cells was considered as 1. The levels of LAP and LIP mRNA in the other experimental condition were expressed as fold-enrichment versus normoxic cells, using the Bio-Rad Software Gene Expression Quantitation (Bio-Rad Laboratories).

Chromatin immunoprecipitation (ChIP). Cells were lysed and sonicated as reported previously [18]. 200µl of the samples were used as inputs. The remaining lysates were pretreated for 2h at 4°C with the Magna ChIP™ Protein A+G Magnetic Beads (Sigma Aldrich), as per manufacturer’s instructions. Samples were incubated overnight with an anti-C/EBP-β antibody (clone C-19, Santa Cruz Biotechnology Inc.) or without antibody, as a blank. The recovered DNA was washed, eluted with the elution buffer (0.1M NaHCO₃, 1% w/vSDS), heated at 65°C for 6h and incubated with proteinase K for 1h at 55°C. Samples were cleaned by Qiaquick spin columns (Qiagen, Venlo, The Netherlands). The CAAT sites on ABCB1, ABCC1 and ABCA1 promoters were identified using the Transfac® Database (http://genexplain.com/transfac/). Inputs and immunoprecipitated samples were analysed by RT-PCR, using primers designed to amplify the sequence around the CAAT sites in ABCB1, ABCC1 and ABCA1 promoters, as well as nonspecific primers, used as negative internal controls (Supplemental Table S2).

Cell transfection. 1µg pcDNA4/TO expression vectors (Invitrogen Life Technologies, Milan, Italy) for C/EBP-β LAP and LIP, produced as reported previously [24], were co-transduced with 1µg pcDNA6/TR vector (Invitrogen Life Technologies) in 1×10⁶ cells. Stable TetON clones were generated by selecting cells with 2μg/ml blasticidin S (ThermoFisher, A1113903) and 100μg/ml zeocin (InvivoGen, San Diego, CA; ant-zn-05). LAP and LIP induction was activated by adding 1μg/ml doxycycline (D9891, Sigma Aldrich) in the culture medium. LAP and LIP expression was analyzed by immunoblotting 24h after doxycycline treatment. For silencing, 1×10⁶ cells were transduced with 1µg of a green fluorescence protein (GFP)-lentiviral plasmid containing a non-effective 29-mer scrambled shRNA cassette (Origene, Rockville, MD), two different sequences targeting HIF-1α (TL320380, Origene), two different sequences targeting C/EBP-β (TL320301, Origene). Stably silenced clones were generated by selecting cells with 0.25μg/ml puromycin (ant-pr-1, InvivoGen) for 4 weeks. The silencing was verified by RT-PCR and immunoblotting.

Release of IPP. The efflux of IPP was measured by radiolabelling 1×10⁶ cells for 1h with 0.02 mCi of [¹⁴C]-IPP (50 mCi/mmol; Amersham International, Piscataway, NJ), extracting lipids, isolating the IPP by thin layer chromatography and counting radiolabelled IPP by liquid scintillation [18]. Results were expressed as nanomoles/ml, according to the relative calibration curves.

Vγ9Vδ2 T-lymphocytes activation and tumor killing. Blood samples were obtained from healthy blood donors (Blood Bank of the AOU Città della Salute e della Scienza, Torino, Italy; DG 767/2015). After isolation on a Ficoll-Hypaque density gradient, peripheral blood mononuclear cells (PBMC) were sorted using the TCRγ/δ⁺T Cell Isolation Kit (Miltenyi Biotec.). The presence of Vγ9Vδ2 T-lymphocytes was confirmed by staining 5×10⁵isolated cells with anti-TCR Vγ9 (VioBlue conjugated; Miltenyi Biotec) and anti-CD3 (fluorescein-isothiocyanate FITC conjugated, Miltenyi Biotec) antibodies. Samples with >80% Vγ9⁺/CD3⁺ cells were incubated 48 h with 1μM zoledronic acid (Sigma Aldrich) and 10IU/ml IL-2 (Sigma Aldrich), to expand Vγ9Vδ2 T-lymphocytes [19], then 5×10⁵Vγ9Vδ2 T-lymphocytes were cultured overnight with NCI-H2228 cells at 1:1 ratio. After this incubation period, the amount of active and proliferating Vγ9Vδ2 T-lymphocytes was measured by staining cells present in the supernatnats with anti-Ki67 (FITC conjugated) and anti-INF-γ (allophycocyanin APC conjugated) antibodies (Miltenyi Biotec), and quantified with a Guava® easyCyte flow cytometer (InCyte software). Results were expressed as percentage of Vγ9⁺Ki67⁺IFNγ⁺over Vγ9⁺cells. Vγ9Vδ2 T-lymphocytes killing was measured as reported [15]. After Vγ9Vδ2 T-lymphocytes/NCI-H2228 cells co-incubation, adherent (i.e. NSCLC cells) were washed twice with PBS, detached by gentle scraping and stained with the Annexin V/Propidium Iodide kit (Sigma Aldrich.), as per manufacturer’s instruction. The fluorescence was acquired using a Guava® easyCyte flow cytometer (InCyte software). The percentage of Annexin V⁺/Propidium Iodide⁺ cancer cells was considered as an index of Vγ9Vδ2 T-lymphocytes killing.

