Hijacking the expression of KDM4B-dependent metabolic genes potentiates curcumin antitumor effects

Hijacking the expression of KDM4B-dependent metabolic genes potentiates curcumin antitumor effects | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hijacking the expression of KDM4B-dependent metabolic genes potentiates curcumin antitumor effects Rosa Sirianni, Marta Claudia Nocito, Alice Amico, Tarig Hamad, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7328223/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cancer therapies often cause significant side effects, driving the search for more tolerable and effective treatments. Natural products, such as the polyphenol curcumin, exhibit promising anticancer properties by modulating cell death pathways. We demonstrated that curcumin inhibits growth and migration of adrenocortical cancer (ACC) cells, a rare tumor with no available targeted therapy. In these cells curcumin caused a shift in cell metabolism toward a glycolytic phenotype and an increased glutamine/glutamate dependency. However, why cells adapt their metabolism promoting glutamate synthesis remains unclear. Curcumin acts as a pro-oxidant in ACC cells, elevating reactive oxygen species (ROS) and, consequently, cells adopt a defense mechanism increasing glutathione, a glutamate-containing antioxidant, and lipid droplet (LD) content, preventing oxidative stress. Key genes in this metabolic adaptation are SLC1A5, SLC27A2, ELOVL5, whose expression is positively regulated by curcumin and abrogated by the ROS scavenger N-acetyl-cysteine (NAC). Mechanistically, we evidenced that curcumin-induced ROS increase transcription factor HIF1α, which drives the upregulation of nuclear receptor PPARγ and the Jumonji histone demethylases (KDMs) family member KDM4B, affecting ACC cell metabolism and epigenome. The use of a KDM4B inhibitor potentiated curcumin anti-proliferative effects in vitro and, more importantly, on ex vivo cultures of H295R xenografts tissue grown in a 3D bioreactor. Collectively these data prove ACC cells’ ability to change their metabolism to counteract curcumin-induced oxidative stress through epigenetic modifications. The identification of KDM4B as the master regulator behind the changes provides novel insights into combinatorial strategies for improving ACC patients’ outcome. Health sciences/Diseases/Cancer/Cancer metabolism Biological sciences/Cancer/Cancer therapy/Targeted therapies Curcumin ROS Epigenetic KDM4B PPARG Lipid metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION The Indian spice curcumin, a polyphenol compound found in Curcuma longa, has gained attention worldwide for its wide range of health beneficial properties including anticancer [ 1 ], which rely on its ability to activate different cell death mechanisms [ 2 ]. The interest for curcumin as antitumor drug is demonstrated by its evaluation in several clinical trials (NCT03980509; NCT03072992; NCT03769766; NCT03598309; NCT03192059, NCT03769766, NCT02321293). Adrenocortical carcinoma (ACC) is a rare endocrine malignancy that carries a poor prognosis with late stage disease. Unfortunately, a majority of patients present with advanced disease at the time of diagnosis and once metastatic, the disease has a low (10–20%) five-year survival. For patients with metastatic disease, the only current FDA approved therapeutic is the adrenolytic agent mitotane, which still challenges patients for its high toxicity and low response rates [ 3 ]. Then, new and better approaches for ACC management are needed. Recently we demonstrated that curcumin reduced ACC cells growth and migration [ 4 ] and affected cell metabolism, favoring a shift toward a glycolytic phenotype where pyruvate is preferentially converted into alanine by the alanine aminotransferase (ALT/GPT), which was also upregulated by curcumin. Our observation of an increase in alanine/glutamine antiport transporter - SLC1A5 - and glutaminase (GLS1) gene expression, led to the hypothesis that SLC1A5, could be engaged to export alanine in exchange for the import of glutamine, acting as substrate for GLS1 to produce glutamate. We proved that limiting glutamine availability in the cell culture medium, potentiated curcumin anti-tumor effects [ 4 ]. In synthesis, our previous study highlighted a metabolic response limiting curcumin anti-tumor effects, but left an open question regarding the meaning of glutamate synthesis. A further definition of the molecular mechanisms activated by curcumin in ACC cells would help identifying some additional targets that could potentiate curcumin antitumor effects. Curcumin is a known antioxidant molecule, however, in several cancer phenotypes it acts as a potent pro-oxidant, causing ROS production responsible for activating cell death mechanisms [ 5 ]. Elevated ROS production and oxidative stress have been observed in melanoma [ 6 ], colorectal cancer [ 7 ], responsive and treatment-resistant breast, liver and lung cancer cells [ 8 ]. High ROS production occurs in cancer cells in response to therapy, adaptation to ROS can favor the selection of aggressive tumor cells. Concomitant to ROS production, cancer cells enhance lipid droplets (LDs) formation which contribute to remove ROS and reduce cellular stress, pointing to LDs as important organelle for the survival of cancer cells [ 9 ]. Elevated LDs are a well-known alteration of lipid reprogramming associated with tumor progression and therapy resistance [ 10 , 11 ]. Several studies have demonstrated the ability of curcumin to influence lipid metabolism, free fatty acids (FFA) availability, fatty acid synthesis, desaturation and β-oxidation [ 12 ]. To increase FA availability, curcumin was shown to influence the expression of FA transporters [ 13 ], such as SLC27A2/FATP2 in rats liver [ 14 ]. Metabolic adaptation are often achieved by inducible changes in gene expression programs [ 15 ]. To rapidly reach this aim, cells engage epigenetic modifiers which reversibly change chromatin accessibility by DNA and histone (de)methylation and (de)acetylation. ROS can influence cell epigenome by affecting either expression or activity of histone demethylases (HDMs). The Jumonji family of HDMs, KDM, consists of 19 members, several of which are involved in the regulation of metabolic genes [ 16 , 17 ]. The expression of several KDMs, such as KDM3A, KDM4B, KDM4C and KDM6B, is regulated by ROS/HIF1α axis [ 18 , 19 ]. Then, a KDM member could be a plausible regulator of the metabolic rewiring occurring in ACC cells exposed to curcumin. This study aimed to investigate curcumin effects on the antioxidant system and lipid metabolism in ACC cells, to identify an epigenetic modifier, acting as a master regulator of the metabolic rewiring, to be co-targeted in a curcumin-based therapy. This becomes particularly relevant for a rare cancer, as ACC, associated with limited treatment options and poor patients’ survival. MATERIALS AND METHODS Cell Cultures H295R cell line were obtained from the American Type Culture Collection (ATCC, Rockville, MD). MUC-1 cells [20] were obtained from Constanze Hantel (University of Zurich-Switzerland). ACC cells lines were grown in a humidified 5% CO2 at 37 °C and were cultured as previously described [4]. Cell Viability Assays Cell viability was measured using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) colorimetric assay, which measures mitochondrial activity in viable cells. Cells were plated in a 48-well plate and treated for 48h with curcumin and ML324. After treatment, fresh MTT resuspended in phosphate buffered saline (PBS), was added to each well (final concentration 0.33 mg / mL) and the plate was incubated at 37° C for 2 hours in a humidified incubator to 5% of CO2. Medium was then removed and the formazan crystals dissolved in 200 µl of DMSO (Sigma-Aldrich) with gentle agitation. The optical density was measured at 570 nm (Synergy H1 plate reader, BioTek Instruments, Inc., Winooski, VT, USA). Each experiment was performed in six replicates and repeated three times. RNA extraction, reverse transcription and real-time PCR Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA). One microgram of total RNA was reverse transcribed into a final volume of 50 μl using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Foster City, CA, USA). cDNA was diluted 1:3 in nuclease-free water and used for real-time PCR. The primer sequences are listed in Table S1. PCR reactions were performed in the QuantStudioTM 3, Real-Time PCR System (Thermo Fisher) using 0.2 μM of each primer. PowerUp™ SYBR™ Green Master Mix (Thermo Fisher) with the dissociation protocol was used for gene amplification; negative controls contained water instead of first-strand cDNA. Each sample was normalized to its 18S rRNA (18S) content. Final results were expressed as n-fold differences relative to a calibrator and calculated using the ΔΔCt method. Lipid peroxidation H295R and MUC-1 cells were plated into 6-well plates containing coverslips. After 48 h, cells were treated for additional 48 h with curcumin. At the end of the treatment, cells were washed two times with PBS and then incubated for 20 min with 5 μM BODIPY™ C11 581/591. Cells were washed two times with PBS and fixed with 4% PFA for 15 min at room temperature. Coverslips were mounted onto glass slides and fluorescence was visualized using an FV3000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). Lipid staining Cells were plated into 6-well plates containing coverslips at a confluence of 30-50%. After 48h, cells were treated for additional 48h with curcumin and N-Acetyl-L-Cysteine (NAC) in growth media and with curcumin without fetal bovine serum. At the end of the treatment cells were washed two times with PBS and then incubate for 20 min with 10μM BODIPY™ 493/503. Cells were washed two times with PBS and fixed with 4% PFA for 15 min at room temperature and permeabilized with Triton X-100 (0.2% in PBS) for 3 min. Cells were then washed again, and incubated with DAPI solution (0.2 mg/ml in PBS) for 5 min. After a final wash, coverslips were mounted onto glass slides. Fluorescent signal was analyzed using an FV3000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan). Determination of oxidative stress Oxidative stress was evaluated using Muse Oxidative Stress Kit (Luminex). H295R and MUC-1 cells were plated on a 6-well plate containing growth medium and then treated for 48h with curcumin and N-Acetyl-L-Cysteine (NAC). Cells were trypsinized, counted and 1x106 cells resuspended in 1 mL of the reagent 1X Assay Buffer. In a test tube, 10 µl of cells (~ 1x104) from each sample were added to 190 µl of Muse Oxidative Stress Reagent (working solution) and incubated for 30 min at 37 °C. Guava® Muse® Cell Analyzer - Luminex instrument was used to analyze 5000 events. Positive control was obtained by treating cells with H2O2 (3 mM final concentration) for 2 h. Glutathione Determination Total Glutathione (GSH) content was evaluated using the Glutathione Colorimetric Detection Kit (Invitrogen). H295R cells were plated on a 6-well plate containing growth medium and treated for 48h with curcumin. Cells were trypsinized and resuspended in 1ml of ice cold 5% aqueous solution of 5-sulfo-salicylic acid dihydrate (SSA) (1x106). Cells were lysed by vigorous vortexing, incubated for 10 min at 4 °C and centrifuged at 14,000 rpm for 10 min at 4 °C. Supernatants were diluted by adding 4X v/v of 1X Assay Buffer. Cell extracts were then incubated with the Reaction Mixture for 20 min and used to detect total GSH according to the