Dihydropyrimidine dehydrogenase predicts survival and response to interferon-α in hepatocellular carcinoma

Abstract

Metastasis and recurrence contribute to poor prognosis of hepatocellular carcinoma (HCC). Recently, we reported that interferon-α (IFN-α) can suppress metastasis of HCC; however, the underlying mechanism has not been fully described. In this study, we demonstrated that expression of dihydropyrimidine dehydrogenase (DPYD), a pyrimidine catabolic enzyme, was dose-dependently downregulated by IFN-α in HCC tissues from nude mice. Notably, DPYD expression was found to be significantly increased in HCC cell lines with higher metastatic potentials compared with their controls. Moreover, upregulation of DPYD in HCC cells could promote in vitro migration, invasion, and in vivo lung metastasis, and inducing changes characteristic of epithelial-mesenchymal transition (EMT). In contrast, knockdown of DPYD inhibited these processes. Mechanistically, DPYD functioned as a positive regulator of EMT in HCC by targeting the p38/NF-κB/Snail1 pathway. Clinically, tissue microarray analysis showed that high DPYD expression was positively associated with aggressive tumor characteristics, including larger tumor size, tumor recurrence, and advanced tumor node metastasis (TNM) stage, and independently correlated with poorer overall survival times after curative resection. HCC patients with low DPYD expression have better response to IFN-α therapy. Taken together, our findings elucidate that IFN-α could downregulate DPYD expression to inhibit EMT and HCC metastasis, and suggest that DPYD might be a potential prognostic biomarker and a therapeutic target for HCC.

Fig. 1. Identification of DPYD as a therapeutic target of IFN-α

a HCCLM3 orthotopic mouse models were successfully established, and then treated with or without IFN-α (3 × 10⁷ U/kg/day) for 35 days. Altered metabolic gene profiling of these two groups was analyzed by RNA sequencing, and the top 10% of statistically differently expressed genes (P < 0.001) were shown by heat map. b The lead gene, DPYD, was dose-dependently downregulated by IFN-α both in HCC samples from either HCCLM3 or MHCC97H orthotopic mouse models by qRT-PCR analysis. Data, mean  ±S.D. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 2. DPYD expression is upregulated in various human HCC cell lines

The mRNA and protein expression of DPYD in five HCC cell lines (SMMC7721, HepG2, MHCC97L, MHCC97H, and HCCLM3) were compared with a nontransformed hepatic cell line (L0-2). a, b qRT-PCR analysis confirmed that the mRNA expression of DPYD in HCC cell lines is upregulated compared to hepatic cell line (L0-2). The DPYD mRNA expression was significantly increased in the HCC cell lines with high metastatic potentials (MHCC97L, MHCC97H, and HCCLM3) than those with low metastatic potentials (SMMC7721 and HepG2). Data, mean ± S.D. ***P < 0.001. c, d Western blot analysis showed a significant increase of DPYD in the HCC cells lines. The increased levels of DPYD protein were observed in the HCC cell lines with high metastatic potentials (MHCC97L, MHCC97H, and HCCLM3) compared to those with low metastatic potentials (SMMC7721 and HepG2). Data, mean ± S.D. *P < 0.05, **P < 0.01

Fig. 3. DPYD expression is upregulated in human HCC samples and correlated with HCC progression

a Relative DPYD mRNA levels among peritumoral and tumoral tissues of HCC patients (n = 24) with distant metastasis after curative resection. b qRT-PCR analysis of DPYD levels in tumor samples (n = 24). The mRNA expression of DPYD is significantly higher in HCC tissues than in peritumor tissues. Data, mean ± S.D. *P < 0.05. c Western blot analysis showed the protein expression of DPYD in HCC tissues. d Expression of DPYD was examined by immunohistochemical staining analyses in 185 HCC specimens, and representative images are shown. (scale bar, 50 μm; original magnification for right panel: ×400). e Prognostic significance of DPYD in HCC patients was evaluated by Kaplan–Meier analysis. Upregulation of DPYD in HCC predicts lower overall survival times. f, g Association of DPYD expression with IFN-α therapeutic response in HCC patients. Kaplan–Meier analysis indicated that HCC patients with low DPYD expression have better response to IFN-α therapy

