Kynurenine analog 3-HAA is a ligand for transcription factor YY1
Zhaopeng Shi Shanghai Jiao Tong University School of Medicine Guifang Gan Shanghai Jiao Tong University School of Medicine Xiang Xu Shanghai Jiao Tong University School of Medicine Jieying Zhang Shanghai Jiao Tong University School of Medicine Yuan Yuan Shanghai Jiao Tong University School of Medicine Bo Bi Shanghai Jiao Tong University School of Medicine Xianfu Gao Shanghai Pro eader Biotech Co., Ltd Pengfei Xu Xiangya School of Pharmaceutical Sciences, Central South University Wenbin Zeng Xiangya School of Pharmaceutical Sciences, Central South University Jixi Li Fudan University https://orcid.org/0000-0003-3463-3175 Youqiong Ye Shanghai Jiao Tong University School of Medicine https://orcid.org/0000-0001-8332-4710 Aiwu Zhou Shanghai Jiao Tong University School of Medicine https://orcid.org/0000-0002-2555-5091 Naixia Zhang Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China Wen Liu Xiamen University https://orcid.org/0000-0003-3434-4162 Shu-hai Lin Xiamen University Jun Mi ( [email protected] ) Shanghai Jiao Tong University https://orcid.org/0000-0001-5788-3965 Article
Keywords: 3-hydroxyanthronic acid (3-HAA), kynurenine, YY1, DUSP6, Hepatocellular Carcinoma (HCC), tryptophan metabolism
Posted Date: October 21st, 2020
DOI: https://doi.org/10.21203/rs.3.rs-92311/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Kynurenine analog 3-HAA is a ligand for transcription factor YY1
Zhaopeng Shi 1,#, Guifang Gan 1,#, Xiang Xu 1, Jieying Zhang 1, Yuan Yuan 1, Bo Bi 2, Xianfu Gao 3,
Pengfei Xu 4, Wenbin Zeng 4, Jixi Li 5, Youqiong Ye 1, Aiwu Zhou 1, Naixia Zhang 6, Wen Liu 7,*, Shuhai
Lin 7,*, Jun Mi 1,2,*
1 Basic Medical Institute; Key Laboratory of Cell Differentiation and Apoptosis of the Chinese Ministry
of Education; Hongqiao International Institute of Medicine, Tongren Hospital, Shanghai Jiao Tong
University School of Medicine
2 Department of Nuclear Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine
3 Shanghai Profleader Biotech Co., Ltd
4 Xiangya School of Pharmaceutical Sciences, Central South University
5 Fudan University School of Life Science
6 CAS Key Laboratory of Receptor Research, Department of Analytical Chemistry, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai, China
7 School of Pharmaceutical Sciences, State Key Laboratory of Cellular Stress Biology, Xiamen University
# These authors contributed equally to this work
* Correspondence to: Jun Mi, [email protected]; Shuhai Lin, [email protected] ; Wen Liu,
Key words: 3-hydroxyanthronic acid (3-HAA); kynurenine; YY1; DUSP6; Hepatocellular Carcinoma (HCC); tryptophan metabolism ABTRACT
Kynurenine, a metabolite of tryptophan, promotes immune tolerance in development and tumor evasion by binding to the aryl hydrocarbon receptor (AHR). However, the IDO inhibitors, blocking kynurenine generation, fail in stage III of clinical trials in several tumors for unknown reasons. Here, we report that 3- hydroxyanthranilic acid (3-HAA) induces apoptosis and synergizes with IDO inhibitors by increasing the suppression of IDO inhibitors on HCC xenograft growth. The content of 3-HAA, a catabolite of kynurenine, is lower in tumor cells by downregulating its synthetic enzyme KMO/KYNU and/or upregulating its catalytic enzyme HAAO. Overexpression of KMO suppresses tumor formation and tumor growth by increasing endogenous 3-HAA while adding exogenous 3-HAA also inhibits tumor growth.
Moreover, we found that 3-HAA directly binds transcription factor YY1 rather than AHR and increasing
the PKCζ phosphorylation of YY1 at the Thr 398 in response to 3-HAA; YY1 phosphorylation at T398
increases the YY1 binding to chromatin. 3-HAA-induced Thr398 phosphorylation of YY1 upregulates the
expression of dual-specificity phosphatase 6 (DUSP6), etc. DUSP6 overexpression induces apoptosis of
hepatocellular carcinoma (HCC) cells and suppresses the HCC growth in vitro and in vivo. The T398
phosphorylation of YY1 is critical for the 3-HAA-induced apoptosis in tumors. These findings
demonstrate that kynurenine analog 3-HAA is a functional metabolite associating YY1 as an endogenous
ligand, downregulation of 3-HAA is necessary for the rapid growth of tumor cells, suggesting its
promising approach in HCC therapy.
Highlights:
1. 3-HAA selectively downregulated in tumor cells;
2. 3-HAA induced apoptosis of HCC cells by upregulating DUSP6;
3. 3-HAA directly binds transcription factor YY1;
4. PKCζ phosphorylates T398 of YY1 in response to 3-HAA.
The essential amino acid tryptophan is catabolized mainly through the serotonin pathway in the brain
or the kynurenine pathway in the liver (Chen and Guillemin, 2009; Schrocksnadel et al., 2006).
Tryptophan metabolism is enhanced in various tumors by upregulating the expression of indoleamine 2,3- dioxygenase 1/2 (IDO1/2), the rate-limiting enzyme in the kynurenine pathway. Kynurenine, a catabolite of tryptophan, increases immune tolerance in development and disease by directly binding to the aryl hydrocarbon receptor (AHR) (Opitz et al., 2011; Sharma et al., 2007), and enhances tumor immune evasion to promote tumor growth (Opitz et al., 2011) (Li et al., 2011) (Schwarcz et al.). The 3- hydroxyanthranilic acid (3-HAA), a derivative of kynurenine was reported to exert anti-inflammatory effects by selectively inducing the apoptosis of activated T cells (Krause et al., 2011; Lee et al., 2010).
However, the function of other kynurenine derivatives largely remains unclear. Here, we report 3-HAA, selectively decreased in various tumors, regulates the activity of transcription factor YY1 by directly binding and consequently induces tumor cell apoptosis and suppresses HCC growth in vitro and in vivo.
3-HAA is decreased in tumor cells
To comprehensively understand the effect of kynurenine derivatives on tumor, tryptophan catabolites were first 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 significant difference in 3-hydroxykynurenine (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). Immuno- histochemical analysis further confirmed lower 3-HAA content in clinical HCC tissues than in adjacent non-cancerous tissues (Fig. 1C).
Metabolic flux analysis using labeled tryptophan revealed catabolism into kynurenine but not HK or 3-HAA in HCC cells, and the newly generated kynurenine was secreted into the culture medium (Fig.
1D). Although the expression of 3-HAA degrading enzyme hydroxyanthranilate 3,4-dioxygenase
(HAAO) varied in HCC cells, both western blotting and immuno-histochemical analysis showed that kynurenine 3-monooxygenase (KMO) and kynureninase (KYNU), enzymes converting kynurenine into
3-HAA, were downregulated in HCC cells (Fig. 1E &S1B). The analysis of HCC expression profile in the
TCGA database also supported our finding (Fig. S1C), suggesting tumor cells reprogram the tryptophan metabolism by modulating the upstream/downstream catalytic enzymes.
Furthermore, overexpression of KMO or knockdown of HAAO significantly increased the concentration of 3-HAA in HCC SMMC7721 cells, 6 times more than control cells, but not the HK, picolinate (PA), or quinolinate (QA) (Fig. 1F & S1D). These observations suggest that 3-HAA is selectively downregulated in cancer cells by downregulating KMO expression and/or upregulating
HAAO.
3-HAA inhibits tumor growth by inducing apoptosis
To determine whether 3-HAA could inhibit tumor growth, exogenous 3-HAA was added to HCC cultures, and cell proliferation was assessed. Indeed, 3-HAA significantly inhibited HCC cell growth and colony formation (Figs. 2A and S2A). In contrast, the other tryptophan metabolites Kyn, PA, and QA did not substantially affect growth or colony formation of HepG2 or SMMC7721 cells. To verify that this effect was caused by 3-HAA and not its substrate, the cellular concentrations of 3-HAA and kynurenine 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, and the downstream substrate but not kynurenine (Fig. S2B).
To understand the mechanism by which 3-HAA inhibited HCC cell growth, gene expression profiling of 3-HAA-treated HCC cells was analyzed by the gene ontology (GO) enrichment. The GO analysis revealed that the cell death-related pathways were highly activated in 3-HAA-treated HCC cells
(Fig. 2B). To further determine what signal mediates 3-HAA-induced cell death, tumor cells were treated with 3-HAA and then analyzed for markers of apoptosis, autophagy, and necrosis (Fig. S2C). Apoptosis was increased in a dose-dependent manner by TUNEL assay in the SMMC7721 cells (Fig. 2C). Levels of cleaved caspase 3 and cleaved PARP were increased in a dose- and time-dependent manner in the
SMMC7721 and HepG2 cells (Fig. 2D). However, 3-HAA treatment did not significantly alter levels of autophagy markers LC3-II and p62 or necrosis marker RIP3 in the SMMC7721 or HepG2 cells (Fig.
S2C).
Furthermore, 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 (Fig. 2E).
This effect was observed at ZVAD doses of 50 and 100 μM. 3-HAA treatment also increased apoptosis in
HCC xenografts, as observed based on TUNEL foci and flow cytometry of Annexin V (Figs. 2F and 2G).
Consistent with these results, 3-HAA slowed tumor growth (Fig. S2D).
To determine the effect of endogenous 3-HAA on HCC tumor formation, the cell growth was
analyzed in SMMC7721 cells overexpressing KMO or knocking down HAAO enzymes. Either overexpression of KMO or knock down of HAAO inhibited cell growth of HCC cells (Fig. 2H & S2E).
Moreover, the tumor formation assay showed KMO overexpression suppressed tumor formation and
tumor growth in HCC xenograft model, (9/10) versus (4/10) (Fig. 2I, S2F & S2G). The flow cytometry
analysis showed that overexpression of KMO increased apoptosis of HCC cells, two times more than
control cells (Fig. 2J & S2H). Remarkably, the Kaplan-Meier survival analysis showed that HCC patients
with high KMO expression had longer disease-free survival than patients with low KMO expression (Fig.
