Acetyl-Coa Synthetase 3 Promotes Bladder Cancer Cell Growth Under Metabolic Stress Jianhao Zhang1, Hongjian Duan1, Zhipeng Feng1,Xinweihan1 and Chaohui Gu2

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Zhang et al. Oncogenesis (2020) 9:46 https://doi.org/10.1038/s41389-020-0230-3 Oncogenesis

ARTICLE Open Access Acetyl-CoA synthetase 3 promotes bladder cancer cell growth under metabolic stress Jianhao Zhang1, Hongjian Duan1, Zhipeng Feng1,XinweiHan1 and Chaohui Gu2

Abstract Cancer cells adapt to nutrient-deprived tumor microenvironment during progression via regulating the level and function of metabolic enzymes. Acetyl-coenzyme A (AcCoA) is a key metabolic intermediate that is crucial for cancer cell metabolism, especially under metabolic stress. It is of special signiﬁcance to decipher the role acetyl-CoA synthetase short chain family (ACSS) in cancer cells confronting metabolic stress. Here we analyzed the generation of lipogenic AcCoA in bladder cancer cells under metabolic stress and found that in bladder urothelial carcinoma (BLCA) cells, the proportion of lipogenic AcCoA generated from glucose were largely reduced under metabolic stress. Our results revealed that ACSS3 was responsible for lipogenic AcCoA synthesis in BLCA cells under metabolic stress. Interestingly, we found that ACSS3 was required for acetate utilization and histone acetylation. Moreover, our data illustrated that ACSS3 promoted BLCA cell growth. In addition, through analyzing clinical samples, we found that both mRNA and protein levels of ACSS3 were dramatically upregulated in BLCA samples in comparison with adjacent controls and BLCA patients with lower ACSS3 expression were entitled with longer overall survival. Our data revealed an oncogenic role of ACSS3 via regulating AcCoA generation in BLCA and provided a promising target in metabolic pathway for BLCA treatment. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,;

Introduction acetyl-CoA synthetase short chain family (ACSS), which In cancer cells, considerable number of metabolic ligates acetate and CoA6. ACSS family contains three enzymes and intermediates are deregulated1. Acetyl- family members, ACSS1, ACSS2, and ACSS3. ACSS1 was coenzyme A (AcCoA) is a key metabolic intermediate in originally found to be required for acetate uptake and cell anabolic and catabolic pathways, and plays a critical role in survival in hepatocellular carcinoma7. In addition, ACSS1 biomass production2,3. AcCoA homeostasis directly affects was involved in cell survival and tumor growth of mela- the level of histone acetylation, which contributes to noma cells8. Most recently, it has been proposed that transcriptional and posttranslational gene expression reg- ACSS family proteins distinct heterogeneity of hepatocel- ulations4. Hence, AcCoA biosynthesis is crucial for cancer lular carcinoma in a metabolic manner9. Among the three cell metabolism, especially under metabolic stress5. ACSSs, ACSS2 was most extensively studied. Reports AcCoA were basically generated from two pathways: one showed that ACSS2 precipitated in lipid metabolism and is mediated by ATP citrate lyase, which cleaved citrate into regulated carcinogenesis in hepatocellular carcinoma7,10, oxaloacetate and acetyl-CoA, and the other is mediated by glioblastoma11, breast cancer6, prostate cancer6, and bladder cancer12. On the other hand, little is known about the function of newly identified ACSS member, ACSS3. It Correspondence: Chaohui Gu ([email protected]) 1 fi has been proposed that ACSS3 acts as an important Department of Interventional Radiology, First Af liated Hospital of Zhengzhou 13 University, Zhengzhou City, Henan Province 450052, China prognosis biomarker in gastric cancer and ACSS3 serves 2Department of Urology, Henan Institute of Urology and Zhengzhou Key as a biomarker to stratify subtypes of hepatocellular car- fi Laboratory for Molecular Biology of Urological Tumor Research, First Af liated cinoma9. However, the biological function of ACSS3 has Hospital of Zhengzhou University, Zhengzhou City, Henan Province 450052, China not been verified in cancer cells yet. These authors contributed equally: Jianhao Zhang, Hongjian Duan

