Retinoic Acid-Induced 2 (RAI2) Is a Novel Antagonist of Wnt/β-Catenin Signaling Pathway and Potential Biomarker of Chemosensitivity in Colorectal Cancer.

Weitao Zhang,Weitao Zhang,Weitao Zhang,Weitao Zhang,Weiwei Zhang,Weiwei Zhang,Shurong Zhang,Shurong Zhang,Niansong Qian,Niansong Qian,Bin Yan,Bin Yan,Zhi Cui,Zhi Cui,Quanli Han,Xindan Kang,Xindan Kang,Wenji Yan,Hongbin Zhu,Decong Sun,Guanghai Dai,Guanghai Dai,Lu Kong

doi:10.3389/fonc.2022.805290

Abstract

ObjectiveAberrant activation of Wnt/β-catenin signaling contributes to the maintenance of cancer stem cells and chemoresistance in colorectal cancer (CRC). Retinoic acid-induced 2 (RAI2) was proved to be a tumor suppressor in CRC in our previous report. In this study, the role of RAI2 in Wnt/β-catenin signaling was further investigated.MethodsAs a transcriptional co-regulator, C-terminal Binding Protein 2 (CtBP2) was reported to be involved in Wnt signaling in multiple and complex ways. The correlation of RAI2 and CtBP2 in CRC was analyzed by TCGA dataset, and the interaction between RAI2 and CtBP2 was explored by co-immunoprecipitation (Co-IP) in CRC cells. The effect of RAI2 on the activity of Wnt signaling and the location of β-catenin was detected by Dual-Luciferase reporter assay and Immunofluorescence respectively. Western blotting analysis was performed to detect the expression of target genes involved in Wnt signaling. Sphere formation assay was employed to detect the effect of RAI2 on stem cell like properties. Cell viability assay was used to detect the chemosensitivity of cells before and after transfection of RAI2.ResultsThe interaction between RAI2 and CtBP2 was confirmed by Co-IP in CRC cells. Besides, the negative correlation of RAI2 and CtBP2 in CRC was found by analyzing the TCGA dataset. Re-expression of RAI2 in human colon cancer cells (HCT116 and LoVo) suppressed the fluorescent activity of Wnt signaling, increased the phosphorylation and inhibited nuclear translocation of β-catenin, with down-regulation of target genes like c-Myc, CyclinD1, ASCL2, and LGR5. In contrast, the mutated RAI2, which can’t interact with CtBP2, has no above effects. We observed low expression of RAI2 in 33.89% (101/298) of CRC patients, which was significantly associated with reduced phosphorylation of β-catenin (r=0.8866, P<0.0001), poor 5-year relapse-free survival (RFS) (P = 0.0029) and overall survival (OS) (P = 0.0102). Restoration of RAI2 in HCT116 and LoVo cells inhibited stem cell-like properties of CRC cells and increased chemosensitivity of these cells to oxaliplatin and fluorouracil.ConclusionLow expression of RAI2 can serve as an independent poor prognostic marker. RAI2 inhibits Wnt signaling by interacting with or down-regulating CtBP2, resulting in repression of stem cell-like properties and increased chemosensitivity of CRC cells.

Highlights

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with more than 1.9 million new cases and almost 935,000 deaths in 2020 [1]. 5-fluorouracil has always been the basis of all chemotherapy schedules, used alone or combined with other agents
By searching The Cancer Genome Atlas (TCGA) databases and analyzing RNA sequencing (RNASeq) data in 434 cases of CRC samples, we found that the expression of CtBP2 was negatively correlated with the expression of RAI2 (p
We found that RAI2 suppressed the expression of achaete-scute complex homolog 2 (ASCL2) and Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) by Western blot in CRC cells, and we found a negative correlation between RAI2 and ASCL2/LGR5 in 298 cases of cancer tissues from CRC patients by IHC detection (Figure 4A)

Summary

Introduction

Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide, with more than 1.9 million new cases and almost 935,000 deaths in 2020 [1]. 5-fluorouracil has always been the basis of all chemotherapy schedules, used alone or combined with other agents. New strategies to improve chemotherapy sensitivity may help improve the treatment and prognosis of colorectal cancer. 90% of the colorectal tumors have a mutation in one of the key regulatory factors of the Wnt/b-catenin signaling pathway, resulting in aberrant activation of this pathway, and up to 80% of tumors exhibit nuclear accumulation of b-catenin [6–8]. Transcriptional co-regulation by CtBPs has been found to play an important role in several diseases, including human cancer [11–14]. CtBPs intersects with Wnt signaling in multiple and complex ways as revealed by work in both model organisms and human cancer cells and tumors [15, 16]. In mutant APC setting, CtBPs promotes oligomerization of truncated APC by binding to 15 amino acid-repeats [17], facilitating the release of b-catenin into the nucleus and activation of downstream oncogenic b-catenin transcriptional targets, such as Cyclin D1. It was proved that CtBP2 reduced chemosensitivity of non-small cell lung cancer cells to cisdiamminedichloroplatinum (CDDP) via promoting the Wnt/bcatenin pathway [20]

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Conclusion