BACKGROUND:Metabolic dysfunctions are associated with increased cancer morbidity and rapid tumor progression. The alpha1-oleate complex has been used successfully to treat bladder cancer in a placebo-controlled Phase II study. Studies of the related BAMLET complexes recently identified effects on metabolism, suggesting additional functions of the "HAMLET family" of tumoricidal complexes. AIMS:To investigate if intravesical alpha1-oleate treatment affects metabolism in bladder cancer tissue. MATERIALS AND METHODS:Samples were obtained from patients with NMIBC, enrolled in a placebo-controlled study of intravesical alpha1-oleate treatment [1]. Cells shed into the urine were harvested at each instillation and biopsies obtained at TURBT after six instillations of alpha1-oleate. The shed cells and biopsies were subjected to RNA sequencing and genome-wide transcriptomic analysis, and urine samples were analyzed using adipokine arrays. RESULTS:RNA sequencing detected significant inhibition of metabolic genes and networks in shed cells and tissue biopsies from alpha1-oleate treated patients, compared to the placebo group. Genes and gene networks regulating glucose and lipid synthesis were inhibited in alpha1-oleate treated patients, including ADIPOQ, which encodes the lipid- and glucose-regulating protein adiponectin and LEP, which encodes the metabolism and fat storage regulator Leptin. Leptin was further identified as an upstream regulator of the adiponectin response to alpha1-oleate and LEP and ADIPOQ networks showed strong interconnection in treated tissues. Analysis of shed tumor cells, suggested rapid kinetics of the metabolic response and urine levels of adiponectin and leptin were increased post-treatment, compared to pre-treatment samples and the shed tumor cells in urine contained adiponectin and leptin, as shown by immunohistochemistry. CONCLUSIONS:The findings identify potent local effects of alpha1-oleate treatment on tumor metabolism, after intravesical instillation. The metabolic regulators adiponectin and leptin were affected in tumor tissue and cells containing adiponectin and leptin were shed into the urine, potentially depleting the tumor of cells with high adipokine levels. These potent effects suggest a new molecular approach for targeting and inhibiting key regulators of cancer metabolism in superficial tumors. TRIAL REGISTRATION:Doubleblinded Phase I/II clinical trial (EudraCT 201600426914 NCT03560479, Registration Date 20180521).
Small cell lung cancer (SCLC) is a deadly cancer with few treatment options and poor prognosis, creating a dire need for improving therapies. Poly (ADP-ribose) polymerase inhibitors (PARPi) have been tested as a treatment strategy, but patient response varies. We aimed to identify novel approaches to sensitize SCLC to PARPi through a genome-wide CRISPR dropout screen. Genome-wide CRISPR dropout screening was conducted in two SCLC cell lines using the PARPi, olaparib, as the selection pressure. Stable shRNA-mediated knockdown cell lines were validated by Western blotting and tested for olaparib sensitivity by assaying for cell viability. Synergy between PARPi and autophagy inhibition was tested by treating SCLC cell lines and analyzing cell viability using SynergyFinder+. The therapeutic strategy combining AZD5305 (PARPi) and GNS561 (novel autophagy inhibitor) was tested in cell line-derived xenograft mouse models. CRISPR screening identified the loss of mTOR negative regulators as a mechanism of PARPi sensitivity in SCLC, and knockdown of TSC1 and TSC2 sensitized SCLC cell lines to olaparib. Therapeutic strategies combining PARPi and autophagy inhibition demonstrated synergy in SCLC cell lines, and combination therapy with AZD5305 and GNS561 was effective in cell line-derived xenograft mouse models. Autophagy inhibition downstream of the mTOR pathway is a mechanism of PARPi sensitivity in SCLC. This suggests that a therapeutic combination of autophagy inhibition and PARPi is a promising treatment strategy in SCLC, paving the way for the adoption of novel treatments in this disease context.