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As solid tumors usually confronted unfavorable envir- −20 °C for 10 min. To extract fatty acids, we scraped onments where oxygen and nutrition were limited, cells and transferred to glass tubes, added 0.5 mL cold alterative metabolic pathways were aberrantly activated to chloroform, and vortexed for 1 min and dried under meet the demands for biomass production in cancer nitrogen gas. Then, add toluene (80 μl), methanol cells14. Under hypoxia status, AcCoA production from (600 μl), and methanolic-HCl (120 μl) to each tube, glucose was diminished and molecular tools have been vortex, and incubate at 100 °C for 60 min. Water (0.4 ml) developed to trace the metabolism of AcCoA15,16. It has and hwxane (0.3 ml) were added to samples after redu- been illustrated that acetate serve as an alternative carbon cing to room temperature, vortex and analyzed the top origin under metabolic stress conditions6,17. Therefore, it hexane phase by Gas Chromatography-Mass Spectro- is of special significance to further validate the participa- meter (GC-MS). The carbon sources of lipogenic AcCoA tion of ACSS proteins in tumorigenesis and evaluate the was determined by computed binomial distributions as role ACSS proteins in alternative utilization of carbon previously described21. sources. Bladder urothelial carcinoma (BLCA) is the most Quantification of medium acetate common malignancy in the urinary tract, which lead to For acetate derivatization, 200 μl cell culture medium ~150,000 death worldwide each year18. Even though only was collected into a 2 mL microfuge tube, followed by ~25% of BLCA progressed into muscle invasive BLCA, 40 μl 2H3-acetate (1 mM), 1-propanol (50 μl), and pyr- half of the advanced cancers further metastasize19. What idine (50 μl). Tube was put on ice for 5 min and 100 μl make the situation worse is that no well-defined prog- NaOH (1 M), 30 μl methyl-chloroformate were added. nostic marker for BLCA is currently available20. Hence, After overtaxing for 20 s, add 300 μl of tert-butyl methyl identification of proteins, especially metabolic enzymes, ether (MTBE) to the tube and vortex again. The samples facilitates carcinogenesis of BLCA provides novel insights were then centrifuged at 10,000 × g for 5 min. The upper and potential therapeutic targets for BLCA. layer was transferred to GC-MS vials and analyzed with Here we quantified the AcCoA metabolism in bladder an Agilent 7890B GC system and 7000 Triple Quadrupole cancer (BLCA) cells under normoxia and hypoxia, and GC-MS system. Data were collected and analyzed as evaluated the role ACSS3 in AcCoA metabolism during previously described22. metabolic stress in bladder cancer cell lines. Moreover, we further dissected the function of ACSS3 in bladder cancer Histones and histone-bound acetate extraction cell growth in vitro and in vivo. Importantly, we analyzed Cells were collected and washed with cold PBS sup- our clinical data and revealed an oncogenic role of ACSS3 plemented with sodium butyrate (10 mM) and nicotina- in bladder cancer. mide (50 mM). Extraction of nuclei was followed as described previously23. Histones were separated with Material and methods SDS-polyacrylamide gel electrophoresis (PAGE) and Cell culture detected with acetyl-histone-specific antibodies. Isolated SV-HUC-1, UMUC3, T24 cells were purchased from histones were placed at 95 °C overnight with 10 M NaOH, the American Type Culture Collection. SV-HUC-1 cell then added with hydrochloric acid for GC-MS. line was maintained in F-12K Medium (HyClone). UMUC3 cell line was cultured in Eagle’s Minimum Transfection of small interfering RNA and small hairpin Essential Medium (HyClone). T24 cell line was cultured RNA in McCoy’s 5a Medium (HyClone). All the cell lines were To construct inducible knockdown cell lines, two dif- cultured in base medium supplemented with 10% fetal ferent small hairpin RNA (shRNA) sequences target bovine serum (Gibco) and 1% penicillin/streptomycin ACSS3 and a control shRNA were cloned into pLKO- (Gibco) at 37 °C and 5% CO2. Puro plasmids. The sequences of shRNA are as follows: shACSS3-1: 5′-GGGTTACCTAAGGGTGTGGAAtt-3′, 13C Enrichment in lipogenic AcCoA detection shACSS3-2: 5′-GAAAAGATATAAATGCAAGAAtt-3′. Fatty acid labeling assay was performed as previously Lentivirus production and infection were generated as described21.Briefly, cells were maintained in culture previously described in 293T cells. 293T cells were seeded medium containing 2 mM glutamine. Cells were plated at 105 cells per well and were transfected with plasmids. in six-well plates overnight before changing medium. Viral supernatant was harvested 48 h after transfection. Medium were changedto Dulbecco’smodified Eagle Cells were infected for 12 h and cultured for another 24 h medium with 13C-labeled glucose (10 mM), 13C- and collected. Same sequences were synthesized as small glutamine (2 mM), and 13C-acetate for 48–72 h. Cells interfering RNA (siRNA) and siRNAs were transfected were washed with phosphate-buffered saline (PBS), add with Lipofectamine 2000 (Invitrogen). Cells were col- 0.75 mL of methanol : PBS (1 : 1), and place the plate at lected 2 days post siRNA transfection.