2K). These observations suggest that 3-HAA is a negative regulator for tumor formation and tumor
growth.
3-HAA suppressed tumor growth by upregulating DUSP6 expression
To determine the mechanism by which 3-HAA induces tumor cell apoptosis, RNA sequencing was used to profile gene expression in SMMC7721 or HepG2 cells after 0, 1, 8, or 24 h treatment with 3-HAA according to the screening strategy previously applied (Opitz et al., 2011). At all three time points after the start of 3-HAA treatment, the top 21 upregulated genes were selected (Fig. 3A). The expression of the top seven of these genes was individually verified 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. 3B). These results suggest that 3-HAA alters the gene expression profile 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 were longer than patients expressing a low
level of DUSP6 (p < 0.05) (Fig. 3C and S3A). The multivariate analysis showed that DUSP6 was a major
factor in the HCC patients’ survival among the top 4 upregulated genes (Fig. S3B), and the corrective
analysis with the clinical characteristics also supported this finding (Fig. S3C), indicating that DUSP6 is
important to 3-HAA-induced apoptosis.
To demonstrate whether DUSP6 mediates 3-HAA-induced tumor cell apoptosis, the effects of
DUSP6 on HCC cell growth were first analyzed in HCC cells. DUSP6 knockdown restored growth of
HepG2 and SMMC7721 cells inhibited by 3-HAA (Figs. 3D and S3D), whereas it did not affect the
growth of untreated HCC cells (Fig. S3E). 3-HAA induced apoptosis to a smaller extent in DUSP6-
depleted SMMC7721 cells than in the control cells, based on flow cytometry using Annexin V (Fig. 3E).
DUSP6 depletion was also associated with reduced levels of cleaved caspase-3 and cleaved PARP, as
well as enhanced ERK activity (Fig. 3F). In fact, DUSP6 knockdown restored ERK activity that had been
suppressed by 3-HAA (Fig. 3F). ERK enhancement inhibits apoptosis via BAD/BCL2/BCL-XL signaling
(Piya et al., 2012). More impressively, DUSP6 knockdown reversed 3-HAA-mediated suppression of
tumor growth in SMMC7721 xenografts (Fig. 3G).
3-HAA upregulated DUSP6 expression by binding with transcription factor YY1
To determine which transcription factor or co-activator mediates 3-HAA regulation of gene expression, the 38 common transcription factors or co-activators were first selected from those proteins potentially bind to the promoter region (-5000 to +1) of top 4 genes (http://gtrd.biouml.org) (Yevshin et al., 2019) (Fig. 4A and S4A). Moreover, levels of chromatin proteins in 3-HAA-treated SMMC7721 and
HepG2 cells were quantified by tandem mass-tagged quantitative proteomics analysis (TMT). The 91 proteins consistently and increasingly bound to chromatin at the 1st hour and the 8th hour post 3-HAA treatment (Fig. S4B), and YY1 was the only protein overlapped with the predicted transcription factors
that potentially bind to the promoter region of the top 4 genes (Figs. 4B). Both the immunoblotting of nuclear fraction and immunofluorescent staining also showed that 3-HAA increased YY1 nuclear accumulation (Fig. 4C & S4C). Moreover, analysis on gene expression profile showed that about 16% of
3-HAA-regulating genes were the same with YY1-targeting genes, including DUSP6 (Fig. S4D), suggesting transcription factor YY1 could regulate DUSP6 expression after 3-HAA treatment.
Indeed, YY1 knockdown abolished 3-HAA-induced upregulation of DUSP6 in SMMC7721 cells
(Fig. 4D). After 3-HAA treatment, the level of cleaved Caspase 3 and cleaved PARP was decreased in
SMMC7721 cells depleted of YY1, and recovered by DUSP6 overexpression (Fig. 4E). The TUNEL
assay and flow cytometry analysis 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. S4E, S4F & 4F). Eventually, YY1 knockdown restored SMMC7721 cell growth
suppressed by 3-HAA, and overexpressing DUSP6 inhibited YY1-depleted cell growth again (Fig. 4G), suggesting YY1 mediates the 3-HAA upregulation of DUSP6. Also, adding 3-HAA did not affect the expression of CYP1A1, a target gene of AHR (aryl hydrocarbon receptor) (Fig. S4G), suggesting that 3-
HAA functionally overlaps with YY1, but not the AHR, which was distinct from kynurenine.
Since YY1 is a transcription factor, 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) (Khan et al., 2018a; Khan et al., 2018b) revealed a novel potential YY1 binding DNA fragment at positions -1,145 to -1,134
(TCCATCCGGCTT), which is distinct from the reported consensuses binding sequence
(CAANATGGCGGC) (Kim and Kim, 2009). 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 specific sequence as well as the consensus YY1 binding sequence. Luciferase activity decreased when mutations involving this novel binding site (mut2) occurred (Fig. 4H). 3-HAA increased YY1 binding to this novel binding sequence in a dose-dependent manner, as evidenced by an in vitro electrophoretic mobility shift assay (Fig. 4I).
Moreover, the quantitative PCR analysis following chromatin immunoprecipitation of YY1 revealed that 3-HAA promoted YY1 binding to the consensus sequence of p53 promoter region (positive control)
and the novel binding sequence in the DUSP6 promoter region, as reflected by 3-HAA-induced YY1
enrichment (Figs. 4J and S4H). ChIP sequencing analysis further confirmed that 3-HAA induced the
union peak formation of YY1 on the promoter region of DUSP6, IGFBP1, NR0B2 and IER3 genes (Fig.
4K & S4I).
To determine whether 3-HAA promotes DUSP6 expression by binding YY1, we first tested whether
3-HAA directly associates with YY1 in vitro using nuclear magnetic resonance. Dose-dependent signal attenuation was observed in the T1r NMR spectrum, and positive saturation transfer difference (STD) signals were detected in the STD spectrum, suggesting that YY1 interacts with 3-HAA (Fig. 4L). Surface plasmon resonance experiments suggested a moderate binding affinity with KD of 121.7 μM (Fig. 4M).
This binding was further confirmed by microscale thermophoresis (MST) analysis (Fig. S4J).
PKCζ phosphorylates YY1 at Thr 398 in response to 3-HAA
Previous studies showed that phosphorylation regulated YY1 DNA binding activity (Daub et al.,
2008; Kaludov et al., 1996). Thus, the phosphorylation of YY1 were first analyzed 2 hours after 3-HAA
treatment to determine whether 3-HAA induced YY1 phosphorylation. The immunoblotting following
phosphorylated protein enrichment showed that 3-HAA treatment also increased YY1 phosphorylation,
and mass spectrometry analysis revealed that the T398 of YY1 were phosphorylated by 3-HAA (Fig. 5A).
The T398A mutation diminished the 3-HAA-induced YY1 nuclear accumulation and YY1 T398 phosphorylation (Fig. 5B & 5C). The T398 phosphorylation specific antibody was developed for this detection.
The function analysis displayed that the T398A mutation of YY1 suppressed 3-HAA-upregulated
DUSP6 expression and reduced the level of cleaved Caspase 3/cleaved PARP, whereas the mimic phosphorylation of T398E mutation promoted DUSP6 expression even without 3-HAA treatment (Fig. 5D). The TUNEL assay and the flow cytometry analysis demonstrated that the T398A mutation of YY1
suppressed 3-HAA-induced apoptosis (Fig. 5E & S5A), suggesting T398 phosphorylation of YY1 is
critical for 3-HAA-induced apoptosis.
To identify the kinase for the 3-HAA-induced T398 phosphorylation of YY1, the kinase screening
assay was performed on the peptide of FAQSTNLK. Three candidate enzymes AKT, mTOR, and PKCζ were chosen from predicted kinases based on the online software (www.cbs.dtu.dk; gps.biocuckoo.cn)
(Mok et al.; Xue et al., 2011). Furthermore, proteomics analysis by mass spectrometry following YY1
immunoprecipitation showed that 3-HAA increased the association of YY1 with PKCζ, which were
further confirmed by immunoblotting, suggesting 3-HAA recruits PKCζ to phosphorylate YY1 (Figs. 5F,
S5B & 5G). The kinase PKCζ significantly increased the peptide phosphorylation, reflected by the
autoradiogram on dot blot (Fig. 5H). Also, only the PKCζ inhibitor markedly decreased YY1
phosphorylation (Fig. 5I), suggesting PKCζ is the kinase for T398phosphorylation of YY1 induced by 3-
HAA.
Moreover, the PKCζ inhibitor markedly decreased the mRNA level and protein level of DUSP6 in
the SMMC7721 cells depleted of endogenous YY1 and expressing exogenous wild type YY1, but not in
the cells expressing T398A/T398E mutant YY1 (Fig. 5J & 5K). Eventually, the PKCζ inhibitor decreased
the level of cleaved Caspase 3 and cleaved PARP in the cells expressing exogenous wild type YY1 but
not T398A/T398E mutant YY1 (Fig. 5K). The clinical data that the PKCζ expression level is closely
correlated with the overall survival of the grade I HCC patients (Fig. S5C), further supports these
findings.
Using computational software, we propose a binding model that 3-HAA binds to the site
encompassing Gln396 to Thr398 of YY1 (Fig. 5L), which is consistent with our above finding that the
Thr398 phosphorylation increasing the YY1 binding to the promoter of DUSP6 gene.
T398 phosphorylation of YY1 is critical for 3-HAA-suppressed HCC xenografts growth
To further evaluate the effects of T398 phosphorylation of YY1 on 3-HAA-suppressed HCC cell growth, the apoptosis and tumor growth were determined in HCC xenograft mice. In mice with control HCC xenografts, 3-HAA (100 mg/kg•day) slowed xenograft growth, leading to smaller tumors, whereas the same dose of 3-HAA had no remarkable effect on YY1 depleting xenograft growth (Fig. 6A). Both
the TUNEL assay and flow cytometry revealed a noticeably higher percentage of apoptotic cells only in
YY1 expressing HCC xenografts, but not in YY1 depleting HCC xenografts (Fig. 6B-C & S6A). This result was obtained with SMMC7721 and HepG2 xenografts.
