Melanoma is an aggressive type of skin cancer that is responsible for the greatest number of skin cancer-related deaths worldwide. Discovery of the BRAF mutation in more than 50% of melanomas has significantly changed the therapeutic management of metastatic melanoma in the past decade. BRAF proto-oncogene is a component of the mitogen activated protein kinase (MAPK) signaling pathway that leads to cell proliferation, differentiation and survival upon activation. Following the development and application of the highly selective BRAF inhibitors, many unanticipated adverse effects were reported. Cutaneous squamous cell carcinoma (cSCC) is the most common side effect of these drugs and can lead to premature termination or dose reduction of therapy. Even though the underlying mechanisms by which BRAF inhibitors induce cSCC in normal cells still remains unclear, it is largely believed that BRAF inhibitors paradoxically activate MAPK signaling pathway in wild type BRAF cells. Pre-existing tumor-driving mutations, such as RAS mutation, or epigenetic changes in these cells contribute to the BRAF inhibitor- induced paradoxical activation of MAPK pathway. BRAF inhibitors have also exhibited an off-target effect on other signaling pathways involved in tumor suppression. This review summarizes the proposed mechanisms by which BRAF inhibitors induce cSCC initiation and progression in normal cells in melanoma patients. A better understanding of the underlying mechanisms that drive BRAF inhibitor-induced cSCC is crucial for the development of future therapeutic interventions to improve the quality of life for the patients receiving targeted cancer therapy.

Introduction 

Melanoma is the most aggressive form of skin cancer arising from melanocytes, which are specialised cells producing pigments in the skin, hair and eyes. Melanocytes provide protection from damaging effects of ultraviolet radiation.1 Even though melanomas account for only 4% of all skin cancers, they are responsible for the greatest number of skin cancer-related deaths.1 Melanoma can be cured by surgical resection if diagnosed early, with 5 year survival rates of up to 25% in patients with isolated metastases. However, it is largely ineffective in patients with distant metastases.2 Understanding the molecular biology and role of underlying mutations is crucial for development of genome-specific anti- cancer agents.

Several gene mutations have been associated with an increased risk of melanoma. In 2002, the discovery of a high incidence of BRAF point mutations significantly changed the therapeutic management of this cancer.3 Activating BRAF mutations account for more than 50% of all melanomas and are more common in skins cells following intermittent exposure to UV radiation when compared to chronic exposure.1,4,6 BRAF is a serine/ threonine protein kinase that activates the MAP kinase/ERK signaling pathway.4 The MAPK pathway regulates cell proliferation, differentiation and survival by activating gene expression.1 Activating mutations in key effectors of MAPK signalling have been found in up to 90% of melanoma cases. The most common activating mutation is a single amino acid substitution from valine (V) to glutamate (E) at position 600 of the BRAF protein (BRAF V600E).5-8 BRAF inhibition blocks cell proliferation and induces apoptosis in both in vitro and in vivo models.1 BRAF V600E therefore provides an efficacious therapeutic target in treating melanomas.1,7

Although BRAF inhibitor therapy has resulted in a 74% decrease in risk of death and disease progression, the development of cutaneous squamous cell carcinoma (cSCC) is an unanticipated adverse effect of BRAF inhibition. cSCC has been reported in 18-31% of treated patients, resulting in early termination of treatment in some cases.8,9 Currently, the mechanism by which BRAF inhibitors drive cSCC is not fully understood.

This review briefly summarises the role of activating BRAF mutations in melanoma and its inhibition as a therapeutic intervention. It also outlines the proposed mechanisms by which BRAF inhibitors drive cSCC in wild type BRAF cells.

1. Mitogen-activated protein kinase (MAPK) pathway

The mitogen-activated protein kinase (MAPK) pathway connects extracellular signalling to the nucleus leading to increased expression of genes required for cell cycle entry.10 Binding of a proper ligand, such as a growth factor or a hormone, to receptor tyrosine kinase (RTK) on the surface of the cell membrane activates the RAS protein.
The RAS protein then acts on its major cytosolic effector: the RAF protein family. RAF protein signals through phosphorylation and downstream activation of the MEK kinase protein which subsequently phosphorylates and activates ERK mitogen activated proteins. The ERK protein, upon activation, is translocated to the nucleus and regulates gene expression.11 In normal cells, this signaling transduction pathway regulates cell proliferation, differentiation, senescence, and survival. In cancer cells this pathway is constitutively activated, driving tumor growth.12 Three RAF proteins (ARAF, BRAF, and CRAF) have been characterized; activating mutations in any of the RAF proteins have been implicated in cancer cell proliferation.4 Given the high frequency of BRAF mutations in melanoma, the BRAF proto-oncogene was found to be a promising and potentially effective therapeutic target to treat advanced melanoma.

