Proteinase-activated receptor 2 (PAR2) in hepatic stellate cells – evidence for a role in hepatocellular carcinoma growth in vivo

Molecular Cancer

Research

Franziska Mußbach¹  Roland Kaufmann¹  Utz Settmacher¹  Martin Westermann²  Hendrik Ungefroren³  Susanne Sebens⁴  Lennart Lenk⁴  Petra Henklein⁵  Jörg P. Müller⁶  Frank-Dietmar Böhmer⁶  Kathrin Katenkamp⁷  Bernd Günther⁸ 

[1] Department of General, Visceral and Vascular Surgery, Jena University Hospital, Erlanger Allee 101, D-07747, Jena, Germany;Electron Microscopy Center, Jena University Hospital, Jena, Germany;First Department of Medicine, UKSH and University of Lübeck, Lübeck, Germany;Group Inflammatory Carcinogenesis, Institute for Experimental Cancer Research, Christian-Albrechts-University Kiel and University Hospital Schleswig-Holstein (UKSH), Campus Kiel, Kiel, Germany;Institute of Biochemistry, Charité, Berlin, Germany;Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Jena University Hospital, Jena, Germany;Institute of Pathology, Jena University Hospital, Jena, Germany;Service Unit Small Animal, Research Center Lobeda (FZL), Jena University Hospital, Jena, Germany;

关键词: Proteinase-activated receptor 2;    PAR;    Hepatocellular carcinoma;    HCC;    Hepatic stellate cells;    HSCs;    LX-2;    Cell migration;    Receptor tyrosine kinase;    Met;    Src;    HCC xenograft;    Angiogenesis;   

DOI  :  10.1186/s12943-016-0538-y

 received in 2016-02-08, accepted in 2016-07-18,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundPrevious studies have established that proteinase-activated receptor 2 (PAR2) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR2 in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model.MethodsPAR2 expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca2+]i measurements, respectively. The impact of LX-2-expressed PAR2 on tumour growth in vivo was monitored using HCC xenotransplantation experiments in SCID mice, in which HCC-like tumours were induced by coinjection of LX-2 cells and Hep3B cells. To characterise the effects of PAR2 activation in LX-2 cells, various signalling pathways were analysed by immunoblotting and proteome profiler arrays.ResultsFollowing verification of functional PAR2 expression in LX-2 cells, in vivo studies showed that these cells promoted tumour growth and angiogenesis of HCC xenografts in mice. These effects were significantly reduced when F2RL1 (encoding PAR2) was downregulated by RNA interference (RNAi). In vitro studies confirmed these results demonstrating RNAi mediated inhibition of PAR2 attenuated Smad2/3 activation in response to TGF-β1 stimulation in LX-2 cells and blocked the pro-mitotic effect of LX-2 derived conditioned medium on Hep3B cells. Furthermore, PAR2 stimulation with trypsin or a PAR2-selective activating peptide (PAR2-AP) led to activation of different intracellular signalling pathways, an increased secretion of pro-angiogenic and pro-mitotic factors and proteinases, and an enhanced migration rate across a collagen-coated membrane barrier. Silencing F2RL1 by RNAi or pharmacological inhibition of Src, hepatocyte growth factor receptor (Met), platelet-derived growth factor receptor (PDGFR), p42/p44 mitogen activated protein kinase (MAPK) or matrix-metalloproteinases (MMPs) blocked PAR2-AP-induced migration.ConclusionPAR2 in HSCs plays a crucial role in promoting HCC growth presumably by mediating migration and secretion of pro-angiogenic and pro-mitotic factors. Therefore, PAR2 in stromal HSCs may have relevance as a therapeutic target of HCC.

【 授权许可】

CC BY   
© The Author(s). 2016