Adenosine triphosphate citrate lyase alters cellular myelocytomatosis oncogene signaling and fatty acid oxidation in residual breast cancer tumor tissues after neoadjuvant treatment

Adenosine triphosphate citrate lyase alters cellular myelocytomatosis oncogene signaling and fatty acid oxidation in residual breast cancer tumor tissues after neoadjuvant treatment | 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 Research Article Adenosine triphosphate citrate lyase alters cellular myelocytomatosis oncogene signaling and fatty acid oxidation in residual breast cancer tumor tissues after neoadjuvant treatment Ju-Ha Kim, Woo-Ju Song, In-Wook Hwang, Jinho Kang, Ji Hyung Hong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9173952/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background Metabolic adaptation enables breast cancer cells to develop neoadjuvant chemoendocrine therapy (NAC) resistance through metabolic alterations. Fatty acid oxidation (FAO) is a key metabolic pathway that facilitates the growth of resistant breast cancers. However, the mechanisms of FAO rewiring in NAC-resistant breast cancers remain unclear. Therefore, this study aimed to investigate metabolic signal reprogramming in residual tumor tissues after chemoendocrine treatment. Methods Residual tumor tissues from 11 patients with hormone receptor-positive (HR+) breast cancer receiving NAC therapy were analyzed for adenosine triphosphate citrate lyase (ACLY), myelocytomatosis oncogene (MYC), and fatty acid synthase mRNA levels. Moreover, MCF-7-long-term estrogen deprivation (LTED) and T47D-LTED cell lines were developed to model endocrine-tolerant residual cells. ACLY and cellular MYC (c-MYC) protein expression were confirmed, and FAO rates were measured via mitostress tests and BODIPY-C16 fluorescence. ACLY activity was inhibited via small interfering RNAs or NDI-091143 (ACLY inhibitor), and exogenous ACLY expression was induced via the FLAG-ACLY plasmid. Mechanistic studies included proliferation assays, western blotting, immunoprecipitation, and quantitative reverse transcription polymerase chain reaction, and NDI-091143 toxicity was assessed in animal models. Results ACLY mRNA levels were increased in patients with HR+ breast cancer. FAO-related gene sets increased after chemoendocrine therapy, and ACLY and MYC mRNA levels were increased. ACLY protein depletion disrupted c-MYC stability in breast cancer cells. MCF7-LTED cells exhibited metabolic characteristics similar to those of residual breast cancer cells in patients post-NAC. ACLY depletion also interrupted c-MYC protein stability in MCF7-LTED cells, thereby regulating FAO. NDI-091143 suppressed cancer cell proliferation and FAO by blocking ACLY phosphorylation (S455) and regulating c-MYC phosphorylation (S62). Notably, NDI-091143 enhanced cellular sensitivity and exhibited synergistic effects with etomoxir (carnitine palmitoyltransferase 1 inhibitor). Conclusions Overall, residual HR+ cancer cells shifted from lipid biosynthesis to FAO as a survival strategy. Moreover, the role of ACLY in regulating FAO was elucidated, thereby highlighting its role in residual cancer cell survival. Altogether, changes in metabolism via ACLY/c-MYC signaling are related to the survival of HR+ breast cancer cells after chemoendocrine treatment. In addition, ACLY inhibition has promising potential as a metabolic therapeutic strategy to overcome endocrine resistance in HR+ breast cancer. Adenosine triphosphate citrate lyase Cellular myelocytomatosis oncogene Fatty acid oxidation Neoadjuvant chemoendocrine therapy Breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Among all cancer types worldwide, breast cancer has the highest incidence and second-highest mortality rate [ 1 ]. Approximately 70% of breast cancer cases are hormone receptor-positive (HR+). In cases of locally advanced breast cancer, neoadjuvant treatment via chemotherapy or endocrine therapy has been introduced to reduce tumor size in patients with HR+ breast cancer. Despite efforts to increase treatment efficacy, the complete remission rate varies widely, ranging from 5–20% in patients with HR+ breast. A phase II clinical trial was conducted to evaluate the ability of neoadjuvant chemoendocrine therapy (NAC) to improve treatment outcomes. Although the response rate increased, the pathological complete response (pCR) rate was only 3.8% [ 2 ]. Despite advancements in antihormone therapies and chemotherapeutic agents, some breast cancer cells persist after treatment. Metabolic adaptation is a critical mechanism that facilitates cancer cell survival. Metabolic reprogramming underlies endocrine resistance, with single-cell subpopulations contributing to drug tolerance and residual cells exhibiting lipid metabolism signatures [ 3 , 4 ]. Lipids, including fatty acids, triglycerides, and lipoids, play vital roles in cancer metabolism [ 5 ]. Under certain conditions, cancer cells utilize fatty acids as an energy source to produce adenosine triphosphate (ATP) via oxidative phosphorylation (OXPHOS) [ 4 , 6 – 8 ]. Fatty acid oxidation (FAO) is crucial for drug resistance and cancer aggressiveness [ 6 , 7 , 9 ]. Moreover, FAO was identified as a key metabolic pathway involved in the growth of triple-negative breast cancers (TNBC) [ 10 ], particularly in myelocytomatosis oncogene (MYC)-high TNBC [ 11 ]. In patients with HR+ breast cancer, cellular MYC (c-MYC) amplification is correlated with a high pCR rate after NAC therapy [ 12 ]. Moreover, high c-MYC scores are considerably correlated with reduced recurrence-free survival in patients with long-term estrogen deprivation (LTED), whereas c-MYC depletion suppresses LTED-related breast cancer cell growth; therefore, c-MYC mediates antiestrogen resistance [ 13 ]. In addition, c-MYC regulates androgen receptors, which are aromatase substrates, through epigenetic factors, such as disruptor of telomeric silencing 1-like [ 14 ]. Moreover, c-MYC inhibition and overexpression induce notable metabolic changes in tumor cells [ 4 , 15 ]. Overexpression of c-MYC stimulates FAO via calcium signaling [ 16 ], whereas c-MYC inhibition causes the accumulation of lipid droplets in tumor cells [ 17 ], regulates lipid signaling, and influences mitochondrial acetyl-CoA levels [ 18 ]. ATP citrate lyase (ACLY) is upregulated in human breast cancer cells, and its depletion inhibits tumor growth [ 19 , 20 ] and promotes apoptosis [ 21 , 22 ]. ACLY regulates the acetylation of histones H3 [ 23 ] and H4 [ 24 ] after their translocation to the nucleus. DNA damage signaling through ionizing radiation increases ACLY and phosphorylated ACLY (p-ACLY) (S455) expression in the nucleus [ 24 ]. Although ACLY catalyzes acetyl-CoA production from citrate and transfers it to fatty acid synthase (FASN), new roles for ACLY have been revealed, including lipid peroxidation and OXPHOS [ 25 , 26 ]. ACLY increases histone acetylation via EP300 at the melanocytic lineage oncogenic factor locus, thereby promoting mitochondrial OXPHOS, cancer growth, and drug resistance [ 27 ]. Conversely, ACLY inhibition reduces OXPHOS under low-glucose conditions [ 28 ]. Despite these discoveries, the mechanisms of FAO rewiring in NAC-resistant-HR+ breast cancers are unclear. Therefore, this study aimed to investigate the metabolic signal reprogramming in residual tumor tissues after chemoendocrine treatment. Overall, the study revealed that the action of ACLY and c-MYC during FAO is crucial for the survival of HR+ breast cancer cells. Methods Public data analysis The mRNA microarray data were downloaded from [ 29 ]. The data specifications were patients with breast cancer treated with neoadjuvant therapy (letrozole oral administration, 2.5 mg/day), and mRNA samples were taken sequentially from biopsies of non-treated cancer and the same cancers after 90 days. ACLY mRNA levels were analyzed using Gene Expression Omnibus (GEO) data (accession number: GSE20181, Affymetrix probe ID: 201128_s_at, NCT02296801) on the Affymetrix Human Genome U133A Array platform. CEL data were also downloaded, normalized, and analyzed via the Bioconductor (version 3.14) package (BiocManger 1.30.16) in R studio (version 4.1.0). ACLY mRNA levels were compared between 112 normal controls and 1100 patients with breast cancer reported in The Cancer Genome Atlas database (TCGA, version 2016.01.28). Survival plot data were downloaded via the Kaplan-Meier plotter [ 30 ] from the GEO database (GSE7390) [ 31 ] to identify whether patient prognosis was affected by ACLY mRNA levels in node-negative, estrogen-positive, and human epidermal growth factor receptor 2 (HER2)-negative breast cancers. In 123 patients, overall survival (OS) rates were calculated with a cutoff value of 3134 (Affymetrix probe ID: 201128_at) between the upper and lower quartiles. Patient tissue sample analysis Formalin-fixed paraffin-embedded (FFPE) residual tumor tissues from patients with HER2-positive breast cancer and those with TNBC after neoadjuvant treatment were obtained from the National Cancer Center Bio Bank (NCCTTR-18005; Goyang, ROK). HR+ residual tissues were derived from a phase II neoadjuvant trial, which aimed to assess the efficacy of a chemoendocrine combination treatment strategy (IRB No. NCC2017-0110, Phase II Study of Neo-adjuvant Chemotherapy with Letrozole in Patients with Estrogen Receptor Positive/HER-2 Negative Breast Cancer, NCT03497702. Registered 18 March 2018, https://clinicaltrials.gov/study/NCT03497702 ) [ 2 ]. Eleven biopsy and surgery samples from the cohort were stored as FFPE samples and used for mRNA analysis. The patients were administered doxorubicin and cyclophosphamide four times followed by paclitaxel and docetaxel four times as neoadjuvant cytotoxic therapy. Letrozole was continuously administered to the patients undergoing treatment. After surgery and tissue collection, 11 paired tumor samples were stored as FFPE tissues and used for NanoString analysis. For mRNA quantification, 201 metabolism-related genes and 12 housekeeping genes were used. The mRNA levels were calculated using digital transcript counting (nCounter metabolic pathways panel assay, NanoString, Seattle, WA, USA). Total RNA (100 ng) was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The samples were assayed on an nCounter Digital Analyzer (NanoString) according to the manufacturer’s instructions. The data were normalized to the housekeeping gene probes for each sample. The heatmap was plotted using Multiple Experiment Viewer software. The gene set enrichment score graph was analyzed via gene set enrichment analysis (GSEA) software, version 4.1.0 (Broad Institute and UCSD, MA and CA, USA). Cell culture All human breast cancer cell lines, including MCF7, T47D, and MDA-MB-231, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). T47D and MDA-MB-231 breast cancer cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). MCF7 parent and MCF7-LTED cell lines were cultured in Dulbecco's modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12, Gibco, MA, USA) supplemented with GlutaMAX (Gibco). All cell lines, excluding LTED cells, were supplemented with 10% fetal bovine serum (FBS; Gibco). LTED cell lines were generated with 10% charcoal-stripped FBS (csFBS; Gibco) instead of FBS and were maintained under these conditions until the number of culture passages surpassed 75; these cell lines were subsequently used for all experiments. The letrozole-resistant (Let R ) MCF7 cell line (MCF7-Let R ) was purchased from the European Collection of Authenticated Cell Cultures (MCF7/LetR-1; Salisbury, UK). The MCF7-Let R cell line was cultured in DMEM/F12 supplemented with GlutaMAX, 0.1 µM testosterone, and 1 µM letrozole. The parental cell line was maintained under the same conditions as the MCF7-Let R cell line but without letrozole. All the cell lines were cultured with a 1% antibiotic solution comprising penicillin at 10,000 units/mL, streptomycin at 10,000 µg/mL, and amphotericin B at 25 µg/mL (Gibco) at 37℃ under 5% CO 2 in a humidified chamber. Western blotting The cells were lysed in RIPA buffer (Merck KGaA, Darmstadt, Germany) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitors (Merck KGaA). Lysates were quantified using a BCA protein assay kit (Thermo Fisher Scientific). The protein samples were electrophoresed on 8% handmade sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes with a pore size of 0.4 µm. The membranes were blocked with Tris-buffered saline with 0.1% Tween® 20 detergent (TBST), diluted with 3% skim milk, or bovine serum albumin (BSA) for 1–2 h at room temperature. The membranes were then incubated with primary antibodies and β-actin (Cat. no. A2228, Merck KGaA) diluted in 3% BSA in TBST overnight at 4 ℃. Subsequently, the membranes were washed three times for 10 min with TBST and incubated with horse radish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA) for 2 h at room temperature. ACLY (Cat. No. 4332, Cell Signaling Technology, Danvers, MA, USA or Cat. no. ab40793, Abcam, Cambridge, UK), p-ACLY S455 (Cat. no. 4331, Cell Signaling Technology), c-MYC (Cat. no. 5605, Cell Signaling Technology), phosphorylated c-MYC (p-c-MYC S62; Cat. no. ab185656, Abcam), FASN (Cat. no. 3180, Cell Signaling Technology), carnitine palmitoyltransferase (CPT)1A (Cat. no. 12252, Cell Signaling Technology), type I topoisomerase (Cat. no. sc-32736, Santa Cruz Biotechnology, Dallas, TX, USA), ɑ-tubulin (Cat. no. sc-32293, Santa Cruz Biotechnology), β-actin (Cat. no. 4970, Cell Signaling Technology), FLAG (Cat. no. 8146, Cell Signaling Technology), and green fluorescent protein (GFP; Cat.no.sc-8834, Santa Cruz Biotechnology) were used for the experiments. Expression was visualized using enhanced chemiluminescence Immunoblotting Detection Reagent (Thermo Fisher Scientific), and fluorescence images were captured with an Invitrogen iBright FL1500 system (Thermo Fisher Scientific). RNA interference and plasmid transfection The cells were seeded onto culture plates overnight at 40% confluence and transfected with a mixture of ACLY small interfering RNA (siRNA) or negative control siRNA purchased from Bioneer (Daejeon, ROK) and Thermo Fisher Scientific. The siACLY #1 sequence was RNA- (GUGUA)CAGCGAUACCAUC U/RNA-AGAUGGUAUCGCUG (UACAC). The siACLY #2 sequence was RNA-GGGUGUCAACGAGCUGGCAAACUAU/RNA-AUAGUUUGCCAGCUCGUUGACACCC. INTERFERin transfection reagent (40 nM; Polyplus, Illkirch, France) was used according to the manufacturer’s protocol. The transfected cells were then incubated for 72 h. FASN (Cat. No. SC122268, pCMV6-XL5) and ACLY (Cat. No. RC200508, pCMV6-Entry) were purchased from OriGene (Rockville, MD, USA). The pcDNA 3.0 and c-MYC-GFP (Cat. no. 42142) antibodies were purchased from Addgene (Watertown, MA, USA). V5-c-MYC has been described previously [ 32 ]. The cells were transfected with TurboFect transfection reagent (Thermo Fisher Scientific) and incubated for 36 h for further study. Cycloheximide (CHX) assay The cells transfected with