Electron transport chain (ETC) activity. Mitochondria were isolated from 10×10⁶cells. 50 µl aliquots were sonicated and used for the measurement of protein content by the BCA Protein Assay kit (Sigma). 200µl were used to measure electron flux from complex I to complex III, taken as an index of the mitochondrial respiratory activity, as reported [25]. Results were expressed as nanomoles of reduced cytochrome c/min/mg mitochondrial proteins.

Mitochondrial ATP. ATP levels in mitochondrial extracts were measured with the ATP Bioluminescent Assay Kit (FLAA; Sigma Aldrich), as per manufacturer’s instructions. Results were expressed as nanomoles/mg mitochondrial proteins.

Mitochondrial depolarization. 1×10⁶ cells were washed with PBS, detached by gentle scraping and incubated for 30 minutes at 37°C with 2μM of the fluorescent probe JC-1 (Biotium Inc., Hayward, CA). The relative fluorescence units (RFUs) were used to calculate the percentage of green (depolarized)/red (polarized) mitochondria, considered an index of damaged mitochondria [26].

Total and mitochondrial ROS. 10×10⁶cells were washed with PBS and detached by gentle scraping. One 50 µl aliquot was sonicated and used to measure cellular proteins. The remaining cells were treated for 30 min at 37^oC with 5µM of the ROS-sensitive fluorescent probes 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate (CM-H₂DCFDA) (ThermoFisher) or with 5µM MitoSOX (ThermoFisher), to measure total and mtROS, respectively. The RFUs were converted into nanomoles ROS/mg proteins, according to a titration curve performed with serial dilutions of H₂O₂.

Patient enrolment and immunohistochemical analyses. 60 patients with confirmed histological diagnosis of NSCLC, candidate to receive cisplatin/carboplatin as first-line therapy, were prospectively enrolled at San Lugi Gonzaga Hospital, Orbassano, and Città della Salute e della Scienza Hospital, Torino, Department of Oncology, University of Torino, Italy (March 2018-January 2020). Each patient was anonymized and indicated as “unknown patient number” (UPN). The pathological features, the smoking habits, the clinical follow-up (progression free survival, PFS; overall survival, OS) of patients, performed at the Thoracic Oncology Unit, San Luigi Gonzaga Hospital, are reported in the Supplemental Table S3. Formalin-fixed paraffin-embedded (FFPE) samples of patients were analyzed for the presence of hypoxic areas using the pimonidazole-based Hypoxyprobe^TM Kit (Hypoxyprobe Inc., Burlington, MA), according to the manufacturer’s instructions. The same sections were also stained with an antibody recognizing only the N-terminal portion of C/EBP-β, corresponding to LAP (clone 21A1, ThermoFisher), followed by peroxidase-conjugated horseradish antibody (Dako, Glostrup, Denmark). Nuclei were counterstained with hematoxylin (Sigma Aldrich). C/EBP-β LAP was considered positive when a weak-to strong nuclear or cytosolic positivity was shown. The tumor proportion positivity was recorded. Patients were divided into LAP^low and LAP^high, if the tumor proportion of LAP staining was respectively below or equal/above the median value. The Ethics Committee of San Luigi Gonzaga Hospital, Orbassano, Italy approved the study (#73/2018).

