ATF3-SLC7A7 Axis Regulates mTORC1 Signaling to Suppress Lipogenesis and Tumorigenesis in Hepatocellular Carcinoma

doi:10.21203/rs.3.rs-5195664/v1

Download PDF

Research Article

ATF3-SLC7A7 Axis Regulates mTORC1 Signaling to Suppress Lipogenesis and Tumorigenesis in Hepatocellular Carcinoma

https://doi.org/10.21203/rs.3.rs-5195664/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background

Hepatocellular carcinoma (HCC) remains a major global health challenge with poor prognosis and high mortality. Dysregulated lipid metabolism is increasingly recognized as a key driver of HCC progression. While the transcription factor ATF3 has been implicated in tumorigenesis and lipid regulation, its precise role in HCC development and lipid biosynthesis is not fully understood.

Methods

We generated ATF3-overexpression and ATF3-knockdown HCC cell lines via lentiviral infection. To investigate ATF3’s role in HCC, we performed in vitro assays, including MTT, transwell migration, wound healing, and colony formation assays, alongside an in vivo mouse xenograft model to assess tumor growth. The correlation between ATF3 expression and lipid synthesis was analyzed using RNA-Seq data from HCC patients and further validated by qRT-PCR, Nile Red staining, lipid quantification, correlation analysis, and immunohistochemistry. The impact of ATF3 on mTORC1 signaling was evaluated through western blotting, confocal microscopy, Oil Red O staining, and lipid quantification assays. SLC7A7, identified as a potential downstream effector of ATF3, was analyzed using qRT-PCR, and its influence on mTORC1 signaling was examined in the same manner as ATF3. The binding of ATF3 to the enhancer region of SLC7A7 was identified via ChIP-Seq and confirmed through ChIP-qPCR, luciferase reporter assays, and DNA pull-down assays.

Results

We demonstrate that mTORC1 inhibition markedly reduces lipogenesis in HCC and identify a regulatory axis involving ATF3 and the amino acid transporter SLC7A7. Transcriptomic analysis reveals an inverse correlation between ATF3 expression and lipid synthesis, a finding supported by experimental data. Mechanistically, ATF3 represses mTORC1 signaling, inhibiting lipid biosynthesis, with SLC7A7 acting as a key mediator. ATF3 directly binds to the enhancer region of SLC7A7, activating its transcription and subsequently limiting mTORC1 activity. Functional assays in ATF3-overexpression and -knockdown HCC cell lines confirm ATF3’s tumor-suppressive role.

Conclusion

This study uncovers a novel ATF3-SLC7A7-mTORC1 regulatory axis that mitigates lipogenesis and tumorigenesis in HCC, highlighting a critical link between lipid metabolism and liver cancer progression. These findings present promising therapeutic targets for HCC treatment.

Cellular Metabolism

Lipid metabolism

transcriptional regulation

mTORC1 signaling

Hepatocellular carcinoma (HCC) stands as the predominant form of liver cancer, constituting approximately 90% of all liver cancer cases worldwide ¹. Despite advancements in therapeutic modalities such as transplantation and resection, HCC remains a formidable challenge, ranking sixth among the most diagnosed cancers and fourth among cancer-related causes of mortality ^2,3. Notably, the incidence of HCC attributed to nonalcoholic fatty liver disease (NAFLD) has increased sharply in recent years, with NAFLD emerging as the fastest-growing etiology of HCC in western nations ^3,4. Central to NAFLD pathology is the excessive accumulation of triglycerides (TG), primarily fueled by heightened de novo fatty acid (FA) synthesis within hepatic cells ⁵. Consequently, aberrant upregulation of lipid synthesis emerges as a pivotal risk factor in NAFLD progression, with implications extending to HCC development ^5,6. A deeper exploration of the underlying mechanisms governing this process holds promise for enhancing our comprehension of HCC pathogenesis and may unveil novel therapeutic targets for HCC management.

Enhanced de novo lipid synthesis is a critical driver of HCC progression ⁷. The process begins with the conversion of citrate into acetyl-CoA, which is subsequently transformed into palmitic acid and monounsaturated FAs by ATP citrate lyase (ACLY), fatty acid synthase (FASN), and stearoyl-CoA desaturase (SCD). FAs are then converted into TGs, which can be stored in lipid droplets as an energy reserve to support the unchecked proliferation of cancer cells ^8,9. Elevated expression of ACLY, FASN, and SCD has been observed in HCC patients, indicating a link between increased lipogenesis and tumor development ^10–12. Importantly, activated mTORC1 signaling enhances lipid biosynthesis by promoting the stability and splicing of transcripts encoding ACLY, FASN, and SCD via activation of SRPK2 ¹³. Moreover, mTORC1 stimulates the processing and transcription of sterol regulatory element-binding proteins (SREBPs), key transcription factors that regulate FA synthesis ^14,15. Identifying novel pathways that modulate mTORC1 activity could offer new therapeutic opportunities to target dysregulated lipid metabolism in HCC.

Typical activators of mTORC1 include growth factors and amino acids such as arginine, leucine, and glutamine ¹³. Transporters from the solute carrier (SLC) family, which facilitate the import of these amino acids, including SLC1A5 and SLC38A5, are upregulated in various cancers ¹⁶. Therefore, further investigation of SLC family members, particularly those involved in amino acid transport, may reveal novel regulatory mechanisms governing mTORC1 signaling.

In addition to nutrient signaling, mTORC1 is mediated by transcription factors. Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding (CREB) family, plays a multifaceted role in regulating immunity, metabolism, and tumorigenesis. Elimination of ATF3 has been shown to activate mTORC1-p70S6K signaling in liver inflammatory injury ¹⁷. In the context of hepatocellular carcinoma (HCC), ATF3 predominantly functions as a tumor suppressor, with decreased expression levels observed in HCC patients compared to healthy individuals ^18,19. Furthermore, beyond its involvement in oncogenesis, ATF3 has been implicated in mediating lipid anabolism, a process tightly associated with the progression of NAFLD and HCC. Previous studies have revealed that ATF3 binds to the promoter region of ChREBP and suppresses its transcription, thereby inhibiting lipogenesis in mouse white adipocytes ²⁰. Despite these findings, the role of ATF3, the SLC family, and mTORC1 in lipid synthesis within the context of HCC remains poorly understood.

Here, we provide evidence that ATF3 inhibits lipid accumulation in HCC by modulating the SLC7A7-mTOR signaling axis, utilizing a combination of bioinformatics and experimental approaches. Mechanistically, we demonstrate that ATF3 directly binds to the enhancer region of SLC7A7, promoting its transcriptional activation and consequently suppressing mTOR signaling and lipid synthesis. Our findings elucidate a novel regulatory circuit involving mTORC1 in lipid metabolism and HCC tumorigenesis, establishing a crucial link between lipid homeostasis and cancer progression.

Cell Maintenance

The human HCC cell lines Huh7, PLC/PRF/5, HepG2 and the human kidney cell line 293T were acquired from the Japanese Collection of Research Bioresources (JCRB) Cell Bank, Osaka, Japan. Huh7, PLC/PRF/5, and 293T cells were grown in high-glucose Dulbecco’s modified Eagle medium (DMEM, Gibco), while HepG2 cells were cultured in William's E Medium (Gibco). Both media were supplemented with 10% fetal bovine serum (FBS, qualified, Gibco) and an antibiotic-antimycotic solution (Gibco). All cell lines were incubated at 37°C in a humidified atmosphere of 5% CO₂ using a Heracell VIOS 160i incubator (Thermo).

