3-HAA Metabolic Pathway Regulates HCC Growth

metabolism


ChIP analysis
Chromatin was isolated from HCC cells treated with or without 3-HAA and fragmented to a size range from 150 to 400 bp. The solubilized chromatin fragments were immunoprecipitated with antibodies against YY1 (Active Motif, cat: 61779). The recovered DNA fragments were processed for DNA sequencing by the Illumina Genome Analyzer. The generated short reads were mapped onto the genome, and the peak calling program was used to identify peaks with the mapped reads. Western blotting assays Appropriate cells were lyzed in RIPA lysis buffer containing a cocktail of protease inhibitors (Roche) and PMSF. Total protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Ding Guo Biotechnology, cat: BCA02). For nuclear and cytoplasmic protein analysis, the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, cat: P0028) was used according to the instructions. For immunoprecipitation, HCC cells were lyzed in lysis buffer (50mM Tris, 150mM NaCl, 1% TritonX-100, and 1mM EDTA) containing a cocktail of protease inhibitors and PMSF. Protein A/G beads, HA/YY1 antibodies were added to soluble protein and incubated overnight at 4℃, with gentle agitation. Immunoprecipitated materials were washed three times, eluted with loading buffer at 95℃ for 5 min, and analyzed by western blotting. Antibodies against the following proteins were used for immunoblotting: The immunoblots were scanned using an Odyssey infrared imaging system (LI-COR). Immunolabeling was detected using the ECL reagent (Sigma). Protein expression was normalized against β-actin.

Real-time quantitative PCR
Total cellular RNA was prepared using the TRIzol reagent (Invitrogen, cat: 15596018) as instructed by the manufacturer and was reverse transcribed using a reverse transcription reagent kit (TAKARA, cat: RR036A). After cDNA synthesis, real-time quantitative polymerase chain reaction (PCR) was performed in triplicate in a 96-well plate with an ABI7500 real-time PCR system (Life Technologies, Grand Island, NY, USA) using SYBR Green mixture (AG, cat: AG11702). CYP1A1, CYP1B1, and TIPARP expression were normalized against β-actin. The primer sequences were as follows:

Quantitative proteomics
Cell samples were sonicated three times on ice using a high-intensity ultrasonic processor (Scientz, Ninbo, Zhejiang, China) in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail). The supernatant was collected, and proteins were reduced with ve mM dithiothreitol for 30 min at 56°C, then alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. Following the addition of 100 mM TEAB to dilute the urea to < 2 M, trypsin was added to the protein samples rst at a trypsin-to-protein mass ratio of 1:50 for digestion overnight, then at a ratio of 1:100 for second digestion lasting four h.
After trypsin digestion, peptides were desalted on a Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer's protocol for the TMT kit/iTRAQ kit. The tryptic peptides dissolved in 0.1% formic acid (solvent A) were directly loaded onto a custom-made reverse-phase analytical column (15-cm length, 75 µm i.d.) on an EASY-nLC 1000 UPLC system. The gradient to solvent B (0.1% formic acid in 98% acetonitrile) increased from 6-23% over 26 min, then from 23-35% in 8 min and then to 80% in 3 min, after which it remained at 80% for 3 min. The ow rate was constant at 400 nL/min.
Peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Orbitrap Fusion™ Tribrid™ (Thermo, CA, USA) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 60,000. Peptides were selected for MS/MS by NCE at 35; ion fragments were detected in the Orbitrap at a resolution of 30,000. A data-dependent procedure that alternated between one MS scan followed by 10 MS/MS scans was applied for the top 10 precursor ions above a threshold intensity, which were greater than 5 x 10 3 in the MS survey scan with dynamic exclusion of 30.0 s. The electrospray voltage applied was 2.0 kV. Automatic gain control was used to prevent the orbitrap from over lling; 5 x 10 4 ions were accumulated to generate MS/MS spectra. For MS scans, the m/z scan range was from 350 to 1550. The xed rst mass was set as 100 m/z. MS/MS data were processed using the Maxquant search engine (version 1.5.2.8). Carbamidomethyl on cysteine was speci ed as a xed modi cation, while oxidation on methionine was speci ed as a variable modi cation. FDR was adjusted to < 1% and the minimum score for peptides was set to > 40.