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Tumor xenografts 4 °C overnight and followed by secondary antibody for 1 h. All animals were maintained in specific pathogen-free The primary antibodies used were as follows: anti-ACSS1 conditions according to the recommendation of Guide for (Invitrogen, PA5-59392), anti-ACSS2 (Cell Signaling the Care and Use of Laboratory Animals of the National Technology, 3568 S), anti-ACSS3 (Invitrogen, PA5- Institutes of Health and approved by the Ethics Com- 80305), anti-Tubulin (Proteintech, 66031–1), anti-acetyl- mittee of First Affiliated Hospital of Zhengzhou Uni- histone H4 (Millipore, 06–598), anti-histone H3 (CST, versity (Approval number 2019-KY-174). 4499), anti-acetyl-histone H3 (Millipore, 06–599), and For tumor xenograft model, 2 × 106 indicated UMUC3 anti-histone H4 (CST, 13919). and T24 cells were injected subcutaneously on the right side of the dorsum (n = 6 for each group). The tumor Clinical samples and immunohistochemistry diameters were measured each week. Tumor volumes This project was approved by the Ethics Committee of were calculated with the formula: V = 1/2 × A × B2. The First Affiliated Hospital of Zhengzhou University mice were sacrificed after 6 weeks and tumors were (Approval number 2019-KY-174) and patients enrolled analyzed. were informed of the scientific usage of the samples. Serial sections of formalin-fixed, paraffin-embedded Cell growth curve and colony-formation assay tissues from 60 patients were obtained between Sep- Two thousand indicated cells were seeded in each well tember 2014 and March 2017. The routine immuno- of a six-well plate and cultured for 10 days. The colonies histochemistry were performed as previously were fixed with 4% paraformaldehyde and stained with described25. Slices were immunostained using anti- crystal violet. ACSS3 (Invitrogen, PA5-80305) antibody. DAB (diami- nobenzidine) stainings were performed with an HRP kit RNA extraction and quantitative reverse-transcriptase PCR (UltraTek, USA) according to the manufacturer’s Cell total RNA was extracted using RNeasy kit (Qia- instructions. Stained specimens were visualized using a gen) as manufacturer’s instructions. First strand cDNA generalopticalmicroscope with a camera (Axiovert was synthesis using QuantiTect Reverse Transcription 100 M, Germany). kit (Qiagen). qRT-PCR wasperformedusingSBBR GREEN PCR Master Mix (Applied Biosystems) and Immunofluorescence and confocal microscopy detected with ABI 7900 System. Relative mRNA Cells were planted on the glass coverslips a day in expression was calculated using the ΔΔCt equations. advance. After transfected with indicated plasmids, the The primers used for ACSS genes detection by RT- cells were fixed with 4% paraformaldehyde. The cell qPCR are as following: membranes were p permeabilized with NP40, then ACSS3: blocked with blocking buffer for 1 h. After incubation Forward primer: 5′-TGGACCAAAACGCTGGAGAA with primary antibody against Myc-tag (Proteintech, C-3′ 16286-1-AP) for overnight, cells were washed with Reverse primer: 5′-ACGATCAACGGCATTGTAACA-3′ Phosphate-Buffered Saline (PBS)-Tween and incubated ACSS2: with Alexa Fluor 488 donkey anti-rabbit secondary anti- Forward primer: 5′-AAAGGAGCAACTACCAACAT body (Invitrogen, R37118). Mitochondrial were stained CTG-3′ with MitoTracker (Invitrogen, M22426) for 30 min and Reverse primer: 5′-GCTGAACTGACACACTTGGAC-3′ nuclei were stained with DAPI (Beyotime, C1002) for ACSS1: 10 min. The slides were observed using the Lecia Tcs SP8 Forward primer: 5′-CACAGGACAGACAACAAG confocal microscope. GTC-3′ Reverse primer: 5′-CCTGGGTATGGACGATGCC-3′ Spheroid growth assay Spheroid formation assay were performed as previously Western blotting described26, cells were digested with trypsin, and cultured Western blot experiments was performed as previously with 2% matrigel in 96-well ultralow attachment plates described24. Briefly, protein was extracted with RIPA (Costar). Spheroid formation was initiated by centrifuga- buffer (Byotime) supplemented with protease inhibitor tion at 850 × g for 10 min and the spheroids were analyzed cocktail (Roche). Total protein concentration was quan- at the indicated time points. Spheroids were treated with tified with BCA Protein Assay Kit (Thermo). An equal ethanol (control) or doxycycline 48 h. At indicated time amount of total proteins were separated by 10% SDS- points, images were captured by a general optical micro- PAGE and transferred to polyvinylidene difluoride scope with a camera (Axiovert 100 M, Germany). Spher- membrane (Millipore). Then block the membrane with oid volume was calculated based on image analysis by area 5% non-fat milk, incubated with primary antibodies at determination using image J software.