siNC and ACLY siRNA for 72 h were exposed to 50 µg/mL CHX (Merck KGaA) at the indicated concentrations and for the indicated durations (0, 10, 20, and 40 min). Cycloheximide (CHX was used to verify whether ACLY regulated c-MYC stability. Immunofluorescence The cells transfected with siRNAs were fixed with 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) for 20 min at room temperature and permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 2 min on ice. The cells were labeled with primary antibodies diluted in 1% BSA/phosphate-buffered saline (PBS) overnight at 4°C. The cells were then exposed to secondary Alexa Fluor (Invitrogen, Thermo Fisher Scientific) diluted with 1% BSA/PBS for 2 h at room temperature. The samples were mounted with mounting medium (VectorLabs, Newark, CA, USA) containing 4′,6-diamidino-2-phenylindole, detected with fluorescence, and imaged using an Axio Observer Z1 (Carl Zeiss, Oberkochen, Germany). Immunoprecipitation MCF7 cells transfected with the plasmids were lysed and quantified according to western blotting protocols. A total of 2 µg of antibodies against DYKDDDDK (FLAG; Cat. No. 8146, Cell Signaling Technology) was added to 500 µg lysate, which was subsequently incubated at 4°C in a rotator overnight. A total of 30 µL of protein A/G agarose beads (Santa Cruz Biotechnology) was added, and the mixture was rotated at 4°C for 4 h. The resulting lysates were washed three times with NP40 lysis buffer (Thermo Fisher Scientific). The bound proteins were immunoblotted as previously described. The protein input amount was 10% of the immunoprecipitated samples. Transient stimulation of the c-MYC protein The c-MYC and ACLY protein levels in breast cancer cells were determined after stimulation with 20% FBS (Thermo Fisher Scientific). To artificially stimulate c-MYC, 20% FBS was added at the indicated time points following serum starvation for 36 h. Protein levels were evaluated via western blotting. Next-generation sequencing (NGS) analysis Total RNA was extracted from MCF7-parent and MCF7-LTED cells using QIAzol lysis reagent (Qiagen), and RNA quality was assessed by Macrogen (Seoul, ROK). The samples were processed via next-generation sequencing (NGS) sequence analysis (NextSeq 500; Illumina, San Diego, CA, USA). The raw NGS data were deposited in the National Centre for Biotechnology Information BioProject database (accession number: PRJNA802345). Mitochondrial respiration and FAO assay The cells were seeded onto an XF96 plate using the Seahorse Mito-Stress Kit and Seahorse XF Palmitate Oxidation Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA). The cell growth medium was changed to substrate-limited medium (Agilent Technologies), and the cells were incubated with the medium overnight. Each reagent was placed in a cartridge according to the manufacturer’s protocol (Agilent Technologies). Basal respiration values were calculated three times at intervals. Nonmitochondrial respiratory consumption was measured after treatment with rotenone (complex I inhibitor) or antimycin A (complex I inhibitor). The ATP-linked respiration rate was calculated using oligomycin, which is an inhibitor of the electron transport channel complex V. Maximum respiration was artificially induced using the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). When palmitate-conjugated BSA was used as the energy source, instead of BSA alone, FAO was calculated using spare capacity values, which were calculated by subtracting basal respiration from maximum respiration. The following equations were used: nonmitochondrial oxygen consumption = minimum rate measurement after rotenone / antimycin A injection; basal respiration = last rate measurement before first injection − nonmitochondrial respiration rate; maximal respiration = maximum rate measurement after FCCP injection − nonmitochondrial respiration; spare respiratory capacity = maximal respiration − basal respiration; and spare respiratory capacity (%) = maximal respiration / basal respiration * 100 BODIPY-C16 assay The cells were grown in Operetta 96-well culture plates to measure lipid degradation. BODIPY-C16 (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid, Thermo Fisher Scientific) was added to the medium (final concentration = 1 µM), incubated for the designated duration, and exchanged immediately before detection with Hoechst staining. Fluorescence was detected using an Operetta CLS real-time imaging machine (Perkin-Elmer, Waltham, MA, USA) every 4 h and calculated per nucleus stained with Hoechst. The fluorescent cells were traced via the Alexa 488 and Hoechst channels designated in Harmony software v4.8 (Perkin-Elmer). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) RNAs isolated from the cells were lysed using QIAzol reagent (Qiagen) according to the manufacturer’s protocol. A total of 2 µg of each RNA sample was synthesized into complementary DNA (cDNA) with oligo-deoxythymidines, deoxynucleoside triphosphates (Takara, Shiga, Japan), M-MLV reverse transcriptase, and its buffer (Enzynomics, Daejeon, ROK) following the manufacturer’s protocol. The cDNA samples (100 ng) were placed in the wells of a qRT-PCR plate for each reaction. The primers used were synthesized by Bioneer (Daejeon, ROK). The primers for MYC , CPT1 , FASN , ACLY , and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) were as follows: MYC , 5’-AAGAGGGTCAAGTTGGACAGT-3’ (Forward) and 5’-CGTTTTAGCTCGTTCCTCCTC-3’ (Reverse); CPT1 , 5’-TCTGCCTTTACGTGGTGTCTA-3’ (Forward) and 5’-CTGGACACGTACTCTGGGTTA-3’ (Reverse); FASN , 5’-CGTTGACCTGGTCTTGAACTC-3’ (Forward) and 5’-CGTGGAATGTCACGTTCTTCA-3’ (Reverse); ACLY , 5’-CTGTGATCTAGGGGGTGTCAA-3’ (Forward) and 5’-TGCCTCCAATGATGAGGATCT-3’ (Reverse); and GAPDH , 5’-GACGGTGCCATGGAATTTGC-3’ (Forward) and 5’-ATGGGGAAGGTGAAGGTCGG-3’ (Reverse). Nucleus and cytosol isolation The cells were harvested using trypsin-ethylenediamine tetraacetic acid and centrifuged at 1000 rpm for 5 min. The cell samples were washed by suspending the cell pellets in PBS. Following the manufacturer’s protocol (NE-PER Nuclear and Cytoplasmic Extraction Reagents; Thermo Fisher Scientific), ice-cold cytoplasmic extraction reagent (CER) I was added to the cell pellet, vortexed for 15 s, and incubated for 10 min. Ice-cold CER II was then added, and the mixture was vortexed for 5 s, incubated for 15 min, and centrifuged at 15000 rpm for 5 min. Thereafter, the supernatant was collected as the cytosol, and the nuclear extraction reagent solution was added to the pellet. The sample was then vortexed for 15 s and incubated for 10 min; this process was repeated four times. The mixture was then centrifuged at 15000 rpm for 10 min, and the supernatant was collected and used as the nucleus for western blotting. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) Metabolites involved in energy metabolism were analyzed using LC-MS/MS with a 1290 HPLC system (Agilent Technologies), Qtrap 5500 (ABSciex, Framingham, MA, USA), and a reverse phase column (Synergi fusion RP 50 × 2 mm). The sample (3 µL) was injected into the LC-MS/MS system and ionized with a turbo spray ionization source. Mobile phases A and B comprised 5 mM of ammonium acetate in H 2 O and 5 mM of ammonium acetate in methanol, respectively. The separation gradient was as follows: maintained at 0% B for 5 min, 0–90% B for 2 min, 90% B for 8 min, 90–0% B for 1 min, and 0% B for 9 min. The liquid chromatography flow rate was 70 µL/min but was increased to 140 µL/min between 7–15 min, and the column temperature was maintained at 23°C. Multiple reaction monitoring was used in negative ion mode, and the extracted ion chromatogram corresponding to the specific transition for each metabolite was used for quantification. The area under the curve of each extracted ion chromatogram was normalized to that of the internal standard chromatogram. The ratio of the peak area of each metabolite to that of the internal standard was normalized using the protein content of each sample and was used for relative comparison. Data analysis was performed using Analyst 1.5.2 software (SCIEX, Framingham, MA, USA). Mouse experiments Two animal studies were performed in accordance with the Animal Use Protocol (IACUC numbers: NCC-21-660 and NCC-21-704) approved by the National Cancer Center IACU Committee. BALB/c female athymic nude mice (four weeks old, Orient Bio, Seongnam, ROK) were used. For drug toxicity identification before the xenograft model was established, 68 mice were allowed to rest for one week after receiving the animal at the animal facility. The body weights of four mice per group were measured every 2–3 days over a 21-day period. For additional studies, the mice were anesthetized via inhalation with 2% isoflurane, and a 17β-estradiol pellet (Cat. No. E8875, Sigma Aldrich) was implanted on the lateral side of the neck between the ear and the shoulder. The drugs were injected intraperitoneally into mice one day after implantation of the 17β-estradiol pellet. For the xenograft experiments, all procedures were performed in the same manner as those used for the drug toxicity experiments before the cancer cells were injected. Cancer cells (5 × 10 6 cells) suspended in 50% saline/50% Matrigel (Cat. No. 354234, BD Biosciences, Franklin Lakes, NJ, USA) were injected into the fourth mammary fat pad. Tumor size was monitored while the drugs were injected intraperitoneally into the mice, and the mice were euthanized via 2–5% isoflurane inhalation. Statistical analysis The data are expressed as the means ± standard deviations from at least three independent experiments. Student’s t test for two-group comparisons and one-way analysis of variance, followed by Tukey’s post-hoc test, were conducted for multigroup comparisons via GraphPad Prism software (version 5.0; San Diego, CA, USA). Differences were considered significant if the p value was less than 0.05. Results ACLY mRNA was increased in breast cancers and upregulated after antihormonal therapy Abnormal amplification of ACLY in patients with breast cancer has been reported in several cohorts, including African-American [ 33 ] and Chinese women [ 21 ]. To investigate whether this pattern was also common in other groups, public and clinical data were analyzed. The TCGA data revealed that ACLY mRNA levels in breast cancer tissues were markedly increased in 1100 patients with invasive breast cancer compared with those in 112 patients with normal breast tissues (Fig. 1 A). To identify the differences in ACLY expression in patients with HR+ breast cancer, OS was analyzed in 123 patients with estrogen receptor-positive (ER+) breast cancer using the GEO database. Compared with the low ACLY -expression group, the high ACLY -expression group had notably different OS rates, indicating a poor prognosis (Fig. 1 B). To determine whether antiestrogen treatment affects ACLY expression, a clinical trial (ClinicalTrials.gov ID NCT02296801) of patients with ER+ breast cancer treated with letrozole was conducted. ACLY mRNA expression was markedly upregulated 90 days after letrozole administration (p < 0.001) (Fig. 1 C). Moreover, GSEA revealed that lipid metabolic processes and FAO pathways were upregulated 90 days after letrozole administration (Fig. 1 D). ACLY and FASN expression levels in various clinical samples were investigated. The clinical data [ 2 ] included 11 paired samples from patients with HR+/HER2 − breast cancer before and after NAC (Fig. 1 E, see Additional file 1 Fig. S1 ). The ACLY and MYC mRNA expression levels were markedly increased after NAC (Fig. 1 F). In contrast, FASN mRNA levels decreased, which opposed the ACLY pattern; however, ACLY is an upstream gene of FASN , thereby inducing fatty acid synthesis [ 34 ]. When gene expression levels were analyzed via GSEA, lipid metabolic processes and the regulation of FAO gene sets in the post-NAC group were upregulated (shown as B in Fig. 1 G) compared with those in the pre-NAC group. To elucidate the importance of MYC levels in metabolic gene patterns, the changes before and after NAC were analyzed and presented, with a focus on genes whose p values were less than 0.05 (Fig. 1 H). As shown in Fig. 1 I, a similar pattern was observed within the MYC- high group, which was distinct from the pattern observed in the MYC- low group, thereby indicating that c-MYC may mediate metabolism in this cohort. ACLY depletion disrupted c-MYC protein stability in breast cancer cells To investigate whether ACLY regulates c-MYC to regulate metabolism in the cells, ACLY was suppressed using two siRNAs targeting the citrate-binding sites, which are the main sites that block enzyme activity and acetyl-CoA genesis. The decrease in c-MYC expression was dependent on ACLY levels, whereas FASN showed the opposite pattern (Fig. 2 A, see Additional file 1 Fig. S2 A). In addition, c-MYC protein stabilization and stimulation assays were performed to determine whether ACLY post-transcriptionally regulates c-MYC protein expression in breast cancer cells. ACLY knockdown disrupted c-MYC protein stability, rendering the half-life of c-MYC in breast cancer cells to be shorter than that of the control (Fig. 2 B–C). ACLY depletion also blocked FBS-induced stimulation of the c-MYC protein in breast cancer cells (Fig. 2 D), and immunoprecipitation suggested an interaction between ACLY and c-MYC (Fig. 2 E). Moreover, the immunofluorescence assay revealed the colocalization of ACLY and c-MYC (yellow color), which suggests the merging of c-MYC and ACLY (Fig. 2 F). Depletion of ACLY also downregulated c-MYC protein in the nucleus, indicating that ACLY may mediate c-MYC protein regulation in the nucleus (see Additional file 1 Fig. S2 B). Effects of ACLY on altered metabolism in csFBS-treated model cells The csFBS removed hormones and growth factors from cells but retained metabolites, such as cholesterol (Fig. 3 A) [ 35 ]. LTED cells adapted to csFBS show altered gene signatures and paclitaxel resistance [ 36 , 37 ]; hence, these cells are considered effective models of NAC resistance [ 13 , 38 , 39 ]. The estrogen independence of LTED cells was also confirmed via a cell viability assay (see Additional file 1 Fig. S2 C). Gene profiles of LTED cells are associated with MYC activation [ 13 ]. Both c-MYC and ACLY expression levels were upregulated in correlation with the duration of hormone deprivation (Fig. 3 A, right panel). Elevated c-MYC and ACLY levels were also confirmed in MCF7-Let R cells (Fig. 3 B, see Additional file 1 Fig. S2 D and S2E). To determine whether ACLY mediates c-MYC expression in MCF7-LTED cells, ACLY was depleted via two siRNAs, as in MCF7 cells. ACLY depletion downregulated c-MYC expression, whereas FASN had the opposite effect, which was consistent with the results in MCF7 and MCF7-Let R cells (Fig. 3 C, see Additional file 1 Fig. S2 F). Whole RNA sequencing was conducted, and the mRNA values were analyzed with GSEA to determine whether these gene patterns affect metabolic characteristics. MCF7-LTED