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Background: Lung cancer (LC) is Europe's primary cause of cancer-related mortality largely due to its historically low survival rates. The aim of this study was to analyze 26-year survival trends in the province of Girona, Spain, and to identify key prognostic factors., Methods: Population-based study of LC cases collected between 1994 and 2019, with follow-up until December 31, 2021. Variables included date of diagnosis, sex, age, histology, and tumor stage (the latter since 2010). Diagnosis dates were categorized into three periods (1994-2002, 2003-2011, and 2012-2019). Multivariate flexible parametric models, incorporating age as a non-linear, time-varying covariate, were used to analyze net survival (NS) and trends. Annual absolute change in survival (AAC_S) was calculated using 3-year NS., Results: The analysis of 9,113 LC cases showed a NS improvement between the first and last period (7.1 months (95 %CI: 6.5;7.6) to 8.5 months (95 %CI: 7.9;9.1)). Squamous cell carcinoma (NSC-SCC) showed the greatest improvement with an AAC_S of 0.32 % (95 % CI: 0.21; 0.43), while survival for non-small cell lung cancer not otherwise specified declined (AAC_S of -0.19 % (95 %CI: -0.26; -0.12)). Prognostic analysis of the 3,642 cases (2010-2019) indicated a lower LC death risk for adenocarcinoma and NSC-SCC compared to LC not otherwise specified (HR 0.52 and 0.62, respectively). Increasing tumor stage correlated with higher LC mortality risk (1.8-, 4.0-, and 10.1-fold increase for stage II, III, and IV, respectively, compared to stage I)., Conclusions: LC survival has notably improved, particularly for NSC-SCC. Survival is influenced by sex, age, date of diagnosis, tumor histology and especially by stage, underscoring comprehensive data collection's importance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fatty acid synthase (FASN)-catalyzed endogenous lipogenesis is a hallmark of cancer metabolism. However, whether FASN is an intrinsic mechanism of tumor cell defense against T cell immunity remains unexplored. To test this hypothesis, here we combined bioinformatic analysis of the FASN-related immune cell landscape, real-time assessment of cell-based immunotherapy efficacy in CRISPR/Cas9-based FASN gene knockout (FASN KO) cell models, and mathematical and mechanistic evaluation of FASN-driven immunoresistance. FASN expression negatively correlates with infiltrating immune cells associated with cancer suppression, cytolytic activity signatures, and HLA-I expression. Cancer cells engineered to carry a loss-of-function mutation in FASN exhibit an enhanced cytolytic response and an accelerated extinction kinetics upon interaction with cytokine-activated T cells. Depletion of FASN results in reduced carrying capacity, accompanied by the suppression of mitochondrial OXPHOS and strong downregulation of electron transport chain complexes. Targeted FASN depletion primes cancer cells for mitochondrial apoptosis as it synergizes with BCL-2/BCL-X L -targeting BH3 mimetics to render cancer cells more susceptible to T-cell-mediated killing. FASN depletion prevents adaptive induction of PD-L1 in response to interferon-gamma and reduces constitutive overexpression of PD-L1 by abolishing PD-L1 post-translational palmitoylation. FASN is a novel tumor cell-intrinsic metabolic checkpoint that restricts T cell immunity and may be exploited to improve the efficacy of T cell-based immunotherapy., (© 2024. The Author(s).)