RNA Extraction and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was isolated from the cell lines using RNAzol (Sigma), and its concentration was measured with a Nanodrop2000 spectrophotometer (Fisher). The isolated RNA was then reverse transcribed into cDNA using the Reverse Transcription Kit (Qiangen). Quantitative real-time PCR (qRT-PCR) was carried out on the ABI StepOne Plus system (Applied Biosystems) using iTaq Universal SYBR Green Supermix (Bio-Rad). The primer information are provided in Supplementary Table 1. Gene expression was normalized to the internal control genes GAPDH or RPL13A, and relative mRNA levels were calculated using the 2-ΔΔCT method.

Vector Construction

ATF3 coding sequence (CDS) was amplified from pRK-ATF3 (26115, Addgene), and SLC7A7 CDS was amplified from Huh7 cDNA. Sequences of ATF3 promoter and SLC7A7 enhancer were amplified from Huh7 genome DNA, which was extracted and purified by using Monarch Genomic DNA Purification Kit (New England Biolabs, NEB). Above sequences were amplified by PCR. For construction of ATF3 and SLC7A7 expression vectors, CDS of ATF3 or SLC7A7 was inserted between the 2 Esp3I cutting sites of a modified lenti-3×Flag plasmid generated from lentiCas9-Blast (52962, Addgene) using homemade hot fusion solution ²¹; For construction of luciferase vectors, sequences of ATF3 promoter or SLC7A7 enhancer were inserted between the MluI and XhoI cutting sites of pGL3-promoter (Promega) with T4 ligase (NEB). ATF3 and SLC7A7 shRNA oligos were obtained from Integrated DNA Technologies (IDT). Each of the oligos was annealed and inserted into the Esp3I cutting site of pLV-H1TetO-GFP-Bsd (Biosettia). For DNA affinity assay, ATF3 cDNA was inserted into the BamHI cutting site of a modified pET-28a (+) vector (Novagen) with a SUMO tag to generate recombinant ATF3 expression vector. Information of the primers and oligo utilized in cloning are provided in Supplementary Table 2.

Lentivirus Production and Infection

The 293T cells were seeded in 60 mm plates (Corning) and co-transfected with lentiviral vectors for ATF3, SLC7A7, shATF3, or shSLC7A7, along with psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) using Lipo2000 (Vazyme). Viral supernatants were harvested at 48- and 72-hours post-transfection and concentrated using PEG-it Virus Precipitation Solution (System Biosciences). The concentrated virus was then used to infect target cells in the presence of 8 µg/ml polybrene. Twenty-four hours after infection, the cells were processed for subsequent experiments. To induce lentiviral vector expression, 1 µg/ml doxycycline was supplied to the culture media.

Transwell Assay

8µm-pore-transwell inserts with were pre-coated with Geltrex (Gibco) and placed into 24-well plates (Corning). The lower chamber of each well was filled with 700 µl of DMEM supplemented with 10% FBS. Cells (1×10⁵) were resuspended in 300 µl of serum-free DMEM, introduced into the upper layer, and kept for 24 hours. Non-migratory cells on the upper side of the membrane were erased using a cotton swab. Inserts were then collected, fixed with methanol for 10 minutes and stained with crystal violet for another 10 minutes. Migrated cells were visualized and counted using an inverted microscope at 100× magnification.

Wound Healing Assay

Cells were seeded into a 6-well plate (Corning). Once the confluency reached 80%-100%, scratch was manually created, and cells were washed twice with Phosphate-buffered saline (PBS). Medium was changed into DMEM with 2% FBS and 1mM thymidine. Scratch was photographed at 0h, 24h and 48h. The scratch area was measured by ImageJ and the wound closure ratio was calculated.

Colony Formation Assay

Cells were trypsinized, counted, and plated at a density of 2000 cells per well in 6-well plates (Corning). The cells were then cultured for 14–21 days to allow colony formation. Once colonies containing more than 50 cells became visible, the plates were processed by fixing the cells with 4% paraformaldehyde for 10 minutes, followed by staining with crystal violet for 10 minutes. The colonies were subsequently photographed and manually counted.

MTT Assay

3000 cells were counted, resespended in 100 µl of complete DMEM medium, and seeded into 96-well plates (Corning). The plates were maintained for 4–5 days, and cell proliferation was measured every day. 10 µl of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was supplied into the wells, following with incubation at 37°C for 3 hours. The media was abandoned, and 100 µl of DMSO was supplied into the wells to dissolve insoluble formazan. The plates were shaken for 15 minutes in avoid of light. Absorbance was then recorded at 490 nm.

Immunoblotting

Cells were lysed in RIPA buffer for 30 minutes to obtain whole-cell lysates. Tumor tissues were similarly lysed in RIPA buffer and homogenized with a glass homogenizer to produce the lysate. The resulting lysate was centrifuged at 12,000 rcf for 3 minutes. 5× Laemmli buffer was supplied to the supernatant, and the samples were kept at 95°C for 20 minutes. The samples were then resolved with SDS-PAGE. The condition is 80V for 18 minutes followed by 140V for 1 hour. Proteins were transferred to 0.45 µm polyvinylidene difluoride membranes at 20V for 40 minutes. Membranes were blocked with 5% bovine serum albumin for 30 minutes and then incubated with primary antibody overnight at 4°C. After washing three times with Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) for 5 minutes each, the membranes were exposed to a secondary antibody at room temperature for 1 hour. Following another round of washing with TBST for 5 minutes 3 times, protein bands were visualized. Antibodies information is provided in Supplementary Table 3.

Confocal Assay

Cells were seeded into 12-well plate (Corning). Once the confluency reached 50–60%, cells were transfected with pcDNA3-TORCAR plasmid (Addgene, 64927) and maintained for 24h. After transfection, cells were photographed with Carl Zeiss LSM 980 Confocal Laser Scanning Microscope. The reporter activity was calculated by the intensity ratio of CFP/YFP.

Nile Red Staining Assay

Cells were seeded into 12-well plate (Corning). Once the confluency reached 50–60%, cells were washed twice with PBS and were fixed with 4% Paraformaldehyde (PFA) for 10min. After fixing, cells were washed three times with PBS. Nile Red (Invitrogen) was dissolved in acetone with the final concentration of 1mg/ml. Cells were stained with PBS supplied with 1ug/ml Nile Red and 500ng/ml Hoechst 33342 solution (Dojindo Laboratories) and were incubated on a shaker for 10min in dark. After incubation, cells were washed three times with PBS and were photographed with BioTek Cytation1 Cell Imaging Multimode Reader. GFP fluorescence intensity was quantified using Image J.

Oil Red O Staining Assay

Cells were seeded into 6-well plate (Corning). Once the confluency reached 70%, cells were washed twice with PBS and were fixed with 4% Paraformaldehyde (PFA) for 10min. After fixing, cells were washed twice with PBS. Oil Red O (Dieckmann) was dissolved in 60% isopropanol with the final concentration of 3.5mg/ml. Cells were stained with Oil Red O solution and incubated for 10min. After incubation, cells were washed three times with PBS and were photographed using an inverted microscope.

Triglyceride Quantification Assay

Cells were seeded into 96-well plate or 10cm plate (Corning). Once the confluency reached 90–100%, cells were subjected to Triglyceride-Glo™ Assay Kit (Promega, for 96-well plate) or Triglyceride Assay Kit (Nanjing Jiancheng Bioengineering Institude, for 10cm plate) to make samples. Luminescence or absorbance at 500nm was measured using BioTek Cytation1 Cell Imaging Multimode Reader.

