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Int. J. Mol. Sci. 2017, 18(3), 585; doi:10.3390/ijms18030585

Strategies for Overcoming Resistance in Tumours Harboring BRAF Mutations
Department of Pathology, Dalhousie University, Halifax, NS B3H 4R2, Canada
Department of Pathology, Nova Scotia Health Authority, Halifax, NS B3H 1V8, Canada
Author to whom correspondence should be addressed.
Academic Editor: Terrence Piva
Received: 10 January 2017 / Accepted: 23 February 2017 / Published: 8 March 2017


The development of resistance to previously effective treatments has been a challenge for health care providers and a fear for patients undergoing cancer therapy. This is an unfortunately frequent occurrence for patients undergoing targeted therapy for tumours harboring the activating V600E mutation of the BRAF gene. Since the initial identification of the BRAF mutation in 2002, a series of small molecular inhibitors that target the BRAFV600E have been developed, but intrinsic and acquired resistance to these drugs has presented an ongoing challenge. More recently, improvements in therapy have been achieved by combining the use of BRAF inhibitors with other drugs, such as inhibitors of the downstream effector mitogen activated protein kinase (MAPK)/extracellular-signal regulated kinase (ERK) kinase (MEK). Despite improved success in response rates and in delaying resistance using combination therapy, ultimately, the acquisition of resistance remains a concern. Recent research articles have shed light on some of the underlying mechanisms of this resistance and have proposed numerous strategies that might be employed to overcome or avoid resistance to targeted therapies. This review will explore some of the resistance mechanisms, compare what is known in melanoma cancer to colorectal cancer, and discuss strategies under development to manage the development of resistance.
BRAF; acquired resistance

1. Introduction

Beginning with the discovery of the retroviral oncogene RAF (originally named from rapidly accelerated fibrosarcoma) in 1983, the family of RAF proto-oncogenes has emerged as an important set of signaling molecules that play a role in the development of cancer [1]. Molecules such as growth factors and cytokines that promote cell proliferation can activate a signaling cascade initiated through receptor tyrosine kinases at the surface and transmit a signal through a series of protein modifications passing from RAS (originally named from rat sarcoma) to RAF to mitogen activated kinase (MAPK) to extracellular signal regulated kinase (ERK) and ultimately to the nucleus, where gene expression changes lead to cell proliferation [2,3]. Shortly after the discovery of the RAF gene family, it was found that activating mutations in the BRAF gene, encoding the B-raf serine-threonine protein kinase, play an oncogenic role in cancer development [4]. Currently, it is estimated that eight percent of all cancers have mutations in the BRAF gene, and they are present in a wide range of malignant tumours including ~50% of melanomas [5], ~40% of papillary thyroid cancer (PTC) [6], ~30% of serous ovarian cancer [6,7,8,9], ~10% of colorectal cancers (CRC) [10], and ~2%–3% of lung cancers [7,11]. Mutations in BRAF have also been found in a pre-malignant colon polyps [12], as well as benign skin lesions [13,14]. This finding of oncogenic mutations in lesions that have not yet advanced to cancer can be attributed to the ability of BRAF mutations (like other oncogenic mutations) to induce growth arrest and senescence [15].
To date, more than fifty distinct mutations in the BRAF gene have been described [7]. The BRAFV600E mutation accounts for approximately 90% of the BRAF mutations found in cancer [1,16,17,18]. This variant (rs113488022) represents an acquired mutation, and is observed only extremely rarely in genomic DNA samples, with a frequency of 0.0016% in the ExAC sequence database. The single nucleotide change from a thymine (T) to adenine (A) at position 1799 of the messenger RNA (NM_004333.4: c.1799T>A) results in the substitution of a valine (V) to a glutamic acid (E) (p.Val600Glu), thereby inducing a conformational change into an activated form of the BRAF protein [7]. Patients with tumours that carry the BRAFV600E mutation display a less promising prognosis compared to those with a wild type BRAF, in particular, in melanoma, colorectal cancer, and thyroid cancer [19,20,21]. Since the discovery of BRAF mutations, efforts have been underway to understand the mechanisms by which these mutations lead to cancer progression, and to identify potential therapeutic strategies to target BRAF mutation positive tumours.

2. Involvement of BRAF in the MAPK Pathway

BRAF is part of the MAPK pathway, a signaling pathway present in all eukaryotic cells. MAPK signaling controls and regulates numerous essential cellular mechanisms including cell proliferation, differentiation, development, survival, and apoptosis [22,23,24,25]. In normal cells, extracellular signals such as cytokines, hormones, and growth factors bind to their receptor on the cells’ surface [26,27]. This binding leads to the activation of receptor tyrosine kinase (RTK) domains on the portion of the receptor on the inside of the cell. This in turn initiates activation of the kinase domain on the intracellular portion of the receptor, and a signaling cascade as sequential phosphorylation events pass from one protein to the next (i.e., RAS-RAF-MEK1/2-ERK1/2). Ultimately, a signal is passed to the nucleus, leading to transcriptional changes that promote cell proliferation. The V600E mutation in BRAF results in this signaling pathway being activated even in the absence of the cytokine, hormone, or growth factor stimulation, leading to unregulated cell proliferation and ultimately cancer [28].
It is worth noting that active ERK1/2 distributes a signal by phosphorylating and/or interacting with a vast array of cytosolic and nuclear substrates, each designated to initiate a specific set of functions. There are more than 150 protein substrates found in both the nucleus and the cytoplasm where activation by ERK influences transcription [27]. Therefore, there is a broad range of outcomes following activation of ERK1/2, depending on which of its broad set of targets become activated, for how long, and to what extent. A list of some of ERKs’ substrates is summarized in Table 1. Cell signaling through this pathway is not a simple on and off switch, but rather consists of multiple targets, and multiple mechanisms of feedback inhibition to modulate the pathway.

Regulation of the MAPK Pathway

Homeostatic balance is essential for almost every physiological process in the human body. In non-transformed cells the MAPK pathway is balanced by inhibitory regulators, which provide a negative feedback signal. The MAPK pathway is in part regulated through a classical negative feedback loop, which is controlled by ERK activation of the dual specificity phosphatases (DUSPs) [58], and other molecules such as sprouty proteins (SPRYs) [59], kinase suppressor of RAS1 (KSR1) [46,60], and RAF kinase inhibitor protein (RKIP) [61,62]. DUSPs can inhibit ERK directly while SPRYs proteins inhibit the MAPK pathway at an upstream level through inhibiting RAS activation. ERK itself can also directly inactivate the MAPK pathway at several levels by directly inhibiting RTKs, RAS activation, and RAF dimerization. One of these inhibitory mechanisms is through regulation BRAF itself. Activated ERK can phosphorylate BRAF in two sites: Ser750 and Thr753, resulting in its inhibition [63]. In BRAFV600E mutations, the negative feedback mechanisms can be impaired. In some cases, the target of the inhibition is for steps upstream of the activated BRAF, and inhibition is ineffective. The mutation itself can also impair the feedback inhibition. For example, negative feedback inhibition through SPRYs is impaired because the SPRYs are unable to bind to BRAF due to disruption by mutation [64]. Constitutive activation of mutant BRAF results in hyperactive ERK that in turn can increase the expression of DUSPs and SPRYs [65]. However, this surge in expression of inhibitory regulators no longer acts as efficiently as in healthy cells.

3. Conferred Resistance Mechanisms in BRAFV600E Tumours

The development of drugs to target the hyperactivation of the BRAF-MAPK-ERK signaling pathway has led to substantial advances in patients’ overall survival and progression-free survival for melanoma, and the further addition of MEK inhibitors given in combination has improved response rates and survival compared to monotherapy [66]. Unfortunately, the story of BRAF inhibitors is not entirely one of success. While most melanoma cancers initially respond well to therapy, most patients will relapse with tumours that are now resistant [67,68]. For tumours other than melanoma, the combined targeted therapy is not always effective. For example, while some success with combination BRAF-MEK1/2 inhibition was observed in colorectal cancer [69], the efficacy of this combination strategy is still far less than is observed for BRAF mutant melanoma. In addition, there are some circumstances where therapy can actually result in increased tumour growth. This is a result of the inhibitors’ ability to induce a paradoxical activation of downstream signaling in BRAF-WT cells and in cell harboring RAS mutations [70,71,72,73]. Here we will discuss the main mechanisms for the resistance to therapy.