Ovarian cancer (OV) is a leading cause of cancer-related mortality, with cisplatin resistance being a major clinical challenge. This study investigates the role of the Notch ligand DLL1 in mediating ferroptosis resistance and its impact on cisplatin sensitivity in OV. Multi-omics data, clinical samples, and OV cell lines were used to assess DLL1 expression, its link to prognosis, and its effect on the Nrf2/GPX4 axis. CCK-8, clone formation, and ferroptosis assays evaluated the impact of DLL1 on cell behavior and cisplatin sensitivity. DLL1 was upregulated in OV tissues and correlated with poor prognosis. Its knockdown inhibited cell proliferation, migration, and EMT, while inducing ferroptosis, evidenced by increased lipid peroxidation and mitochondrial dysfunction. Mechanistically, DLL1 activated the Nrf2/GPX4 antioxidant axis. DLL1 depletion sensitized OV cells to cisplatin. In vivo, combining DLL1-targeted therapy with a ferroptosis inducer significantly reduced tumor growth in cisplatin-resistant models. DLL1 drives cisplatin resistance in OV by enhancing ferroptosis resistance via the Notch-Nrf2/GPX4 axis. Targeting DLL1 alongside ferroptosis induction represents a promising therapeutic strategy, positioning DLL1 as a potential biomarker and target in drug-resistant OV.
The incidence of colorectal cancer (CRC) is closely associated with prolonged exposure to fine particulate matter (PM 2.5) and tobacco carcinogens, 4-methylnitrosamino-l-3-pyridyl-butanone (NNK). However, due to a lack of understanding of its intricate pathological processes, there is currently no approved clinical medication or successful treatment plan. Also, tobacco carcinogens and PM 2.5 are implicated in the progression of CRC by the onset of a metabolic disorder. Nonetheless, direct evidence connecting the metabolic disorder to NNK/PM 2.5-mediated CRC development has yet to be developed. We investigated CRC cells treated with NNK/PM 2.5 to investigate the function of metabolic alterations in CRC (NNK/PM 2.5)-induced tumor growth. The results were verified using human CRC cancer samples, cell-derived xenografts, and online survival analysis. To clarify the mechanism of regulation of changes in metabolism, metabolic tracking, western blotting, massarray assay, real-time PCR, and reporter assays were conducted. SPSS version 20.0 or GraphPad Prism version 10.0 were used for all statistical analyses. Our research indicates that NNK/PM 2.5, elicit characteristics of metabolic syndrome, notably hyperglycemia, decrease in reactive oxygen species (ROS) contents, and a substantial upregulation of anti-oxidative stress-related gene expression, thereby facilitating the progression of colon cancer in a murine model. By increasing the expression of glucose transporter 3 (GLUT3) and glucose transporter 1 (GLUT1), NNK/PM 2.5 increases the uptake of glucose in tumor-associated macrophages. This leads to the increased expression of insulin-like growth factor 2 (IGF2), which in turn paracrinely hyper-activates the insulin receptor (IR) in colorectal cancer cells, facilitating its nuclear import. Through IR/NPM1 mediation, nuclear IR and nucleophosmin (NPM1) interact to activate the CD274 promoter and produce programmed death ligand-1 (PD-L1). Inhibiting glycolysis, diminishing macrophages, or obstructing PD-L1 impedes NNK/PM 2.5-induced colorectal cancer growth. Analysis of patient samples and public databases reveals elevated expression of tumoral PD-L1 and phosphorylated insulin-like growth factor 1 receptor/insulin receptor (pIGF-1R/IR), as well as elevated levels of IGF2 and GLUT1 in tumor-associated macrophages, suggesting possible negative prognostic biomarkers for patients with colorectal cancer. Our results provided new insights into the mechanisms of intestinal cancer progresses by paracrine IR-PD-L1 signaling, which is aided by the deregulation of oxidative stress and macrophage communication brought on by smoking carcinogen-induced the metabolic syndrome.