manufacturer’s instructions. Absorbance was measured at 405 nm (Synergy H1 plate reader, BioTek Instruments, Inc., Winooski, VT, USA). MUFA and PUFA determination H295R cells (1X106 cells) were plated into petri dishes and treated with curcumin for 48 h. Cells were then lifted and washed 3 times by centrifugation in ice-cold PBS prior to lysis. Each cell pellet was subjected to extraction (200 µL of chloroform:isopropanol:NP-40 in a ratio of 7:11:0.1) and centrifugation (10 minutes at 15,000 x g). The organic phase was transferred to a new tube, air dried at 50 ºC to remove the chloroform and subjected to vacuum for 30 minutes to remove the trace amounts of organic solvent. The dried lipids were dissolved in 200 μL of DMSO by vortex. MUFA and PUFA determination was measured using a colorimetric Lipid Quantification Kit (Cell Biolabs) according to the manufacturer’s instruction. Molecular Docking Molecular docking studies were carried out using the crystallographic structure of human Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) retrieved from the Protein Data Bank (PDB) and corresponding to entry 7AWC [21]. In this structure the receptor is in complex with the known agonist rosiglitazone. Ligand structures (rosiglitazone, GW9662, DHA, and curcumin) were constructed and optimized using Avogadro software [22]. Docking simulations were performed with AutoDock Vina 1.1.2 [23], and preliminary file preparation and conversion were conducted by using the graphical interface AutoDock Tools 1.5.6 [24]. Polar hydrogens were added for the crystallographic protein, and apolar hydrogens of all compounds were merged to the carbon atom they were attached to. Full flexibility was guaranteed for the ligands, resulting in 7, 3, 15, and 12 rotatable dihedral angles for rosiglitazone, GW9662, DHA, and curcumin, respectively. A single simulation run was carried out in each case at very high exhaustiveness, 16 times larger than the default value. The binding modes of the ligand were analysed through visual inspection by using VMD [25]. The Molecular Graphics System PyMOL has been used to visualise protein structure. Ligand binding intermolecular interactions were evaluated using the automated protein–ligand interaction profiler (PLIP) [26]. Ex vivo experiments All animal experiments were performed in accordance with the national (Ministero della Salute) and institutional (University of Calabria Animal Welfare Committee; OPBA) guidelines and regulations (protocol n. 170/2024-PR). H295R cells (5 × 10 6 ) were inoculated subcutaneously in the interscapular region of a 8-week-old female Foxn1nu mouse (Harlen Envigo). When tumor reached the volume of 300 mm 3 , was explanted, washed with PBS, minced to obtain small sections, to be transferred in the ClinoReactor™ that was placed in a CelVivo ClinoStar™ system at standard culturing conditions. The rotation speed was monitored to accommodate tumoroid growth by maintaining a microgravity environment. Culture media was exchanged every second day, and ClinoReactor™ replaced after 10 days. Treatment was maintained for two weeks before acquisition of microphotographs under Olympus CKX53 inverted microscope with a 2X objective. Patients’ data analysis Bioinformatics analyses from transcriptome data were performed in R (http://www.R-project.org) using software packages from the Bioconductor portal (www.bioconductor.org). To assess the effect of gene expression on survival, we used transcriptome data from the ACC-TCGA dataset [27]. We downloaded legacy RNA-seq counts data from the Genomic Data Commons (GDC) portal using TCGA biolinks [28] and performed log2-cpm normalization using edgeR, after correcting for library size using the TMM method [29]. For overall survival analysis, we split the ACC-TCGA cohort into quartiles according to the expression of each gene. We used Kaplan-Meier plots to depict survival times, and the log-rank test to assess statistical significance. To evaluate the combined effects of KDM4B and PPARγ or ELOVL5 expression on survival, we stratified the cohort into two groups based on the median expression of KDM4B and PPARγ or ELOVL5. Kaplan-Meier plots were utilized to illustrate survival times, and the log-rank test was employed to determine statistical significance. Furthermore, we applied Cox regression models to estimate the impact of KDM4B, both independently, and in combination with other genes, on overall survival. To assess whether incorporating a second variable (PPARγ or ELOVL5) into the univariate model with KDM4B improved the model’s fit, we performed likelihood-ratio tests. Statistical Analysis All experiments were performed at least three times. Data are expressed as mean values ± standard error (SE). The statistical significance was analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). Groups were compared using the analysis of variance (ANOVA) with Bonferroni’s post hoc testing. Significance was defined as p < 0.05. Results Curcumin increases glutamine metabolism to sustain glutathione synthesis. Our previous study demonstrated that ACC cells treated with curcumin, rewire their metabolism toward glutamine (Gln) uptake [ 4 ]. To address Gln fate, we investigated the expression of its metabolizing enzyme glutaminase-1 (GLS1) in response to curcumin, and evidenced its upregulation (Fig. 1 A). GLS1 mediates conversion of Gln into glutamate (Glu), one of the three amino acids that together with cysteine and glycine constitutes the tripeptide glutathione (GSH), the most abundant intracellular antioxidant molecule, previously shown to be upregulated by curcumin [ 30 ]. Indeed, we found that in ACC cells curcumin was able to increase the expression of SLC7A11 and SLC6A9, transporters for cystine and glycine, respectively (Fig. 1 B, C), and glutathione synthase (GSS), the GSH synthesis enzyme, (Fig. 1 D), raising remarkably the amount of glutathione (GSH) (Fig. 1 E). We then evidenced that the increased amount of GSH is paralleled by a significant rise in ROS production. Indeed, in H295R cells we found a 3- and 6-fold increase in ROS content in response to curcumin 20 and 40 µM, respectively (Fig. 1 F). In MUC-1 cells ROS levels rose by 3-fold in response to both 20 and 40 µM curcumin (Fig. 1 G). Despite the increase in ROS production, we did not evidence the presence of peroxidized lipids in the two ACC cell lines (Fig. 1 H, 1 I), supporting the hypothesis that curcumin-treated cells activate an antioxidant mechanism to limit membrane damage. ROS/HIF1α/SLC27A2 axis sustains lipid droplets formation Elevated ROS levels can trigger the formation of lipid droplets in several contexts, to counteract cell oxidative stress [ 31 , 32 ]. This protective mechanism is activated in also ACC cells treated with inhibitors for EZH2, a histone modifier, as demonstrated by our recent publication. This protective mechanism involves increased glutathione synthesis and increased formation of lipid droplets (LDs) [ 33 ]. Similarly, curcumin-treated cells increase the abundance of LDs (Fig. 2 A) and, accordingly, the amount of triglycerides (TAG) (Fig. 2 B). Analysis of LDs-associated enzymes evidenced a decrease in the lipase ATGL (Fig. 2 C), which converts TAG to DAG, and a concomitant decrease in LIPE (Fig. 2 D), the gene for hormone sensitive lipase, avoiding the degradation of cholesterol esters (CE). Additionally, we also noticed an increase in G0S2 (Fig. 2 E), which blocks the residual ATGL activity. Curcumin downregulated ACACA and FASN mRNA (Fig. 2 F, 2 G), involved in the synthesis of palmitate, a saturated FA. These data suggest that FA derive from uptake, as a proof-of-principle curcumin-treated cells grown in serum free medium were unable to accumulate LDs (Fig. 2 H). To specify the pathway associated to FA uptake, we analyzed mRNA expression for genes involved in this process, evidencing the ability of curcumin to decrease CD36, LDLR and SLC27A4 (Fig. 3 A, 3 B, 3 C) while selectively increasing SLC27A2 (Fig. 3 D). Accordingly, expression of SREBP1 and SREBP2, transcription factors (TFs) for CD36 and LDLR decreased (Fig. 3 E, 3 F). By contrast, HIF1α, TF for SLC27A2, increased (Fig. 3 G). Importantly, HIF1α and SLC27A2 expression increased also in curcumin-treated MUC-1 cells (Fig. 3 H, 3 I). It’s known that ROS are able to increase HIF1α expression in cancer cells [ 34 , 35 ]. To prove a role for ROS in the regulation of HIF1α gene expression in our experimental settings, we used the antioxidant N-acetyl-cysteine (NAC). NAC as ROS scavenger, prevented curcumin effects on HIF1α mRNA and, accordingly, on SLC27A2 ( Supplementary Fig. 1A, 1B ). We also evaluated ROS amounts in the presence of NAC, demonstrating its ability to counteract curcumin effects ( Supplementary Fig. 1C ). NAC prevented also curcumin-dependent LDs accumulation in both cell lines ( Supplementary Fig. 1D, 1E ). Curcumin activates PPARγ to increase intracellular unsaturated FA SLC27A2 mediates FA cell entry, converting long-chain, branched-chain and very-long-chain fatty acids containing 22 or more carbons to their CoA derivatives, with a preference for ω-3 fatty acids [ 36 ]. We evidenced an increase in unsaturated FAs (MUFA-PUFA) in the presence of curcumin (Fig. 4 A). Evaluation of mRNA for genes involved in FA desaturation and elongation evidenced a decrease in SCD1, which converts saturated C16 and C18 FA to their unsaturated form (Fig. 4 B). Elongation of very long-chain fatty acid (ELOVL) 1 and 5 were upregulated (Fig. 4 C, D). Instead, FA desaturase FADS2 was decreased (Fig. 4 E). Importantly, ELOVL1,5 are under the transcriptional control of peroxisome proliferator-activated receptor gamma (PPARγ), that we found to be dose-dependently upregulated by curcumin (Fig. 4 F). SCD1 and FADS2 are instead under SREBP1 transcriptional control, then, their downregulation is consistent with the decreased expression of the transcription factor (Fig. 3 E). ELOVL5 and PPARγ upregulation was observed also MUC-1 cells (Fig. 4 G, H). ELOVL family enzymes catalyze fatty acid elongation, with ELOVL5 specifically involved in the elongation of PUFA [ 37 ]. We hypothesized that unsaturated FA would enter the cells through SLC27A2 and then be used as substrate by ELOVL5. Importantly, PPARγ is under HIF1α transcriptional control [ 38 ], then, so far our data suggest that HIF1α activates PPARγ expression, which, in turn, would increase expression of FA elongation genes. The central role played by ROS/HIF1α activation, was proved by the ability of NAC to prevent curcumin-induced PPARγ transcription (Fig. 4 J), and consequently the effects on its target genes ELOVL5 (Fig. 4 K ) and SLC1A5 (Fig. 4 L). The transcriptional activity of PPARs is finely regulated by co-activators and co-repressors, which modulate signaling and interaction with the basal transcription machinery [ 39 ]. In the absence of ligands (ligand-independent repression), PPARs bind the promoters of their target genes and repress transcription by recruiting co-repressors (e.g., NCoR and SMRT). Upon ligand activation (ligand-dependent transactivation), PPARs undergo conformational changes that provoke the displacement of co-repressors and recruitment of co-activators such as p300/CBP and p160, activating transcription [ 39 ]. Similarly to our data, previous studies showed that some of the effects of curcumin rely on PPARγ activation [ 40 ]. To assess the binding affinity and interaction profile of curcumin toward PPARγ, molecular docking simulations were performed on the crystallographic structure of the receptor. Given