3.1. Resistance Through MAPK Pathway Reactivation

In the case of BRAFV600E, the reactivation of the MAPK signaling pathway accounts for the majority of acquired resistance mechanisms [74]. In a study of 100 primary and 134 follow-up samples from melanoma patients (where 87% were BRAFV600E positive), resistance mechanisms in the recurrent section could be identified in approximately 58% of the cases. These largely represented BRAF splice variants (29%) or BRAF gene amplification (8%) [75,76], however, secondary mutations in other genes in the RAS-RAF-MEK-ERK pathway, such as neuroblastoma RAS viral oncogene homologue (NRAS) [77], and MAPK/ERK kinase (MEK) [78,79] can lead to resistance to therapy. These mechanisms involve BRAF-independent activation of the MAPK pathway.
Secondary mutations within the BRAF gene have only rarely been linked to the resistance to BRAF inhibitors [80,81,82]. One exception to this is the identification of an alternative splice form of the BRAFV600E which lacks the dimerization domain and has been observed as a mechanism of resistance [83]. Resistance to BRAF inhibitors can be a result of BRAFV600E amplification [80]. Whole-exome sequencing of 20 melanoma patients before and after treatment with BRAF inhibitors identified that four patients with disease progression had BRAFV600E copy-number gain relative to baseline tumours from the same patient. Quantitative PCR confirmed an increase in BRAFV600E expression in these patients, and a cell culture model was used to demonstrate that the copy-number gain of BRAFV600E did indeed induce resistance to BRAF inhibitors while sensitivity was restored by its knockdown [80].
Acquired mutations in NRAS have been associated with acquired resistance to BRAF inhibitors. Comparing melanoma tumours collected before BRAF inhibitor therapy to resistant tumours in the same patient after therapy identified acquired NRAS mutations in many of these tumours, including in tumours that continue to harbor the BRAF mutations [84]. The Kristen rat sarcoma viral oncogene homologue (KRAS) mutation G12D has been identified in many tumour types, including colorectal cancers. The acquisition of this activating mutation following BRAF inhibitor exposure has been linked to the development of resistance in the BRAFV600E mutant parathyroid cancer cell line [85]. Similarly, resistance in a colorectal cell line has been linked to the appearance KRAS G12D and G13D mutations [86], suggesting activating mutations in this RAS pathway may contribute to intrinsic and acquired resistance. Post treatment acquisition of MEK1 and MEK2 mutations have also been associated with acquired resistance [75,86].
Besides secondary mutations to elements of the MAPK pathway, changes in gene expression level for elements of the MAPK pathway have been linked to resistance. By screening the effect of overexpressing 597 kinases, MAP3K8 (COT) kinase and C-RAF emerged as among the genes that could confer resistance to BRAF inhibitor therapy. BRAFV600E positive cancer cell lines that express higher levels of MAP3K8 tended to be less sensitive to BRAF inhibitor drugs; MAP3K8 expression increased in the tumours of patients treated with BRAF inhibitors, and was even further elevated in drug resistance relapse tumour samples [87]. Similarly, Montagut et al. found that elevated CRAF expression was observed in cells resistant to the RAF inhibitor AZ628 compared to their sensitive parental cell, and that elevated CRAF can activate the MAPK pathway independent of BRAF activity [88].
Both MAP3K8 and CRAF elevations can confer resistance either as primary or acquired resistance mechanisms. One approach that has been employed in an attempt to overcome resistance resulting from elevated expression was the use of agents that bind to and inhibit heat shock protein 90 (HSP90) [88]. HSP90 is required for the conformational stability of mutant BRAFV600E and RAF related family members [89,90,91], making blockade of HSP90 a potential strategy for overcoming resistance [92,93]. HSP90 inhibitor therapy has been included in some cancer treatment combinations [94], and has been tried in clinical phase II trials for the treatment melanoma, however, the studies either showed little effect [95] or were inconclusive [96]. Further research into this approach is required.

3.2. Resistance Involving Insensitivity to MAPK Regulators

Negative feedback regulators of the MAPK pathway, including DUSPs and SPRYs, have been linked to the development of acquired resistance to BRAF inhibitors. Ordinarily, a balance emerges between the activation of the RAS-RAF-MEK-ERK pathway, and negative feedback imposed by ERK-induced expression of DUSPs. Activated phosphor-ERK directly inhibits the upstream pathway, dampening the signal, and elevation in DUSPs leads to dephosphorylation of ERK, further dampening the signal cascade. Pratilas and colleagues revealed that despite elevated feedback inhibition signals, BRAFV600E is insensitive to negative feedback regulation by DUSPs [65,97]. The cell falls into a new, distorted balance with elevated ERK and elevated DUSP, but the negative feedback components are overwhelmed by persistent signaling. Similarly, SPRY2 and SPRY4 can provide negative feedback to wild type BRAF, but are unable to inhibit the BRAFV600E mutation [64]. It has been proposed that resistance to treatment may be related to further disruption in the balance between the negative feedback mechanisms and the activation [98].

3.3. Other Mechanisms of Resistance

The cross-talk that exists between signaling pathways activated by receptor tyrosin kinases (RTK)s, such the RAS-RAF-MEK-ERK and the PI3K-PTEN-AKT pathway, was first identified in 1994 by Chung and colleagues [99]. Overexpression of RTKs could be expected to elevate the signaling in both of these arms. Elevations in epidermal growth factor receptor (EGFR) [100], PDGFR [77,101], and IGF1-R [102] have been observed in resistance. Release of hepatocyte growth factor (HGF) from the surrounding stromal cells to activate MET, the HGF RTK on the tumour cell, has also been described as a resistance mechanism [103,104].
The integration between these two signaling pathways and the fact that both are sharing the same upstream RTKs raise the possibility of involvement of activated PI3K pathway in resistant tumour cells. Shi et al. [105] have identified BRAF inhibitor resistant melanomas with gain-of-function mutations in AKT. This AKT-mediated resistance mechanism results in P13K up-regulation. Their data suggested that, in spite of MAPK pathway inhibition through BRAF inhibitors, the BRAF mutated cells evade treatment by adapting to the use of PI3K signaling to survive. In addition to AKT mutations, PTEN mutations are found in 15.2% in metastatic melanoma leading to a similar resistance mechanism [106]. PTEN loss of function promotes AKT activation, which in turn can lead to dysregulation of the pro-apoptotic Bcl-2 like proteins. The resulting impairment of the apoptotic pathway was associated with resistance to BRAF inhibitors, vemurafenib and dabrafenib [107,108,109].
Figure 1 illustrates how aberrant signaling resulting from the V600E mutation in BRAF gene led to uncontrolled growth, and summarizes hypothesized mechanisms of resistance.
More recently, other mechanisms have been proposed. Treatment with inhibitors that inhibit MEK and ERK phosphorylation prevent the phosphorylation and stabilization of the transcriptional regulator MYC, leading to rapid degradation [110]. MYC promotes modifications to histones that influence transcription, and the loss of MYC following MEK inhibition has been found to cause epigenetic modifications to gene expression through histones and altered binding of regulatory molecules to enhancer regions [111]. While this was not specifically tested in the context of BRAFV600E resistance, this mechanism warrants consideration.
Another emerging mechanism of resistance to BRAF inhibition is through altered expression of microRNAs. MicroRNAs are small non-protein coding RNAs that bind to the transcripts of other genes and promote their degradation. Recently, the loss of microRNA miR-579-3P has been identified as a potential mechanism of both primary and acquired resistance to BRAF and MEK inhibitor drugs [112]. The mechanism by which loss of miR-579-3p leads to resistance is not fully understood, but Fattore et al. observed that this loss results in increases for both BRAF and the MDM2 pathway. MDM2 is an important negative regulator of the tumour suppressor p53, so elevation in MDM2 would reduce this protective tumour suppression pathway.

4. Challenges Encountered by Colorectal Cancer (CRC) Patients with BRAFV600E Mutation

The BRAFV600E mutation is found in 10% of colorectal cancer (CRC) cases [10]. Those patients progress rapidly and tend to not respond well to therapy. This subgroup of patients is distinct from other forms of CRC, and has its own molecular and genetic profile. However, the response rate to vemurafenib was only 5% in CRCs exhibiting BRAFV600E mutation compared to 60% to 80% of melanoma patients harboring the same mutation [80,106].