and MCF7-Let R cells presented higher enrichment scores for fatty acid derivative metabolism, very long-chain fatty acid metabolism, and beta-oxidation (acyl-CoA oxidase gene sets) than MCF7-parent cells did (Fig. 3 D). These results indicate that MCF7-LTED and MCF7-Let R cells share metabolic signatures similar to those of NAC clinical cases, thereby reflecting adaptation to hormone removal. These findings provide a basis for studying metabolic reprogramming. The metabolic gene sets have been validated via biochemical experiments. To confirm fatty acid (beta)oxidation, the FAO rate of the mitochondria was analyzed in MCF7-parent and MCF7-LTED cells. FAO activation was greater in the MCF7-LTED cells than in the parent cells, as indicated by the markedly higher spare respiratory rate for palmitate:BSA in MCF7-LTED cells than in the parent cells (Fig. 3 E). Total mitochondrial oxidative respiration was also greater in MCF7-LTED, T47D-LTED, and MCF7-Let R cells than in the parent cells (see Additional file 1 Fig. S2 G). The expression of CPT1A, a pivotal regulator of FAO [ 40 ], was also increased in MCF7-LTED and MCF7-Let R cells (Fig. 3 F, see Additional file 1 Fig. S2 D). ACLY depletion interrupts c-MYC protein stability, thereby regulating FAO in MCF7 LTED cells Next, whether ACLY mediates c-MYC expression and whether metabolic reprogramming occurs when ACLY regulates c-MYC in the presence of csFBS were investigated. ACLY knockdown attenuated c-MYC stability in MCF7-LTED cells (Fig. 4 A), and c-MYC expression decreased in the nucleus with the depletion of ACLY, which is translocated into the nucleus [ 23 ]. The lower-sized band was similar to the band size reported by Sivanand et al. and was confirmed with p-ACLY (S455) levels in the nucleus [ 24 ] (Fig. 4 B). FAO assays were performed to investigate the role of ACLY in FAO upregulation in LTED cells. ACLY depletion disrupted FAO gene and protein expression in MCF7-LTED cells (Fig. 4 C). To investigate the mechanism by which ACLY differentially regulates OXPHOS and metabolite profiles in the absence of fatty acid stimulation in MCF7-parent and MCF7-LTED cells, a mitochondrial respiration assay and LC-MS/MS were conducted. Although ACLY manipulation had no considerable effect on mitochondrial spare respiration or energy metabolites in MCF7 cells, LTED cells exhibited a slight reduction following ACLY depletion (Fig. S3 A–B). A key FAO gene ( CPT1 ) increased protein levels in LTED cells (Fig. 3 F); therefore, whether ACLY signaling regulates FAO through CPT1 was investigated. When ACLY expression was reduced, the decrease in CPT1 levels in LTED or Let R cells was less pronounced than that in parent cells (Fig. 4 D). These findings suggest that FAO in LTED or Let R cells may be considerably influenced by other proteins rather than solely by those affecting parent cells. To clarify whether c-MYC mediates this mechanism in LTED cells, an FAO assay was performed following the transfection of LTED cells with the c-MYC or FASN plasmid. Deregulation of ACLY decreased FAO, and FAO was notably recovered by c-MYC, but not by FASN (Fig. 4 E and S3C). These findings suggest that csFBS-treated cells, which mimic post-NAC conditions, exhibit FAO activation mediated by the ACLY/c-MYC pathway. Moreover, these findings indicate that metabolic adaptation through ACLY/c-MYC signaling occurs in residual cancer cells, which may promote cancer recurrence [ 3 , 4 , 8 , 22 , 41 – 43 ]. In Fig. 4 F, the left panel shows that FAO was blocked by malonyl-CoA, a product of acetyl-CoA carboxylase (ACC), in primary cancer cell metabolism. ACLY binds to citrate to produce acetyl-CoA, which activates ACC and FASN [ 22 ]. ACC binds to acetyl-CoA to produce malonyl-CoA, thereby inhibiting CPT1A activation. Therefore, FASN synthesizes palmitate, leading to the production of acyl-CoA, and acyl-CoA and carnitine react with CPT1A, thereby enabling transport into the mitochondria for β-oxidation. This activates the tricarboxylic acid (TCA) cycle through acetyl-CoA to generate ATP [ 43 ]. In residual cancer cells (right panel), ACLY in the cytosol translocates to the nucleus when its serine 455 site is phosphorylated, which mediates c-MYC activation to induce FAO. Therefore, metabolism was altered by FAO activation, primarily through c-MYC/ACLY signaling, rather than the negative feedback of malonyl-CoA (Fig. 4 F). ACLY inhibition suppressed cancer cell proliferation and FAO by regulating c-MYC To determine the effects of ACLY inhibition on cell viability, the cells were treated with ACLY inhibitors (NDI-091143, SB-204990, and ETC-1002). The assays revealed that the inhibitors reduced the proliferation of MCF7-parent, -LTED, and -Let R cells (Fig. 5 A and S3D–E). NDI-091143 treatment suppressed p-ACLY (Ser455) expression, which regulates ACLY nuclear translocation, and p-c-MYC (Ser62), which mediates c-MYC ubiquitination at 1 and 24 h (Fig. 5 B and S3F). FAO was measured fluorescence-labeled palmitate (BODIPY-C16) and showed a slower decrease in fluorescence in the NDI-091143-treated groups than in the control groups; this decrease was more pronounced at approximately 96 h (Fig. 5 C–D). These findings suggest that ACLY plays a key role in fatty acid consumption through lipid droplet degradation [ 3 ]. NDI-091143 treatment had a synergistic effect with etomoxir and increased cell sensitivity to letrozole To determine the effect of NDI-091143 on letrozole resistance in MCR7-Let R cells, a cell viability assay was performed. NDI-091143 (1.25 µM) markedly enhanced the drug sensitivity of Let R cells that were simultaneously treated with letrozole (Fig. 6 A). Moreover, etomoxir (CPT1 inhibitor) had a pronounced synergistic effect with NDI-091143 in LTED cells (Fig. 6 B), and MCF7-Let R cells responded well to etomoxir at the same dose (Fig. S3 G). To evaluate the toxicity of NDI-091143, which was not assessed in normal breast cells, a mouse body weight test was performed. Neither NDI-091143 nor combination treatment with etomoxir caused substantial changes in body weight (Fig. 6 C). The synergistic effect was confirmed by targeting LTED cells and comparing the results with those in the PBS control group in orthotopic breast cancer xenograft models. However, mice injected with LTED cells exhibited metastatic responses rather than subcutaneous proliferation, thereby supporting the aggressive characteristics of LTED cells (data not shown). Overall, NDI-091143/etomoxir combination therapy, which suppresses ACLY and CPT1, may be effective against NAC-resistant breast cancers (Fig. 6 D). Discussion Concurrent chemoendocrine therapy is generally not recommended as a neoadjuvant treatment for HR+ breast cancer [ 44 ]. In a previous clinical trial, although the response rate to chemoendocrine therapy was 83%, the pCR rate was only 4% [ 2 ]. Despite the addition of endocrine therapy to conventional chemotherapy, many cancer cells have adapted to these therapies and survived. Therefore, this study explored the metabolic adaptations of cancer cells to chemoendocrine treatments. The effects of NAC therapy have been investigated on the basis of transcription [ 45 ], genetic mutations [ 29 ], and lipid profiles [ 46 ]. Consistent with these studies, this study analyzed the mRNA profiles from the GEO database (NCT02296801) of paired breast tissues before and after letrozole treatment (90 days). After treatment in the clinical trial of this study (NCC2017-0110), ACLY mRNA expression was markedly increased in patients with HR+ breast cancer (Fig. 1 A–D). In paired FFPE tissues from 11 new patients who underwent NAC, metabolic gene analysis revealed that ACLY mRNA levels were consistently elevated despite considerable downregulation of FASN. This pattern was similar to that of c-MYC (Fig. 1 E–G). Furthermore, ACLY knockdown reduced c-MYC protein expression and stability in HR+ breast cancer cells, thereby shortening its half-life, whereas FASN had the opposite effect (Fig. 2 A–D, see Additional file 1 Fig. S2 A). Although ACLY is mainly present in the cytosol, it translocates to the nucleus upon Ser455 phosphorylation [ 23 ]. Suitably, the study findings revealed the molecular interaction and nuclear colocalization of ACLY and c-MYC (Fig. 2 , see Additional file 1 Fig. S2 B). This study hypothesized that specific mediators regulate downstream ACLY pathways to reprogram lipid metabolism in response to endocrine therapy during NAC. Although aromatase inhibitors, a type of endocrine therapy, may promote cancer aggressiveness and drug resistance [ 10 ] in breast cancer cells via amplification in breast cancer 1/ERα [ 47 ], epidermal growth factor receptor/erythroblastic leukemia viral oncogene/protein kinase B [ 38 ], or ER/phosphoinositide 3-kinase mechanisms [ 39 ], the exact factors driving resistance remain unclear [ 48 ]. Several aromatase inhibitor-resistant breast cancer cell lines have been developed and have revealed a metabolic shift from glycolysis, which is the main form of cancer cell metabolism [ 49 – 51 ]. In this study, chemoendocrine-tolerant cell lines (LTED cells) were generated, and LTED cells overexpressed c-MYC and ACLY at both the protein and mRNA levels (Fig. 3 A–B, see Additional file 1 Fig. S2 D). Notably, ACLY regulated c-MYC protein expression in LTED cells (Fig. 3 C, see Additional file 1 Fig. S2 A). ACLY inhibition downregulates the TCA cycle in acute myeloid leukemia through mitochondrial OXPHOS, a noncanonical TCA cycle [ 26 ]. Hence, this study aimed to determine whether ACLY signaling regulates OXPHOS in LTED cells when fatty acids are utilized as an energy source instead of glycolysis, as aberrant FAO activation contributes to drug resistance via breast cancer stem cell regulation [ 8 ], mitochondrial membrane alteration [ 42 ], and genetic alterations via the acyl-CoA oxidase 1 [ 3 ] and rat sarcoma virus pathways [ 52 ]. The MCF7-LTED and MCF7-Let R cells presented greater FAO activity than that of the parent cells, showing activity similar to that of the NAC clinical cases (Fig. 3 D–E). CPT1A, a key regulator of FAO, was also upregulated in MCF7-LTED cells (Fig. 3 F). Furthermore, c-MYC plays an important role in FAO in breast cancer cells [ 11 , 16 ]. Accordingly, ACLY depletion disrupted c-MYC protein stability, as detected in the nucleus of MCF7-LTED cells (Fig. 4 A–B). Moreover, deregulation of ACLY reduced FAO, which was considerably restored by c-MYC but not by FASN or CPT1, in LTED cells (Fig. 4 C–E). These findings suggest that residual cancer cells activate FAO following NAC therapy, and this is mediated by the ACLY/c-MYC pathway. This phenomenon is distinct from that in primary cells [ 43 ], thereby indicating that p-ACLY (S455) translocates into the nucleus and activates c-MYC to induce FAO in residual cells (Fig. 4 F). NDI-091143, an ACLY inhibitor, suppresses cancer cell proliferation and FAO by regulating the c-MYC protein. The inhibitor allosterically binds to the citrate-binding domain of ACLY at residues R378 and G380 [ 53 ]. In this study, NDI-091143 reduced the viability and proliferation of MCF7-parent, -LTED, and -Let R cells and inhibited fatty acid degradation, thereby supporting the hypothesis that ACLY stimulates FAO in LTED cells (Fig. 5 A and 5 C–D). This inhibitor also suppressed p-ACLY (S455) and regulated the nuclear translocation of ACLY and p-c-MYC (S62) (Fig. 5 B and S3F). Moreover, NDI-091143 sensitized cells to letrozole and had a synergistic effect with etomoxir, a CPT1 inhibitor, in LTED cells. Furthermore, the combination did not notably affect mouse body weight, confirming that blocking both the ACLY and CPT1 genes may be a potential treatment strategy for NAC-resistant breast cancers (Fig. 6 ). Conclusion Following NAC for HR+ breast cancer, residual cells survive via metabolic adaptation. This manifests as a shift to FAO, which is mediated by the ACLY and c-MYC pathways. Therefore, targeting the FAO metabolic pathway by inhibiting ACLY serves as a promising strategy for overcoming endocrine resistance and achieving good neoadjuvant outcomes. Abbreviations HR+ hormone receptor-positive NAC neoadjuvant chemoendocrine therapy ATP adenosine triphosphate ACLY ATP citrate lyase FAO fatty acid oxidation FASN fatty acid synthase p-ACLY phosphorylated ACLY MYC myelocytomatosis oncogene c-MYC cellular MYC p-c-MYC phosphorylated c-MYC pCR pathological complete response LTED long-term estrogen deprivation TNBC triple-negative breast cancer GEO Gene Expression Omnibus HER2 human epidermal growth factor receptor 2 FFPE formalin-fixed paraffin-embedded GSEA gene set enrichment analysis DMEM/F12 Dulbecco's Modified Eagle’s Medium/Nutrient Mixture F-12 FBS fetal bovine serum csFBS charcoal-stripped FBS Let R letrozole-resistant MCF7-Let R letrozole-resistant MCF7 cell line TBST Tris-buffered saline with 0.1% Tween® 20 detergent BSA bovine serum albumin CPT carnitine palmitoyltransferase GFP green fluorescent protein siRNA small interfering RNA PBS phosphate-buffered saline NGS next generation sequencing FCCP carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone qRT-PCR quantitative reverse transcription polymerase chain reaction cDNA complementary DNA OS overall survival TCGA The Cancer Genome Atlas database ER+ estrogen receptor-positive ACC acetyl-CoA carboxylase TCA tricarboxylic acid OXPHOS oxidative phosphorylation CHX cycloheximide GAPDH glyceraldehyde-3-phosphate dehydrogenase CER cytoplasmic extraction reagent LC-MS/MS liquid chromatography-tandem mass spectrometry. Declarations Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Acknowledgments We thank the NCC Bio Bank of the National Cancer Center, Korea for providing the tissue and blood samples data. Funding This work was supported by the National Cancer Center of Korea (Grant no. 2510771-1). Authors’ information Authors' contributions JHK, SHS, and KSL designed the project; JHK, WS, IH, and JK performed the experiments; JHK, WS, JHH, SHS, IH, JK, and KL analyzed the data; JHK and SHS wrote the manuscript. All the authors have read and agreed to the published version of the manuscript. Corresponding author Corresponding author: Sung Hoon Sim. 