Free Fatty Acids Quantification Assay

Cells were seeded into 10cm plate (Corning). Once the confluency reached 90–100%, cells were subjected to Free Fatty Acids (FFA) Content Assay Kit (Solarbio) to make samples. Absorbance at 550nm was measured using BioTek Cytation1 Cell Imaging Multimode Reader.

Chromatin Immunoprecipitation (ChIP) Assay

Huh7 cells that had been infected with ATF3 lentivirus were cultured in 15 cm plates (Corning). When the cells reached 90–100% confluency, they were washed twice with PBS and treated with DMEM containing 1% formaldehyde, followed by a 5-minute incubation on a shaker. After this step, 900 µl of 2.5 M Glycine was added, and the cells were incubated for an additional 5 minutes on a shaker. The cells were then washed twice with PBS, scraped off, and collected by centrifugation at 500 g for 5 minutes at 4°C. They were resuspended in lysis buffer (85 mM KCl, 0.5% CA-630, 5 mM PIPES pH 8, 1 mM PMSF, and Pierce Protease and Phosphatase Inhibitor from Thermo) and sonicated for 150 seconds using the M220 Focused-ultrasonicator (Covaris) at a peak power of 75 W, duty factor of 2%, 200 cycles/burst, and temperature maintained at 4°C. Following sonication, the lysate was centrifuged at 1,000 g for 5 minutes at 4°C. The nuclear pellet was then resuspended in shearing buffer (10 mM Tris-HCl pH 8, 0.1% SDS, 1 mM EDTA pH 8, 1 mM PMSF, and Protease and Phosphatase Inhibitor) and subjected to sonication for 1560 seconds under the same conditions as before. After another centrifugation at 10,000 g for 10 minutes at 4°C, the supernatant was collected. This supernatant was either stored as input or mixed with antibodies and incubated overnight on a shaker at 4°C. After incubation, pre-blocked ChIP-grade Protein A/G Magnetic Beads (Thermo) were added and incubated on a shaker at 4°C for 4 hours. After this step, the beads were precipitated, and the supernatant was discarded. The beads were washed three times with sequential wash buffers: wash buffer I (150 mM NaCl, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 0.1% SDS, 1% Triton), wash buffer II (500 mM NaCl, 20 mM Tris-HCl pH 8, 2 mM EDTA pH 8, 0.1% SDS, 1% Triton), and wash buffer III (250 mM LiCl, 10 mM Tris-HCl pH 8, 1 mM EDTA pH 8, 1% sodium deoxycholate, 1% CA-630). The proteins were then eluted using elution buffer (1% SDS, 100 mM NaHCO₃). All samples received a 5 M NaCl solution to achieve a final concentration of 0.2 M and were incubated overnight at 65°C. After this incubation, the samples were treated with RNase A and Proteinase K, purified using the Monarch PCR & DNA Cleanup Kit (NEB), and subsequently analyzed by qRT-PCR.

Luciferase Reporter Assay

Huh7 cells infected with empty vector (EV) and ATF3 lentivirus were seeded into 12-well plate (corning). Once the confluency reached 60–70%, cells were co-transfected with either pGL3-SLC7A7 enhancer or pGL3-ATF3 promoter plasmids and pRL-TK plasmid (Promega) and maintained for 24h. After transfection, cells were washed twice with PBS and subjected to Dual-Glo Luciferase Assay System Kit (Promega) to make samples. The luciferases activities were measured using GloMax 20/20 Luminometer (Promega).

DNA Affinity Assay

Recombinant ATF3 expression vector was transformed into TOP10 Ecoli strain and single colony was picked up and cultured in LB medium overnight. 0.5mol/L IPTG was then added into the culture to induce the expression of recombinant ATF3, and the final concentration of IPTG was 0.1mmol/L. After 6h of induction, the culture was centrifuged, and the pellet was suspended in PBS with Pierce Protease and Phosphatase Inhibitor. The suspension was then sonicated for 15min using Branson Ultrasonics Sonifier 250 under the condition of Duty Cycle 30%, Output control 4. The sonicated product was subjected to HisPur™ Cobalt Spin Columns (Thermo) to purify recombinant ATF3 following the manufacturer’s instructions. 1ug of recombinant ATF3 was incubated along or with 2ug biotin-labeled SLC7A7-enhancer oligo (5’-biotin-CTT TCT TTG GTT TGG AAT CTG GAA GCT GAT GCA ACA GCT ACC CCG-3’) or with 2ug biotin-labeled SLC7A7-enhancer oligo and 50ug unlabeled SLC7A7-enhancer oligo in incubation buffer (25mM Tris-HCl, pH8, 2mM EDTA, 1mM EGTA, 0.1% NP-40, 100mM NaCl, 1 mM DTT, and 5% glycerol) on a shaker at 4°C for 1h. 20ul of Dynabeads™ M-280 Streptavidin (Thermo) was added into each sample and incubated on a shaker at 4°C for 2h. After incubation, beads were washed with wash buffer (25mM Tris-HCl, pH8, 2mM EDTA, 1mM EGTA, 0.5% NP-40, 150mM NaCl, 1mM DTT, and 5% glycerol) for 5 times and was eluted with 20ul 5× Laemmli buffer at 100°C for 15min. Samples were then subjected to western blot.

Mouse xenograft model

This study was conducted following the ARRIVE guidelines, with the experimental protocol receiving approval from the Committee on the Use of Live Animals in Teaching and Research (CULATR) at The University of Hong Kong (CULATR No. 5712-21). License of animal experiments was approved by Hong Kong SAR Government, Department of Health. The Huh7 shATF3 cell line was suspended in sterilized PBS with a final density of 5×10⁷ cells/ml, and cell suspension was injected subcutaneously into the flanks of 4- to 6-week-old female nude mice. Each mouse was inoculated with 5×10⁶ cells. Mice body weight and tumor volume were recorded every three days. 5 weeks after inoculation, mice were anesthetized via intraperitoneal injection of 100 µl of 12.5 mg/ml tribromoethanol, followed by dissection for sacrifice, and the tumor xenografts were acquired.

Immunohistochemistry

Tumor tissues obtained from the mouse xenograft model were collected and fixed in 4% paraformaldehyde (PFA) for 48 hours. Following fixation, the tissues were dehydrated using 30% sucrose, embedded in OCT, and sectioned at -20°C. The sections underwent Oil Red O staining and hematoxylin-eosin (H&E) staining. For Oil Red O staining, the sections were fixed in 4% PFA for 10 minutes, washed with 60% isopropanol, and then stained with Oil Red O solution for 10 minutes. After staining, the sections were rinsed with 60% isopropanol and distilled water (ddH₂O), followed by a 2-minute incubation in hematoxylin solution (Beyotime). For H&E staining, sections were fixed in 95% ethanol for 20 minutes and washed twice with PBS. They were subsequently stained with hematoxylin solution for 2 minutes, rinsed with ddH₂O, and stained with eosin solution (Beyotime) for 1 minute. All sections were allowed to air dry, sealed with neutral gum, and examined under a microscope. The positive area for Oil Red O and lipid droplets was quantified using Image J.

RNA-Seq analysis

RNA-Seq data for hepatocellular carcinoma (HCC) patients were obtained from cBioPortal (Liver Hepatocellular Carcinoma, TCGA Firehose Legacy), as detailed in Supplementary Table 4. The samples were categorized into high and low expression groups based on the median expression levels of ATF3. Subsequently, the RNA-Seq data underwent Gene Set Enrichment Analysis (GSEA) using multiple gene sets, along with a summary analysis of the results.

