Functional characterization of cancer-associated fibroblasts in the regulation of cancer stem cell-like properties in hepatocellular carcinoma

Abstract

Hepatocellular Carcinoma (HCC) is one of the most common and deadliest malignancies worldwide. The poor prognosis of HCC is attributed to refractory to chemotherapy and high rate of recurrence. Increasing evidence showed that tumor-initiating cells (T-ICs), which are capable of tumor initiation, self-renewal, differentiation and chemoresistance, are responsible for treatment failure and tumor relapse. Identification of the signaling pathways that maintain the functions of T-ICs provides potential targets for treatment of HCC.

Similar to normal stem cells, T-ICs are also regulated extrinsically by cells within the tumor microenvironment. Cancer-associated fibroblasts (CAFs), being the major cell population within the tumor stroma, were found to play a prominent role in the progression of various cancers, including HCC. However, their role in the regulation of liver T-ICs has not been studied. In this study, we aim to examine the functional role of CAFs in regulating the properties of liver T-ICs. For this purpose, CAFs from fresh HCC samples were isolated and cultured. After confirming their "fibroblastic identity", conditioned medium (CM) of CAFs was collected to examine its effect on functional properties of liver T-ICs. CM of CAFs enriched the population of liver T-IC as evidenced by the enhanced tumorigenicity, spheroid forming ability, expression of T-IC markers, tumor invasiveness, and chemoresistance through paracrine secretion, which could be further augmented by the reciprocal interaction between CAFs and HCC cells.

By cytokine profiling, we identified several cytokines that were preferentially secreted by CAFs and further increased in response to HCC cell stimulation. Subsequent assessment revealed hepatocyte growth factor (HGF) to be the most potent cytokine in stimulating spheroid formation of HCC cells. The effect of HGF on liver T-IC properties was further confirmed by administration of recombinant HGF at a level comparable to that secreted by CAFs. Through blocking the activation of HGF/c-Met pathway induced by CM of CAFs using HGF neutralizing antibody or c-Met kinase inhibitor, we confirmed that the effect of CAFs on liver T-IC function was due to the secretion of HGF.

To delineate the downstream mechanism by which HGF/c-Met signaling regulates liver T-ICs, a cDNA microarray analysis was employed and we identified FOS-like antigen 1 (FOSL1/FRA1) to be significantly upregulated upon HGF treatment. This finding is also supported by the clinical observation that expressions of HGF and FOSL1 were positively correlated in HCC clinical specimens. Stable knockdown of FRA1 abolished HGF-induced T-IC properties, which further confirmed the role of FRA1 as a downstream effector of HGF/c-Met signaling. Clinically, FRA1 overexpression was significantly correlated with poor patient outcome. By utilizing the 〖STAM〗^TM non alcoholic steatohepatitis (NASH)-HCC mouse model, HGF-induced FRA1 activation was found to associate with fibrosis-dependent HCC development. Further investigation by RNA-sequencing analysis identified HEY1, a downstream target of Notch pathway, as a direct downstream effector of FRA1, which was further confirmed by subsequent rescue experiments and promoter activation analysis.

In conclusion, our study delineated the novel mechanism by which CAFs regulate liver T-ICs through HGF/c-Met/FRA1/HEY1 pathway, which provides an attractive target for the development of therapeutic strategies against HCC.