Society for Endocrinology
Lipid synthesis seems to drive proliferation in Men1 mouse adrenals and human adrenocortical cell lines
Adam Edholm1, Su-Chen Li1, Xia Chu1, Hanna Wargelius1, Masoud Razmara2, Anna Widgren3, S J Kumari A Ubhayasekera3, Jonas Bergquist3, Peter Stålberg4, Azita Monazzam 01,* and Britt Skogseid1,*
1Department of Medical Sciences, Uppsala University, Uppsala, Sweden
2Department of Clinical Pathology, Akademiska University Hospital, Uppsala, Sweden
3Department of Chemistry - BMC, Analytical Chemistry and Neurochemistry, Uppsala University, Uppsala, Sweden
4Department of Surgical Sciences, Uppsala University, Uppsala, Sweden
Correspondence should be addressed to A Monazzam: azita.monazzam@medsci.uu.se
*(A Monazzam and B Skogseid contributed equally to this work)
Abstract
Adrenocortical carcinoma (ACC) is a devastating disease with few effective treatments. The underlying molecular pathways remain largely unknown. To identify potential pathways and drivers relevant to ACC pathogenesis, we utilized histologically normal adrenals from heterozygous multiple endocrine neoplasia type 1 (Men1) mice to study early adrenocortical tumorigenesis. Employing mass spectrometry-based proteomic profiling, we identified 681 proteins, of which 52 displayed significant differential regulation in the adrenal tissues of heterozygous Men1 mice in comparison with their wild-type counterparts. Among these were fatty acid synthase (FASN) and ATP-citrate lyase (ACLY), two enzymes previously shown to be upregulated in several other types of tumors. To assess the functional impact of ACLY and FASN in ACC, we used H295R cells as the primary model. Cells were treated with SB-204990 (ACLY inhibitor) or C75 (FASN inhibitor), which both showed a dose-dependent antiproliferative effect. Lipidomic analysis revealed a significant reduction in palmitic acid and palmitoleic acid in treated cells compared to controls, supporting a mechanistic link between ACLY/FASN activity and lipid biosynthesis. Finally, data from The Cancer Genome Atlas showed significantly diminished survival outcomes among ACC patients exhibiting high ACLY or FASN expression. These findings underscore the potential importance of exploring the inhibition of lipid synthesis as a promising avenue for further research in the context of human ACC.
Keywords: MEN1; adrenocortical carcinoma; ACLY; FASN
Introduction
Multiple endocrine neoplasia type 1 (MEN1) is a rare, autosomal dominant inherited condition, which is characterized by tumors in endocrine organs, primarily the parathyroid, pituitary, and pancreas (1). One-third of
gene carriers have adrenocortical enlargements, usually without accompanying hormone overproduction (2). These adrenal glands may develop adenomas and, in rare cases, even adrenocortical carcinoma (ACC) (3).
For nearly two decades, both conventional and conditional Men1 knockout mice models, with close phenotypic resemblance to the human disease, have been available for researchers (4, 5). The benign MEN1 adrenal lesions retain the wild-type allele of the MEN1 gene (2), and its protein menin (6) is expressed to what seems to be the same level and subcellular localization as in normal adrenals in both MEN1 patients and Men1 knockout mice (7).
ACC is an ultra-rare malignancy characterized by a dismal prognosis and a scarcity of effective treatments. While significant progress has been made in studying genetic alterations and molecular markers in sporadic human ACC, such as the loss of heterozygosity (LOH) of the MEN1 locus (8, 9) and the identification of several driver genes, for example, ZNRF3, PRKAR1A, TP53, and CTNNB1 (10, 11), this has not yet translated into improved treatments or clinical outcome. Consequently, there is an urgent and unmet need for identifying new therapeutic targets in ACC. There is still a knowledge gap regarding early events in adrenocortical transformation, and identifying these could represent a promising avenue to identify pathways and molecular changes of relevance in ACC. A complicating factor for this research is the relative paucity of representative pre-neoplastic models for the adrenal cortex, although recent efforts have resulted in novel cell lines (12). The MEN1-syndrome offers a unique window of opportunity to study this pre-neoplastic phase in the adrenals. This trait is one of the few associated with an increased prevalence of ACC and also provides an established pre-clinical mouse model. To elucidate the specific proteins and pathways affected in early MEN1 adrenal tumorigenesis, we use a Men1 knockout (Men1+/-) mouse model. In a previously published study, we investigated miRNA expression in histologically normal adrenals from Men1+/- mice compared to adrenals from wild-type (Men1+/+) mice (7); 31 miRNAs were differently expressed, some of which were known oncogene or tumor suppressor miRNAs.
In a parallel effort presented in this paper, we pursued mass spectrometry-based proteomic profiling and western blot on Men1+/- mice adrenals. As in the miRNA study, we chose to study the adrenals of adult mice aged 10-13 months, possibly before long-term proliferation results in further, Men1-unrelated, molecular alterations. In the present study, we compared protein expression patterns in Men1+/- vs Men1+/+ mice adrenals, identifying changes that might drive adrenal proliferation. After identifying differentially expressed proteins, we selected a subset for further investigation in a human context; to determine whether increased expression of these proteins confers proliferation advantages to human ACC cells, we studied the effects of inhibition of the selected proteins in human cell lines. Finally, we were also interested in exploring whether the level of expression of these proteins has any effect in patients
with sporadic ACC. The prognostic impact of increased expression of the chosen proteins in ACC was evaluated using data from The Cancer Genome Atlas (TCGA).