Immuno-xenografts. 1×10⁶ NCI-H2228 C/EBP-β LAP-overexpressing cells, mixed with 100 μl Matrigel (Sigma Aldrich), were injected subcutaneously (s.c.) in female NOD SCID-γ (NSG) mice engrafted with human hematopoietic CD34⁺ cells (Hu-CD34⁺; The Jackson Laboratories, Bar Harbor, MA). Mice were housed (5 per cage) under 12h light/dark cycle, with food and drinking provided ad libitum. When indicated, doxycycline (1mg/ml) was added daily to the drinking water to induce C/EBP-β LAP intratumorally. Tumor growth was measured daily by caliper, according to the equation (LxW²)/2, where L=tumor length and W=tumor width. When tumors reached the volume of 50mm³, animals (6/group) were randomized and treated for 6 weeks as it follows: 1) vehicle group, treated with 0.1ml saline solution intravenously (i.v.), once a week; 2) cisplatin group, treated with 2mg/kg cisplatin i.v., once a week; 3) mitoquinol (mitoQ) group, treated with 100μg/kg daily via oral gavage; 4) cisplatin+mitoQ group, treated with 2mg/kg cisplatin i.v., once a week, and 100μg/kg mitoQ daily via oral gavage. Tumor volumes were monitored by caliper and animals were euthanized at day 49 after randomization with zolazepam (0.2ml/kg) and xylazine (16mg/kg). Animal weights were monitored throughout the study. Tumors were excised, weighted and photographed. Tumor sections, fixed in 4% v/v paraformaldehyde, were stained with hematoxylin/eosin (Sigma Aldrich) or immunostained for: Hypoxyprobe^TM Kit, LAP (clone 21A1, ThermoFisher), C/EBP-β LAP (ThermoFisher), ABCB1 (Novus Biologicals, Centennial, CO), ABCC1 (MyBioSource, San Diego, CA), ABCA1 (Abcam), cleaved (Asp175) caspase-3 (Cell Signaling Technology, Danvers, MA, diluted 1/400), followed by a peroxidase-conjugated secondary antibody (Dako). Nuclei were counterstained with hematoxylin (Sigma Aldrich). Nuclei were counter-stained with hematoxylin. Sections were examined with a Leica DC100 microscope. To evaluate the intratumour Vγ9δ2 T-lymphocytes, the tumors were digested with 1mg/ml collagenase (Sigma Aldrich) and 0.2mg/ml hyaluronidase (Sigma Aldrich) for 1h at 37 °C and filtered using a 70μm cell strainer to obtain a single cell suspension. Infiltrating immune cells were collected by centrifugation on Ficoll-Hypaque density gradient and immunostained with the following antibodies (Miltenyi Biotec): anti-CD3, anti-Vγ9, anti-Ki67, anti-INF-γ, as indicated in the “Vγ9Vδ2 T-lymphocytes activation” section. Cells were quantified with using Guava® easyCyte flow cytometer and InCyte software. Results were expressed as percentage of Vγ9⁺Ki67⁺IFNγ⁺ over CD3⁺ cells. At 3.5 weeks and immediately after the euthanasia, 200 µL blood were collected to measure the following parameters: red blood cells (RBC), white blood cells (WBC), haemoglobin (Hb), platelets (PLT), as indexes of bone marrow function; lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP), as indexes of liver function; creatinine, as index of kidney function; creatine phosphokinase (CPK) as index of muscle/heart damage, using commercially available kits from Beckman Coulter Inc. (Miami, FL).

Animal care and experimental procedures were approved by the Italian Ministry of Health (#627/2018-PR, 10/08/2018).

Statistical analysis. All data in the text and figures are provided as means ± SD. The results were analyzed by a one-way analysis of variance (ANOVA), using Statistical Package for Social Science (SPSS) software (IBM SPSS Statistics v.19). p<0.05 was considered significant. The Kaplan-Meier method was used to calculate the PFS (survival from the beginning of chemotherapy to the first sign of disease’s progression) and OS (survival from the beginning of chemotherapy until patients’ death). Log rank test was used to compare the outcome of LAP^low and LAP^high groups. The patient and animal sample sizes were calculated with the G*Power software (www.gpower.hhu.de), setting α<0.05 and 1-β=0.80. Researchers analyzing the results were unaware of the treatments received.

Intermittent hypoxia up-regulates ABCB1 and ABCC1, inducing chemoresistance in non-small cell lung cancer cells

To compare the effect of normoxia, continuous and intermittent hypoxia, we grew cells in the following experimental conditions: normoxia (24h incubation at 20% O₂), hypoxia (24h incubation at 1% O₂), hypoxia/normoxia (12h at 1% O₂, followed by 12h at 20% O₂), hypoxia/normoxia/hypoxia, termed intermittent hypoxia (12h at 1% O₂, followed by 12h at 20% O₂and 12h at 1% O₂) (Figure 1A).

To evaluate chemosensitivity, after each time-point, the viability of 28 human NSCLC cell lines, incubated with increasing concentrations of cisplatin and docetaxel for 48h, was measured. Notwithstanding the different drug sensitivity among each cell line, the IC₅₀ of both drugs progressively increased moving from cells grown in normoxia to cells grown in hypoxia, hypoxia/normoxia and intermittent hypoxia (Figure 1B). Since cisplatin and docetaxel are substrates of ABCB1 and ABCC1 [10], both up-regulated by hypoxia [4], we next measured the amount of these transporters on the cell surface. Both ABCB1 and ABCC1 increased progressively in cells subjected to hypoxia, hypoxia/reoxygenation and intermittent hypoxia, compared to the normoxic counterpart (Figure 1C). In line with other chemoresistant tumors [15], high levels of ABCB1 and ABCC1 were paralleled by low levels of ABCA1 in normoxic NSCLC cell lines. This divergence in the expression of ABCB1/ABCC1 versus ABCA1 was further amplified in NSCLC subjected to hypoxia, hypoxia/normoxia and intermittent hypoxia (Figure 1C).