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Fig. 1 AcCoA is generated through alternative pathway in BLCA cells under metabolic stress. a Sensitivity of normal bladder urothelial cells (SV-HUC-1) and bladder cancer cells to FASN inhibitor C75 in high (10%) or low (1%) serum levels. b Sensitivity of bladder cancer cells to FASN

inhibitor AZ22 in high (10%) or low (1%) serum levels as revealed by pIC50. c Schematic of lipogenic AcCoA production. d Percentage of lipogenic 13 13 AcCoA generation from acetate (labeled by C) under normoxia (21% O2) or hypoxia (1% O2) culture. e Sources of lipogenic AcCoA (labeled by C) under normoxia (21% O2) or hypoxia (1% O2) culture.

Statistical analysis (SV-HUC-1) cultured with 10% serum (full serum) or 1% Statistical analysis was performed with GraphPad Prism serum (low serum). Although the growth of BLCA cells 5.0. (GraphPad Software). Experiments were performed at (UMUC3 and T24) was not affected by C75 treatment in least in triplicates and error bars stands for SD. Two- full serum, we observed that the growth of BLCA cells tailed Student’s t-test was performed to determine the cultured in low serum was significantly inhibited by C75 significance of paired data. One-way analysis of variance administration (Fig. 1a). Moreover, we found that BLCA for quantitative data from grouped DataSets. P-value < cells cultured with low serum were more sensitive to 0.05 was considered significant. *P < 0.05; **P < 0.01, and another FASN inhibitor, AZ22, as revealed by ***P < 0.001. A log-rank test was performed to compare improvement of IC50 (Fig. 1b). As AcCoA is originated tumor-free survival. P-values less than 0.05 were con- from alternative pathways (Fig. 1c) and hypoxic status sidered statistically significant. mimics the in vivo lipid metabolic conditions5,6,wenext evaluated the fatty acids metabolism in BLCA under Results hypoxia. We first analyzed the contribution of various Lipogenic AcCoA metabolism is altered in BLCA under precursors of lipogenic AcCoA and found that utiliza- stress tion of acetate (13C labeled) was dramatically increased As FASN is closely related to the carcinogenesis, we in BLCA cells under hypoxia (Fig. 1d). Moreover, via therefore analyzed the role FASN in BLCA. We first isotopic labeling of glucose, glutamine, and acetate, our analyzed the growth of BLCA cells treated with FASN results revealed that the proportion of lipogenic AcCoA inhibitor C75. Our data revealed that C75 did not affect generated from glucose were largely reduced, whereas the growth of normal bladder urothelial cells the proportion of lipogenic AcCoA generated from