Materials and methods
Men1+/- mice
The Men1+/- mouse (C57BL/6J) is a conventional heterozygous Men1 knockout mouse, which was a kind gift from Professor Hayward of the Queensland Institute of Medical Research, Herston, Australia. The deletion of the second exon of the Men1 gene produces heterozygous non-sense mutation. Men1+/- mice at 9 months of age have developed lesions in the endocrine pancreas, parathyroid, and pituitary (13). Adrenal enlargement, sometimes bilateral, was observed in both male and female mice. The most frequent adrenal phenotype was enlargements of the cortex, but occasionally, the medulla also seemed to be affected (13). Hence, Men1+/- mice were utilized as a model to study early tumorigenesis in adrenals. All mice were housed in a barrier facility at Uppsala University with a 12 h light:12 h darkness cycle. The maintenance of animals and the experimental design were in agreement with both the Swedish animal protection legislation and European regulations and were approved by the animal ethics committee (Uppsala djurförsöksetiska nämnd) in Uppsala, Sweden, permit number: C187/14.
Adrenals
In the present study, adrenals of ten Men1+/- mice with a mean age of 10.3 months ± 0.95 (SD) months were used, and adrenals of ten Men1+/+ mice of the same age range served as controls. The sex distribution was balanced between groups (Men1+/ -: 4 males, 6 females; Men1+/ *: 6 males, 4 females). From each animal, one gland was subjected to protein profiling and the contralateral gland was either formalin-fixed/paraffin-embedded for immunohistochemical analysis or biobanked for future research. Morphology and proliferation were evaluated to exclude possible adrenocortical tumors that could interfere with the interpretation of the proteomic results.
Size, morphology, menin immunoreactivity, and Ki67 of the dissected adrenals were assessed. The size was assessed as the product of the length of the adrenal body long-axis and the maximum perpendicular width. For morphology and menin immunoreactivity, adrenal sections were deparaffinized, were rehydrated in alcohol, and underwent heat-mediated antigen retrieval in Tris-EDTA, pH 9.0. Endogenous peroxidase activity was blocked by incubating the slides with Peroxidazed 1 (Histolab, Sweden). After blocking non-specific staining
(Background Sniper, Histolab, Sweden), primary antibody menin dilution 1:400 was applied (Rabbit anti-menin, 1:800, Bethyl Laboratories, USA). Incubation with secondary antibody (Rabbit-on-Rodent HRP Polymer, Histolab, Sweden) was followed by chromogen Betazoid DAB staining. All sections were counterstained with Mayer’s hematoxylin, mounted, and evaluated under an Axioplan 2 light microscope, using an AxioCam HRm camera and AxioVision imaging software, version 4.8.1 (Carl Zeiss, Germany, Oberkochen).
For Ki67 immunohistochemistry, sections were deparaffinized, rehydrated, and epitope heat-retrieved in citrate buffer (pH 6.0) (Dako, Denmark, Glostrup). Sections were stained at room temperature with primary antibody Ki67 (Cell Signaling Technology, USA) dilution 1:200 for 1 h, followed by washing and incubation with peroxidase for 30 min. Slices were then stained with an EnVision kit (Dako, Denmark, Glostrup) to visualize the targeted proteins.
Protein preparation
Dissected adrenal glands, 10 from each genotype, were minced and homogenized; protein was extracted, denatured, reduced, and digested as previously described (14). In brief, the samples were homogenized in the presence of octyl-B-D-glucopyranoside, Tris-HCI, NaCl, and EDTA in PBS solution. A protease cocktail inhibitor (Sigma-Aldrich, Germany) was added during lysis. After incubation and centrifugation of the lysate, the clear supernatant was collected for mass spectrometry. The total protein content was determined using the DC Protein Assay Kit (BioRad Laboratories, USA) with bovine serum as standard.
For mass spectrometry analysis, 35 µg of protein were dissolved in 8 M urea and acetonitrile (ACN) (1:1) containing NH4HCO3 (50 mM). A volume of 10 uL of 45 mM DTT was added to the samples for incubation at 50℃ for 15 min. After cooling, 10 uL of 100 mM IAA were added to the samples for incubation at room temperature in a dark room. The samples were then transferred to 3 kDa spin filters (Pall Life Sciences, USA) for three steps of purification. A volume of 100 uL of 50 mM NH4HCO3 was added to the filter, followed by centrifugation at 14,000 g for 10 min. Progressively, 250 uL of 1:1 2% ACN/50 mM NH4HCO3 and 150 µL of 50 mM NH4HCO3 were added separately, and then the filter centrifugation as above followed. Spin filters were transferred to new vials, and 50 µL of 5% trypsin were applied for digestion at 37℃ overnight in dark room. Samples were centrifuged, and tryptic peptides were collected. Residual peptides on the filters were collected by applying 100 uL of 50% ACN and 1% HAc, and the eluate was pooled with the first filtrate. Eluted peptides were dried using the SpeedVac system ISS110 (Thermo Scientific, USA), and peptides were re-dissolved in 30 uL 0.1% formic acid (FA) before mass spectrometry analysis.
Liquid chromatography and tandem mass spectrometry (LS-MS/MS)
The nano-LC-MS/MS system EASY-nLC II nanoLC (Thermo Fisher Scientific, USA) and LTQ-Orbitrap Velos Pro EDT mass spectrometer were used. Five microliters of protein sample were injected into the EASY-nLC II nanoLC system for separation of the tryptic peptides in reversed phase on a C18-A2 column (Thermo Fisher Scientific, USA, 75 um, 10 cm) using mobile phase A = 0.1% FA, 99.9% ACN. Separation started with a slow gradient 4-50% B for 60 min, followed by a steep gradient to 80% B. The separated peptides were electrosprayed into the LTQ-Orbitrap Velos Pro ETD mass spectrometer, which was used for a high-resolution survey of mass spectrum (resolving power 100,000 FWHM). Tandem mass-spectrometry was performed with collision-induced dissociation.
