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. 2018 May;31:287-298.
doi: 10.1016/j.ebiom.2018.05.004.

Hydroxylase Activity of ASPH Promotes Hepatocellular Carcinoma Metastasis Through Epithelial-to-Mesenchymal Transition Pathway

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Free PMC article

Hydroxylase Activity of ASPH Promotes Hepatocellular Carcinoma Metastasis Through Epithelial-to-Mesenchymal Transition Pathway

Qifei Zou et al. EBioMedicine. .
Free PMC article

Abstract

Over-expression of aspartyl (asparagynal)-β-hydroxylase (ASPH) contributes to hepatocellular carcinoma (HCC) invasiveness, but the role of ASPH hydroxylase activity in this process remains to be defined. As such, the current study investigated the role of ASPH hydroxylase activity in downstream signalling of HCC tumorgenesis and, specifically, metastasis development. Over-expression of wild-type ASPH, but not a hydroxylase mutant, promoted HCC cell migration in vitro, as well as intrahepatic and distant metastases in vivo. The enhanced migration and epithelial to mesenchymal transition (EMT) activation was notably absent in response to hydroxylase activity blockade. Vimentin, a regulator of EMT, interacted with ASPH and likely mediated the effect of ASPH hydroxylase activity with cell migration. The enhanced hydroxylase activity in tumor tissues predicted worse prognoses of HCC patients. Collectively, the hydroxylase activity of ASPH affected HCC metastasis through interacting with vimentin and regulating EMT. As such, ASPH might be a promising therapeutic target of HCC.

Keywords: ASPH; Epithelial-mesenchymal transition; Hepatocellular carcinoma; Hydroxylase; Metastasis; Vimentin.

