Kynurenine derivative 3-HAA is an agonist ligand for transcription factor YY1

The 3-hydroxyanthranilic acid (3-HAA), a derivative of kynurenine, was reported to suppress tumor growth. However, the function of 3-HAA largely remains unclear. Here, we report that 3-hydroxyanthranilic acid (3-HAA) is lower in tumor cells, while adding exogenous 3-HAA induces apoptosis in hepatocellular carcinoma by binding YY1. This 3-HAA binding of YY1 leads to phosphorylation of YY1 at the Thr 398 by PKCζ, concomitantly enhances YY1 chromatin binding activity to increase expression of target genes. These findings demonstrate that 3-HAA is a ligand of YY1, suggesting it is a promising therapeutic candidate for HCC. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-021-01165-4.

TFs predicted on 4 genes' promoters E Increased proteins binding to chromatin post-3-HAA treatment Time ( Cleaved PARP Figure S2 PKCζ phosphorylates YY1 at Thr398 in response to 3-HAA. Related to Figure2 Figure S2 legend A. The apoptotic SMMC7721 cells determined by the flow cytometry analysis. The concentration of 3-HAA was 100 μM. B. The representative YY1 binding proteins identified by protein mass-spect analysis following coimmunoprecipitation. C. The effect of PKCζ expression on grade 1 liver cancer overall survival. The total patient number was 55 and devided into two group by the mean, p < 0.05.

Colony formation
HCC cells were seeded into 6-well dishes at a cell density of 1000 cells/well and treated with drugs for 10-14 days until clones were visible. PBS-washed cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The stained clones were counted.

Cell proliferation assay
Cell proliferation was measured using the Cell Counting Kit-8 reagent (CCK-8, Dojindo, cat: CK04). HCC cells were seeded into a 96-well plate at 2000 cells/well, were treated with 3-HAA (Sigma, cat: 148776) and ZVAD (TargetMol, cat: T6013) at appropriate doses as indicated in the figure legend. CCK-8 assays were performed in triplicates as instructed by the manufacturer for 2 hours at indicated time points. Absorbance was measured at 450 nm using a microplate reader, and cell viability was normalized to control, and the mean of at least three independent experiments was calculated.

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 18505-1-AP), TIPARP (Sigma, cat:SAB2102431) and β-actin (Santa Cruz, cat: 47778). 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

Expression and Purification of HIS-YY1 Fusion Protein
A plasmid containing N-terminal 6×His-tagged human YY1 coding sequence was NaCl). Subsequently, proteins were loaded onto a Hitrap heparin column and eluted by a NaCl gradient (0.1-1.5 M). The peak fractions were collected and dialyzed against PBS buffer. Proteins were then concentrated for use. TGGAAGCCGGATGGAGGCGT), before labeling at 5' terminal of the primers using FAM.

Dual-luciferase reporter
For luciferase assay, the promoter region of human DUSP6 was moved from pGL2-DUSP6 (a gift from Dr. Norton, University of Florida) by digestion, gel purified, and inserted into a KpnI/XhoI digested pGL4 vector. We constructed some mutants using primers (DUSP6- 293T cells were co-transfected with 100ng different pGL4-DUSP6-promoter vectors, 800ng pSG5-YY1, or pSG5, and 1 ng pRL-TK-Renilla luciferase plasmids. After 48 hours, the cells were lysed, and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.

RNA-Seq
RNA was extracted from HCC cells with the TRIzol reagent (Invitrogen, cat: 15596018) according to the instructions. RNA-seq libraries were prepared with the Ovation RNASeq Systems 1-16 (Nugen), and indexed libraries were multiplexed in a single flow cell and underwent 75 base pair single-end sequencing on an Illumina NextSeq500 using the High Output kit v2 (75 cycles) at BGI Group.

Lentivirus Production
293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco) and were maintained at 37℃ in a humidified atmosphere with 5% CO2. For virus production, 8 μg of the appropriate plasmid and 3.2 mg of helper plasmids (2μg pMD2.G and 6μg psPAX2) were transfected into 293T cells cultured at 80% confluence in a 10 cm dish using Lipoplus (SAGE) according to the manufacturer's instructions. Viral supernatants were collected 48 hours after transfection and filtered through a 0.45 mm filter.

Silver Stain
HCC cells were treated with 100μM of 3-HAA for 24 hours. Cells were collected and lysed by lysis buffer. Proteins were separated by SDS-PAGE and stained by using Fast Silver Stain Kit (Beyotime, cat: P0017S) according to the instructions.

Metabolism flux analysis by LC-MS/MS
For the flux experiment of tryptophan catabolism, tryptophan in the medium was replaced by

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 five 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 first 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% to 23% over 26 min, then from 23% to 35% in 8 min and then to 80% in 3 min, after which it remained at 80% for 3 min. The flow rate was constant at 400 nL/min.
Peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Orbitrap Fusion TM Tribrid TM (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 datadependent 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 overfilling; 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 fixed first 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 specified as a fixed modification, while oxidation on methionine was specified as a variable modification. FDR was adjusted to < 1% and the minimum score for peptides was set to > 40.

Surface plasmon resonance and microscale thermophoresis
In order to measure the affinity of 3-HAA to YY1 protein, the dissociation constants were measured using a BIAcore T200 instrument (GE Healthcare, CA, USA) with a CM5 sensor chip (GE Healthcare). The YY1 protein was immobilized on a CM5 sensor chip in sodium acetate buffer (1 μg/mL, pH 5.5), and 3-HAA was gradually titrated at the indicated concentrations. The 3-HAA was injected at a flow rate of 30 μL/min. The association time was 120 s and the dissociation time was 420 s. The binding constant was calculated using a 1:1 Langmuir binding model via the BIAevaluation software.
Measurements were made using the Monolith NT.115 (NanoTemper), and data were analyzed using MO. Affinity analysis (×64) software. Graphs were plotted using Prism 5 software.

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 fix 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 fluorescence microscope. After two weeks, subcutaneously transplanted tumors were removed, and the volume was measured, and the tumors were photographed. Following homogenization or tissue slicing, the flow cytometry analysis and TUNEL assay were performed to determine the ratio of apoptotic cells in xenografts. For flow cytometry analysis, cells were collected in binding buffer and stained with Annexin V-APC and PI, and apoptotic signals were detected by flow 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 specificpathogen-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 [23]. 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 [24][25][26]. Briefly, 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 filtered 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 specific time after hydrodynamic transfection (HDT). The six mice in each group were used for survival analysis.