Proteomic data analysis and gene ontology (GO)
The acquired data (RAW-files) were processed in MaxQuant, and database searches were performed using the implemented Andromeda search engine. MS/MS spectra were correlated to a FASTA database containing proteins from Mus Musculus extracted from the UniProt database. A decoy search database, including common contaminants and a reverse database, was used to estimate the identification false discovery rate (FDR). An FDR of 1% was accepted. The search parameters included maximum 10 ppm and 0.6 Da error tolerances for the survey scan and MS/MS analysis, respectively; enzyme specificity was trypsin; a maximum of one missed cleavage site was allowed; cysteine carbamidomethylation was set as static modification; and oxidation (M) was set as variable modification. The search criteria for protein identification were set to at least two matching peptides of 95% confidence level per protein. Label-free quantification was applied for comparative proteomics.
The results were exported into MS Excel (Microsoft, USA) for manual data interpretation and statistical analysis. The significantly regulated proteins in the dataset were pin-pointed by a search rule stating that the protein had to be identified in all samples that it should show a significant difference (P < 0.05 according to Welch’s unequal variances t-test) in a comparison between the compared groups and also a significant change (P < 0.05 according to paired t-test) in the investigated groups. To establish the rank lists, we used the ratio between the Men1+/- and Men1+/+ samples.
For the interpretation of the proteomic result, we performed enrichment analysis for GO by using the STRING protein-protein interaction database (https://string-db.org).
| No. protein name | Ratio | P-value | Regulation |
|---|---|---|---|
| 1 Pyruvate carboxylase | 1.09 | 9.9E-20 | Up |
| 2 ATP citrate lyase (ACLY) | 1.34 | 9.9E-20 | Up |
| 3 Fatty acid synthase (FASN) | 1.67 | 9.9E-20 | Up |
| 4 Aldose reductase-related protein 1 | 1.55 | 9.9E-20 | Up |
| 5 Alcohol dehydrogenase 1 | 1.76 | 9.9E-20 | Up |
| 6 Microsomal glutathione S-transferase 1 | 2.00 | 2.2E-16 | Up |
| 7 Steroid 21-hydroxylase | 1.59 | 5.8E-13 | Up |
| 8 cAMP-dependent protein kinase catalytic subunit a | 1.42 | 7.4E-13 | Up |
| 9 Aldose reductase-related protein 2 | 1.41 | 9.7E-13 | Up |
| 10 Aldehyde dehydrogenase | 1.45 | 1.9E-11 | Up |
| 11 Keratin, type I cytoskeletal 28 | 1.51 | 1.0E-09 | Up |
| 12 ATP synthase subunit O | 1.56 | 1.4E-09 | Up |
| 13 60 kDa heat shock protein | 1.21 | 3.7E-08 | Up |
| 14 Short-chain specific acyl-CoA dehydrogenase | 1.70 | 9.3E-08 | Up |
| 15 E3 ubiquitin protein ligase UBR4 | 1.70 | 4.0E-07 | Up |
| 16 Galactose-3-O-sulfotransferase 3 | 2.80 | 6.1E-07 | Up |
| 17 Ribosome-binding protein 1 | 1.15 | 2.5E-06 | Up |
| 18 Aldose reductase | 1.40 | 9.2E-06 | Up |
| 19 Neutral cholesterol ester hydrolase 1 | 1.12 | 2.8E-05 | Up |
| 20 Apoptosis-inducing factor 1 | 1.33 | 4.1E-05 | Up |
| 21 Elongation factor 1 alpha 1 | 1.58 | 9.1E-05 | Up |
| 22 Fatty acid-binding protein | 2.10 | 2.0E-04 | Up |
| 23 Keratin, type I cuticular Ha3-II | 2.14 | 2.3E-04 | Up |
| 24 Glycerol-3-phosphate dehydrogenase | 1.15 | 2.4E-04 | Up |
| 25 DNA repair endonuclease XPF | 1.29 | 4.2E-04 | Up |
| 26 Alcohol dehydrogenase class 3 | 2.02 | 5.9E-04 | Up |
| 27 Keratin, type II cuticular Hb6 | 2.14 | 0.001 | Up |
| 28 Keratin, type II cytoskeletal 8 | 1.47 | 0.002 | Up |
| 29 Keratin, type I cytoskeletal 13 | 1.22 | 0.003 | Up |
| 30 3-beta-hydroxysteroid dehydrogenase | 1.26 | 0.005 | Up |
| 31 Succinyl-CoA ligase | 1.43 | 0.005 | Up |
| 32 S-formylglutathione hydrolase | 1.80 | 0.006 | Up |
| 33 Leukotriene A4 hydrolase | 1.47 | 0.008 | Up |
| 34 Aspartate-tRNA ligase | 0.99 | 0.010 | Down |
| 35 Nucleoside diphosphate kinase B | 0.75 | 0.011 | Down |
| 36 Coatomer subunit 8 | 1.35 | 0.014 | Up |
| 37 60S ribosomal protein L12 | 1.25 | 0.015 | Up |
| 38 Citrate synthase | 1.15 | 0.017 | Up |
| 39 Low affinity immunoglobulin epsilon Fc receptor | 1.32 | 0.017 | Up |
| 40 Keratin, type II cytoskeletal 2 oral | 1.20 | 0.017 | Up |
| 41 Adrenodoxin | 0.84 | 0.019 | Down |
| 42 6-phosphogluconate dehydrogenase | 1.20 | 0.020 | Up |
| 43 Protein transport protein Sec61 subunit ß | 1.05 | 0.022 | Up |
| 44 3-oxo-5ß-steroid 4-dehydrogenase | 1.44 | 0.025 | Up |
| 45 Hydrocephalus-inducing protein | 1.08 | 0.025 | Up |
| 46 Prohibitin 2 | 1.26 | 0.028 | Up |
| 47 Transmembrane protein 20 | 1.31 | 0.031 | Up |
| 48 Glutathione S-transferase kappa 1 | 1.35 | 0.032 | Up |
| 49 Histidine triad nucleotide-binding protein 1 | 1.77 | 0.036 | Up |
| 50 Thioredoxin-dependent peroxide reductase (PRDX3) | 1.44 | 0.043 | Up |
| 51 Reticulon 4 | 1.78 | 0.045 | Up |
| 52 NADH-ubiquinone oxidoreductase 75 kDa subunit | 1.21 | 0.049 | Up |
Based upon the proteomic results, we selected ATP citrate lyase (ACLY) and fatty acid synthase (FASN) for further evaluation. These proteins ranked among the most significantly upregulated hits in our dataset (2nd and
3rd, respectively; Table 1, Fig. 1). Moreover, the availability of pharmacological inhibitors against ACLY and FASN strengthened the rationale for their selection in our further investigations.
