Oncogenic Mutation BRAF V600E Changes Phenotypic Behavior of THLE-2 Liver Cells through Alteration of Gene Expression.

Magdalena Śmiech,Mariusz Pierzchała,Hiroaki Taniguchi,Piotr Poznański,Christopher Wardell,Paweł Leszczyński

doi:10.3390/ijms23031548

Abstract

The accumulation of mutations in cancer driver genes, such as tumor suppressors or proto-oncogenes, affects cellular homeostasis. Disturbances in the mechanism controlling proliferation cause significant augmentation of cell growth and division due to the loss of sensitivity to the regulatory signals. Nowadays, an increasing number of cases of liver cancer are observed worldwide. Data provided by the International Cancer Genome Consortium (ICGC) have indicated many alterations within gene sequences, whose roles in tumor development are not well understood. A comprehensive analysis of liver cancer (virus-associated hepatocellular carcinoma) samples has identified new and rare mutations in B-Raf proto-oncogene (BRAF) in Japanese HCC patients, as well as BRAF V600E mutations in French HCC patients. However, their function in liver cancer has never been investigated. Here, using functional analysis and next generation sequencing, we demonstrate the tumorigenic effect of BRAF V600E on hepatocytes (THLE-2 cell line). Moreover, we identified genes such as BMP6, CXCL11, IL1B, TBX21, RSAD2, MMP10, and SERPIND1, which are possibly regulated by the BRAF V600E-mediated, mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) signaling pathway. Through several functional assays, we demonstrate that BRAF L537M, D594A, and E648G mutations alone are not pathogenic in liver cancer. The investigation of genome mutations and the determination of their impact on cellular processes and functions is crucial to unraveling the molecular mechanisms of liver cancer development.

Highlights

Nowadays, an increasing number of chronic liver diseases and cancer cases is observed and has a high mortality rate, amounting to 2 million precocious deaths annually worldwide [1]
The expression of B-Raf proto-oncogene (BRAF) (FLAG), ERK, and P-ERK proteins were measured in THLE2 cells overexpressed with p3XFLAG-CMV empty plasmid (EM) or plasmid containing
ERK), FLAG (BRAF), and ERK proteins in THLE-2 cells overexpressed with p3XFLAG-CMV empty plasmid (EM), or plasmid containing BRAF V600E-transfected THLE-2 cells (V600E)-overexpressed THLE-2 cells pared with control (BRAF WT-transfected THLE-2 cells (WT)) (WT) and BRAF mutations (V600E, E648G, L537M, plasmid (EM), or plasmid containing BRAF WT (WT) and BRAF mutations (V600E, E648G, L537M, D594A)

Summary

Introduction

An increasing number of chronic liver diseases and cancer cases is observed and has a high mortality rate, amounting to 2 million precocious deaths annually worldwide [1]. Several main risk factors predispose patients to developing liver cancer, including the following: long-term viral infection [1], alcohol abuse [5], exposure to toxins, such as aflatoxin [6,7], and metabolic disorders, such as obesity, diabetes, and fatty liver disease [8,9]. These elements may lead to the accumulation of mutations in cells and to malignant hepatocyte transformation [10]

Objectives

Methods

Results

Conclusion