Evidence of Specific Resistance Mechanisms in BRAFV600E Mutated CRC

The small subset of BRAFV600E mutant CRC displays different tumour biology and different clinical behaviors compared to RAS mutant CRC [113]. In a meta-analysis by Pietrantonio et al. [114], BRAF mutated CRC patients had limited benefit from any of the “available standard-of-care therapies”. These findings have raised the attention of many groups to understand why BRAF inhibitor treatment showed little or no response [67,115]. In 2012, two independent groups recognized the involvement of EGFR in CRC resistance to BRAF inhibitors. Prahallad et al. proposed that the inhibition of mutant BRAF led to a powerful feedback activation of EGFR triggering a secondary reactivation of the MAPK pathway [116]. This feedback activation of EGFR increased the activation of not only the MAPK pathway but also the parallel pathway PI3K generating growth renewal. The group studied the involvement of cell division cycle 25C (CDC25C), which is a downstream substrate of ERK that when activated can bind to and deactivate EGFR [117]. Treatment with BRAF inhibitors resulted in decreased activation of MEK1/2 and ERK1/2, consequently a failure of ERK to phosphorylate CDC25C. This failure to activate the negative feedback signal of CDC25C leads to a prolonged EGFR activation and greater activation of the P13K pathway [116]. Corcoran et al. proposed a slightly different mechanism for the prolonged EGFR activation [80]. This group postulated that negative feedback regulators such as SPRYs participated in EGFR reactivation. SPRYs comprise a key regulatory function for the MAPK pathway and transcribe in an ERK-dependent manner [118]. SPRYs negatively regulate upstream MAPK at the RTKs and RAS level. BRAF targeted therapy led to decreases in the level of SPRYs, enabling EGFR to rebound and reactivate the MAPK pathway [80]. Both groups showed that the efficacy of BRAF inhibitor is improved greatly in vitro when combined with an EGFR inhibitor and that this combined treatment leads to tumour regression in vivo. They further examined EGFR levels in clinical biopsies from patients with the BRAFV600E mutation and compared across CRC, melanoma, and PTC. The majority of BRAF mutated CRC showed high levels of active EGFR compared to other tumour types [80,116]. Moreover, single agent treatment with either inhibitor (BRAF or EGFR) produced little to poor response, indicating a combination strategy might be more appropriate for patients with BRAF mutated CRC.
Several studies that have been exploring new therapeutic approaches aimed to target resistance-conferring mutations are providing promising treatment options for patients harboring the BRAFV600E mutation. For example, Mao et al. showed that BRAF inhibitor combined with PI3K inhibitors hindered the growth of BRAF mutated CRC cell lines [119]. In addition, epigenetic factors may be playing a role in drug resistance in colorectal cancer. Hypermethylation of CpG islands is observed in colorectal tumours with the BRAFV600E mutation, and results in gene silencing of multiple target genes. Mao et al. found that the efficacy of BRAF inhibitor improves after treatment with demethylating agents [119].
Triple targeted inhibitor combinations are also being examined, combining BRAF and EGFR inhibitors with additional targets, including P13K and MEK1/2 inhibitors [120]. A more robust response rate was observed compared to monotherapy or BRAF-MEK combination therapy [121,122]. These advances illustrate the importance of understanding the underlying mechanisms of resistance in specific tumour types. New potential therapies may emerge for BRAFV600E positive CRC tumours that failed to respond to therapies designed for melanoma tumours.

5. Conclusions

Discovery of BRAF mutations in cancer allowed many scientists to link BRAFV600E with poor prognosis and overall survival in comparison to BRAF-WT and or RAS mutations [7,123,124,125]. The identification of this challenging subgroup of patients has shed light in the search for a broader concept of tumour progression and has helped further the investigation of therapeutic targets for cancers exhibiting the BRAFV600E mutation.
Emerging understanding of the direct and adaptive effects of BRAFV600E allow for the discovery and development of therapeutic agents that aid in reducing kinase activity of mutant BRAF and the development of strategies to overcome the resistance to such treatments. Although many individuals with recurrent melanomas or primary colon cancers fail to respond to currently available treatments, there are subpopulations that would in fact benefit from existing therapies. There has been ongoing research for robust biomarkers that can identify the activated pathways causing intrinsic resistance and acquired resistance [126]. This would potentially identify those who would benefit from treatments, and also may point the way towards preventing or reversing drug resistance.


Funding for Nourah Mohammad Obaid was kindly provided by the Ministry of Higher Education of Saudi Arabia.

Conflicts of Interest

The authors declare no conflict of interest.


AKTprotein kinase B
ATPadenosine triphosphate
BRAFv-RAF murine sarcoma viral oncogene homolog B
CRCcolorectal cancer
EGFRepidermal growth factor receptor
ERKextracellular signal-regulated kinase
HSP90heat shock protein 90
LKB1liver kinase B1
MAPKmitogen activated protein kinase
MEKmitogen-activated protein kinase kinase
mTORmammalian target of rapamycin
PCRpolymerase chain reaction
PTENphosphatase and tensin homolog
RTKreceptor tyrosine kinase
TSC2tuberous sclerosis complex 2


