Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastrointest Oncol. May 15, 2020; 12(5): 514-525
Published online May 15, 2020. doi: 10.4251/wjgo.v12.i5.514
Free fatty acids receptors 2 and 3 control cell proliferation by regulating cellular glucose uptake
Saeed Al Mahri, Amal Al Ghamdi, Maaged Akiel, Monira Al Aujan, Sameer Mohammad, Mohammad Azhar Aziz
Saeed Al Mahri, Amal Al Ghamdi, Maaged Akiel, Monira Al Aujan, Sameer Mohammad, Mohammad Azhar Aziz, King Abdullah International Medical Research Center, Riyadh 11426, Saudi Arabia
Saeed Al Mahri, Amal Al Ghamdi, Maaged Akiel, Monira Al Aujan, Sameer Mohammad, Mohammad Azhar Aziz, King Saud bin Abdulaziz University for Health Sciences, Riyadh 11426, Saudi Arabia
Saeed Al Mahri, Amal Al Ghamdi, Monira Al Aujan, Sameer Mohammad, Mohammad Azhar Aziz, Ministry of the National Guard - Health Affairs, Riyadh 11426, Saudi Arabia
Saeed Al Mahri, Sameer Mohammad, Department of Experimental Medicine, King Abdullah International Medical Research Center, Riyadh 11426, Saudi Arabia
Amal Al Ghamdi, Mohammad Azhar Aziz, Colorectal Cancer Research Program, King Abdullah International Medical Research Center, Riyadh 11426, Saudi Arabia
Maaged Akiel, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud bin Abdulaziz University for Health Sciences, Riyadh 11426, Saudi Arabia
Maaged Akiel, Monira Al Aujan, Department of Medical Genomics, King Abdullah International Medical Research Center, Riyadh 11426, Saudi Arabia
Maaged Akiel, Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23284, United States
Author contributions: Mohammad S and Aziz MA conceived the idea and designed research; Al Mahri S and Akiel M generated knockdown cell lines; Al Mahri S and Al Ghamdi A performed qRT-PCR assays; Al Mahri S and Mohammad S performed glucose uptake, cAMP signalling and immunofluorescence studies; Al Ghamdi A performed western blots and cell proliferation assay; Akiel M designed primers; Al Aujan M Assisted in maintaining knockdown cells; Aziz MA, Al Mahri S, Akiel M and Mohammad S analyzed data; Aziz MA, Akiel M and Mohammad S prepared final figures, wrote and edited the paper; Aziz MA carried out microarray and qRT-PCR analyses; All authors reviewed and endorsed all contents of the manuscript.
Institutional review board statement: This study was approved by Institutional Research Board of King Abdullah International Medical Research Center through protocol number RC15/153.
Conflict-of-interest statement: The authors declare that they have no conflict of interest.
Data sharing statement: This patient microarray dataset is available on Gene Expression Omnibus, National Center for Biotechnology Information, National Institute of Health, United States (Accession: GSE 50421).
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: Mohammad Azhar Aziz, BSc, MSc, PhD, Research Scientist, Colorectal Cancer Research Program, King Abdullah International Medical Research Center, P.O. Box 3660, Riyadh 11426, Saudi Arabia. azizmo@ngha.med.sa
Received: December 17, 2019
Peer-review started: December 17, 2019
First decision: February 20, 2020
Revised: March 18, 2020
Accepted: March 28, 2020
Article in press: March 28, 2020
Published online: May 15, 2020
Abstract
BACKGROUND

Colorectal cancer (CRC) is a worldwide problem, which has been associated with changes in diet and lifestyle pattern. As a result of colonic fermentation of dietary fibres, short chain free fatty acids are generated which activate free fatty acid receptors (FFAR) 2 and 3. FFAR2 and FFAR3 genes are abundantly expressed in colonic epithelium and play an important role in the metabolic homeostasis of colonic epithelial cells. Earlier studies point to the involvement of FFAR2 in colorectal carcinogenesis.

AIM

To understand the role of short chain FFARs in CRC.

METHODS

Transcriptome analysis console software was used to analyse microarray data from CRC patients and cell lines. We employed short-hairpin RNA mediated down regulation of FFAR2 and FFAR3 genes, which was validated using quantitative real time polymerase chain reaction. Assays for glucose uptake and cyclic adenosine monophosphate (cAMP) generation was done along with immunofluorescence studies to study the effects of FFAR2/FFAR3 knockdown. For measuring cell proliferation, we employed real time electrical impedance-based assay available from xCELLigence.

RESULTS

Microarray data analysis of CRC patient samples showed a significant down regulation of FFAR2 gene expression. This prompted us to study the FFAR2 in CRC. Since, FFAR3 shares significant structural and functional homology with FFAR2, we knocked down both these receptors in CRC cell line HCT 116. These modified cell lines exhibited higher proliferation rate and were found to have increased glucose uptake as well as increased level of glucose transporter 1. Since, FFAR2 and FFAR3 signal through G protein subunit (Gαi), knockdown of these receptors was associated with increased cAMP. Inhibition of protein kinase A (PKA) did not alter the growth and proliferation of these cells indicating a mechanism independent of cAMP/PKA pathway.

CONCLUSION

Our results suggest role of FFAR2/FFAR3 genes in increased proliferation of colon cancer cells via enhanced glucose uptake and exclude the role of PKA mediated cAMP signalling. Alternate pathways could be involved that would ultimately result in increased cell proliferation as a result of down regulated FFAR2/FFAR3 genes. This study paves the way to understand the mechanism of action of short chain FFARs in CRC.

Keywords: Cell proliferation, Glucose transporter 1, Colorectal cancer, Free fatty acids receptor 2, Free fatty acids receptor 3, Glucose uptake

Core tip: Free fatty acid receptors (FFAR) have been reported to be associated with colorectal cancer (CRC). In this report, we studied short chain FFAR2 and FFAR3 and have provided preliminary evidence about the possible involvement of increased glucose uptake in FFAR2 and FFAR3 knockdown clones of CRC cell line. We generated double knock down for FFAR2 and FFAR3 genes in a CRC cell line (HCT116) and studied possible mechanisms of increased cell proliferation in these cells.