Mutant BRAF induces DNA strand breaks, activates DNA damage response pathway, and up-regulates glucose transporter-1 in nontransformed epithelial cells.
Although the oncogenic functions of activating BRAF mutations have been clearly demonstrated in human cancer, their roles in nontransformed epithelial cells remain largely unclear. Investigating the cellular response to the expression of mutant BRAF in nontransformed epithelial cells is fundamental to the understanding of the roles of BRAF in cancer pathogenesis. In this study, we used two nontransformed cyst108 and RK3E epithelial cell lines as models in which to compare the phenotypes of cells expressing BRAF(WT) and BRAF(V600E). We found that transfection of the BRAF(V600E), but not the BRAF(WT), expression vector suppressed cellular proliferation and induced apoptosis in both cell types. BRAF(V600E) generated reactive oxygen species, induced DNA double-strand breaks, and caused subsequent DNA damage response as evidenced by an increased number of pCHK2 and γH2AX nuclear foci as well as the up-regulation of pCHK2, p53, and p21. Because BRAF and KRAS (alias Ki-ras) mutations have been correlated with GLUT1 up-regulation, which encodes glucose transporter-1, we demonstrated here that expression of BRAF(V600E), but not BRAF(WT), was sufficient to up-regulate GLUT1. Taken together, our findings provide new insights into mutant BRAF-induced oncogenic stress that is manifested by DNA damage and growth arrest by activating the pCHK2-p53-p21 pathway in nontransformed cells, while it also confers tumor-promoting phenotypes such as the up-regulation of GLUT1 that contributes to enhanced glucose metabolism that characterizes tumor cells.
Human Genetic Center, China Medical University Hospital, Taichung, Taiwan; School of Chinese Medicine, China Medical University, Taichung, Taiwan. Electronic address: jimsheu@mail.cmu.edu.tw.
Jim Jinn-Chyuan
JJ
Sheu
Departments of Pathology, Oncology, Gynecology and Obstetrics, Johns Hopkins Medical Institutions, Baltimore, Maryland.
Bin
B
Guan
Human Genetic Center, China Medical University Hospital, Taichung, Taiwan.
Fuu-Jen
FJ
Tsai
Human Genetic Center, China Medical University Hospital, Taichung, Taiwan.
Erin Yi-Ting
EY
Hsiao
Human Genetic Center, China Medical University Hospital, Taichung, Taiwan.
Chih-Mei
CM
Chen
Institute of Molecular Pathology and Immunology, The University of Porto, Porto, Portugal.
Raquel
R
Seruca
Departments of Pathology, Oncology, Gynecology and Obstetrics, Johns Hopkins Medical Institutions, Baltimore, Maryland.
Tian-Li
TL
Wang
Departments of Pathology, Oncology, Gynecology and Obstetrics, Johns Hopkins Medical Institutions, Baltimore, Maryland. Electronic address: ishih@jhmi.edu.
Ie-Ming
IM
Shih
D017209:Apoptosis; D049109:Cell Proliferation; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D053960:DNA Breaks; D004847:Epithelial Cells; D005260:Female; D051272:Glucose Transporter Type 1 / Q000235:genetics*; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D051272:Glucose Transporter Type 1; D016159:Tumor Suppressor Protein p53; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf