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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    • "Phosphorylation of p53 is classically regarded as a crucial step of p53 stabilisation, particularly at Ser15 that has been reported to stabilise p53 by inhibiting the interaction between p53 and MDM2[36]. Of note, Ser15 is phosphorylated after DNA damage and other types of stress such as mutant BRAF inducing DNA strand breaks and activating DNA damage response pathway[38]. Thus, in BRAF mutated cells, Ser15 phosphorylation may be viewed as a marker of functional reactivation of p53. "
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    ABSTRACT: Intrinsic and acquired resistance of metastatic melanoma to V600E/KBRAF and/or MEK inhibitors, which is often caused by activation of the PI3K/AKT survival pathway, represents a major clinical challenge. Given that p53 is capable of antagonising PI3K/AKT activation we hypothesised that pharmacological restoration of p53 activity may increase the sensitivity of BRAF-mutant melanoma to MAPK-targeted therapy and eventually delay and/or prevent acquisition of drug resistance. To test this possibility we exposed a panel of vemurafenib-sensitive and resistant (innate and acquired) V600E/KBRAF melanomas to a V600E/KBRAF inhibitor (vemurafenib) alone or in combination with a direct p53 activator (PRIMA-1Met/APR-246). Strikingly, PRIMA-1Met synergised with vemurafenib to induce apoptosis and suppress proliferation of V600E/KBRAF melanoma cells in vitro and to inhibit tumour growth in vivo. Importantly, this drug combination decreased the viability of both vemurafenib-sensitive and resistant melanoma cells irrespectively of the TP53 status. Notably, p53 reactivation was invariably accompanied by PI3K/AKT pathway inhibition, the activity of which was found as a dominant resistance mechanism to BRAF inhibition in our lines. From all various combinatorial modalities tested, targeting the MAPK and PI3K signalling pathways through p53 reactivation or not, the PRIMA-1Met/vemurafenib combination was the most cytotoxic. We conclude that PRIMA-1Met through its ability to directly reactivate p53 regardless of the mechanism causing its deactivation, and thereby dampen PI3K signalling, sensitises V600E/KBRAF-positive melanoma to BRAF inhibitors.
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    • "S1H,I). Similar to HRAS-induced OIS, previous data suggest that BRAF-induced OIS leads to cellular hyperproliferation in vitro and in vivo (Zhu et al. 1998; Dankort et al. 2007) and that in vivo human nevi display features of an activated DDR (Gorgoulis et al. 2005; d'Adda di Fagagna 2008; Sheu et al. 2012). Thus, we performed MLL1 knockdown in diBRAF melanocytes, and, similar to the fibroblast system, this led to a reduction of both MLL1 and SASP gene expression (Supplemen- talFig. "

    Full-text · Article · Feb 2016 · Genes & Development
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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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