Background: ID3 (inhibitor of DNA binding/differentiation-3) is a transcription factor that enables metastasis by promoting stem cell-like properties in endothelial and tumor cells. The milk thistle flavonolignan silibinin is a phytochemical with anti-metastatic potential through largely unknown mechanisms., Hypothesis/purpose: We have mechanistically investigated the ability of silibinin to inhibit the aberrant activation of ID3 in brain endothelium and non-small cell lung cancer (NSCLC) models., Methods: Bioinformatic analyses were performed to investigate the co-expression correlation between ID3 and bone morphogenic protein (BMP) ligands/BMP receptors (BMPRs) genes in NSCLC patient datasets. ID3 expression was assessed by immunoblotting and qRT-PCR. Luciferase reporter assays were used to evaluate the gene sequences targeted by silibinin to regulate ID3 transcription. In silico computational modeling and LanthaScreen TR-FRET kinase assays were used to characterize and validate the BMPR inhibitory activity of silibinin. Tumor tissues from NSCLC xenograft models treated with oral silibinin were used to evaluate the in vivo anti-ID3 effects of silibinin., Results: Analysis of lung cancer patient datasets revealed a top-ranked positive association of ID3 with the BMP9 endothelial receptor ACVRL1/ALK1 and the BMP ligand BMP6. Silibinin treatment blocked the BMP9-induced activation of the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Constitutive, acquired, and adaptive expression of ID3 in NSCLC cells were all significantly downregulated in response to silibinin. Silibinin blocked ID3 transcription via BMP-responsive elements in ID3 gene enhancers. Silibinin inhibited the kinase activities of BMPRs in the micromolar range, with the lower IC 50 values occurring against ACVRL1/ALK1 and BMPR2. In an in vivo NSCLC xenograft model, tumoral overexpression of ID3 was completely suppressed by systematically achievable oral doses of silibinin., Conclusions: ID3 is a largely undruggable metastasis-promoting transcription factor. Silibinin is a novel suppressor of ID3 that may be explored as a novel therapeutic approach to interfere with the metastatic dissemination capacity of NSCLC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The initial excitement generated more than two decades ago by the discovery of drugs targeting fatty acid synthase (FASN)-catalyzed de novo lipogenesis for cancer therapy was short-lived. However, the advent of the first clinical-grade FASN inhibitor (TVB-2640; denifanstat), which is currently being studied in various phase II trials, and the exciting advances in understanding the FASN signalome are fueling a renewed interest in FASN-targeted strategies for the treatment and prevention of cancer. Here, we provide a detailed overview of how FASN can drive phenotypic plasticity and cell fate decisions, mitochondrial regulation of cell death, immune escape and organ-specific metastatic potential. We then present a variety of FASN-targeted therapeutic approaches that address the major challenges facing FASN therapy. These include limitations of current FASN inhibitors and the lack of precision tools to maximize the therapeutic potential of FASN inhibitors in the clinic. Rethinking the role of FASN as a signal transducer in cancer pathogenesis may provide molecularly driven strategies to optimize FASN as a long-awaited target for cancer therapeutics., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Epithelial-to-mesenchymal transition (EMT) is key to tumor aggressiveness, therapy resistance, and immune escape in breast cancer. Because metabolic traits might be involved along the EMT continuum, we investigated whether human breast epithelial cells engineered to stably acquire a mesenchymal phenotype in non-tumorigenic and H-Ras V12 -driven tumorigenic backgrounds possess unique metabolic fingerprints. We profiled mitochondrial-cytosolic bioenergetic and one-carbon (1C) metabolites by metabolomic analysis, and then questioned the utilization of different mitochondrial substrates by EMT mitochondria and their sensitivity to mitochondria-centered inhibitors. "Upper" and "lower" glycolysis were the preferred glucose fluxes activated by EMT in non-tumorigenic and tumorigenic backgrounds, respectively. EMT in non-tumorigenic and tumorigenic backgrounds could be distinguished by the differential contribution of the homocysteine-methionine 1C cycle to the transsulfuration pathway. Both non-tumorigenic and tumorigenic EMT-activated cells showed elevated mitochondrial utilization of glycolysis end-products such as lactic acid, β-oxidation substrates including palmitoyl-carnitine, and tricarboxylic acid pathway substrates such as succinic acid. Notably, mitochondria in tumorigenic EMT cells distinctively exhibited a significant alteration in the electron flow intensity from succinate to mitochondrial complex III as they were highly refractory to the inhibitory effects of antimycin A and myxothiazol. Our results show that the bioenergetic/1C metabolic signature, the utilization rates of preferred mitochondrial substrates, and sensitivity to mitochondrial drugs significantly differs upon execution of EMT in non-tumorigenic and tumorigenic backgrounds, which could help to resolve the relationship between EMT, malignancy, and therapeutic resistance in breast cancer.

