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

Abstract

Background

Previous studies have established that proteinase-activated receptor 2 (PAR ₂) promotes migration and invasion of hepatocellular carcinoma (HCC) cells, suggesting a role in HCC progression. Here, we assessed the impact of PAR ₂ in HCC stromal cells on HCC growth using LX-2 hepatic stellate cells (HSCs) and Hep3B cells as model.

Methods

PAR ₂ expression and function in LX-2 cells was analysed by RT-PCR, confocal immunofluorescence, electron microscopy, and [Ca ²⁺] _i measurements, respectively. The impact of LX-2-expressed PAR ₂ 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 PAR ₂ activation in LX-2 cells, various signalling pathways were analysed by immunoblotting and proteome profiler arrays.

Results

Following verification of functional PAR ₂ 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 PAR ₂) was downregulated by RNA interference (RNAi). In vitro studies confirmed these results demonstrating RNAi mediated inhibition of PAR ₂ 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, PAR ₂ stimulation with trypsin or a PAR ₂-selective activating peptide (PAR ₂-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 PAR ₂-AP-induced migration.

Conclusion

PAR ₂ in HSCs plays a crucial role in promoting HCC growth presumably by mediating migration and secretion of pro-angiogenic and pro-mitotic factors. Therefore, PAR ₂ in stromal HSCs may have relevance as a therapeutic target of HCC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12943-016-0538-y) contains supplementary material, which is available to authorized users.