Mutant BRAF Induces DNA Strand Breaks, Activates DNA Damage Response Pathway, and Up-Regulates Glucose Transporter-1 in Nontransformed Epithelial Cells

Human Genetic Center, China Medical University Hospital, Taichung, Taiwan.
American Journal Of Pathology (Impact Factor: 4.59). 03/2012; 180(3):1179-88. DOI: 10.1016/j.ajpath.2011.11.026
Source: PubMed


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.

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    • "Second, while the formal criteria for a radiographic partial response (by RECIST) were not met, achieving a transient response in such an aggressive version of LCH is striking and we find the metabolic response at least noteworthy (Figure 4). The similarities of our data and those from xenograft models are impressive[26] and indicate a metabolic function of mutant-BRAF in histiocytic neoplasia[26-31]. We interpret the stable radiographic disease as an apparent lack of ‘oncogenic shock’[32, 33]. "
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    ABSTRACT: For a growing number of tumors the BRAF V600E mutation carries therapeutic relevance. In histiocytic proliferations the distribution of BRAF mutations and their relevance has not been clarified. Here we present a retrospective genotyping study and a prospective observational study of a patient treated with a BRAF inhibitor. Genotyping of 69 histiocytic lesions revealed that 23/48 Langerhans cell lesions were BRAF-V600E-mutant whereas all non-Langerhans cell lesions (including dendritic cell sarcoma, juvenile xanthogranuloma, Rosai-Dorfman disease, and granular cell tumor) were wild-type. A metareview of 29 publications showed an overall mutation frequency of 48.5%; and with N=653 samples, this frequency is well defined. The BRAF mutation status cannot be predicted based on clinical parameters and outcome analysis showed no difference. Genotyping identified a 45 year-old woman with an aggressive and treatment-refractory, ultrastructurally confirmed systemic BRAF-mutant LCH. Prior treatments included glucocorticoid/vinblastine and cladribine-monotherapy. Treatment with vemurafenib over 3 months resulted in a dramatic metabolic response by FDG-PET and stable radiographic disease; the patient experienced progression after 6 months. In conclusion, BRAF mutations in histiocytic proliferations are restricted to lesions of the Langerhans-cell type. While for most LCH-patients efficient therapies are available, patients with BRAF mutations may benefit from the BRAF inhibitor vemurafenib.
    Oncotarget 06/2014; 5(12). DOI:10.18632/oncotarget.2061 · 6.36 Impact Factor
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    • "B- Raf V600E activates the downstream MEK/ERK signaling pathway independently of Ras-GTP and its expression is required to maintain the proliferative and oncogenic characteristics of B-Raf V600E -expressing human tumor cell lines [14- 16]. Recent evidence suggests that mutant B-Raf also causes chromosome instability via phosphorylation of Mps1; up-regulates GLUT-1 which enhances glucose metabolism in tumor cells; and causes DNA strand breakage and damage [17] [18]. Mutated B-Raf V600E is also no longer repressed by SPRY2-an inhibitor of MAPK signaling in epithelial and fibroblast cell lines [19] which only binds to wild-type B-Raf to initiate the inhibition of MEK/ERK signaling, but will not bind to B-Raf V600E [20]. "
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    ABSTRACT: The Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) pathway mediates cellular responses to different growth signals and is frequently deregulated in cancer. There are three Raf kinases-A-Raf, B-Raf, and C-Raf; however, only B-Raf is frequently mutated in various cancers. The most common B-Raf mutation involves a substitution of a glutamic acid residue to a valine moiety at codon 600. Subsequently, the MAPK pathway is constitutively activated, even in the absence of any growth signals. Although early attempts to target Ras have not yielded any viable drug candidates, many novel compounds inhibiting the activities of B-Raf and MEK have been developed and investigated in clinical trials in recent years and have shown promising result. The first MEK inhibitor (CI-1040) lacked efficacy in clinical trials, but its low toxicity encouraged the search for novel compounds-now there are over a hundred open clinical trials employing various B-Raf and MEK inhibitors. Several of these trials are now in Phase III. In this chapter, we will discuss new patents and patent applications related to inhibitors of the Ras/Raf/MEK/ERK pathway and some recent clinical trial results.
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    ABSTRACT: Serous borderline tumor also known as atypical proliferative serous tumor (APST) is the precursor of ovarian low-grade serous carcinoma (LGSC). In this study, we correlated the morphologic and immunohistochemical phenotypes of 71 APSTs and 18 LGSCs with the mutational status of KRAS and BRAF, the most common molecular genetic changes in these neoplasms. A subset of cells characterized by abundant eosinophilic cytoplasm (EC), discrete cell borders, and bland nuclei was identified in all (100%) 25 BRAF-mutated APSTs but in only 5 (10%) of 46 APSTs without BRAF mutations (P<0.0001). Among the 18 LGSCs, EC cells were found in only 2, and both contained BRAF mutations. The EC cells were present admixed with cuboidal and columnar cells lining the papillae and appeared to be budding from the surface, resulting in individual cells and clusters of detached cells "floating" above the papillae. Immunohistochemistry showed that the EC cells always expressed p16, a senescence-associated marker, and had a significantly lower Ki-67 labeling index than adjacent cuboidal and columnar cells (P=0.02). In vitro studies supported the interpretation that these cells were undergoing senescence, as the same morphologic features could be reproduced in cultured epithelial cells by ectopic expression of BRAF. Senescence was further established by markers such as SA-β-gal staining, expression of p16 and p21, and reduction in DNA synthesis. In conclusion, this study sheds light on the pathogenesis of this unique group of ovarian tumors by showing that BRAF mutation is associated with cellular senescence and the presence of a specific cell type characterized by abundant EC. This "oncogene-induced senescence" phenotype may represent a mechanism that impedes progression of APSTs to LGSC.
    American Journal of Surgical Pathology 09/2014; 38(12). DOI:10.1097/PAS.0000000000000313 · 5.15 Impact Factor
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