TUNEL assay
The cover glass was placed in the 24-well plate, and the HCC cells were inoculated on the cover glass overnight. DMSO or 100 µM 3-HAA was added to the culture medium for 48 hours. The cells were washed with PBS for three times. Add 0.5mL of 4% paraformaldehyde and x cells at room temperature for 10 minutes. Cells were treated with 0.4%Triton X-100 for 5 minutes and washed with PBS. Cells on the cover glass were treated with TUNEL staining solution and incubated in a wet box at 37℃ for 1 hour. DAPI staining solution was used to stain the nuclear for 5 minutes in dark. Cells were observed and photographed under a uorescence microscope.

HCC-PCX mouse model
Six-week-old male BALB/c nude mice or male immune-competent C57BL/6 mice were purchased from Lingchang, Shanghai, China. Xenograft mouse model of HCC was generated by injecting SMMC-7721 HCC cells (1.5×10^6) or mouse live cancer Hep1-6 cells (1 × 10^6) subcutaneously into the armpit of the rear limb. These SMMC-7721 cells were overexpressing KMO or T398E/T398A mutant YY1 or depleted of HAAO, DUSP6, or YY1. Mice were injected intraperitoneally with 100 mg/kg of 3-HAA or an equal amount of DMSO every day. The PKC inhibitor Go6983 was injected intravenously every two days. The dose of Go6983 was 0.8 mg/kg.day, and the IDO1 inhibitor Epacadostat was orally administered at the dose of 100 mg/kg.day.
After two weeks, subcutaneously transplanted tumors were removed, and the volume was measured, and the tumors were photographed. Following homogenization or tissue slicing, the ow cytometry analysis and TUNEL assay were performed to determine the ratio of apoptotic cells in xenografts. For ow cytometry analysis, cells were collected in binding buffer and stained with Annexin V-APC and PI, and apoptotic signals were detected by ow cytometry.

HCC-PDX mouse models
This study received ethics board approval at the Shanghai Jiao Tong University School of Medicine. The HCC-PDX models (LIV#031, #046, and #057) were initially isolated from patients and were stored in liquid nitrogen. Mice were maintained under speci c-pathogen-free (SPF) conditions. Once the recovered tumors grew to 250 mm 3 in mice, tumor tissues were cut into 2×2 mm pieces and implanted subcutaneously into SCID mice [24]. The 3-HAA were intraperitoneally administered every day when the tumor volume reached approximately 200 mm 3 . Tumor size and mice body weight were monitored for up to 4 weeks, and tumor volume (TV) was calculated.

Transposon HCC mouse Model
This induced HCC mouse model was adopted from the literature [25][26][27]. Brie y, HCC inducing oncogenes β-Catenin and MET in pT2 vector along with Sleeping Beauty transposon (SB100) was introduced with GFP, pT2-shDUSP6, or pT2-shYY1 (also in pT2 vector) into C57BL/6 mice. Thirty micrograms of the oncogene plasmids and three micrograms SB100 plasmids were diluted in 2 ml of a ltered 0.9% NaCl solution and followed by an injection into the lateral tail veins of 6-week-old mice. Livers of some mice were harvested to determine tumor burden at a speci c time after hydrodynamic transfection (HDT). The six mice in each group were used for survival analysis.

Statistical analysis
Data were presented as means ± SD. All data were representative of at least three independent experiments. The unpaired two-tailed Student's t-test and the Two-way ANOVA were used as indicated. All Page 9/24 presented differences were P < 0.05 unless otherwise stated.

3-HAA is decreased in tumor cells
To comprehensively understand the effect of kynurenine derivatives on tumors, tryptophan catabolites were rst analyzed in clinical HCCs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS). The concentration of kynurenine catabolite 3-HAA decreased in 37 cases of HCC (p < 0.01) and 42 cases of esophageal carcinomas (p < 0.01) compared to the matched paratumor tissues (Figs. 1A and S1A). Conversely, the concentration of tryptophan and kynurenine was higher in these HCCs and esophageal carcinomas than in the matched paratumor tissues, respectively. There was no signi cant difference in 3-hydroxykynurenine (3-HK) between tumors and adjacent non-cancerous tissues. Consistent with this observation, the concentration of 3-HAA was also lower in seven HCC cell lines tested than in normal hepatic LO2 cells, whereas the content of tryptophan and kynurenine increased in these tested HCC cell lines (Fig. 1B). The immunohistochemistry analysis further con rmed lower 3-HAA content in clinical HCC tissues than in adjacent non-cancerous tissues (Fig. 1C).
Metabolic ux analysis revealed tryptophan metabolized to kynurenine but not 3-HK or 3-HAA in HCC cells, and the newly generated kynurenine was secreted into the culture medium (Fig. 1D), suggesting 3-HAA is decreased in tumors, at least in HCCs and esophageal carcinomas.
In addition, overexpression of KMO signi cantly increased the concentration of 3-HAA in HCC SMMC7721 cells, six times more than control cells, but not the hydroxykynurenine (3-HK), picolinate (PA), or quinolinate (QA) (Fig. 2E). The HAAO knockdown had similar effects on the levels of these metabolites (Fig. 2F). These observations suggested that 3-HAA is decreased in cancer cells by upregulating KMO expression and/or downregulating HAAO.

3-HAA inhibits tumor formation by inducing apoptosis
In order to determine the effect of 3-HAA on tumor growth, exogenous 3-HAA was added to HCC cultures, and cell proliferation was assessed. Indeed, 3-HAA signi cantly inhibited HCC cell growth and colony formation (Figs. 3A and S3A). In contrast, the other tryptophan metabolites, kynurenine (KYN), 3hydroxykynurenine (3-HK), and quinolinate acid (QA) did not substantially affect the growth and colony formation of HepG2 and SMMC7721 cells. Moreover, 3-HAA treatment also slowed tumor growth in a CDX (cell-derived xenograft) model and in a PDX (patient-derived xenograft) model while the same dose of kynurenine had negligible effects on xenografts' growth (Figs. S3B and 3B).
In order to verify that this effect was caused by 3-HAA and not its precursor kynurenine or 3-HK, the cellular concentrations of 3-HAA, kynurenine, and 3-HK were analyzed by LC-MS/MS in 3-HAA-treated tumor cells. Although the content of endogenous 3-HAA varied among different HCC cell lines, 3-HAA treatment increased the cellular concentration of 3-HAA, particularly in SMMC7721 and HepG2, but not kynurenine (Fig. S3C). Moreover, the cell growth was analyzed in SMMC7721 cells overexpressing KMO or knocking down HAAO enzymes. Either overexpression of KMO or knockdown of HAAO inhibited cell growth of HCC cells in vitro (Fig. 3C).
To determine what signal mediates 3-HAA-induced cell death, we treated tumor cells with 3-HAA and speci c inhibitors to apoptosis, autophagy, and necrosis, respectively. As shown in Fig. 3D, the apoptosis inhibitor ZVAD, but not the necrosis inhibitor Nec1 or autophagy inhibitor 3-MA, restored growth of HepG2 and SMMC7721 cells following 3-HAA treatment. This effect was observed at ZVAD doses of 50 and 100 µM. The KMO overexpression increased apoptosis in HCC cells, and the apoptosis inhibitor ZVAD restored the HCC cell growth (Fig. 3E & 3F). Moreover, the tumor formation assay showed that KMO overexpression suppressed tumor formation and tumor growth in HCC xenograft nude mice model. After inoculation of 0.5 × 10 6 SMMC7721 cells, the tumor formation rates within three weeks were 90% (9/10) versus 40% (4/10) in the control group and KMO overexpression group, respectively (Figs. 3G). When increasing the inoculation number of tumor cells to 1.5 × 10 6 , the tumor volumes in KMO overexpressing group were apparently small than the control group by the end of 4 weeks (Fig. S3D). Remarkably, the Kaplan-Meier survival analysis showed that HCC patients with high KMO expression had a more prolonged disease-free survival than patients with low KMO expression (Fig. 3H). These observations suggested that 3-HAA is a negative regulator for tumor formation and tumor growth.

3-HAA induces apoptosis by upregulating DUSP6 expression
In order to determine the mechanism by which 3-HAA induces tumor cell apoptosis, RNA sequencing was used to pro le gene expression in SMMC7721 or HepG2 cells after 1, 8, or 24 h treatment with 3-HAA according to the screening strategy previously applied [7]. At all three time points after the start of 3-HAA treatment, the top 6 upregulated genes were selected (Fig. 4A). The expression of these 6 genes was individually veri ed by real-time PCR. Immunoblotting of the top two upregulated genes also showed elevated levels of DUSP6 and IGFBP1 in SMMC7721 and HepG2 cells (Fig. 4B). These results suggested that 3-HAA altered the gene expression pro le of HCC cells, with DUSP6 and IGFBP1 as two of the most upregulated genes. However, the clinical data showed that the overall survival of HCC patients was only associated with the expression level of DUSP6 (p < 0.05), but not IGFBP1 (p > 0.05). Patients expressing a high level of DUSP6 showed a more prolonged overall survival than patients expressing a low level of DUSP6 (p < 0.05) (Fig. 4C), and the corrective analysis with the clinical characteristics also supported this nding (Fig. S4A), indicating that DUSP6 might mediate the 3-HAA-induced apoptosis.
To demonstrate whether DUSP6 mediating 3-HAA-induced tumor cell apoptosis, the effects of DUSP6 on HCC cell growth were rst analyzed in HCC cells. DUSP6 knockdown restored growth of HepG2 and SMMC7721 cells inhibited by 3-HAA (Figs. 4D and S4B). 3-HAA induced apoptosis to a smaller extent in DUSP6-depleted SMMC7721 cells than in the control cells, based on ow cytometry using Annexin V (Fig.  4E & S4C). DUSP6 depletion was also associated with reduced levels of cleaved caspase-3 and cleaved PARP; in the meantime, DUSP6 knockdown facilitated ERK enhancing signal on cell survival, which was consistent with the previous study [Piya, 2012] (Fig. 4F). In fact, DUSP6 knockdown restored ERK activity that had been suppressed by 3-HAA (Fig. 4F). ERK activation inhibited apoptosis via BAD/BCL2/BCL-XL signaling, consistent with the previous reports [13] [14].
Based on the previous nding that 3-HAA activates transcription factor YY1 [Shi, 2021], we speculated that 3-HAA could regulate YY1 transcription activity on DUSP6 gene promoter. Closer analysis of the DUSP6 promoter region using online-based prediction tools (jaspar.genereg.net and ecrbrowser.dcode.org) [15,16] revealed a novel potential YY1 binding DNA fragment at positions − 1145 to -1134 (TCCATCCGGCTT), which was distinct from the reported consensuses binding sequence (CAANATGGCGGC) [17]. In order to determine whether YY1 regulates DUSP6 expression by binding this novel sequence, each DNA fragment was added to a luciferase reporter gene, and YY1-driven luciferase expression was measured by its enzyme activity. Higher luciferase activity was observed with the full length or partial DUSP6 promoter containing this novel speci c sequence as well as the consensus YY1 binding sequence. Luciferase activity decreased when mutations involving this novel binding site (mut2) occurred (Fig. 4G). Moreover, the quantitative PCR analysis following chromatin immunoprecipitation of YY1 revealed that 3-HAA promoted YY1 binding to the consensus sequence of the P53 promoter region (positive control) and the novel binding sequence in the DUSP6 promoter region, as re ected by 3-HAAinduced YY1 enrichment (Figs. 4H & S4D). The TUNEL assay demonstrated that 3-HAA-induced apoptosis was also reduced in SMMC7721 cells depleted of YY1, overexpression of DUSP6 restored the apoptosis suppressed by YY1 depletion (Fig. 4I), suggesting DUSP6 mediates 3-HAA-induced HCC apoptosis.

3-HAA synergizes with IDO1 inhibitor on HCC growth
To further evaluate the potential application of 3-HAA in clinics, the various HCC mouse models were implemented in this study. As shown in Fig. 5A, DUSP6 knockdown reversed 3-HAA-mediated suppression of tumor growth in SMMC7721 xenografts, of which the SMMC7721 cells were depleted of DUSP6 by shRNAs before inoculation. More impressively, 3-HAA treatment reduced the tumor numbers and prolonged the survival in a transposon HCC mouse model. DUSP6 depletion promoted tumor formation and shorten mice survival, and 3-HAA treatment had little effect on tumor formation and mice survival after DUSP6 knockdown (Fig. 5B).
Most importantly, 3-HAA synergized with IDO1 inhibitor Epacadostat to suppress HCC xenograft growth in an immune-competent mouse model (Fig. 5C). In the meantime, the combination of 3-HAA with IDO1 inhibitor Epacadostat also inhibited the genetic HCC tumor growth and prolonged the survival of mice bearing transposon-induced HCCs (Fig. 5D).
In order to evaluate the stability of 3-HAA in vivo, the concentration of 3-HAA was analyzed at the time course of administration in the plasma of mice. As shown in Fig. S5A, the half-life of 3-HAA was 3.89 hours in the serum of mice. The concentration of 3-HAA reached 71.3 µmol per gram in tumors post seven days of treatment. The seven days' administration of 3-HAA at the dose of 100 mg/kg.day did not induce apoptosis of naïve T lymphocytes in the spleen (Fig. S5B). These results highlighted the promise of 3-HAA as a potential HCC therapy.