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Fig. 2 ACSS3 is induced under metabolic stress and facilitate BLCA cell growth. a ACSS1, ACSS2, and ACSS3 mRNA expression levels in BLCA

cells under normoxia (21% O2) or hypoxia (1% O2) culture with 10% or 1% serum levels. b ACSS1, ACSS2, and ACSS3 protein expression levels in BLCA cells under normoxia (21% O2) or hypoxia (1% O2) culture with 10% or 1% serum levels. c ACSS1, ACSS2, and ACSS3 protein expression levels in BLCA cells transfected with control of ACSS3 siRNA. d Colony formed from BLCA cells transfected with control of ACSS3 siRNA under indicated culture situations. e Cell number of BLCA cells transfected with control of ACSS3 siRNA under indicated culture situations after 48 h incubation. f Percentage 13 of lipogenic AcCoA generation from acetate (labeled by C) in BLCA cells transfected with control of ACSS3 siRNA under hypoxia (1% O2) culture. g Percentage of lipogenic AcCoA generation from glutamine or glucose (labeled by 13C) in BLCA cells transfected with control of ACSS3 siRNA under

hypoxia (1% O2) culture. h Subcellular localization of ACSS3-Myc expressed in T24 cells. ACSS3 was detected with anti-myc antibody. Mitochondria was stained with MitoTracker. glutamine and acetate were increased significantly in and ACSS2 were not changed when ACSS3 was knocked BLCA cells (Fig. 1e). down by siRNA (Fig. 2c). Colony-formation experiments revealed that downregulation of ACSS3 attenuated the ACSS3 is responsible for lipogenic AcCoA synthesis in number of colonies formed in both normoxia and hypoxia BLCA cells under metabolic stress conditions in UMUC3 and T24 cells (Fig. 2d). Cell Previous study demonstrated that ACSS2 participated number counting revealed that ACSS3 knockdown sig- in lipid metabolism in breast cancer cells under hypoxic nificantly inhibited cell proliferation in both normoxia conditions5. Here we analyzed the role another ACSS and hypoxia conditions (Fig. 2e), suggesting that ACSS3 family member plays, ACSS3, in BLCA. We first analyzed was responsible for BLCA cell growth under metabolic the expression levels of ACSS3 cultured with 10% serum stress. Importantly, a significant reduction was found in or 1% serum under normal oxygen concentration, and the lipogenic AcCoA from 13C-acetate (Fig. 2f). Mean- found that the expression of all three members of ACSS while, labeling from 13C-glucose and 13C-glutamine were family were not dramatically changed (Fig. 2a, b). How- both increased (Fig. 2g) under indicated conditions. In ever, when BLCA cells were cultured under hypoxia order to clarify the localization of ACSS3, ACSS3-Myc condition, the expression level of ACSS2 and ACSS3 in plasmid was constructed and expressed in T24 cells. The BLCA cells cultured with 1% serum were significantly expression of ACSS3 was detected through immuno- increased in comparison with the expression level in fluorescence staining assay using antibody against Myc- BLCA cells cultured with 10% serum (Fig. 2a, b), indi- tag. As shown in Fig. 2h, myc signals were observed co- cating that metabolic stress, especially hypoxia, triggered localized with MitoTracker, suggesting the colocalization the expression of ACSS2 and ACSS3 in BLCA cells. As with mitochondria. ACSS2 has been reported to response to metabolic stress, we focused on the function of ACSS3. We utilized siRNA ACSS3 is crucial for histone acetylation in BLCA cells to downregulated ACSS3 expression in BLCA cells and To determine the net uptake of acetate by cancer cells, a both of the two siRNAs achieved considerable knockdown delicate measurement has been developed previously5 and efficiency (Fig. 2c). Meanwhile, the protein level of ACSS1 we utilized this measurement to study the role ACSS3 in

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Fig. 3 ACSS3 is required for acetate utilization and histone acetylation. a Consumption of medium acetate and release of cytoplasmic acetate in

BLCA cells transfected with control of ACSS3 siRNA under hypoxia (1% O2) culture. b Ratio of acetate release and consumption in BLCA cells transfected with control of ACSS3 siRNA under hypoxia (1% O2) culture. c Level of histone H3, acetylated histone H3, histone H4, and acetylated histone H4 in BLCA cells cultured in indicated conditions. Right panel are quantiﬁed results of protein expression levels.

