BRAF Mutation in Colorectal Cancers: From Prognostic Marker to Targetable Mutation

Abstract

The Raf murine sarcoma viral oncogene homolog B (BRAF) mutation is detected in 8-12% of metastatic colorectal cancers (mCRCs) and is strongly correlated with poor prognosis. The recent success of the BEACON CRC study and the development of targeted therapy have led to the determination of BRAF-mutated mCRCs as an independent category. For nearly two decades, a growing body of evidence has established the significance of the BRAF mutation in the development of CRC. Herein, we overview both basic and clinical data relevant to BRAF-mutated CRC, mainly focusing on the development of treatment strategies. This review is organized into eight sections, including clinicopathological features, molecular features, prognosis, the predictive value of anti-epidermal growth factor receptor (EGFR) therapy, resistant mechanisms for BRAF-targeting treatment, the heterogeneity of the BRAF mutation, future perspectives, and conclusions. A characterization of the canonical mitogen-activated protein kinase (MAPK) pathway is essential for controlling this malignancy, and the optimal combination of multiple interventions for treatments remains a point of debate.

Keywords: BRAF inhibitor; RAF–MEK–ERK signaling pathway; cancer precision medicine.

Figure 1

Signal transduction in the EGF/EGFR–RAS–RAF–MEK–MAPK pathway. (A) Regulated signal transduction in RAS/BRAF wt cells. EGFR is activated by the binding of EGF, a specific ligand to become an active homodimer. Homodimerization stimulates the autophosphorylation of tyrosine residues in the EGFR domain, which initiates the downstream signal cascade via RAS–RAF–MEK–MAPK signal transduction. Activated RAS-induced dimerization of RAFs is necessary for phosphorylating downstream effectors, MEK and ERK (left). This signal transduction pathway comprises a negative feedback loop, i.e., elevated pERK in turn causes the de-phosphorylation of EGFR and thereby attenuates this signal (right). (B) The constitutive activation of the RAF–MEK–MAPK signal in BRAF^V600E cells. BRAF^V600E-mutated cells constitutively activate its downstream effectors as a monomer in the absence of upstream signals. Given this independence, the activation of the MAPK pathway is refractory to the pERK-mediated negative feedback regulation. This explains why RAF–MEK–MAPK signal transduction is constitutively active in BRAFV600E cells. (C) BRAF inhibition with ATP competitive RAF inhibitors in BRAF^V600E. ATP-competitive inhibitors such as vemurafenib act on monomeric BRAF and suppress downstream signal transduction. These kind of kinase inhibitors can initially control the constitutive activation; however, because of the reduction of pERK, they attenuate the negative feedback. EGFR-mediated signal transduction will be reactivated before long in CRCs, as activated RAS induces RAF dimerization, between BRAF and CRAF, which is refractory to vemurafenib. Therefore, BRAF-mutated CRCs can easily overcome the monomer inhibition treatment. (D) The concept of the vertical blockade strategy: blocking both upstream and downstream of BRAF using anti-EGFR mAb and MEK inhibitor in EGFR signal axis, respectively, were developed to overcome the resistance mechanism. Theoretically, this vertical blockade can suppress the growth signaling if and only if the EGFR–RAS–MEK–MAPK pathway remains the prime pathway throughout treatment. (E) A possible signaling pathway providing resistance to the vertical blockade triplet therapy. The EGFR signal axis is not the only pathway for the proliferation of BRAF-mutated CRCs. Reactivation of other receptor tyrosine kinases (RTKs), such as HER2 and MET, was pointed out in vitro. Moreover, the RAS–RAF–MEK–MAPK pathway is known to interact with the PI3K/AKT pathway. BRAF-mutated CRCs can therefore evade the vertical blockade with triplet therapy through these alternative means. EGFR: epidermal growth factor receptor, EGF: epidermal growth Factor, Wt: wild type, pERK: phosphorylated ERK, mAb: monoclonal antibody, CRC: colorectal cancer.

Figure 1

Signal transduction in the EGF/EGFR–RAS–RAF–MEK–MAPK pathway. (A) Regulated signal transduction in RAS/BRAF wt cells. EGFR is activated by the binding of EGF, a specific ligand to become an active homodimer. Homodimerization stimulates the autophosphorylation of tyrosine residues in the EGFR domain, which initiates the downstream signal cascade via RAS–RAF–MEK–MAPK signal transduction. Activated RAS-induced dimerization of RAFs is necessary for phosphorylating downstream effectors, MEK and ERK (left). This signal transduction pathway comprises a negative feedback loop, i.e., elevated pERK in turn causes the de-phosphorylation of EGFR and thereby attenuates this signal (right). (B) The constitutive activation of the RAF–MEK–MAPK signal in BRAF^V600E cells. BRAF^V600E-mutated cells constitutively activate its downstream effectors as a monomer in the absence of upstream signals. Given this independence, the activation of the MAPK pathway is refractory to the pERK-mediated negative feedback regulation. This explains why RAF–MEK–MAPK signal transduction is constitutively active in BRAFV600E cells. (C) BRAF inhibition with ATP competitive RAF inhibitors in BRAF^V600E. ATP-competitive inhibitors such as vemurafenib act on monomeric BRAF and suppress downstream signal transduction. These kind of kinase inhibitors can initially control the constitutive activation; however, because of the reduction of pERK, they attenuate the negative feedback. EGFR-mediated signal transduction will be reactivated before long in CRCs, as activated RAS induces RAF dimerization, between BRAF and CRAF, which is refractory to vemurafenib. Therefore, BRAF-mutated CRCs can easily overcome the monomer inhibition treatment. (D) The concept of the vertical blockade strategy: blocking both upstream and downstream of BRAF using anti-EGFR mAb and MEK inhibitor in EGFR signal axis, respectively, were developed to overcome the resistance mechanism. Theoretically, this vertical blockade can suppress the growth signaling if and only if the EGFR–RAS–MEK–MAPK pathway remains the prime pathway throughout treatment. (E) A possible signaling pathway providing resistance to the vertical blockade triplet therapy. The EGFR signal axis is not the only pathway for the proliferation of BRAF-mutated CRCs. Reactivation of other receptor tyrosine kinases (RTKs), such as HER2 and MET, was pointed out in vitro. Moreover, the RAS–RAF–MEK–MAPK pathway is known to interact with the PI3K/AKT pathway. BRAF-mutated CRCs can therefore evade the vertical blockade with triplet therapy through these alternative means. EGFR: epidermal growth factor receptor, EGF: epidermal growth Factor, Wt: wild type, pERK: phosphorylated ERK, mAb: monoclonal antibody, CRC: colorectal cancer.