20
PC
AKR1BZ
FASN
Up
ACLY
ADH1
Down
NS
MGST1
15.
AKR1B8 CYP21A1
-log10 p-value
PRKACA
ALDH1A1
10
KRT28
ATP50
HSPD1
ACADS
UBR4
GAL3ST3
· RRBP1
5
AKR1B1
NCEHAIFM1
0
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
log2 fold change
Adrenocortical cancer cell line
The human ACC cell line H295R was used as the primary in vitro model. SW13 cells, which have historically been used in ACC research but have limited clinical relevance, were included only for comparison and are presented in the Supplementary Data (see section on Supplementary materials given at the end of the article). Both cell lines, H295R and SW13 (ATCC, USA), were maintained in a standard humidified incubator at 37°℃ in a 5% CO2 atmosphere. H295R cells were cultured in Dulbecco’s modified Eagle’s medium/Ham F12, supplemented with 1% ITS liquid media supplement, 100 units/mL of penicillin, 100 µg/mL streptomycin, and 2% Nu-serum. Sw13 cells were cultured in Dulbecco’s modified Eagle’s medium/Ham F12, supplemented with 100 units/mL of penicillin, 100 µg/mL streptomycin, and 10% fetal bovine serum. All reagents were purchased from Thermo Fisher Scientific (USA).
Inhibition of ACLY and FASN in ACC cell line
H295R cells were plated at an initial seeding density of 50,000 cells per well in 24-well plates. After 72 h, the medium was replaced with a complete culture medium containing either the ACLY inhibitor SB-204990 (Tocris, UK; 0, 15, 30, or 60 µM) or the FASN inhibitor C75 (Sigma-Aldrich, Sweden; 0, 1, 4, or 16 uM). Concentrations were selected based on prior studies in
cancer cell lines (3, 15) and on preliminary toxicity experiments in H295R and SW13 cells. From these experiments, approximate IC50 values were obtained (Supplementary Fig. 1), confirming effective but non- toxic ranges. The reported IC50 values in the literature are 10-30 µM for SB-204990 (16) and 35-200 uM for C75 depending on the assay system (Selleckchem; Abcam, UK; (17)). The concentrations applied in this study were therefore well below these reported values, supporting specificity of the inhibitory effects.
The incubation medium was renewed daily, and cells were harvested by trypsinization for counting after 1, 2, 3, 4, or 5 days of treatment. Cell numbers were determined using a NucleoCounter® NC-100™M (Chemometec, Denmark). The experiment was repeated three times independently for each concentration and time point.
The experiment was repeated three times independently for each concentration and time point.
Parallel experiments were also performed in SW13 cells for comparison; these data are presented only in the Supplementary Figures as SW13 is not recognized to be of adrenocortical origin.
Cell viability assay
In addition to proliferation experiments, cell viability was assessed using a BioRad TC20 automated cell counter.
Both adherent and non-adherent cells were collected after treatment. However, the viability of adherent cells is reported, as this most directly reflects treatment effects on proliferation.
Lipidomic analysis
Free fatty acids
For lipidomic profiling, H295R cells were seeded into 10 cm culture dishes at a density of 1.25 x 105 cells/dish. After 48 h, cultures were treated for 4 days with vehicle control, 15 uM SB-204990 (ACLY inhibitor), or 1 µM C75 (FASN inhibitor). The medium was renewed daily. At the end of treatment, cells were trypsinized and counted; 2 x 106 cells per condition were washed with cold PBS, dissolved in 500 uL of isopropanol, and transferred to DURAN tubes. Lipid extraction was performed with a solvent mixture of isopropanol/heptane/1 M HCl (40:10:1, v/v/v), including internal standards (C9:0, C17:0, and C23:0) and butylated hydroxytoluene (0.04 mg/mL) to prevent oxidation. After equilibration, 1 mL water was added, and lipids were extracted into 2 mL heptane. The heptane phase was evaporated under nitrogen, and lipids were re-dissolved in 100 µL of a hexane-methanol mixture before storage at -80℃. Analyses were performed using ultra-performance supercritical fluid chromatography tandem mass spectrometry.
Lipidomic experiments were also performed in parallel for SW13 cells but are presented only in the Supplementary Data.