Discussion
Tryptophan metabolism plays a critical role in development and tumor progression. It not only provides critical intermediates for anabolism but also regulates cell signaling. IDO converts tryptophan into kynurenine, which is a well-known functional metabolite of tryptophan and could be further catabolized to 3-HAA. Kynurenine binds AHR to induce immune suppression (tolerance), which is associated with successful embryo implantation but also with poor prognosis in various malignancies. The inhibition of IDO1/2 suppressed tumor formation in animal models but failed in stage III of clinical trials. This is probably due to the expression of the other kynurenine generating enzyme TDO, which may not be suppressed by the IDO inhibitor. However, the 3-HAA treatment reverses the tumor-promoting effect of kynurenine and signi cantly improves the e cacy of IDO1/2 inhibitors on HCC xenografts, suggesting 3-HAA as a negative feedback regulator reverses the tumor cell-hijacked tryptophan metabolism.
The 3-HAA exerted anti-in ammatory and neuroprotective effects by selectively inducing the apoptosis of activated T cells or suppressing microglia/astrocytes that expressing cytokines and chemokines [11,12]. The 3-HAA induced the expression of cytoprotective enzyme hemeoxygenase-1 in astrocytes and microglia; the latter is an enzyme with proven anti-in ammatory and cytoprotective activities [18,19]. The 3-HAA-induced apoptosis of activated T lymphocytes was linked to oxidative stress and induction of caspases [20,21]. These results indicated that 3-HAA has a distinct biological function in a different type of cells. However, the biological function of 3-HAA largely remained unclear.
In this study, we report that 3-HAA decreases in HCC cells and HCC tissues due to the downregulation of KMO and KYNU enzymes as well as the upregulation of the HAAO enzyme. The excessive kynurenine generated from tryptophan, in return, regulates these enzyme expressions by autocrine or paracrine of HCC cells [22,23]. Also, we observe that the sensitivity of HCC cell to 3-HAA, to some extent, are correlated with the cellular 3-HAA concentration after 3-HAA treatment. However, the increase of intracellular 3-HAA concentration post-3-HAA treatment may not completely associate with expression levels of KMO, KYNU, or HAAO enzymes in HCC cells, since the 3-HAA transporters and drug-resistant genes may also involve in the regulation of HCC sensitivity to 3-HAA. This observation suggests that the heterogeneity of tryptophan metabolism in HCC cells may determine their sensitivity to 3-HAA.

Conclusion
In brief, both the increase of 3-HAA and upregulation of 3-HAA synthetic enzyme KMO suppresses HCC growth in the PDX and transposon-induced HCC mouse model by inducing apoptosis of HCC cell, suggesting the 3-HAA metabolic pathway is important to HCC growth and downregulation of 3-HAA appears to be essential for HCC growth. Exogenous 3-HAA or KMO overexpression induces HCC cell apoptosis, sequentially reduces the formation and growth of HCC; moreover, the 3-HAA treatment improves the e cacy of IDO1/2 inhibitors on HCC growth, suggesting its potential use in HCC therapy.

Availability of data and material
The data used to support the ndings of this study are available from the corresponding author upon request.

Competing interests
The authors declare that they have no competing interests.     K-M plotter. The total patient number was 415. The HCC patients were divided into two groups by the median value of KMO expression. D. Effects of DUSP6 knockdown on the growth of HCC cells. Cells were treated with 100 μM 3-HAA for the indicated time. DUSP6 was stably knocked down in SMMC7721 cells using lentivirus-generated shRNA. **: P<0.01. E. Effects of DUSP6 knockdown on HCC cell apoptosis. SMMC7721 cells were treated with 100 μM of 3-HAA for 12 h, stained for Annexin V, and analyzed by ow cytometry. *: P<0.05. F. Effects of DUSP6 knockdown on 3-HAA-activated apoptotic signaling. SMMC7721 cells depleted of DUSP6 were treated with 100 μM 3-HAA for 24 h. G. Transcription activity of YY1 on the DUSP6 promoter, as determined in a luciferase reporter assay. The schematic depicts the plasmid encoding luciferase under the control of the DUSP6 promoter, which was truncated or mutated as indicated. H. The ChIP-PCR analysis. The positive control (PC) is TP53. I. The apoptosis analysis by TUNEL assay in SMMC7721 cells depleted of YY1 and/or overexpressing DUSP6. Figure 5 3-HAA synergizes with IDO1 inhibitor on HCC growth A. DUSP6 knockdown recovered 3-HAA-suppressed xenograft growth. Animals were treated with 3-HAA (100 mg/kg•day) for seven days, then sacri ced on