To investigate the mechanism underlying, we next focused on NCI-H2228 cells, i.e. the cells with the highest IC₅₀values of cisplatin and docetaxel (Figure 1B, Supplemental Figure S1A-B), and the highest ratio between ABCB1/ABCB1 versus ABCA1 in hypoxic conditions (Figure 1C-D).

Intermittent hypoxia increases HIF-1α-mediated stabilization of C/EBP-β LAP mRNA

As expected, hypoxia increases the amount of HIF-1α protein, which was reduced by hypoxia/normoxia cycle. By contrast, intermittent hypoxia increased again HIF-1α (Figure 2A). The levels of typical HIF-1α target genes, such as erythropoietin (EPO1), vascular endothelial growth factor A (VEGFA), glucose transporter 1 (GLUT1), varied accordingly to the changes in HIF-1α. By contrasts, ABCB1 and ABCC1 mRNA progressively increased, while ABCA1 mRNA progressively decreased in hypoxia, hypoxia/normoxia and intermittent hypoxia (Figure 2B). This trend was limited these three genes, since other ABC transporters mRNAs displayed minor variations or no significant changes (Supplemental Figure S2).

To explain the transcriptional changes of ABCB1, ABCC1 and ABCA1, we investigated if there were specific transcription factors, commonly binding the promoters of all these genes, showing their same modulation in hypoxia, hypoxia/normoxia and intermittent hypoxia. A targeted transcriptomic analysis, focused on the 38 transcription factors predicted to bind the promoters of ABCB1, ABCC1 and ABCA1 genes (Figure 2C, Supplemental Table S2), unveiled that C/EBP-β was the transcription factor whose mRNA showed the progressively higher increase in hypoxia, hypoxia/normoxia and intermittent hypoxia (Figure 2D), as ABCB1 and ABCC1.

Among the two splicing isoforms of C/EBP-β, LIP protein was undetectable in each condition, while LAP progressively increased in hypoxia, hypoxia/normoxia and intermittent hypoxia (Figure 3A). Co-immunoprecipitation between HIF-1α and C/EBP-β in hypoxic NCI-H2228 cells did not show any interaction between the two proteins even in hypoxia, where HIF-1α and C/EBP-β LAP were present (Figure 3B), neither in wild-type cells nor in cells selectively overexpressing LAP or LIP (Supplemental Figure S3). This experimental set excluded that HIF-1α binds and eventually modulates the amount of C/EBP-β LAP at protein level.

Interestingly, both the C/EBP-β LAP (Supplemental Figure S4A) and LIP (Supplemental Figure S4B) mRNAs contained a hypoxia responsive element (HRE). RNA-IP experiments indicated that HIF-1α interacted with C/EBP-β mRNA, increasing the level of LAP and decreasing the levels of LIP, with progressively higher effects in hypoxia, hypoxia/normoxia and intermittent hypoxia (Figure 3C). Consistently, the binding of LAP to the CAAT elements of ABCB1 and ABCC1 promoter (Supplemental Figure S5A-B) was progressively increased (Figure 3D), suggesting a transcriptional activation of these genes mediated by LAP. To the contrary, the binding of LAP to ABCA1 promoter (Figure 3D), which also has a CAAT sequence (Supplemental Figure S5C), was progressively decreased.

As proof of concept that LAP mRNA was increased upon its interaction with HIF-1α and modulated the expression of ABCB1, ABCC1 and ABCA1, we silenced HIF-1α in NCI-H2228 cells (Figure 3E). In silenced cells, the amount of LAP mRNA (Figure 3F), as well the LAP transcriptional activity on ABCB1, ABCC1 and ABCA1 promoters (Figure 3G), did not differ in normoxia, hypoxia, hypoxia/normoxia and intermittent hypoxia.

C/EBP-β LAP determines chemo-immuno-resistance in intermittent hypoxia and is a negative prognostic factor

To further prove that C/EBP-β LAP was the main responsible for changes in ABCB1, ABCC1 and ABCA1 under hypoxic conditions, we generated two clones of NCI-H2228 cells silenced for LAP (Figure 4A). As expected, in these cells hypoxia, hypoxia/normoxia and intermittent hypoxia did not increase LAP protein (Figure 4B). At the same time, LAP-silenced cells showed a marked decrease in the IC₅₀ of cisplatin and docetaxel (Figure 4C, Supplemental Figure S6A-B), indicating that preventing the increase of LAP reduces the hypoxia-induced chemoresistance.