Identification and quantification of free fatty acids
An Ultra-Performance Convergence Supercritical Chromatography (UPC2) system, coupled with tandem mass spectrometry, was employed (Waters ACQUITY® UPC2TM and XEVOR TQ-S, respectively, both from Waters, USA). A specialized Acquity UPC2 HSS C18 SB column (100 mm × 3.0 mm, 1.8 um) was used at a temperature of 40℃. Mobile phase A consisted of 99.99% pure compressed CO2, while mobile phase B was comprised of methanol with 0.1% FA. The mobile phase was delivered at a flow rate of 0.8 mL/min, with a gradient starting at 98% A for 5 min, transitioning to 96% A over 8 min, and finally reaching 80% A in 8 additional minutes. The total analytical runtime was 9 min, and the backpressure was maintained at 1,900 psi. The make-up flow of 0.2 mL of methanol in 0.1% NH4OH FA was employed. The mass spectrometry analysis was conducted using negative ion electrospray ionization. Quantification of free fatty acids (FFA) involved the use of appropriate internal standards based on the carbon chain length of the FFA. Each sample was analyzed in triplicate, and the average values were reported with a
coefficient of variation (CV) less than 5%. Identification of FFA compounds was carried out using multiple reaction monitoring with authentic FFA standards. The experiment was replicated five times.
Proteomic data analysis
Data visualization was performed using GraphPad Prism, version 10.6.0. A volcano plot was generated by plotting log2 fold change against -log10 P-value (Student’s t-test). Proteins uniquely detected in one genotype (present in ≥3 biological replicates of one group and none in the other) were recorded separately but not included in the statistical analysis to avoid potential bias from low- abundance or sporadically detected proteins.
The Cancer Genome Atlas (TCGA)
Public clinical and RNA-Seq data from ACC patients were obtained from TCGA data accessible via the NCI Genomic Data Commons (GDC) data portal, GDC Legacy Archive (https://portal.gdc.cancer.gov/). There were 77 ACC patients with available mRNA expression and survival data. For these 77 patients, information on disease stage and gender was also retrieved.
Statistical analysis
All calculations were performed using GraphPad Prism, version 10 (GraphPad Software, USA). All values are expressed as mean + SEM. Probabilities (P) of chance differences between groups in cell growth and lipidomic experiments were calculated using two-way ANOVA. Post hoc analysis was performed using Fisher’s least significant difference (LSD) test (uncorrected), selected for its sensitivity in detecting differences in small experimental groups. Differences at the 95% confidence level (P < 0.05) were considered significant.
For TCGA data, a univariate survival analysis for ACLY and FASN gene expression as a prognostic variable on overall survival was performed according to the Kaplan-Meier method. The terminal event was death, and the data were censored when it was unknown what happened to the subject. We aimed to investigate whether it was possible to identify groups where high and low expression of ACLY/FASN correlated with a significant difference in survival. We identified a cutoff for both ACLY and FASN expression, in both cases close to the median, where high expression correlated with a shorter survival. The statistical significance of the differences in survival distribution between the groups was evaluated by the log-rank Mantel-Cox test. P-values of 0.05 were regarded as statistically significant in two-tailed tests.
| #Pathway ID | Pathway description | Observed gene count | False discovery rate | Matching proteins in your network (labels) |
|---|---|---|---|---|
| GO.1901564 | Organonitrogen compound metabolic process | 17 | 0.000 | ACADSB, ACLY, AKR1B1, EEF1A1, FASN, GAL3ST3, GPD1, GSTK1, HINT1, LTA4H, NDUFS1, PC, PGD, RPL12, RRBP1, SEC61B, SUCLA2 |
| GO.0010033 | Response to organic substance | 15 | 0.018 | ADH5, AKR1B1, ALDH3A1, AVP, EEF1A1, FASN, GPD1, HSPD1, KRT13, KRT8, LTA4H, PRDX3, PRKACA, RTN4, SEC61B |
| GO.0055114 | Oxidation-reduction process | 13 | 0.000 | ACADSB, ACLY, AKR1B1, AKR1D1, ALDH3A1, CS, GSTK1, MGST1, NDUFS1, PGD, PRDX3, PRKACA, SUCLA2 |
| GO.0009056 | Catabolic process | 13 | 0.013 | ACADSB, ADH5, AKR1D1, ESD, FABP2, GPD1, HINT1, LTA4H, PRDX3, PRKACA, RPL12, SEC61B, UBR4 |
| GO.0044248 | Cellular catabolic process | 12 | 0.011 | ACADSB, ADH5, AKR1D1, ESD, FABP2, HINT1, LTA4H, PRDX3, PRKACA, RPL12, SEC61B, UBR4 |
| GO.1901575 | Organic substance catabolic process | 12 | 0.015 | ACADSB, ADH5, AKR1D1, ESD, FABP2, GPD1, HINT1, LTA4H, PRKACA, RPL12, SEC61B, UBR4 |
| GO.1901700 | Response to oxygen- containing compound | 11 | 0.019 | ADH5, AKR1B1, ALDH3A1, AVP, GPD1, HSPD1, KRT13, KRT8, LTA4H, PRDX3, PRKACA |
| GO.0006629 | Lipid metabolic process | 10 | 0.018 | ACADSB, ADH5, AKR1B1, AKR1D1, FABP2, FASN, GPD1, LTA4H, PC, PRKACA |