2. BRAF inhibitors in treatment of melanoma

BRAF was first pursued as a therapeutic target in 2003 when an allele-specific siRNA was used
to suppress BRAF V600E expression in cultured melanoma cells.5 Suppression of mutated BRAF in human melanoma cells resulted in inhibition of cell proliferation and increased apoptosis.5 Characterisation of activating BRAF mutation and its stability led to the development of selective small molecule BRAF inhibitors.7

2.1. Sorafenib

Sorafenib was the first drug used to inhibit BRAF activity in patients with melanoma. This drug is not highly selective for BRAF protein kinase and can also target several other protein kinases. Sorafenib has shown little efficacy in increasing survival of melanoma patients.1

2.2. Vemurafenib

Vemurafenib is a potent inhibitor of BRAF with high selectivity for mutant BRAF compared to wild type BRAF.1 This drug was approved by the FDA in 2011 for treating candidate metastatic melanoma patients with BRAF V600 mutation.4 Vemurafenib was the first drug to result in an improvement in the survival of melanoma patients carrying BRAF V600E mutation in clinical trials.4

2.3. Dabrafenib

Dabrafenib has demonstrated a significantly higher selectivity than vemurafenib for mutant BRAF over wild type to the extent that only patients with BRAF mutation at position 600 respond to this therapy. Dabrafenib was approved by the FDA in 2013 for the treatment of patients with metastatic melanoma harboring BRAF V600E.13

Even though the majority of patients treated with BRAF inhibitors exhibited improved rates of progression-free survival, evidence of disease progression and drug resistance appeared in a number of the patients. The emergence of resistance called for clinical trials to test the BRAF inhibitors in combination with other therapies such as MEK inhibitors.4 BRAF inhibitors in combination with MEK inhibitor therapy results in significantly reduced resistance as well as reduced skin side effects such as cSCC.1

3. BRAF inhibitor-associated cutaneous squamous cell carcinoma lesions

Cutaneous squamous cell carcinoma (cSCC) is one of the most recognised skin side effects of all the BRAF inhibitors and has been reported in 15-30% of the patients.4,23 cSCC is a common malignant skin tumor that is mostly related to sun exposure or existence of a precancerous lesion on the skin. However, BRAF inhibitor-induced cSCC can appear on both sun exposed and non-sun exposed skin areas.14 Despite similar histological appearances, there are clear differences at the molecular level between the cSCC lesions induced by BRAF inhibitors and other types of cSCCs.1 5 To date, metastatic progression of cSCC has not been documented as a result of BRAF inhibitor therapy and lesions have been easily removed by surgical resection.4 With newer and more specific drugs such as vemurafenib, lesions can appear as early as two weeks after initiation of therapy.14

4. Paradoxical activation of MAPK pathway by BRAF inhibitors in Wild type BRAF cells

4.1. The effect of BRAF inhibitors on CRAF activation
Many molecular mechanisms have been proposed for the paradoxical activation of MAPK signaling pathway following BRAF inhibition in cells with wild type BRAF. Despite this, there is still no clear evidence of the exact underlying mechanism that leads to the development of cSCC lesions. One model focuses on the activation of CRAF, another member of the RAF family, following BRAF inhibition in normal cells.14 BRAF inhibitors can induce homo- and hetero dimerisation of wild type CRAF and prime its activity.16 When BRAF is inhibited it is recruited to RAS protein where it dimerises with CRAF and activates it leading to hyperactivation of MAPK signaling pathway.17,18 Based on this model, preventing CRAF activation can prevent the undesirable side effects of BRAF inhibitors in normal cells.16 Activated RAS provides a scaffold for formation of RAF dimers and activating mutations in RAS are required to cause BRAF-mediated CRAF activation.

4.2. RAS mutation in paradoxical activation of MAPK pathway
Enhanced RAS activity has been reported in cSCC lesions from more than 50% of patients treated with BRAF inhibitors.19 Although BRAF inhibition is sufficient for the paradoxical activation of MAPK pathway, it is not enough to initiate cSCC.20 This provides an explanation for the development of cSCC lesions in only a subset of patients with pre-existing mutations following treatment.21 Even though there have been no reported cases of malignant BRAF inhibitor-induced cSCC, development of these lesions is still concerning when it is considered that RAS mutations have been found in 30% of all human cancers and have the potential to promote malignant tumour growth. Moreover, BRAF inhibitors may lead to cell proliferation and transformation in other organs harboring the mutant RAS, such as the lung, colon and pancreas.19 RAS-driven leukaemia, colon cancer and polyp formation have been previously reported during RAF inhibitor treatment.8 RAS mutations were still not detected in up to 40% of the BRAF inhibitor-induced cSCC lesions. Thus, RAS-mediated models are still not able to explain a significant portion of these skin lesions.8

