Bone morphogenetic protein 4 provides cancer-supportive phenotypes to liver fibroblasts in patients with hepatocellular carcinoma

Background: Cancer-associated fibroblasts (CAFs) are essential constituents of cancer-supportive microenvironments. The high incidence of hepatocellular carcinoma (HCC) in advanced fibrosis patients implies that fibroblasts have a promoting effect on HCC development. We aimed to explore the regulators of phenotypes and function of CAFs in the liver. Methods: We established primary cancer-associated fibroblasts (CAFs) and non-cancerous liver fibroblasts (NFs) from 15 patients who underwent HCC resection. We compared phenotypes, capacity of cytokine/chemokine production and gene expression profiles between pairs of CAFs and NFs from the same donors. We examined resected tissue from additional 50 patients with HCC for immunohistochemical analyses. Results: The CAFs expressed more ACTA2 and COL1A1 than the NFs, suggesting that CAFs are more activated phenotype. The CAFs produced larger amounts of IL-6, IL-8 and CCL2 than the NFs, which led to invasiveness of HuH7 in vitro. We found that Bone Morphogenetic Protein-4 (BMP4) is up-regulated in CAFs compared to NFs. The CAF phenotype and function were gained by BMP4 over-expression or recombinant BMP4 given to fibroblasts, all of which decreased with BMP4 knockdown. In tissues obtained from the patients, BMP4-positive cells are mainly observed in encapsulated fibrous lesions and HCC. Positive expression of BMP4 in HCC in resected tissues, not in fibroblasts, was associated with poorer postoperative overall survival in patients with HCC. Conclusion: Endogenous and exogenous BMP4 activate liver fibroblasts to gain capacity of secreting cytokines and enhancing invasiveness of cancer cells in the liver. BMP4 is one of the regulatory factors of CAFs functioning in the microenvironment of HCC.