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Supplementary Files Supplementaryfigure1.pdf Supplementaryfigure2.pdf Supplementaryfigure3.pdf SupplementaryInformationFinal.docx Supplementaryuncroppedblotimages.pptx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 24 Apr, 2026 Reviewers agreed at journal 15 Apr, 2026 Reviewers invited by journal 15 Apr, 2026 Editor assigned by journal 24 Mar, 2026 Submission checks completed at journal 24 Mar, 2026 First submitted to journal 19 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9173952","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626594640,"identity":"c95a0cfd-6846-45a8-823d-da9f07656d5d","order_by":0,"name":"Ju-Ha Kim","email":"","orcid":"","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Ju-Ha","middleName":"","lastName":"Kim","suffix":""},{"id":626594641,"identity":"580cc863-9ee0-4602-a132-e7cc6dfc9d93","order_by":1,"name":"Woo-Ju Song","email":"","orcid":"","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Woo-Ju","middleName":"","lastName":"Song","suffix":""},{"id":626594642,"identity":"48703683-38c6-4cf5-89e3-7fafe1fdfe91","order_by":2,"name":"In-Wook Hwang","email":"","orcid":"","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"In-Wook","middleName":"","lastName":"Hwang","suffix":""},{"id":626594643,"identity":"3123816d-e063-4789-9948-e1a8690ed73c","order_by":3,"name":"Jinho Kang","email":"","orcid":"","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Jinho","middleName":"","lastName":"Kang","suffix":""},{"id":626594645,"identity":"910999ce-04f2-4ab4-89fc-6b0809207ba6","order_by":4,"name":"Ji Hyung Hong","email":"","orcid":"","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Ji","middleName":"Hyung","lastName":"Hong","suffix":""},{"id":626594647,"identity":"e2b54a30-70c6-4106-95b6-a14e87dee3b5","order_by":5,"name":"Sung Hoon Sim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArElEQVRIiWNgGAWjYDACZhCqgPEOEK3lDElaQLoY20jRIt/Onfy5cN4def4G3ocPGM7cI6zF4DDvNumZ254ZzjjAbmzAcKOYCC3MvNuA6HCCAQMbmwTDhwQiHNbMu/kz7xywFvYfRGlhOMy7QZq3AWILA8MNIrSA/cJz7LDhjMNszBIJZ4hxWP/ZzZ95ag7L87e3MX74cIwYh8EBME4ZSNIwCkbBKBgFowA3AAD01zMPXmyZugAAAABJRU5ErkJggg==","orcid":"","institution":"National Cancer Center","correspondingAuthor":true,"prefix":"","firstName":"Sung","middleName":"Hoon","lastName":"Sim","suffix":""},{"id":626594648,"identity":"ead892e1-234c-4002-8641-918abab62cfe","order_by":6,"name":"Keun Seok Lee","email":"","orcid":"","institution":"National Cancer Center","correspondingAuthor":false,"prefix":"","firstName":"Keun","middleName":"Seok","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2026-03-20 02:23:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9173952/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9173952/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107636114,"identity":"a83b69a9-e800-4735-a614-475aafb47d03","added_by":"auto","created_at":"2026-04-23 12:37:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":648247,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eACLY \u003c/em\u003emRNA levels are increased in breast cancer and upregulated via FAO after antihormonal therapy. \u003cstrong\u003e(A)\u003c/strong\u003e Using TCGA data, the \u003cem\u003eACLY\u003c/em\u003e mRNA levels in the tissues of 1100 patients with invasive breast cancer and 112 patients with normal breast tissues were analyzed (***p \u0026lt; 0.001). \u003cstrong\u003e(B)\u003c/strong\u003e OS according to the ACLY levels was analyzed using Kaplan-Meier analysis in 123 patients with ER+ subtypes of breast cancer in the GEO database (Affymetrix ID: 201128_s_at). \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eACLY\u003c/em\u003e mRNA levels were analyzed using the paired breast tissues collected prior to/after letrozole treatment for 90 days through the clinical trial (NCT02296801); paired t-test, p = 5.3669 × 10\u003csup\u003e-12\u003c/sup\u003e. \u003cstrong\u003e(D)\u003c/strong\u003e GSEA was performed with the prior and post-letrozole treatment groups, and lipid metabolic processes and FAO pathways were examined. \u003cstrong\u003e(E)\u003c/strong\u003e Flow diagram of the study treatment regimen for patients with breast cancer, consisting of neoadjuvant therapy with letrozole, surgery, and adjuvant therapy before recurrence. \u003cstrong\u003e(F)\u003c/strong\u003e This study included 11 paired FFPE samples from patients with HR+/HER2− breast cancer before and after neoadjuvant therapy and analyzed the mRNA levels using the NanoString nCounter metabolic panel. \u003cstrong\u003e(G)\u003c/strong\u003e GSEA was conducted on the basis of the mRNA values associated with lipid metabolic processes and the regulation of FAO gene sets. \u003cstrong\u003e(H)\u003c/strong\u003e The mRNAs of total genes were organized according to the fold change values with statistical significance (p \u0026lt; 0.05) between pre-NAC and post-NAC. \u003cstrong\u003e(I)\u003c/strong\u003e Z scores of the mRNAs were analyzed hierarchically between nine \u003cem\u003eMYC\u003c/em\u003e-high patients and two \u003cem\u003eMYC\u003c/em\u003e-low patients by clustering heatmap analysis to identify differences between these two groups. Abbreviations: ACLY, adenosine triphosphate citrate lyase; TCGA, The Cancer Genome Atlas database; ER+, estrogen receptor-positive; GEO, Gene Expression Omnibus; GSEA, gene set enrichment analysis; FFPE, formalin-fixed paraffin-embedded. HR+, hormone receptor-positive; HER2−, human epidermal growth factor receptor 2 negative; FAO, fatty acid oxidation; OS, overall survival; NAC, neoadjuvant chemoendocrine therapy.\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/4bff695eb93664ed080447d9.jpg"},{"id":107636116,"identity":"cb4ba913-6538-45ee-8339-ff50c748bb13","added_by":"auto","created_at":"2026-04-23 12:37:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":336526,"visible":true,"origin":"","legend":"\u003cp\u003eACLY depletion disrupts c-MYC protein stability in breast cancer cells. \u003cstrong\u003e(A)\u003c/strong\u003e The c-MYC, p-ACLY (S455), and ACLY protein levels were measured in MDA-MB-231 cells after transfection with siNC or siACLY. The protein fold changes were calculated and are shown as bar graphs. \u003cstrong\u003e(B–C)\u003c/strong\u003eUsing #1 siRNA, a c-MYC protein stabilization assay and western blot were performed, and the c-MYC fold change was calculated per the amount of β-actin in MCF7 and MDA-MB-231 cells. \u003cstrong\u003e(D)\u003c/strong\u003e A c-MYC stimulation assay was performed via 20% FBS stimulation, and a western blot analysis was conducted with c-MYC, p-ACLY, ACLY, and β-actin antibodies. \u003cstrong\u003e(E)\u003c/strong\u003e MCF7 cells were transfected with the pcDNA3.0, FLAG-ACLY and GFP-c-MYC plasmids. FLAG protein was precipitated, and the FLAG-binding proteins were lysed and detected via the western blot assay. \u003cstrong\u003e(F)\u003c/strong\u003e Colocalization of ACLY and c-MYC was also assessed using an immunofluorescence assay in which the FLAG tag of ACLY and c-MYC wild-type plasmids were detected. The nuclear portions are indicated by red arrows, and the merged expression of ACLY and c-MYC is shown in yellow. Abbreviations: ACLY, adenosine triphosphate citrate lyase; p-ACLY, phosphorylated ACLY; MYC, myelocytomatosis oncogene; c-MYC; cellular MYC; p-c-MYC, phosphorylated c-MYC; siRNA, small interfering RNA; FBS, fetal bovine serum; GFP, green fluorescent protein.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/6787d6cf7cae9117caf55187.jpg"},{"id":107706285,"identity":"4c798317-616c-4c3b-aba9-1a73fb61e230","added_by":"auto","created_at":"2026-04-24 09:17:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":580323,"visible":true,"origin":"","legend":"\u003cp\u003eMCF7-LTED cells have metabolic characteristics similar to those in clinical post-NAC cases. \u003cstrong\u003e(A)\u003c/strong\u003e HR+ breast cancer cells were cultured for \u0026gt;75 passages without hormones and adapted to hormone (h)-, growth factor (g)-, and cytokine (c)-free conditions with charcoal-stripped FBS (left panel). ACLY and c-MYC expression was evaluated at three time points with 0, 40, and 75 passages after hormone removal and western blot assays. Protein fold changes are shown as bar graphs. \u003cstrong\u003e(B)\u003c/strong\u003e Western blot analysis was performed with c-MYC, ACLY, and β-actin antibodies for MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells. Protein fold changes are shown as bar graphs. \u003cstrong\u003e(C)\u003c/strong\u003e ACLY was suppressed in MCF7-LTED cells with two siRNAs (#1 and #2) and western blot analysis was performed with antibodies against c-MYC, FASN, ACLY, and β-actin antibodies. Protein fold changes are shown as bar graphs. \u003cstrong\u003e(D)\u003c/strong\u003e Whole RNA sequencing was performed using NGS, and mRNA values were analyzed via GSEA to compare the resistant cells and MCF7 parent cells. \u003cstrong\u003e(E)\u003c/strong\u003e The mitochondrial FAO rate was analyzed in MCF7-parent and MCF7-LTED cells using BSA and palmitate:BSA conjugate agents. Each respiration value was measured three times at specific time intervals. The statistical significance of the differences between the spare capacity (FAO) of palmitate:BSA-MCF7-parent and palmitate:BSA-MCF7-LTED cells was analyzed via the student’s t-test (***p \u0026lt; 0.001). \u003cstrong\u003e(F)\u003c/strong\u003e The CPT1A, ACLY, MYC, and β-actin protein expression levels were measured in MCF7-parent and -LTED cells via western blot analysis. Protein fold changes are shown as bar graphs. Abbreviations: LTED, long-term estrogen deprivation; NAC, neoadjuvant chemotherapy; HR+, hormone receptor-positive; FBS, fetal bovine serum; ACLY, adenosine triphosphate citrate lyase; MYC, myelocytomatosis oncogene; c-MYC; cellular MYC; MCF7-Let\u003csup\u003eR\u003c/sup\u003e, letrozole-resistant MCF7 cell line; siRNA, small interfering RNA; FASN, fatty acid synthase; NGS, next generation sequencing; GSEA, gene set enrichment analysis; FAO, fatty acid oxidation; BSA, bovine serum albumin; CPT1A, carnitine palmitoyltransferase 1A.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/48478a2581511f2633272c42.jpg"},{"id":107707391,"identity":"5bce28a6-2dae-4b27-a5df-c5c1b9c4b4a4","added_by":"auto","created_at":"2026-04-24 09:20:13","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":540382,"visible":true,"origin":"","legend":"\u003cp\u003eACLY depletion disrupts c-MYC protein stability, thereby regulating FAO in MCF7-LTED cells. \u003cstrong\u003e(A)\u003c/strong\u003e Using #1 siRNA, c-MYC protein stabilization and western blot assays were performed and the c-MYC fold change was calculated according to the amount of β-actin in MCF7-LTED cells. \u003cstrong\u003e(B)\u003c/strong\u003e MCF7-LTED cells were separated into the cytosol and the nucleus. A western blot analysis was performed with c-MYC, ACLY, topo-1 (nuclear marker), and ɑ-tubulin (cytosolic maker) antibodies. The protein fold changes were calculated and are shown as bar graphs. \u003cstrong\u003e(C)\u003c/strong\u003e Mitochondrial FAO rate was analyzed in #1 siRNA-transfected MCF7-LTED cells using BSA and palmitate:BSA conjugate agents. The following drugs were added: rotenone, a complex I inhibitor; antimycin A, a complex I inhibitor; and oligomycin, an inhibitor of electron transport channel (ETC) complex V. The FAO rate was measured via the spare respiratory capacity values. The statistical significance of the differences was analyzed via Student’s t test (**p \u0026lt; 0.01). \u003cstrong\u003e(D)\u003c/strong\u003e MCF7-parent, -LTED, and -Let\u003csup\u003eR \u003c/sup\u003ecells were transfected with siACLY #1, and western blot assays were performed with CPT1, c-MYC, ACLY, and β-actin antibodies. The protein fold changes were calculated and are shown under the bands. \u003cstrong\u003e(E)\u003c/strong\u003e V5-c-MYC WT and FASN WT plasmids were transfected, followed by siRNA transfection, and FAO assays were subsequently performed. The statistical significance of the differences was analyzed via Student’s t test (*p \u0026lt; 0.05 and **p \u0026lt; 0.01). \u003cstrong\u003e(F)\u003c/strong\u003e Metabolic adaptation through ACLY/c-MYC signaling occurs in the residual cancer cells rather than in primary cancer cells. Abbreviations: ACLY, adenosine triphosphate citrate lyase; MYC, myelocytomatosis oncogene; c-MYC; cellular MYC; FAO, fatty acid oxidation; LTED, long-term estrogen deprivation; siRNA, small interfering RNA; topo-1, type I topoisomerase; BSA, bovine serum albumin; Let\u003csup\u003eR\u003c/sup\u003e, letrozole-resistant; CPT1, carnitine palmitoyltransferase 1; WT, wild-type.\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/904118bc06c7b61626cd91fe.jpg"},{"id":107636121,"identity":"7f6c045a-a4df-4b65-bf81-fcddf2eb9e91","added_by":"auto","created_at":"2026-04-23 12:37:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":425963,"visible":true,"origin":"","legend":"\u003cp\u003eThe ACLY inhibitor NDI-091143 suppresses cancer cell proliferation and FAO, thereby regulating the c-MYC protein. \u003cstrong\u003e(A)\u003c/strong\u003e A cytotoxicity assay was performed with the ACLY inhibitor NDI-091143 (NDI). The statistical significance of the differences was analyzed via Student’s t test (*p \u0026lt; 0.05). \u003cstrong\u003e(B)\u003c/strong\u003e Protein expression was identified using western blot assays with p-c-MYC (S62), c-MYC, p-ACLY (S455), ACLY, and β-actin antibodies after 1 h of NDI treatment. The protein fold changes were calculated and are shown under the bands. \u003cstrong\u003e(C–D)\u003c/strong\u003e FAO was measured via the fluorescence-labeled palmitate BODIPY-C16. The fluorescence was detected in real time using an imaging machine every 4 h and was calculated per nucleus amount via Hoechst staining. The cells after 0 h and 96 h of incubation are displayed. The statistical significance of the differences was analyzed via Student’s t test (**p \u0026lt; 0.01). Abbreviations: ACLY, adenosine triphosphate citrate lyase; p-ACLY, phosphorylated ACLY; FAO, fatty acid oxidation; MYC, myelocytomatosis oncogene; c-MYC; cellular MYC; p-c-MYC, phosphorylated c-MYC.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/07cb97c889349e1b3431a3fe.jpg"},{"id":107636123,"identity":"a38e339e-32de-4aff-a71a-5181bc3627dc","added_by":"auto","created_at":"2026-04-23 12:37:35","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":620661,"visible":true,"origin":"","legend":"\u003cp\u003eNDI-091143 (NDI) increased cell sensitivity to letrozole and had a synergistic effect with etomoxir (Eto). \u003cstrong\u003e(A)\u003c/strong\u003e A cell viability assay was performed in MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells treated with NDI and letrozole. The statistical significance of the differences was analyzed via Student’s t test (*p \u0026lt; 0.05). \u003cstrong\u003e(B)\u003c/strong\u003e A cell viability assay was performed in MCF7-parent and MCF7-LTED cells treated with NDI or Eto. The synergistic effect was calculated using Calcusyn software (bottom panel). The statistical significance of the difference was analyzed using via Student’s t-test (***p \u0026lt; 0.001). \u003cstrong\u003e(C)\u003c/strong\u003e \u003cem\u003eIn vivo\u003c/em\u003e, the toxicity of the combination treatment of NDI and Eto was verified by determining the changes in body weight. \u003cstrong\u003e(D)\u003c/strong\u003e FAO activation through c-MYC/ACLY signaling is inhibited by NDI treatment, which inhibits ACLY. Eto, a CPT1 inhibitor, exerts a synergistic effect with NDI treatment by simultaneously blocking dual cancer metabolism. Abbreviations: CPT1, carnitine palmitoyltransferase 1; MCF7-Let\u003csup\u003eR\u003c/sup\u003e, letrozole-resistant MCF7 cell line; LTED, long-term estrogen deprivation; FAO, fatty acid oxidation; ACLY, adenosine triphosphate citrate lyase; MYC, myelocytomatosis oncogene; c-MYC; cellular MYC.