Epithelial-to-mesenchymal transition (EMT) may drive the escape of ALK-rearranged non-small-cell lung cancer (NSCLC) tumors from ALK-tyrosine kinase inhibitors (TKIs). We investigated whether first-generation ALK-TKI therapy-induced EMT promotes cross-resistance to new-generation ALK-TKIs and whether this could be circumvented by the flavonolignan silibinin, an EMT inhibitor. ALK-rearranged NSCLC cells acquiring a bona fide EMT phenotype upon chronic exposure to the first-generation ALK-TKI crizotinib exhibited increased resistance to second-generation brigatinib and were fully refractory to third-generation lorlatinib. Such cross-resistance to new-generation ALK-TKIs, which was partially recapitulated upon chronic TGFβ stimulation, was less pronounced in ALK-rearranged NSCLC cells solely acquiring a partial/hybrid E/M transition state. Silibinin overcame EMT-induced resistance to brigatinib and lorlatinib and restored their efficacy involving the transforming growth factor-beta (TGFβ)/SMAD signaling pathway. Silibinin deactivated TGFβ-regulated SMAD2/3 phosphorylation and suppressed the transcriptional activation of genes under the control of SMAD binding elements. Computational modeling studies and kinase binding assays predicted a targeted inhibitory binding of silibinin to the ATP-binding pocket of TGFβ type-1 receptor 1 (TGFBR1) and TGFBR2 but solely at the two-digit micromolar range. A secretome profiling confirmed the ability of silibinin to normalize the augmented release of TGFβ into the extracellular fluid of ALK-TKIs-resistant NSCLC cells and reduce constitutive and inducible SMAD2/3 phosphorylation occurring in the presence of ALK-TKIs. In summary, the ab initio plasticity along the EMT spectrum may explain the propensity of ALK-rearranged NSCLC cells to acquire resistance to new-generation ALK-TKIs, a phenomenon that could be abrogated by the silibinin-driven attenuation of the TGFβ/SMAD signaling axis in mesenchymal ALK-rearranged NSCLC cells.

The mitokine MOTS-c is a mitochondrially-encoded "exercise-mimetic peptide" expressed in multiple tissues, particularly skeletal muscles, which can be detected as a circulating hormone in the blood. MOTS-c mechanisms of action (MoA) involve insulin sensitization, enhanced glucose utilization, suppression of mitochondrial respiration, and targeting of the folate-AICAR-AMPK pathway. Although MOTS-c MoA largely overlap those of the anti-diabetic biguanide metformin, the putative regulatory actions of metformin on MOTS-c have not yet been evaluated in detail. Here, we measured circulating MOTS-c in paired baseline and post-treatment sera obtained from HER2-positive breast cancer patients randomized to receive either metformin combined with neoadjuvant chemotherapy and trastuzumab or an equivalent regimen without metformin. We failed to find any significant alteration of circulating MOTS-c -as measured using the commercially available competitive ELISA CEX132Hu- in response to 24 weeks of a neoadjuvant chemotherapy/trastuzumab regimen with or without daily metformin. Changes in circulating MOTS-c levels failed to reach statistical significance when comparing patients achieving pathological complete response (pCR), irrespective of metformin treatment. The inability of metformin to target skeletal muscle, the major tissue for MOTS-c production and secretion, might limit its regulatory effects on circulating MOTS-c. Further studies are needed to definitely elucidate the nature of the interaction between metformin and MOTS-c in cancer and non-cancer patients.

Therapy-induced senescence (TIS) is a state of stable proliferative arrest of both normal and neoplastic cells that is triggered by exposure to anticancer treatments. TIS cells acquire a senescence-associated secretory phenotype (SASP), which is pro-inflammatory and actively promotes tumor relapse and adverse side-effects in patients. Here, we hypothesized that TIS cells adapt their scavenging and catabolic ability to overcome the nutritional constraints in their microenvironmental niches. We used a panel of mechanistically-diverse TIS triggers (i.e., bleomycin, doxorubicin, alisertib, and palbociclib) and Biolog Phenotype MicroArrays to identify (among 190 different carbon and nitrogen sources) candidate metabolites that support the survival of TIS cells in limiting nutrient conditions. We provide evidence of distinguishable TIS-associated nutrient consumption profiles involving a core set of shared (e.g., glutamine) and unique (e.g., glucose-1-phosphate, inosine, and uridine) nutritional sources after diverse senescence-inducing interventions. We also observed a trend for an inverse correlation between the intensity of the pro-inflammatory SASP provoked by different TIS agents and diversity of compensatory nutritional niches utilizable by senescent cells. These findings support the detailed exploration of the nutritional niche as a new metabolic dimension to understand and target TIS in cancer.