4.3. BRAF inhibitors can induce cSCC by inhibition of JNK signaling
BRAF inhibitors can also cause cSCC by off- target inhibition of several kinases in the JNK signaling pathway.22 Damage from exposure to UV radiation activates JNK signaling-induced cell apoptosis. In one study, BRAF-inhibitors were shown to suppress stress-induced JNK signaling and cell apoptosis leading to development of cSCC, particularly in sun exposed areas of the skin.22 JNK signaling suppression has also been reported in BRAF-inhibitor induced cSCC lesions in melanoma patients. It has been estimated that inhibition of the JNK signaling accounts for 17.6-40% of the BRAF inhibitor-induced cSCC lesions that cannot be explained by paradoxical MAPK pathway activation.22 Different BRAF inhibitors show significant differences in their off-target suppression of JNK signaling. For example, dabrafenib appears to have very little effect in off- target suppression of apoptosis, which is consistent with its significantly lower rate of cSCC incidence (60%) compared to vemurafenib (26%).22 This finding suggests that while both drugs can cause paradoxical MAPK pathway activation, vemurafenib can also promote tumour growth and progression by inhibiting JNK signaling pathway.

4.4. Viral involvement in MAPK pathway activation
BRAF inhibitor-induced cSCC lesions have similar morphological characteristics to those caused by viruses e.g. cutaneous warts caused by human papilloma viruses (HPVs).8 Recent advances in virus detection technology has enabled the detection of several human HPVs and human polyomaviruses (HPyVs) in BRAF inhibitor-induced cSCC. Whilst this finding suggests that HPV and HPyV may contribute to the development of cSCC lesions, their precise role has not yet been proven. It is known however that these viruses promote cell proliferation through activation of MAPK signaling pathway.11 Viral infection likely increases the frequency of pre- malignant mutations contributing to the paradoxical activation of MAPK pathway in the presence of BRAF inhibitors.8

Conclusion

Activating mutations in BRAF proto-oncogene have been implicated in about 50% of melanomas. BRAF is a protein kinase that activates the MAPK signaling pathway resulting in cell proliferation, differentiation and cell survival.1 Development of BRAF inhibitors to target and inactivate this signalling pathway has revolutionised the treatment of metastatic melanoma for the past decade; however, this therapy has resulted in the development of other cutaneous adverse events. BRAF inhibitors are known to reduce cell proliferation and cause cell apoptosis in melanoma cells with BRAF mutations. However, they can have opposite effects on the wild type cells by targeting the same signalling pathway leading to development of cutaneous Squamous Cell Carcinoma in up to 30% of the patients within three months of the treatment.23

Development of cSCC following BRAF inhibition therapy is caused by paradoxical activation of MAPK signalling pathway in keratinocytes with pre- existing activating RAS mutations. BRAF inhibitors can increase MAPK signaling in wild type cells by formation of RAF dimers and activation of CRAF.14-18 Pre-existing RAS mutation is also necessary for paradoxical activation of MAPK pathway and has been reported in up to 60% of the cSCCs associated with BRAF inhibitor therapy.19-21 BRAF inhibitors can also cause off-target suppression of JNK signaling pathway inhibiting cell apoptosis in response to damage from exposure to UV radiation.22 Viral involvement, other pre-existing genetic mutations and epigenetic changes in the wild type cells also contribute to the paradoxical effect of BRAF inhibitors.8 , 1 1

To date, cSCC remains the most concerning side effect of BRAF inhibitor treatments. Surgical resection is the only treatment for BRAF inhibitor- induced cSCC. With large number of these cutaneous lesions developed, the high number of subsequent surgical procedures may severely affect a patient’s quality of life. Combined BRAF/ MEK inhibitor therapies were found to achieve more effective and durable inhibition of MAPK signalling and are currently the standard of practice for the treatment of melanoma patients.24 In patients treated with BRAF inhibitor only, the treatment is often interrupted or the delivered dose is reduced in order to control the development of cSCC lesions. The combination of BRAF and MEK inhibitor regimens has improved the skin condition allowing clinicians to continue treatment for a longer period.24,25 Although the combined therapies overcame the paradoxical activation, they resulted in increased adverse effects and drug resistance still remains an issue. Recent efforts focus on developing next-generation BRAF inhibitors that avoid paradoxical MAPK activation, therefore dubbed “paradox breakers”. Better understanding of the underlying mechanisms of paradoxical effects of BRAF inhibitors in normal cells is crucial for development of the “paradox breakers” with improved safety and efficacy.26

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