Gene Expression Analysis

mRNA expression data for normal and hepatocellular carcinoma (HCC) patients were sourced from cBioPortal (Liver Hepatocellular Carcinoma, TCGA PanCancer Atlas), as presented in Supplementary Table 5.

Kaplan-Meier (KM) Survival analysis

The KM plot was conducted on the website (https://kmplot.com/analysis/) ²².

Correlation analysis

The correlation analysis of gene expression level in liver cancer was conducted using Correlation AnalyzeR ²³. Data is listed in Supplementary Table 6–12.

Statistical analysis

Gene expression analysis was conducted using the Kruskal–Wallis test. For other assays, results are expressed as the mean ± standard deviation (S.D.) from at least three independent experiments. Statistical comparisons of various groups were conducted with unpaired two-tailed t-tests using Prism 9. P-values of < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****) were considered statistically significant.

ATF3 suppresses the amplification and invasion of HCC cells

The impact of ATF3 on hepatocellular carcinoma (HCC) cell behavior, encompassing proliferation, invasion, migration, and colony formation, was investigated to delineate its role, given its reported dual functions in HCC pathogenesis ^24,25. Lentiviral vectors encoding ATF3 were engineered by incorporating human ATF3 cDNA into a modified lenti-3×Flag plasmid. Stable ATF3-overexpressing (ATF3-oe) cell lines were constructed by lentiviral transduction of HCC cell lines. Robust elevation of ATF3 mRNA levels, exceeding 15-fold compared to empty vector (EV)-infected cell lines, was confirmed via qRT-PCR (Fig. 1A), affirming the successful establishment of stable cell lines. Subsequently, these ATF3-oe cell lines were subjected to a battery of assays, including MTT, transwell, scratch, and colony formation assays. Notably, MTT assay results revealed a marked reduction in HCC cell proliferation upon ATF3 overexpression (Fig. 1B). Similarly, transwell and scratch assays demonstrated a significant attenuation of invasion and migration capacities in ATF3-oe cells, respectively (Fig. 1C-F). Consistently, colony formation assay data indicated a notable decrease in the clonogenic potential of HCC cells following ATF3 overexpression (Fig. 1G-H). Collectively, these findings underscore the suppressive role of ATF3 overexpression in modulating the oncogenic phenotype of HCC cells.

To further corroborate the tumor-suppressive role of ATF3 in HCC, we employed an ATF3 short hairpin RNA (shRNA) oligo to knock down ATF3 expression. The shRNA was cloned into the pLV-H1TetO-GFP-Bsd plasmid, packaged into lentivirus, and used to infect HCC cell lines. Given that the vector's promoter is inducible by doxycycline, we introduced doxycycline 24 hours post lentivirus infection to induce stable ATF3 knockdown (ATF3-kd) cell lines. Concurrently, infected cell lines were maintained without doxycycline as controls. Both control and ATF3-kd cell lines underwent qRT-PCR analysis to ascertain the effectiveness of knockdown. Results indicated a substantial reduction of ATF3 mRNA levels, by approximately 40% in Huh7 ATF3-kd cell line and 50% in PLC/PRF/5 ATF3-kd cell line compared to the control group (Fig. 1I), affirming the efficacy of ATF3 knockdown. Following successful knockdown, ATF3-kd cell lines were subjected to the same functional assays as ATF3-oe cell lines. MTT assay results unveiled a significant enhancement in HCC cell proliferation upon ATF3 knockdown (Fig. 1J). Transwell and scratch assays demonstrated an augmented invasion and migration capacity in ATF3-kd cell lines, respectively (Fig. 1K-N), while colony formation assays revealed an increased colony formation ability in these cells (Fig. 1O-P). By integrating the outcomes of phenotype assays conducted on both ATF3-overexpressing and ATF3-knockdown HCC cell lines, we provide compelling evidence supporting the tumor-suppressive role of ATF3 in HCC in vitro.

ATF3 is correlated with better prognosis and downregulated lipid synthesis in HCC

After establishing the suppressive effect of ATF3 on the malignant traits of HCC cells in vitro, we delved deeper into the relationship between ATF3 expression and the occurrence and progression of HCC by comparing its expression levels in normal and HCC samples. To this end, we analyzed ATF3 mRNA expression data from normal samples (n = 50) and HCC samples (n = 316) sourced from the TCGA database. Our analysis unveiled a significant downregulation of ATF3 mRNA levels in HCC samples compared to normal samples, suggesting a potential association between ATF3 downregulation and the onset and progression of HCC (Fig. 2A). These findings are consistent with both our functional assays results and previous research findings ^19,24.

Moreover, we conducted Kaplan-Meier survival analysis using an online tool developed by Gyorffy B²². HCC patients were stratified into two groups based on their ATF3 mRNA expression levels: high expression (n = 249) and low expression (n = 115). Subsequently, survival curves were plotted and compared between the two patient cohorts. Our analysis revealed that patients with high ATF3 expression exhibited a higher probability of survival compared to those with low ATF3 expression within 80 months. However, in the period spanning 80–120 months, the survival probability of the ATF3 high expression group showed a slight decline, likely attributable to the larger sample size within this timeframe (Fig. 2B). Overall, these results corroborate the tumor suppressive role of ATF3, indicating that HCC patients with high ATF3 expression levels have a more favorable prognosis.

To elucidate the cellular events and signaling pathways influenced by ATF3 expression in HCC patients, we accessed the RNA-Seq data of HCC patients from the TCGA database. These patients were categorized into two groups based on their ATF3 expression levels: ATF3 high expression group (n = 183) and ATF3 low expression group (n = 182). Subsequently, we conducted Gene Set Enrichment Analysis (GSEA) on the RNA-Seq data. Our analysis, summarizing multiple GSEA gene sets including KEGG gene sets, gene ontology biological process (GOBP) gene sets, and hallmark gene sets, revealed a consistent pattern: the ATF3 high expression group exhibited an overall decrease in lipid metabolism processes compared to the ATF3 low expression group (Fig. 2C). Further scrutiny through individual gene set analyses within the KEGG, GOBP, and hallmark gene sets corroborated this observation, indicating a downregulation of fatty acid metabolism in ATF3 high expression patients (Fig. 2D). Notably, the GOBP gene set analysis unveiled a decrease in both fatty acid catabolism and lipid synthesis in the ATF3 high expression group.

Given the well-documented propensity of cancer cells to upregulate de novo fatty acid synthesis to fuel their uncontrolled growth ^9,26, and considering the pivotal role of fatty acids as substrates for various lipid species essential for cancer progression, including diglycerides, triglycerides, and phosphoglycerides ²⁷, our findings suggest a compelling association between ATF3 high expression, attenuated lipid biosynthesis, and improved prognosis in HCC patients.

ATF3 inhibits lipid biosynthesis in HCC cells

To further corroborate the findings from RNA-Seq analysis of HCC patients, we conducted qRT-PCR to assess the expression levels of a panel of lipid metabolism-related genes in both ATF3-overexpressing (ATF3-oe) and ATF3-knockdown (ATF3-kd) HCC cell lines. This panel encompassed genes encoding key enzymes involved in fatty acid synthesis (FASN, ACLY, and SCD) ²⁷, components of lipoproteins (APOA1, APOB, and APOC3) ^28–30, and regulators of lipid synthesis (mTORC1 and SREBF2) ^31,32. In ATF3-oe Huh7 cell lines, we observed a significant decrease in the expression levels of all examined genes except mTORC1 (Fig. 3A). Conversely, in ATF3-kd Huh7 cell lines, the expression levels of FASN, ACLY, SCD, APOB, and APOC3 were elevated, while mTORC1 expression remained unchanged (Fig. 3B). These findings suggest that ATF3 negatively regulates lipid synthesis and transportation in HCC cells.

