Cancer Susceptibility Candidate 9 (CASC9) Promotes Colorectal Cancer Carcinogenesis via mTOR-Dependent Autophagy and Epithelial-Mesenchymal Transition Pathways.

Md Zahirul Islam Khan,Helen Ka Wai Law

doi:10.3389/fmolb.2021.627022

Abstract

BackgroundColorectal cancer (CRC) is the third most common cancer worldwide. Many recent studies have demonstrated that different long non-coding RNAs (lncRNAs) are involved in the initiation, advancement, and metastasis of many cancers including CRC. Cancer susceptibility candidate 9 (CASC9) is an lncRNA that has been reported in many cancers, but its role in CRC is poorly understood. In this study, we aimed to examine the expression of CASC9 in CRC cell lines and to determine the mechanism of action of CASC9 in CRC carcinogenesis.MethodsThe expression of CASC9 in CRC tissues was compared with normal samples from publicly available datasets in The Cancer Genome Atlas (TCGA) and The Encyclopedia of RNA Interactomes (ENCORI). CASC9 expression was further verified in four CRC cell lines (DLD1, HT-29, SW480, and HCT-116) and normal colorectal cell line (CCD-112CoN) by real-time quantitative polymerase chain reaction (RT-qPCR). After gene silencing in HCT-116 and SW480, Cell Counting Kit-8 assay, clonogenic assay, and wound healing assay were performed to evaluate cell proliferation, viability, and migration index of cells. Western blotting was used to explore the key pathways involved.ResultsCASC9 was significantly upregulated as analyzed from both public datasets TCGA and ENCORI where its overexpression was associated with poor survival of CRC patients. Similarly, CASC9 was significantly overexpressed in the CRC cell lines compared with normal cells studied. The silencing of CASC9 in HCT-116 and SW480 attenuated cell proliferation and migration significantly. Furthermore, pathways investigations showed that silencing of CASC9 significantly induced autophagy, promoted AMP-activated protein kinase (AMPK) phosphorylation, inhibited mTOR and AKT signaling pathways, and altered epithelial–mesenchymal transition (EMT) marker protein expression.ConclusionWe demonstrated that silencing of CASC9 contributes to the reduced CRC cell proliferation and migration by regulating autophagy and AKT/mTOR/EMT signaling. Therefore, CASC9 plays an important role in carcinogenesis, and its expression may act as a prognostic biomarker and a potential therapeutic target of CRC management.

Highlights

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy worldwide (Bray et al, 2018; Rawla et al, 2019)
Our present study aimed to explore the expression of Cancer susceptibility candidate 9 (CASC9) in CRC cell lines and to determine the roles of CASC9 in mTOR-dependent autophagy and epithelial– mesenchymal transition (EMT), which are associated with CRC progression
We plotted the survival curve of CRC patients according to their CASC9 expression level, number of transcript per million (TPM), and hazard ratio (HR)% using The Cancer Genome Atlas (TCGA)-colon adenocarcinoma (COAD) dataset in Gene Expression Profiling Interactive Analysis bioinformatics tool2

Summary

Introduction

Colorectal cancer (CRC) is the third most commonly diagnosed malignancy worldwide (Bray et al, 2018; Rawla et al, 2019). Long non-coding RNAs (lncRNAs) are fragments of RNA that lack protein coding transcript. They are members of noncoding RNAs (ncRNAs). In recent years, accumulating evidence suggested that lncRNAs sometimes behaved like regulatory molecules to control gene expressions. They are involved in the signaling pathways responsible for cell growth, development, and metabolic processes (Lin and He, 2017; Sparber et al, 2019). Many recent studies have demonstrated that different long non-coding RNAs (lncRNAs) are involved in the initiation, advancement, and metastasis of many cancers including CRC. We aimed to examine the expression of CASC9 in CRC cell lines and to determine the mechanism of action of CASC9 in CRC carcinogenesis

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