Abstract 2317: Cancer-associated fibroblast-derived HGF regulates cancer stem cell-like properties in hepatocellular carcinoma through c-Met/FRA1/HEY1 signaling pathway
Authors: Lau Yuen Ting , Lo Jessica , Ng Irene Oi Lin , Lee Terence Kin Wah 
Published: August 2015

Liver cancer (HCC) remains one of the most deadliest malignancies in the world. Increasing evidence showed the existence of a subset of cancer cells called tumor-initiating cells (T-ICs) within the tumor bulk which are resistant to conventional treatments. Similar to normal stem cells, T-ICs are also regulated extrinsically within tumor microenvironment. Since HCC is mostly developed with cirrhotic background with the enrichment of activated fibroblasts, we hypothesized that cancer-associated fibroblasts (CAFs) would regulate liver T-ICs. For this purpose, we first isolated CAFs from fresh HCC clinical specimens, cultured and propagated them in vitro. After confirming the fibroblastic identity of CAFs, we subcutaneously injected CAFs, conditioned medium (CM) of CAFs, together with HCC tumor cells into NOD/SCID mice to assess their role in regulating tumorigenicity. Both CAFs and CM of CAFs were shown to have a similar ability in promoting the tumorigenicity of HCC cells, suggesting that CAFs regulate HCC probably by paracrine regulation. Therefore, we collected the CM of CAFs and examined its effect on the properties of liver T-ICs. We found that CM of CAFs enhanced liver T-IC properties, and HCC cells could stimulate CAFs to further aggravate its potential.

By cytokine profiling, we identified hepatocyte growth factor (HGF) to be the most potent cytokine in CM of CAFs critical for promotion of T-IC properties, and this effect was further confirmed by the administration of recombinant HGF at a level comparable to that secreted by CAFs. Upon cDNA microarray analysis, we identified FRA1, to be mediator for the effect of HGF-induced T-IC properties. Further characterization showed that activation of both Erk1/2 and Akt pathways are important for HGF-mediated FRA1 activation and induction. By IHC staining, we demonstrated that overexpression of FRA1 is significantly correlated with poorer cellular differentiation, as well as poor disease-free and overall survival in HCC patients. In view of the functional significance of FRA1 in regulating liver T-ICs, and the upregulation of FRA1 by CAF-derived HGF, we next investigated whether FRA1 activation is associated with fibrosis-dependent hepatocarcinogenesis. Using STAM™ non-alcoholic steatohepatitis (NASH) HCC mouse model, in which HCC is developed from liver fibrosis, we found that the activation of HGF/c-Met/FRA1 signaling pathway might be associated with the regulation of T-ICs during liver carcinogenesis. Lastly, we identified HEY1 to be the direct transcriptional target of FRA1. Rescue experiments by overexpression of HEY1 in FRA1 knockdown HCC cells, suggest that FRA1 promotes liver T-IC properties, at least in part, through HEY1 regulation. In conclusion, our study suggests the role of CAFs in regulating liver T-IC properties through the secretion of HGF, which is mediated through a novel c-Met/FRA1/HEY1 pathway.

Citation Format: Yuen Ting Lau, Jessica Lo, Irene Oi Lin Ng, Terence Kin Wah Lee. Cancer-associated fibroblast-derived HGF regulates cancer stem cell-like properties in hepatocellular carcinoma through c-Met/FRA1/HEY1 signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2317. doi:10.1158/1538-7445.AM2015-2317