  1. Niault, T.S.; Baccarini, M. Targets of RAF in tumorigenesis. Carcinogenesis 2010, 31, 1165–1174. [Google Scholar] [CrossRef] [PubMed]
  2. Marshall, C.J. Specificity of receptor tyrosine kinase signaling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 1995, 80, 179–185. [Google Scholar] [CrossRef]
  3. Perrimon, N. The torso receptor protein-tyrosine kinase signaling pathway: An endless story. Cell 1993, 74, 219–222. [Google Scholar] [CrossRef]
  4. Ikawa, S.; Fukui, M.; Ueyama, Y.; Tamaoki, N.; Yamamoto, T.; Toyoshima, K. BRAF, a new member of the RAF family, is activated by DNA rearrangement. Mol. Cell. Biol. 1988, 8, 2651–2654. [Google Scholar] [CrossRef] [PubMed]
  5. Ascierto, P.A.; Kirkwood, J.M.; Grob, J.; Simeone, E.; Grimaldi, A.M.; Maio, M.; Palmieri, G.; Testori, A.; Marincola, F.M.; Mozzillo, N. The role of BRAF V600 mutation in melanoma. J. Transl. Med. 2012, 10, 85. [Google Scholar] [PubMed]
  6. Cohen, Y.; Xing, M.; Mambo, E.; Guo, Z.; Wu, G.; Trink, B.; Beller, U.; Westra, W.H.; Ladenson, P.W.; Sidransky, D. BRAF mutation in papillary thyroid carcinoma. J. Natl. Cancer Inst. 2003, 95, 625–627. [Google Scholar] [CrossRef] [PubMed]
  7. Davies, H.; Bignell, G.R.; Cox, C.; Stephens, P.; Edkins, S.; Clegg, S.; Teague, J.; Woffendin, H.; Garnett, M.J.; Bottomley, W. Mutations of the BRAF gene in human cancer. Nature 2002, 417, 949–954. [Google Scholar] [CrossRef] [PubMed]
  8. Singer, G.; Oldt, R.; Cohen, Y.; Wang, B.G.; Sidransky, D.; Kurman, R.J.; Ie, M.S. Mutations in BRAF and KRAS characterize the development of low-grade ovarian serous carcinoma. J. Natl. Cancer Inst. 2003, 95, 484–486. [Google Scholar] [CrossRef] [PubMed]
  9. Nakayama, K.; Nakayama, N.; Kurman, R.J.; Cope, L.; Pohl, G.; Samuels, Y.; Velculescu, V.E.; Wang, T.; Shih, I. Sequence mutations and amplification of Pik3ca and Akt2 genes in purified ovarian serous neoplasms. Cancer Biol. Ther. 2006, 5, 779–785. [Google Scholar] [CrossRef] [PubMed]
  10. Santarpia, L.; Lippman, S.M.; El-Naggar, A.K. Targeting the MAPK-RAS-RAF signaling pathway in cancer therapy. Expert Opin. Ther. Targets 2012, 16, 103–119. [Google Scholar] [CrossRef] [PubMed]
  11. Brose, M.S.; Volpe, P.; Feldman, M.; Kumar, M.; Rishi, I.; Gerrero, R.; Einhorn, E.; Herlyn, M.; Minna, J.; Nicholson, A. BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res. 2002, 62, 6997–7000. [Google Scholar] [PubMed]
  12. Yuen, S.T.; Davies, H.; Chan, T.L.; Ho, J.W.; Bignell, G.R.; Cox, C.; Stephens, P.; Edkins, S.; Tsui, W.W.; Chan, A.S. Similarity of the phenotypic patterns associated with BRAF and KRAS mutations in colorectal neoplasia. Cancer Res. 2002, 62, 6451–6455. [Google Scholar] [PubMed]
  13. Pollock, P.M.; Harper, U.L.; Hansen, K.S.; Yudt, L.M.; Stark, M.; Robbins, C.M.; Moses, T.Y.; Hostetter, G.; Wagner, U.; Kakareka, J. High frequency of BRAF mutations in nevi. Nat. Genet. 2002, 33, 19–20. [Google Scholar] [CrossRef] [PubMed]
  14. Yazdi, A.S.; Palmedo, G.; Flaig, M.J.; Puchta, U.; Reckwerth, A.; Rütten, A.; Mentzel, T.; Hügel, H.; Hantschke, M.; Schmid-Wendtner, M. Mutations of the BRAF gene in benign and malignant melanocytic lesions. J. Investig. Dermatol. 2003, 121, 1160–1162. [Google Scholar] [CrossRef] [PubMed]
  15. Braig, M.; Schmitt, C.A. Oncogene-induced senescence: Putting the brakes on tumor development. Cancer Res. 2006, 66, 2881–2884. [Google Scholar] [CrossRef] [PubMed]
  16. Xing, M.; Tufano, R.P.; Tufaro, A.P.; Basaria, S.; Ewertz, M.; Rosenbaum, E.; Byrne, P.J.; Wang, J.; Sidransky, D.; Ladenson, P.W. Detection of BRAF mutation on fine needle aspiration biopsy specimens: A new diagnostic tool for papillary thyroid cancer. J. Clin. Endocrinol. Metab. 2004, 89, 2867–2872. [Google Scholar] [CrossRef] [PubMed]
  17. Tiacci, E.; Trifonov, V.; Schiavoni, G.; Holmes, A.; Kern, W.; Martelli, M.P.; Pucciarini, A.; Bigerna, B.; Pacini, R.; Wells, V.A. BRAF mutations in hairy-cell leukemia. N. Engl. J. Med. 2011, 364, 2305–2315. [Google Scholar] [CrossRef] [PubMed]
  18. Mercer, K.E.; Pritchard, C.A. RAF proteins and cancer: BRAF is identified as a mutational target. Biochim. Biophys. Acta Rev. Cancer 2003, 1653, 25–40. [Google Scholar] [CrossRef]
  19. Houben, R.; Becker, J.C.; Kappel, A.; Terheyden, P.; Bröcker, E.; Goetz, R.; Rapp, U.R. Constitutive Activation of the RAS-RAF signaling pathway in metastatic melanoma is associated with poor prognosis. J. Carcinog. 2004, 3, 6. [Google Scholar] [CrossRef] [PubMed]
  20. Ogino, S.; Shima, K.; Meyerhardt, J.A.; McCleary, N.J.; Ng, K.; Hollis, D.; Saltz, L.B.; Mayer, R.J.; Schaefer, P.; Whittom, R. Predictive and prognostic roles of BRAF mutation in stage III colon cancer: Results from intergroup trial CALGB 89803. Clin. Cancer Res. 2012, 18, 890–900. [Google Scholar] [CrossRef] [PubMed]
  21. Xing, M.; Alzahrani, A.S.; Carson, K.A.; Viola, D.; Elisei, R.; Bendlova, B.; Yip, L.; Mian, C.; Vianello, F.; Tuttle, R.M. Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. JAMA 2013, 309, 1493–1501. [Google Scholar] [CrossRef] [PubMed]
  22. Robinson, M.J.; Cobb, M.H. Mitogen-activated protein kinase pathways. Curr. Opin. Cell Biol. 1997, 9, 180–186. [Google Scholar] [CrossRef]
  23. Keshet, Y.; Seger, R. The MAP kinase signaling cascades: A system of hundreds of components regulates a diverse array of physiological functions. In MAP Kinase Signaling Protocols; Springer: New York, NY, USA, 2010; pp. 3–38. [Google Scholar]
  24. Shaul, Y.D.; Seger, R. The MEK/ERK cascade: From signaling specificity to diverse functions. Biochim. Biophys. Acta Mol. Cell Res. 2007, 1773, 1213–1226. [Google Scholar] [CrossRef] [PubMed]
  25. Kolch, W. Meaningful relationships: The regulation of the RAS/Raf/MEK/ERK pathway by protein interactions. Biochem. J. 2000, 351, 289–305. [Google Scholar] [CrossRef] [PubMed]
  26. Troppmair, J.; Bruder, J.T.; App, H.; Cai, H.; Liptak, L.; Szeberenyi, J.; Cooper, G.M.; Rapp, U.R. RAS controls coupling of growth factor receptors and protein kinase C in the membrane to RAF-1 and BRAF protein serine kinases in the cytosol. Oncogene 1992, 7, 1867–1873. [Google Scholar] [PubMed]
  27. Chen, R.H.; Sarnecki, C.; Blenis, J. Nuclear localization and regulation of ERK- and RSK-encoded protein kinases. Mol. Cell. Biol. 1992, 12, 915–927. [Google Scholar] [CrossRef] [PubMed]
  28. Karnoub, A.E.; Weinberg, R.A. RAS oncogenes: Split personalities. Nat. Rev. Mol. Cell Biol. 2008, 9, 517–531. [Google Scholar] [CrossRef] [PubMed]
  29. Eblen, S.T.; Slack-Davis, J.K.; Tarcsafalvi, A.; Parsons, J.T.; Weber, M.J.; Catling, A.D. Mitogen-activated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion. Mol. Cell. Biol. 2004, 24, 2308–2317. [Google Scholar] [CrossRef] [PubMed]
  30. Anderson, N.G.; Li, P.; Marsden, L.; Williams, N.; Roberts, T.M.; Sturgill, T.W. RAF-1 is a potential substrate for mitogen-activated protein kinase in vivo. Biochem. J. 1991, 277, 573–576. [Google Scholar] [CrossRef] [PubMed]
  31. Dougherty, M.K.; Müller, J.; Ritt, D.A.; Zhou, M.; Zhou, X.Z.; Copeland, T.D.; Conrads, T.P.; Veenstra, T.D.; Lu, K.P.; Morrison, D.K. Regulation of RAF-1 by direct feedback phosphorylation. Mol. Cell 2005, 17, 215–224. [Google Scholar] [CrossRef] [PubMed]
  32. Brummer, T.; Naegele, H.; Reth, M.; Misawa, Y. Identification of novel ERK-mediated feedback phosphorylation sites at the C-terminus of BRAF. Oncogene 2003, 22, 8823–8834. [Google Scholar] [CrossRef] [PubMed]