The decrease of ABCA1 was associated to a progressive decrease in the efflux of IPP (Figure 4D). Consistently, Vγ9Vδ2 T-cells, immune-purified from PBMC and co-cultured with NCI-H2228 cells, previously grown in normoxia, hypoxia, hypoxia/normoxia and intermittent hypoxia (Figure 4E), were progressively less proliferating and less cytolytic, as indicated by the lower percentage of Ki67⁺IFNγ⁺ Vγ9Vδ2 T-cells (Figure 4F) and by the lower amount of necro-apoptotic (i.e. annexin V⁺/PI⁺) NCI-H2228 cells (Figure 4G). C/EBP-β LAP was responsible for this immune-resistance, because LAP silencing restored IPP efflux (Figure 4D), Vγ9Vδ2 T-cells proliferation and activation (Figure 4F), and killing of hypoxic tumor cells (Figure 4G) to levels comparable to normoxic cells.

To validate if also in patients C/EBP-β LAP was related to hypoxic areas and may have a clinical meaning, we analyzed a cohort of 60 prospectively recruited NSCLC patients at stage III/IV, candidate to receive adjuvant chemotherapy with cisplatin/carboplatin as first-line treatment (Supplemental Table S3). Interestingly, C/EBP-β LAP was detected in the FFPE samples of all patients, with different intensity (Supplemental Table S3), and was higher in the most hypoxic tumors (Figure 4H). The median of LAP staining intensity was used to dichotomize patients in LAP^low and LAP^high groups. As shown in Figure 4I, the LAP^high group had a significantly lower PFS and OS.

Mitochondrial ROS promote C/EBP-β LAP stabilization during intermittent hypoxia

One of the most frequent events associated with hypoxia, particularly with repeated cycles of hypoxia and reoxygenation, is the reduction of OXPHOS, caused by the impairment of the ETC that induces a dramatic drop in ATP synthesis and an increase in mtROS [4]. Since mtROS mediate part of HIF-1α physiological effects [27], we next investigated if they also increase the stabilization of LAP protein observed during intermittent hypoxia.

As expected, the electron flux through ETC dropped after 24h of hypoxia, was rescued by 12h in normoxia following 12h in hypoxia, but it was dramatically low after intermittent hypoxia (Figure 5A). Mitochondrial depolarization (Figure 5B) and mtROS (Figure 5C) progressively increased in hypoxia, hypoxia/normoxia and intermittent hypoxia, while ATP levels progressively decreased (Figure 5C), indicating the impairment of mitochondrial metabolism in these conditions. The increase of mtROS was mirrored by an increase in total ROS (Figure 5D), indicating a diffusion of ROS from the mitochondria to the cytosol and/or the generation of radical species within the cytosol.

To assess the functional effect of mtROS on C/EBP-β LAP stabilization during intermittent hypoxia, we challenged normoxic NCI-H2228 cells with the ETC-targeting and pro-oxidant agent elesclomol [28], at a concentration that increased mtROS at the same level detected in intermittent hypoxia (Supplemental Figure S7A). In a complementary set of experiments, we treated hypoxic NCI-H2228 cells with the mtROS scavenger mitoquinol (mitoQ) [29], currently under clinical evaluation in different chronic diseases associated with oxidative damage [30], to reduce mtROS to the same levels of normoxic cells (Supplemental Figure S7B). Notably, mitoQ prevented the stabilization of LAP mRNA mediated by HIF-1α in hypoxia, hypoxia/normoxia and intermittent hypoxia (Figure 5E), and reduced LAP protein to the level of normoxic cells (Figure 5F). Consistently, mitoQ abrogated the hypoxia-induced changes in the binding of LAP to ABCB1, ABCC1 and ABCA1 promoters (Figure 5G), reduced ABCB1 and ABCC1 mRNAs, and increased ABCA1 mRNA (Figure 5H) to levels comparable to cells growing in normoxia. By contrast, normoxic cells treated with elesclomol displayed the same amount of LAP, the same LAP binding activity to ABC gene promoters and the same expression profile of ABC transporters observed in hypoxic cells (Figure 5E-H).

Scavenging mitochondrial ROS induces chemo-immuno-sensitization of hypoxic cells

Based on the previous results, we next explored whether preventing the increase of mtROS may be an effective strategy to chemo-immuno-sensitize cells exposed to repeated cycles of hypoxia. As shown in Figure 6A, mitoQ effectively reduced the IC₅₀ of cisplatin and docetaxel in cells subjected to hypoxia, hypoxia/normoxia or intermittent hypoxia, in line with the observed decrease in ABCB1 and ABCC1. In agreement with the increase of ABCA1, mitoQ maintained the amount of active and proliferating Vγ9Vδ2 T-cells (Figure 6B), and the Vγ9Vδ2 T-cell-mediated tumor killing (Figure 6C) in all the hypoxic conditions at the same levels of normoxic cultures, indicating an effective immuno-sensitization.