The third-generation anaplastic lymphoma tyrosine kinase inhibitor (ALK-TKI) lorlatinib has a unique side effect profile that includes hypercholesteremia and hypertriglyceridemia in >80% of lung cancer patients. Here, we tested the hypothesis that lorlatinib might directly promote the accumulation of cholesterol and/or triglycerides in human hepatic cells. We investigated the capacity of the hepatoprotectant silibinin to modify the lipid-modifying activity of lorlatinib. To predict clinically relevant drug−drug interactions if silibinin were used to clinically manage lorlatinib-induced hyperlipidemic effects in hepatic cells, we also explored the capacity of silibinin to interact with and block CYP3A4 activity using in silico computational descriptions and in vitro biochemical assays. A semi-targeted ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-ESI-QTOF-MS/MS)-based lipidomic approach revealed that short-term treatment of hepatic cells with lorlatinib promotes the accumulation of numerous molecular species of cholesteryl esters and triglycerides. Silibinin treatment significantly protected the steady-state lipidome of hepatocytes against the hyperlipidemic actions of lorlatinib. Lipid staining confirmed the ability of lorlatinib to promote neutral lipid overload in hepatocytes upon long-term exposure, which was prevented by co-treatment with silibinin. Computational analyses and cell-free biochemical assays predicted a weak to moderate inhibitory activity of clinically relevant concentrations of silibinin against CYP3A4 when compared with recommended (rosuvastatin) and non-recommended (simvastatin) statins for lorlatinib-associated dyslipidemia. The elevated plasma cholesterol and triglyceride levels in lorlatinib-treated lung cancer patients might involve primary alterations in the hepatic accumulation of lipid intermediates. Silibinin could be clinically explored to reduce the undesirable hyperlipidemic activity of lorlatinib in lung cancer patients.

The HER3/4 ligand heregulin-β2 (HRG) is a secreted growth factor that transactivates the ligand-less receptor HER2 to promote aggressive phenotypes in breast cancer. HRG can also localize to the nucleus of breast cancer cells, but both the nuclear translocation mechanism and the physiological role of nuclear HRG remain elusive. Here we show that nucleolin-driven nuclear moonlighting of HRG uncouples its role as a driver of endocrine resistance from its canonical HER network-activating role in breast cancer. Tandem affinity purification coupled to mass spectrometry identified the intracellular transporter nucleolin as a major HRG-binding protein. HRG interacts with nucleolin via a nuclear localization signal motif located at the N-terminal extracellular domain of HRG. Nucleolin interacts with HRG via aspartate/glutamate-rich acidic stretches located at the N-terminal domain of nucleolin. Depletion of nucleolin abolishes HRG nuclear translocation and decreases HRG mRNA and protein expression. Isolated deficiency of nuclear HRG abolishes the HRG-driven endocrine resistance phenotype in vitro and in mouse xenograft models, while preserving its capacity to activate the HRG/HER/MAPK autocrine signaling axis. Conversely, isolated deficiency of secreted HRG to bind HER2/3 receptors does not impair endocrine resistance. The discovery that the functions of dual compartment-resident HRG do not depend on the same effector (i.e., activation of HER2/3 receptors) establishes a new paradigm for the functional and therapeutic relevance of nuclear HRG in breast cancer., Competing Interests: None., (AJCR Copyright © 2022.)

Triple-negative/basal-like breast cancer (BC) is characterized by aggressive biological features, which allow relapse and metastatic spread to occur more frequently than in hormone receptor-positive (luminal) subtypes. The molecular complexity of triple-negative/basal-like BC poses major challenges for the implementation of targeted therapies, and chemotherapy remains the standard approach at all stages. The matricellular protein cysteine-rich angiogenic inducer 61 (CCN1/CYR61) is associated with aggressive metastatic phenotypes and poor prognosis in BC, but it is unclear whether anti-CCN1 approaches can be successfully applied in triple-negative/basal-like BC. Herein, we first characterized the prevalence of CNN1 expression in matched samples of primary tumors and metastatic relapse in a series of patients with BC. We then investigated the biological effect of CCN1 depletion on tumorigenic traits in vitro and in vivo using archetypal TNBC cell lines. Immunohistochemical analyses of tissue microarrays revealed a significant increase of the highest CCN1 score in recurrent tissues of triple-negative/basal-like BC tumors. Stable silencing of CCN1 in triple-negative/basal-like BC cells promoted a marked reduction in the expression of the CCN1 integrin receptor α v β 3 , inhibited anchorage-dependent cell growth, reduced clonogenicity, and impaired migration capacity. In an orthotopic model of triple-negative/basal-like BC, silencing of CCN1 notably reduced tumor burden, which was accompanied by decreased microvessel density and concurrent induction of the luminal epithelial marker E-cadherin. Thus, CNN1/CYR61-targeting strategies might have therapeutic value in suppressing the biological aggressiveness of triple-negative/basal-like BC., Competing Interests: None., (AJCR Copyright © 2022.)

