Cholangiocarcinoma (CCA) is a rare, but highly malignant primary hepatobiliary cancer with a very poor prognosis and limited treatment options. Our recent studies reported that conjugated bile acids (CBAs) promote the invasive growth of CCA via activation of sphingosine 1-phosphate receptor 2 (S1PR2). Cyclooxygenase-2 (COX-2)-derived prostaglandin E₂ (PGE₂) is the most abundant prostaglandin in various human malignancies including CCA. Previous studies have indicated that COX-2 was highly expressed in CCA tissues, and the survival rate of CCA patients was negatively associated with high COX-2 expression levels. It has also been reported that CBAs induce COX-2 expression, whereas free bile acids inhibit COX-2 expression in CCA mouse models. However, the underlying cellular mechanisms and connection between S1PR2 and COX-2 expression in CCA cells have still not been fully elucidated. In the current study, we examined the role of S1PR2 in conjugated bile acid (taurocholate, (TCA))-induced COX-2 expression in a human HuCCT1 CCA cell line and further identified the potential underlying cellular mechanisms. The results indicated that TCA-induced invasive growth of human CCA cells was correlated with S1PR2-medated up-regulation of COX-2 expression and PGE₂ production. Inhibition of S1PR2 activation with chemical antagonist (JTE-013) or down-regulation of S1PR2 expression with gene-specific shRNA not only reduced COX-2 expression, but also inhibited TCA-induced activation of EGFR and the ERK1/2/Akt-NF-κB signaling cascade. In conclusion, S1PR2 plays a critical role in TCA-induced COX-2 expression and CCA growth and may represent a novel therapeutic target for CCA.

Osteopontin (OPN) is a multifunctional cytokine that can impact cancer progression. Therefore, it is crucial to determine the key factors involved in the biological role of OPN for the development of treatment. Here, we investigated that OPN promoted hepatocellular carcinoma (HCC) cell proliferation and migration by increasing Reactive oxygen species (ROS) production and disclosed the underlying mechanism. Knockdown of OPN suppressed ROS production in vitro and in vivo, whereas treatment with human recombinant OPN produced the opposite effect. N-Acetyl-L-cysteine (NAC, ROS scavenger) partially blocked HCC cell proliferation and migration induced by OPN. Mechanistically, OPN induced ROS production in HCC cells by upregulating the expression of NADPH oxidase 1 (NOX1). NOX1 knockdown in HCC cells partially abrogated the cell proliferation and migration induced by OPN. Moreover, inhibition of JAK2/STAT3 phosphorylation effectively decreased the transcription of NOX1, upregulated by OPN. In addition, NOX1 overexpression increased JAK2 and STAT3 phosphorylation by increasing ROS production, creating a positive feedback loop for stimulating JAK2/STAT3 signaling induced by OPN. This study for the first time demonstrated that HCC cells utilized OPN to generate ROS for tumor progression, and disruption of OPN/NOX1 axis might be a promising therapeutic strategy for HCC.