the availability on the Protein Data Bank (PDB) of several crystallographic structures of PPARγ, we selected the entry with the code 7AWC, one of the most recently deposited structures, which corresponds to the ligand-binding domain of the receptor. In this structure the protein is co-crystallized with rosiglitazone, a well-established PPARγ agonist approved by the Food and Drug Administration for the treatment of type II diabetes [ 41 ]. Firstly, rosiglitazone was re-docked into the receptor’s ligand binding site, resulting in a predicted binding energy (BE) of -7.8 kcal/mol, which was taken as a reference for the following docking analyses. Next, we performed molecular docking of curcumin, GW9662, a selective and potent PPARγ antagonist, and docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid an endogenous PPARγ ligand [ 42 ]. All ligands occupied the same ligand-binding pocket ( Fig. S2 ), and notably curcumin exhibited the most favourable binding score (BE = -8.6 kcal/mol), outperforming all the other ligands (BE= -8.2 and − 7.5 kcal/mol for GW9662 and DHA, respectively). These findings are consistent with previously reported studies [ 43 ]. Despite the absence of hydrogen bonding with Tyr473, a key residue involved in full agonist activity and engaged by rosiglitazone, curcumin established an extensive network of hydrophobic interactions with key residues such as Arg288, Ile326, Tyr327, Leu330, Ile341, and Phe363, many of which are also involved in stabilizing classical PPARγ ligands. Interestingly, curcumin shares a similar interaction pattern with GW9662, relying primarily on hydrophobic contacts within the same residues, with the addition of an extra interaction involving Ile326. Conversely, DHA, although capable of forming hydrogen bonds with Tyr327 and Tyr473, exhibited a less extensive hydrophobic network, as reflected in its lower binding affinity ( Table S1 ). Taken together, these findings underscore the strong binding affinity of curcumin and its ability to interact with residues that are crucial for the binding of known ligands, providing compelling structural evidence for its modulatory role on PPARγ. The histone demethylase KDM4B is the master regulator of curcumin/PPARγ-mediated lipid metabolism gene expression Activation of selective gene expression in response to curcumin treatment led us to hypothesize the involvement of a ROS-mediated epigenetic regulation. ROS, are known to increase the expression of several member of the Jumanji family of histone demethylase (KDMs) [ 44 , 45 ]. Members of this family enhance transcription by demethylating specific lysine residues of histone H3. We focused our attention on KDM4B, which was shown to increase glucose metabolism in colorectal cancer [ 17 ], similarly to what we observed following curcumin treatment. Additionally, KDM4B is under the transcriptional control of HIF1α [ 18 ], upregulated by curcumin. Importantly, high expression levels of KDM4B is associated with decreased overall survival in ACC patients (Fig. 5 A). To assess whether the addition of a second variable (PPARγ or ELOVL5) improved the fitness of the univariate model (KDM4B), we performed likelihood-ratio tests comparing the univariate model (EZH2 only) and each of the bivariate models. The addition of a second variable (PPARγ or ELOVL5), improved the fitness of the univariate model, suggesting it provided further information relevant for overall survival. To visualize this effect, Kaplan-Meier curves were obtained dividing samples into four groups, based on the median expression of KDM4B and PPARγ or ELOVL5 (Fig. 5 B, C). Log-rank tests of each pair-wise comparison were performed. As demonstrated in the Kaplan-Meier curves, the addition further stratification according to the expression of PPARγ or ELOVL5, can further improve risk stratification by identifying a subgroup of KDM4B-high patients with very high risk. Analysis of KDM4B mRNA evidenced curcumin ability to increase its expression (Fig. 6 A), that was prevented by NAC (Fig. 6 B). We tested the effect of curcumin in combination with KDM4B inhibitor, ML324, on the expression of two PPARγ target genes modulated by curcumin. ML324 abrogated curcumin effects on ELOVL5 expression (Fig. 6 C), while no influence was seen for SLC1A5 (Fig. 6 D). The combination of the two drugs was tested on cell growth using concentrations of the single agents that inhibited by no more than 25%. The combined treatment produced a more potent inhibitory effect on both H295R and MUC-1(Fig. 6 E). Importantly, we evidenced the combined treatment was also effective on H295R tumoroids, that appeared smaller in diameter, more compact and with a reduced translucency (Fig. 6 F). DISCUSSION Changes in metabolic pathways and fluctuations in intermediate metabolites transmit information about the intracellular metabolic status to the nucleus by regulating the activity of epigenetic enzymes. This modulation reshapes the epigenetic landscape, thereby eliciting transcriptional responses tailored to diverse metabolic demands. The interplay between cell metabolism and epigenome becomes particularly relevant in the context of cancer therapy, when cancer cells adapt their metabolism to overcome drug-induced cell death. Recently, we and others have recently proved curcumin effectiveness in reducing ACC cell growth [ 4 , 46 ]. We demonstrated that curcumin caused a metabolic rewiring, characterized by increased glutamine (Gln) metabolism, and lowering glutamine availability potentiated curcumin inhibitory effects. The current study demonstrates that glutamine is used to sustain a 20-fold increase in the content of glutathione (GSH), the most powerful endogenous antioxidant. To sustain glutathione synthesis, we observed that curcumin caused a transcriptional up-regulation of SLC7A11 and SLC6A9, cell membrane carriers for cystine and glycine (Gly), respectively. Cystine is used by cells for the synthesis of cysteine (Cys), which together with Glu and Gly are incorporated in the tripeptide GSH by glutathione synthase (GSS), also upregulated by curcumin. Glutathione exists in the reduced GSH form and oxidized GSSG form. The protocol we used evaluated the reduced form only, it is then possible that the cell amount of glutathione could be even higher than what we found. Curcumin-induced antioxidant response was previously reported for other cell types [ 47 , 48 ], however, several studies indicate that curcumin-dependent anticancer mechanisms are mediated by the induction of oxidative stress [ 5 ]. Indeed, ROS levels were increased upon curcumin exposure, then, ACC cells activate an antioxidant response to counteract the pro-oxidant effects exerted by the polyphenol, with GSH being used to counteract ROS-mediated damages, and in fact, no lipid peroxidation was observed. We need to underline that the doses of curcumin used in the current study are not associated with activation of apoptosis, which instead is executed at the dose of 40 µM as shown in our previous report[ 4 ]. The discovered antioxidant mechanism could help explaining the limited cell death-inducing ability of the lower doses of curcumin. MUC-1 cells used as multidrug resistant ACC phenotype [ 20 ] show high levels of TAG-containing LDs compared to the drug-sensitive H295R cells, which instead preferentially accumulate cholesterol esters-containing LDs [ 49 ]. We recently demonstrated that an increase in TAG-containing LDs occurs in ACC cells treated with tazemetostat, an inhibitor for the methyl transferase EZH2, which similarly to curcumin causes oxidative stress [ 33 ]. Lipid droplets act as antioxidant organelles that control polyunsaturated fatty acids storage in triglycerides in order to reduce membrane lipid peroxidation, preserving organelle function. This defense mechanism would reduce cellular stress, favoring cancer cell survival and therapy resistance [ 9 – 11 ]. Accordingly, evaluation of LDs amount in ACC models treated with curcumin evidenced a remarkable increase. Moreover, curcumin specifically increased TAG and PUFA content. The increase in LDs depends on a reduced metabolism as demonstrated by decreased expression of ATGL, the lipase localized on LDs surface and involved in the TAG degradation, and LIPE, responsible for DAG and cholesterol esters degradation. We further evidenced an increased expression of G0S2, which prevents any residual ATGL activity. FA synthesis was not involved in LDs accumulation since ACACA and FASN, and their transcription factor SREBP1 (sterol-responsive element binding protein-1) were downregulated. Genes involved in FA uptake, LDLR and CD36, and their transcription factors SREBP1 and SREBP2[ 50 ], were downregulated, instead, FA transporter SLC27A2 and its transcription factor HIF1α were significantly upregulated. HIF1α is known to be responsive to hypoxic conditions and to increased ROS production in both hypoxia and normoxia [ 34 ]. The use on the ROS scavenger N-acetyl-cysteine by neutralizing curcumin-induced ROS, prevented HIF1α upregulation, and consequently, transcription of its target gene, SLC27A2, and LDs formation. The SLC27A2 gene encodes for proteins known as FATP2 that was shown to preferentially mediate cell entry of ω3-PUFA [ 36 , 51 ]. Dietary PUFA drastically decrease the mature, cleaved form, of SREBP-1 protein, consequently less lipogenic enzymes are transcribed [ 52 ] Collectively these data draw an adaptive metabolic response which favors an enhanced LDs formation through the ROS/HIF1α/SLC27A2 axis, used by ACC cells to prevent the detrimental effects exerted by curcumin-mediated ROS production. Evaluation of mRNA for FA modifying enzymes such as elongase (ELOVL1 and ELOVL5) and desaturase (SCD1, FADS2) evidenced preferential FA modifications. ELOVL1 and 5 are targets for the transcription factor PPARγ, a member of the nuclear receptor family of metabolic TFs [ 53 ], that we evidenced to be upregulated by curcumin. Notably, HIF1α regulates PPARγ transcription [ 38 ], and accordingly the use of NAC prevented activation of its transcription, and consequently of its targets, in curcumin-treated ACC cells. PPARγ is a TF also for ATGL and LIPE, which however, we evidenced to be downregulated by curcumin. These divergent effects could be explained by changes in the cofactors recruited by PPARγ and/or by differences in the localization of PPRE sites within the 5’UTR (untranslated region) on target genes, allowing ligand-dependent transcriptional activation or repression. An intriguing possibility is a direct interaction between PPARγ and curcumin, which can cause a change in the protein 3D structure and allow interaction with specific co-factors. This possibility was screened by docking analysis which proved a strong interaction between curcumin and PPARγ, similar to known PPAR ligands. This observation deserves further investigations through additional experiments as PPAR/curcumin interaction still represents a debatable question [ 43 , 54 ]. For example, PPARγ was found to associate with G protein suppressor 2 (GPS2), a TFs interacting protein, and regulate transcription of a subset of PPARγ target genes. Specifically, GPS2 acted as a priming factor for PPARγ recruitment to ATGL and LIPE and stabilized interaction with the histone demethylase KDM4A/JMJD2 [ 55 ]. Additionally, PPARγ/GPS2 binding was stronger at promoter regions than at enhancers or other genomic locations [ 55 ]. Histone lysine demethylases (KDMs) play important roles in chromatin remodeling and gene regulation by catalyzing the demethylation