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Abstract

Objective: Activated Hepatic Stellate Cells (HSCs) promote Hepatocellular Carcinoma (HCC) cell growth and migration. Senescent and cancer-associated fibroblasts express various factors contributing to tumor progression, collectively called the Senescence-Associated Secretory Phenotype (SASP). Herein, we investigated the mechanism underlying the bile acid-mediated induction of HSC activation via SASP expression in HCC cells.

Methods: Serum samples from 296 patients with HCC were used to analyze IL-6 levels and survival. IL-6 mRNA levels were quantified using real-time PCR. A mouse xenograft model was used to confirm the role of the SASP. MTT and invasion assays were performed to evaluate the invasive ability of HCC cells (Huh-BAT, SNU-761, and Huh-SR) co-cultured with HSCs (LX-2 cells). Western blotting was performed to investigate the levels of survival signals.

Results: Bile acid increased the expression of mesenchymal markers and Myeloid cell leukemia-1 (Mcl-1) and Cyclooxygenase-2 (COX-2) proteins in HCC cells and HSCs. Inhibiting Mcl-1 induction or COX-2 activity decreased bile acid-mediated HCC invasion. Takeda G protein-coupled Receptor 5 (TGR5) activation-dependent Mcl-1 and COX-2 expression enhanced gene transcription. Bile acid significantly increased IL-6 levels and HCC cell invasion in HSC/HCC cell co-cultures compared to HCC cell monocultures. The IL-6+MoRAL score also showed significant discrimination in overall survival (Harrell’s c-index 0.765, p<0.0001) of HCC patients. A mouse xenograft model revealed that Mcl-1 short hairpin RNA transfection, not celecoxib treatment, significantly suppressed Huh-SR cell growth.

Conclusion: Bile acid-mediated activation of HSCs enhances the proliferation and invasion of HCC cells via the expression of SASP proteins, including IL-6. TGR-5-dependent Mcl-1 overexpression may be a key factor in hepatocarcinogenesis

Keywords: Hepatocellular carcinoma, Senescence-associated secretory phenotype, Invasion, Mcl-1