  33. Sturgill, T.W.; Ray, L.B.; Erikson, E.; Maller, J.L. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature 1988, 334, 715–718. [Google Scholar] [CrossRef] [PubMed]
  34. Mukhopadhyay, N.K.; Price, D.J.; Kyriakis, J.M.; Pelech, S.; Sanghera, J.; Avruch, J. An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the P70 S6 kinase. J. Biol. Chem. 1992, 267, 3325–3335. [Google Scholar] [PubMed]
  35. Wang, L.; Gout, I.; Proud, C.G. Cross-talk between the ERK and P70 S6 kinase (S6k) signaling pathways. MEK-dependent activation of S6k2 in cardiomyocytes. J. Biol. Chem. 2001, 276, 32670–32677. [Google Scholar] [CrossRef] [PubMed]
  36. Camps, M.; Nichols, A.; Gillieron, C.; Antonsson, B.; Muda, M.; Chabert, C.; Boschert, U.; Arkinstall, S. Catalytic activation of the phosphatase MKP-3 by ERK2 mitogen-activated protein kinase. Science 1998, 280, 1262–1265. [Google Scholar] [CrossRef] [PubMed]
  37. Marchetti, S.; Gimond, C.; Chambard, J.; Touboul, T.; Roux, D.; Pouysségur, J.; Pagès, G. Extracellular signal-regulated kinases phosphorylate mitogen-activated protein kinase phosphatase 3/DUSP6 at serines 159 and 197, two sites critical for its proteasomal degradation. Mol. Cell. Biol. 2005, 25, 854–864. [Google Scholar] [CrossRef] [PubMed]
  38. Masuda, K.; Shima, H.; Katagiri, C.; Kikuchi, K. Activation of ERK induces phosphorylation of MAPK phosphatase-7, a JNK specific phosphatase, at Ser-446. J. Biol. Chem. 2003, 278, 32448–32456. [Google Scholar] [CrossRef] [PubMed]
  39. Ozaki, K.; Miyazaki, S.; Tanimura, S.; Kohno, M. Efficient suppression of FGF-2-induced ERK activation by the cooperative interaction among mammalian sprouty isoforms. J. Cell Sci. 2005, 118, 5861–5871. [Google Scholar] [CrossRef] [PubMed]
  40. Hacohen, N.; Kramer, S.; Sutherland, D.; Hiromi, Y.; Krasnow, M.A. Sprouty encodes a novel antagonist of FGF signaling that patterns apical branching of the drosophila airways. Cell 1998, 92, 253–263. [Google Scholar] [CrossRef]
  41. Alvarez, E.; Northwood, I.C.; Gonzalez, F.A.; Latour, D.A.; Seth, A.; Abate, C.; Curran, T.; Davis, R.J. Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-Myc and c-Jun proteins by an epidermal growth factor receptor threonine 669 protein kinase. J. Biol. Chem. 1991, 266, 15277–15285. [Google Scholar] [PubMed]
  42. Arnaud, M.; Crouin, C.; Deon, C.; Loyaux, D.; Bertoglio, J. Phosphorylation of GRB2-associated binder 2 on serine 623 by ERK/MAPK regulates its association with the phosphatase SHP-2 and decreases STAT5 activation. J. Immunol. 2004, 173, 3962–3971. [Google Scholar] [CrossRef]
  43. Langlois, W.J.; Sasaoka, T.; Saltiel, A.R.; Olefsky, J.M. Negative feedback regulation and desensitization of insulin-and epidermal growth factor-stimulated P21RAS activation. J. Biol. Chem. 1995, 270, 25320–25323. [Google Scholar] [CrossRef] [PubMed]
  44. Andreozzi, F.; D’Alessandris, C.; Federici, M.; Laratta, E.; del Guerra, S.; del Prato, S.; Marchetti, P.; Lauro, R.; Perticone, F.; Sesti, G. Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-Kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in rin pancreatic Β-Cells. Endocrinology 2004, 145, 2845–2857. [Google Scholar] [CrossRef] [PubMed]
  45. Balaban, R.S.; Nemoto, S.; Finkel, T. Mitochondria, oxidants, and aging. Cell 2005, 120, 483–495. [Google Scholar] [CrossRef] [PubMed]
  46. Kato, K.; Ito, H.; Kamei, K.; Inaguma, Y.; Iwamoto, I.; Saga, S. Phosphorylation of Αb-crystallin in mitotic cells and identification of enzymatic activities responsible for phosphorylation. J. Biol. Chem. 1998, 273, 28346–28354. [Google Scholar] [CrossRef] [PubMed]
  47. Saal, S.K.G.; Parsons, R. Is the small heat shock protein Αb-crystallin an oncogene? J. Clin. Investig. 2006, 116, 30–32. [Google Scholar] [CrossRef] [PubMed]
  48. Cruzalegui, F.H.; Cano, E.; Treisman, R. ERK activation induces phosphorylation of ELK-1 at multiple S/TP motifs to high stoichiometry. Oncogene 1999, 18, 7948–7957. [Google Scholar] [CrossRef] [PubMed]
  49. Murphy, L.O.; MacKeigan, J.P.; Blenis, J. A network of immediate early gene products propagates subtle differences in mitogen-activated protein kinase signal amplitude and duration. Mol. Cell. Biol. 2004, 24, 144–153. [Google Scholar] [CrossRef] [PubMed]
  50. Morton, S.; Davis, R.J.; McLaren, A.; Cohen, P. A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun. EMBO J. 2003, 22, 3876–3886. [Google Scholar] [CrossRef] [PubMed]
  51. Milne, D.M.; Campbell, D.G.; Caudwell, F.B.; Meek, D.W. Phosphorylation of the tumor suppressor protein P53 by mitogen-activated protein kinases. J. Biol. Chem. 1994, 269, 9253–9260. [Google Scholar] [PubMed]
  52. Yeh, P.Y.; Chuang, S.; Yeh, K.; Song, Y.C.; Chang, L.L.; Cheng, A. Phosphorylation of P53 on Thr55 by ERK2 is necessary for doxorubicin-induced P53 activation and cell death. Oncogene 2004, 23, 3580–3588. [Google Scholar] [CrossRef] [PubMed]
  53. Biswas, S.C.; Greene, L.A. Nerve growth factor (NGF) down-regulates the Bcl-2 homology 3 (BH3) domain-only protein BIM and suppresses its proapoptotic activity by phosphorylation. J. Biol. Chem. 2002, 277, 49511–49516. [Google Scholar] [CrossRef] [PubMed]
  54. Allan, L.A.; Morrice, N.; Brady, S.; Magee, G.; Pathak, S.; Clarke, P.R. Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK/MAPK. Nat. Cell Biol. 2003, 5, 647–654. [Google Scholar] [CrossRef] [PubMed]
  55. Scheid, M.P.; Schubert, K.M.; Duronio, V. Regulation of bad phosphorylation and association with Bcl-Xl by the MAPK/ERK kinase. J. Biol. Chem. 1999, 274, 31108–31113. [Google Scholar] [CrossRef] [PubMed]
  56. Garnovskaya, M.N.; Mukhin, Y.V.; Vlasova, T.M.; Grewal, J.S.; Ullian, M.E.; Tholanikunnel, B.G.; Raymond, J.R. Mitogen-induced rapid phosphorylation of serine 795 of the retinoblastoma gene product in vascular smooth muscle cells involves ERK activation. J. Biol. Chem. 2004, 279, 24899–24905. [Google Scholar] [CrossRef] [PubMed]
  57. Yang, X.; Gabuzda, D. Mitogen-activated protein kinase phosphorylates and regulates the HIV-1 VIF protein. J. Biol. Chem. 1998, 273, 29879–29887. [Google Scholar] [CrossRef] [PubMed]
  58. Bermudez, O.; Pagès, G.; Gimond, C. The dual-specificity MAP kinase phosphatases: Critical roles in development and cancer. Am. J. Physiol. Cell Physiol. 2010, 299, 189–202. [Google Scholar] [CrossRef] [PubMed]
  59. Rubin, C.; Litvak, V.; Medvedovsky, H.; Zwang, Y.; Lev, S.; Yarden, Y. Sprouty fine-tunes EGF signaling through interlinked positive and negative feedback loops. Curr. Biol. 2003, 13, 297–307. [Google Scholar] [CrossRef]
  60. Therrien, M.; Chang, H.C.; Solomon, N.M.; Karim, F.D.; Wassarman, D.A.; Rubin, G.M. KSR, a novel protein kinase required for RAS signal transduction. Cell 1995, 83, 879–888. [Google Scholar] [CrossRef]
  61. Keller, E.T.; Fu, Z.; Brennan, M. The role of RAF kinase inhibitor protein (RKIP) in health and disease. Biochem. Pharmacol. 2004, 68, 1049–1053. [Google Scholar] [CrossRef] [PubMed]
  62. Park, S.; Yeung, M.L.; Beach, S.; Shields, J.M.; Yeung, K.C. RKIP downregulates BRAF kinase activity in melanoma cancer cells. Oncogene 2005, 24, 3535–3540. [Google Scholar] [CrossRef] [PubMed]