| GO.0043603 | Cellular amide metabolic process | 9 | 0.004 | EEF1A1, FASN, GSTK1, LTA4H, MGST1, PC, RPL12, RRBP1, SEC61B |
| GO.0044712 | Single-organism catabolic process | 9 | 0.024 | ACADSB, ADH5, AKR1D1, ESD, FABP2, HINT1, PRDX3, PRKACA, SEC61B |
| GO.0006091 | Generation of precursor metabolites and energy | 8 | 0.004 | ACLY, AVP, CS, FASN, GPD1, NDUFS1, PRKACA, SUCLA2 |
| GO.0015980 | Energy derivation by oxidation of organic compounds | 7 | 0.005 | ACLY, CS, FASN, GPD1, NDUFS1, PRKACA, SUCLA2 |
| GO.0006518 | Peptide metabolic process | 7 | 0.018 | EEF1A1, GSTK1, LTA4H, MGST1, RPL12, RRBP1, SEC61B AKR1B1, GAL3ST3, GPD1, PC, PGD, PRKACA |
| GO.0005996 | Monosaccharide metabolic process | 6 | 0.005 | |
| GO.0046364 | Monosaccharide biosynthetic process | 5 | 0.001 | AKR1B1, GPD1, PC, PGD, PRKACA |
| GO.0006641 | Triglyceride metabolic process | 5 | 0.004 | ACLY, FABP2, FASN, GPD1, PRKACA |
| GO.0044262 | Cellular carbohydrate metabolic process | 5 | 0.011 | ACLY, AKR1B1, CS, GAL3ST3, PGD |
ACADSB, acyl-CoA dehydrogenase short/very short chain; ACLY, ATP citrate lyase; AKR1B1, aldo-keto reductase family 1 member B; EEF1A1, eukaryotic translation elongation factor 1 alpha 1; FASN, fatty acid synthase; GAL3ST3, galactose-3-O-sulfotransferase 3; GPD1, glycerol-3-phosphate dehydrogenase 1; GSTK1, glutathione S-transferase kappa 1; HINT1, histidine triad nucleotide-binding protein 1; LTA4H, leukotriene A4 hydrolase; NDUFS1, NADH dehydrogenase (ubiquinone) iron-sulfur protein 1; PC, pyruvate carboxylase; PGD, phosphogluconate dehydrogenase; RPL12, ribosomal protein L12; RRBP1, ribosome-binding protein 1; SEC61B, Sec61 translocon beta subunit; SUCLA2, succinate-CoA ligase (ADP/GDP-forming) subunit beta; GO, gene ontology.
Results
Adrenal glands of Men1+/- mice
There was no statistically significant difference in the size of the adrenals of Men1+/- mice compared to the wild-type Men1+/+ mice; the size of the adrenals of the Men1+/- mice was 7.0 ± 1.4 (mean + standard deviation) mm2 compared to 5.8 ± 1.9 mm2 in the Men1++ mice (P = 0.07). The weight of the adrenals of the Men1+/- mice was 4.264 + 1.367 g (median ± SD), with the Men1+/+ adrenals weighing 3.96 ± 1.04 g (P = 0.61). No significant difference in adrenal size between males and females was observed (P = 0.45). Normal adrenal morphology and menin immunoreactivity were maintained in the adrenals of
both genotypes, with the zones of the adrenal cortex clearly distinguishable (Li et al. (7)). The proliferation rate, assessed by the percentage of Ki67 immunoreactive cells, was very low, less than 0.2%, and similar between the adrenal glands of the two genotypes (data not shown).
Differentially expressed proteins in the adrenal glands of Men1+/- mice, enrichment analysis
Tandem mass spectrometry identified a total of 681 proteins across all datasets. Of these, 662 were commonly detected in both genotypes, while 8 were
A
B
ACLY
100
100
FASN
Percent Survival
Percent Survival
< 2000
< 4000
50
50
> 2000
J
> 4000
0
0
1000
2000
3000
4000
5000
Survival (days)
Patients at risk: FASN<4000
50
29
11
5
2
FASN >4000
27
10
4
2
0
| 0 0 | 1000 | 2000 | 3000 | 4000 | 5000 | |
|---|---|---|---|---|---|---|
| Survival (days) | ||||||
| Patients at risk: | ||||||
| ACLY<2000 | 32 | 17 | 5 | 2 | 2 | |
| ACLY >2000 | 45 | 22 | 10 | 5 | 1 | |
Figure 2 Association between (A) ACLY and (B) FASN mRNA expression and ACC patient survival. mRNA expression reported as transcripts-per-million. Kaplan-Meier analyses for mRNA expression of ACLY and FASN in the ACC patient cohort consisting of 77 patients. In both A and B, the dashed line indicates patients with high expression of ACLY and FASN, respectively. There was a significant difference in survival between patients with high and low expression of both ACLY (P = 0.033) and FASN (P = 0.006). The statistical significance of the differences in survival was evaluated by the log-rank Mantel-Cox test.
detected only in Men1+/- adrenals and 11 were detected only in Men1+/+ adrenals (defined as detection in ≥3 biological replicates of one group and none in the other; Supplementary Table 1). Among the 662 shared proteins, 541 (81.7%) showed higher abundance and 121 (18.3%) lower abundance in Men1+/- compared with Men1+/+ adrenals. Statistical analysis (t-test) identified 52 significantly differentially expressed proteins (P < 0.05) (Table 1). To visualize these findings, we generated a volcano plot (Fig. 1), highlighting the 20 most significant proteins and indicating the unique proteins in an inset. The expression ratios of the 52 significantly regulated proteins between Men1+/- and Men1+/+ were generally low, with a mean of 1.4.
Several important proteins in glucose and lipid metabolism were upregulated; among them were proteins in the PPARa pathway, such as fatty acid synthase (FASN) and ATP-citrate lyase (ACLY). No difference was seen in the expression of FASN or ACLY between males and females of either genotype (P = 0.9).