The efficacy of mitoQ was validated in NSG Hu-CD34⁺ cells, bearing a reconstituted human immune system including Vγ9Vδ2 T-cells in peripheral blood [31], and NCI-H2228 tumors, engineered with a doxycycline-inducible expression vector of LAP [13]. In the absence of LAP inductions, cisplatin slightly reduced tumor growth (Figure 7A-B; Supplemental Figure S8A), without producing however any significant effect, in agreement with the chemoresistant phenotype of this cell line (Figure 1B). The induction of LAP made tumors even more resistant to cisplatin (Figures 7A-B, Supplementary Figure S8A). MitoQ alone did not exert anti-tumor effects, but the combination of cisplatin and mitoQ dramatically reduced tumor growth (Figures 7A-B, Supplementary Figure S8A). NCI-H2228 tumors, which were hypoxic (Figure 7C), had detectable levels of ABCB1 and ABCC1, and very low levels of ABCA1 protein, not modified by cisplatin (Figure 7C, Supplemental Figure 8B). In agreement with the findings obtained in vitro (Figure 3D), the intratumor induction of LAP increased ABCB1 and ABCC1, and decreased ABCA1. Notably, mitoQ reduced LAP, ABCB1 and ABCC1, and increased ABCA1, both alone or with vehicle or cisplatin (Figure 7C, Supplemental Figure 8B). We observed a moderate increase in the activated cleaved caspase 3 in NCI-H2228 tumors treated with cisplatin. This effect was abrogated in cisplatin-treated LAP-overexpressing tumors, but it was restored by mitoQ (Figure 7C, Supplemental Figure 8B). In line with the modulation of ABCA1 levels, intratumor activated (Ki67⁺IFNγ⁺) Vγ9Vδ2 T-cells were lower upon LAP induction and their percentage increased by mitoQ (Figure 7D).

Furthermore, mitoQ, either alone or in combination with cisplatin, was not toxic, according to the post-mortem analysis of liver, kidney and spleen (Supplemental Figure 8C) and to the hematochemical parameters measured (Supplemental Table S4): no signs of bone marrow, liver and muscle/heart toxicities were detected. At the end of the treatment cisplatin increased creatine, both in animals with or without induced LAP, but the presence of mitoQ reduced such increase (Supplemental Table S4).

The growth in stably hypoxic conditions determines chemoresistance [4] and impairs the activity of effector T-lymphocytes [32], but less is known about the effects of intermittent hypoxia on chemo-immuno-resistance. Two orders of intermittent hypoxia have been reported in tumors: a rapid intermittent hypoxia, occurring 2-5 times/h, and a prolonged intermittent hypoxia, with a duration of hours or days, caused by vascular reshaping [2]. In animal models of NSCLC this chronic intermittent hypoxia induces the development of tumors with high aggressiveness, characterized by the activation of HIF-1α transcriptional program, apoptosis inhibition, stemness, epithelial mesenchymal transition and drug resistance [33,34].

In the present work we set up a model of prolonged intermittent hypoxia that induces durable changes in gene transcription and metabolism, with the aim of analyzing how the resistance to chemotherapy and to immune-killing was affected. We found that intermittent hypoxia progressively increased the expression of the drug efflux transporters ABCB1 and ABCC1, and decreased the immune-sensitizer transporter ABCA1, exacerbating the chemo-immuno-resistant phenotype induced by constant hypoxia.

Since the changes in ABC transporters occurred at transcriptional level, we hypothesized a transcriptional effect of HIF-1α on these genes. HIF-1α protein was increased after chronic intermittent hypoxia more than after one shot of hypoxia, but it was reduced after one hypoxia/normoxia cycle, likely because of the activity of PHD2 enzyme, which is induced by hypoxia and is active during the re-oxygenation [35]. Intriguingly, while the expression of HIF-1α target genes (EPO1, VEGFA, GLUT1) followed HIF-1α levels, the levels of ABCB1, ABCC1 and ABCA1 mRNAs did not vary in parallel to HIF-1α protein, but their mRNAs were progressively accumulated (for ABCB1 and ABCC1) or reduced (for ABCA1) in chronic hypoxia, hypoxia/normoxia and intermittent hypoxia. We thus hypothesized the involvement of other transcription factors, related to hypoxia. Indeed, in brain blood barrier exposed to chronic intermittent hypoxia ABCB1 was up-regulated by both HIF-1α and Nrf-2, which is stabilized by the increased ROS generated by hypoxia [36]. Among the 38 transcription factors commonly binding the promoters of ABCB1, ABCC1 and ABCA1, C/EBP-β emerged as the protein most progressively increasing in chronic hypoxia, hypoxia/normoxia and intermittent hypoxia. Notably, in human endothelial cells intermittent hypoxia increases C/EBP-β, which cooperates with HIF-1α in realizing its transcriptional program [37]. We did not find a direct interaction between HIF-1α and C/EBP-β proteins, but HIF-1α was progressively more associated to C/EBP-β LAP mRNA in cells growing under hypoxia, hypoxia/normoxia and intermittent hypoxia. This data was consistent with the high levels of HIF-1α in cells growing in chronic hypoxia and intermittent hypoxia. It was less expected after one hypoxic/normoxic cycle, where cells had the same LAP mRNA levels of cells subjected to one shot of hypoxia, but HIF-1α was lower. We hypothesize that the low residual levels of HIF-1α were sufficient to stabilize LAP mRNA. Indeed, the knock-out experiments of HIF-1α proved that the downstream biological effects mediated by C/EBP-β LAP were HIF-1α-dependent.