A combination of chemical carcinogens and Western diet have been used to induce hepatocellular carcinoma (HCC) in mice, but these models show low incidence of HCC in female mice, while mice that do develop HCC show a wide range of HCC stage development. Therefore, a more reliable method that induces advanced stage HCC in both male and female mice is warranted. The purpose of this study was to create a simple, yet tolerable and reliable method that utilizes chemical carcinogens and a Western diet to induce advanced stage HCC in both male and female mice. Mice were provided with a low-fat control diet or Western diet at weaning and for the remainder of their 30-week lifetime. To induce HCC, mice given the Western diet received the sequential administration of low dose diethylnitrosamine (DEN), thioacetamide (TAA), and sucrose water. This method produced stage 2–3 HCC by 30 weeks of age in 100
Cholesterol (CHO)/bile acids (BAs) homeostasis is critically involved in hepatocellular carcinoma (HCC), yet the underlying mechanisms and the specific regulatory role of CYP8B1-a key enzyme maintaining this balance-remain poorly defined. This study seeks to clarify the roles of CYP8B1 and its regulated CHO/BA homeostasis in the initiation and progression of HCC driven by aberrant RAS/ERK signaling. Utilizing TCGA data analysis, clinical sample examination (including protein/mRNA level assessments and serum measurements), in vitro cell line studies, an HCC animal model (with histopathological classification and inhibitor treatments), and dietary interventions (a high-CHO diet and Taurodeoxycholic acid [TDCA, a main secondary BA] treatment) to investigate the roles of Ras/ERK and CYP8B1 in CHO/BA homeostasis and HCC progression. Clinical HCC data and cell lines frequently show ERK hyperactivation with concomitant CYP8B1 reduction and CHO accumulation. In Hras12V transgenic mice (Ras-Tg), CHO was accumulated in hepatic tumor tissues (T), peri-tumoral tissues (P), and serum compared with wild-type (WT) mice, and this accumulation predominantly resulted from the attenuation of BA biosynthesis induced by reduced CYP8B1 activity. Inhibition of ERK activity by AZD6244 (a p-ERK inhibitor) notably elevated CYP8B1 activity and BAs levels and reduced CHO level in Ras-Tg. Further investigation revealed that SREBP2 is critically involved in the ERK-mediated regulation of CYP8B1 and CHO/BA homeostasis. Intriguingly, although CYP8B1 activity were extremely down-regulated in T compared with P, CHO was significantly lower in T than in P, and the combination of the extreme up-regulation of apolipoprotein (APO) A1 and APOB in T with disordered expression of serum high-density lipoprotein (HDL) and low-density lipoprotein (LDL) might reflect active efflux of CHO from hepatoma cells. Notably, further elevation of the CHO level with a high-CHO diet perturbed multiple signaling pathways (mTOR, NF-κB, GSK3β/β-catenin, caspase 3, and BAX/BCL2) and suppressed hepatic tumor progression but not tumorigenesis. Intriguingly, feeding TDCA significantly inhibits hepatic tumorigenesis and development by inhibiting ERK and mTOR pathways. CYP8B1 repressed by Ras/ERK plays crucial roles in CHO/BA homeostasis which facilitate hepatic tumor progression. These findings provide a novel theoretical foundation and therapeutic perspective for HCC treatment.
Background Gut microbiota (GM) regulates the tumor microenvironment through microbial metabolites. Indole 3-propionic acid (3-IPA) is one such metabolite that regulates gastrointestinal barrier function. In this study, we investigated the effects of 3-IPA on the progression of lymph node metastasis of gastric cancer (GC) and the molecular mechanisms that underlie them.Methods The microbial metabolites were identified using a fecal metabolomic assay in GC patients. Lymphangiogenesis was evaluated using tube formation and wound healing assays in vitro. The expression of aryl hydrocarbon receptor (AHR), CYP1A1, and vascular endothelial growth factor receptor 3 (VEGFR3) were assayed using quantitative real-time PCR (qRT-PCR) and western blot (WB) analyses. Matrigel plug and popliteal lymph node metastasis model were employed to validate the influence on lymphangiogenesis and lymph node metastasis in vivo.Results Fecal metabolomic and microbiome profiling was drastically different between GC patients with lymph node metastasis (GC-LM) and those without metastasis. The GC-LM group showed high 3-IPA expression in the feces; 3-IPA had no significant effect on GC cells; Human lymphatic endothelial cells showed greater tube formation and promoted migration after 3-IPA administration. Also, upregulation of AHR, CYP1A1, and VEGFR3 was observed. Moreover, administration of the AHR inhibitor suppressed tube formation and lymph node metastasis both in vitro and in vivo.Conclusions Our findings suggest that gut microbiota-derived 3-IPA functions as a lymph node metastasis promoter through the AHR/CYP1A1-VEGFR3 axis in GC. 3-IPA could serve as a prognostic biomarker and conceivably a therapeutic target for GC lymph node metastasis.
