No abstract

BiR melanoma cells overexpress NAMPT, which acts as a connecting element between BRAF oncogenic signaling and metabolism, becoming an actionable target for this subset of MM patients.

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.

MicroRNAs (miRNAs) are small, non-coding RNAs about 22 nucleotides in length that regulate the expression of target genes post-transcriptionally, and are highly involved in cancer progression. They are able to impact a variety of cell processes such as proliferation, apoptosis and differentiation and can consequently control tumor initiation, tumor progression and metastasis formation. miRNAs can regulate, at the same time, metabolic gene expression which, in turn, influences relevant traits of malignancy such as cell adhesion, migration and invasion. Since the interaction between metabolism and adhesion or cell movement has not, to date, been well understood, in this review, we will specifically focus on miRNA alterations that can interfere with some metabolic processes leading to the modulation of cancer cell movement. In addition, we will analyze the signaling pathways connecting metabolism and adhesion/migration, alterations that often affect cancer cell dissemination and metastasis formation.

Gender is an important stratification and prognostic factor in oncology in the response to treatments, particularly to the immunotherapy. The anti-PD-1/PD-L1 immune-checkpoint inhibitors (Pembrolizumab, Nivolumab, Atezolizumab) have improved the prognosis of non-small cell lung cancer (NSCLC) patients in 20-35% cases, but with a lower efficacy in females. The different steroid hormones - estrogens, progesterone or androgens - present in both gender have often contrasting effects on the activity of the host immune system, partly explaining the differences observed. It is not known if metabolic features of cancer cells of male and female patients may impact on the immune system response and immunotherapy efficacy. We set up an analytical pipeline based on patient-derived samples, cell lines and humanized male and female mice bearing gender-matched NSCLC xenografts. NanoString analysis in a cohort of 42 NSCLC patients of both genders treated with Pembrolizumab, identified the estrogen receptor α (ERα)/ESR1 gene as one of the most significantly associated with the response to Pembrolizumab, particularly in female patients. A panel of 30 human NSCLC cell lines of female and male origin, that reproduced the ERα/gender association observed in clinical samples, the amount of ERα and 17-β-estradiol was directly related to the expression of PD-L1, suggesting a potential direct linkage. ERα transcriptionally up-regulated CD274/PD-L1 gene, with higher effects in females. EGFR downstream effectors Akt and ERK1/2, increased the active form phospho(Ser118)ERα, upregulating PD-L1. Inhibiting the estrogen synthesis or switching off EGFR activity down-regulated PD-L1 levels. Consistently, the aromatase inhibitor letrozole increased the efficacy of Pembrolizumab in humanized NSCLC xenografts, by reducing tumor growth and tumor-associated PD-L1, increasing the infiltrating CD8+T-lymphocytes, NK cells and Vγ9Vδ2 anti-tumor T-lymphocytes. The benefit was maximal in 17-β-estradiol/ERα high female xenografts, intermediate in 17-β-estradiol/ERα medium female and male xenografts, minimal in 17-β-estradiol/ERα low male xenografts. Additionally, an untargeted lipidome analysis revealed that letrozole decreased the saturated fatty acids (SFAs)/mono- or poly-unsaturated fatty acids (MUFAs-PUFAs) ratio, and the cholesterol/cholesterol esters ratio. These changes increased plasma-membrane fluidity, an event that in turns reduced the affinity between PD-L1 and PD-1. As further proof of concept, ω6- or ω3-PUFA treated cells, displayed an increased membrane fluidity and a change in lipid metabolism that affect the response to anti-PD-1/PD-L1 as letrozole did. We propose a prototypical in vitro 3D spheroids culture gender-tailored platform that allows to investigate the metabolic circuitries causing such differences and to repurpose metabolic modifiers as novel immune-adjuvant agents in oncology. Citation Format: Sofia La Vecchia, Dario Pasquale Anobile, Fabrizio Tabbò, Iris C. Salaroglio, Joanna Kopecka, Luca Primo, Luisella Righi, Giorgio Vittorio Scagliotti, Silvia Novello, Cyril Corbet, Chiara Riganti. Gender and estrogens as key factors in the response to immunotherapy in non small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2163.

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.

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 transcriptomic identified efflux transporters and nuclear factors involved in this chemo-immuno-resistant phenotype. 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 and depolarization were measured spectrofluorimetrically. The role of ROS/HIF-1α/LAP axis was validated in HIF-1α/LAP 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, 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 implanted in Hu-CD34+NSG mice recapitulated the chemo-immuno-resistant phenotype of patients. Interestingly, the ROS scavenger mitoquinol induced chemo-immuno-sensitization in 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.

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 transcriptomic identified efflux transporters and nuclear factors involved in this chemo-immuno-resistant phenotype. 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 and depolarization were measured spectrofluorimetrically. The role of ROS/HIF-1α/LAP axis was validated by HIF-1α/LAP 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, 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., in Hu- LAP-overexpressing tumors implanted in CD34+NSG mice recapitulated the chemo-immuno-resistant phenotype of patients. Interestingly, the ROS scavenger mitoquinol induced chemo-immuno-sensitization in 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.