Moreover, in mice with HCC xenograft depleting endogenous YY1 and expressing T398A mutant
YY1, 3-HAA (100 mg/kg•day) had no effect on tumor growth. Whereas, the same dose of 3-HAA
significantly decreased xenograft growth expressing wild type YY1 (Fig. 6D). The TUNEL assay and
flow cytometry also demonstrated a markedly higher percentage of apoptotic cells in xenografts
expressing wild type YY1, but not in T398A mutant xenografts (Fig. 6E, 5F & S6B). In mice with HCC
xenografts expressing T398E mutant YY1, the growth of xenografts was obviously slower than the
xenografts expressing wild type YY1, and 3-HAA had no effects on these xenograft growth (Fig. 6E).
And, the PKCζ inhibitor (0.8mg/kg.day) treatment restored the growth of xenografts suppressed by 3-
HAA (Fig. 6G), and the flow cytometry analysis also displayed a decreased percentage of apoptotic cells
in HCC xenografts treated with the PKCζ inhibitor, compared to the 3-HAA alone-treated xenografts
(Fig. 6H). The GC-MS/MS analysis showed that the 3-HAA content was indeed dramatically increased in
3-HAA-treated xenografts (Fig. 6I). Most importantly, 3-HAA synergizes with IDO inhibitors to suppress
HCC xenograft growth (Fig. 6J).
To determine the pharmacokinetic of 3-HAA, the concentration of 3-HAA was analyzed at the time
course of administration in the plasma of mice. As shown in Fig. S6D, the half-time 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 7 days treatment. Moreover, 3-HAA did not disrupt liver/renal function in Balb/c mice at a dose of 800 mg/kg.day, which was six-fold higher than the tested dose (Fig. S6E). These results highlight the promise of 3-HAA as a potential HCC therapy.
Discussion
Tryptophan metabolism plays a very important 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 poor prognosis in various malignancies. The inhibition of IDO1/2 suppresses 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 reversed the tumor-promoting effect of kynurenine and significantly improved the efficacy 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 exerts anti-inflammatory and neuroprotective effects by selectively inducing the apoptosis of activated T cells or suppressing microglia/astrocytes that express cytokines and chemokines
(Krause et al., 2011; Lee et al., 2010). The 3-HAA induced the expression of cytoprotective enzyme hemeoxygenase-1 in astrocytes and microglia, the latter is an enzyme with proven anti-inflammatory and cytoprotective activities (Brusko et al., 2005; Ryter and Choi, 2009). The 3-HAA-induced apoptosis of activated T lymphocytes are linked to oxidative stress and induction of caspases (Hayashi et al., 2007;
Hiramatsu et al., 2008). These results indicate that 3-HAA- has a distinct biological function in different type of cells. However, the biological function of 3-HAA largely remains unclear.
In this study, we report that 3-HAA is downregulated in HCC cells and HCC tissues due to the downregulation of KMO and KYNU enzymes as well as the upregulation of the HAAO enzyme, which are regulated by kynurenine signal. The excessive kynurenine generated from tryptophan in HCCs is exported. We also observed that the HCC cell sensitivity to 3-HAA, to some extent, were correlated with the cellular 3-HAA concentration post exogenous 3-HAA treatment. However, the variation of the cellular 3-HAA concentration post exogenous 3-HAA treatment and the endogenous 3-HAA concentration may not completely associate with expression levels of KMO, KYNU, and HAAO enzymes in HCC cells (Fig. 1E & S2B), since the 3-HAA transporters and drug resistant genes may also involve in the regulation of HCC cell sensitivity to 3-HAA. This observation suggests that the heterogeneity of tryptophan metabolism in HCC cells may differ in their sensitivity to 3-HAA.
Moreover, we also discovered that 3-HAA is a ligand of transcription factor YY1 and that binding of the two molecules leads to 3-HAA-mediated alterations in gene expression. 3-HAA up- or downregulated a number of genes via YY1 binding, including DUSP6, IGFBP1, and NR0B2 etc. YY1 may bind to two motifs in the DUSP6 promoter; the known consensus binding site, as well as a novel binding site, was identified in this study. The T398 phosphorylation of YY1 may change its structure conformation associating with DNA binding, promoting YY1 binding to its target sequence.
Although the in vitro measurement showed a higher dissociation constant of YY1 with 3-HAA (the
KD50 of 3-HAA was 121.7 µM), this may not reflect the binding affinity of YY1 with 3-HAA in cells since the cellular contents of 3-HAA were less than 20 µM, even in HCC cells treated by 3-HAA or expressing KMO. Moreover, the expression level of solute carriers determines the cellular uptake of 3-
HAA, thus, the contents of 3-HAA in 3-HAA-treated HCC cells were only 5-10 times more than untreated cells, similar to the level of endogenous 3-HAA in cells expressing KMO (Fig. 1F & 2B).
Taken together, our results have determined that 3-HAA is a functional metabolite regulating tumor cell fate by binding to and activating the transcription factor YY1 (Fig. 6K). Selective downregulation of
3-HAA by kynurenine signal appears to be essential for HCC growth. Exogenous 3-HAA induces tumor cell apoptosis and inhibits HCC growth, especially improves the efficacy of IDO1/2 inhibitors, suggesting its potential use in HCC therapy.
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Acknowledgments
We thank Profs. Lei Shen (Shanghai Jiao Tong University School of Medicine), Qunying
Lei (Fudan University School of Medicine) and Chief Editor Dangsheng Li (Journal of Cell
Research) for useful discussions. This study was supported by grants from the Ministry of
Science and Technology of the People’s Republic of China (2018YFC1313205), the Shanghai
Committee of Science and Technology (11DZ2260200) and the National Natural Science
Foundation of China (81572300) (81872342) to Dr. Mi.
Author contributions
G.G., Z.S., S.L. and X.X. performed most of experiments; Y.Y., J.Z. and B.B. performed some of animal experiments; X.G. performed metabolites analysis, P.X. and W.Z. synthesize the 3-
HAA derivatives; N.Z. and A.Z. performed the 3-HAA and YY1 binding experiments; W.L. performed ChIP-Seq analysis and kinase screening; J.M. initiated the project, led the project team, designed experiments, analyzed results and wrote the paper with input from all authors.
Conflict of interest
No conflict of interest to declare. Figure legends
Figure 1 3-hydroxyanthranilic acid (3-HAA) is selectively decreased in hepatocellular carcinoma
A. Quantitative analysis of tryptophan metabolites by LC-MS/MS and GC-MS in HCC and corresponding paratumor tissues.
B. Quantitative analysis of tryptophan metabolites by LC-MS/MS and GC-MS in normal hepatic cells and
HCC cells.
C. Immunohistochemistry staining of 3-HAA in clinical HCC samples.
D. Metabolic flux analysis of tryptophan metabolites in Huh7 and HepG2 cells. L-Tryptophan was completely 13C-labelled. The content of tryptophan catabolites in cells and medium was assessed using
LC-MS/MS. The M0 stands for no carbon in tryptophan was 13C-labelled, M11 stands for all 11 carbon in
tryptophan were 13C-labelled.
E. Expression analysis of metabolic enzymes involved in 3-HAA generation.
F. The effect of KMO overexpression on the concentration of endogenous 3-HAA and tryptophan catabolites in SMMC7721 cells.
Figure 2 3-hydroxyanthranilic acid (3-HAA) induces apoptosis of hepatocellular carcinoma cells
A. Analysis of the growth of HCC cells treated for 4 days with one of four tryptophan metabolites (100
μM).
B. Gene Ontology (GO) enrichment analysis of HCC SMMC7721 cells treated by 3-HAA for 8 hours.
C. Apoptosis analysis by TUNEL assay in SMMC7721 cells treated for 24 h with 3-HAA at the indicated dose.
D. Analysis of apoptosis signaling in HCC cells treated with 100 μM 3-HAA for indicated time or treated for 12 h with 3-HAA at the indicated dose.
E. The effects of three types of inhibitors on 3-HAA-induced HCC cell death. ZVAD: apoptosis inhibitor
(20 μM); Nec1: necrosis inhibitor (100 μM); 3-MA: autophagy inhibitor (5 mM). The dose of 3-HAA was
100 μM. Cells were treated for 4 days. F and G. Apoptosis analysis by TUNEL assay and flow cytometry in SMMC7721 xenografts with and without 3-HAA treatment. The dose of 3-HAA was 100 mg/kg•day. Mice were treated for 7 days and sacrificed on day 10.
H. The influence of endogenous 3-HAA on cell growth and apoptosis signaling of HCC cells. The KMO was overexpressed in SMMC 7721. The **: p < 0.01. The concentration of 3-HAA and ZVAD was 100
μM and 20 μM, respectively.
I. Tumor formation analysis of HCC cells overexpressing KMO. The effect of endogenous 3-HAA on xenografted tumor formation.
J. The effect of KMO overexpression on apoptosis of SMMC7721 cells.
K. The effect of KMO expression on the disease-free survival of HCC patients. The patients were divided by the median of KMO expression value.
Figure 3 3-HAA induces tumor cell apoptosis by upregulating DUSP6 expression
A. The Heatmap of the top consistently upregulated genes in SMMC7721, Hep3B, and SMMC97H. Deep sequencing was used to profile gene expression in HCC SMMC7721, Hep3B, and SMMC97H cells. HCC cells were treated with 100 μM 3-HAA for 1, 8, or 24 h.
B. Top consistently upregulated genes were individually verified using quantitative PCR and immunoblotting.
C. DUSP6 expression reversely correlated with overall survival of HCC patients, as analyzed by the K-M plotter. The total patient number was 415. The HCC patients were divided by the median of KMO expression value.
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.
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 flow cytometry. F. Effects of DUSP6 knockdown on 3-HAA-activated apoptosis signaling. SMMC7721 cells depleted of
DUSP6 were treated with 100 μM 3-HAA for 24 h.
G. DUSP6 knockdown recovered 3-HAA-suppressed xenograft growth. Animals were treated with 3-
HAA (100 mg/kg•day) for seven days, then sacrificed on day 10. SMMC7721 cells were subcutaneously
injected into mice. The middle graph shows xenograft weights in different groups. Photographs on the
right show representative tumors in different groups.