Subsequently, we assessed the lipid accumulation levels in ATF3-oe and ATF3-kd HCC cell lines using Nile Red staining. Consistently, ATF3-oe cell lines exhibited reduced lipid accumulation, while ATF3-kd cell lines showed elevated lipid accumulation (Fig. 3C-F). Since lipid de novo synthesis seems to have more direct impact on lipid accumulation in HCC cells than lipid transport in HCC cell lines, we examined the correlation between ATF3 expression and the key lipid metabolism genes (FASN, ACLY, and SCD) in liver cancer patients. The results revealed a negative correlation between ATF3 expression and the expression of FASN, ACLY, and SCD, consistent with our RNA-Seq and qRT-PCR findings (Fig. 3G).

Furthermore, we quantified triglyceride levels in both ATF3-oe and ATF3-kd cell lines, demonstrating that ATF3 overexpression significantly reduces cellular triglyceride levels, whereas ATF3 knockdown leads to an increase in triglyceride levels (Fig. 3H). Taken together, these results underscore the role of ATF3 in suppressing lipid anabolism by inhibiting the expression of key enzymes involved in fatty acid synthesis in HCC cells.

ATF3 represses lipid biosynthesis by inhibiting mTORC1 signaling

To elucidate the mechanism underlying ATF3-mediated inhibition of lipid synthesis, we investigated the signaling pathways related to lipid synthesis that could be modulated by ATF3. Given previous findings indicating that activated mTORC1-S6K phosphorylates SRPK2, leading to enhanced splicing of FASN, ACLY, and SCD mRNA ³³, the relationship between ATF3 and mTORC1 signaling is pertinent to our observation of ATF3-mediated downregulation of FASN, ACLY, and SCD mRNA levels. Thus, we hypothesized that mTORC1 might be a potential signaling pathway influenced by ATF3.

Although the mRNA levels of mTORC1 were not significantly altered in either ATF3-oe or ATF3-kd HCC cell lines, mTORC1 functions predominantly through the phosphorylation of S6K to exert its downstream effects ^13,33. Therefore, we assessed the levels of phosphorylated S6K (pS6K) in both ATF3-oe and ATF3-kd HCC cell lines via western blot analysis. Remarkably, pS6K levels were markedly decreased in ATF3-oe cell lines and conversely elevated in ATF3-kd cell lines (Fig. 4A-B). Besides, we adopted a well-defined molecular biosensor, TORCAR, to measure the mTORC1 activity in single living cells ³⁴. Since activated mTORC1 kinase leads to the conformational change of TORCAR and results in an increased CFP/YFP ratio, the results further proved that ATF3 downregulates mTORC1 activity in HCC cells, while ATF3 knockdown exhibits the opposite effect (Fig. 4C-F). These findings suggest that ATF3-mediated inhibition of mTORC1 signaling is involved in the repression of lipid anabolism.

To further corroborate the role of mTORC1 as the main signaling pathway in ATF3-mediated suppression of lipid biosynthesis, we treated ATF3-kd HCC cell lines with rapamycin, an inhibitor of mTORC1 ³⁵. Remarkably, while ATF3 knockdown increased cellular lipid levels, inhibition of mTORC1 reversed the elevated lipid levels induced by ATF3 knockdown (Fig. 4G), as confirmed by Nile Red staining (Fig. 4H-I). Additionally, quantification of triglyceride (TG) levels in ATF3-kd cell lines with or without rapamycin treatment revealed that mTORC1 inactivation abolished the upregulation of triglycerides caused by ATF3 knockdown in HCC cells (Fig. 4J). Free fatty acids (FFA) quantification indicated that while ATF3 alone can reduce FFA synthesis like rapamycin, it strikingly enhanced the sensitivity of HCC cells to rapamycin (Fig. 4K). The inhibitory effect of rapamycin on lipid synthesis related genes was confirmed by qRT-PCR. Notably, rapamycin leads to decreased ATF3 expression level, which is probably a negative feedback regulatory mechanism of HCC cells to maintain necessary lipid synthesis under the stress of rapamycin (Fig. 4L). Collectively, these results provide compelling evidence that ATF3 inhibits lipid synthesis by repressing mTORC1 signaling.

SLC7A7 is involved in the inhibition of ATF3 on mTORC1 signaling

To delineate the mechanism by which ATF3 inhibits mTORC1 signaling in HCC, we turned our attention to the role of amino acids, particularly glutamine, leucine, and arginine, as activators of mTORC1 signaling. These amino acids induce lysosomal translocation of mTORC1, thereby activating its signaling cascade ¹³. Given that the SLC3 and SLC7 families are known amino acid transporters, and SLC7A5 has been implicated in mediating leucine influx and mTOR signaling activation, we sought to identify other potential members of the amino acid transporter family that could influence mTORC1 signaling or lipid synthesis ^36,37.

Previous studies have implicated SLC7A7 in the activation of mTOR signaling and the regulation of left-right asymmetry in medaka ³⁸, while loss of SLC7A10 has been associated with lipid accumulation in zebrafish ³⁹. Therefore, we assessed the mRNA expression levels of SLC7A7 and SLC7A10 in ATF3-oe Huh7 cell lines. Strikingly, ATF3 overexpression led to a 2-fold upregulation of SLC7A7 expression, while SLC7A10 expression remained unchanged. Subsequent analysis in ATF3-oe PLC/PRF/5 cell lines revealed a similar trend (Fig. 5A). Conversely, in ATF3-kd HCC cell lines, SLC7A7 expression was significantly decreased (Fig. 5B). These results collectively suggest that ATF3 upregulation promotes SLC7A7 expression, implicating it as a potential mediator of ATF3's inhibitory effects on mTORC1 signaling.

To ascertain whether SLC7A7 contributes to ATF3-induced downregulation of lipid synthesis, we generated Huh7 SLC7A7-kd cell lines using the same approach as for ATF3-kd cell lines and assessed the expression of genes related to lipid anabolism and transportation. The results confirmed the efficacy of SLC7A7 shRNA and revealed that, akin to ATF3 knockdown, SLC7A7 knockdown led to increased expression of FASN, ACLY, SCD, APOA1, APOB, and APOC3 (Fig. 5C). Notably, SLC7A7 knockdown did not affect ATF3 expression, suggesting that SLC7A7 could be a downstream effector of ATF3, negatively regulating lipid biosynthesis. Correlation analysis between SLC7A7 and ATF3, FASN, ACLY, and SCD further supported these findings, showing a positive correlation with ATF3 and negative correlations with FASN, ACLY, and SCD, consistent with the qRT-PCR results (Fig. 5D).