\u003c/p\u003e","description":"","filename":"fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/3b39efc54af8a65206a15377.jpg"},{"id":107709225,"identity":"fea69a7f-644e-416b-b373-4dab9684b4e5","added_by":"auto","created_at":"2026-04-24 09:35:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3573025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/fee227a4-37df-4191-9bf9-336fdd795e6b.pdf"},{"id":107636113,"identity":"b793f118-92cd-4856-a8d0-4082ab7191ec","added_by":"auto","created_at":"2026-04-23 12:37:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":56753,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/ed27fd72dbec4b741e6b7da2.pdf"},{"id":107707568,"identity":"e13126ad-7958-4c85-bb85-82961b0a9157","added_by":"auto","created_at":"2026-04-24 09:20:36","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":317667,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/cf79a89a3955ec1b807c2e7b.pdf"},{"id":107706026,"identity":"91159832-c282-4e09-8358-c09884c1d0b6","added_by":"auto","created_at":"2026-04-24 09:17:10","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":484709,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/b2bd43b0669c0eb4c81bacc9.pdf"},{"id":107707503,"identity":"c3b75ed9-e217-4319-aca1-5000d2863d69","added_by":"auto","created_at":"2026-04-24 09:20:28","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17713,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationFinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/585aed8830483829851633ce.docx"},{"id":107636118,"identity":"c7231c3f-cb5b-4b4e-9be9-2d9987804715","added_by":"auto","created_at":"2026-04-23 12:37:34","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":11545536,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryuncroppedblotimages.pptx","url":"https://assets-eu.researchsquare.com/files/rs-9173952/v1/c7d47201a564e86e41fb8d6d.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Adenosine triphosphate citrate lyase alters cellular myelocytomatosis oncogene signaling and fatty acid oxidation in residual breast cancer tumor tissues after neoadjuvant treatment","fulltext":[{"header":"Background","content":"\u003cp\u003eAmong all cancer types worldwide, breast cancer has the highest incidence and second-highest mortality rate [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Approximately 70% of breast cancer cases are hormone receptor-positive (HR+). In cases of locally advanced breast cancer, neoadjuvant treatment via chemotherapy or endocrine therapy has been introduced to reduce tumor size in patients with HR+ breast cancer. Despite efforts to increase treatment efficacy, the complete remission rate varies widely, ranging from 5\u0026ndash;20% in patients with HR+ breast. A phase II clinical trial was conducted to evaluate the ability of neoadjuvant chemoendocrine therapy (NAC) to improve treatment outcomes. Although the response rate increased, the pathological complete response (pCR) rate was only 3.8% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite advancements in antihormone therapies and chemotherapeutic agents, some breast cancer cells persist after treatment. Metabolic adaptation is a critical mechanism that facilitates cancer cell survival. Metabolic reprogramming underlies endocrine resistance, with single-cell subpopulations contributing to drug tolerance and residual cells exhibiting lipid metabolism signatures [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLipids, including fatty acids, triglycerides, and lipoids, play vital roles in cancer metabolism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Under certain conditions, cancer cells utilize fatty acids as an energy source to produce adenosine triphosphate (ATP) via oxidative phosphorylation (OXPHOS) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Fatty acid oxidation (FAO) is crucial for drug resistance and cancer aggressiveness [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, FAO was identified as a key metabolic pathway involved in the growth of triple-negative breast cancers (TNBC) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], particularly in myelocytomatosis oncogene (MYC)-high TNBC [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn patients with HR+ breast cancer, cellular MYC (c-MYC) amplification is correlated with a high pCR rate after NAC therapy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Moreover, high c-MYC scores are considerably correlated with reduced recurrence-free survival in patients with long-term estrogen deprivation (LTED), whereas c-MYC depletion suppresses LTED-related breast cancer cell growth; therefore, c-MYC mediates antiestrogen resistance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, c-MYC regulates androgen receptors, which are aromatase substrates, through epigenetic factors, such as disruptor of telomeric silencing 1-like [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, c-MYC inhibition and overexpression induce notable metabolic changes in tumor cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Overexpression of c-MYC stimulates FAO via calcium signaling [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], whereas c-MYC inhibition causes the accumulation of lipid droplets in tumor cells [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], regulates lipid signaling, and influences mitochondrial acetyl-CoA levels [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eATP citrate lyase (ACLY) is upregulated in human breast cancer cells, and its depletion inhibits tumor growth [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and promotes apoptosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. ACLY regulates the acetylation of histones H3 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and H4 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] after their translocation to the nucleus. DNA damage signaling through ionizing radiation increases ACLY and phosphorylated ACLY (p-ACLY) (S455) expression in the nucleus [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Although ACLY catalyzes acetyl-CoA production from citrate and transfers it to fatty acid synthase (FASN), new roles for ACLY have been revealed, including lipid peroxidation and OXPHOS [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. ACLY increases histone acetylation via EP300 at the melanocytic lineage oncogenic factor locus, thereby promoting mitochondrial OXPHOS, cancer growth, and drug resistance [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Conversely, ACLY inhibition reduces OXPHOS under low-glucose conditions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite these discoveries, the mechanisms of FAO rewiring in NAC-resistant-HR+ breast cancers are unclear. Therefore, this study aimed to investigate the metabolic signal reprogramming in residual tumor tissues after chemoendocrine treatment. Overall, the study revealed that the action of ACLY and c-MYC during FAO is crucial for the survival of HR+ breast cancer cells.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePublic data analysis\u003c/h2\u003e \u003cp\u003eThe mRNA microarray data were downloaded from [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The data specifications were patients with breast cancer treated with neoadjuvant therapy (letrozole oral administration, 2.5 mg/day), and mRNA samples were taken sequentially from biopsies of non-treated cancer and the same cancers after 90 days. ACLY mRNA levels were analyzed using Gene Expression Omnibus (GEO) data (accession number: GSE20181, Affymetrix probe ID: 201128_s_at, NCT02296801) on the Affymetrix Human Genome U133A Array platform. CEL data were also downloaded, normalized, and analyzed via the Bioconductor (version 3.14) package (BiocManger 1.30.16) in R studio (version 4.1.0).\u003c/p\u003e \u003cp\u003eACLY mRNA levels were compared between 112 normal controls and 1100 patients with breast cancer reported in The Cancer Genome Atlas database (TCGA, version 2016.01.28).\u003c/p\u003e \u003cp\u003eSurvival plot data were downloaded via the Kaplan-Meier plotter [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] from the GEO database (GSE7390) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] to identify whether patient prognosis was affected by ACLY mRNA levels in node-negative, estrogen-positive, and human epidermal growth factor receptor 2 (HER2)-negative breast cancers. In 123 patients, overall survival (OS) rates were calculated with a cutoff value of 3134 (Affymetrix probe ID: 201128_at) between the upper and lower quartiles.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePatient tissue sample analysis\u003c/h3\u003e\n\u003cp\u003eFormalin-fixed paraffin-embedded (FFPE) residual tumor tissues from patients with HER2-positive breast cancer and those with TNBC after neoadjuvant treatment were obtained from the National Cancer Center Bio Bank (NCCTTR-18005; Goyang, ROK). HR+ residual tissues were derived from a phase II neoadjuvant trial, which aimed to assess the efficacy of a chemoendocrine combination treatment strategy (IRB No. NCC2017-0110, Phase II Study of Neo-adjuvant Chemotherapy with Letrozole in Patients with Estrogen Receptor Positive/HER-2 Negative Breast Cancer, NCT03497702. Registered 18 March 2018, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://clinicaltrials.gov/study/NCT03497702\u003c/span\u003e\u003cspan address=\"https://clinicaltrials.gov/study/NCT03497702\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Eleven biopsy and surgery samples from the cohort were stored as FFPE samples and used for mRNA analysis. The patients were administered doxorubicin and cyclophosphamide four times followed by paclitaxel and docetaxel four times as neoadjuvant cytotoxic therapy. Letrozole was continuously administered to the patients undergoing treatment. After surgery and tissue collection, 11 paired tumor samples were stored as FFPE tissues and used for NanoString analysis.\u003c/p\u003e \u003cp\u003eFor mRNA quantification, 201 metabolism-related genes and 12 housekeeping genes were used. The mRNA levels were calculated using digital transcript counting (nCounter metabolic pathways panel assay, NanoString, Seattle, WA, USA). Total RNA (100 ng) was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The samples were assayed on an nCounter Digital Analyzer (NanoString) according to the manufacturer\u0026rsquo;s instructions. The data were normalized to the housekeeping gene probes for each sample. The heatmap was plotted using Multiple Experiment Viewer software. The gene set enrichment score graph was analyzed via gene set enrichment analysis (GSEA) software, version 4.1.0 (Broad Institute and UCSD, MA and CA, USA).\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eAll human breast cancer cell lines, including MCF7, T47D, and MDA-MB-231, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). T47D and MDA-MB-231 breast cancer cells were cultured in Roswell Park Memorial Institute 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). MCF7 parent and MCF7-LTED cell lines were cultured in Dulbecco's modified Eagle\u0026rsquo;s medium/nutrient mixture F-12 (DMEM/F12, Gibco, MA, USA) supplemented with GlutaMAX (Gibco). All cell lines, excluding LTED cells, were supplemented with 10% fetal bovine serum (FBS; Gibco). LTED cell lines were generated with 10% charcoal-stripped FBS (csFBS; Gibco) instead of FBS and were maintained under these conditions until the number of culture passages surpassed 75; these cell lines were subsequently used for all experiments. The letrozole-resistant (Let\u003csup\u003eR\u003c/sup\u003e) MCF7 cell line (MCF7-Let\u003csup\u003eR\u003c/sup\u003e) was purchased from the European Collection of Authenticated Cell Cultures (MCF7/LetR-1; Salisbury, UK). The MCF7-Let\u003csup\u003eR\u003c/sup\u003e cell line was cultured in DMEM/F12 supplemented with GlutaMAX, 0.1 \u0026micro;M testosterone, and 1 \u0026micro;M letrozole. The parental cell line was maintained under the same conditions as the MCF7-Let\u003csup\u003eR\u003c/sup\u003e cell line but without letrozole. All the cell lines were cultured with a 1% antibiotic solution comprising penicillin at 10,000 units/mL, streptomycin at 10,000 \u0026micro;g/mL, and amphotericin B at 25 \u0026micro;g/mL (Gibco) at 37℃ under 5% CO\u003csub\u003e2\u003c/sub\u003e in a humidified chamber.\u003c/p\u003e\n\u003ch3\u003eWestern blotting\u003c/h3\u003e\n\u003cp\u003eThe cells were lysed in RIPA buffer (Merck KGaA, Darmstadt, Germany) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitors (Merck KGaA). Lysates were quantified using a BCA protein assay kit (Thermo Fisher Scientific). The protein samples were electrophoresed on 8% handmade sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membranes with a pore size of 0.4 \u0026micro;m. The membranes were blocked with Tris-buffered saline with 0.1% Tween\u0026reg; 20 detergent (TBST), diluted with 3% skim milk, or bovine serum albumin (BSA) for 1\u0026ndash;2 h at room temperature. The membranes were then incubated with primary antibodies and β-actin (Cat. no. A2228, Merck KGaA) diluted in 3% BSA in TBST overnight at 4 ℃. Subsequently, the membranes were washed three times for 10 min with TBST and incubated with horse radish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA, USA) for 2 h at room temperature. ACLY (Cat. No. 4332, Cell Signaling Technology, Danvers, MA, USA or Cat. no. ab40793, Abcam, Cambridge, UK), p-ACLY S455 (Cat. no. 4331, Cell Signaling Technology), c-MYC (Cat. no. 5605, Cell Signaling Technology), phosphorylated c-MYC (p-c-MYC S62; Cat. no. ab185656, Abcam), FASN (Cat. no. 3180, Cell Signaling Technology), carnitine palmitoyltransferase (CPT)1A (Cat. no. 12252, Cell Signaling Technology), type I topoisomerase (Cat. no. sc-32736, Santa Cruz Biotechnology, Dallas, TX, USA), ɑ-tubulin (Cat. no. sc-32293, Santa Cruz Biotechnology), β-actin (Cat. no. 4970, Cell Signaling Technology), FLAG (Cat. no. 8146, Cell Signaling Technology), and green fluorescent protein (GFP; Cat.no.sc-8834, Santa Cruz Biotechnology) were used for the experiments. Expression was visualized using enhanced chemiluminescence Immunoblotting Detection Reagent (Thermo Fisher Scientific), and fluorescence images were captured with an Invitrogen iBright FL1500 system (Thermo Fisher Scientific).