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

Abstract

Liver cancer stem cells (LCSCs) play a critical role in hepatocellular carcinoma (HCC) by virtue of their aggressive behaviour and association with poor prognoses. Aquaporin-9 (AQP9) is a transmembrane protein that transports water and reportedly transports H 2 O 2 . 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 CD133cells were isolated by flow cytometry. Real-time quantitative PCR (RT-qPCR), western blot 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. Further, 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 on August 31, 2021.

The Role of IGF/IGF-1R Signaling in Hepatocellular Carcinomas: Stemness-Related Properties and Drug Resistance

“…Overexpression/suppression of lncRNA H19 induced/reduced, respectively, the expression of stemness markers including Lin28 and Notch1 [173]. Pre-treatment with IGF-2 for 48 h upregulated mRNA and protein expression of CD133 and Bmi-1 as well as sphere formation in Huh7 cells [174]. In addition, studies have demonstrated that the expression of IGF-1R positively correlates with expression of both OCT4 and NANOG [133,134,138] and CD133 [132] in human HCC tissues and HCC cell lines.…”

Wnt/β-Catenin Signaling as a Driver of Hepatocellular Carcinoma Progression: An Emphasis on Molecular Pathways

“…In this way, AQP9 induces ROS overgeneration to suppress β-catenin signaling, leading to a significant decrease in CSC features. 105 Besides, activation of β-catenin signaling can mediate transformation of hepatic progenitor cells to CSCs. 106 Hence, molecular pathways are divided into two major kinds, including tumor-suppressor and tumor-promoting factors that regulate β-catenin signaling in affecting CSC features of HCC cells.…”

Low expression of AQP9 and its value in hepatocellular carcinoma

“…AQP9 acts as a tumor suppressor in HCC invasion via the regulation of HIF-1α expression in the tumor hypoxic microenvironment. 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 liver cancer stem cells (LCSCs) (12,(14)(15)(16)(17); However, the prognostic value of AQP9 and the specific mechanism underlying its role in HCC remain to be further elucidated. Therefore, in this study, we conducted a bioinformatics analysis of AQP9 in liver cancer.…”

“…Liver steatosis is characterized by ectopic accumulation of fat and altered glycerol uptake, and interestingly, AQP9 are dysregulated in liver steatosis mice (11)(12)(13). In humans, there is evidence to suggest that AQP9 inhibits the progression of HCC via the Wnt/β-catenin pathway (14), as well as epithelialto-mesenchymal transition (15) and the ROS-HIF-1α signaling pathway (16,17). However, the value of AQP9 in the diagnosis and prognosis of HCC as well as the changes in genes and pathways related to AQP9, immune infiltration levels and the reason for the decrease of AQP9 expression in HCC remain to be elucidated.…”