We then explored whether SLC7A7 knockdown elevated lipid synthesis by influencing mTORC1 signaling in SLC7A7-kd HCC cell lines. The results revealed that SLC7A7 knockdown activated mTORC1, mirroring the effect of ATF3 knockdown (Fig. 5E). To further substantiate that SLC7A7 is a downstream inhibitor of mTORC1 and lipid synthesis regulated by ATF3, we infected Huh7 SLC7A7-kd cell lines with either empty vector (EV) or ATF3 lentivirus for 24 hours and assessed the lipid accumulation level. The findings demonstrated that while ATF3 alone suppressed lipid accumulation, SLC7A7 knockdown reversed this inhibitory effect (Fig. 5F-G). Similarly, western blot assays on EV- or ATF3-infected Huh7 and PLC/PRF/5 cell lines revealed that ATF3 inhibited mTORC1 activation, while SLC7A7 knockdown reversed ATF3-induced mTORC1 inactivation (Fig. 5H). Both TG and FFA quantification results indicated that ATF3 represses FFA and TG synthesis, which can be reversed by SLC7A7 knockdown, consistent with Nile Red staining results (Fig. 5I-J). Overall, these results underscore the role of SLC7A7 in ATF3-mediated inhibition of mTORC1 signaling and lipid biosynthesis.

ATF3 binds to SLC7A7 enhancer and activates its transcription

Next, we intend to enclose the mechanism of how ATF3 upregulates SLC7A7 expression. Since ATF3 is a transcription factor which can both activates and suppresses gene transcription ⁴⁰, we assume that ATF3 could binds to promoter or enhancer of SLC7A7 to promotes its transcription. Utilizing ATF3 ChIP-Seq data from the Encode database (ENCSR402ZCY) in the HepG2 cell line, we identified a peak approximately 3000bp upstream of the SLC7A7 promoter, suggesting ATF3 binding to this region (Fig. 6A). Sequence analysis revealed the presence of a known ATF3 binding site (TTGCATCA) within this region (Fig. 6B) ¹⁸. Thus, we hypothesized this region as a potential enhancer of SLC7A7 and performed ChIP-qPCR assays in the Huh7 ATF3-oe cell line to confirm ATF3 binding. We included the ATF3 promoter as a positive control, as ATF3 can bind to and inhibit its own promoter ⁴¹. The results validated ATF3 binding to the potential enhancer of SLC7A7 (Fig. 6C-D).

To further validate this region as the enhancer of SLC7A7, we cloned its sequence into a pGL3-promoter vector (Fig. 6E), alongside the ATF3 promoter sequence as a control. We co-transfected these plasmids with a pRL-TK plasmid into Huh7 cell lines infected with either EV or ATF3 lentivirus, conducted luciferase reporter assays after 24 hours. The findings demonstrated that ATF3 bound to the SLC7A7 potential enhancer and promoted luciferase transcription, while also repressing its own promoter, consistent with prior research (Fig. 6F).

Given that H3K27ac and H3K4me1 are known histone modifications of enhancers ⁴², we analyzed H3K27ac and H3K4me1 histone ChIP-Seq data from the Encode database (ENCSR678LND and ENCSR642HII). Both modifications exhibited peaks at the same position as the potential SLC7A7 enhancer (Fig. 6G). ChIP-qPCR assays further confirmed the enrichment of H3K27ac and H3K4me1 on the potential SLC7A7 enhancer (Fig. 6H). DNA-pull down assay indicated that purified ATF3 protein bound to biotin-labeled SLC7A7 enhancer oligos, which was impaired by the competitive binding of unlabeled oligos (Fig. 6I).

In summary, these findings reveal a newly characterized SLC7A7 enhancer upstream of its promoter, flanking the ATF3 binding site. ATF3 binds to this enhancer to activate SLC7A7 transcription, thereby inhibiting lipid synthesis through the ATF3-SLC7A7-mTORC1 axis.

ATF3 represses tumor growth and lipogenesis in vivo

Building on our in vitro findings that ATF3 suppresses lipid synthesis and tumorigenesis, we sought to confirm these effects in vivo. We subcutaneously injected Huh7 cells with shRNA-mediated ATF3 knockdown (shATF3) into the flanks of nude mice, dividing them into two groups. The control group received water containing 5% sucrose, while the experimental group was administered water with 5% sucrose and 2mg/ml doxycycline to induce ATF3 shRNA expression. Tumor growth was monitored, revealing a significant increase in both tumor volume and weight in the experimental group compared to controls (Fig. 7A-C), suggesting that ATF3 knockdown promotes tumor growth in HCC, consistent with our in vitro results.

To investigate whether SLC7A7 plays a role in this effect, we examined its expression in tumor tissues. Given the lack of a sensitive SLC7A7 antibody for immunohistochemistry, we analyzed tumor lysates via western blotting. Results showed that ATF3 knockdown also reduced SLC7A7 expression (Fig. 7D), implying that downregulation of SLC7A7 may contribute to the enhanced tumor growth observed upon ATF3 knockdown. This hypothesis was further supported by transwell and MTT assays, where HCC cell lines overexpressing both ATF3 and SLC7A7 exhibited greater suppression of invasion and proliferation compared to cells overexpressing ATF3 alone (Fig. 7E-G). Collectively, these findings demonstrate that ATF3 inhibits tumorigenesis in vivo through regulation of SLC7A7 expression.

We next examined the impact of ATF3 on lipid synthesis in vivo. Oil Red O staining of tumor tissues revealed a significant increase in lipid accumulation in the ATF3 knockdown group compared to controls (Fig. 7H-I). Furthermore, H&E staining indicated that tumors with ATF3 knockdown exhibited more cytoplasmic vacuoles, attributable to the increased lipid droplets (Fig. 7J-K). These results indicate that ATF3 represses lipogenesis in HCC in vivo.

Tumor cells exhibit a voracious demand for energy and biomass to sustain their relentless proliferation. Metabolic reprogramming stands out as a hallmark of these cells, enabling them to generate the necessary energy and intermediates for biomass production ⁴³. In addition to the well-known reprogramming of glucose metabolism, characterized by the Warburg effect, recent attention has also turned towards the reprogramming of lipid metabolism in cancer ^9,26. Lipids serve as both essential components of cellular and organelle membranes and key signaling molecules within cells. In cancer cells, lipogenesis is upregulated, leading to the accumulation of excessive lipids stored as lipid droplets, which can be metabolized via β-oxidation to produce ATP ^44–46. Investigating pathways related to the reprogramming of tumor lipid metabolism holds promise for identifying potential therapeutic targets and advancing our theoretical understanding of cancer treatment.

Hepatocellular carcinoma (HCC) stands out as a leading cause of malignancy-related mortality globally. Despite advancements in treatment, approximately 70% of late-stage HCC patients face the grim reality of recurrence and metastasis following surgical resection ⁴⁷. The urgent need for effective therapies to combat HCC is underscored by its poor prognosis. Notably, enzymes involved in fatty acid synthesis, such as FASN, ACLY, and SCD, are dysregulated in HCC, highlighting elevated lipogenesis as a potential therapeutic target ⁴⁸. However, the precise association between HCC development and lipid synthesis pathways remains incompletely understood.

ATF3, a transcription factor implicated in mediating various biological processes including metabolism, immunity, and tumorigenesis, presents an intriguing subject of study. Despite its known roles in regulating both oncogenesis and lipogenesis, the precise contribution of ATF3 to HCC development remains contentious ¹⁸. Moreover, the impact of ATF3 on lipid metabolism in HCC has not been thoroughly investigated. Therefore, our study seeks to elucidate the role of ATF3 in HCC progression and lipid metabolism.

Through functional assays conducted in both ATF3-overexpressing (ATF3-oe) and ATF3-knockdown (ATF3-kd) cell lines, we confirmed the tumor suppressor role of ATF3 in HCC development. Transcriptome analysis of HCC patients revealed that high ATF3 expression affects lipid metabolism by downregulating lipid anabolism, a finding corroborated by experimental evidence. We discovered that ATF3 inhibits lipid synthesis in HCC cells by suppressing mTORC1 signaling, with SLC7A7 playing a crucial role in this process. ATF3 binds to the enhancer region of SLC7A7, activating its transcription, which, in turn, inhibits mTORC1 activation. Our findings unveil the ATF3-SLC7A7-mTORC1 axis as a critical regulator of lipid synthesis and HCC development (Fig. 8).