\u003c/p\u003e\n\u003ch3\u003eRNA interference and plasmid transfection\u003c/h3\u003e\n\u003cp\u003eThe cells were seeded onto culture plates overnight at 40% confluence and transfected with a mixture of ACLY small interfering RNA (siRNA) or negative control siRNA purchased from Bioneer (Daejeon, ROK) and Thermo Fisher Scientific. The siACLY #1 sequence was RNA- (GUGUA)CAGCGAUACCAUC U/RNA-AGAUGGUAUCGCUG (UACAC). The siACLY #2 sequence was RNA-GGGUGUCAACGAGCUGGCAAACUAU/RNA-AUAGUUUGCCAGCUCGUUGACACCC. INTERFERin transfection reagent (40 nM; Polyplus, Illkirch, France) was used according to the manufacturer\u0026rsquo;s protocol. The transfected cells were then incubated for 72 h. FASN (Cat. No. SC122268, pCMV6-XL5) and ACLY (Cat. No. RC200508, pCMV6-Entry) were purchased from OriGene (Rockville, MD, USA). The pcDNA 3.0 and c-MYC-GFP (Cat. no. 42142) antibodies were purchased from Addgene (Watertown, MA, USA). V5-c-MYC has been described previously [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The cells were transfected with TurboFect transfection reagent (Thermo Fisher Scientific) and incubated for 36 h for further study.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCycloheximide (CHX) assay\u003c/h2\u003e \u003cp\u003eThe cells transfected with siNC and ACLY siRNA for 72 h were exposed to 50 \u0026micro;g/mL CHX (Merck KGaA) at the indicated concentrations and for the indicated durations (0, 10, 20, and 40 min). Cycloheximide (CHX was used to verify whether ACLY regulated c-MYC stability.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eThe cells transfected with siRNAs were fixed with 4% paraformaldehyde\u003c/p\u003e \u003cp\u003e(Sigma Aldrich, St. Louis, MO, USA) for 20 min at room temperature and permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 2 min on ice. The cells were labeled with primary antibodies diluted in 1% BSA/phosphate-buffered saline (PBS) overnight at 4\u0026deg;C. The cells were then exposed to secondary Alexa Fluor (Invitrogen, Thermo Fisher Scientific) diluted with 1% BSA/PBS for 2 h at room temperature. The samples were mounted with mounting medium (VectorLabs, Newark, CA, USA) containing 4\u0026prime;,6-diamidino-2-phenylindole, detected with fluorescence, and imaged using an Axio Observer Z1 (Carl Zeiss, Oberkochen, Germany).\u003c/p\u003e\n\u003ch3\u003eImmunoprecipitation\u003c/h3\u003e\n\u003cp\u003eMCF7 cells transfected with the plasmids were lysed and quantified according to western blotting protocols. A total of 2 \u0026micro;g of antibodies against DYKDDDDK (FLAG; Cat. No. 8146, Cell Signaling Technology) was added to 500 \u0026micro;g lysate, which was subsequently incubated at 4\u0026deg;C in a rotator overnight. A total of 30 \u0026micro;L of protein A/G agarose beads (Santa Cruz Biotechnology) was added, and the mixture was rotated at 4\u0026deg;C for 4 h. The resulting lysates were washed three times with NP40 lysis buffer (Thermo Fisher Scientific). The bound proteins were immunoblotted as previously described. The protein input amount was 10% of the immunoprecipitated samples.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTransient stimulation of the c-MYC protein\u003c/h2\u003e \u003cp\u003eThe c-MYC and ACLY protein levels in breast cancer cells were determined after stimulation with 20% FBS (Thermo Fisher Scientific). To artificially stimulate c-MYC, 20% FBS was added at the indicated time points following serum starvation for 36 h. Protein levels were evaluated via western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNext-generation sequencing (NGS) analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from MCF7-parent and MCF7-LTED cells using QIAzol lysis reagent (Qiagen), and RNA quality was assessed by Macrogen (Seoul, ROK). The samples were processed via next-generation sequencing (NGS) sequence analysis (NextSeq 500; Illumina, San Diego, CA, USA). The raw NGS data were deposited in the National Centre for Biotechnology Information BioProject database (accession number: PRJNA802345).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial respiration and FAO assay\u003c/h2\u003e \u003cp\u003eThe cells were seeded onto an XF96 plate using the Seahorse Mito-Stress Kit and Seahorse XF Palmitate Oxidation Stress Test Kit (Agilent Technologies, Santa Clara, CA, USA). The cell growth medium was changed to substrate-limited medium (Agilent Technologies), and the cells were incubated with the medium overnight. Each reagent was placed in a cartridge according to the manufacturer\u0026rsquo;s protocol (Agilent Technologies).\u003c/p\u003e \u003cp\u003eBasal respiration values were calculated three times at intervals. Nonmitochondrial respiratory consumption was measured after treatment with rotenone (complex I inhibitor) or antimycin A (complex I inhibitor). The ATP-linked respiration rate was calculated using oligomycin, which is an inhibitor of the electron transport channel complex V. Maximum respiration was artificially induced using the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). When palmitate-conjugated BSA was used as the energy source, instead of BSA alone, FAO was calculated using spare capacity values, which were calculated by subtracting basal respiration from maximum respiration. The following equations were used: nonmitochondrial oxygen consumption\u0026thinsp;=\u0026thinsp;minimum rate measurement after rotenone / antimycin A injection; basal respiration\u0026thinsp;=\u0026thinsp;last rate measurement before first injection\u0026thinsp;\u0026minus;\u0026thinsp;nonmitochondrial respiration rate; maximal respiration\u0026thinsp;=\u0026thinsp;maximum rate measurement after FCCP injection\u0026thinsp;\u0026minus;\u0026thinsp;nonmitochondrial respiration; spare respiratory capacity\u0026thinsp;=\u0026thinsp;maximal respiration\u0026thinsp;\u0026minus;\u0026thinsp;basal respiration; and spare respiratory capacity (%) = maximal respiration / basal respiration * 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eBODIPY-C16 assay\u003c/h2\u003e \u003cp\u003eThe cells were grown in Operetta 96-well culture plates to measure lipid degradation. BODIPY-C16 (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid, Thermo Fisher Scientific) was added to the medium (final concentration\u0026thinsp;=\u0026thinsp;1 \u0026micro;M), incubated for the designated duration, and exchanged immediately before detection with Hoechst staining. Fluorescence was detected using an Operetta CLS real-time imaging machine (Perkin-Elmer, Waltham, MA, USA) every 4 h and calculated per nucleus stained with Hoechst. The fluorescent cells were traced via the Alexa 488 and Hoechst channels designated in Harmony software v4.8 (Perkin-Elmer).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eRNAs isolated from the cells were lysed using QIAzol reagent (Qiagen) according to the manufacturer\u0026rsquo;s protocol. A total of 2 \u0026micro;g of each RNA sample was synthesized into complementary DNA (cDNA) with oligo-deoxythymidines, deoxynucleoside triphosphates (Takara, Shiga, Japan), M-MLV reverse transcriptase, and its buffer (Enzynomics, Daejeon, ROK) following the manufacturer\u0026rsquo;s protocol. The cDNA samples (100 ng) were placed in the wells of a qRT-PCR plate for each reaction. The primers used were synthesized by Bioneer (Daejeon, ROK). The primers for \u003cem\u003eMYC\u003c/em\u003e, \u003cem\u003eCPT1\u003c/em\u003e, \u003cem\u003eFASN\u003c/em\u003e, \u003cem\u003eACLY\u003c/em\u003e, and glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) were as follows: \u003cem\u003eMYC\u003c/em\u003e, 5\u0026rsquo;-AAGAGGGTCAAGTTGGACAGT-3\u0026rsquo; (Forward) and 5\u0026rsquo;-CGTTTTAGCTCGTTCCTCCTC-3\u0026rsquo; (Reverse); \u003cem\u003eCPT1\u003c/em\u003e, 5\u0026rsquo;-TCTGCCTTTACGTGGTGTCTA-3\u0026rsquo; (Forward) and 5\u0026rsquo;-CTGGACACGTACTCTGGGTTA-3\u0026rsquo; (Reverse); \u003cem\u003eFASN\u003c/em\u003e, 5\u0026rsquo;-CGTTGACCTGGTCTTGAACTC-3\u0026rsquo; (Forward) and 5\u0026rsquo;-CGTGGAATGTCACGTTCTTCA-3\u0026rsquo; (Reverse); \u003cem\u003eACLY\u003c/em\u003e, 5\u0026rsquo;-CTGTGATCTAGGGGGTGTCAA-3\u0026rsquo; (Forward) and 5\u0026rsquo;-TGCCTCCAATGATGAGGATCT-3\u0026rsquo; (Reverse); and \u003cem\u003eGAPDH\u003c/em\u003e, 5\u0026rsquo;-GACGGTGCCATGGAATTTGC-3\u0026rsquo; (Forward) and 5\u0026rsquo;-ATGGGGAAGGTGAAGGTCGG-3\u0026rsquo; (Reverse).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNucleus and cytosol isolation\u003c/h2\u003e \u003cp\u003eThe cells were harvested using trypsin-ethylenediamine tetraacetic acid and centrifuged at 1000 rpm for 5 min. The cell samples were washed by suspending the cell pellets in PBS. Following the manufacturer\u0026rsquo;s protocol (NE-PER Nuclear and Cytoplasmic Extraction Reagents; Thermo Fisher Scientific), ice-cold cytoplasmic extraction reagent (CER) I was added to the cell pellet, vortexed for 15 s, and incubated for 10 min. Ice-cold CER II was then added, and the mixture was vortexed for 5 s, incubated for 15 min, and centrifuged at 15000 rpm for 5 min. Thereafter, the supernatant was collected as the cytosol, and the nuclear extraction reagent solution was added to the pellet. The sample was then vortexed for 15 s and incubated for 10 min; this process was repeated four times. The mixture was then centrifuged at 15000 rpm for 10 min, and the supernatant was collected and used as the nucleus for western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLiquid chromatography-tandem mass spectrometry (LC-MS/MS)\u003c/h2\u003e \u003cp\u003eMetabolites involved in energy metabolism were analyzed using LC-MS/MS with a 1290 HPLC system (Agilent Technologies), Qtrap 5500 (ABSciex, Framingham, MA, USA), and a reverse phase column (Synergi fusion RP 50 \u0026times; 2 mm). The sample (3 \u0026micro;L) was injected into the LC-MS/MS system and ionized with a turbo spray ionization source. Mobile phases A and B comprised 5 mM of ammonium acetate in H\u003csub\u003e2\u003c/sub\u003eO and 5 mM of ammonium acetate in methanol, respectively. The separation gradient was as follows: maintained at 0% B for 5 min, 0\u0026ndash;90% B for 2 min, 90% B for 8 min, 90\u0026ndash;0% B for 1 min, and 0% B for 9 min. The liquid chromatography flow rate was 70 \u0026micro;L/min but was increased to 140 \u0026micro;L/min between 7\u0026ndash;15 min, and the column temperature was maintained at 23\u0026deg;C. Multiple reaction monitoring was used in negative ion mode, and the extracted ion chromatogram corresponding to the specific transition for each metabolite was used for quantification. The area under the curve of each extracted ion chromatogram was normalized to that of the internal standard chromatogram. The ratio of the peak area of each metabolite to that of the internal standard was normalized using the protein content of each sample and was used for relative comparison. Data analysis was performed using Analyst 1.5.2 software (SCIEX, Framingham, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMouse experiments\u003c/h2\u003e \u003cp\u003e Two animal studies were performed in accordance with the Animal Use Protocol (IACUC numbers: NCC-21-660 and NCC-21-704) approved by the National Cancer Center IACU Committee. BALB/c female athymic nude mice (four weeks old, Orient Bio, Seongnam, ROK) were used. For drug toxicity identification before the xenograft model was established, 68 mice were allowed to rest for one week after receiving the animal at the animal facility. The body weights of four mice per group were measured every 2\u0026ndash;3 days over a 21-day period.\u003c/p\u003e \u003cp\u003eFor additional studies, the mice were anesthetized via inhalation with 2% isoflurane, and a 17β-estradiol pellet (Cat. No. E8875, Sigma Aldrich) was implanted on the lateral side of the neck between the ear and the shoulder. The drugs were injected intraperitoneally into mice one day after implantation of the 17β-estradiol pellet. For the xenograft experiments, all procedures were performed in the same manner as those used for the drug toxicity experiments before the cancer cells were injected. Cancer cells (5 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells) suspended in 50% saline/50% Matrigel (Cat. No. 354234, BD Biosciences, Franklin Lakes, NJ, USA) were injected into the fourth mammary fat pad. Tumor size was monitored while the drugs were injected intraperitoneally into the mice, and the mice were euthanized via 2\u0026ndash;5% isoflurane inhalation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations from at least three independent experiments. Student\u0026rsquo;s t test for two-group comparisons and one-way analysis of variance, followed by Tukey\u0026rsquo;s post-hoc test, were conducted for multigroup comparisons via GraphPad Prism software (version 5.0; San Diego, CA, USA). Differences were considered significant if the p value was less than 0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eACLY\u003c/b\u003e \u003cb\u003emRNA was increased in breast cancers and upregulated after antihormonal therapy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAbnormal amplification of \u003cem\u003eACLY\u003c/em\u003e in patients with breast cancer has been reported in several cohorts, including African-American [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and Chinese women [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. To investigate whether this pattern was also common in other groups, public and clinical data were analyzed. The TCGA data revealed that \u003cem\u003eACLY\u003c/em\u003e mRNA levels in breast cancer tissues were markedly increased in 1100 patients with invasive breast cancer compared with those in 112 patients with normal breast tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To identify the differences in \u003cem\u003eACLY\u003c/em\u003e expression in patients with HR+ breast cancer, OS was analyzed in 123 patients with estrogen receptor-positive (ER+) breast cancer using the GEO database. Compared with the low \u003cem\u003eACLY\u003c/em\u003e-expression group, the high \u003cem\u003eACLY\u003c/em\u003e-expression group had notably different OS rates, indicating a poor prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether antiestrogen treatment affects \u003cem\u003eACLY\u003c/em\u003e expression, a clinical trial (ClinicalTrials.gov ID NCT02296801) of patients with ER+ breast cancer treated with letrozole was conducted. \u003cem\u003eACLY\u003c/em\u003e mRNA expression was markedly upregulated 90 days after letrozole administration (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, GSEA revealed that lipid metabolic processes and FAO pathways were upregulated 90 days after letrozole administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003cem\u003eACLY\u003c/em\u003e and \u003cem\u003eFASN\u003c/em\u003e expression levels in various clinical samples were investigated. The clinical data [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] included 11 paired samples from patients with HR+/HER2\u0026thinsp;\u0026minus;\u0026thinsp;breast cancer before and after NAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, see Additional file 1 Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The \u003cem\u003eACLY\u003c/em\u003e and \u003cem\u003eMYC\u003c/em\u003e mRNA expression levels were markedly increased after NAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). In contrast, \u003cem\u003eFASN\u003c/em\u003e mRNA levels decreased, which opposed the \u003cem\u003eACLY\u003c/em\u003e pattern; however, \u003cem\u003eACLY\u003c/em\u003e is an upstream gene of \u003cem\u003eFASN\u003c/em\u003e, thereby inducing fatty acid synthesis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. When gene expression levels were analyzed via GSEA, lipid metabolic processes and the regulation of FAO gene sets in the post-NAC group were upregulated (shown as B in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG) compared with those in the pre-NAC group. To elucidate the importance of \u003cem\u003eMYC\u003c/em\u003e levels in metabolic gene patterns, the changes before and after NAC were analyzed and presented, with a focus on genes whose p values were less than 0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI, a similar pattern was observed within the \u003cem\u003eMYC-\u003c/em\u003ehigh group, which was distinct from the pattern observed in the \u003cem\u003eMYC-\u003c/em\u003elow group, thereby indicating that c-MYC may mediate metabolism in this cohort.