Figures

Fig. 1
Fig. 1
ASPH hydroxylase activity is required for HCC cell migration and adhesion. (a) Validation of enforced expression of ASPH (wild-type) and its enzymatic mutant (H679A) in MHCC-97L, EHBC-512 and Huh-7 blotted by ASPH antibody specific for C-terminus. endo-ASPH, the lower band around 120kD is the endogenously expressed ASPH; exo-ASPH, the upper band is the exogenously expressed ASPH that is fused by a GFP tag. (b) The ASPH hydroxylase activity of wild-type and enzymatic mutant of ASPH measured by α-ketoglutarate (α-KG) consumption in in vitro Asp/Asn β-hydroxylation assay in 293 cells transfected with indicated construct. (c) The statistical results of cell migration of MHCC-97L, EHBC-512 and Huh-7 transfected with indicated constructs in the transwell assay. (d) The statistical results of MHCC-97L, EHBC-512 and Huh-7 cell migration upon administration of hydroxylase inhibitor DIPY (1 μM) and DMOG (100 nM). (e) The statistical results of cell adhesion of MHCC-97L, EHBC-512 and Huh-7 transfected with indicated constructs. (f) The silencing of ASPH in MHCC-97L and EHBC-512 cells by lenti-virus mediated shRNA (sh-1 and sh-2) that is testified by immunoblot. The relative quantification of blotting results was shown below. Effective RNAi constructs of sh-2 were used for later studies. (g) and (h) The statistical results of cell migration or cell adhesion for MHCC-97L and EHBC-512 cells transfected with indicated constructs in the transwell or cell adhesion assay, respectively. All data are shown as average ± SD based on at least three independent experiments after normalization to the control group. *P < 0.05, **P < 0.01 vs. control. Abbreviations: ctl or sh-ctl, vector only control group; WT, wild-type of ASPH; H679A, enzymatic mutant of ASPH.
Fig. 2
Fig. 2
Blockade of cell migration by a novel antibody FE1 that targets the catalytic domain of ASPH. (a) Validation of the specificity of FE1 by the immunoblot. Upper: the peptide competition assay using EHBC-512 and MHCC-97L cell lysate in which FE1 were pre-incubated with the antigen peptide before used in immunoblot. Bottom: the specific recognition to wild-type but not enzymatic mutant of ASPH by FE1 using MHCC-97L transfected by indicated constructs. endo-ASPH, the endogenously expressed ASPH; exo-ASPH, the exogenously expressed ASPH that was fused by a GFP tag. (b) Validation of the specificity of FE1 by the immunostaining. Co-localization of positive signal stained by FE1 and anti-GFP antibodies in Huh-7 cells over-expressed with GFP-tagged ASPH (400×). (c) Cell surface expression of ASPH. Upper: immunostaining of ASPH by FE1 in impermeable EHBC-512 and MHCC-97L cells without triton X-100 treatment. The cell morphology was characterized by F-actin presence through phalloidin staining. Bottom: the presence of cell subsets with membrane or intracellular ASPH expression in EHBC-512 and MHCC-97L cells with or without triton X-100 treatment measured by flow cytometers. (d) The ASPH hydroxylase activity in 293 cells transfected with ASPH upon administrating 100 μg/ml of FE1 antibody or isotype IgG measured by α-ketoglutarate (α-KG) consumption in in vitro Asp/Asn β-hydroxylation assay. (e) and (f) The statistical results of cell migration in Huh-7 cells transfected with indicated constructs, EHBC-512 and MHCC-97L cells that were treated by 100 μg/ml of FE1 antibody or isotype IgG, otherwise the FE1 concentration was designated. All data are shown as average ± SD based on at least three independent experiments after normalization to the control group. *P < 0.05, **P < 0.01 vs. IgG treatment. Abbreviations: ctl, vector only control group; WT, wild type of ASPH; H679A, enzymatic mutant of ASPH.
Fig. 3
Fig. 3
ASPH hydroxylase activity regulate epithelial-to-mesenchymal transition of HCC cells. (a) The relative up- or down-regulation of EMT biomarkers and regulatory genes in EHBC-512 cells transfected with indicated constructs by the EMT PCR-array normalized by control cells transfected with vector only. (b) The validation of gene expression based on PCR-array results in EHBC-512 cells transfected with indicated constructs. (c) and (d) The immunoblot and immunostaining of EMT biomarker including γ-catenin, α-catenin, E-cadherin and vimentin in Huh-7 cells transfected with indicated constructs. Fluorescent images were taken under 600× magnification. (e) The activation of notch pathway genes in EHBC-512 cells transfected with indicated constructs. Empty vector served as control. *P < 0.05, **P < 0.01 vs. control. All data are shown as average ± SD based on at least three independent experiments after normalization to the control group. *P < 0.05, **P < 0.01 vs. IgG treatment. Abbreviations: ctl, vector only control group; WT, wild type of ASPH; H679A, enzymatic mutant of ASPH.
Fig. 4
Fig. 4
The role of ASPH-vimentin interaction in promoting HCC cell migration. (a) and (b) Identification of exogenous ASPH-vimentin interaction. Left: The base-peak plot of mass spectrometry analysis of protein complex from pull-down assay in 293 cells over-expressed with FLAG-fusion ASPH or HA-fusion vimentin using protein tag antibodies. Right: identified peptide sequence belonging to vimentin and ASPH in the protein complex. (c) Validation of endogenous ASPH-vimentin interaction. The immunoblot (IB) of the protein immuno-precipitated (IP) with FE1 and anti-vimentin in MHCC-97  cells. (d) Validation of manipulated vimentin expression in MHCC-97L and Huh-7 cells. Left: complementary over-expression of vimentin control and ASPH-silenced MHCC-97L cells. Right: complementary silencing vimentin in control and ASPH-over-expressed Huh-7 cells. The relative quantification of blotting results is shown below. (e) The indispensable role of vimentin for ASPH in regulating cell migration. Left: functional blockade of cell migration by silencing vimentin in ASPH-over-expressed Huh-7 cells. Right: functional rescue cell migration by over-expressing vimentin in ASPH-silenced in MHCC-97L cells. (f) Effect of hydroxylase inhibition to vimentin-dependent cell migration. Left y axis: the statistical results of cell migration in vimentin-over-expressed MHCC-97L cells that is treated by DIPY (1 μM) and DMOG (100 nM). right y axis: the corresponding increased fold of cell migration by vimentin over-expression in comparison to control group. All data are shown as average ± SD based on at least three independent experiments after normalization to the control group. *P < 0.05, **P < 0.01 vs. control. Abbreviations: ctl or sh-ctl, vector only control group; WT, wild type of ASPH; VIM: vimentin; sh-ASPH, ASPH silencing; sh-VIM, vimentin silencing.
Fig. 5
Fig. 5
ASPH hydroxylase activity promotes HCC formation and metastasis in vivo. (a) Visible liver tumor formation or intrahepatic metastatic nodules in nude mice transplanted with Huh-7 cells over-expressing control vector, wild type and H679A in the liver (n = 25 for each group). Scale bar, 1 cm. (b) The quantification of intrahepatic metastasis in the mice with liver tumor formation. Left y axis: the number of mice with or without visible intrahepatic metastasis in each group as indicated; right y axis: the total number of observed metastatic nodules in each group as indicated. (c) Histological or immunohistochemical examination of the liver tumor. a, b, c, liver tumor formed by Huh-7 cells transfected with control, wild type and H679A, respectively, 100× magnification. d, e, microsatellite tumor and stromal infiltration found in the liver tumor in the group of ASPH, 200× magnification. f, g, h, immunostaining of vimentin in the liver tumors of indicated groups, 400× magnification. (d) Histological or immunohistochemical examination of metastatic nodules detected in the lungs of mice subcutaneously injected with MHCC-97L cells with ASPH silenced or transfected with control vector only (n = 25 for each group). a, metastatic nodules in the lungs of mice injected with MHCC-97L cells, 200× magnification. b, lungs without metastatic lesion in mice injected with control MHCC-97L cells, 200× magnification. c, immunostaining of vimentin in the lung metastatic nodules, 400× magnification. d, immunostaining of vimentin in the primary subcutaneous tumor, 400× magnification. (e) The counting of lung metastasis in the mice with subcutaneous tumor formation. Left y axis: the number of mice with or without detectable metastasis in each group as indicated; right y axis: the total number of observed micro-metastatic lesion in each group as indicated.
Fig. 6
Fig. 6
The ASPH hydroxylase activity is associated with prognosis in HCC patients. (a) Representative images (upper, 40× magnifications; bottom, 400× magnification) of immunostaining by FE1 antibody in HCC tissue array. a, d, non-tumor tissues. b, e, tumor tissues with weak ASPH staining. c, f, tumor tissue with intense ASPH staining. (b) Kaplan-Meier curve of tumor recurrence and OS after hepatectomy for patients with low (blue curve) or high (green curve) ASPH hydroxylase activity in the training cohort. a, b, tumor recurrence in the whole cohort and its BCLC 0/A subgroup. c, d, OS in the whole cohort and its BCLC 0/A subgroup. (c) Kaplan-Meier curve of tumor recurrence and OS after hepatectomy for patients with low (blue curve) or high (green curve) ASPH hydroxylase activity in the validation cohort. a, b, tumor recurrence in the whole cohort and its BCLC 0/A subgroup; c, d, OS in the whole cohort and its BCLC 0/A subgroup.

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