Furthermore, we speculate on the potential dual role of SLC7A7, a transmembrane arginine-leucine transporter, in mTORC1 activation, given the role of leucine and arginine as mTORC1 activators ^13,49,50. While our study demonstrates an inhibitory role for SLC7A7 in mTORC1 signaling in HCC, further investigation into its structure and function, including measurements of arginine efflux and leucine influx, is warranted. Additionally, ATF3's reported interaction with the promoter of SLC7A11 raises the possibility of broader effects on the transcriptional regulation of the SLC7 family ⁵¹. Exploring this aspect may deepen our understanding of ATF3's regulatory mechanisms and offer insights for clinical therapy across various diseases.

HCC

Hepatocellular Caricinoma

qRT-PCR

Quantitative Reverse Transcription Polymerase Chain Reaction

RNA-Seq

RNA sequencing

ChIP-Seq

Chromatin Immunoprecipitation Sequencing

ChIP-qPCR

Chromatin Immunoprecipitation- Quantitative Polymerase Chain Reaction

mTORC1

Mammalian Target of Rapamycin Complex 1

ATF3

Activating Transcription Factor 3

SLC7A7

Solute Carrier Family 7 Member 7

SRPK2

Serine/arginine-rich Protein Kinase 2

Availability of data and materials

All the data generated during this study are included in this article (and its Supplementary Information files).

Authors’ contributions

JZ conceived the project and designed the experiments. QZ performed the experiments, survival analysis, correlation analysis, conducted the data analysis and wrote the manuscript; FZ performed the gene expression analysis and RNA-Seq analysis of public databases. YH performed the transwell assay of ATF3-oe Huh7 cell line. JZ and YT revised the manuscript.

Consent for publication

All the authors read and approved the final manuscript.

Competing interests

The authors claim that they do not have competing interests.

Additional Information

Supplementary File 1: Table S1: Sequences of the qRT‒PCR primers. Table S2: Sequences of the cloning primers and oligos. Table S3: Antibody Information. Table S4: RNA-Seq data of HCC patients in Liver Hepatocellular Carcinoma (TCGA, Firehose Legacy). Table S5: RNA-Seq data of HCC patients in Liver Hepatocellular Carcinoma (TCGA, PanCancer Atlas). Table S6-S12: Gene expression data for correlation analysis.

Funding

This work was supported by Research Grants Council of Hong Kong China [HKU 17123819 General Research Fund (GRF) to JZ].

Ethics approval and consent to participate

Not Applicable.

Acknowledgements

Not Applicable.

