Endothelial fatty liver binding protein 4: a new targetable mediator in hepatocellular carcinoma related to metabolic syndrome - PubMed (original) (raw)
. 2019 Apr;38(16):3033-3046.
doi: 10.1038/s41388-018-0597-1. Epub 2018 Dec 21.
Aurélie Sannier 1, Emma Norkowski 1, François Cauchy 1 2, Sabrina Doblas 1, Pierre Emmanuel Rautou 3, Miguel Albuquerque 4, Philippe Garteiser 1, Laura Sognigbé 1, Jerôme Raffenne 1, Bernard E van Beers 1, Olivier Soubrane 2, Pierre Bedossa 1, Jerôme Cros 1 4, Valérie Paradis 5 6
Affiliations
- PMID: 30575815
- PMCID: PMC6484689
- DOI: 10.1038/s41388-018-0597-1
Endothelial fatty liver binding protein 4: a new targetable mediator in hepatocellular carcinoma related to metabolic syndrome
Samira Laouirem et al. Oncogene. 2019 Apr.
Abstract
Metabolic syndrome (MS) is becoming the leading risk factor for hepatocellular carcinoma (HCC). HCC development related to MS may occur in advanced or non-advanced liver fibrosis, suggesting specific molecular pathways. Among these pathways, basal inflammatory state and adipokines production are involved. The aim of this study was to evaluate the role of fatty acid-binding protein 4 (FABP4). In this study, we demonstrate the specific overexpression of FABP4 in human HCC samples from patients with MS compared to other risk factors for chronic liver disease with FABP4 expression restricted to peritumoral endothelial cells. In vitro, glucose, insulin, VEGFA and hypoxia upregulated endothelial FABP4, which was reversed by metformin through mTOR pathway inhibition. FABP4 exerts oncogenic effects on hepatoma cell lines by upregulating the angiogenesis gene signature and pathways involved in the cell cycle, leading to increased cell proliferation and migration, and downregulating HIF1 pathway; effects were reversed in the presence of a specific FABP4 inhibitor (BMS309403). We showed the role of microvesicles as FABP4 vectors between endothelial and tumor cells. In vivo, BMS309403 significantly reduces tumor growth in heterotopic and orthotopic xenografted mice model. In conclusion, this study demonstrates the emerging oncogenic role of liver endothelial cells through FABP4 in HCC related to MS, and highlights new anti-neoplastic mechanism of metformin.
Conflict of interest statement
The authors declare that they have no conflict of interest.
Figures
Fig. 1
FABP4 is overexpressed in HCC from patients with MS-related chronic liver disease. Representative western blot analysis of FABP4 expression in HCC (a) and non-tumoral liver (b) from MS (with and without metformin treatment) and non-MS patients (HCV Hepatitis C Virus, HBV Hepatitis B Virus). c Quantitative analysis of FABP4 immunostaining in HCC and paired non-tumoral liver samples from MS patients. d Circulating FABP4 levels quantified by ELISA in HCC patients with MS or HCV infection
Fig. 2
FABP4 is mostly expressed in endothelial peritumoral cells in HCC related to MS. a HCC/MS without metformin treatment. b HCC/MS with metformin treatment. c HCC/HCV. Three panels include hematoxylin & eosin staining of HCC (left), FABP4 immunostaining in HCC (middle) and FABP4 immunostaining in non-tumoral liver (right). Arrows indicate positive vessels in portal tracts and fibrous bands. (Scale bars = 100 μm). d FABP4 immunostaining in HCC/MS without metformin. (left) higher magnification showing slight staining was observed in tumoral hepatocytes; (middle) double immunostaining with FABP4 (pink) and ERG (endothelial cells, brown); (right) double immunostaining with FABP4 (brown) and CD68 (macrophages, pink)
Fig. 3