of methylated lysine residues on the N-terminal tails of histones. The histone demethylase activity of KDM4s requires α-ketoglutarate (α-KG), O 2 , and Fe 2+ , to catalyze the demethylation of tri- and di-methylated forms of both histone H3 lysine 9 (H3K9me3/me2) and lysine 36 (H3K36me3/me2) [ 16 , 17 ]. It is worth highlighting that curcumin causes an increase in glutamine [ 4 ] that could also be metabolized into α-KG, pointing to the idea of an interplay between cell metabolism and epigenetic regulation. This intriguing possibility was proved by demonstrating that curcumin increased the expression of KDM4B and the use of its inhibitor, ML324, at a concentration that did only minimally affect cell proliferation, potentiated curcumin anti-proliferative effects. This was evidenced in vitro by MTT assay and ex-vivo using tumoroids derived from H295R xenografts and grown in a bioreactor. KDM4B is under HIF1α transcriptional regulation [ 18 ], and the use of NAC, by preventing HIF1α activation, abrogated KDM4B transcription. To ascertain the role of KDM4B in the epigenetic regulation of ELOVL5 in response to curcumin, the polyphenol was combined with ML324 and QPCR analysis evidenced the loss of curcumin-induced up-regulation. On the contrary, expression of PPARγ evaluated in the same experimental settings was unaffected. These data underline that the epigenetic modulation in response to metabolic adaptation is the result of a fine tuning in expression and activity of epigenetic modifiers. Indeed, a cluster of cluster (COC) analysis of ACC-TCGA data identified three molecular ACC subtypes COC1, COC2, and COC3, which, on an epigenetic level, respectively bear low, intermediate and high levels of genome-wide CpG island methylation [ 27 , 56 ]. Of the three COC clusters, COC3 shows the higher disease progression rate, highlighting a tight connection between the worst clinical outcome profound epigenetic modifications. In conclusion, our study further proves curcumin to be a promising molecule for the treatment of ACC. Having more in detail dissected curcumin effects on metabolic and epigenetic genes we demonstrated a more potent effect when combined with KDM4B inhibitors. Importantly, suppressing a broad spectrum of epigenetic enzymes can cause potential deleterious side effects, while the use of specific KDM4B inhibitors could help overcoming this issue. Declarations COMPETING INTERESTS The authors declare no competing interests. AUTHOR CONTRIBUTIONS IC and RS conceived and designed this project; MCN, AA, AC and MGS conducted the experiment and analyzed results; FM and CM performed and analyzed metabolomic studies; FG and MAO performed docking analysis; TAH performed patients’ databases analyses; CH, CI, MFS, and ML contributed reagents/analytic tools; CH, MFS, CI, SG, ML, VP, IC, and RS participated in data analysis and results discussion; IC and RS wrote the paper. 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Department of Pharmacy and Health and Nutritional Sciences, Cell Biology Lab","correspondingAuthor":false,"prefix":"","firstName":"Vincenzo","middleName":"","lastName":"Pezzi","suffix":""},{"id":512177369,"identity":"f4216c85-5eaa-47fd-ac6e-f844f7969872","order_by":16,"name":"Ivan Casauri","email":"","orcid":"","institution":"University of Calabria - Department of Pharmacy and Health and Nutritional Sciences, Cell Biology Lab","correspondingAuthor":false,"prefix":"","firstName":"Ivan","middleName":"","lastName":"Casauri","suffix":""}],"badges":[],"createdAt":"2025-08-08 14:31:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7328223/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7328223/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90895465,"identity":"7dd26c38-cc29-47e3-96ce-aaf245198aae","added_by":"auto","created_at":"2025-09-09 11:33:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":457957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlutamine metabolism favors GSH synthesis to counteract ROS-dependent lipid peroxidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH295R cells were treated for 48h with curcumin (20 and 40 µM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-D.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003emRNA\u003c/em\u003e expression\u003cem\u003e \u003c/em\u003eof genes related to glutamine metabolism (n=3, means ± SEM, **p \u0026lt; 0.01; ****p-value \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE.\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003eGlutathione (GSH) content normalized to the number of cells. (\u003cem\u003en\u003c/em\u003e = 3, means ± SEM, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF, G. \u003c/strong\u003eCells were labeled with oxidative stress reagent and analyzed by Muse Cell Analyzer. In histogram profiles, cells positive for reactive oxygen species (ROS) are shown in red. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used as a positive control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH, I. \u003c/strong\u003eConfocal images of lipid peroxidation detected by BODIPY-C11 fluorescent dye in H295R (H) and MUC-1 cells (F) (scale bar 50 µm).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/b4e70bfcd94e4219d4dc4410.png"},{"id":90895469,"identity":"cb0428ab-00a4-4b9d-97c7-829aebf02088","added_by":"auto","created_at":"2025-09-09 11:33:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":398064,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCurcumin-induced LDs formation is dependent on FA uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA. \u003c/strong\u003eConfocal images of neutral lipids in 48h treated H295R and MUC-1 cells. Nuclei were stained by DAPI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB. \u003c/strong\u003eIntracellular triglycerides quantification normalized to the number of cells in H295R cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-G. \u003c/strong\u003emRNA expression of LDs-associated genes in 48h treated H295R cells (n=3, means ± SEM, **p \u0026lt; 0.01; ****p-value \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH. \u003c/strong\u003eConfocal images of neutral lipids in 48h treated H295R cells with or without fetal bovine serum (FBS). Nuclei were stained by DAPI.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/0dffe020b20bf6e5c79e1512.png"},{"id":90895470,"identity":"f213455a-f8d1-45e9-8fb0-12e866ddee60","added_by":"auto","created_at":"2025-09-09 11:33:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":249146,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSLC27A2 is selectively increased by curcumin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-G.\u003c/strong\u003e\u003cem\u003e m\u003c/em\u003eRNA expression of genes related to FA uptake in H295R cells treated with curcumin (20 and 40µM) (n=3, means ± SEM, **p \u0026lt; 0.01; ****p-value \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH, I.\u003c/strong\u003e\u003cem\u003e m\u003c/em\u003eRNAexpression\u003cem\u003e \u003c/em\u003eof HIF1α and SLC27A2 genes in MUC-1 cells treated with curcumin (20µM) (n=3, means ± SEM, **p \u0026lt; 0.01; ****p-value \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/2d3dc8bc4023f783346c4cfb.png"},{"id":90897336,"identity":"791b4928-987b-4090-b7a5-0295ecc3c60b","added_by":"auto","created_at":"2025-09-09 11:41:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":296414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCurcumin treated ACC cells modify their lipidome according to changes in the expression of FA-modifying genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Unsaturated FA (MUFA and PUFA) quantification normalized to the number of cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB-G.\u003c/strong\u003e mRNA expression of genes related to FA desaturation and elongation in 48h treated H295R cells (n=3, means ± SEM, **p \u0026lt; 0.01; ****p-value \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH, I.\u003c/strong\u003e mRNA expression of ELOVL5 and PPARγ genes in 48h treated MUC-1 cells with curcumin (20µM) (n=3, means ± SEM, **p \u0026lt; 0.01; ****p-value \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJ-L.\u003c/strong\u003e Cells were treated for 48h with curcumin (20µM) and N-Acetyl-L-Cysteine (NAC) (3mM). mRNA expression of PPARγ, ELOVL5 and SLC1A5 genes.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/a7425bd956587335087d9efb.png"},{"id":90897755,"identity":"aebcfc73-31be-4836-b9c4-8234ddf60805","added_by":"auto","created_at":"2025-09-09 11:49:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":590318,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B expression impacts ACC patients’ survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Kaplan-Meier survival curves for KDM4B expression in the ACC-TCGA dataset [26] divided in quartiles. Overall survival for the quartile group with highest KDM4B expression compared with the other groups. High and low level of expression are indicated by red and blue colors, respectively. \u0026nbsp;Log-rank p-values are shown at the bottom of plot. \u003cstrong\u003eB, C \u003c/strong\u003eKaplan-Meier curves showing overall survival (in days) of subgroups of patients stratified according to expressions of KDM4B and PPARγ (\u003cstrong\u003eB\u003c/strong\u003e) or KDM4B and ELOVL5 (\u003cstrong\u003eC\u003c/strong\u003e). The total number of patients in each subgroup is shown below the curve. The p-value on top of the plot represents the overall result of the log-rank test including all groups.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/14439147624427fcd515da9b.png"},{"id":90895475,"identity":"f6bc2867-c319-4329-956a-eede4a4f8ae3","added_by":"auto","created_at":"2025-09-09 11:33:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":348428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKDM4B is involved in the epigenetic regulation of curcumin-induced genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, B. \u003c/strong\u003emRNA expression of KDM4B gene in 48h treated H295R (A) and MUC-1 (B) cells (n=3, means ± SEM, **p \u0026lt; 0.01; ****p-value \u0026lt; 0.0001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC. \u003c/strong\u003emRNA expression of KDM4B gene in H295R cells treated for 48h with curcumin (20µM) and N-Acetyl-L-Cysteine (NAC) (3mM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD-F. \u003c/strong\u003eH295R cells were treated for 48h with curcumin (20µM) and ML324 (10µM).\u003cem\u003e mRNA\u003c/em\u003e expression\u003cem\u003e \u003c/em\u003eof PPARγ, ELOVL5 and SLC1A5 genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG, H. \u003c/strong\u003eCell viability of tumor cell lines treated for 48 h with curcumin (20μM) and ML324 (10µM) and their combinations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI. \u003c/strong\u003eSchematic representation of ex vivo experimental procedure. Representative images of tumoroids under the different treatment conditions. Graph represents the mean + SE of the surface area of 4 different tumoroids.