Hypoxia-inducible factors (HIF1α, HIF2α) influence radiotherapy responses in non-small cell lung cancer (NSCLC) and glioblastoma (GBM), tumors characterized by oxygen and HIF expression heterogeneity. As the function of HIFs in normoxic metabolic function remained unexplored, we investigated how loss of HIF1α or HIF2α affects metabolism, redox homeostasis, and radiotherapy sensitivity in normoxia, aiming to identify opportunities for combined metabolic inhibition. NSCLC HIF1α or HIF2α knockout (KO) and HIFα wildtype (WT) models were analyzed using 13C-glucose mass spectrometry tracing before and after radiotherapy treatment. Metabolic phenotypes were validated using serine/glycine (ser/gly) synthesis enzyme expression by immunoblot and quantitative PCR, redox by ROS flow cytometry analysis, and DNA methylation by 5mC dot-blot assessment. Pharmacological inhibition of ser/gly metabolism was performed in both NSCLC and GBM models using the repurposed serine-glycine conversion inhibitor sertraline using incucyte confluency monitoring. Both HIF1α and HIF2α KO cells displayed reduced glycolysis and compensatory ser/gly pathway hyperactivation. HIF1α KO cells channeled ser/gly into nucleotide (particularly TTP) synthesis and glutathione (GSH)-mediated antioxidant defense, conferring radiotherapy resistance. In contrast, HIF2α KO cells preferentially used serine for α-ketoglutarate (α-KG) production, the enhanced NADH/methionine-dependent redox system and the methionine cycle to support enhanced DNA methylation. Subsequently, following irradiation, only the radiation resistant HIF1α KO cells further enhanced ser/gly metabolism, increasing AMP/ATP and GSH/GSSG (oxidized GSH) ratios, whereas HIF2α KO cells failed to adapt and accumulated oxidative stress. HIF1α KO cells were more sensitive to pharmacological inhibition of ser/gly metabolism by sertraline, particularly in combination with irradiation, which abrogated their radioresistant phenotype in both NSCLC and GBM models. HIF1α-deficient cells rely on ser/gly synthesis for nucleotide production and antioxidant defense, promoting radiotherapy resistance while creating vulnerability to sertraline plus irradiation. HIF2α-deficient cells favor α-KG production and methionine-driven alternative redox and methylation pathways. Targeting ser/gly synthesis may overcome HIF-gradient–dependent radiotherapy resistance.
Aberrant cholesterol accumulation in diverse cancers is associated with intratumoral hypoxia, whereas the specific mechanistic connection underlying hypoxia-driven cholesterol metabolic dysregulation in prostate cancer (PCa) remains undefined. Our previous findings indicate that the positive feedback loop involving SUMO-specific protease 1 (SENP1) and hypoxia-inducible factor-1α (HIF-1α) is critical to maintaining hypoxia-mediated metabolic reprogramming, while whether SENP1 modulates hypoxia-driven cholesterol metabolism remains unclear. Correlations among SENP1 expression, hypoxic status, and cholesterol metabolic profiles were analyzed using TCGA/GEO datasets, PCa tissue microarrays, and PCa cell lines. A series of molecular biology approaches, including co-immunoprecipitation, SUMOylation assay, proximity ligation assay (PLA), Western blotting, and luciferase reporter assay, were performed in HEK293T and PCa cells to clarify the regulatory mechanism of SENP1 in cholesterol metabolism. Patient-derived organoids (PDOs) and xenograft mouse models were utilized to evaluate the anti-tumor efficacy and safety of the SENP1 inhibitor Momordin Ic in vitro and in vivo. SENP1 expression was positively correlated with the expression of key cholesterogenic enzymes in PCa, and SENP1 was essential for hypoxia-induced intracellular cholesterol accumulation. Mechanistically, SENP1 directly catalyzed the deSUMOylation of sterol regulatory element-binding protein 2 (SREBP2) at the lysine 464 (K464) residue, which disrupted the interaction between SREBP2 and the E3 ubiquitin ligase FBXW7 and thereby stabilized the SREBP2 protein. Therapeutically, targeted inhibition of SENP1 by Momordin Ic significantly suppressed de novo cholesterogenesis, impaired the viability of PCa PDOs, and robustly inhibited tumor growth in xenograft models without inducing obvious systemic toxicity. Our study uncovers a previously unrecognized SENP1–SREBP2 regulatory axis that mediates hypoxia-induced de novo cholesterol biosynthesis in PCa, and identifies SENP1 as a promising therapeutic target for metabolic intervention in PCa treatment.