\u003c/p\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eACLY depletion disrupted c-MYC protein stability in breast cancer cells\u003c/h2\u003e \u003cp\u003eTo investigate whether ACLY regulates c-MYC to regulate metabolism in the cells, ACLY was suppressed using two siRNAs targeting the citrate-binding sites, which are the main sites that block enzyme activity and acetyl-CoA genesis. The decrease in c-MYC expression was dependent on ACLY levels, whereas FASN showed the opposite pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). In addition, c-MYC protein stabilization and stimulation assays were performed to determine whether ACLY post-transcriptionally regulates c-MYC protein expression in breast cancer cells. ACLY knockdown disrupted c-MYC protein stability, rendering the half-life of c-MYC in breast cancer cells to be shorter than that of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;C). ACLY depletion also blocked FBS-induced stimulation of the c-MYC protein in breast cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and immunoprecipitation suggested an interaction between ACLY and c-MYC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Moreover, the immunofluorescence assay revealed the colocalization of ACLY and c-MYC (yellow color), which suggests the merging of c-MYC and ACLY (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). Depletion of ACLY also downregulated c-MYC protein in the nucleus, indicating that ACLY may mediate c-MYC protein regulation in the nucleus (see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eEffects of ACLY on altered metabolism in csFBS-treated model cells\u003c/h2\u003e \u003cp\u003eThe csFBS removed hormones and growth factors from cells but retained metabolites, such as cholesterol (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. LTED cells adapted to csFBS show altered gene signatures and paclitaxel resistance [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]; hence, these cells are considered effective models of NAC resistance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The estrogen independence of LTED cells was also confirmed via a cell viability assay (see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene profiles of LTED cells are associated with \u003cem\u003eMYC\u003c/em\u003e activation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Both c-MYC and ACLY expression levels were upregulated in correlation with the duration of hormone deprivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, right panel). Elevated c-MYC and ACLY levels were also confirmed in MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD and S2E). To determine whether ACLY mediates c-MYC expression in MCF7-LTED cells, ACLY was depleted via two siRNAs, as in MCF7 cells. ACLY depletion downregulated c-MYC expression, whereas FASN had the opposite effect, which was consistent with the results in MCF7 and MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eF). Whole RNA sequencing was conducted, and the mRNA values were analyzed with GSEA to determine whether these gene patterns affect metabolic characteristics. MCF7-LTED and MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells presented higher enrichment scores for fatty acid derivative metabolism, very long-chain fatty acid metabolism, and beta-oxidation (acyl-CoA oxidase gene sets) than MCF7-parent cells did (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These results indicate that MCF7-LTED and MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells share metabolic signatures similar to those of NAC clinical cases, thereby reflecting adaptation to hormone removal. These findings provide a basis for studying metabolic reprogramming.\u003c/p\u003e \u003cp\u003eThe metabolic gene sets have been validated via biochemical experiments. To confirm fatty acid (beta)oxidation, the FAO rate of the mitochondria was analyzed in MCF7-parent and MCF7-LTED cells. FAO activation was greater in the MCF7-LTED cells than in the parent cells, as indicated by the markedly higher spare respiratory rate for palmitate:BSA in MCF7-LTED cells than in the parent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Total mitochondrial oxidative respiration was also greater in MCF7-LTED, T47D-LTED, and MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells than in the parent cells (see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eG). The expression of CPT1A, a pivotal regulator of FAO [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], was also increased in MCF7-LTED and MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eACLY depletion interrupts c-MYC protein stability, thereby regulating FAO in MCF7 LTED cells\u003c/h2\u003e \u003cp\u003eNext, whether ACLY mediates c-MYC expression and whether metabolic reprogramming occurs when ACLY regulates c-MYC in the presence of csFBS were investigated. \u003cem\u003eACLY\u003c/em\u003e knockdown attenuated c-MYC stability in MCF7-LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and c-MYC expression decreased in the nucleus with the depletion of ACLY, which is translocated into the nucleus [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The lower-sized band was similar to the band size reported by Sivanand et al. and was confirmed with p-ACLY (S455) levels in the nucleus [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFAO assays were performed to investigate the role of ACLY in FAO upregulation in LTED cells. ACLY depletion disrupted FAO gene and protein expression in MCF7-LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). To investigate the mechanism by which ACLY differentially regulates OXPHOS and metabolite profiles in the absence of fatty acid stimulation in MCF7-parent and MCF7-LTED cells, a mitochondrial respiration assay and LC-MS/MS were conducted. Although ACLY manipulation had no considerable effect on mitochondrial spare respiration or energy metabolites in MCF7 cells, LTED cells exhibited a slight reduction following ACLY depletion (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA\u0026ndash;B).\u003c/p\u003e \u003cp\u003eA key FAO gene (\u003cem\u003eCPT1\u003c/em\u003e) increased protein levels in LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF); therefore, whether ACLY signaling regulates FAO through \u003cem\u003eCPT1\u003c/em\u003e was investigated. When ACLY expression was reduced, the decrease in CPT1 levels in LTED or Let\u003csup\u003eR\u003c/sup\u003e cells was less pronounced than that in parent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings suggest that FAO in LTED or Let\u003csup\u003eR\u003c/sup\u003e cells may be considerably influenced by other proteins rather than solely by those affecting parent cells. To clarify whether c-MYC mediates this mechanism in LTED cells, an FAO assay was performed following the transfection of LTED cells with the c-MYC or FASN plasmid. Deregulation of ACLY decreased FAO, and FAO was notably recovered by c-MYC, but not by FASN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and S3C). These findings suggest that csFBS-treated cells, which mimic post-NAC conditions, exhibit FAO activation mediated by the ACLY/c-MYC pathway. Moreover, these findings indicate that metabolic adaptation through ACLY/c-MYC signaling occurs in residual cancer cells, which may promote cancer recurrence [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, the left panel shows that FAO was blocked by malonyl-CoA, a product of acetyl-CoA carboxylase (ACC), in primary cancer cell metabolism. ACLY binds to citrate to produce acetyl-CoA, which activates ACC and FASN [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. ACC binds to acetyl-CoA to produce malonyl-CoA, thereby inhibiting CPT1A activation. Therefore, FASN synthesizes palmitate, leading to the production of acyl-CoA, and acyl-CoA and carnitine react with CPT1A, thereby enabling transport into the mitochondria for β-oxidation. This activates the tricarboxylic acid (TCA) cycle through acetyl-CoA to generate ATP [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In residual cancer cells (right panel), ACLY in the cytosol translocates to the nucleus when its serine 455 site is phosphorylated, which mediates c-MYC activation to induce FAO. Therefore, metabolism was altered by FAO activation, primarily through c-MYC/ACLY signaling, rather than the negative feedback of malonyl-CoA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eACLY inhibition suppressed cancer cell proliferation and FAO by regulating c-MYC\u003c/h2\u003e \u003cp\u003eTo determine the effects of ACLY inhibition on cell viability, the cells were treated with ACLY inhibitors (NDI-091143, SB-204990, and ETC-1002). The assays revealed that the inhibitors reduced the proliferation of MCF7-parent, -LTED, and -Let\u003csup\u003eR\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and S3D\u0026ndash;E). NDI-091143 treatment suppressed p-ACLY (Ser455) expression, which regulates ACLY nuclear translocation, and p-c-MYC (Ser62), which mediates c-MYC ubiquitination at 1 and 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and S3F). FAO was measured fluorescence-labeled palmitate (BODIPY-C16) and showed a slower decrease in fluorescence in the NDI-091143-treated groups than in the control groups; this decrease was more pronounced at approximately 96 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;D). These findings suggest that ACLY plays a key role in fatty acid consumption through lipid droplet degradation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eNDI-091143 treatment had a synergistic effect with etomoxir and increased cell sensitivity to letrozole\u003c/h2\u003e \u003cp\u003eTo determine the effect of NDI-091143 on letrozole resistance in MCR7-Let\u003csup\u003eR\u003c/sup\u003e cells, a cell viability assay was performed. NDI-091143 (1.25 \u0026micro;M) markedly enhanced the drug sensitivity of Let\u003csup\u003eR\u003c/sup\u003e cells that were simultaneously treated with letrozole (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Moreover, etomoxir (CPT1 inhibitor) had a pronounced synergistic effect with NDI-091143 in LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), and MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells responded well to etomoxir at the same dose (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG). To evaluate the toxicity of NDI-091143, which was not assessed in normal breast cells, a mouse body weight test was performed. Neither NDI-091143 nor combination treatment with etomoxir caused substantial changes in body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe synergistic effect was confirmed by targeting LTED cells and comparing the results with those in the PBS control group in orthotopic breast cancer xenograft models. However, mice injected with LTED cells exhibited metastatic responses rather than subcutaneous proliferation, thereby supporting the aggressive characteristics of LTED cells (data not shown). Overall, NDI-091143/etomoxir combination therapy, which suppresses ACLY and CPT1, may be effective against NAC-resistant breast cancers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eConcurrent chemoendocrine therapy is generally not recommended as a neoadjuvant treatment for HR+ breast cancer [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In a previous clinical trial, although the response rate to chemoendocrine therapy was 83%, the pCR rate was only 4% [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite the addition of endocrine therapy to conventional chemotherapy, many cancer cells have adapted to these therapies and survived. Therefore, this study explored the metabolic adaptations of cancer cells to chemoendocrine treatments. The effects of NAC therapy have been investigated on the basis of transcription [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], genetic mutations [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and lipid profiles [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Consistent with these studies, this study analyzed the mRNA profiles from the GEO database (NCT02296801) of paired breast tissues before and after letrozole treatment (90 days). After treatment in the clinical trial of this study (NCC2017-0110), \u003cem\u003eACLY\u003c/em\u003e mRNA expression was markedly increased in patients with HR+ breast cancer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u0026ndash;D). In paired FFPE tissues from 11 new patients who underwent NAC, metabolic gene analysis revealed that \u003cem\u003eACLY\u003c/em\u003e mRNA levels were consistently elevated despite considerable downregulation of FASN. This pattern was similar to that of c-MYC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;G). Furthermore, ACLY knockdown reduced c-MYC protein expression and stability in HR+ breast cancer cells, thereby shortening its half-life, whereas FASN had the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D, see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). Although ACLY is mainly present in the cytosol, it translocates to the nucleus upon Ser455 phosphorylation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Suitably, the study findings revealed the molecular interaction and nuclear colocalization of ACLY and c-MYC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThis study hypothesized that specific mediators regulate downstream ACLY pathways to reprogram lipid metabolism in response to endocrine therapy during NAC. Although aromatase inhibitors, a type of endocrine therapy, may promote cancer aggressiveness and drug resistance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] in breast cancer cells via amplification in breast cancer 1/ERα [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], epidermal growth factor receptor/erythroblastic