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/1342522cef4c4b5f7a7b8cd1.png"},{"id":90899767,"identity":"ed621d2c-7323-4183-a33b-4f4dca9951f8","added_by":"auto","created_at":"2025-09-09 12:05:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3114690,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/fbfb4555-12af-4766-a483-f32b148911d2.pdf"},{"id":90895464,"identity":"3ff7fd69-f87b-4f13-8830-9f4ce498c4c1","added_by":"auto","created_at":"2025-09-09 11:33:02","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":147209,"visible":true,"origin":"","legend":"Supplementary figure 1","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/fdaf412a8ae3381eeb2c8673.pdf"},{"id":90897753,"identity":"237b79d7-068d-419a-97f0-180f04a539bf","added_by":"auto","created_at":"2025-09-09 11:49:02","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":119533,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary figure 2\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/f3ea71830d1d43c98d32edf0.pdf"},{"id":90895463,"identity":"f16e7e89-d03e-4749-9204-4e513167c62f","added_by":"auto","created_at":"2025-09-09 11:33:02","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":34463,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7328223/v1/6cb037267bf287d8339bfc18.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Hijacking the expression of KDM4B-dependent metabolic genes potentiates curcumin antitumor effects","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe Indian spice curcumin, a polyphenol compound found in Curcuma longa, has gained attention worldwide for its wide range of health beneficial properties including anticancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], which rely on its ability to activate different cell death mechanisms [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The interest for curcumin as antitumor drug is demonstrated by its evaluation in several clinical trials (NCT03980509; NCT03072992; NCT03769766; NCT03598309; NCT03192059, NCT03769766, NCT02321293).\u003c/p\u003e\u003cp\u003eAdrenocortical carcinoma (ACC) is a rare endocrine malignancy that carries a poor prognosis with late stage disease. Unfortunately, a majority of patients present with advanced disease at the time of diagnosis and once metastatic, the disease has a low (10\u0026ndash;20%) five-year survival. For patients with metastatic disease, the only current FDA approved therapeutic is the adrenolytic agent mitotane, which still challenges patients for its high toxicity and low response rates [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Then, new and better approaches for ACC management are needed.\u003c/p\u003e\u003cp\u003eRecently we demonstrated that curcumin reduced ACC cells growth and migration [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and affected cell metabolism, favoring a shift toward a glycolytic phenotype where pyruvate is preferentially converted into alanine by the alanine aminotransferase (ALT/GPT), which was also upregulated by curcumin. Our observation of an increase in alanine/glutamine antiport transporter - SLC1A5 - and glutaminase (GLS1) gene expression, led to the hypothesis that SLC1A5, could be engaged to export alanine in exchange for the import of glutamine, acting as substrate for GLS1 to produce glutamate. We proved that limiting glutamine availability in the cell culture medium, potentiated curcumin anti-tumor effects [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In synthesis, our previous study highlighted a metabolic response limiting curcumin anti-tumor effects, but left an open question regarding the meaning of glutamate synthesis. A further definition of the molecular mechanisms activated by curcumin in ACC cells would help identifying some additional targets that could potentiate curcumin antitumor effects.\u003c/p\u003e\u003cp\u003eCurcumin is a known antioxidant molecule, however, in several cancer phenotypes it acts as a potent pro-oxidant, causing ROS production responsible for activating cell death mechanisms [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Elevated ROS production and oxidative stress have been observed in melanoma [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], colorectal cancer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], responsive and treatment-resistant breast, liver and lung cancer cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. High ROS production occurs in cancer cells in response to therapy, adaptation to ROS can favor the selection of aggressive tumor cells. Concomitant to ROS production, cancer cells enhance lipid droplets (LDs) formation which contribute to remove ROS and reduce cellular stress, pointing to LDs as important organelle for the survival of cancer cells [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Elevated LDs are a well-known alteration of lipid reprogramming associated with tumor progression and therapy resistance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Several studies have demonstrated the ability of curcumin to influence lipid metabolism, free fatty acids (FFA) availability, fatty acid synthesis, desaturation and β-oxidation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To increase FA availability, curcumin was shown to influence the expression of FA transporters [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], such as SLC27A2/FATP2 in rats liver [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMetabolic adaptation are often achieved by inducible changes in gene expression programs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To rapidly reach this aim, cells engage epigenetic modifiers which reversibly change chromatin accessibility by DNA and histone (de)methylation and (de)acetylation. ROS can influence cell epigenome by affecting either expression or activity of histone demethylases (HDMs). The Jumonji family of HDMs, KDM, consists of 19 members, several of which are involved in the regulation of metabolic genes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The expression of several KDMs, such as KDM3A, KDM4B, KDM4C and KDM6B, is regulated by ROS/HIF1α axis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Then, a KDM member could be a plausible regulator of the metabolic rewiring occurring in ACC cells exposed to curcumin.\u003c/p\u003e\u003cp\u003eThis study aimed to investigate curcumin effects on the antioxidant system and lipid metabolism in ACC cells, to identify an epigenetic modifier, acting as a master regulator of the metabolic rewiring, to be co-targeted in a curcumin-based therapy. This becomes particularly relevant for a rare cancer, as ACC, associated with limited treatment options and poor patients\u0026rsquo; survival.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eCell Cultures\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH295R cell line were obtained from the American Type Culture Collection (ATCC, Rockville, MD). MUC-1 cells [20] were obtained from Constanze Hantel (University of Zurich-Switzerland). ACC cells lines were grown in a humidified 5% CO2 at 37 \u0026deg;C and were cultured as previously described [4]. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Viability Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell viability was measured using the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) colorimetric assay, which measures mitochondrial activity in viable cells. Cells were plated in a 48-well plate and treated for 48h with curcumin and ML324. After treatment, fresh MTT resuspended in phosphate buffered saline (PBS), was added to each well (final concentration 0.33 mg / mL) and the plate was incubated at 37\u0026deg; C for 2 hours in a humidified incubator to 5% of CO2. Medium was then removed and the formazan crystals dissolved in 200 \u0026micro;l of DMSO (Sigma-Aldrich) with gentle agitation. The optical density was measured at 570 nm (Synergy H1 plate reader, BioTek Instruments, Inc., Winooski, VT, USA). Each experiment was performed in six replicates and repeated three times. \u0026nbsp; \u0026nbsp;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction, reverse transcription and real-time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA). One microgram of total RNA was reverse transcribed into a final volume of 50 \u0026mu;l using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher, Foster City, CA, USA). cDNA was diluted 1:3 in nuclease-free water and used for real-time PCR. The primer sequences are listed in Table S1. PCR reactions were performed in the QuantStudioTM 3, Real-Time PCR System (Thermo Fisher) using 0.2 \u0026mu;M of each primer. PowerUp\u0026trade; SYBR\u0026trade; Green Master Mix (Thermo Fisher) with the dissociation protocol was used for gene amplification; negative controls contained water instead of first-strand cDNA. Each sample was normalized to its 18S rRNA (18S) content. Final results were expressed as n-fold differences relative to a calibrator and calculated using the \u0026Delta;\u0026Delta;Ct method. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid peroxidation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH295R and MUC-1 cells were plated into 6-well plates containing coverslips. After 48 h, cells were treated for additional 48 h with curcumin. At the end of the treatment, cells were washed two times with PBS and then incubated for 20 min with 5 \u0026mu;M BODIPY\u0026trade; C11 581/591. Cells were washed two times with PBS and fixed with 4% PFA for 15 min at room temperature. Coverslips were mounted onto glass slides and fluorescence was visualized using an FV3000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLipid staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were plated into 6-well plates containing coverslips at a confluence of 30-50%. After 48h, cells were treated for additional 48h with curcumin and N-Acetyl-L-Cysteine (NAC) in growth media and with curcumin without fetal bovine serum. At the end of the treatment cells were washed two times with PBS and then incubate for 20 min with 10\u0026mu;M BODIPY\u0026trade; 493/503. Cells were washed two times with PBS and fixed with 4% PFA for 15 min at room temperature and permeabilized with Triton X-100 (0.2% in PBS) for 3 min. Cells were then washed again, and incubated with DAPI solution (0.2 mg/ml in PBS) for 5 min. After a final wash, coverslips were mounted onto glass slides. Fluorescent signal was analyzed using an FV3000 confocal laser scanning microscope (Olympus Corporation, Tokyo, Japan).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of oxidative stress\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOxidative stress was evaluated using Muse Oxidative Stress Kit (Luminex). H295R and MUC-1 cells were plated on a 6-well plate containing growth medium and then treated for 48h with curcumin and N-Acetyl-L-Cysteine (NAC). Cells were trypsinized, counted and 1x106 cells resuspended in 1 mL of the reagent 1X Assay Buffer. In a test tube, 10 \u0026micro;l of cells (~ 1x104) from each sample were added to 190 \u0026micro;l of Muse Oxidative Stress Reagent (working solution) and incubated for 30 min at 37 \u0026deg;C. Guava\u0026reg; Muse\u0026reg; Cell Analyzer - Luminex instrument was used to analyze 5000 events. Positive control was obtained by treating cells with H2O2 (3 mM final concentration) for 2 h.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlutathione Determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal Glutathione (GSH) content was evaluated using the Glutathione Colorimetric Detection Kit (Invitrogen). H295R cells were plated on a 6-well plate containing growth medium and treated for 48h with curcumin. Cells were trypsinized and resuspended in 1ml of ice cold 5% aqueous solution of 5-sulfo-salicylic acid dihydrate (SSA) (1x106). Cells were lysed by vigorous vortexing, incubated for 10 min at 4 \u0026deg;C and centrifuged at 14,000 rpm for 10 min at 4 \u0026deg;C. Supernatants were diluted by adding 4X v/v of 1X Assay Buffer. Cell extracts were then incubated with the Reaction Mixture for 20 min and used to detect total GSH according to the manufacturer\u0026rsquo;s instructions. Absorbance was measured at 405 nm (Synergy H1 plate reader, BioTek Instruments, Inc., Winooski, VT, USA). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMUFA and PUFA determination\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH295R cells (1X106 cells) were plated into petri dishes and treated with curcumin for 48 h. Cells were then lifted and washed 3 times by centrifugation in ice-cold PBS prior to lysis. Each cell pellet was subjected to extraction (200 \u0026micro;L of chloroform:isopropanol:NP-40 in a ratio of 7:11:0.1) and centrifugation (10 minutes at 15,000 x g). The organic phase was transferred to a new tube, air dried at 50 \u0026ordm;C to remove the chloroform and subjected to vacuum for 30 minutes to remove the trace amounts of organic solvent. The dried lipids were dissolved in 200 \u0026mu;L of DMSO by vortex. MUFA and PUFA determination was measured using a colorimetric Lipid Quantification Kit (Cell Biolabs) according to the manufacturer\u0026rsquo;s instruction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular Docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking studies were carried out using the crystallographic structure of human Peroxisome Proliferator-Activated Receptor Gamma (PPAR\u0026gamma;) retrieved from the Protein Data Bank (PDB) and corresponding to entry 7AWC [21]. In this structure the receptor is in complex with the known agonist rosiglitazone. Ligand structures (rosiglitazone, GW9662, DHA, and curcumin) were constructed and optimized using Avogadro software [22]. Docking simulations were performed with AutoDock Vina 1.1.2 [23], and preliminary file preparation and conversion were conducted by using the graphical interface AutoDock Tools 1.5.6 [24]. Polar hydrogens were added for the crystallographic protein, and apolar hydrogens of all compounds were merged to the carbon atom they were attached to. Full flexibility was guaranteed for the ligands, resulting in 7, 3, 15, and 12 rotatable dihedral angles for rosiglitazone, GW9662, DHA, and curcumin, respectively. A single simulation run was carried out in each case at very high exhaustiveness, 16 times larger than the default value. The binding modes of the ligand were analysed through visual inspection by using VMD [25]. The Molecular Graphics System PyMOL has been used to visualise protein structure. Ligand binding intermolecular interactions were evaluated using the automated protein\u0026ndash;ligand interaction profiler (PLIP) [26].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEx vivo experiments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the national (Ministero della Salute) and institutional (University of Calabria Animal Welfare Committee; OPBA) guidelines and regulations (protocol n. 170/2024-PR). H295R cells (5\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e) were inoculated subcutaneously in the interscapular region of a 8-week-old female Foxn1nu mouse (Harlen Envigo). When tumor reached the volume of 300 mm\u003csup\u003e3\u003c/sup\u003e, was explanted, washed with PBS, minced to obtain small sections, to be transferred in the ClinoReactor\u0026trade; that was placed in a CelVivo ClinoStar\u0026trade; system at standard culturing conditions. The rotation speed was monitored to accommodate tumoroid growth by maintaining a microgravity environment. Culture media was exchanged every second day, and ClinoReactor\u0026trade; replaced after 10 days. Treatment was maintained for two weeks before acquisition of microphotographs under Olympus CKX53 inverted microscope with a 2X objective.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients\u0026rsquo; data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBioinformatics analyses from transcriptome data were performed in R (http://www.R-project.org) using software packages from the Bioconductor portal (www.bioconductor.org). To assess the effect of gene expression on survival, we used transcriptome data from the ACC-TCGA dataset [27]. We downloaded legacy RNA-seq counts data from the Genomic Data Commons (GDC) portal using TCGA biolinks [28] and performed log2-cpm normalization using edgeR, after correcting for library size using the TMM method [29]. For overall survival analysis, we split the ACC-TCGA cohort into quartiles according to the expression of each gene. We used Kaplan-Meier plots to depict survival times, and the log-rank test to assess statistical significance.\u003c/p\u003e\n\u003cp\u003eTo evaluate the combined effects of KDM4B and PPAR\u0026gamma; or ELOVL5 expression on survival, we stratified the cohort into two groups based on the median expression of KDM4B and PPAR\u0026gamma; or ELOVL5. Kaplan-Meier plots were utilized to illustrate survival times, and the log-rank test was employed to determine statistical significance. Furthermore, we applied Cox regression models to estimate the impact of KDM4B, both independently, and in combination with other genes, on overall survival. To assess whether incorporating a second variable (PPAR\u0026gamma; or ELOVL5) into the univariate model with KDM4B improved the model\u0026rsquo;s fit, we performed likelihood-ratio tests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed at least three times. Data are expressed as mean values \u0026plusmn; standard error (SE). The statistical significance was analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). Groups were compared using the analysis of variance (ANOVA) with Bonferroni\u0026rsquo;s post hoc testing. Significance was defined as p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCurcumin increases glutamine metabolism to sustain glutathione synthesis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur previous study demonstrated that ACC cells treated with curcumin, rewire their metabolism toward glutamine (Gln) uptake [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. To address Gln fate, we investigated the expression of its metabolizing enzyme glutaminase-1 (GLS1) in response to curcumin, and evidenced its upregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). GLS1 mediates conversion of Gln into glutamate (Glu), one of the three amino acids that together with cysteine and glycine constitutes the tripeptide glutathione (GSH), the most abundant intracellular antioxidant molecule, previously shown to be upregulated by curcumin [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Indeed, we found that in ACC cells curcumin was able to increase the expression of SLC7A11 and SLC6A9, transporters for cystine and glycine, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C), and glutathione synthase (GSS), the GSH synthesis enzyme, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), raising remarkably the amount of glutathione (GSH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe then evidenced that the increased amount of GSH is paralleled by a significant rise in ROS production. Indeed, in H295R cells we found a 3- and 6-fold increase in ROS content in response to curcumin 20 and 40 \u0026micro;M, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In MUC-1 cells ROS levels rose by 3-fold in response to both 20 and 40 \u0026micro;M curcumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Despite the increase in ROS production, we did not evidence the presence of peroxidized lipids in the two ACC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), supporting the hypothesis that curcumin-treated cells activate an antioxidant mechanism to limit membrane damage.\u003c/p\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eROS/HIF1α/SLC27A2 axis sustains lipid droplets formation\u003c/h2\u003e\u003cp\u003eElevated ROS levels can trigger the formation of lipid droplets in several contexts, to counteract cell oxidative stress [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This protective mechanism is activated in also ACC cells treated with inhibitors for EZH2, a histone modifier, as demonstrated by our recent publication. This protective mechanism involves increased glutathione synthesis and increased formation of lipid droplets (LDs) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Similarly, curcumin-treated cells increase the abundance of LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and, accordingly, the amount of triglycerides (TAG) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Analysis of LDs-associated enzymes evidenced a decrease in the lipase ATGL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), which converts TAG to DAG, and a concomitant decrease in LIPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), the gene for hormone sensitive lipase, avoiding the degradation of cholesterol esters (CE). Additionally, we also noticed an increase in G0S2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), which blocks the residual ATGL activity. Curcumin downregulated ACACA and FASN mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), involved in the synthesis of palmitate, a saturated FA. These data suggest that FA derive from uptake, as a proof-of-principle curcumin-treated cells grown in serum free medium were unable to accumulate LDs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo specify the pathway associated to FA uptake, we analyzed mRNA expression for genes involved in this process, evidencing the ability of curcumin to decrease CD36, LDLR and SLC27A4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) while selectively increasing SLC27A2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Accordingly, expression of SREBP1 and SREBP2, transcription factors (TFs) for CD36 and LDLR decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). By contrast, HIF1α, TF for SLC27A2, increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Importantly, HIF1α and SLC27A2 expression increased also in curcumin-treated MUC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIt\u0026rsquo;s known that ROS are able to increase HIF1α expression in cancer cells [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. To prove a role for ROS in the regulation of HIF1α gene expression in our experimental settings, we used the antioxidant N-acetyl-cysteine (NAC). NAC as ROS scavenger, prevented curcumin effects on HIF1α mRNA and, accordingly, on SLC27A2 (\u003cb\u003eSupplementary Fig.\u0026nbsp;1A, 1B\u003c/b\u003e). We also evaluated ROS amounts in the presence of NAC, demonstrating its ability to counteract curcumin effects (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C\u003c/b\u003e). NAC prevented also curcumin-dependent LDs accumulation in both cell lines (\u003cb\u003eSupplementary Fig.