Abstract Background Isocitrate dehydrogenase-wildtype glioblastoma (IDHwtGB) is the most common primary malignant brain tumor in adults, with a universally poor prognosis. For survival and growth under conditions of the tumor microenvironment, glioblastoma cells require antioxidant glutathione (GSH) and its metabolic precursor cystathionine (Cth) to maintain redox balance. We aimed to characterize GSH and Cth in vivo, in IDHwtGB patients, using edited MR spectroscopy (MRS). Our goal was to evaluate their tumor-molecular-status-dependent alterations and assess their potential as biomarkers for therapies targeting redox imbalance, following a reproducibility assessment of the measurement protocol in healthy subjects. Methods In this prospective study (January 2023 - July 2025), 5 healthy subjects and 27 patients with MRI-suspected glioma were scanned on a 3T MR scanner using single-voxel MEGA-sLASER MRS. Tumoral and contralateral metabolite concentrations were compared using linear mixed models. Associations between tumoral GSH and other metabolite concentrations, as well as tumor subregion fractions, were assessed via multiple linear regression. Spearman correlation was used to evaluate the association between GSH and p53 immunoreactivity. The GSH concentration was compared across MGMT status and molecular subtypes with the Wilcoxon rank-sum test. Results A good scan-rescan reproducibility was observed in metabolite quantification in healthy subjects. Fifteen patients with IDHwtGB and high-quality spectra (mean age 59 ± 11 years; 9 men) were included in the final analysis. Tumor tissue exhibited significantly elevated Cth (1.17 ± 1.30 mM vs. 0.63 ± 0.59 mM, p = 0.03) and lower gamma-aminobutyric acid levels (2.36 ± 0.70 mM vs. 3.04 ± 0.89 mM, p = 0.006) compared to contralateral. Tumoral GSH concentrations correlated positively with Cth (p < 0.001) and enhancing-tumor fraction (p < 0.001), and negatively with p53 accumulation (p = 0.008). No difference in GSH levels was observed with respect to MGMT status (p = 0.66), whereas lower GSH concentrations were found in the mesenchymal subtype (p = 0.04). Conclusions GSH and Cth show promise as in vivo MRS biomarkers for therapies aimed at modulating redox balance. Trial registration German Clinical Trials Register (DRKS00032097), retrospectively registered on 25 November 2024.
Objective Hepatocellular carcinoma (HCC) poses a substantial health burden globally. We explored the mechanism of PFKFB4 affecting M2 polarization of tumor-associated macrophages (TAMs) in HCC. Methods The correlation between PFKFB4 expression and macrophage infiltration was analyzed by TIMER database. HCC and adjacent tissues from 30 HCC patients were collected to analyze the PFKFB4 positive expression rate, and the infiltration percentages of the M1-and M2-phenotype TAMs via immunohistochemistry and flow cytometry. PFKFB4 was knocked down or overexpressed in HCC cells, with cell glycolytic, proliferation, and migration assessed. M0 macrophages were co-cultured with HCC cells, with lactate level and TAM M2 polarization detected. H3K18 lactylation (H3K18la) level in TAMs, and its enrichment on the M2 polarization-related gene promoters (arginase 1 [Arg-1], CD206) were assessed by western blot and ChIP. Arg-1 and CD206 levels were tested by RT-qPCR and western blot. In vivo validations were performed in nude mice. Results PFKFB4 expression was increased in HCC, and was correlated with poor prognosis. The positive expression rate of PFKFB4 was positively correlated with M2-polarized TAM infiltration. PFKFB4 was up-regulated in HCC cells, which promoted the proliferation and migration of HCC cells via the glycolytic pathway and stimulated lactate production. Co-culture ofTAMs with PFKFB4-overexpressing HCC cells promoted TAM M2 polarization. PFKFB4 stimulated tumor growth by promoting TAM M2 polarization via the glycolysis/lactate/H3K18la pathway in vivo. Conclusion PFKFB4 promoted lactate accumulation in the tumor microenvironment via glycolysis, stimulated the H3K18la/M2 polarization regulatory axis in TAMs, thus regulating the immune microenvironment and promoting HCC growth.