  63. Brummer, T.; Martin, P.; Herzog, S.; Misawa, Y.; Daly, R.J.; Reth, M. Functional analysis of the regulatory requirements of BRAF and the BRAF V600E oncoprotein. Oncogene 2006, 25, 6262–6276. [Google Scholar] [CrossRef] [PubMed]
  64. Tsavachidou, D.; Coleman, M.L.; Athanasiadis, G.; Li, S.; Licht, J.D.; Olson, M.F.; Weber, B.L. Spry2 is an inhibitor of the RAS/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599e mutant. Cancer Res. 2004, 64, 5556–5559. [Google Scholar] [CrossRef] [PubMed]
  65. Pratilas, C.A.; Solit, D.B. Targeting the mitogen-activated protein kinase pathway: Physiological feedback and drug response. Clin. Cancer Res. 2010, 16, 3329–3334. [Google Scholar] [CrossRef] [PubMed]
  66. Robert, C.; Karaszewska, B.; Schachter, J.; Rutkowski, P.; Mackiewicz, A.; Stroiakovski, D.; Lichinitser, M.; Dummer, R.; Grange, F.; Mortier, L.; et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N. Engl. J. Med. 2015, 372, 30–39. [Google Scholar] [CrossRef] [PubMed]
  67. Chapman, P.B.; Hauschild, A.; Robert, C.; Haanen, J.B.; Ascierto, P.; Larkin, J.; Dummer, R.; Garbe, C.; Testori, A.; Maio, M.; et al. Improved survival with vemurafenib in melanoma with BRAF V600e mutation. N. Engl. J. Med. 2011, 364, 2507–2516. [Google Scholar] [CrossRef] [PubMed]
  68. Gibney, G.T.; Messina, J.L.; Fedorenko, I.V.; Sondak, V.K.; Smalley, K.S. Paradoxical oncogenesis—The long-term effects of braf inhibition in melanoma. Nat. Rev. Clin. Oncol. 2013, 10, 390–399. [Google Scholar] [CrossRef] [PubMed]
  69. Corcoran, R.B.; Atreya, C.E.; Falchook, G.S.; Infante, J.R.; Hamid, O.; Messersmith, W.A.; Daud, A.; Kwak, E.L.; Ryan, D.; Kurzrock, R. Phase 1–2 trial of the BRAF inhibitor dabrafenib (D) plus MEK inhibitor trametinib (T) in BRAF V600 mutant colorectal cancer (CRC): Updated efficacy and biomarker analysis. In Proceedings of the ASCO Annual Meeting, Chicago, IL, USA, 30 May–3 June 2014.
  70. Hatzivassiliou, G.; Song, K.; Yen, I.; Brandhuber, B.J.; Anderson, D.J.; Alvarado, R.; Ludlam, M.J.; Stokoe, D.; Gloor, S.L.; Vigers, G.; et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 2010, 464, 431–435. [Google Scholar] [CrossRef] [PubMed]
  71. Heidorn, S.J.; Milagre, C.; Whittaker, S.; Nourry, A.; Niculescu-Duvas, I.; Dhomen, N.; Hussain, J.; Reis-Filho, J.S.; Springer, C.J.; Pritchard, C.; et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 2010, 140, 209–221. [Google Scholar] [CrossRef] [PubMed]
  72. Poulikakos, P.I.; Zhang, C.; Bollag, G.; Shokat, K.M.; Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 2010, 464, 427–430. [Google Scholar] [CrossRef] [PubMed]
  73. Oberholzer, P.A.; Kee, D.; Dziunycz, P.; Sucker, A.; Kamsukom, N.; Jones, R.; Roden, C.; Chalk, C.J.; Ardlie, K.; Palescandolo, E. RAS mutations are associated with the development of cutaneous squamous cell tumors in patients treated with RAF inhibitors. J. Clin. Oncol. 2012, 30, 316–321. [Google Scholar] [CrossRef] [PubMed]
  74. Alcala, A.M.; Flaherty, K.T. BRAF inhibitors for the treatment of metastatic melanoma: Clinical trials and mechanisms of resistance. Clin. Cancer Res. 2012, 18, 33–39. [Google Scholar] [CrossRef] [PubMed]
  75. Rizos, H.; Menzies, A.M.; Pupo, G.M.; Carlino, M.S.; Fung, C.; Hyman, J.; Haydu, L.E.; Mijatov, B.; Becker, T.M.; Boyd, S.C.; et al. BRAF inhibitor resistance mechanisms in metastatic melanoma: Spectrum and clinical impact. Clin. Cancer Res. 2014, 20, 1965–1977. [Google Scholar] [CrossRef] [PubMed]
  76. Johnson, D.B.; Menzies, A.M.; Zimmer, L.; Eroglu, Z.; Ye, F.; Zhao, S.; Rizos, H.; Sucker, A.; Scolyer, R.A.; Gutzmer, R.; et al. Acquired BRAF inhibitor resistance: A multicenter meta-analysis of the spectrum and frequencies, clinical behaviour, and phenotypic associations of resistance mechanisms. Eur. J. Cancer 2015, 51, 2792–2799. [Google Scholar] [CrossRef] [PubMed]
  77. Nazarian, R.; Shi, H.; Wang, Q.; Kong, X.; Koya, R.C.; Lee, H.; Chen, Z.; Lee, M.K.; Attar, N.; Sazegar, H.; et al. melanomas acquire resistance to BRAF V600E inhibition by RTK or NRAS upregulation. Nature 2010, 468, 973–977. [Google Scholar] [CrossRef] [PubMed]
  78. Long, G.V.; Fung, C.; Menzies, A.M.; Pupo, G.M.; Carlino, M.S.; Hyman, J.; Shahheydari, H.; Tembe, V.; Thompson, J.F.; Saw, R.P.; et al. Increased MAPK reactivation in early resistance to dabrafenib/trametinib combination therapy of BRAF-mutant metastatic melanoma. Nat. Commun. 2014, 5, 5694. [Google Scholar] [CrossRef] [PubMed]
  79. Wagle, N.; Emery, C.; Berger, M.F.; Davis, M.J.; Sawyer, A.; Pochanard, P.; Kehoe, S.M.; Johannessen, C.M.; Macconaill, L.E.; Hahn, W.C.; et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. J. Clin. Oncol. 2011, 29, 3085–3096. [Google Scholar] [CrossRef] [PubMed]
  80. Corcoran, R.B.; Ebi, H.; Turke, A.B.; Coffee, E.M.; Nishino, M.; Cogdill, A.P.; Brown, R.D.; della Pelle, P.; Dias-Santagata, D.; Hung, K.E.; et al. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov. 2012, 2, 227–235. [Google Scholar] [CrossRef] [PubMed]
  81. Hoogstraat, M.; Hooijdonk, C.G.G.; Ubink, I.; Besselink, N.J.; Pieterse, M.; Veldhuis, W.; van Stralen, M.; Meijer, E.F.; Willems, S.M.; Hadders, M.A.; et al. Detailed imaging and genetic analysis reveal a secondary BRAF L505h resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib. Pigment Cell Melanoma Res. 2015, 28, 318–323. [Google Scholar] [CrossRef] [PubMed]
  82. Wagenaar, T.R.; Ma, L.; Roscoe, B.; Park, S.M.; Bolon, D.N.; Green, M.R. Resistance to vemurafenib resulting from a novel mutation in the BRAF V600E kinase domain. Pigment Cell Melanoma Res. 2014, 27, 124–133. [Google Scholar] [CrossRef] [PubMed]
  83. Poulikakos, P.I.; Persaud, Y.; Janakiraman, M.; Kong, X.; Ng, C.; Moriceau, G.; Shi, H.; Atefi, M.; Titz, B.; Gabay, M.T.; et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF V600E. Nature 2011, 480, 387–390. [Google Scholar] [CrossRef] [PubMed]
  84. Raaijmakers, M.I.; Widmer, D.S.; Narechania, A.; Eichhoff, O.; Freiberger, S.N.; Wenzina, J.; Cheng, P.F.; Mihic-Probst, D.; Desalle, R.; Dummer, R.; et al. Co-existence of BRAF and NRAS driver mutations in the same melanoma cells results in heterogeneity of targeted therapy resistance. Oncotarget 2016, 7, 77163–77174. [Google Scholar] [CrossRef] [PubMed]
  85. Danysh, B.P.; Rieger, E.Y.; Sinha, D.K.; Evers, C.V.; Cote, G.J.; Cabanillas, M.E.; Hofmann, M.C. Long-term vemurafenib treatment drives inhibitor resistance through a spontaneous KRAS G12D mutation in a BRAF V600E papillary thyroid carcinoma model. Oncotarget 2016, 7, 30907–30923. [Google Scholar] [CrossRef] [PubMed]
  86. Ahronian, L.G.; Sennott, E.M.; van Allen, E.M.; Wagle, N.; Kwak, E.L.; Faris, J.E.; Godfrey, J.T.; Nishimura, K.; Lynch, K.D.; Mermel, C.H.; et al. Clinical acquired resistance to RAF inhibitor combinations in BRAF-mutant colorectal cancer through MAPK pathway alterations. Cancer Discov. 2015, 5, 358–367. [Google Scholar] [CrossRef] [PubMed]
  87. Johannessen, C.M.; Boehm, J.S.; Kim, S.Y.; Thomas, S.R.; Wardwell, L.; Johnson, L.A.; Emery, C.M.; Stransky, N.; Cogdill, A.P.; Barretina, J.; et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 2010, 468, 968–972. [Google Scholar] [CrossRef] [PubMed]
  88. Montagut, C.; Sharma, S.V.; Shioda, T.; McDermott, U.; Ulman, M.; Ulkus, L.E.; Dias-Santagata, D.; Stubbs, H.; Lee, D.Y.; Singh, A.; et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res. 2008, 68, 4853–4861. [Google Scholar] [CrossRef] [PubMed]