\u0026nbsp;1D, 1E\u003c/b\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eCurcumin activates PPARγ to increase intracellular unsaturated FA\u003c/h2\u003e\u003cp\u003eSLC27A2 mediates FA cell entry, converting long-chain, branched-chain and very-long-chain fatty acids containing 22 or more carbons to their CoA derivatives, with a preference for ω-3 fatty acids [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We evidenced an increase in unsaturated FAs (MUFA-PUFA) in the presence of curcumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Evaluation of mRNA for genes involved in FA desaturation and elongation evidenced a decrease in SCD1, which converts saturated C16 and C18 FA to their unsaturated form (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Elongation of very long-chain fatty acid (ELOVL) 1 and 5 were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Instead, FA desaturase FADS2 was decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Importantly, ELOVL1,5 are under the transcriptional control of peroxisome proliferator-activated receptor gamma (PPARγ), that we found to be dose-dependently upregulated by curcumin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). SCD1 and FADS2 are instead under SREBP1 transcriptional control, then, their downregulation is consistent with the decreased expression of the transcription factor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). ELOVL5 and PPARγ upregulation was observed also MUC-1 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H). ELOVL family enzymes catalyze fatty acid elongation, with ELOVL5 specifically involved in the elongation of PUFA [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. We hypothesized that unsaturated FA would enter the cells through SLC27A2 and then be used as substrate by ELOVL5. Importantly, PPARγ is under HIF1α transcriptional control [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], then, so far our data suggest that HIF1α activates PPARγ expression, which, in turn, would increase expression of FA elongation genes. The central role played by ROS/HIF1α activation, was proved by the ability of NAC to prevent curcumin-induced PPARγ transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ), and consequently the effects on its target genes ELOVL5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK\u003cb\u003e)\u003c/b\u003e and SLC1A5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe transcriptional activity of PPARs is finely regulated by co-activators and co-repressors, which modulate signaling and interaction with the basal transcription machinery [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the absence of ligands (ligand-independent repression), PPARs bind the promoters of their target genes and repress transcription by recruiting co-repressors (e.g., NCoR and SMRT). Upon ligand activation (ligand-dependent transactivation), PPARs undergo conformational changes that provoke the displacement of co-repressors and recruitment of co-activators such as p300/CBP and p160, activating transcription [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Similarly to our data, previous studies showed that some of the effects of curcumin rely on PPARγ activation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. To assess the binding affinity and interaction profile of curcumin toward PPARγ, molecular docking simulations were performed on the crystallographic structure of the receptor. Given the availability on the Protein Data Bank (PDB) of several crystallographic structures of PPARγ, we selected the entry with the code 7AWC, one of the most recently deposited structures, which corresponds to the ligand-binding domain of the receptor. In this structure the protein is co-crystallized with rosiglitazone, a well-established PPARγ agonist approved by the Food and Drug Administration for the treatment of type II diabetes [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Firstly, rosiglitazone was re-docked into the receptor\u0026rsquo;s ligand binding site, resulting in a predicted binding energy (BE) of -7.8 kcal/mol, which was taken as a reference for the following docking analyses. Next, we performed molecular docking of curcumin, GW9662, a selective and potent PPARγ antagonist, and docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid an endogenous PPARγ ligand [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAll ligands occupied the same ligand-binding pocket (\u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e), and notably curcumin exhibited the most favourable binding score (BE = -8.6 kcal/mol), outperforming all the other ligands (BE= -8.2 and \u0026minus;\u0026thinsp;7.5 kcal/mol for GW9662 and DHA, respectively). These findings are consistent with previously reported studies [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Despite the absence of hydrogen bonding with Tyr473, a key residue involved in full agonist activity and engaged by rosiglitazone, curcumin established an extensive network of hydrophobic interactions with key residues such as Arg288, Ile326, Tyr327, Leu330, Ile341, and Phe363, many of which are also involved in stabilizing classical PPARγ ligands. Interestingly, curcumin shares a similar interaction pattern with GW9662, relying primarily on hydrophobic contacts within the same residues, with the addition of an extra interaction involving Ile326. Conversely, DHA, although capable of forming hydrogen bonds with Tyr327 and Tyr473, exhibited a less extensive hydrophobic network, as reflected in its lower binding affinity (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eTaken together, these findings underscore the strong binding affinity of curcumin and its ability to interact with residues that are crucial for the binding of known ligands, providing compelling structural evidence for its modulatory role on PPARγ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eThe histone demethylase KDM4B is the master regulator of curcumin/PPARγ-mediated lipid metabolism gene expression\u003c/h2\u003e\u003cp\u003eActivation of selective gene expression in response to curcumin treatment led us to hypothesize the involvement of a ROS-mediated epigenetic regulation. ROS, are known to increase the expression of several member of the Jumanji family of histone demethylase (KDMs) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Members of this family enhance transcription by demethylating specific lysine residues of histone H3. We focused our attention on KDM4B, which was shown to increase glucose metabolism in colorectal cancer [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], similarly to what we observed following curcumin treatment. Additionally, KDM4B is under the transcriptional control of HIF1α [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], upregulated by curcumin.\u003c/p\u003e\u003cp\u003eImportantly, high expression levels of KDM4B is associated with decreased overall survival in ACC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To assess whether the addition of a second variable (PPARγ or ELOVL5) improved the fitness of the univariate model (KDM4B), we performed likelihood-ratio tests comparing the univariate model (EZH2 only) and each of the bivariate models. The addition of a second variable (PPARγ or ELOVL5), improved the fitness of the univariate model, suggesting it provided further information relevant for overall survival. To visualize this effect, Kaplan-Meier curves were obtained dividing samples into four groups, based on the median expression of KDM4B and PPARγ or ELOVL5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). Log-rank tests of each pair-wise comparison were performed. As demonstrated in the Kaplan-Meier curves, the addition further stratification according to the expression of PPARγ or ELOVL5, can further improve risk stratification by identifying a subgroup of KDM4B-high patients with very high risk.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of KDM4B mRNA evidenced curcumin ability to increase its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), that was prevented by NAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We tested the effect of curcumin in combination with KDM4B inhibitor, ML324, on the expression of two PPARγ target genes modulated by curcumin. ML324 abrogated curcumin effects on ELOVL5 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), while no influence was seen for SLC1A5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The combination of the two drugs was tested on cell growth using concentrations of the single agents that inhibited by no more than 25%. The combined treatment produced a more potent inhibitory effect on both H295R and MUC-1(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Importantly, we evidenced the combined treatment was also effective on H295R tumoroids, that appeared smaller in diameter, more compact and with a reduced translucency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eChanges in metabolic pathways and fluctuations in intermediate metabolites transmit information about the intracellular metabolic status to the nucleus by regulating the activity of epigenetic enzymes. This modulation reshapes the epigenetic landscape, thereby eliciting transcriptional responses tailored to diverse metabolic demands. The interplay between cell metabolism and epigenome becomes particularly relevant in the context of cancer therapy, when cancer cells adapt their metabolism to overcome drug-induced cell death.\u003c/p\u003e\u003cp\u003eRecently, we and others have recently proved curcumin effectiveness in reducing ACC cell growth [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. We demonstrated that curcumin caused a metabolic rewiring, characterized by increased glutamine (Gln) metabolism, and lowering glutamine availability potentiated curcumin inhibitory effects. The current study demonstrates that glutamine is used to sustain a 20-fold increase in the content of glutathione (GSH), the most powerful endogenous antioxidant. To sustain glutathione synthesis, we observed that curcumin caused a transcriptional up-regulation of SLC7A11 and SLC6A9, cell membrane carriers for cystine and glycine (Gly), respectively. Cystine is used by cells for the synthesis of cysteine (Cys), which together with Glu and Gly are incorporated in the tripeptide GSH by glutathione synthase (GSS), also upregulated by curcumin. Glutathione exists in the reduced GSH form and oxidized GSSG form. The protocol we used evaluated the reduced form only, it is then possible that the cell amount of glutathione could be even higher than what we found.\u003c/p\u003e\u003cp\u003eCurcumin-induced antioxidant response was previously reported for other cell types [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], however, several studies indicate that curcumin-dependent anticancer mechanisms are mediated by the induction of oxidative stress [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Indeed, ROS levels were increased upon curcumin exposure, then, ACC cells activate an antioxidant response to counteract the pro-oxidant effects exerted by the polyphenol, with GSH being used to counteract ROS-mediated damages, and in fact, no lipid peroxidation was observed. We need to underline that the doses of curcumin used in the current study are not associated with activation of apoptosis, which instead is executed at the dose of 40 \u0026micro;M as shown in our previous report[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The discovered antioxidant mechanism could help explaining the limited cell death-inducing ability of the lower doses of curcumin.\u003c/p\u003e\u003cp\u003eMUC-1 cells used as multidrug resistant ACC phenotype [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] show high levels of TAG-containing LDs compared to the drug-sensitive H295R cells, which instead preferentially accumulate cholesterol esters-containing LDs [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. We recently demonstrated that an increase in TAG-containing LDs occurs in ACC cells treated with tazemetostat, an inhibitor for the methyl transferase EZH2, which similarly to curcumin causes oxidative stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Lipid droplets act as antioxidant organelles that control polyunsaturated fatty acids storage in triglycerides in order to reduce membrane lipid peroxidation, preserving organelle function. This defense mechanism would reduce cellular stress, favoring cancer cell survival and therapy resistance [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Accordingly, evaluation of LDs amount in ACC models treated with curcumin evidenced a remarkable increase. Moreover, curcumin specifically increased TAG and PUFA content.