Figure 4 3-HAA upregulated DUSP6 expression by binding with transcription factor YY1
A. The transcription factors potentially binding with promoters of top 4 genes upregulated by 3-HAA.
B. The common proteins between 3-HAA-increased chromatin binding proteins and predicted transcription factors binding to the DUSP6 promoter region.
C. 3-HAA increases YY1 nuclear translocation. SMMC7721 cells were treated with 100 μM of 3-HAA for 8 hours.
D. YY1 knockdown abolished 3-HAA-induced DUSP6 expression. SMMC7721 cells were treated with
100 μM of 3-HAA for the indicated time.
E. The effect of YY1 knockdown on 3-HAA-activated apoptosis signaling. The concentration of 3-HAA was 100 μM.
F. The effect of YY1 knockdown on 3-HAA-suppressed HCC cell growth. The concentration of 3-HAA was 100 μM. The **: P < 0.01.
G. The effect of YY1 knockdown on 3-HAA-induced HCC apoptosis, analyzed by flow cytometry. The concentration of 3-HAA was 100 μM. The **: P < 0.01.
H. 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.
I. 3-HAA binding to YY1 determined by electrophoretic mobility shift assay (EMSA). The synthesized oligonucleotide was labeled at the 5’ terminus with FAM, and YY1 was purified using a Hitrap heparin column. J. 3-HAA increased YY1 enrichment on the DUSP6 promoter, analyzed by chromatin immunoprecipitation (ChIP) and quantitative PCR (QPCR). The dose of 3-HAA was 100 μM.
K. The ChIP-sequencing analysis of YY1 on the DUSP6 gene. The HCC cells were treated with the indicated dose of 3-HAA prior to ChIP-sequencing.
L. NMR measurement of direct binding between 3-HAA and YY1. T1r NMR spectra for 6b (red) alone or in the presence of SPOP at 2.5 mM (blue), 5 mM (cyan), or 10 mM (green). The STD spectrum for 6b was recorded in the presence of 5 mM SPOP.
M. 3-HAA bound YY1 protein as shown by surface plasmon resonance. Graphs of equilibrium unit response versus concentrations are shown. The estimated Kd was 121.7 μM.
Figure 5 PKCζ phosphorylates YY1 at Thr 398 in response to 3-HAA
A. The YY1 phosphorylation was analyzed by the immunoblotting and the mass-spectrometry. The YY1
was blotted on the enriched phospho-proteins from SMMC7721 cells. The YY1 modification was
analyzed by the mass-spectrometry following YY1 immunoprecipitation.
B. The YY1 mutation of T398A attenuates the 3-HAA-increased YY1 nuclear accumulation. The
concentration of 3-HAA was 100 μM and treated for 24 h.
C. The T398A but not S247A mutation abolished 3-HAA-induced YY1 phosphorylation. The YY1 was conjugated with HA tag. The YY1 phosphorylation was detected by the T398 phospho-specific antibody.
D. The mutation of T398E in YY1 promoted DUSP6 expression, whereas the T398A mutation suppressed DUSP6 upregulation. The YY1 were fused with HA tag.
E. The YY1 mutation of T398A reduces the 3-HAA-induced apoptosis, analyzed by the TUNEL assay.
F. 3-HAA increased PKCζ binding to YY1, analyzed by co-immunoprecipitation and mass-spec.
G. 3-HAA increased PKCζ binding to YY1, analyzed by immunoblots.
H. The kinase screening for T398 phosphorylation of YY1. The kinase candidates were predicted by online tools NetPhos 3.1 (www.cbs.dtu.dk/services/NetPhos) (Blom et al., 2004) and GPS 5.0
(gps.biocuckoo.cn)(Song et al., 2012). I. The effect of kinase inhibitors on 3-HAA-induced YY1 phosphorylation. The YY1 phosphorylation
was detected by the T398 phospho-specific antibody. The concentration of 3-HAA was 100 μM. AKT inhibitor, MK2206 (0.5 μM); PKC inhibitor, Go6983 (0.5 μM); mTOR inhibitor, rapamycin (0.1 μM).
L. The proposed 3-HAA binding model with YY1.
Figure 6 T398 phosphorylation of YY1 is critical for 3-HAA-suppressed HCC xenografts growth
A. YY1 knockdown recovered the 3-HAA-inhibited HCC xenografts growth.
B. & C. The apoptosis analysis in xenografts by TUNEL assay and the flow cytometry. The histogram
presents as mean ± SD (*: P < 0.05; **: P < 0.01).
D. The T398A mutation of YY1 abolished the 3-HAA-inhibited HCC xenografts growth.
E. & F. The apoptosis analysis in xenografts by TUNEL assay and the flow cytometry. The histogram
presents as mean ± SD (*: P < 0.05; **: P < 0.01.).
G. The PKCζ inhibitor reverted the HCC xenografts growth inhibited by 3-HAA.
H. The apoptosis analysis in xenografts by the flow cytometry. The histogram presents as mean ± SD (*:
P < 0.05; **: P < 0.01.).
I. The concentration of 3-HAA in tumors post-administration. The 3-HAA content in xenografts was
detected by LC-MS/MS.
J. 3-HAA synergizes with IDO inhibitors to HCC xenograft growth. The dose of both 3-HAA and
Epacadostat (IDO inhibitor) were 100 mg/kg.day, respectively.
K. The working model of 3-HAA-inducing apoptosis.
Note: The mouse xenografts were generated by the inoculation of 1.5 x 10^6 of SMMC7721 cells into the armpit of rear limb. The 3-HAA (100 mg/kg.day) was administered by intraperitoneal injection for 7 days. Tumor volumes are presented as mean ± SD (*: P < 0.05; **: P < 0.01.). Photographs on the right show representative xenografts in different groups.
STAR Methods
Metabolism flux analysis by LC-MS/MS
For the flux experiment of tryptophan catabolism, tryptophan in medium was replaced by fully 13C-
13 labeled tryptophan (tryptophan- C11). Sample preparation and LC-MS/MS analysis were the same as for quantitation of tryptophan catabolic products, except that the parameters of MRM transitions were
13 different. MRM transitions for catabolic products of tryptophan- C11 (M11: 320 > 273) were set as 5-
hydroxyindoleacetic acid (M10: 323 > 155), kynurenine (M10: 427 > 122), kynurenic acid (M10: 304 >
105), 3-hydroxykynurenine (M10: 547 > 05), xanthurenic acid (M10: 424 > 05), 3-hydroxyanthranilic
acid (M7: 369 > 247), cinnabarinic acid (M14: 419 > 105). Simultaneously, the MRM transitions (M0) of
unlabeled tryptophan and its catabolic products were acquired using the same settings as in quantitation
experiments.
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 5 mM dithiothreitol for 30 min at 56 °C, then alkylated with 11
mM iodoacetamide for 15 min at room temperature in darkness. Following 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 a second digestion lasting 4 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 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 FusionTM TribridTM (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 103 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 104 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.
NMR detection of 3-HAA-YY1 interaction
NMR experiments based on the Carr-Purcell-Meiboom-Gill (CPMG) sequence and saturation transfer difference (STD) were applied to investigate 3-HAA-YY1 interactions. All NMR spectra were acquired at 25 °C on a 600 MHz Bruker Avance III spectrometer equipped with a cryogenically cooled probe (Bruker Biospin, Germany). Samples containing 200 µM 3-HAA and 200 µM 3-HAA in the presence of 5 µM protein were dissolved in Tris-HCl buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl,
5% DMSO, 95% D2O) and then used in NMR data acquisition. CPMGs were recorded using the pulse sequence of solvent-suppressed 1D 1H CPMG (cpmgPr1d). The 90° pulse length was adjusted to approximately 11.82 μs. A total of 4 dummy scans and 64 free induction decays (FIDs) were collected into 13 K acquisition points, covering a spectral width of 8 kHz (13.3 ppm) and giving an acquisition time of 3 s. STD data were acquired using 4 dummy scans and a relaxation delay of 3 s, followed by 40-dB
pulsed irradiation at a frequency of -1.0 ppm or alternatively 33 ppm. Total time to acquire the STD
spectrum was 21 min with 128 FIDs.
Surface plasmon resonance and microscale thermophoresis 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.
The dissociation constant of 3-HAA for YY1 was also measured by microscale thermophoresis assay according to manufacturer’s protocol (Nanotemper Technologies Ltd., Shanghai, China). YY1 protein was labeled using NT650-NHS labeling kit (Nanotemper Technologies Ltd., Shanghai, China), and the concentration of labeled YY1 protein was set to 50 nM. The unlabelled 3-HAA was gradually diluted from 2500 μM to 76.3 nM as indicated. YY1 and 3-HAA interacted in PBS buffer containing 2.5%
DMSO, 0.05% Tween-20, 85mM NaCl. Measurements were made using the Monolith NT.115
(NanoTemper), and data were analyzed using MO.Affinity analysis (×64) software. Graphs were blotted 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 culture medium for 48 hours. The cells were
washed with PBS for 3 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.
Xenograft HCC mouse model Six-week-old male BALB/c mice were purchased form Lingchang, Shanghai, China. Xenograft
mouse model of HCC was generated by injecting SMMC-7721 HCC cells (1.5×10^6) subcutaneously into the armpit of rear limb. Mice were injected intraperitoneally with 100 mg/kg 3-HAA or equal amount
of DMSO every day. when tumors reaching 1 cm3, the PKC inhibitor Go6983 was injected intravenously every two days and the dose of PKC inhibitor was administered at the dose of 0.8 mg/kg.day.
