Conjugated bile acids promote esophageal adenocarcinoma cell invasive growth via activation of sphingosine 1‐phosphate receptor 2 and sphingosine kinase 2
Authors: Liu Runping , Li Xiaojiaoyang , Li Yunzhou , Hylemon Phillip B. , Zhou Huiping 
Published: November 2023

The esophageal adenocarcinoma (EA) is a highly lethal malignancy with an extremely low survival rate. Effective therapy for EA is currently unavailable due to the limited understanding of its pathophysiology. Barrett's esophagus (BE) is a premalignant condition that is strongly associated with an increased risk for EA. It has been reported that BE and EA patients reflux high concentrations (>150 μM) of bile acids into the esophagus as compared to normal individuals (<20 μM). Bile acids are now recognized as important regulatory molecules, which activate different cell signaling pathways based on conjugation state and hydrophobicity. Our previous studies reported that conjugated bile acids (CBA), taurocholic acid (TCA) and glycocholic acid (GCA), but not unconjugated bile acids are able to activate growth promoting and tumorigenic cell signaling pathways by activating the sphingosine‐1‐phosphate receptor 2 (S1PR2) in cholangiocarcinoma cells. S1PR2 also has been linked to activation of sphingosine kinase 2 (sphK2). However, the role of S1PR2/SphK2 in bile acid‐mediated proliferation and migration of EA cells is not clear and is the focus of this study.

Methods

Studies were conducted in the human normal esophagus cell line Het‐1A, BE cell line CP‐A and EA cell lines, OE‐19 and OE‐33. The expression of individual S1PR and SphK was determined by real‐time RT‐PCR and Western blot analysis and confirmed by DNA sequencing. The chemical antagonist of S1PR2, JTE‐013, Sphk2 inhibitor, K145, and gene‐specific shRNAs were used to inhibit S1PR2/SphK2 activation and expression. Cell proliferation was determined using a CCK‐8 kit. The Wound‐Healing assay and invasion assay were used to examine cell migration and invasion. Anchorage independent growth was examined using soft agar colony formation assay. TCGA and GEO databases were used to investigate the clinical relevance of S1PR2/SphK2 in EA disease progression.

Results

The expression levels of S1PR2 and SphK2 in aggressive EA cell line, OE‐33, were significantly higher than those in normal (Het‐1A) or BE (CP‐A) or the lower grade EA cell line (OE‐19). In addition, S1PR2 was the predominant S1PR in OE‐33 cells. TCA, GCA and S1P significantly induced proliferation, migration, invasion and anchorage‐independent growth of EA cells, which were inhibited by JTE‐013 and K145 as well as gene specific shRNAs targeting S1PR2 and SphK2. Compared to OE‐19, OE‐33 had a stronger response to TCA and S1P. This is consistent with clinical data as EA patients with high SphK2 expression had poor prognosis and high grade dysplasia.

Discussion/Conclusion

High bile acid level is associated with EA. S1PR1, S1PR2, S1PR3, SphK1 and SphK2 are all expressed in human EA cancer cells. However, S1PR2 and SphK2 expression levels are much higher than those of S1PR1, S1PR3 and SphK1. In this study, by using chemical antagonists and gene‐specific shRNAs, we were able to show that S1PR2 and SphK2 play critical roles in conjugated bile acid/S1P‐mediated invasive growth of EA cells. The results in this study suggest that targeting S1PR2/SphK2 may represent a novel therapy for EA.