Fig. 4 ACSS3 is required for spheroid and xenografts formation. a Induction of ACSS3 knockdown by doxycycline in BLCA cells. b Spheroid formation in BLCA cells stably transfected with control of ACSS3 shRNA with or without doxycycline administration under high serum or low serum culture. c Xenografts formed by BLCA cells stably transfected with control of ACSS3 shRNA with or without doxycycline administration under high serum or low serum culture. Right panels are measurements of tumor volumes at indicated time points.

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Table 1 Correlation between ACSS3 expression and acetylated histone H3 and H4 (Fig. 3c). However, under clinicopathological characteristics of bladder cancer metabolic stress, ACSS3 was required for maintaining the patients. levels of acetylated histone H3 and H4 (Fig. 3c). More- over, our data also revealed that metabolic stress induced Parameters Total ACSS3 expression P-value a significant increase in histone acetylation in BLCA cells, Low High which was aborted in ACSS3 knockdown cells (Fig. 3c), (n = 30) (n = 30) further indicating that ACSS3 is crucial for histone acetylation in BLCA cells. Gender 0.194 Male 33 14 19 ACSS3 silencing inhibits spheroid and tumor growth To further analyze the role ACSS3 in BCLA, we gen- Female 27 16 11 erated stable cell lines in which ACSS3 could be silenced Age (years) 0.519 by doxycycline administration. We first validated that <59 12 7 5 doxycycline was able to induce ACSS3 silence in the ≥59 48 23 25 stable cell lines (Fig. 4a). We then cultured these cells as spheroids in 10% or 1% serum with or without the Tumor stage 0.793 administration of doxycycline. Importantly, our results Ta,T1 11 5 6 showed that only in low serum culture, downregulation of T2–T4 49 25 24 ACSS3 resulted in significant impairment of spheroids TNM stage 0.010* formation in UMUC3 and T24 cells (Fig. 4b). In addition, BLCA cells were inoculated subcutaneously into nude S-I 28 19 9 mice and half of the mice were treated with doxycycline S-II + III 32 11 21 on the 7th day post inoculation. The tumor growth of Grade 0.004* the xenografts formed by BLCA cells expressing fi G1 33 22 11 ACSS3 shRNA was signi cantly inhibited in comparison with xenografts formed by BLCA cells expressing control G2–G3 27 8 19 shRNA or xenografts formed by BLCA cells without Tumor size 0.781 doxycycline treatment (Fig. 4c). Hence, our data indicated <3 cm 19 9 10 an oncogenic role of ACSS3 in BLCA. ≥3cm 41 21 20 We then analyzed the expression pattern of ACSS3 in clinical samples (Table 1). We found that the both mRNA Lymph node metastasis 0.000* and protein levels of ACSS3 were significantly upregu- Absent 32 9 23 lated in BLCA samples compared with adjacent controls Present 28 21 7 (Fig. 5a, b). We analyzed the staining intensity of the Distant metastasis 0.028* immunochemistry staining of ACSS3 and found that the staining intensity of ACSS3 was significantly higher in Absent 48 21 27 samples from BLCA patients in both stage I and stage II Present 12 9 3 plus stage III (Fig. 5c, d). Moreover, BLCA patients with lower ACSS3 expression were entitled with longer overall survival time (Fig. 5e). Taken together, our analysis illu- lipid metabolism in BLCA cancer cells. Our results strated that the expression of ACSS3 was statistically showed that ACSS3 knockdown attenuated acetate con- elevated in BLCA patients and high ACSS3 level predicted sumption from the medium (Fig. 3a). Meanwhile, more poor outcome of BCLA prognosis. endogenous acetate was released to the medium suggesting that ACSS3 (Fig. 3a) is required for the utilization Discussions of endogenous acetate in cancer cells. We next calculated Cancer cells adapt to nutrient-deprived tumor micro- the ratio of acetate release and consumption in BLCA environment during progression via adjusting the level cells transfected with control siRNA or ACSS3 siRNA and and function of metabolic enzymes26. Several metabolic found that this ratio was markedly increased in BLCA altered pathways are involved in bladder tumorigenesis27. cells transfected with ACSS3 siRNA (Fig. 3b), further With the advancement of multi-omics analysis, de novo suggesting that ACSS3 facilitated acetate metabolism in identification of oncogenes have been carried out in var- cancer cells. We then studied the role of ACSS3 under ious types of cancers, including BLCA28. A most recent metabolic stress. Interestingly, under normal culture sta- multi-omics study has shown that fatty acid metabolism tus, downregulation of ACSS3 did not affect the levels of plays a central role in facilitating the progress of BLCA29.