After two weeks, subcutaneous transplanted tumors were removed, and the volume were measured and the tumors were photographed. Following homogenation 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. Figure 1 A L-TRP Kynurenine 3-HK IDO1 >95% Tryptophan 3-HAA 300 **300 **400 ** 2.0 # KYN 300 KMO 200 200 1.5 3-HK 200 1.0 KYNU 100 100 3HAA 100 0.5 HAAO 0 0 0 0.0 Concentration (nmol/g) Paratumor Tumor Paratumor Tumor Paratumor Tumor Paratumor Tumor
PIC QUIN B C ) Adjacent tissues Liver tumor % LO2 ( 15 3-HAA Try Kyn 3-HK 3-HAA
n HepG2 5 3 SMMC7721 SMMC97H Case1 tratio SMMC97L n
e PLC8024 0
nc Hep3B
o Huh7 c -3 Case2 -10 -30 Relative
D Cellular Medium Tryptophan Kynurenine 3-HAA 3-HK Kynurenine 100 M0 100 M0 100 M0 100 M0 100 M0 M11 M10 M7 M10 M10
50 50 50 50 50
0 (%) content Relative 0 0 0 (%) content Relative
Relative content (%) content Relative 0 Relative content (%) content Relative Relative content (%) content Relative 0 12 24 48 0 12 24 48 0 12 24 48 0 12 24 48 0 48 Time afer labeling (hrs) Time afer labeling (hrs) Time afer labeling (hrs) Time afer labeling (hrs) Time afer labeling (hrs)
E F
n 8 Ctrl KMO OE
LO2 SMMC-97HSMMC-97LHepG2SMMC-7721PLC8024Hep3BHuh7 tratio **
n 6
IDO1 e
nc #
KYNU o 4
c Ctrl KMO OE KMO 2 # # KMO HAAO β-actin
β-actin Relative 0 3-HK3-HAA PA QA Figure 2 A B transcription factor activity, RNA HepG2 SMMC7721 transcriptional factor activity, RNA RNA polymerase II transcription 8 ** 10 ** RNA polymerase II regulatory region DNA binding RNA polymerase II regulatory padj 8 response to molecule of bacteria origin 1.00 6 response to mechanical stimulus 0.75 response to bacterium 6 positive regulation of programmed cell death 0.50 4 positive regulation of cell death 0.25 positive regulation of apoptotic process 4 negative regulation of cell proliferation 0.00 2 MAP kinase tyrosine/serine/threonine phosphatase 2 MAP kinase phosphatase activity Count inactivation of MAPK activity 10 0 cytokine activity 20 0 core promoter sequence-specific DNA binding
Cell number(10*4) Cell 30 cellular response to lipid Ctrl PA QA PA QA 0.03 0.06 0.09 0.12 KYN Ctrl KYN 3-HAA 3-HAA Gene Ratio C D SMMC7721 SMMC7721 Time (h) Dose (μM) 150 ** HepG2 SMMC7721 HepG2 SMMC7721 0
(μM) ** 0 4 8 0 4 8 0 100 150 0 100 150 100 ** 100 Caspase3
150 50 Cleaved Caspase3 200 0 PARP Concentration Tunel positive rate (%) positive Tunel 0 100 150 200 Cleaved PARP TUNEL Merge Concentration (μM) β-actin w/ DAPI E F HepG2 SMMC721 Xenografts 25 1.5 1.5 3HAA * Ctrl 3-HAA ** ** 3HAA+ZVAD 20 * 3HAA+Nec1 1.0 ** 1.0 3HAA+3-MA 15 TUNEL 0.5 0.5 10 Cell viability Cell Cell viability Cell MERGE 5 0.0 0.0 W/ DAPI
50 100 50 100 rate (%) positive Tunel 0 Concentration (μM) Concentration (μM) Ctrl 3-HAA G H Ctrl 3-HAA (100mg/kg.day) SMMC7721 Ctrl 1500 KMO OE 10 * KMO OE Ctrl KMO OE +ZVAD KMO OE+ZVAD 8 ** Cleaved 1000 PARP 6 Cleaved 4 Caspase3 500 2 KMO Apoptosis rate(%) Apoptosis
0 (*104) number Cell 0 β-actin Ctrl 3-HAA 0 2 4 6 8 Days I J K Disease Free Survival
150 15 1.0 HR=0.58 (0.43-0.78) ** * P=0.00034 0.8 100 10 0.6 0.4 50 5
0.2 KMO > 403
Apoptosis rate(%) KMO < 403
Tumor Formation Rate (%) Formation Tumor 0 Survival probabily
0 0.0 Ctrl KMO OE Ctrl KMO OE Time 0 20 40 60 80 100 120 (months) Figure 3 A B C Time (h) 0 1 8 24 IGFBP1 25 IGFBP1 DUSP6 DUSP6 DUSP6 20 NROB2 NR0B2 EGLN3 HR = 0.93 (0.13 − 1.91) EGLN3 15 PFKFB4 PFKFB4 IER3 P = 0.0023 IER3 10 FKBP1C 5 PLCXD1 PLIN2 4.0 0 SMIM3 mRNA level Relative 0 1 8 0 1 8 0 1 8 0 1 8 0 1 8 0 1 8 STC2 24 24 24 24 24 24 NDRG1 3.0 Time (h) NEDD9 RNASET2 SMMC7721 HepG2 Expression SERPINE1 2.0 Time (h) 0 4 8 12 24 48 0 4 8 12 24 48
Survival fraction low C8orf4 DUSP6 high
PSORS1C3 0.0 0.2 0.4 0.6 0.8 1.0 AC093642.5 1.0 IGFBP1 LRRK1 0 20 40 60 80 100 120 MAFF β-actin Time (months) APOL2 0.0 D F SMMC7721 HepG2 Ctrl KD1 KD2 150 50 Ctrl ** Ctrl DUSP6-KD1 40 DUSP6-KD1 ** Ctrl KD1 KD2- + - + - + 3-HAA 100 DUSP6-KD2 DUSP6-KD2 ** 30 ** p-ERK1/2 20 50 ** ERK1/2 10 ** 0 0 BAD Cell Number(10*4) Cell 0 1 4 7 0 1 4 7 BCL2 Time(day) Time (day) BCL-XL E 5 Casp3 * Ctrl 4 DUSP6-KD1 DUSP6-KD2 3 rate (%) NS s i NS Cleaved Casp3 s 2 PARP 1 Cleaved PARP 0 Apopto DUSP6
Ctrl 3HAA Ctrl 3HAA Ctrl 3HAA β-actin
G Ctrl-Ctrl ** 600 Ctrl-3-HAA Ctrl 0.6 ** DUSP6 KD-Ctrl ** DUSP6 KD-3-HAA 3-HAA 400 0.4 ** Control+ 200 0.2 DUSP6 KD 3-HAA+ Tumor volume (mm3) volume Tumor 0 Tumor weight (g) weight Tumor 0.0 DUSP6 KD 0 2 4 6 8 10 Time (day) HAA ontrol 3- Ctrl+ HAA+ C 3- DUSP6 KD1DUSP6 KD1 Figure 4 A BC 3-HAA-regulated TFs on 4 genes chromatin proteins promoter region cytoplasm nuclear 3-HAA - + - + YY1 91 YY1 38 β-actin H3
D E DMSO 3-HAA F ** WT YY1-KD 15 # ** 3-HAA(h) 0 6 12 24 48 0 6 12 24 48 ** YY1 KD + YY1 KD + 10 Ctrl YY1 KD DUSP6Ctrl OE YY1 KD DUSP6 OE DUSP6 Cleaved # Casp3 5 YY1 Cleaved PARP
β-actin rate(%) Apoptosis 0 DUSP6 CTRL CTRL YY1 YY1 KDYY1 KD+ YY1 KDYY1 KD+ G DUSP6 OE DUSP6 OE DMSO+Ctrl β-actin Ctrl 3-HAA 15 DMSO+YY1 KD L DMSO+YY1 KD+DUSP6 OE I 3-HAA+Ctrl ** 10 3-HAA+YY1 KD 3-HAA+YY1 KD+DUSP6 OE ProbeYY1 5μM YY1+Probe7.5μM YY1+Probe10μM YY1+Probe12.5μMYY1+SC YY1+ProbeYY1+NSC 5
Cell number (10^6) number Cell 0 0 2 4 6 8 Days 7.127.10 6.74 6.72 6.52 6.50 6.48 1H (ppm) H J 2.5 AAATTTCCCGGG (mut1: -1598 to -1587) Ctrl GCCGCCTTTTTT (nonsense sequence) ** ** 2.0 3-HAA -1665 +1 DUSP6 promoter luciferase 1.5 pGL4-basic vector TCCATCCGGCTT (YY1 binding site) 1.0
CTTCCTTTTTCC (mut2: -1145 to -1134) 0.5 80 Enrichment (times) Enrichment 10 9 8 7 6 5 4 3 2 1 0 0.0 1H (ppm) 60 Neg-Ctrl Pos-Ctrl DUSP6
40 K M YY1 ChIP-Sequencing 20 KD=121.7µM 140 5Kb 500µM 120 250µM Luciferase activity Luciferase 0 125µM 100 62.5µM 150μM 80 31.25µM + Vector 60 50μM 40 DUSP6 20 Positive(-1665 ctrl + -YY1 +1) + YY1 (-1115 - +1) + YY1 0 Ctrl (RU) ResponseUnit (-1665 - -1085) + YY1 -20 DUSP6 (-1665 -(-1665 -1085)mut1DUSP6 --1085)mut2 + YY1 + YY1 DUSP6 -50 0 50 100 150 200 DUSP6 gene TSS Time (s) DUSP6 DUSP6 Figure 5
A B Nuclear C 394-FAQSTNLK-401 Ctrl 3-HAA 3-HAA WT T398A
b(2)++ 3-HAA - - 0 100 150 + + WT S247AT398A WT S247AT398A p-YY1 HA p-YY1-398 b(3)++ b(4)-98++,y(3)++ b(7)-196++ β-actin YY1 y(5)-196++,y*(2),a*(6)-196++ H3 100 200 300400 500 Whole lysate HA HA YY1 D E DMSO 3-HAA Ctrl 3-HAA TUNELMerge w/ DAPI TUNEL Merge w/ DAPI
YY1-WT YY1-WTYY1-T398AYY1-T398EYY1-WTYY1-T398AYY1-T398E DUSP6 Cleaved Casp3 YY1-T398A HA YY1 β-actin YY1-T398E
FG3-HAA H I YY1 peptide 3-HAA - + 250 1 2 4 8 16 150 YY1 Ctrl Input IgG - + 3-HAA 100 PKCζ YY1 PKCζ IP: HA YY1-WTYY1-T398AYY1-WT+mTORYY1-WT+AKTYY1-WT+PKC i i i 70 YY1 AKT IP YY1 p-YY1-398 50 PKCζ mTOR H3 HA 40 Autoradiogram 35 YY1 25 20 Ctrl 3-HAA J K L DMSO 3 *** WT T398A T398E WT T398A T398E 3-HAA ** *** PKC i -+-+-+-+-+-+ 2 ** DUSP6 ** Cleaved T398 PARP 1 Cleaved Casp3
Enrichment (times) 0 HA YY1 WT+Ctrl WT+PKCi WT+CtrlLWT+PKCi T398E+Ctrl T398A+CtrlT398E+Ctrl T398A+CtrlLT398A+PKCiT398E+PKCi T398A+PKCiT398E+PKCi β-actin Figure 6
A 12 ** ** B C # # ) DMSO 3-HAA
3 20 Ctrl+DMSO 8 Merge Merge ** TUNEL w/ DAPI TUNEL w/ DAPI 15 # Ctrl+3-HAA 10 YY1 KD+DMSO 4 Ctrl Volume (cm Volume 5 YY1 KD+3-HAA
