Modulation of ATP8B1 Gene Expression in Colorectal Cancer Cells Suggest its Role as a Tumor Suppressor

Abstract

Aim: The study aims to understand the role of tumor suppressor genes in colorectal cancer initiation and progression. <P> Background: Sporadic colorectal cancer (CRC) develops through distinct molecular events. Loss of the 18q chromosome is a conspicuous event in the progression of adenoma to carcinoma. There is limited information regarding the molecular effectors of this event. Earlier, we had reported ATP8B1 as a novel gene associated with CRC. ATP8B1 belongs to the family of P-type ATPases (P4 ATPase) that primarily function to facilitate the translocation of phospholipids. <P> Objective: In this study, we attempt to implicate the ATP8B1 gene located on chromosome 18q as a tumor suppressor gene. <P> Methods: Cells culture, Patient data analysis, Generation of stable ATP8B1 overexpressing SW480 cell line, Preparation of viral particles, Cell Transduction, Generation of stable ATP8B1 knockdown HT29 cell line with CRISPR/Cas9, Generation of stable ATP8B1 knockdown HT29 cell line with shRNA, Quantification of ATP8B1 gene expression, Real-time cell proliferation and migration assays, Cell proliferation assay, Cell migration assay, Protein isolation and western blotting, Endpoint cell viability assay, Uptake and efflux of sphingolipid, Statistical and computational analyses. <P> Results: We studied indigenous patient data and confirmed the reduced expression of ATP8B1 in tumor samples. CRC cell lines were engineered with reduced and enhanced levels of ATP8B1, which provided a tool to study its role in cancer progression. Forced reduction of ATP8B1 expression either by CRISPR/Cas9 or shRNA was associated with increased growth and proliferation of CRC cell line - HT29. In contrast, overexpression of ATP8B1 resulted in reduced growth and proliferation of SW480 cell lines. We generated a network of genes that are downstream of ATP8B1. Further, we provide the predicted effect of modulation of ATP8B1 levels on this network and the possible effect on fatty acid metabolism-related genes. <P> Conclusion: Tumor suppressor gene (ATP8B1) located on chromosome 18q could be responsible in the progression of colorectal cancer. Knocking down of this gene causes an increased rate of cell proliferation and reduced cell death, suggesting its role as a tumor suppressor. Increasing the expression of this gene in colorectal cancer cells slowed down their growth and increased cell death. These evidences suggest the role of ATP8B1 as a tumor suppressor gene.

Pathological image profiling identifies onco‐microbial, tumor immune microenvironment, and prognostic subtypes of colorectal cancer

“…Several studies have reported that Enterococcus produces hydroxyl radicals and extracellular superoxide, leading to DNA fragmentation, promotion of chromosomal instability, and increased inflammation [27, 28]. Some studies have investigated the role of host gene regulation in patients with CRC [29, 30]. We found that the FABP4 gene expression was upregulated in HSS2.…”

ATP8B1: A prognostic prostate cancer biomarker identified via genetic analysis

“…or short hairpin RNA causes increased cell growth, whereas its overexpression results in reduced cell proliferation. 49 Following pathway analysis, it was discovered that under-expression of ATP8B1 was associated with the dysregulation of multiple biological processes with oncogenic potential, including phospholipid transport, phospholipid metabolism, cell-cell adhesion, and epithelial-mesenchymal transition. 48…”

“…Gene expression studies have shown that ATP8B1 is significantly downregulated in colorectal cancer 47 and that high expression of this gene is a biomarker of favorable patient prognosis 48 . Furthermore, knocking down of ATP8B1 expression in colorectal cancer cells by either clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 or short hairpin RNA causes increased cell growth, whereas its overexpression results in reduced cell proliferation 49 . Following pathway analysis, it was discovered that under‐expression of ATP8B1 was associated with the dysregulation of multiple biological processes with oncogenic potential, including phospholipid transport, phospholipid metabolism, cell‐cell adhesion, and epithelial‐mesenchymal transition 48 .…”

Type IV P-Type ATPases: Recent Updates in Cancer Development, Progression, and Treatment

“…Its prognostic value was also validated with the data from the Gene Expression Omnibus (GEO) [46]. However, in another study, ATP8B1 was suggested as a tumor suppressor for CRC since the forced reduction in ATP8B1 expression either by CRISPR/Cas9 or shRNA was associated with increased growth and proliferation of the CRC cell line HT29, while an overexpression of ATP8B1 resulted in reduced growth and proliferation of the SW480 CRC cell line [47]. Gene expression for another P4-ATPase, ATP11A, was assessed in 7 colorectal cancer cell lines and 95 paired cases of colorectal cancer and non-cancerous regions.…”