Both ACLY and FASN have been shown to be of importance in several human cancers (18, 19, 20, 21, 22, 23). Network enrichment analysis of upregulated genes identified 17 GO categories with at least five of the differentially expressed proteins involved in each (Table 2). All the identified biological processes were classified as related to metabolism, e.g., lipid metabolism-associated mechanisms.
Prognostic impact of ACLY/FASN expression in human ACC patients
TCGA database had survival data, as well as mRNA expression data for ACLY and FASN, for 77 patients
(47 females and 30 males) (Supplementary Table 2). A significantly poorer survival and a higher risk of death were found for ACC patients bearing tumors that expressed high levels of ACLY and FASN enzymes compared to patients with tumors expressing low levels (P= 0.010 and 0.006, respectively) (Fig. 2). In addition, data on disease stage were available for 76 of the patients (Supplementary Fig. 2).
Antiproliferative effect of inhibition of ACLY and FASN in ACC cells
Two of the highest ranked proteins in the proteomics results were ACLY and FASN. To investigate whether these enzymes affect proliferation in ACC, we treated H295R cells with pharmacological inhibitors of ACLY (SB-204990) and FASN (C75). Cells were exposed daily for 5 days to 15, 30, or 60 µM SB-204990, or to 1, 4, or 16 uM C75. Both compounds displayed a clear dose-dependent inhibitory effect on proliferation (Fig. 3).
Exposing H295R to 60 µM SB-204990 resulted in a statistically significant inhibition of proliferation already after 1 day (P < 0.0001). By day 2, a significant effect was also seen for 30 µM (P < 0.0001) and 15 µM (P < 0.001). After 5 days, the antiproliferative effect was complete at 60 uM (0 cells remaining). Cultures treated with 30 and 15 uM contained 4 and 56% of control cell numbers, respectively. A significant difference was observed between 15 and 30 uM (P < 0.0001) (Fig. 3A).
For C75, a significant inhibitory effect was observed after 1 day with 16 uM (P < 0.01), after 2 days with 4 uM (P < 0.0001), and after 3 days with 1 µM (P < 0.01).
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On day 5, the number of treated cells was reduced to 4, 5, and 42% of control levels for 16, 4, and 1 uM, respectively. A significant difference was seen between 1 and 4 uM (P < 0.0001) and between 1 and 16 uM (P < 0.0001), although not between 4 and 16 uM (P = 0.72) (Fig. 3B).
Parallel experiments were also performed in SW13 cells, which showed qualitatively similar but overall,
less pronounced effects. These data are provided in Supplementary Fig. 3A and B.
At day 4 of treatment, cell viability normalized to control was 81 ± 9.0% following FASN inhibition and 55 ± 10.0% following ACLY inhibition. The difference was statistically significant for ACLY inhibition (P < 0.01), whereas the reduction after FASN inhibition did not reach significance (P = 0.08). These results suggest that the observed
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Relative intensity (% of control) of selected fatty acids in H295Rcells treated with ACLY-inhibitor (SB204990)
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Relative intensity (% of control) of selected fatty acids in H295Rcells treated with FASN-inhibitor (C75)
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decrease in cell number and lipid production mainly reflects reduced proliferation rather than general cytotoxicity.
Inhibiting ACLY and FASN in ACC cell line: effects on fatty acid synthesis
To investigate whether ACLY and FASN inhibition in H295R cells directly affected lipid metabolism, we quantified intracellular levels of palmitic acid (a FASN product) and palmitoleic acid after treatment. H295R cells were treated daily for 4 days with 15 uM SB- 204990 or 1 µM C75, conditions chosen to avoid complete growth arrest while ensuring effective inhibition of proliferation.
Lipidomic analysis revealed a significant reduction in both palmitic acid and palmitoleic acid after treatment with either SB-204990 or C75 compared to controls (Fig. 4).
For comparison, lipidomic experiments in SW13 cells were also performed and are shown in Supplementary Fig. 4.
Discussion
Adrenocortical carcinoma (ACC) is a rare and aggressive malignancy with poor prognosis and limited treatment options (24, 25). To contribute to this field, we studied proteomic changes in Men1+/- adrenals, hypothesizing that early proteomic alterations detectable before tumor development may play an important role in later transformation.
The present study aimed to investigate the pre-tumor stage in Men1+/- adrenals. It is recognized from studies on both human MEN1 patients and Men1+/- mice that menin haploinsufficiency results in the development of adrenocortical adenomas and, in rare cases, ACC (3, 5, 26). The tumor suppressor role of menin has been studied in detail in pancreatic islets of Men1+/- mice, where hyperplasia predictably developed, followed by tumors after loss of heterozygosity and additional somatic mutations (4). While molecular events in pancreatic islets are increasingly understood, early proteomic changes in MEN1 adrenals remain largely unexplored. Adrenal lesions in human MEN1 patients are usually identified during the 5th and 6th decades of life (27), and adrenal lesions in Men1+/- mice appears relatively late, compared to pancreatic adenomas. In Loffler et al. (5), it was reported that 30% of Men1+/- mice aged ≥ 18 months (n = 49) had adrenal lesions, while only one case of adrenocortical tumor was found among 6- to 12-month-old mice (n = 17). Based on these observations, we used mice aged 10.3 +/-0.95 months, representing a pre-neoplastic stage devoid of macroscopic tumors but predisposed to adrenocortical transformation. While these mice are not young per se, they represent a relatively early stage in the process of
tumorigenesis in Men1 adrenals. Using an older age span would increase the risk of including adrenocortical tumors with accrued mutations, while a younger cohort instead risks missing the relevant changes, particularly due to the relatively late presentation of adrenal lesions in MEN1. Adrenal size, weight, Ki67, and gross pathology were comparable between Men1+/- and Men1+/+ littermates, and menin expression remained intact, supporting that those proteomic changes reflect haploinsufficiency rather than overt tumorigenesis. It is worth noting that the whole adrenal, including the medulla, was used for proteomics. While adrenal medullary lesions are rare in Men1 mice (5), this is a possible confounder. Recent investigations have revealed new insights regarding a possible immunological mechanism behind sex-based differences in the prevalence of adrenal disorders (28), but a full investigation of these mechanisms in Men1+/- mice is beyond the scope of this project. In this study, no difference was seen in the expression of FASN or ACLY between male and female mice.