  89. Grbovic, O.M.; Basso, A.D.; Sawai, A.; Ye, Q.; Friedlander, P.; Solit, D.; Rosen, N. V600e BRAF requires the Hsp90 chaperone for stability and is degraded in response to Hsp90 inhibitors. Proc. Natl. Acad. Sci. USA 2006, 103, 57–62. [Google Scholar] [CrossRef] [PubMed]
  90. Da Rocha Dias, S.; Friedlos, F.; Light, Y.; Springer, C.; Workman, P.; Marais, R. Activated BRAF is an Hsp90 client protein that is targeted by the anticancer drug 17-allylamino-17-demethoxygeldanamycin. Cancer Res. 2005, 65, 10686–10691. [Google Scholar] [CrossRef] [PubMed]
  91. Maloney, A.; Clarke, P.A.; Workman, P. Genes and proteins governing the cellular sensitivity to Hsp90 inhibitors: A mechanistic perspective. Curr. Cancer Drug Targets 2003, 3, 331–341. [Google Scholar] [CrossRef] [PubMed]
  92. Acquaviva, J.; Smith, D.L.; Jimenez, J.P.; Zhang, C.; Sequeira, M.; He, S.; Sang, J.; Bates, R.C.; Proia, D.A. Overcoming acquired BRAF inhibitor resistance in melanoma via targeted inhibition of Hsp90 with ganetespib. Mol. Cancer Ther. 2014, 13, 353–363. [Google Scholar] [CrossRef] [PubMed]
  93. Paraiso, K.H.; Haarberg, H.E.; Wood, E.; Rebecca, V.W.; Chen, Y.A.; Xiang, Y.; Ribas, A.; Lo, R.S.; Weber, J.S.; Sondak, V.K.; et al. The Hsp90 inhibitor Xl888 overcomes BRAF inhibitor resistance mediated through diverse mechanisms. Clin. Cancer Res. 2012, 18, 2502–2514. [Google Scholar] [CrossRef] [PubMed]
  94. Wang, H.; Lu, M.; Yao, M.; Zhu, W. Effects of treatment with an Hsp90 inhibitor in tumors based on 15 phase II clinical trials. Mol. Clin. Oncol. 2016, 5, 326–334. [Google Scholar] [CrossRef] [PubMed]
  95. Solit, D.B.; Osman, I.; Polsky, D.; Panageas, K.S.; Daud, A.; Goydos, J.S.; Teitcher, J.; Wolchok, J.D.; Germino, F.J.; Krown, S.E.; et al. Phase II trial of 17-allylamino-17-demethoxygeldanamycin in patients with metastatic melanoma. Clin. Cancer Res. 2008, 14, 8302–8307. [Google Scholar] [CrossRef] [PubMed]
  96. Pacey, S.; Gore, M.; Chao, D.; Banerji, U.; Larkin, J.; Sarker, S.; Owen, K.; Asad, Y.; Raynaud, F.; Walton, M.; et al. A phase II trial of 17-allylamino, 17-demethoxygeldanamycin (17-Aag, Tanespimycin) in patients with metastatic melanoma. Investig. New Drugs 2012, 30, 341–349. [Google Scholar] [CrossRef] [PubMed]
  97. Pratilas, C.A.; Taylor, B.S.; Ye, Q.; Viale, A.; Sander, C.; Solit, D.B.; Rosen, N. BRAF V600e is associated with disabled feedback inhibition of RAF/MEK signaling and elevated transcriptional output of the pathway. Proc. Natl. Acad. Sci. USA 2009, 106, 4519–4524. [Google Scholar] [CrossRef] [PubMed]
  98. Spagnolo, F.; Ghiorzo, P.; Queirolo, P. Overcoming resistance to BRAF inhibition in BRAF-mutated metastatic melanoma. Oncotarget 2014, 5, 10206–10221. [Google Scholar] [CrossRef] [PubMed]
  99. Chung, J.; Grammer, T.C.; Lemon, K.P.; Kazlauskas, A.; Blenis, J. PDGF- and insulin-dependent PP70S6K activation mediated by phosphatidylinositol-3-OH kinase. Nature 1994, 370, 71–75. [Google Scholar] [CrossRef]
  100. Sun, C.; Wang, L.; Huang, S.; Heynen, G.J.; Prahallad, A.; Robert, C.; Haanen, J.; Blank, C.; Wesseling, J.; Willems, S.M.; et al. Reversible and adaptive resistance to BRAF V600e inhibition in melanoma. Nature 2014, 508, 118–122. [Google Scholar] [CrossRef] [PubMed]
  101. Shi, H.; Kong, X.; Ribas, A.; Lo, R.S. Combinatorial treatments that overcome PDGFRβ-driven resistance of melanoma cells to BRAF V600e inhibition. Cancer Res. 2011, 71, 5067–5074. [Google Scholar] [CrossRef]
  102. Villanueva, J.; Vultur, A.; Lee, J.T.; Somasundaram, R.; Fukunaga-Kalabis, M.; Cipolla, A.K.; Wubbenhorst, B.; Xu, X.; Gimotty, P.A.; Kee, D.; et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 2010, 18, 683–695. [Google Scholar] [CrossRef] [PubMed]
  103. Wilson, T.R.; Fridlyand, J.; Yan, Y.; Penuel, E.; Burton, L.; Chan, E.; Peng, J.; Lin, E.; Wang, Y.; Sosman, J.; et al. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature 2012, 487, 505–509. [Google Scholar] [CrossRef] [PubMed]
  104. Straussman, R.; Morikawa, T.; Shee, K.; Barzily-Rokni, M.; Qian, Z.R.; Du, J.; Davis, A.; Mongare, M.M.; Gould, J.; Frederick, D.T.; et al. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 2012, 487, 500–504. [Google Scholar] [CrossRef] [PubMed]
  105. Shi, H.; Hugo, W.; Kong, X.; Hong, A.; Koya, R.C.; Moriceau, G.; Chodon, T.; Guo, R.; Johnson, D.B.; Dahlman, K.B.; et al. Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov. 2014, 4, 80–93. [Google Scholar] [CrossRef] [PubMed]
  106. Aguissa-Toure, A.H.; Li, G. Genetic alterations of PTEN in human melanoma. Cell Mol. Life Sci. 2012, 69, 1475–1491. [Google Scholar] [CrossRef] [PubMed]
  107. Turajlic, S.; Furney, S.J.; Stamp, G.; Rana, S.; Ricken, G.; Oduko, Y.; Saturno, G.; Springer, C.; Hayes, A.; Gore, M.; et al. Whole-genome sequencing reveals complex mechanisms of intrinsic resistance to BRAF inhibition. Ann. Oncol. 2014, 25, 959–967. [Google Scholar] [CrossRef] [PubMed]
  108. Rebecca, V.W.; Alicea, G.M.; Paraiso, K.H.; Lawrence, H.; Gibney, G.T.; Smalley, K.S. Vertical inhibition of the MAPK pathway enhances therapeutic responses in NRAS-mutant melanoma. Pigment Cell Melanoma Res. 2014, 27, 1154–1158. [Google Scholar] [CrossRef] [PubMed]
  109. Paraiso, K.H.; Xiang, Y.; Rebecca, V.W.; Abel, E.V.; Chen, Y.A.; Munko, A.C.; Wood, E.; Fedorenko, I.V.; Sondak, V.K.; Anderson, A.R.A. PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression. Cancer Res. 2011, 71, 2750–2760. [Google Scholar] [CrossRef] [PubMed]
  110. Marampon, F.; Ciccarelli, C.; Zani, B.M. Down-regulation of C-Myc following MEK/ERK inhibition halts the expression of malignant phenotype in rhabdomyosarcoma and in non muscle-derived human tumors. Mol. Cancer 2006, 5, 31. [Google Scholar] [CrossRef] [PubMed]
  111. Zawistowski, J.S.; Bevill, S.M.; Goulet, D.R.; Stuhlmiller, T.J.; Beltran, A.S.; Olivares-Quintero, J.F.; Singh, D.; Sciaky, N.; Parker, J.S.; Rashid, N.U.; et al. Enhancer remodeling during adaptive bypass to MEK inhibition is attenuated by pharmacological targeting of the P-TEFB complex. Cancer Discov. 2017. [Google Scholar] [CrossRef] [PubMed]
  112. Fattore, L.; Mancini, R.; Acunzo, M.; Romano, G.; Lagana, A.; Pisanu, M.E.; Malpicci, D.; Madonna, G.; Mallardo, D.; Capone, M.; et al. Mir-579-3p controls melanoma progression and resistance to target therapy. Proc. Natl. Acad. Sci. USA 2016, 113, 5005–5013. [Google Scholar] [CrossRef] [PubMed]
  113. Clarke, C.N.; Kopetz, E.S. BRAF mutant colorectal cancer as a distinct subset of colorectal cancer: Clinical characteristics, clinical behavior, and response to targeted therapies. J. Gastrointest. Oncol. 2015, 6, 660–667. [Google Scholar] [PubMed]
  114. Pietrantonio, F.; Petrelli, F.; Coinu, A.; di Bartolomeo, M.; Borgonovo, K.; Maggi, C.; Cabiddu, M.; Iacovelli, R.; Bossi, I.; Lonati, V.; et al. Predictive role of BRAF mutations in patients with advanced colorectal cancer receiving cetuximab and panitumumab: A meta-analysis. Eur. J. Cancer 2015, 51, 587–594. [Google Scholar] [CrossRef] [PubMed]
  115. Shi, H.; Moriceau, G.; Kong, X.; Lee, M.K.; Lee, H.; Koya, R.C.; Ng, C.; Chodon, T.; Scolyer, R.A.; Dahlman, K.B.; et al. Melanoma whole-exome sequencing identifies BRAF V600e amplification-mediated acquired BRAF inhibitor resistance. Nat. Commun. 2012, 3, 724. [Google Scholar] [CrossRef] [PubMed]