Background Gemcitabine (GEM) is a standard chemotherapy for pancreatic cancer, but resistance limits its clinical benefit. The role of SRRM1, a splicing regulator, in GEM resistance remains unclear. Methods The expression and prognostic significance of SRRM1 were analyzed in clinical datasets and validated by Western blotting and immunohistochemistry. GEM-resistant pancreatic cancer cell lines were established, and functional assays including colony formation, apoptosis, and CFDA staining were conducted to assess SRRM1's role in chemoresistance. RNA-seq and KEGG enrichment analyses were performed to explore downstream pathways. Ferroptosis was evaluated by C11-BODIPY staining, iron/MDA/GSH quantification, and mitochondrial function assays. NRF2's regulatory effect on SRRM1 was assessed using gain- and loss-of-function experiments. A pancreatic cancer xenograft model was used to validate the therapeutic relevance in vivo. Results SRRM1 was significantly upregulated in pancreatic cancer and associated with poor prognosis and GEM resistance. Knockdown of SRRM1 suppressed tumor growth, enhanced GEM sensitivity, and induced ferroptosis, as evidenced by increased lipid peroxidation and mitochondrial damage. Rescue experiments confirmed the ferroptosis-suppressive function of SRRM1. Notably, we identified NRF2 as an upstream transcriptional activator of SRRM1, forming a pro-survival NRF2-SRRM1 axis that suppresses ferroptosis and promotes GEM resistance. Combining GEM with the ferroptosis inducer RSL3 yielded synergistic antitumor effects in vivo, especially in SRRM1-high tumors. Conclusion This study suggests that SRRM1 may serve as a predictive biomarker for GEM response in pancreatic cancer. Targeting SRRM1 in conjunction with ferroptosis inducers could offer a promising strategy to overcome GEM resistance.
Endometrial cancer (EC) is characterized by metabolic reprogramming, with cholesterol biosynthesis playing a critical role. However, the upstream transcriptional regulation of this process requires further elucidation. In this study, we identified POU2F1 as a key oncogenic transcription factor that drives cholesterol biosynthesis and tumor progression in EC. Integrative bioinformatics and clinical analyses revealed that POU2F1 is upregulated in EC and predicts poor prognosis. Mechanistically, POU2F1 directly activates DHCR24 and ELOVL2 transcription, thereby promoting DHCR24-mediated cholesterol biosynthesis and ELOVL2-associated lipid metabolic remodeling in EC. Functional assays demonstrated that POU2F1 promotes proliferation, migration, invasion, and xenograft growth in a DHCR24/ELOVL2-dependent manner. Clinically, POU2F1 expression was positively correlated with DHCR24 and ELOVL2 levels and served as an independent prognostic factor. Together, these findings establish the POU2F1-DHCR24/ELOVL2 axis as a critical driver of cholesterol-associated lipid metabolic reprogramming and cancer progression in EC, highlighting a potential therapeutic target for metabolic intervention.
Background High sensitivity to mitochondria damage belongs to established differences between stem cells of acute myeloid leukemia (AML) and their healthy counterparts. Mitophagy, as the key mitochondria quality control, is a possible therapy target in AML. However, research in this field requires simple and robust methods for functional measurement of mitochondria clearance. Methods We developed protocols to analyze (i) the rate of mitochondria clearance and (ii) total mitochondrial mass and mitochondria polarization using MitoTracker dyes and flow cytometry. Substantial improvement was achieved by incorporating (i) a proliferation dye to account for the effect of cell division on mitochondria content and (ii) reference cells to compensate for variable cell density. These protocols were tested in 12 leukemia cell lines with different variants of the tumor suppresor TP53, as well as the adherent cell line HS-5. Results Repeated analyses confirmed high sensitivity and reproducibility of our protocols.The mitophagy rate in p53-null cells was higher compared to cells with the wild-type p53. Mitophagy inhibition was associated with an increase of the total mitochondria mass. The impact of mitophagy inhibition on mitochondria polarization was context-dependent: mitochondria depolarization was associated with cell death induction whereas hyperpolarization was observed during cell recovery in resistant cell lines. Conclusion Our optimized protocols represent a useful tool for analysis of mitophagy and mitochondria content in various cell types.
Circadian rhythm disruption is increasingly linked to tumor progression and therapy resistance, but its role in immune evasion remains unclear. Here, we demonstrate that knockdown of the core circadian gene PER2 (PER2-KD) drives resistance to immune checkpoint inhibitors (ICIs) in gastric cancer (GC) by promoting monocytic myeloid-derived suppressor cell (M-MDSC) accumulation and metabolic reprogramming. RNA-seq and scRNA-seq revealed that PER2-KD upregulates immunosuppressive genes (e.g., PD-L1, IL-10) while suppressing T cell activation signals. Mechanistically, PER2-KD GC cells secrete elevated VEGF and IL-6, inducing STAT3 phosphorylation in M-MDSCs and enhancing their immunosuppressive function. Untargeted metabolomics identified glycolysis and fatty acid metabolism as key pathways dysregulated in PER2-KD M-MDSCs, with lactate accumulation further amplifying CD8 + T cell dysfunction. In vivo, PER2-KD tumors exhibited accelerated growth, reduced PD-1 blockade efficacy, and shorter survival, accompanied by increased M-MDSC infiltration and lactate levels. Neutralizing VEGF/IL-6 or inhibiting lactate metabolism restored CD8+ T cell activity, suggesting actionable therapeutic targets. Our study unveils PER2-KD as a novel regulator of metabolic-immune crosstalk in GC, providing a mechanistic basis for ICI resistance and highlighting circadian-metabolic interventions to improve immunotherapy outcomes.