Forner A, Reig M, Bruix J (2018) Hepatocellular carcinoma. Lancet 391:1301–1314. https://doi.org:10.1016/S0140-6736(18)30010-2
Brown ZJ et al (2023) Management of Hepatocellular Carcinoma: A Review. JAMA Surg 158:410–420. https://doi.org:10.1001/jamasurg.2022.7989
Ganesan P, Kulik LM (2023) Hepatocellular Carcinoma: New Developments. Clin Liver Dis 27:85–102. https://doi.org:10.1016/j.cld.2022.08.004
Huang DQ, El-Serag HB, Loomba R (2021) Global epidemiology of NAFLD-related HCC: trends, predictions, risk factors and prevention. Nat Rev Gastroenterol Hepatol 18:223–238. https://doi.org:10.1038/s41575-020-00381-6
Guri Y et al (2017) mTORC2 Promotes Tumorigenesis via Lipid Synthesis. Cancer Cell 32, 807–823 e812 https://doi.org:10.1016/j.ccell.2017.11.011
Ning Z et al (2022) USP22 regulates lipidome accumulation by stabilizing PPARgamma in hepatocellular carcinoma. Nat Commun 13:2187. https://doi.org:10.1038/s41467-022-29846-9
Calvisi DF et al (2011) Increased lipogenesis, induced by AKT-mTORC1-RPS6 signaling, promotes development of human hepatocellular carcinoma. Gastroenterology 140:1071–1083. https://doi.org:10.1053/j.gastro.2010.12.006
Du W et al (2017) HIF drives lipid deposition and cancer in ccRCC via repression of fatty acid metabolism. Nat Commun 8:1769. https://doi.org:10.1038/s41467-017-01965-8
Chen J et al (2021) ACSL4 reprograms fatty acid metabolism in hepatocellular carcinoma via c-Myc/SREBP1 pathway. Cancer Lett 502:154–165. https://doi.org:10.1016/j.canlet.2020.12.019
Li L et al (2016) Inactivation of fatty acid synthase impairs hepatocarcinogenesis driven by AKT in mice and humans. J Hepatol 64:333–341. https://doi.org:10.1016/j.jhep.2015.10.004
Budhu A et al (2013) Integrated metabolite and gene expression profiles identify lipid biomarkers associated with progression of hepatocellular carcinoma and patient outcomes. Gastroenterology 144, 1066–1075 e1061 https://doi.org:10.1053/j.gastro.2013.01.054
Zhou Y, Tao J, Calvisi DF, Chen X (2022) Role of Lipogenesis Rewiring in Hepatocellular Carcinoma. Semin Liver Dis 42:77–86. https://doi.org:10.1055/s-0041-1731709
Mossmann D, Park S, Hall MN (2018) mTOR signalling and cellular metabolism are mutual determinants in cancer. Nat Rev Cancer 18:744–757. https://doi.org:10.1038/s41568-018-0074-8
Owen JL et al (2012) Insulin stimulation of SREBP-1c processing in transgenic rat hepatocytes requires p70 S6-kinase. Proc Natl Acad Sci U S A 109:16184–16189. https://doi.org:10.1073/pnas.1213343109
Peterson TR et al (2011) mTOR complex 1 regulates lipin 1 localization to control the SREBP pathway. Cell 146:408–420. https://doi.org:10.1016/j.cell.2011.06.034
Bhutia YD, Babu E, Ramachandran S, Ganapathy V (2015) Amino Acid transporters in cancer and their relevance to glutamine addiction: novel targets for the design of a new class of anticancer drugs. Cancer Res 75:1782–1788. https://doi.org:10.1158/0008-5472.CAN-14-3745
Zhu Q et al (2018) Loss of ATF3 exacerbates liver damage through the activation of mTOR/p70S6K/ HIF-1alpha signaling pathway in liver inflammatory injury. Cell Death Dis 9:910. https://doi.org:10.1038/s41419-018-0894-1
Ku HC, Cheng CF (2020) Master Regulator Activating Transcription Factor 3 (ATF3) in Metabolic Homeostasis and Cancer. Front Endocrinol (Lausanne) 11:556. https://doi.org:10.3389/fendo.2020.00556
Li L, Song S, Fang X, Cao D (2021) Role of ATF3 as a prognostic biomarker and correlation of ATF3 expression with macrophage infiltration in hepatocellular carcinoma. BMC Med Genomics 14:8. https://doi.org:10.1186/s12920-020-00852-4
Cheng CF et al (2019) Adipocyte browning and resistance to obesity in mice is induced by expression of ATF3. Commun Biol 2:389. https://doi.org:10.1038/s42003-019-0624-y
Fu C, Donovan WP, Shikapwashya-Hasser O, Ye X, Cole RH (2014) Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase. PLoS ONE 9:e115318. https://doi.org:10.1371/journal.pone.0115318
Gyorffy B (2024) Transcriptome-level discovery of survival-associated biomarkers and therapy targets in non-small-cell lung cancer. Br J Pharmacol 181:362–374. https://doi.org:10.1111/bph.16257
Miller HE, Bishop AJR (2021) Correlation AnalyzeR: functional predictions from gene co-expression correlations. BMC Bioinformatics 22:206. https://doi.org:10.1186/s12859-021-04130-7
Chen C et al (2018) ATF3 inhibits the tumorigenesis and progression of hepatocellular carcinoma cells via upregulation of CYR61 expression. J Exp Clin Cancer Res 37:263. https://doi.org:10.1186/s13046-018-0919-8
Lin L et al (2014) Transcriptional regulation of STAT3 by SPTBN1 and SMAD3 in HCC through cAMP-response element-binding proteins ATF3 and CREB2. Carcinogenesis 35:2393–2403. https://doi.org:10.1093/carcin/bgu163
Currie E, Schulze A, Zechner R, Walther TC, Farese RV (2013) Jr. Cellular fatty acid metabolism and cancer. Cell Metab 18:153–161. https://doi.org:10.1016/j.cmet.2013.05.017
Baenke F, Peck B, Miess H, Schulze A (2013) Hooked on fat: the role of lipid synthesis in cancer metabolism and tumour development. Dis Model Mech 6:1353–1363. https://doi.org:10.1242/dmm.011338
Dong J et al (2024) Oncolytic adenovirus encoding apolipoprotein A1 suppresses metastasis of triple-negative breast cancer in mice. J Exp Clin Cancer Res 43:102. https://doi.org:10.1186/s13046-024-03011-0
Ieda A et al (2021) Ellagic Acid Suppresses ApoB Secretion and Enhances ApoA-1 Secretion from Human Hepatoma Cells, HepG2. Molecules 26 https://doi.org:10.3390/molecules26133885
Ren L et al (2022) Systematic pan-cancer analysis identifies APOC1 as an immunological biomarker which regulates macrophage polarization and promotes tumor metastasis. Pharmacol Res 183:106376. https://doi.org:10.1016/j.phrs.2022.106376
Saxton RA, Sabatini DM (2017) mTOR Signaling in Growth, Metabolism, and Disease. Cell 169:361–371. https://doi.org:10.1016/j.cell.2017.03.035
Xu D et al (2020) The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis. Nature 580:530–535. https://doi.org:10.1038/s41586-020-2183-2
Lee G et al (2017) Post-transcriptional Regulation of De Novo Lipogenesis by mTORC1-S6K1-SRPK2 Signaling. Cell 171, 1545–1558 e1518 https://doi.org:10.1016/j.cell.2017.10.037
Zhou X et al (2015) Dynamic Visualization of mTORC1 Activity in Living Cells. Cell Rep 10:1767–1777. https://doi.org:10.1016/j.celrep.2015.02.031
Liu K et al (2022) Lipotoxicity-induced STING1 activation stimulates MTORC1 and restricts hepatic lipophagy. Autophagy 18:860–876. https://doi.org:10.1080/15548627.2021.1961072
Fotiadis D, Kanai Y, Palacin M (2013) The SLC3 and SLC7 families of amino acid transporters. Mol Aspects Med 34:139–158. https://doi.org:10.1016/j.mam.2012.10.007
Nicklin P et al (2009) Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 136:521–534. https://doi.org:10.1016/j.cell.2008.11.044
Asaoka Y, Nagai Y, Namae M, Furutani-Seiki M, Nishina H (2016) SLC7 family transporters control the establishment of left-right asymmetry during organogenesis in medaka by activating mTOR signaling. Biochem Biophys Res Commun 474:146–153. https://doi.org:10.1016/j.bbrc.2016.04.087
Jersin RA et al (2021) Role of the Neutral Amino Acid Transporter SLC7A10 in Adipocyte Lipid Storage, Obesity, and Insulin Resistance. Diabetes 70:680–695. https://doi.org:10.2337/db20-0096
Zhang S et al (2023) ATF3 induction prevents precocious activation of skeletal muscle stem cell by regulating H2B expression. Nat Commun 14:4978. https://doi.org:10.1038/s41467-023-40465-w
Wolfgang CD, Liang G, Okamoto Y, Allen AE, Hai T (2000) Transcriptional autorepression of the stress-inducible gene ATF3. J Biol Chem 275:16865–16870. https://doi.org:10.1074/jbc.M909637199
Kang Y, Kim YW, Kang J, Kim A, Histone (2021) H3K4me1 and H3K27ac play roles in nucleosome eviction and eRNA transcription, respectively, at enhancers. FASEB J 35:e21781. https://doi.org:10.1096/fj.202100488R
Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674. https://doi.org:10.1016/j.cell.2011.02.013
Beloribi-Djefaflia S, Vasseur S, Guillaumond F (2016) Lipid metabolic reprogramming in cancer cells. Oncogenesis 5, e189 https://doi.org:10.1038/oncsis.2015.49
Cheng C, Geng F, Cheng X, Guo D (2018) Lipid metabolism reprogramming and its potential targets in cancer. Cancer Commun (Lond) 38:27. https://doi.org:10.1186/s40880-018-0301-4
Koundouros N, Poulogiannis G (2020) Reprogramming of fatty acid metabolism in cancer. Br J Cancer 122:4–22. https://doi.org:10.1038/s41416-019-0650-z
Ishizawa T et al (2008) Neither multiple tumors nor portal hypertension are surgical contraindications for hepatocellular carcinoma. Gastroenterology 134:1908–1916. https://doi.org:10.1053/j.gastro.2008.02.091
Pope ED 3 et al (2019) Aberrant lipid metabolism as a therapeutic target in liver cancer. Expert Opin Ther Targets 23:473–483. https://doi.org:10.1080/14728222.2019.1615883
Rossi F, Casano AM, Henke K, Richter K, Peri F (2015) The SLC7A7 Transporter Identifies Microglial Precursors prior to Entry into the Brain. Cell Rep 11:1008–1017. https://doi.org:10.1016/j.celrep.2015.04.028
Ji X et al (2018) Function of SLC7A7 in T-Cell Acute Lymphoblastic Leukemia. Cell Physiol Biochem 48:731–740. https://doi.org:10.1159/000491899
Wang L et al (2020) ATF3 promotes erastin-induced ferroptosis by suppressing system Xc(). Cell Death Differ 27:662–675. https://doi.org:10.1038/s41418-019-0380-z

Supplementary Files are not available with this version.

The authors declare no competing interests.

Download PDF

Version 1

posted

You are reading this latest preprint version

ATF3-SLC7A7 Axis Regulates mTORC1 Signaling to Suppress Lipogenesis and Tumorigenesis in Hepatocellular Carcinoma

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Materials and Methods

Statistical analysis

Results

ATF3 suppresses the amplification and invasion of HCC cells

ATF3 is correlated with better prognosis and downregulated lipid synthesis in HCC

ATF3 inhibits lipid biosynthesis in HCC cells

ATF3 represses lipid biosynthesis by inhibiting mTORC1 signaling

SLC7A7 is involved in the inhibition of ATF3 on mTORC1 signaling

ATF3 binds to SLC7A7 enhancer and activates its transcription

ATF3 represses tumor growth and lipogenesis in vivo

Discussion

Abbreviations

Declarations

References

Supplementary Files

Additional Declarations

Status:

Version 1