Of 681 identifiable proteins, 52 were significantly dysregulated in Men1+/- mouse adrenals. Among them, ATP citrate lyase (ACLY) and fatty acid synthase (FASN) were among the most upregulated hits, both central to lipid metabolism. Notably, TCGA data confirm that increased expression of ACLY and FASN in sporadic ACC correlates with a poorer patient survival, underscoring their potential clinical relevance. These findings prompted us to evaluate ACLY and FASN functionally in human ACC models.
To validate these findings, we treated H295R cells, the most widely used and representative ACC model, with pharmacological inhibitors of ACLY (SB-204990) and FASN (C75). SW13 cells were analyzed in parallel, and the results are presented in the Supplementary Data. SW13 was considered an ACC model for decades but has, in recent years, seen less use due to the possibility of it not being of adrenocortical origin. It is still used in some studies and is of interest due to its earlier importance in the field, but the results derived from SW13 are likely of limited clinical relevance in ACC. Inhibition of ACLY and FASN significantly reduced proliferation in a dose-dependent manner. Importantly, additional viability assays demonstrated that the reduction in cell numbers and lipid synthesis following ACLY and FASN inhibition was mainly due to suppressed proliferation rather than nonspecific cytotoxicity, further strengthening the mechanistic link between lipid metabolism and proliferative control in ACC.
Lipidomic analysis provided additional support, showing a significant reduction in downstream products (palmitate and palmitoleic acid) after ACLY and FASN inhibition, consistent with impaired lipid biosynthesis. These findings align with earlier studies of ACLY inhibition in lung adenocarcinoma cell lines, where a 50% reduction in acetyl-CoA was observed (15),
and with reports that exogenous palmitate supplementation mitigates cytotoxic effects observed at higher FASN inhibitor concentrations (29). Under our experimental conditions, the lower concentration used primarily exerted antiproliferative rather than cytotoxic effects.
Our study focused on early changes in a pre-neoplastic, non-human adrenal model, complemented by exploratory experiments in human ACC cells and TCGA patient data. While the correlation between ACLY/FASN expression and patient survival in sporadic ACC is intriguing, confounding factors in retrospective datasets limit causal interpretation. Nevertheless, our results are consistent with broader evidence that many cancers reprogram metabolism to support rapid proliferation, including upregulation of enzymes for de novo lipid synthesis (30, 31).
The precise mechanisms underlying the antiproliferative effects of ACLY and FASN inhibition are not fully understood. The proposed explanations include substrate limitation for fatty acid and cholesterol synthesis, altered membrane composition, or effects on signaling pathways, such as the mevalonate pathway (32). Additional hypotheses include citrate accumulation (33) and altered histone acetylation with downstream effects on glucose metabolism (34). Not all tumor cell phenotypes are expected to be equally sensitive to lipid biosynthesis inhibition, and our Supplementary Data suggest that SW13 cells were less sensitive than H295R cells, consistent with earlier findings that glycolytic phenotypes may be more susceptible to ACLY inhibition (15).
While ACLY and FASN inhibitions are attractive therapeutic strategies, available inhibitors have off-target effects that limit clinical utility (35). TVB-2640, a FASN inhibitor currently in clinical trials, has shown an acceptable safety profile (36) and is being tested in patients with non-small cell lung cancer (https://clinicaltrials.gov/study/NCT02223247), colorectal cancer (https://clinicaltrials.gov/study/NCT02980029), breast cancer (https://clinicaltrials.gov/study/ NCT03179904), and astrocytomas (https://clinicaltrials. gov/study/NCT02990468).
In conclusion, increased expression of ACLY and FASN is an early event in Men1+/- adrenal tumorigenesis. Functional validation in H295R cells and lipidomic analysis support their role in proliferation via lipid biosynthesis. Together with TCGA correlations linking high ACLY/FASN expression to poorer survival in ACC patients, these findings highlight ACLY and FASN as potential therapeutic targets.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ERC-25-0442.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.
Funding
This work was supported by the Swedish Cancer Society (Cancerfonden, 20 1310 PjF).
Author contribution statement
XC, AM, and BS initiated and supervised the project. AE, S-CL, XC, HW, MR, JB, AW, KU, PS, AM, and BS designed the experiments. AE, S-CL, XC, HW, MR, AW, KU, and AM conducted the experiments. AE, S-CL, XC, HW, MR, JB, AW, KU, PS, AM, and BS analyzed the results. AE, AM, and BS wrote the manuscript. All authors reviewed the manuscript.
Data availability
The proteomics dataset supporting this study has been deposited in the SciLifeLab Data Repository (DOI: 10.17044/scilifelab.30138382). The deposited dataset represents the processed SIEVE output used in the manuscript analyses.
Acknowledgments
This study was supported by the Swedish Cancer Society (Cancerfonden). The authors wish to express their gratitude to the Platform for MS-based Proteomics at Uppsala University, Sweden, and particularly Jia Mi for his invaluable support in performing the proteomic data analysis and gene ontology analysis in this research.
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