Fig. 4. Establishment of different DPYD expression levels in various HCC cell lines

qRT-PCR (a, b) and western blot (c, d) analysis showed the mRNA and protein expression of DPYD in SMMC7721 and HCCLM3 cells. Data, mean ± S.D. **P < 0.01, ***P < 0.001. WT wild type, OE overexpression

Fig. 5. DPYD promotes metastatic potential of HCC cells in vivo and in vitro

a In vivo imaging of lung metastasis of the four cell lines (SMMC7721-vector, SMMC7721-DPYD-OE, HCCLM3-vector, and HCCLM3-shDPYD) are shown by fluorescence in nude mice. The number of metastatic nodules in lung is significantly larger in the HCCLM3-vector group compared to the HCCLM3-shDPYD group from 20 days after tail vein injection. Similar results were observed in SMMC7721-DPYD-OE group compared to SMMC7721-vector group. *P < 0.05 and **P < 0.01. b The motility of the indicated cell lines in vitro was detected by scratch wound assay, and statistics are shown with a bar graph (original magnification: ×100; scale bar, 20 μm). c Invasive behavior in vitro was determined by transwell Matrigel invasion assay, and statistics are shown with a bar graph (original magnification: ×200; scale bar, 20 μm). Data, mean ± S.D., and representative of three independent experiments. **P < 0.01 and ***P < 0.001. OE overexpression

Fig. 6. DPYD enhances aggressiveness of HCC cells by promoting EMT

a The mRNA and protein expression changes of E-cadherin, N-cadherin, and Snail1 in SMMC7721-vector and SMMC7721-DPYD-OE cells. b qRT-PCR and western blot assays of these proteins in HCCLM3 cells with different DPYD expression. c Representative immunostaining images of EMT-related molecules from serial sections of human tumor samples (scale bar, 50 μm). Data represent the mean ± S.D. and are representative of three independent experiments. **P < 0.01, ***P < 0.001. OE overexpression

Fig. 7. DPYD facilitates EMT by suppressing the expression of target gene p38

a The EMT-related signaling molecules were evaluated by western blot analysis in HCCLM3 and SMMC7721 cells with different DPYD expression. b Representative images of lung metastasis of HCC cell lines (HCCLM3-vector, HCCLM3-shDPYD, and HCCLM3-shDPYD-shp38) are shown by fluorescence in nude mice. The reduced number of metastatic nodules in lung was reversed by blockade of p38 in HCCLM3-shDPYD cells. Data, mean ± S.D. *P < 0.05, **P < 0.001. c Scratch wound assay showed that blockade of p38 in HCCLM3-shDPYD cells by p38-shRNA could significantly recover decreased cell migration due to DPYD deficiency (original magnification: ×100; scale bar, 20 μm). d Transwell invasion assay revealed that p38-shRNA treatment in HCCLM3-shDPYD cells markedly reversed the reduced cell invasive ability induced by DPYD deficiency (original magnification: ×200; scale bar, 20 μm). Data, mean ± S.D. *P < 0.05, **P < 0.001. e Expression changes of epithelial and mesenchymal markers at protein levels were detected by western blot analysis after p38 blockade in HCCLM3 cells. OE overexpression

Fig. 8. DPYD regulates EMT via the p38/NF-κB/Snail1 signaling pathway

a Western blot assay showed that the expression of phosphorylated NF-κB p65 was regulated by p38 activity both in SMMC7721 and HCCLM3 cells when treated with p38 inhibitor SB203580. b Western blot assay showed simultaneous upregulation of Snail1 and NF-κB p65 phosphorylation in SMMC7721 cells with high DPYD expression. The activity of Snail1 was regulated by the level of NF-κB p65 phosphorylation in SMMC7721 cells when treated with p65 inhibitor BAY11-7082. c Co-immunoprecipitation assay indicated that NF-κB p65 formed a complex with Snail1 in SMMC7721 cells. d Schematic depiction of the mechanism underlying DPYD-mediated HCC metastasis in HCC cells. OE overexpression, IgG immunoglobulin G