BACKGROUND: Untargeted metabolomics has emerged as a powerful approach to uncover metabolic dysregulation associated with cancer progression. When integrated with a machine learning strategy it facilitates the discovery of key metabolic pathways and predictive biomarkers with high diagnostic and prognostic value. METHODS: In this study, we employed liquid chromatography coupled to high-resolution Tribrid Orbitrap mass spectrometry to perform comprehensive metabolic profiling of bladder cancer (BLCA) as well as predict invasiveness of the disease. RESULTS: By leveraging both in-house retention time-based MS/MS spectral libraries and commercial databases, we robustly identify over 2000 metabolites. In addition, this platform allows identification of novel pathways highlighting metabolic vulnerabilities in BLCA. The application of machine learning algorithms and advanced computational modeling uncovered metabolic signatures that differentiate BLCA from adjacent normal/benign samples and distinguish muscle-invasive from non-muscle-invasive bladder cancer. Our integrative analytical pipeline addresses key challenges in metabolomics-including high dimensionality, metabolite annotation, and biological variability-through feature selection and predictive modeling. We identify candidate metabolic markers with strong potential for early detection and characterize invasiveness of the disease and identify potential therapeutic target pathways. CONCLUSIONS: This work highlights the power of combining untargeted metabolomics with machine learning to map the metabolic landscape of BLCA and to accelerate the development of precision diagnostics and future therapeutic strategies.
Immune checkpoint blockade (ICB) targeting PD-1 has transformed cancer therapy, yet many tumors display primary or acquired resistance. Metabolic interactions within the tumor microenvironment are increasingly recognized as key modulators of anti-tumor immunity. To identify tumor-intrinsic metabolic pathways that contribute to resistance to anti-PD1 therapy, we performed an in vivo CRISPR-based negative selection screen using a metabolism-focused sgRNA library in an oncogenic Kras and p53 loss-driven murine lung adenocarcinoma cell line. The IgG control arm revealed essential metabolic dependencies for in vivo tumor growth, including the TCA cycle, electron transport chain, antioxidant pathways, one carbon metabolism and de novo lipogenesis. Differential analysis of anti-PD1–treated tumors uncovered metabolic genes whose loss sensitized cancer cells to PD-1 blockade, highlighting pathways in antigen presentation, metabolite transport, arachidonic acid metabolism, and peroxisomal function. Secondary subpooled screens across multiple lung cancer cell lines validated the prominence of these pathways. Although Pla2g4a emerged as a top candidate across Kras-driven tumors, genetic deletion of this enzyme individually did not enhance responsiveness to PD-1 therapy. Overall, this work defines metabolic vulnerabilities that contribute to primary tumor growth in vivo, while providing a resource for selecting tumor-intrinsic metabolic targets that need further validation for combination strategies with PD-1 blockade.
BACKGROUND: Glucose is an important fuel in cancer cells, however, its availability may be limited in solid tumors. Cell-autonomous, metabolic adaptations of cancer cells and non-malignant cells to glucose deprivation are still incompletely understood. METHODS: Here, we addressed the changes in central carbon metabolism in lung cancer cells and normal lung cells facing glucose limitation using stable isotopic labeling followed by nuclear magnetic resonance spectroscopy and mass spectrometry. RESULTS: Elevated levels and the release of newly synthesized aspartate were among the most prominent changes in low compared to high glucose conditions. The low glucose-induced export of aspartate occurred in different lung cancer cell lines, but also bronchial epithelial cells and cancer-associated fibroblasts. It was accompanied by a reduced use of aspartate in purine synthesis and suppressed by hypoxia. A knockout of the malate-aspartate shuttle (MAS) enzyme mitochondrial aspartate aminotransferase (GOT2) decreased aspartate release. Low glucose conditions diminished reduced nicotinamide adenine dinucleotide (NADH) and restoring NADH reversed aspartate synthesis, suggesting that the distal, NADH-dependent arm of the MAS is compromised under glucose deprivation. CONCLUSIONS: Cells accumulate and release aspartate, a biosynthetic precursor and signaling molecule, under low glucose conditions, largely due to a truncated MAS, as part of their adaptive metabolic response.