0 rate(%) Apoptosis 0 DMSO 3-HAA DMSO 3-HAA YY1 KD Ctrl YY1 KD Ctrl YY1 KD Ctrl YY1 KD Ctrl 3-HAA
D EFDMSO 3-HAA ** Merge Merge ** 12 # TUNEL w/ DAPI TUNEL w/ DAPI 15 # WT ) ** 3 Ctrl T398A * 8 WT 10 # T398E WT 4 5 3-HAA T398A T398A Volume (cm Volume
T398E 0 rate (%) Apoptosis 0 WT T398A T398E WT T398A T398E T398E WT T398A T398EWT T398A T398E Ctrl 3-HAA Ctrl 3-HAA
G # H I 10 20 ** ** ** 10 *** 3-HAA ) ** 3 8 - 15 8 Ctrl 6 + 10 6 4 4 5 - 2 PKCi 2 Apoptosis rate(%) Apoptosis + (cm Volume 0 0 (nmol/g) Concentration 0 DMSO 3-HAA DMSO 3-HAA DMSO 3-HAA DMSO 3-HAA 1cm Ctrl 3-HAA Ctrl PKCi Ctrl PKCi
J K 3-HAA * 8 )
DMSO 3 * * CTRL 6 3-HAA Cytoplasm P 4 P DUSP6 ERK1/2 DMSO 2 YY1 IDOi PKC
Volume(cm P 3-HAA 0 YY1 DUSP6 1cm DMSO 3-HAA DMSO 3-HAA Ctrl IDOi APOPTOSIS Nuclear Figure S1 3-HAA is decreased in tumor cells. Related to Figure 1
A Trypotophan Kynurenine 3-HAA 3-HK 300 400 * 400 ** ** 4 # 300 300 3 200 200 200 2 100 100 100 1
Concentration (nmol/g) 0 0 0 0 Paratumor Tumor Paratumor Tumor Paratumor Tumor Paratumor Tumor
B KYNU KMO IDO1 HAAO Paratumor Tumor Paratumor Tumor Paratumor Tumor Paratumor Tumor
Case1
Case2
Case3
C KYNU 50 KMO IDO1 80 8 40 60 6 30 40 4 20 20 10 2 0 0 0 Transcript per million Transcript Transcript per million Transcript
Transcript per million Transcript -20 -10 -2 Normal Tumor Normal Tumor Normal Tumor (n=50) (n=371) (n=50) (n=371) (n=50) (n=371) D Ctrl 5 HAAO KD 4 * 3 # 2 # # Ctrl HAAO-KD1HAAO-KD2HAAO-KD3 1 HAAO β-actin Relative concentration 0 3-HK3-HAA PA QA
Figure S1 legend A. The quantitative analysis of general tryptophan catabolites in esophagal carcinoma by mass-spec. The esophageal carcinoma patients were 42 cases. The *: p<0.05; the **: p<0.01; the #: p>0.05. B. The immunohistochemistry staining of KYNU, KMO, IDO and HAAO on HCC samples. C. The expression profiling of KYNU, KMO, and IDO in the HCC samples by RNA-seq. D. The quantitative analysis of 3-HK, 3-HAA, PA, and QA in SMMC7721 cells depleted of HAAO. Figure S2 3-HAA inhibits tumor growth by inducing apoptosis. Related to Figure2
A B SMMC7721 HepG2 3-HAA Kynurenine 14 12 Ctrl 5 Ctrl Ctrl 10 3-HAA 3-HAA 8 4 2.0 3 KYN 1.5 2 1.0 3-HAA 0.5 1 0.0 0 Relative concentration (%) PA Relative concentration (%) LO2 LO2 Huh7 Huh7 HepG2 Hep3B HepG2 Hep3B QA PLC8024 SMMC97LPLC8024 SMMC97HSMMC97L SMMC97H SMMC-7721 SMMC-7721 Ctrl C KYN * SMMC7721 HepG2 180 3-HAA PA Time(h) 0 4 8 12 24 48 0 4 8 12 24 48 140 QA 100 * RIP3 30 P62 20 LC3-I 10 LC3-II 0 Colonynumbers β-actin HepG2 SMMC7721
D ) 3 Ctrl 500 0.8 3HAA 400 Kynurenine 0.6 Ctrl 300 0.4 200 3-HAA 100 0.2 Kyn 1 cm 0 (g) weight Tumor
Tumor volume (mm volume Tumor 0.0 0 2 4 6 8 10 Ctrl 3-HAA Kyn Time (days) E F 1200 ** 1200 Ctrl Ctrl HAAO-KD1 * KMO OE ** ** 900 HAAO-KD2 800 HAAO-KD3 600 600 500 400 * 400 300 300 200 Cell number (10*4) number Cell 0 (10*4) number Cell 0 100 0 4 Days 7 Days 4 days 7 days G H
Ctrl KMO OE Ctrl 200 ** KMO OE 107 Q1 Q2 Q1 Q2 3.34 2.08 1.90 2.66 150 106 105 100 104 PI PE-A 3 50 10 Q4 Q3 Q4 Q3 Weight (mg) 2 1 cm 10 90.8 3.77 88.3 7.13 103 104 105 106 103 104 105 106 0 AnnexinV-APC-A AnnexinV-APC-A Ctrl KMO OE Figure S2 A. The effect of four tryptophan metabolites on colony formation of HCC cells. The concentration of kynurenine, 3-HAA, picolinate and quinolinate was 100 μM. The cells were treated for 14 days. B. The concentration of 3-HAA and kynurenine measured by the LC-MS/MS analysis in cells treated with 3-HAA. C. The cell death marker detection in HCC cells treated with 3-HAA at the dose of 100 μM. D. The effects of 3-HAA on the growth of xenograft SMMC7721 tumors. The concentration of both 3-HAA and Kynurenine was 100mg/Kg.Day. E. The effect of HAAO and KMO on the cell growth of SMMC7721 cells . The KMO and HAAO was overexpressed or knocked down in SMMC7721 cells, separately. F. The representative living image of HCC xenografts expressing KMO. G. The effect of KMO overexpression on the growth of HCC SMMC7721 xenografts. The cell numbers were 1.5 X 106 for each injection. H. The flow cytometry analysis of apoptosis. Figure S3 3-HAA suppressed tumor growth by upregulating DUSP6 expression. Related to Figure 3
A IGFBP1 B HR = 0.73 (0.51 − 1.03) logrank P = 0.072 Harzard Ratio P-value 0.4837 DUSP6 (n=485) (0.1873-0.912) 0.037 1.5056 IGFBP1 (n=485) (0.9472-2.393) 0.656 0.8928 NR0B2 (n=485) (0.5749-1.386) 0.616 1.5056 EGLN3 (n=485) 0.084
Survival fraction Expression (0.9472-2.393) low high 0.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.5 1 2 5 0 20 40 60 80 100 120 Number at risk Time (months) low 240 110 57 26 11 4 0 high 243 128 53 28 13 4 1
C DUSP6 Harzard Ratio P-value D 0.7825 Expression (n=485) (0.6163-0.922) 0.0214 1.0144 Age (n=485) (1.0003-1.029) 0.1207 0.9875 Ctrl DUSP6-KD1DUSP6-KD2Ctrl DUSP6-KD1DUSP6-KD2 (n=485) Gender (0.6832-1.416) 0.9288 DUSP6 1.3125 Stage (n=485) 0.0412 (1.0906-1.582) β-actin 0.9459 Grade (n=485) (0.5705-1.568) 0.8293 SMMC7721 HepG2 0.6 0.8 11.2 1.4 1.6 E SMMC7721 HepG2 200 80 Ctrl DUSP6-KD1 150 60 DUSP6-KD2 100 40 50 20 0 0 Cell Number (10*4) 0 1 4 7 0 1 4 7 Time (day) Time (day) Figure S3 legend A. The effect of IGFBP1 expression on liver cancer overall survival. The total patient number was 415, The HCC patients were divided by the median of KMO expression value. B. The multivariate analysis on the HCC patients’ overall survival. C. Corrective analysis on overall survival of HCC patients with clinical characteristics D. Knockdown effeciency detection of DUSP6 in SMMC7721 and HepG2 cells. E. The effects of DUSP6 knockdown on HCC cell growth without 3-HAA. Figure S4 3-HAA upregulated DUSP6 expression by binding YY1. Related to Figure 4
A TFs predicted on 4 genes’ promoters C E DMSO 3-HAA SP1 KMT2B TFAP4 YY1 Merge TUNEL Merge TUNEL Merge ELF3 KAT7 EGR1 w/ DAPI w/ DAPI MNT MAX NFIL3 POLR2A RNF2 IKZF5 Ctrl YY1 MXI1 SMAD4 Ctrl SP5 ZNF580 ARID4B CREM ZBTB33 THAP11 KAT8 SAP130 KLF16 YY1 KD CREB1 GABPB1 KDM1A 3-HAA GATAD2A DRAP1 ETV5 IRF2 SIN3A MXD3 YY1 KD+ GTF2F1 GATAD1 ERF DUSP6 OE ZNF792 ZGPAT
B Increased proteins binding to chromatin D F post-3-HAA treatment Ctrl YY1 KD YY1 KD+DUSP6 OE 107 Q1 Q2 Q1 Q2 Q1 Q2 PGRMC1 PRDX6 GBF1 0.89 1.96 0.97 2.70 0.24 2.10 CYR61 PHB GTF2H4 106 CIT COPB2 FAM111A 5 PDCD5 LONP1 FAM129B 3-HAA YY1 KD 10 TCERG1 MDH2 PSMB7 4
DMSO 10 SEC16A HSD17B4 RNF2 PI PE-A 103 TRIM24 HNRNPM PKP4 Q4 Q3 Q4 Q3 Q4 Q3 RNASEH2A RARS S100A13 2 94.7 2.41 93.9 2.46 89.6 8.02 ATP5H VCP COPS4 10 LUC7L3 EIF3B TUBB6 771 148 2068 107 Q1 Q2 Q1 Q2 Q1 Q2 PGLS EEFSEC SPATA5L1 6 0.18 1.85 0.80 2.91 0.19 1.99 ETHE1 VBP1 UCK2 10 VTN NUTF2 POLR1E 105 ALDOA GNB1 PPA2 ENO1 RAC1 MCCC2 4 3-HAA 10 TUBB HSPG2 DDX21 PI PE-A 103 DLD CKAP4 AAAS Q4 Q3 Q4 Q3 Q4 Q3 EZR AIMP2 GNG12 G 102 89.5 8.43 93.2 3.13 90.4 7.43 UCHL3 DHRS2 KDM5B 103 104 105 106 107103 104 105 106 107103 104 105 106 107 DSP MTA1 LIMA1 5 ** CD44 SNX1 DDX41 - AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A POR TRIM29 GTF3C4 4 + ATP5J PCM1 CEP131 PAICS LAMTOR1 MRPS33 EIF4B FAM83H MRPS17 3 BRD2 PHLDB2 MRPL11 Ctrl 3-HAAKyn YY1 RHPN2 SAMHD1 2 FKBP2 BAP18 MTCH2 CYP1A1 STOM SRRM1 DCK TBC1D15 1 TMOD1 CACTIN β-actin SSFA2 H1FX level mRNA CYP1A1 0 Kyn 3-HAA