Although HIF-1α is known as a transcriptional regulator more than as a splicing controller, one recent work reports that HIF-1α regulates the alternative splicing of SLC35A3 mRNA in pancreatic adenocarcinoma [38]. This pioneer observation paved the way to consider HIF-1α as a post-transcriptional regulator, as it occurs in the case of C/EBP-β splicing. HIF-1α decreased the LIP/LAP mRNA ratio, explaining the progressive up-regulation of ABCB1, which is negatively decreased by LIP and increased by LAP [13]. Also ABCC1 was up-regulated by LAP, a data that was previously unknown but may explain the findings reporting the increase of ABCC1 in hypoxia [9]. In addition, the activation of HIF-1α/LAP axis, particularly pronounced during intermittent hypoxia, down-regulated ABCA1, causing multidrug resistance and lower killing by Vγ2Vδ9 T-cells.

A hypoxic environment impairs the effector functions of γδ T-lymphocytes against glioblastoma cells [39], in line with the general immuno-suppression observed in the hypoxic tumor environment [32,40]. In our system, however, we demonstrated for the first time that a hypoxic pre-conditioning of tumor cells prevents the expansion and activation of Vγ2Vδ9 T-cells even in normoxia. This tumor-dependent immuno-suppression was due to the intratumor activation of HIF-1α/LAP axis that reduced the expression of ABCA1 and the efflux of IPP, the Vγ2Vδ9 T-cells endogenous activator.

The hypoxia-mediated induction of LAP had a significant clinical meaning: the analysis of NSCLC patients, candidate to receive cisplatin/carboplatin as first-line therapy, highlighted that LAP expression correlated well with higher intratumour hypoxia, lower PFS, indicative of poor response to chemotherapy, and lower OS, indicative of bad prognosis.

Notably, a recent work demonstrated that in triple negative breast cancer hypoxia selected ROS-resistant clones that were more prone to produce lung metastases and induced a hypoxic gene reprogramming associated with a lower survival [41]. Since metastastic triple negative breast cancer is often chemoresistant [42], we believe that a similar situation occurred in NSCLC. We thus focused on the role of ROS, which are increased in intermittent hypoxia [43], as potential inducers of the chemo-immuno-resistance mediated by HIF-1α/LAP axis.

As observed in endothelial cells [44] and neurons [45], also in NSCLC cells intermittent hypoxia impaired OXPHOS, increased mtROS and mitochondrial depolarization, indicative of mitochondrial damage [43]. The use of the mtROS generator elesclomol and the mtROS scavenger mitoQ proved that mtROS stabilized C/EBP-β LAP mRNA in intermittent hypoxia, modulated the expression of ABCB1, ABCC1 and ABCA1, the resistance to chemotherapeutic drugs and immune-killing. The chemo-immuno-resistant phenotype induced by the mtROS/LAP axis and the efficacy of mitoQ as chemo-immune-sensitizer was recapitulated in humanized mice, bearing NCI-H2228 xenografts with a diffuse hypoxic phenotype and an inducible C/EBP-β LAP expression system. In these unfavorable conditions, the combination of cisplatin and mitoQ rescued the efficacy of cisplatin and restored the intratumor infiltration and activity of Vγ2Vδ9 T-cells. Notably, the combination therapy was not more toxic than cisplatin alone. MitoQ even limited the increase in creatinine induced by cisplatin, leading to hypothesize a potential protection toward cisplatin-induced nephrotoxicity. The ratio between anti-tumor benefit and safety indicated a good therapeutic window of the combination cisplatin plus mitoQ, noteworthy of further investigations at clinical level.

Our demonstrates that intermittent hypoxia induces multidrug resistance and resistance to immune-killing in NSCLC with a tumor cell-autonomous mechanism. By impairing the efficacy of ETC and increasing the release of mtROS, intermittent hypoxia stabilizes the interaction between HIF-1α and C/EBP-β mRNA, favors the generation of C/EBP-β LAP isoform that up-regulates ABCB1 and ABCC1, causing of chemoresistance, and down-regulates ABCA1, promoting the low anti-tumor activity of Vγ9Vδ2 T-cells. Preventing this cascade with mtROS scavengers as mitoQ induces chemo-immuno-sensitization of hypoxic tumors (Figure 8A-B).