\u003c/p\u003e\u003cp\u003eThe increase in LDs depends on a reduced metabolism as demonstrated by decreased expression of ATGL, the lipase localized on LDs surface and involved in the TAG degradation, and LIPE, responsible for DAG and cholesterol esters degradation. We further evidenced an increased expression of G0S2, which prevents any residual ATGL activity. FA synthesis was not involved in LDs accumulation since ACACA and FASN, and their transcription factor SREBP1 (sterol-responsive element binding protein-1) were downregulated. Genes involved in FA uptake, LDLR and CD36, and their transcription factors SREBP1 and SREBP2[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], were downregulated, instead, FA transporter SLC27A2 and its transcription factor HIF1α were significantly upregulated. HIF1α is known to be responsive to hypoxic conditions and to increased ROS production in both hypoxia and normoxia [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The use on the ROS scavenger N-acetyl-cysteine by neutralizing curcumin-induced ROS, prevented HIF1α upregulation, and consequently, transcription of its target gene, SLC27A2, and LDs formation. The SLC27A2 gene encodes for proteins known as FATP2 that was shown to preferentially mediate cell entry of ω3-PUFA [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Dietary PUFA drastically decrease the mature, cleaved form, of SREBP-1 protein, consequently less lipogenic enzymes are transcribed [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eCollectively these data draw an adaptive metabolic response which favors an enhanced LDs formation through the ROS/HIF1α/SLC27A2 axis, used by ACC cells to prevent the detrimental effects exerted by curcumin-mediated ROS production.\u003c/p\u003e\u003cp\u003eEvaluation of mRNA for FA modifying enzymes such as elongase (ELOVL1 and ELOVL5) and desaturase (SCD1, FADS2) evidenced preferential FA modifications. ELOVL1 and 5 are targets for the transcription factor PPARγ, a member of the nuclear receptor family of metabolic TFs [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], that we evidenced to be upregulated by curcumin. Notably, HIF1α regulates PPARγ transcription [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and accordingly the use of NAC prevented activation of its transcription, and consequently of its targets, in curcumin-treated ACC cells. PPARγ is a TF also for ATGL and LIPE, which however, we evidenced to be downregulated by curcumin. These divergent effects could be explained by changes in the cofactors recruited by PPARγ and/or by differences in the localization of PPRE sites within the 5\u0026rsquo;UTR (untranslated region) on target genes, allowing ligand-dependent transcriptional activation or repression. An intriguing possibility is a direct interaction between PPARγ and curcumin, which can cause a change in the protein 3D structure and allow interaction with specific co-factors. This possibility was screened by docking analysis which proved a strong interaction between curcumin and PPARγ, similar to known PPAR ligands. This observation deserves further investigations through additional experiments as PPAR/curcumin interaction still represents a debatable question [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor example, PPARγ was found to associate with G protein suppressor 2 (GPS2), a TFs interacting protein, and regulate transcription of a subset of PPARγ target genes. Specifically, GPS2 acted as a priming factor for PPARγ recruitment to ATGL and LIPE and stabilized interaction with the histone demethylase KDM4A/JMJD2 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Additionally, PPARγ/GPS2 binding was stronger at promoter regions than at enhancers or other genomic locations [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHistone lysine demethylases (KDMs) play important roles in chromatin remodeling and gene regulation by catalyzing the demethylation of methylated lysine residues on the N-terminal tails of histones. The histone demethylase activity of KDM4s requires α-ketoglutarate (α-KG), O\u003csub\u003e2\u003c/sub\u003e, and Fe\u003csup\u003e2+\u003c/sup\u003e, to catalyze the demethylation of tri- and di-methylated forms of both histone H3 lysine 9 (H3K9me3/me2) and lysine 36 (H3K36me3/me2) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is worth highlighting that curcumin causes an increase in glutamine [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] that could also be metabolized into α-KG, pointing to the idea of an interplay between cell metabolism and epigenetic regulation.\u003c/p\u003e\u003cp\u003eThis intriguing possibility was proved by demonstrating that curcumin increased the expression of KDM4B and the use of its inhibitor, ML324, at a concentration that did only minimally affect cell proliferation, potentiated curcumin anti-proliferative effects. This was evidenced in vitro by MTT assay and ex-vivo using tumoroids derived from H295R xenografts and grown in a bioreactor.\u003c/p\u003e\u003cp\u003eKDM4B is under HIF1α transcriptional regulation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and the use of NAC, by preventing HIF1α activation, abrogated KDM4B transcription. To ascertain the role of KDM4B in the epigenetic regulation of ELOVL5 in response to curcumin, the polyphenol was combined with ML324 and QPCR analysis evidenced the loss of curcumin-induced up-regulation. On the contrary, expression of PPARγ evaluated in the same experimental settings was unaffected. These data underline that the epigenetic modulation in response to metabolic adaptation is the result of a fine tuning in expression and activity of epigenetic modifiers. Indeed, a cluster of cluster (COC) analysis of ACC-TCGA data identified three molecular ACC subtypes COC1, COC2, and COC3, which, on an epigenetic level, respectively bear low, intermediate and high levels of genome-wide CpG island methylation [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Of the three COC clusters, COC3 shows the higher disease progression rate, highlighting a tight connection between the worst clinical outcome profound epigenetic modifications.\u003c/p\u003e\u003cp\u003eIn conclusion, our study further proves curcumin to be a promising molecule for the treatment of ACC. Having more in detail dissected curcumin effects on metabolic and epigenetic genes we demonstrated a more potent effect when combined with KDM4B inhibitors. Importantly, suppressing a broad spectrum of epigenetic enzymes can cause potential deleterious side effects, while the use of specific KDM4B inhibitors could help overcoming this issue.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCOMPETING INTERESTS\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAUTHOR CONTRIBUTIONS\u003c/h2\u003e\u003cp\u003eIC and RS conceived and designed this project; MCN, AA, AC and MGS conducted the experiment and analyzed results; FM and CM performed and analyzed metabolomic studies; FG and MAO performed docking analysis; TAH performed patients\u0026rsquo; databases analyses; CH, CI, MFS, and ML contributed reagents/analytic tools; CH, MFS, CI, SG, ML, VP, IC, and RS participated in data analysis and results discussion; IC and RS wrote the paper.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e\u003cp\u003eThis work was supported by AIRC\u0026mdash;Fondazione AIRC per la Ricerca sul Cancro ETS Investigator Grant IG30896 to RS.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNocito MC, De Luca A, Prestia F, Avena P, La Padula D, Zavaglia L, et al. 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Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC2717748/\u003c/li\u003e\n\u003cli\u003eCardamone MD, Tanasa B, Chan M, Cederquist CT, Andricovich J, Rosenfeld MG, et al. GPS2/KDM4A Pioneering Activity Regulates Promoter-Specific Recruitment of PPAR\u0026gamma;. Cell Rep. 2014;8:163\u0026ndash;76. \u003c/li\u003e\n\u003cli\u003eMohan DR, Lerario AM, Finco I, Hammer GD. New strategies for applying targeted therapies to adrenocortical carcinoma. Curr Opin Endocr Metab Res. 2019;8:72\u0026ndash;9. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Curcumin, ROS, Epigenetic, KDM4B, PPARG, Lipid metabolism","lastPublishedDoi":"10.21203/rs.3.rs-7328223/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7328223/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCancer therapies often cause significant side effects, driving the search for more tolerable and effective treatments. Natural products, such as the polyphenol curcumin, exhibit promising anticancer properties by modulating cell death pathways. We demonstrated that curcumin inhibits growth and migration of adrenocortical cancer (ACC) cells, a rare tumor with no available targeted therapy. In these cells curcumin caused a shift in cell metabolism toward a glycolytic phenotype and an increased glutamine/glutamate dependency. However, why cells adapt their metabolism promoting glutamate synthesis remains unclear. Curcumin acts as a pro-oxidant in ACC cells, elevating reactive oxygen species (ROS) and, consequently, cells adopt a defense mechanism increasing glutathione, a glutamate-containing antioxidant, and lipid droplet (LD) content, preventing oxidative stress. Key genes in this metabolic adaptation are SLC1A5, SLC27A2, ELOVL5, whose expression is positively regulated by curcumin and abrogated by the ROS scavenger N-acetyl-cysteine (NAC). Mechanistically, we evidenced that curcumin-induced ROS increase transcription factor HIF1α, which drives the upregulation of nuclear receptor PPARγ and the Jumonji histone demethylases (KDMs) family member KDM4B, affecting ACC cell metabolism and epigenome. The use of a KDM4B inhibitor potentiated curcumin anti-proliferative effects in vitro and, more importantly, on ex vivo cultures of H295R xenografts tissue grown in a 3D bioreactor. Collectively these data prove ACC cells\u0026rsquo; ability to change their metabolism to counteract curcumin-induced oxidative stress through epigenetic modifications. The identification of KDM4B as the master regulator behind the changes provides novel insights into combinatorial strategies for improving ACC patients\u0026rsquo; outcome.\u003c/p\u003e","manuscriptTitle":"Hijacking the expression of KDM4B-dependent metabolic genes potentiates curcumin antitumor effects","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 11:32:55","doi":"10.21203/rs.3.rs-7328223/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32422b5f-e086-4b8f-b950-adf613c1140b","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54399844,"name":"Health sciences/Diseases/Cancer/Cancer metabolism"},{"id":54399845,"name":"Biological sciences/Cancer/Cancer therapy/Targeted therapies"}],"tags":[],"updatedAt":"2025-09-09T11:32:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 11:32:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7328223","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7328223","identity":"rs-7328223","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}