Title:Modulation of ATP8B1 Gene Expression in Colorectal Cancer Cells
Suggest its Role as a Tumor Suppressor
Volume: 22
Issue: 7
Author(s): Saleh Althenayyan, Amal AlGhamdi, Mohammed H. AlMuhanna, Esra Hawsa, Dalal Aldeghaither, Jahangir Iqbal, Sameer Mohammad and Mohammad A. Aziz*
Affiliation:
- King Abdullah International
Medical Research Center (KAIMRC), King Saud bin Abdulaziz University for Health Sciences, King Abdulaziz Medical
City Hospital, Ministry of National Guard Health Affairs, Riyadh, 31982, Saudi Arabia
Keywords:
Colorectal cancer, flippase, ion transporter, tumor suppressor gene, chromosome 18q, lipid transport.
Abstract:
Aim: The study aims to understand the role of tumor suppressor genes in colorectal cancer
initiation and progression.
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.
Objective: In this study, we attempt to implicate the ATP8B1 gene located on chromosome 18q as
a tumor suppressor gene.
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.
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.
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.