In a translational perspective, pour work has at least two orders of implication. First, C/EBP-β LAP, which is well detected by immunohistochemical analyses, could be considered a new predictive and prognostic factor in NSCLC patients candidate to receive chemotherapy. Second, we propose to repurpose mitochondrial ROS scavengers as mitoQ, currently under evaluation in neurodegenerative and cardiovascular diseases, chronic liver and kidney inflammatory-degenerative diseases, as a chemo immuno-sensitizer agents. Improving chemotherapy efficacy means increasing the efficacy of one of the first-line treatments for advanced NSCLC. At the same time, since γδ T-cells are the most favorable prognostic tumor-infiltrating population in lung cancer, their activation in hypoxic tumors, where other immunotherapy treatments as immune checkpoint inhibitors or CAR T-cells have limited efficacy, may represent an innovative immunotherapeutic approach.

pO₂, O₂ pressure; HIF-α, hypoxia induced factor α; PHD, prolyl hydroxylase domain; OXPHOS, oxidative phosphorylation; ABC, ATP Binding Cassette; ABCB1/Pgp, ABC transporter B1/P-glycoprotein; ABCC1/MRP1, ABC transporter C1/multidrug resistance related protein 1; ER, endoplasmic reticulum; C/EBP, CCAAT/Enhancer Binding Protein; ABCA1, ABC transporter A1; IPP, isopentenyl pyrophosphate; NSCLC, non-small cell lung cancer; mtROS, mitochondrial reactive oxygen species; FBS, featl bovine serum; mitoQ, [10-(2,5-dihydroxy-3,4-dimethoxy-6,ethylphenyl)decyl]triphenyl-phosphonium, monomethanesulfonate or mitoquinol; RLUs, relative luminescence units; EPD, Eukariotic Promoter Database; RNA-IP, RNA-immunoprecipitation; HRE, hypoxia-response element; ChIP, Chromatin immunoprecipitation; GFP, green fluorescence protein; PBMC, peripheral blood mononuclear cells; FITC, fluorescein isothiocyanate; APC, allophycocyanin; ETC, electron transport chain; RFUs, relative fluorescence units; CM-H₂DCFDA, 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate; UPN; unknown patient number; PFS, progression free survival; OS, overall survival; FFPE, formalin-fixed paraffin-embedded; s.c., subcutaneously; i.v., intravenously; RBC, red blood cells; WBC, white blood cells; Hb, haemoglobin; PLT, platelets; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; AP, alkaline phosphatase; CPK, creatine phosphokinase; ANOVA, one-way analysis of variance; EPO1, erythropoietin; VEGFA, vascular endothelial growth factor A; GLUT1, glucose transporter 1.

Acknowledgments

We would like to thank all the creators and curators of the publicly available datasets used in this study and the developers of the open-source software packages, without whom this study would not be possible.

Authors’ contribution statement

ICS and DCB carried out molecular biology experiments, immunoxenografts and data analysis; ICS wrote the first draft; MA performed the immune-sensitivity assays and flow cytometry assays; SLV and MG performed the chemosensitivity and metabolic activity assays; DPA and GO performed the patients’ immunohistochemical analysis and clinical follow-up; SD, SF and CC performed the cell lines initial screening; JK analyzed the data and reviewed the manuscript; CR supervised the work and reviewed the manuscript. All the authors read and approved the final version of the manuscript.

Funding

The work was supported by Italian Association for Cancer Research (IG21408 to CR), Fondazione Cassa di Risparmio di Torino (ID 2018.0568 and ID 2021.05556 to CR; ID 2020.1648 to JK), Intramural Grant Funding 2019 (to JK), Compagnia di San Paolo Funding 2021 (to CR); European Cooperation in Science and Technology (COST) (CA17104 to CR).

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Ethical approvals and consent to participate

All human tissues and clinical information were collected with the approval of the Ethics Committee of the of San Luigi Gonzaga Hospital, Orbassano, Italy. Written informed consent was obtained from each patient. All animal procedures used in the study were approved by the Italian Ministry of Health and performed according to the Guidelines for the Care and Use of Laboratory Animals of University of Torino, Italy.

Consent for publication

Not applicable

Competing interest

ICS, DCB, MA, SLV, MG, DPA, GO, CC, JK declare no conflict of interest. CR has received research support from PharmaMar. The funder had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, in the decision to publish the results.

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Competing interest reported. ICS, DCB, MA, SLV, MG, DPA, GO, CC, JK declare no conflict of interest. CR has received research support from PharmaMar. The funder had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, in the decision to publish the results.

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

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