H DMSO 3HAA I 5 Kb Input IgG YY1 Ab Input IgG YY1 Ab 150M 50M
Marker NC PC DUSP6 NC PC DUSP6 NC PC DUSP6 NC PC DUSP6 NC PC DUSP6 NC PC DUSP6Marker Ctrl IGFBP1 TSS 5 Kb 250bp 150M 50bp 50M Ctrl J Analysis-Set #4 - MST-Traces NR0B2 TSS 1.0 1.10 5 Kb 0.8 KD = 613 μM 1.05 150M 1.00 0.6 50M 0.4 0.95 Ctrl 0.2 0.90 IER3 TSS Fraction Bound [-] 0.0 0.85 Relative Fluorescence [-] 1.0E-08 1.0E-071.0E-06 1.0E-051.0E-041.0E-03 1.0E-021.0E-01 05 10 15 20 Ligand Concentration (M) MST experiment time [s] Figure S4 A. The common transcription factors potentially binding to the promoter region (-5000 to +1) of top 4 genes, which was up-regulated in 3-HAA-treated SMMC7721 cells. B. Proteins increasely binding to chromatin after 3-HAA treatment, analyzed by quantitative proteomics analysis. C. The localization of YY1 with or without 3-HAA treatment. D. The common genes regualted by both 3-HAA and YY1. E. The apoptosis analysis by TUNEL assay in SMMC7721 cells depleted of YY1 and/or overexpressing DUSP6. F. The apoptosis analysis by flow cytometry in SMMC7721 cells depleted of YY1 and/or overexpressing DUSP6. G. 3-HAA dose not upregulate CYP1A1 as kynurenine does. The dose for borh Kyn and 3-HAA was 100 μM. H. The ChIP-PCR analysis. The positive control (PC) is TP53. I. The peak of ChIP-Seq on the promoter of 3 representative upregulated genes. J. The dissociation constant of 3-HAA to YY1 determined by MicroScale Thermophoresis analysis (MST). Figure S5 PKCζ phosphorylates YY1 at Thr398 in response to 3-HAA. Related to Figure 5 A YY1-WT YY1-T398A YY1-T398E 108 Q1 Q2 108 Q1 Q2 108 Q1 Q2 7 0.96 1.43 7 0.65 1.29 7 1.07 1.55 10 10 10 20 ** 106 106 106 105 105 105 # 4 4 4 15 CTRL PI PE-A 10 PI PE-A 10 PI PE-A 10 ** 103 Q4 Q3 103 Q4 Q3 103 Q4 Q3 * 2 95.8 1.80 2 96.2 1.86 89.9 7.49 10 10 102 10 # 103 104 105 106 107 108 103 104 105 106 107 108 103 104 105 106 107 108 AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A
8 108 Q1 Q2 10 Q1 Q2 108 Q1 Q2 5 7 107 1.04 1.28 10 0.57 1.59 107 0.71 2.12 6 106 10 106 5 rate (%) Apoptosis 0 105 10 105 4 4 4 3-HAA
10 PI PE-A PI PE-A 10 PI PE-A 10 WT WT 3 3 3 10 Q4 Q3 10 Q4 Q3 10 Q4 Q3 T398A T398E 2 T398A T398E 102 89.2 8.43 10 95.7 2.15 102 85.0 12.2 103 104 105 106 107 108 103 104 105 106 107 108 103 104 105 106 107 108 Ctrl 3-HAA AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A B C YY1 binding protein Control 3-HAA PPIB HR = 0.34 (0.12 − 0.95) FLG2 logrank P = 0.032 LYZ HSPA5 HSPA9 HSPD1 Exportin 1 HRNR Expression low HNRNPA2B1 high 0.0 0.2 0.4 0.6 0.8 1.0 SLC25A4 SUrvival fraction PDIA6 0 20 40 60 80 100 120 HNRNPA0 Time (months) Number at risk RuvBL1 low 28 12 3 3 0 0 0 RuvBL2 high 27 20 10 5 3 1 1 PKCζ
Figure S5 legend A. The apoptosis of 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 co- immunoprecipitation. C. The effect of PKCζ expression on grade 1 liver cancer overall survival. The total patient number was 55, p < 0.05. Figure S6 The T398 phosphorylation of YY1 is critical for 3-HAA-suppressed HCC xenografts growth. Related to Figure 6
A DMSO B DMSO C Ctrl YY1 KD YY1-WT YY1-T398A YY1-T398E 7 8 8 8 107 Q1 Q2 10 Q1 Q2 10 Q1 Q2 10 Q1 Q2 10 Q1 Q2 4.57 3.90 7.05 5.74 107 107 107 106 106 1.41 2.57 4.30 3.99 1.71 2.99 106 106 106 DMSO 3-HAA 5 5 10 10 105 105 105 DUSP6 4 4 4 104 104 10 10 10 PI PE-A PI PE-A PI PE-A PI PE-A PI PE-A 103 103 103 103 103 Q4 Q3 Q4 Q3 Q4 Q3 WT Q4 Q3 Q4 Q3 102 93.7 2.35 102 90.5 1.21 102 88.3 6.97 2 90.0 1.58 102 86.3 0.93 10 103 104 105 106 107 108 103 104 105 106 107 108 103 104 105 106 107 108 103 104 105 106 107 103 104 105 106 107 AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A 3-HAA 3-HAA T398A
Ctrl YY1 KD 8 YY1-WT 8 YY1-T398A 8 YY1-T398E 107 Q1 Q2 107 Q1 Q2 10 Q1 Q2 10 Q1 Q2 10 Q1 Q2 2.32 4.37 3.33 3.21 107 1.30 2.70 107 1.37 2.10 107 1.30 3.02 106 106 106 106 106 T398E 105 105 105 105 105 4 4 104 104 104 10 10 PI PE-A PI PE-A PI PE-A PI PE-A 103 103 PI PE-A 103 3 3 Q4 Q3 Q4 Q3 Q4 Q3 10 Q4 Q3 10 Q4 Q3 102 86.9 9.14 102 92.5 4.70 102 88.4 7.33 2 10 82.0 11.3 102 89.8 3.70 103 104 105 106 107 108 103 104 105 106 107 108 103 104 105 106 107 108 103 104 105 106 107 103 104 105 106 107 AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A AnnexinV-APC-A
D E F Balb/c Ctrl+DMSO Ctrl+3-HAA 300 8 8 10 Q1 Q2 10 Q1 Q2 0 7 7 200 10 12.9 4.33 10 18.2 11.2 200 6 6 1000 800 10 10 (μM) 3-HAA 5 5
10 10 IU/ml 100 104 104 100 PI PE-A PI PE-A 3 3 10 Q4 Q3 10 Q4 Q3 10 102 82.0 0.74 102 66.6 4.00 3 4 5 6 7 8 3 4 5 6 7 8 0 10 10 10 10 10 10 10 10 10 10 10 10 ALB TP ALT AST ALP UA UREA AnnexinV-APC-A AnnexinV-APC-A 1 Balb/c PKCi+DMSO PKCi+3-HAA 0.1 25
Serum Concentration Serum 0 108 108 0 100 200 300 400 Q1 Q2 Q1 Q2 20 100 107 13.0 4.19 107 16.2 5.61 Time (minutes) 200 106 106 400 Vz 15 105 105 T1/2 Cmax AUC0-t C0-inf CL MRT 800 4 4 hrs µM hr*µM hr*µM mL mL/hr hrs 10 PI PE-A PI PE-A 10 10 Weight(g) 103 Q4 Q3 103 Q4 Q3 3.89708.9 7.34 7.34625 6.73 3.17 5 102 82.2 0.62 102 74.9 3.30 0 103 104 105 106 107 108 103 104 105 106 107 108 1 2 3 4 5 6 7 8 9 10 AnnexinV-APC-A AnnexinV-APC-A Time(day)
Figure S6 legend: A. The apoptosis analysis of tumors by flow cytometry. The concentration of 3-HAA was 100 mg/kg.day. B. The apoptosis analysis of tumors by flow cytometry. The concentration of 3-HAA was 100 mg/kg.day. C. The DUSP6 expression analysis by IHC in xenografts. D. The apoptosis analysis of tumors by flow cytometry. The concentration of PKCi was 0.8 mg/kg.day. E. Plasma concentration versus time in C57 mice following an intraperitoneal injection of 100mg/kg of 3-HAA (mean ± S.D.). Following a single Intraperitoneal dose, the mean plasma T1/2 of 3-HAA were 3.89 hrs and 4.13 hrs, respectively. T1/2, elimination half-life; Cmax, maximum concentration; AUC0-t, area under the curve of a plasma concentration from time zero to time t; AUC0-inf, area under the plasma concentration-time curve from time zero to infinity; Vz, volume of distribution; CL, total plasma clearance of drug; MRT, mean residence time. F. The effect of 3-HAA on the liver/renal function and body wight of Balb/c mice. Figures
Figure 1
3-hydroxyanthranilic acid (3-HAA) is selectively decreased in hepatocellular carcinoma A. Quantitative analysis of tryptophan metabolites by LC-MS/MS and GC-MS in HCC and corresponding paratumor tissues. B. Quantitative analysis of tryptophan metabolites by LC-MS/MS and GC-MS in normal hepatic cells and HCC cells. C. Immunohistochemistry staining of 3-HAA in clinical HCC samples. D. Metabolic ux analysis of tryptophan metabolites in Huh7 and HepG2 cells. L-Tryptophan was completely 13C- labelled. The content of tryptophan catabolites in cells and medium was assessed using LC-MS/MS. The M0 stands for no carbon in tryptophan was 13C-labelled, M11 stands for all 11 carbon in tryptophan were 13C-labelled. E. Expression analysis of metabolic enzymes involved in 3-HAA generation. F. The effect of KMO overexpression on the concentration of endogenous 3-HAA and tryptophan catabolites in SMMC7721 cells.
