Kynurenine catabolic enzyme KMO regulates HCC growth

Dear editor: Our recent research has found that the kynurenine derivative 3-HAA was lower in tumour cells due to the downregulation of its synthetic enzyme kynurenine 3-monooxygenase (KMO), and overexpression of KMO suppressed hepatocellular carcinoma (HCC) tumour formation and tumour growth by increasing endogenous 3-HAA. It is well known that kynurenine promotes tumour growth by directly binding to the aryl hydrocarbon receptor.1,2,3 The 3-hydroxyanthranilic acid (3-HAA), a

derivative of kynurenine, was reported to induce apoptosis by upregulating phosphatases. 4 However, the metabolism and function of kynurenine derivatives largely remain unclear. Here, we report our novel findings related to kynurenine metabolism.
3-HAA is decreased in tumour cells. Tryptophan catabolites were first analysed in clinical HCCs. The concentration of kynurenine catabolite 3-HAA decreased in both HCC and oesophageal carcinomas compared to the matched paratumour tissues ( Figure 1A; Figure S1A). Conversely, the concentration of tryptophan and kynurenine was higher in these HCCs and oesophageal carcinomas than in the matched paratumour tissues, respectively. Consistent with this observation, the concentration of 3-HAA was also lower in seven HCC cell lines tested than in normal hepatic cells, whereas the content of tryptophan and kynurenine increased in these tested HCC cell lines ( Figure 1B). The immunohistochemistry analysis further confirmed lower 3-HAA content in clinical HCC tissues than in adjacent non-cancerous tissues ( Figure 1C).
Metabolic flux analysis revealed tryptophan metabolised to kynurenine but not 3-hydroxykynurenine (3-HK) or 3- HAA in HCC cells, and the newly generated kynurenine was secreted into the culture medium ( Figure 1D), suggesting 3-HAA is decreased in tumours, at least in HCCs and oesophageal carcinomas.

Upregulation of KMO increases 3-HAA.
To determine whether the metabolic enzymes regulate 3-HAA concentration, we assessed the expression of 3-HAArelated enzymes in HCC cells. The immunoblotting and immunohistochemistry analysis showed that KMO and kynureninase (KYNU) were downregulated in HCC cells and tissues. In contrast, the indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2) was upregulated (Figure 2A,B). This finding was consistent with the HCC expression profile in the TCGA database ( Figure 2C). Moreover, both KMO and KYNU expression (www.gtexportal.org) are commonly downregulated in tumours originated from tissues abundantly expressing KMO and KYNU. These tumours include lung, kidney, and liver carcinomas, which are the top 10 tumours worldwide in terms of death ( Figure 2D).
KMO overexpression inhibits tumour formation by inducing apoptosis. Functionally, either overexpression of KMO or knockdown of HAAO inhibited cell growth of HCC cells in vitro by increasing apoptosis (Figure 3A,B). Only the apoptosis inhibitor zVAD restored growth of HCC cells following 3-HAA treatment or overexpressing KMO ( Figure 3C,D). Moreover, KMO overexpression suppressed tumour formation and tumour growth in the HCC xenograft nude mice model ( Figure 3E; Figure S2A). Remarkably, the Kaplan-Meier survival analysis showed that HCC patients with high KMO expression had a prolonged disease-free survival than patients with low KMO expression ( Figure 3F). The 3-HAA treatment significantly inhibited HCC cell growth and colony formation (Figure 3G; Figure S2B). Moreover, 3-HAA but not kynurenine slowed tumour growth in a CDX model and in a patientderived xenograft (PDX) model ( Figures S2C and 3H), suggesting KMO overexpression inhibits tumour formation and tumour growth via its catabolite 3-HAA.
Through gene expression profiling, real-time PCR and immunoblotting, the top two upregulated genes DUSP6 and IGFBP1 were selected for further study (Figure 3I,J). However, the clinical data showed that the overall survival of HCC patients was only associated with the expression level of DUSP6, but not IGFBP1 ( Figure 3K; Figure S2D). Patients expressing a high level of DUSP6 showed a more prolonged overall survival than patients expressing a low level of DUSP6 ( Figure 3K), and the corrective analysis with the clinical characteristics also supported this finding ( Figure S2E). Also, we demonstrated that DUSP6 mediates 3-HAA-induced tumour cell apoptosis via ERK signalling ( Figure 3L,M; Figure S2F,G), which was consistent with our previous finding. 4 According to the fact that 3-HAA activates transcription factor YY1, 4,5 closer analysis of the DUSP6 promoter region using online-based prediction tools 6,7 revealed a novel potential YY1 binding DNA fragment at positions −1145 to −1134, which was distinct from the reported consensuses binding sequence. 8 This finding was further confirmed by a luciferase assay and ChIP-QPCR ( Figure S2H,I). The TUNEL assay demonstrated that 3-HAA-induced apoptosis was reduced in SMMC7721 cells depleted of YY1, overexpression of DUSP6 restored the apoptosis suppressed by YY1 depletion (Figure S2J).
KMO enhances the inhibition effect of IDO1 inhibitor on HCC growth. The various HCC mouse models were implemented to further evaluate the potential application of KMO target in clinics. As shown in Figure 4A, DUSP6 knockdown reversed KMO-mediated suppression of tumour growth in SMMC7721 xenografts.  Figure 4B).
Most importantly, KMO enhances the effect of IDO1 inhibitor Epacadostat to suppress HCC xenograft growth in an immune-competent mouse model (Figure 4C). In the meantime, the combination of KMO overexpression with IDO1 inhibitor Epacadostat also inhibited the HCC tumour growth and prolonged the survival of mice bearing transposon-induced HCCs ( Figure 4D).
In brief, this study reveals that both KMO and its substrate 3-HAA decreases in HCC cells and HCC tissues. The KMO overexpression as well as 3-HAA treatment reverses the tumour-promoting effect of kynurenine and significantly improves the efficacy of IDO1/2 inhibitors on HCC xenografts ( Figure 4E). These findings show that downregulation of KMO appears to be essential for HCC growth, suggesting the kynurenine metabolic enzyme KMO is a promising therapeutic target for HCC.

C O N S E N T F O R P U B L I C AT I O N
Not applicable.

C O N F L I C T O F I N T E R E S T
The authors declare that they have no competing interests.