  116. Prahallad, A.; Sun, C.; Huang, S.; di Nicolantonio, F.; Salazar, R.; Zecchin, D.; Beijersbergen, R.L.; Bardelli, A.; Bernards, R. Unresponsiveness of colon cancer to BRAF V600e inhibition through feedback activation of EGFR. Nature 2012, 483, 100–103. [Google Scholar] [CrossRef] [PubMed]
  117. Wang, R.; He, G.; Nelman-Gonzalez, M.; Ashorn, C.L.; Gallick, G.E.; Stukenberg, P.T.; Kirschner, M.W.; Kuang, J. Regulation of Cdc25C by ERK/MAP kinases during the G2/M transition. Cell 2007, 128, 1119–1132. [Google Scholar] [CrossRef] [PubMed]
  118. Edwin, F.; Anderson, K.; Ying, C.; Patel, T.B. Intermolecular interactions of sprouty proteins and their implications in development and disease. Mol. Pharmacol. 2009, 76, 679–691. [Google Scholar] [CrossRef] [PubMed]
  119. Mao, M.; Tian, F.; Mariadason, J.M.; Tsao, C.C.; Lemos, R.; Dayyani, F.; Gopal, Y.N.V.; Jiang, Z.; Wistuba, I.I.; Tang, X.M. Resistance to BRAF inhibition in BRAF-mutant colon cancer can be overcome with PI3K inhibition or demethylating agents. Clin. Cancer Res. 2013, 19, 657–667. [Google Scholar] [CrossRef] [PubMed]
  120. Yaeger, R.; Cercek, A.; O’Reilly, E.M.; Reidy, D.L.; Kemeny, N.; Wolinsky, T.; Capanu, M.; Gollub, M.J.; Rosen, N.; Berger, M.F.; et al. Pilot trial of combined BRAF and EGFR inhibition in BRAF-mutant metastatic colorectal cancer patients. Clin. Cancer Res. 2015, 21, 1313–1320. [Google Scholar] [CrossRef] [PubMed]
  121. Corcoran, R.B.; Atreya, C.E.; Falchook, G.S.; Kwak, E.L.; Ryan, D.P.; Bendell, J.C.; Hamid, O.; Messersmith, W.A.; Daud, A.; Kurzrock, R.; et al. Combined BRAF and MEK inhibition with dabrafenib and trametinib in BRAF V600-mutant colorectal cancer. J. Clin. Oncol. 2015, 33, 4023–4031. [Google Scholar] [CrossRef] [PubMed]
  122. Kopetz, S.; Desai, J.; Chan, E.; Hecht, J.; O’dwyer, P.; Lee, R.; Nolop, K.; Saltz, L. PLX4032 in metastatic colorectal cancer patients with mutant BRAF tumors. In Proceedings of the ASCO Annual Meeting, Hollywood, FL, USA, 18–20 October 2010; p. 3534.
  123. Samowitz, W.S.; Sweeney, C.; Herrick, J.; Albertsen, H.; Levin, T.R.; Murtaugh, M.A.; Wolff, R.K.; Slattery, M.L. Poor survival associated with the BRAF V600e mutation in microsatellite-stable colon cancers. Cancer Res. 2005, 65, 6063–6069. [Google Scholar] [CrossRef] [PubMed]
  124. Yokota, T.; Ura, T.; Shibata, N.; Takahari, D.; Shitara, K.; Nomura, M.; Kondo, C.; Mizota, A.; Utsunomiya, S.; Muro, K. BRAF mutation is a powerful prognostic factor in advanced and recurrent colorectal cancer. Br. J. Cancer 2011, 104, 856–862. [Google Scholar] [CrossRef] [PubMed]
  125. Long, G.V.; Menzies, A.M.; Nagrial, A.M.; Haydu, L.E.; Hamilton, A.L.; Mann, G.J.; Hughes, T.M.; Thompson, J.F.; Scolyer, R.A.; Kefford, R.F. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J. Clin. Oncol. 2011, 29, 1239–1246. [Google Scholar] [CrossRef] [PubMed]
  126. Prahallad, A.; Heynen, G.J.; Germano, G.; Willems, S.M.; Evers, B.; Vecchione, L.; Gambino, V.; Lieftink, C.; Beijersbergen, R.L.; di Nicolantonio, F.; et al. PTPN11 is a central node in intrinsic and acquired resistance to targeted cancer drugs. Cell Rep. 2015, 12, 1978–1985. [Google Scholar] [CrossRef]
Figure 1. RAS/RAF/MEK aberrant signaling and mechanisms of resistance to inhibition in melanoma. Oncogenic BRAFV600E cells become independent from external growth factors (GF) (triangle symbol marked with a X) and other stimuli leading to constitutive activation of the MAPK pathway. Increased MAPK signalling (four arrows) eventually leads to enhanced gene expression including MAP kinase phosphatases (DUSPs) and sprouty proteins (SPRYs). Despite elevation of those important inhibitory regulators (T lines) of the MAPK pathway, tumour cells adapt and rely on neighbour pathways, such as the PI3K pathway, to grow and survive, Furthermore, negative inhibitory mechanisms of the MAPK pathway, including inactivation of BRAF via ERK1/2, are now lessened (faded T) due to conformational changes in the BRAF. Conferred mechanisms of resistance to BRAF inhibitors including up-regulation of PDGFRB, RAS mutations, elevation of CRAF, BRAFV600E amplification, alternative splicing of BRAFV600E, elevation of COT (MAP3K8), MEK mutation, PTEN loss, PI3K and AKT mutations were highlighted in green.
Figure 1. RAS/RAF/MEK aberrant signaling and mechanisms of resistance to inhibition in melanoma. Oncogenic BRAFV600E cells become independent from external growth factors (GF) (triangle symbol marked with a X) and other stimuli leading to constitutive activation of the MAPK pathway. Increased MAPK signalling (four arrows) eventually leads to enhanced gene expression including MAP kinase phosphatases (DUSPs) and sprouty proteins (SPRYs). Despite elevation of those important inhibitory regulators (T lines) of the MAPK pathway, tumour cells adapt and rely on neighbour pathways, such as the PI3K pathway, to grow and survive, Furthermore, negative inhibitory mechanisms of the MAPK pathway, including inactivation of BRAF via ERK1/2, are now lessened (faded T) due to conformational changes in the BRAF. Conferred mechanisms of resistance to BRAF inhibitors including up-regulation of PDGFRB, RAS mutations, elevation of CRAF, BRAFV600E amplification, alternative splicing of BRAFV600E, elevation of COT (MAP3K8), MEK mutation, PTEN loss, PI3K and AKT mutations were highlighted in green.
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Table 1. Cellular targets of ERK.
Table 1. Cellular targets of ERK.
CategoryProteinEffect of ERK Phosphorylation on Its FunctionsReference
Kinases and phosphatasesMEK1/2Either enhances its activity or reduces it depending on the phosphorylation site[29]
CRAFInhibits its activity[30,31]
BRAFInhibits its activity[32]
RSKActivation and further signal transduction[33]
DUSPsNegative feedback loop-indirectly via dephosphorylating ERK1/2[36,37,38]
SPRYsNegative feedback loop-directly inactivating upstream[39,40]
Signalling proteinsEGFRDownregulation of the MAPK pathway[41]
Gab2 *Reduces its activation[42]
SOS *Negative feedback mechanism via preventing its association with Gab2[43]
IRS1 *Impaired its downstream signalling[44]
TSC2Weakens its ability to pair with TSC1, therefore Impairs its ability to inhibit mTOR signalling[45]
Cytoskeletal proteinsCrystalline αAnti-apoptotic protection[46,47]
Transcription FactorsELK *Transcription of c-Fos[47,48]
c-Fos *Acts as a sensor for ERKs’ signal duration[49]
c-Jun *Transcription of c-Jun[50]
p53Tumour suppressor protein, plays a role in cell cycle[51,52]
c-Myc *Transcription[41]
Apoptotic proteinsBIM *Inhibit its pro-apoptotic function[53]
Caspase9Reduce its pro-apoptotic function[54]
Bad *Inhibit its pro-apoptotic function[55]
Other proteinsRB *Cell cycle progression[56]
Vif *Activates HIV-1 replication[57]
* Abbreviations: Gab2, GRB2 Associated Binding Protein; SOS, Ras/Rac Guanine Nucleotide Exchange Factor; IRS1, Insulin Receptor Substrate 1; ELK, ETS domain-containing protein Elk-1; c-Fos, Fos proto-oncogene; c-Jun, Jun proto-oncogene; c-Myc, v-myc avian myelocytomatosis viral oncogene homolog; BIM, Bcl-2 like proteins; Bad, Bcl-2 Associated Agonist of Cell Death; RB, retinoblastoma; Vif, Virion infectivity factor.
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