Figure 2
3-hydroxyanthranilic acid (3-HAA) induces apoptosis of hepatocellular carcinoma cells A. Analysis of the growth of HCC cells treated for 4 days with one of four tryptophan metabolites (100 μM). B. Gene Ontology (GO) enrichment analysis of HCC SMMC7721 cells treated by 3-HAA for 8 hours. C. Apoptosis analysis by TUNEL assay in SMMC7721 cells treated for 24 h with 3-HAA at the indicated dose. D. Analysis of apoptosis signaling in HCC cells treated with 100 μM 3-HAA for indicated time or treated for 12 h with 3-HAA at the indicated dose. E. The effects of three types of inhibitors on 3-HAA-induced HCC cell death. ZVAD: apoptosis inhibitor (20 μM); Nec1: necrosis inhibitor (100 μM); 3-MA: autophagy inhibitor (5 mM). The dose of 3-HAA was 100 μM. Cells were treated for 4 days. F and G. Apoptosis analysis by TUNEL assay and ow cytometry in SMMC7721 xenografts with and without 3-HAA treatment. The dose of 3-HAA was 100 mg/kg•day. Mice were treated for 7 days and sacri ced on day 10. H. The in uence of endogenous 3-HAA on cell growth and apoptosis signaling of HCC cells. The KMO was overexpressed in SMMC 7721. The **: p < 0.01. The concentration of 3-HAA and ZVAD was 100 μM and 20 μM, respectively. I. Tumor formation analysis of HCC cells overexpressing KMO. The effect of endogenous 3-HAA on xenografted tumor formation. J. The effect of KMO overexpression on apoptosis of SMMC7721 cells. K. The effect of KMO expression on the disease-free survival of HCC patients. The patients were divided by the median of KMO expression value.
Figure 3 3-HAA induces tumor cell apoptosis by upregulating DUSP6 expression A. The Heatmap of the top consistently upregulated genes in SMMC7721, Hep3B, and SMMC97H. Deep sequencing was used to pro le gene expression in HCC SMMC7721, Hep3B, and SMMC97H cells. HCC cells were treated with 100 μM 3-HAA for 1, 8, or 24 h. B. Top consistently upregulated genes were individually veri ed using quantitative PCR and immunoblotting. C. DUSP6 expression reversely correlated with overall survival of HCC patients, as analyzed by the K-M plotter. The total patient number was 415. The HCC patients were divided by the median of KMO expression value. 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. 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. F. Effects of DUSP6 knockdown on 3-HAA-activated apoptosis signaling. SMMC7721 cells depleted of DUSP6 were treated with 100 μM 3-HAA for 24 h. G. 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 day 10. SMMC7721 cells were subcutaneously injected into mice. The middle graph shows xenograft weights in different groups. Photographs on the right show representative tumors in different groups. Figure 4
3-HAA upregulated DUSP6 expression by binding with transcription factor YY1 A. The transcription factors potentially binding with promoters of top 4 genes upregulated by 3-HAA. B. The common proteins between 3-HAA-increased chromatin binding proteins and predicted transcription factors binding to the DUSP6 promoter region. C. 3-HAA increases YY1 nuclear translocation. SMMC7721 cells were treated with 100 μM of 3-HAA for 8 hours. D. YY1 knockdown abolished 3-HAA-induced DUSP6 expression. SMMC7721 cells were treated with 100 μM of 3-HAA for the indicated time. E. The effect of YY1 knockdown on 3-HAA-activated apoptosis signaling. The concentration of 3-HAA was 100 μM. F. The effect of YY1 knockdown on 3-HAA-suppressed HCC cell growth. The concentration of 3-HAA was 100 μM. The **: P < 0.01. G. The effect of YY1 knockdown on 3-HAA-induced HCC apoptosis, analyzed by ow cytometry. The concentration of 3-HAA was 100 μM. The **: P < 0.01. H. 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. I. 3-HAA binding to YY1 determined by electrophoretic mobility shift assay (EMSA). The synthesized oligonucleotide was labeled at the 5’ terminus with FAM, and YY1 was puri ed using a Hitrap heparin column. J. 3-HAA increased YY1 enrichment on the DUSP6 promoter, analyzed by chromatin immunoprecipitation (ChIP) and quantitative PCR (QPCR). The dose of 3-HAA was 100 μM. K. The ChIP- sequencing analysis of YY1 on the DUSP6 gene. The HCC cells were treated with the indicated dose of 3- HAA prior to ChIP-sequencing. L. NMR measurement of direct binding between 3-HAA and YY1. T1r NMR spectra for 6b (red) alone or in the presence of SPOP at 2.5 mM (blue), 5 mM (cyan), or 10 mM (green). The STD spectrum for 6b was recorded in the presence of 5 mM SPOP. M. 3-HAA bound YY1 protein as shown by surface plasmon resonance. Graphs of equilibrium unit response versus concentrations are shown. The estimated Kd was 121.7 μM. Figure 5
PKCζ phosphorylates YY1 at Thr 398 in response to 3-HAA A. The YY1 phosphorylation was analyzed by the immunoblotting and the mass-spectrometry. The YY1 was blotted on the enriched phospho-proteins from SMMC7721 cells. The YY1 modi cation was analyzed by the mass-spectrometry following YY1 immunoprecipitation. B. The YY1 mutation of T398A attenuates the 3-HAA-increased YY1 nuclear accumulation. The concentration of 3-HAA was 100 μM and treated for 24 h. C. The T398A but not S247A mutation abolished 3-HAA-induced YY1 phosphorylation. The YY1 was conjugated with HA tag. The YY1 phosphorylation was detected by the T398 phospho-speci c antibody. D. The mutation of T398E in YY1 promoted DUSP6 expression, whereas the T398A mutation suppressed DUSP6 upregulation. The YY1 were fused with HA tag. E. The YY1 mutation of T398A reduces the 3-HAA-induced apoptosis, analyzed by the TUNEL assay. F. 3-HAA increased PKCζ binding to YY1, analyzed by co- immunoprecipitation and mass-spec. G. 3-HAA increased PKCζ binding to YY1, analyzed by immunoblots. H. The kinase screening for T398 phosphorylation of YY1. The kinase candidates were predicted by online tools NetPhos 3.1 (www.cbs.dtu.dk/services/NetPhos) (Blom et al., 2004) and GPS 5.0 (gps.biocuckoo.cn)(Song et al., 2012). I. The effect of kinase inhibitors on 3-HAA-induced YY1 phosphorylation. The YY1 phosphorylation was detected by the T398 phospho-speci c antibody. The concentration of 3-HAA was 100 μM. AKT inhibitor, MK2206 (0.5 μM); PKC inhibitor, Go6983 (0.5 μM); mTOR inhibitor, rapamycin (0.1 μM). L. The proposed 3-HAA binding model with YY1. Figure 6
T398 phosphorylation of YY1 is critical for 3-HAA-suppressed HCC xenografts growth A. YY1 knockdown recovered the 3-HAA-inhibited HCC xenografts growth. B. & C. The apoptosis analysis in xenografts by TUNEL assay and the ow cytometry. The histogram presents as mean ± SD (*: P < 0.05; **: P < 0.01). D. The T398A mutation of YY1 abolished the 3-HAA-inhibited HCC xenografts growth. E. & F. The apoptosis analysis in xenografts by TUNEL assay and the ow cytometry. The histogram presents as mean ± SD (*: P < 0.05; **: P < 0.01.). G. The PKCζ inhibitor reverted the HCC xenografts growth inhibited by 3-HAA. H. The apoptosis analysis in xenografts by the ow cytometry. The histogram presents as mean ± SD (*: P < 0.05; **: P < 0.01.). I. The concentration of 3-HAA in tumors post-administration. The 3-HAA content in xenografts was detected by LC-MS/MS. J. 3-HAA synergizes with IDO inhibitors to HCC xenograft growth. The dose of both 3-HAA and Epacadostat (IDO inhibitor) were 100 mg/kg.day, respectively. K. The working model of 3-HAA-inducing apoptosis. Note: The mouse xenografts were generated by the inoculation of 1.5 x 10^6 of SMMC7721 cells into the armpit of rear limb. The 3-HAA (100 mg/kg.day) was administered by intraperitoneal injection for 7 days. Tumor volumes are presented as mean ± SD (*: P < 0.05; **: P < 0.01.). Photographs on the right show representative xenografts in different groups.