CCN1/CYR61 promotes angiogenesis, tumor growth and chemoresistance by binding to its integrin receptor α v β 3 in endothelial and breast cancer (BC) cells. CCN1 controls also tissue regeneration by engaging its integrin receptor α 6 β 1 to induce fibroblast senescence. Here, we explored if the ability of CCN1 to drive an endocrine resistance phenotype in estrogen receptor-positive BC cells relies on interactions with either α v β 3 or α 6 β 1 . First, we took advantage of site-specific mutagenesis abolishing the CCN1 receptor-binding sites to α v β 3 and α 6 β 1 to determine the integrin partner responsible for CCN1-driven endocrine resistance. Second, we explored a putative nuclear role of CCN1 in regulating ERα-driven transcriptional responses. Retroviral forced expression of a CCN1 derivative with a single amino acid change (D125A) that abrogates binding to α v β 3 partially phenocopied the endocrine resistance phenotype induced upon overexpression of wild-type (WT) CCN1. Forced expression of the CCN1 mutant TM, which abrogates all the T1, H1, and H2 binding sites to α 6 β 1 , failed to bypass the estrogen requirement for anchorage-independent growth or to promote resistance to tamoxifen. Wild-type CCN1 promoted estradiol-independent transcriptional activity of ERα and enhanced ERα agonist response to tamoxifen. The α 6 β 1 -binding-defective TM-CCN1 mutant lost the ERα co-activator-like behavior of WT-CCN1. Co-immunoprecipitation assays revealed a direct interaction between endogenous CCN1 and ERα, and in vitro approaches confirmed the ability of recombinant CCN1 to bind ERα. CCN1 signaling via α 6 β 1 , but not via α v β 3 , drives an endocrine resistance phenotype that involves a direct binding of CCN1 to ERα to regulate its transcriptional activity in ER+ BC cells.

COVID-19 pathophysiology is caused by a cascade of respiratory and multiorgan failures arising, at least in part, from the SARS-CoV-2-driven dysregulation of the master transcriptional factor STAT3. Pharmacological correction of STAT3 over-stimulation, which is at the root of acute respiratory distress syndrome (ARDS) and coagulopathy/thrombosis events, should be considered for treatment of severe COVID-19. In this perspective, we first review the current body of knowledge on the role of STAT3 in the pathogenesis of severe COVID-19. We then exemplify the potential clinical value of treating COVID-19 disease with STAT3 inhibitors by presenting the outcomes of two hospitalized patients with active cancer and COVID-19 receiving oral Legalon ® -a nutraceutical containing the naturally occurring STAT3 inhibitor silibinin. Both patients, which were recruited to the clinical trial SIL-COVID19 (EudraCT number: 2020-001794-77) had SARS-CoV-2 bilateral interstitial pneumonia and a high COVID-GRAM score, and showed systemic proinflammatory responses in terms of lymphocytopenia and hypoalbuminemia. Both patients were predicted to be at high risk of critical COVID-19 illness in terms of intensive care unit admission, invasive ventilation, or death. In addition to physician's choice of best available therapy or supportive care, patients received 1050 mg/day Legalon ® for 10 days without side-effects. Silibinin-treated cancer/COVID-19+ patients required only minimal oxygen support (2-4 L/min) during the episode, exhibited a sharp decline of the STAT3-regulated C-reactive protein, and demonstrated complete resolution of the pulmonary lesions. These findings might inspire future research to advance our knowledge and improve silibinin-based clinical interventions aimed to target STAT3-driven COVID-19 pathophysiology.