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Fig. 5 ACSS3 is elevated in BLCA patient samples. a ACSS3 mRNA expression levels in BLCA patient samples and adjacent normal tissues. b Representative immunohistochemistry images of ACSS3 staining in BLCA patient samples and adjacent normal tissues. c ACSS3 protein intensities in BLCA patient samples and adjacent normal tissues. d ACSS3 protein intensities in stage I or stage II + III BLCA patient samples and adjacent normal tissues. e Overall survival of BLCA patients with higher or low ACSS3 mRNA expression levels.

Moreover, another report showed that fibroblast growth of ACSS3 in BLCA. Downregulation of factor receptor 3 promotes the cleavage and activation of ACSS3 significantly suppressed the growth of BLCA cells sterol regulatory element-binding protein 1, which is the in vitro and in vivo. Indeed, previous report uncovered a key regulator of lipogenesis, which in turn facilitates the higher expression of ACSS3 in tumor comparing to nor- BLCA cell growth30. Here we first analyzed the utilization mal parental lesions and downregulation of ACSS3 inhi- of acetate in BLCA confronting environmental stress and bits progression of gastric cancer13. In hepatocellular found that BLCA cell lines altered the way of generation, carcinoma, ACSS3 was expressed in mitochondria and which is in line with previous reports showing that specifically recognized a subtype of hepatocellular carci- cancer cells adapted to nutrient-deprived tumor noma (namely iHCC2, which represents poor clinical microenvironment5,17,31. survival rate)9, suggesting that the presence of ACSS3 may As it has been shown that ACSS family members were be noxious in hepatocellular carcinoma as well. Taken critical for alternative utilization of acetate in cancer together, these data indicated an oncogenic role of ACSS3 cells5,26, we suspected the role ACSS3 in BLCA cells even under routine culture conditions. Interestingly, under metabolic stress. As a member of ACSS enzyme methylation level of ACSS3 was found to be associated family, ACSS3 is the least studied. ACSS1 is the main with prognosis of neuroblastoma patients32. It is of special mitochondrial enzyme and ACSS2 mainly functions in significance to further determine the role of methylated nucleus and cytosol. Here we demonstrated that in BLCA ACSS3 in the progression of BLCA. cells, ACSS3 is localized in mitochondria and functions as Interestingly, we found an upregulation of histone an oncogene to promote lipid synthesis and histone acetylation under low serum and hypoxic conditions in acetylation through the use of acetate. BLCA, which is consistent with the notion that fatty acid Importantly, our data showed that the expression level biosynthesis is significantly elevated in hypoxia and low of ACSS3 were significantly upregulated in BLCA cell serum conditions in breast cancer cell lines5, and the lines upon metabolic stress, suggesting an oncogenic role majority of histone acetylation is derived from fatty acid

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33 carbon . Importantly, our results showed that the upre- Publisher’s note gulated acetylation of histone H3 and histone H4 were Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. significantly impaired in BLCA cells with downregulated ACCS3 level. Our data revealed that ACSS3 is required Received: 6 November 2019 Revised: 17 April 2020 Accepted: 21 April 2020 for the utilization of both environmental and intracellular acetate, which is identical to the role of ACSS2 in breast cancer cells5. As ACSS3 is mainly localized in mitochondria, the mechanisms of using acetate to promote References histone acetylation should be different from that of 1. Corbet,C.&Feron,O.Tumouracidosis:fromthepassengertothedriver’sseat. Nat. Rev. Cancer 17, 577–593 (2017). ACSS2, which could directly converts the acetate to 2. Shi, L. & Tu, B. P. Acetyl-CoA and the regulation of metabolism: mechanisms nuclear acetyl-CoA. 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