leukemia viral oncogene/protein kinase B [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], or ER/phosphoinositide 3-kinase mechanisms [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], the exact factors driving resistance remain unclear [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Several aromatase inhibitor-resistant breast cancer cell lines have been developed and have revealed a metabolic shift from glycolysis, which is the main form of cancer cell metabolism [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this study, chemoendocrine-tolerant cell lines (LTED cells) were generated, and LTED cells overexpressed c-MYC and ACLY at both the protein and mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u0026ndash;B, see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD). Notably, ACLY regulated c-MYC protein expression in LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, see Additional file 1 Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eACLY inhibition downregulates the TCA cycle in acute myeloid leukemia through mitochondrial OXPHOS, a noncanonical TCA cycle [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Hence, this study aimed to determine whether ACLY signaling regulates OXPHOS in LTED cells when fatty acids are utilized as an energy source instead of glycolysis, as aberrant FAO activation contributes to drug resistance via breast cancer stem cell regulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], mitochondrial membrane alteration [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and genetic alterations via the acyl-CoA oxidase 1 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] and rat sarcoma virus pathways [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The MCF7-LTED and MCF7-Let\u003csup\u003eR\u003c/sup\u003e cells presented greater FAO activity than that of the parent cells, showing activity similar to that of the NAC clinical cases (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;E). CPT1A, a key regulator of FAO, was also upregulated in MCF7-LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eFurthermore, c-MYC plays an important role in FAO in breast cancer cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Accordingly, ACLY depletion disrupted c-MYC protein stability, as detected in the nucleus of MCF7-LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;B). Moreover, deregulation of ACLY reduced FAO, which was considerably restored by c-MYC but not by FASN or CPT1, in LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC\u0026ndash;E). These findings suggest that residual cancer cells activate FAO following NAC therapy, and this is mediated by the ACLY/c-MYC pathway. This phenomenon is distinct from that in primary cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], thereby indicating that p-ACLY (S455) translocates into the nucleus and activates c-MYC to induce FAO in residual cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eNDI-091143, an ACLY inhibitor, suppresses cancer cell proliferation and FAO by regulating the c-MYC protein. The inhibitor allosterically binds to the citrate-binding domain of ACLY at residues R378 and G380 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In this study, NDI-091143 reduced the viability and proliferation of MCF7-parent, -LTED, and -Let\u003csup\u003eR\u003c/sup\u003e cells and inhibited fatty acid degradation, thereby supporting the hypothesis that ACLY stimulates FAO in LTED cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;D). This inhibitor also suppressed p-ACLY (S455) and regulated the nuclear translocation of ACLY and p-c-MYC (S62) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and S3F). Moreover, NDI-091143 sensitized cells to letrozole and had a synergistic effect with etomoxir, a CPT1 inhibitor, in LTED cells. Furthermore, the combination did not notably affect mouse body weight, confirming that blocking both the ACLY and CPT1 genes may be a potential treatment strategy for NAC-resistant breast cancers (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFollowing NAC for HR+ breast cancer, residual cells survive via metabolic adaptation. This manifests as a shift to FAO, which is mediated by the ACLY and c-MYC pathways. Therefore, targeting the FAO metabolic pathway by inhibiting ACLY serves as a promising strategy for overcoming endocrine resistance and achieving good neoadjuvant outcomes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHR+\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehormone receptor-positive\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNAC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneoadjuvant chemoendocrine therapy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eadenosine triphosphate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACLY\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eATP citrate lyase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFAO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efatty acid oxidation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFASN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efatty acid synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ep-ACLY\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphorylated ACLY\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMYC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emyelocytomatosis oncogene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ec-MYC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecellular MYC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ep-c-MYC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphorylated c-MYC\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003epCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epathological complete response\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLTED\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elong-term estrogen deprivation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNBC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etriple-negative breast cancer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGEO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Expression Omnibus\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHER2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman epidermal growth factor receptor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFFPE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eformalin-fixed paraffin-embedded\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSEA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egene set enrichment analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDMEM/F12\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's Modified Eagle\u0026rsquo;s Medium/Nutrient Mixture F-12\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecsFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echarcoal-stripped FBS\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLet\u003csup\u003eR\u003c/sup\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eletrozole-resistant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCF7-Let\u003csup\u003eR\u003c/sup\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eletrozole-resistant MCF7 cell line\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTBST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTris-buffered saline with 0.1% Tween\u0026reg; 20 detergent\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebovine serum albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarnitine palmitoyltransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGFP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egreen fluorescent protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esiRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esmall interfering RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNGS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enext generation sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFCCP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003equantitative reverse transcription polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ecDNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecomplementary DNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoverall survival\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCGA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Cancer Genome Atlas database\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eER+\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eestrogen receptor-positive\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eACC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacetyl-CoA carboxylase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etricarboxylic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOXPHOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoxidative phosphorylation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecycloheximide\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGAPDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCER\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecytoplasmic extraction reagent\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLC-MS/MS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eliquid chromatography-tandem mass spectrometry.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the NCC Bio Bank of the National Cancer Center, Korea for providing the tissue and blood samples data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Cancer Center of Korea (Grant no.\u0026nbsp;2510771-1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJHK, SHS, and KSL designed the project; JHK, WS, IH, and JK performed the experiments; JHK, WS, JHH, SHS, IH, JK, and KL analyzed the data; JHK and SHS wrote the manuscript. All the authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding author: Sung Hoon Sim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and approved by the institutional review board of the National Cancer Center in Korea (ClinicalTrials. gov Identifier: NCT03497702 and IRB No. NCC2017-0110) on 4 May 2017. Animal studies were performed in accordance with the Animal Use Protocol (IACUC numbers: NCC-21-660 and NCC-21-704) approved by the National Cancer Center IACU Committee.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional File 1.docx: Supplementary figures S1\u0026ndash;S3\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A: Cancer statistics, 2022. 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Nature 2019, 568(7753):566-570.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Adenosine triphosphate citrate lyase, Cellular myelocytomatosis oncogene, Fatty acid oxidation, Neoadjuvant chemoendocrine therapy, Breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-9173952/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9173952/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMetabolic adaptation enables breast cancer cells to develop neoadjuvant chemoendocrine therapy (NAC) resistance through metabolic alterations. Fatty acid oxidation (FAO) is a key metabolic pathway that facilitates the growth of resistant breast cancers. However, the mechanisms of FAO rewiring in NAC-resistant breast cancers remain unclear. Therefore, this study aimed to investigate metabolic signal reprogramming in residual tumor tissues after chemoendocrine treatment.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eResidual tumor tissues from 11 patients with hormone receptor-positive (HR+) breast cancer receiving NAC therapy were analyzed for adenosine triphosphate citrate lyase (ACLY), myelocytomatosis oncogene (MYC), and fatty acid synthase mRNA levels. Moreover, MCF-7-long-term estrogen deprivation (LTED) and T47D-LTED cell lines were developed to model endocrine-tolerant residual cells. ACLY and cellular MYC (c-MYC) protein expression were confirmed, and FAO rates were measured via mitostress tests and BODIPY-C16 fluorescence. ACLY activity was inhibited via small interfering RNAs or NDI-091143 (ACLY inhibitor), and exogenous ACLY expression was induced via the FLAG-ACLY plasmid. Mechanistic studies included proliferation assays, western blotting, immunoprecipitation, and quantitative reverse transcription polymerase chain reaction, and NDI-091143 toxicity was assessed in animal models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eACLY mRNA levels were increased in patients with HR+ breast cancer. FAO-related gene sets increased after chemoendocrine therapy, and ACLY and MYC mRNA levels were increased. ACLY protein depletion disrupted c-MYC stability in breast cancer cells. MCF7-LTED cells exhibited metabolic characteristics similar to those of residual breast cancer cells in patients post-NAC. ACLY depletion also interrupted c-MYC protein stability in MCF7-LTED cells, thereby regulating FAO. NDI-091143 suppressed cancer cell proliferation and FAO by blocking ACLY phosphorylation (S455) and regulating c-MYC phosphorylation (S62). Notably, NDI-091143 enhanced cellular sensitivity and exhibited synergistic effects with etomoxir (carnitine palmitoyltransferase 1 inhibitor).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOverall, residual HR+ cancer cells shifted from lipid biosynthesis to FAO as a survival strategy. Moreover, the role of ACLY in regulating FAO was elucidated, thereby highlighting its role in residual cancer cell survival. Altogether, changes in metabolism via ACLY/c-MYC signaling are related to the survival of HR+ breast cancer cells after chemoendocrine treatment. In addition, ACLY inhibition has promising potential as a metabolic therapeutic strategy to overcome endocrine resistance in HR+ breast cancer.\u003c/p\u003e","manuscriptTitle":"Adenosine triphosphate citrate lyase alters cellular myelocytomatosis oncogene signaling and fatty acid oxidation in residual breast cancer tumor tissues after neoadjuvant treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-23 12:37:27","doi":"10.21203/rs.3.rs-9173952/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-24T21:05:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"279473595754576869014731617753646463689","date":"2026-04-15T15:44:31+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-15T15:20:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-24T04:18:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-24T04:18:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2026-03-20T02:11:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3635c5ba-5586-44a7-af42-8565babfdf6d","owner":[],"postedDate":"April 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-23T12:37:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-23 12:37:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9173952","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9173952","identity":"rs-9173952","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}