Oncogenic potential of BRAF versus RAS

Abstract

Mutations in the ERK pathway occur in approximately one-third of all human cancers and most often involve production of mutant RAS or BRAF. Several studies, including our own, have shown that mutations in the BRAF and RAS genes are generally mutually exclusive. This study was performed to determine the relative oncogenic potential of the BRAF and RAS oncogenes. BRAF^V600E-, H-RAS^G12V-, and N-RAS^Q61R-transfected mouse embryonic fibroblasts (MEFs) that lack p53 (p53^−/−) or contain mutations at codon 172 (p53^R172H and p53^R172P) were able to induce morphologically transformed foci in p53^−/− and p53^R172H MEFs but not in p53^R172P MEFs. Interestingly, BRAF^V600E was less potent than mutant H-RAS^G12V or N-RAS^Q61R was in cooperating with mutant p53 as the numbers and sizes of foci induced by BRAF^V600E were significantly lower and smaller. In vitro growth characteristics and anchorage-independent growth of transfected MEFs corroborated the transformed phenotype, and in vivo tumorigenesis confirmed the results. These results indicate that mutant BRAF^V600E is weakly oncogenic compared with mutant RAS and that they both cooperate with p53^−/− and p53^R172H but not with p53^R172P in oncogenic transformation.

Introduction

Cancer is a disease defined by genomic modification. In fact it is characterized by alterations in the cell’s genetics leading to: self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programmed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis [1]. Dhillon et al. [2] adds to the list that acquisition of drug resistance and avoidance of oncogene-induced senescence is necessary for the transformation of normal cells to that of cancer cells and propagation.

Mitogen-activated protein kinase (MAPK) pathways modulate the extracellular signals to control growth, proliferation, differentiation, migration, and apoptosis. One of the most studied MAPK pathways is the extracellular signal-regulated kinase (ERK) pathway. ERK is a subgroup of MAPKs that is activated by external factors such as growth factors and mitogens. Ligand-mediated activation of receptor tyrosine kinases initiate the cascade of signaling that flows through RAS GTPase to recruit cytosolic dimers of RAF kinases to the plasma membrane, where RAF is activated by phosphorylation. Activated RAF then phosphorylates MEK and ultimately, ERK. Deregulation of the ERK pathway occurs in approximately one-third of all cancers, resulting in constitutive activation of the signaling pathway. A high frequency of deregulation results from mutations at the level of RAS and RAF.

Researchers have found mutations in the K-RAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), H-RAS (v-Ha-ras Harvey rat sarcoma viral oncogene homolog), and N-RAS (neuroblastoma RAS viral oncogene homolog) genes at codon 12/13 and codon 61 [3], which results in RAS being in an active GTP-bound conformation. Activating mutations in one of these isoforms has been reported at high frequency in cancers of the pancreas (90%), thyroid (55–60%), seminoma (45%), lung (nonsmall cell, 35%), liver (30%), melanoma (15%), bladder (10%), and kidney (10%) [4]. Isoform specific RAS signaling can take place at various subcellular compartments and different regions of the plasma membrane. Various RAS inhibitors have been examined for their ability to block signaling in these constitutive active mutants and isoprenylation inhibitors have been proven to be ineffective in clinical trials [5]. Therefore, greater focus has been placed on the downstream RAF molecules.

Recent genome-based oncogene scanning approaches revealed somatic mutations in the signaling gene BRAF, which are prevalent in many neoplasms including melanoma [6], [7]. While melanoma represents the cancer with the greatest mutation rate in BRAF (66–80%) [6], there is a significant frequency in papillary thyroid cancer (69%), colorectal cancer (12%), and serous ovarian cancer [4]. The most common site for mutation in the BRAF gene confers a constitutive activation in the catalytic activity of the molecule at BRAF^V600E, which is as high as 95% of all BRAF mutations [6]. BRAF is a member of the RAF family of serine/threonine kinases, which are immediate effectors of the RAS GTPases. Under normal conditions, BRAF is controlled by mitogens, growth factors, and hormones, such as melanocyte-specific activation of α-melanocyte-stimulating hormone and related peptide binding to melanocortin receptor-1. Among all of the RAFs (A-, B-, and C-Raf), BRAF is the most efficient in phosphorylating MEK kinase. Once activated, RAFs stimulate a cascade of cytosolic kinases by initially phosphorylating and activating mitogen-activated protein kinase (MAPK)/ERK kinase; this leads to phosphorylation of various cytoplasmic and nuclear proteins, such as myc, jun, and fos, which leads to up-regulation of gene expression, cell proliferation, and survival. The BRAF^V600E mutation in the kinase activation domain generates constitutive kinase activity. The functional consequence of this constitutive activation and subsequent phosphorylation is increased proliferation, differentiation, cellular survival, immortalization, and angiogenesis.

BRAF^V600E is a complex mutation. Amplification of the mutant allele is often seen in melanoma [8] and promoter methylation is increased in colorectal carcinoma [9]. Additionally, recent data suggesting a heterodimer formation of BRAF with RAF-1 results in the activation and signaling through the RAF-1 pathway [10]. Interestingly, BRAF mutations have been found in patients with cardio-facio-cutaneous syndrome (CFC) [11]. These mutations can elevate ERK activity to the levels seen with BRAF^V600E, but CFC patients do not typically develop cancer. This parallels the findings that greater than 80% of benign naevi harbor the BRAF^V600E mutation without ever progressing to melanoma [12]. It is possible that a suppressor mechanism is present in these cells such as RKIP [13], [14] or that intense signaling from BRAF results in oncogene-induced senescence [15].

Mutations in RAS and RAF seldom occur with in the same cell [16], [17], [18] although both may be found with in the same tumor [17]. The mutually exclusive property of these mutations suggests that RAF is the main target of RAS and there is recent evidence that suggests that co-expression of both mutant BRAF and mutant N-RAS induces senescence [19]. The oncogenic potential of RAS and BRAF is explored in this study and experiments are performed using mutant p53 MEFs because mutant RAS and BRAF alone is unable to transform normal (wild-type) MEFs. The data presented here indicate that mutant BRAF is relatively less oncogenic than mutant N-RAS is.