miRNA-guided reprogramming of glucose and glutamine metabolism and its impact on cell adhesion/migration during solid tumor progression.

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.

Metabolic pathways regulating colorectal cancer initiation and progression.

Colorectal cancer (CRC) is one of the most common types of cancer worldwide. Despite recent advances in the molecular genetics of CRC, poor treatment outcomes highlight the need for a better understanding of the underlying mechanisms accounting for tumor initiation and progression. Recently, deregulation of cellular metabolism has emerged as a key hallmark of cancer. Reprogramming of core cellular metabolic pathways by cancer cells provides energy, anaplerotic precursors and reducing equivalents required to support tumor growth. Here, we review key findings implicating cancer metabolism as a major contributor of tumor initiation, growth and metastatic dissemination in CRC. We summarize the metabolic pathways governing stem cell fate in the intestine, the metabolic adaptations of proliferating colon cancer cells and their crosstalk with oncogenic signaling, and how they fulfill the energetic demands imposed by the metastatic cascade. Lastly, we discuss how some of these metabolic pathways could represent new vulnerabilities of CRC cells with the potential to be targeted.

Autocrine 17-ß-Estradiol/Estrogen Receptor-α Loop Determines the Response to Immune Checkpoint Inhibitors in Non-Small Cell Lung Cancer.

PURPOSE: The response to immune checkpoint inhibitors (ICI) often differs between genders in non-small cell lung cancer (NSCLC), but metanalyses results are controversial, and no clear mechanisms are defined. We aim at clarifying the molecular circuitries explaining the differential gender-related response to anti-PD-1/anti-PD-L1 agents in NSCLC. EXPERIMENTAL DESIGN: We prospectively analyzed a cohort of patients with NSCLC treated with ICI as a first-line approach, and we identified the molecular mechanisms determining the differential efficacy of ICI in 29 NSCLC cell lines of both genders, recapitulating patients' phenotype. We validated new immunotherapy strategies in mice bearing NSCLC patient-derived xenografts and human reconstituted immune system ("immune-PDXs"). RESULTS: In patients, we found that estrogen receptor α (ERα) was a predictive factor of response to pembrolizumab, stronger than gender and PD-L1 levels, and was directly correlated with PD-L1 expression, particularly in female patients. ERα transcriptionally upregulated CD274/PD-L1 gene, more in females than in males. This axis was activated by 17-ß-estradiol, autocrinely produced by intratumor aromatase, and by the EGFR-downstream effectors Akt and ERK1/2 that activated ERα. The efficacy of pembrolizumab in immune-PDXs was significantly improved by the aromatase inhibitor letrozole, which reduced PD-L1 and increased the percentage of antitumor CD8+T-lymphocytes, NK cells, and Vγ9Vδ2 T-lymphocytes, producing durable control and even tumor regression after continuous administration, with maximal benefit in 17-ß-estradiol/ERα highfemale immune-xenografts. CONCLUSIONS: Our work unveils that 17-ß-estradiol/ERα status predicts the response to pembrolizumab in patients with NSCLC. Second, we propose aromatase inhibitors as new gender-tailored immune-adjuvants in NSCLC. See related commentary by Valencia et al., p. 3832.

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-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-ß (C/EBP-ß) 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-ß mRNA mediated by HIF-1α. HIF-1α/C/EBP-ß mRNA binding increases the splicing of C/EBP-ß 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-ß 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-ß LAP as a new predictive and prognostic factor in NSCLC patients.

Nicotinamide Phosphoribosyltransferase (NAMPT) as a Therapeutic Target in BRAF-Mutated Metastatic Melanoma.

Background: One of the effects of oncogenic signaling is metabolic reprogramming of tumor cells to support anabolic growth, opening the way to therapeutic targeting of metabolic pathways. Methods: We studied NAD biosynthesis in BRAF inhibitor (BRAFi)-resistant (BiR) melanoma cell lines. Data in cell lines were confirmed by immunohistochemistry in biopsies from 17 patients with metastatic melanoma (MM) before and after the acquisition of resistance to BRAFi. Therapeutic potential of NAD biosynthesis inhibitors was determined by invitro monitoring cell growth and death and in mouse xenograft models. Mice (n = 6-10 mice/group) were treated with nicotinamide phosphoribosyltranferase inhibitor (NAMPTi), BRAFi, or their combination, and tumor growth and survival were analyzed. All statistical tests were two-sided. Results: BiR cells had higher NAD levels compared with their BRAFi-sensitive counterparts (P < .001 and P = .001 for M14 and A375, respectively) and with normal melanocytes (P < .001), achieved through transcriptional upregulation of the enzyme NAMPT, which became the master regulator of NAD synthesis. Conversely, treatment with BRAFi or MEK inhibitors decreased NAMPT expression and cellular NAD levels. Robust NAMPT upregulation was documented in tissue biopsies from MM patients after development of resistance to BRAFi (P < .001). Treatment of melanoma cells with NAMPTi depleted NAD and ATP, depolarized mitochondrial membrane, and led to reactive oxygen species production, blocking cells in the G2/M phase and inducing apoptosis. Treatment of BiR xenografts with NAMPTi improved mouse survival (median survival of vehicle-treated mice was 52 days vs 100 days for NAMPTi-treated ones in M14/BiR, while in A375/BiR median survival of vehicle-treated mice was 23.5 days vs 43 days for NAMPTi-treated ones, P < .001). Conclusions: 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.