Aquaporin-9, Mediated by IGF2, Suppresses Liver Cancer Stem Cell Properties via Augmenting ROS/β-Catenin/FOXO3a Signaling.

Xi Zheng,Keqi Yu,Zhechuan Mei,Shasha Shi,Yanzhi Qian,Chuanfei Li,Hongyu Chen

doi:10.1158/1541-7786.mcr-19-1180

Abstract

Liver cancer stem cells (LCSCs) play a critical role in hepatocellular carcinoma (HCC) by virtue of their aggressive behavior and association with poor prognoses. Aquaporin-9 (AQP9) is a transmembrane protein that transports water and reportedly transports H2O2. Recent studies have shown that AQP9 expression has a negative effect on HCC cell invasion by inhibiting the epithelial-to-mesenchymal transition. However, the role of AQP9 in LCSCs remains obscure. We performed spheroid formation assay and flow cytometric analysis to investigate LCSCs stemness. CD133+ and CD133- cells were isolated by flow cytometry. Real-time quantitative PCR (qRT-PCR), Western blot analysis, and immunofluorescence assay were used to estimate gene expression. The protein association of β-catenin with TCF4 and the interaction of β-catenin with FOXO3a were detected by immunoprecipitation (IP). Here, we found that AQP9 was preferentially decreased in LCSCs. Upregulated AQP9 significantly suppressed LCSCs stemness. In contrast, the inhibition of AQP9 had the opposite effect. Mechanistically, AQP9 was shown to be downregulated by insulin-like growth factor 2 (IGF2), which was widely reported to contribute to maintaining CSCs stemness. Furthermore, AQP9 overexpression was found to result in reactive oxygen species (ROS) accumulation, which inhibited β-catenin activity by attenuating the interaction of β-catenin with TCF4 while concurrently enhancing the association of β-catenin with FOXO3a, ultimately inhibiting LCSCs stemness. Our study implies that stimulation of the AQP9 signaling axis may be a novel preventive and/or therapeutic approach for eliminating LCSCs. IMPLICATIONS: Our findings demonstrate that AQP9 signaling axis may be a novel preventive and/or therapeutic approach for eliminating LCSCs.

Highlights

Cancer stem cells (CSC) are a subgroup of cells capable of exclusive tumorigenicity, unlimited self-renewal potential, and drug resistance [1]
The results from hepatocellular carcinoma (HCC) MHCC97H cells showed that the mRNA levels of well-characterized Liver cancer stem cells (LCSCs)-specific markers CD133, CD90, Oct4, Nanog, SOX2, and Bmi-1 increased in the spheroids compared with the monolayer-attached cells (Fig. 1A), and dramatically lower expression of AQP9 mRNA was observed in the spheroids (Fig. 1B)
To confirm that the positivity rate of CD133 was more than 85%, the sorted CD133þ LCSCs were detected by flow cytometry analysis (Fig. 1D)

Summary

Introduction

Cancer stem cells (CSC) are a subgroup of cells capable of exclusive tumorigenicity, unlimited self-renewal potential, and drug resistance [1]. Several studies have confirmed that liver cancer stem cells (LCSC) in hepatocellular carcinoma (HCC) are the major cause of recurrence and metastasis and that standard therapies fail to eliminate the resistant LCSCs [2, 3]. Elucidation of the underlying molecular mechanisms that regulate LCSCs is pivotal for HCC diagnosis and future treatments [4]. Even though LCSCs can be identified separately, they cannot be effectively eliminated because of the ambiguous regulatory mechanism of LCSCs. Recent studies have shown that CSCs are able to survive by maintaining low levels of ROS because they possess a more effective ROS-scavenging system compared with that of non-

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