Figure 1

Signal transduction in the EGF/EGFR–RAS–RAF–MEK–MAPK pathway. (A) Regulated signal transduction in RAS/BRAF wt cells. EGFR is activated by the binding of EGF, a specific ligand to become an active homodimer. Homodimerization stimulates the autophosphorylation of tyrosine residues in the EGFR domain, which initiates the downstream signal cascade via RAS–RAF–MEK–MAPK signal transduction. Activated RAS-induced dimerization of RAFs is necessary for phosphorylating downstream effectors, MEK and ERK (left). This signal transduction pathway comprises a negative feedback loop, i.e., elevated pERK in turn causes the de-phosphorylation of EGFR and thereby attenuates this signal (right). (B) The constitutive activation of the RAF–MEK–MAPK signal in BRAF^V600E cells. BRAF^V600E-mutated cells constitutively activate its downstream effectors as a monomer in the absence of upstream signals. Given this independence, the activation of the MAPK pathway is refractory to the pERK-mediated negative feedback regulation. This explains why RAF–MEK–MAPK signal transduction is constitutively active in BRAFV600E cells. (C) BRAF inhibition with ATP competitive RAF inhibitors in BRAF^V600E. ATP-competitive inhibitors such as vemurafenib act on monomeric BRAF and suppress downstream signal transduction. These kind of kinase inhibitors can initially control the constitutive activation; however, because of the reduction of pERK, they attenuate the negative feedback. EGFR-mediated signal transduction will be reactivated before long in CRCs, as activated RAS induces RAF dimerization, between BRAF and CRAF, which is refractory to vemurafenib. Therefore, BRAF-mutated CRCs can easily overcome the monomer inhibition treatment. (D) The concept of the vertical blockade strategy: blocking both upstream and downstream of BRAF using anti-EGFR mAb and MEK inhibitor in EGFR signal axis, respectively, were developed to overcome the resistance mechanism. Theoretically, this vertical blockade can suppress the growth signaling if and only if the EGFR–RAS–MEK–MAPK pathway remains the prime pathway throughout treatment. (E) A possible signaling pathway providing resistance to the vertical blockade triplet therapy. The EGFR signal axis is not the only pathway for the proliferation of BRAF-mutated CRCs. Reactivation of other receptor tyrosine kinases (RTKs), such as HER2 and MET, was pointed out in vitro. Moreover, the RAS–RAF–MEK–MAPK pathway is known to interact with the PI3K/AKT pathway. BRAF-mutated CRCs can therefore evade the vertical blockade with triplet therapy through these alternative means. EGFR: epidermal growth factor receptor, EGF: epidermal growth Factor, Wt: wild type, pERK: phosphorylated ERK, mAb: monoclonal antibody, CRC: colorectal cancer.

Figure 2

History of basic studies, genetic tests, and clinical trials that together established the significance of the BRAF mutation in the treatment of CRCs. The RAF genes, ARAF, BRAF, and CRAF, were discovered in 1983. The first report of BRAF mutation in human cancers was published in 2002 and included melanoma, lung cancer, and colorectal cancer. Early studies on BRAF mutation in CRC were focused on its characteristic molecular and clinicopathological features. Although BRAF mutation had a strong negative impact on the survival, the presence of the BRAF mutation itself had little influence when making decisions on treatment options at this time. At that stage, the BRAF genetic test was not mandatory in clinical practice. Since the success of clinical trials with anti-EGFR mAbs in 2004, targeting the EGF/EGFR pathway became technically feasible in treating metastatic colorectal cancer (mCRC). Unlike for the KRAS mutation, the BRAF mutation alone was insufficient to determine indication or anti-EGFR mAb therapy. Studies using clinical samples pointed out a loss of EGFR-mediated negative feedback to be the mechanism of resistance to the BRAF inhibitor. Combination therapies suppress not only activated BRAF but the entire RAF–MEK–MAPK signal transduction pathway, successfully controlling the BRAF-mutated mCRC. ARAF, BRAF, CRAF: Raf murine sarcoma viral oncogene homolog A, B, C; EGFR: epidermal growth factor receptor; mAb: monoclonal antibody; CRC: colorectal cancer.