FABP4 is regulated in endothelial cells (HUVEC). FABP4 expression is induced upon stimulation with glucose (a), insulin (b), VEGFA (c) for 4 or 24 h, and hypoxia for 4 and 8 h (d). e Metformin (5 and 10 µM for 24 h) decreased FABP4 expression in HUVEC stimulated by VEGFA (50 ng/mL for 24 h). f Expression of FABP4 and downstream effectors of the mTOR signaling pathway (p-mTOR and p-p70S6K) are decreased in HUVEC stimulated by VEGFA in the presence of metformin (10 μM for 24 h) or everolimus (0.1 μM for 24 h). The ratios FABP4/β-actin, HIF1α/β-actin, p-mTOR/mTOR and p- p70S6K/p70S6K are indicated. The results are expressed as the mean ± SD from three independent experiments
Fig. 4
FABP4 pro-oncogenic effects in hepatoma cell lines. Unsupervised hierarchical clustering using the top 300 up (red) and down (blue) regulated genes from microarray data of HepG2 (a) and HuH7 cells (b) untreated or treated by exogenous FABP4 (eFABP4). Enriched pathways in either condition, derived from the Gene Set Enrichment Analysis stand on the right side. c eFABP4 reduces active caspase 3 expression in HepG2 cells (effect is reversed in presence of BSM309403) while no effect is observed in HuH7 cells. d Effect on cell proliferation of FABP4 inhibitor (BMS309403) at increasing concentrations on stimulated HepG2 and HuH7 by FABP4 [eFABP4 vs. control (++) p < 0.01; BMS309403 vs. eFABP4 **p < 0.01; ***p < 0.001]. Effect of medium (M) and VEGFA stimulated HUVEC CM (CM+) or not (CM-) on HepG2 and HuH7 proliferation (e) and migration (f) in the presence or absence of BMS309403 [CM vs. M *p < 0.05; ***p < 0.001; CM with BMS309403 vs. CM without BMS309403 ++p < 0.01; +++p < 0.001]. The results are expressed as the mean ± SD from 3 independent experiments. g Immunoblot of FABP4 in CM+ or CM− containing (μV+) or not (μV−) microvesicles. h Double immunofluorescence of HepG2 cells incubated with CM**+** containing (μV+) or not (μV-) microvesicles (FABP4: green; red: β-catenin; DAPI: blue)
Fig. 5
FABP4 is regulated in primary liver sinusoidal endothelial cells (LSEC) and exerts pro-oncogenic effects on HepG2 cells. a FABP4, PPARγ, and p-PPARγ expression in LSEC upon stimulation with glucose (5 and 25 mM), insulin (10 and 20 nM) and VEGFA (25 and 50 ng/ml) for 24 h. b FABP4 expression in LSEC subjected to hypoxia for 1 and 2 h. c Effect of medium (M) and conditioned medium of LSEC stimulated (CM+) or not (CM−) by VEGFA on HepG2 proliferation in presence or absence of BMS309403 [eFABP4 or CM vs. control *p < 0.05; **p < 0.01; CM with BMS309403 vs. CM without BMS309403 ++p < 0.01]. The results are expressed as the mean ± SD from three independent experiments
Fig. 6
FABP4 inhibition decreases tumor growth in vivo (Heterotopic model (a-d), Orthotopic model (e-f). a Tumor volume (mm3) was measured twice a week in the 3 groups of mice. The figure indicates the tumor burden volume versus time since xenograft (in days). Data are expressed as mean values and standard deviation for each point. b Morphological aspect (left, Hematoxylin & Eosin) and FABP4 immunostaining (right) of representative tumors from the 3 different groups [control (top), p-BMS309403 (middle) and c-BMS309403 (below)]. c Percentage of tumor necrosis was assessed by hematoxylin & eosin (HE) staining for each group of mice [BMS309403 vs. control *p < 0.05; **p < 0.01]. d Expression of FABP4, Cyclin D1, VEGFA, VEGFR, caspase 3, mTOR, phospho-mTOR, 70S6K and phospho-70S6K in tumors from control and treated (curative BMS309403 treatment) mice (n = 5 each). e Tumor doubling time of intrahepatic tumors (days) was calculated using MR imaging in control (n = 5) and treated mice (n = 6). f T2-weighted morphological MR images obtained in a control (left) and a treated mouse (right) before treatment, at 2 weeks and 3 weeks after treatment initiation. The intrahepatic tumors are circled in red and the total volume of tumors is indicated (mm3)
Fig. 7
Schematic view recapitulating the involvement of endothelial FABP4 in liver carcinogenesis related to metabolic syndrome
References
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