Fanconi anemia complementation group D2 (FANCD2) is a key component of DNA damage repair and has emerged as a potential cancer therapy target. Its role in enzalutamide (ENZ) resistance in prostate cancer remains unclear. FANCD2 expression was examined in prostate cancer cells. Gain- and loss-of-function experiments were performed to assess ferroptosis, mitochondrial morphology, and cell viability. Nrf2 and GPX4 expression were measured to elucidate antioxidant regulation. Combination therapy using the ferroptosis inducer RSL3 and ENZ was evaluated in vitro and in vivo for antitumor efficacy. FANCD2 was significantly upregulated in prostate cancer and promoted ENZ resistance by inhibiting ferrous iron accumulation and lipid peroxidation, thereby suppressing ferroptosis. Mechanistically, FANCD2 stabilized Nrf2, increasing GPX4 expression and maintaining redox homeostasis. FANCD2 downregulation reduced Nrf2 and GPX4 expression, induced mitochondrial damage, and triggered ferroptosis, enhancing sensitivity to ENZ. Furthermore, combined treatment with RSL3 and ENZ synergistically suppressed cell growth, migration, and tumor progression in vivo. FANCD2 regulates ferroptosis through the Nrf2–GPX4 axis, contributing to ENZ resistance in prostate cancer. Targeting FANCD2 or combining enzalutamide with ferroptosis inducers offers a promising strategy to overcome drug resistance and improve therapeutic outcomes.
Adipose tissue metabolic plasticity and inflammation critically influence tumor progression through endocrine signaling. While white adipose tissue (WAT) has been linked to pro-tumorigenic effects in obesity-related cancers, the influence of brown adipose tissue (BAT) and its secretome on breast cancer remains incompletely understood. Furthermore, how caspase-1/11–mediated inflammasome signaling regulates adipose tissue endocrine function in this context is largely unexplored. This study investigated the differential effects of WAT and BAT secretomes on breast cancer aggressiveness and elucidate the impact of caspase-1/11 deficiency on adipose tissue-tumor crosstalk. Conditioned media (CM) were generated from WAT and BAT of wild-type (WT) and caspase-1/11 knockout (KO) C57BL/6 mice, including animals subjected to cold-induced BAT activation. 4T1 breast cancer cells were exposed to these secretomes, and carcinogenic parameters were assessed, including viability (MTT), cell death (Annexin-V/PI), proliferation (CFSE), migration (wound healing assay), lipid droplet biogenesis (BODIPY and Oil Red staining and microscopy), oxidative stress (ROS and nitrite quantification), and cytokine production (ELISA). Additionally, splenocytes were also stimulated with the secretomes to assess their effect on T and NKT cell activation (Flow cytometry). Global proteomic profiling (LC-MS/MS) was performed to identify key molecular pathways affected of exposed breast cancer cells compared to controls. Statistical analyses included ANOVA with Tukey’s or Student’s t-test, as appropriate. WAT-CM promoted lipid droplet accumulation in 4T1 cells. In contrast, BAT-CM reduced tumor cell viability, cell proliferation, and migration further triggering oxidative stress and cell death. Immunophenotypic analysis revealed that BAT-CM modulated immune activation. These antitumor effects were amplified by caspase-1/11 deficiency and cold-induced BAT activation. Proteomic analyses revealed distinct modulation of metabolic, inflammatory, and immune-related pathways in WAT- and BAT-CM-treated tumor cells. Histological and cytokine analyses demonstrated that caspase-1/11 deficiency led to reduced adipocyte size, increased BAT macrophage infiltration, and a softened inflammatory profile. Our findings uncover a novel anti-tumor role for the BAT secretome in breast cancer, modulated by caspase-1/11-dependent inflammasome signaling and cold-induced activation. Targeting adipose tissue plasticity and inflammasome pathways may offer new strategies to reprogram the tumor microenvironment. These results open novel perspectives for exploring BAT-derived factors as metabolic-based therapeutics for breast cancer.
