Melanoma is the deadliest form of skin cancer and the leading cause of death due to skin disease [1]. It has exhibited an increased incidence in the United States, with an average annual percentage change (AAPC) of 2.2% in males and 2.3% in females between 2013 and 2017. Fortunately, the rate of death between 2013 and 2018 has started to trend downward with an AAPC of −5.7% and −4.4% in males and females, respectively [2]. These decreases are likely due to a combination of more successful prevention and early detection strategies, as well as the development of increasingly efficacious therapies such as targeted BRAF or MEK inhibitors, immunotherapy, and all combinations thereof.

Melanomas arise from the malignant transformation of melanocytes, which are dendritic, pigment-producing cells located in the basal layer of the epidermis, hair follicles, inner ear, and uvea of the eye [3]. Normally, melanocytes are tasked with the protection of adjacent skin cells from solar ultraviolet radiation (UVR) by synthesizing and distributing photoprotective melanin pigment [4]. However, melanocytes are still susceptible to UV-induced DNA damage, which can lead to the formation of key driver mutations in oncogenes, including proliferation/survival regulators such as BRAF and NRAS and cell-cycle regulators such as CDK4 [5,6,7]. Additionally, UVR also leads to the rapid accumulation of passenger mutations, which might not be drivers of melanomagenesis themselves, but collectively serve to cause heightened tumor heterogeneity that complicates treatment [8]. Upon metastasis, melanomas are highly aggressive, often migrating to the brain and lungs, and five-year survival rates are 19% for patients diagnosed with stage IV disease [9].

Current treatment modalities for melanoma include surgical resection, radiotherapy, chemotherapy, immunotherapy, biochemotherapy, and targeted therapy [10]. These agents may be used alone or in combination depending on the tumor’s stage, location, and genetic profile, as well as the patient’s goals for treatment. Earlier stage disease (stages I-IIIB) can usually be fully resected [11]. Surgical margins depend on the depth of invasion: 0.5 cm for in situ melanomas, 1 cm for tumors ≤2 mm in thickness, and 2 cm for tumors >2 mm in thickness [10,12]. Surgical excision (metastasectomy) is also part of the standard of care in the treatment of a solitary site of metastasis. Radiotherapy, although rarely used in treatment of the primary tumor, can also be used for metastatic lesions, especially of the skin, bone, and brain [13]. In high-risk melanomas (stages IIC-IV), adjuvant therapy is recommended following a complete resection of the primary tumor [14].

Untargeted chemotherapies such as dacarbazine and temozolomide were the first pharmaceutical options available for advanced melanomas; however, they have been largely unsuccessful and typically do not improve overall survival [15]. Resistance to apoptosis is thought to be the major mechanism of chemotherapeutic drug failure in melanomas [16]. Untargeted chemotherapies have largely been replaced by newer options but may still have some utility in palliative treatment [15].

Approximately 70% of patients with cutaneous melanoma have oncogenic mutations in signaling pathways involving cell proliferation; targeted therapeutic strategies use small molecule inhibitors or antibodies that affect these mutated proteins [17]. FDA-approved targeted therapies for melanoma include BRAF^V600E and MEK inhibitors. Compared to chemotherapy, BRAF inhibitors improve clinical response rates, progression-free survival, and overall survival in metastatic melanoma patients with BRAF mutations [18,19]. However, their efficacy can be limited due to the rapid development of multiple mechanisms of resistance [20]. In addition to possible drug resistance, BRAF inhibitor monotherapy can also be associated with cutaneous toxicities secondary to paradoxical MAPK pathway activation [19,21]. Combined therapeutic strategies are utilized to attempt to overcome these issues. Targeting signals downstream of BRAF, such as MEK, decreases MAPK-driven acquired resistance and toxicity, resulting in a greater duration of any response to therapy and fewer side effects [22,23]. While combined anti-BRAF/MEK therapy is initially highly effective in patients with melanomas harboring mutant BRAF, eventual resistance is not uncommon [24,25,26,27,28].

Improved understanding of the pathophysiology of the role of the immune system in tumorigenesis has led to the development and approval of several immunotherapies that show promising efficacy [29,30,31]. Combination immunotherapies show even greater promise; the CheckMate 067 trial demonstrated that, among patients with advanced melanoma, sustained long-term survival was seen in a greater percentage of patients who received nivolumab (anti-PD-1) plus ipilimumab (anti-CTLA-4) compared to ipilimumab alone [32,33]. Remarkably, nivolumab/ipilimumab combination therapy is the only currently available treatment for metastatic melanoma with a median overall survival greater than five years [33]. While immunotherapies show great promise, it is possible for primary and acquired resistance to occur, especially in the case of monotherapy, which may be due to the lack of recognition by T-cells, interactions with the cancer immune cycle, and/or complex signaling pathways within the tumor microenvironment [34,35,36].

Biochemotherapy combines the apoptotic and DNA-damaging mechanisms of chemotherapy with the immunomodulatory effects of immunotherapy [10]. Compared to chemotherapy monotherapy, biochemotherapy improves median progression-free survival but does not improve overall survival and can be associated with severe toxicity [10]. Therefore, further understanding of the metastatic progression of melanomas could provide insight into novel therapies that contribute to prevention and treatment.

Oxidative stress is an important aspect involved in normal melanocyte physiology, melanoma initiation, and disease progression, as well as a potential source for novel therapies going forward. Even in the absence of disease, melanocytes are subject to a significant degree of oxidative stress that originates both exogenously, from sources such as UVR, and endogenously, from metabolic pathways including mitochondrial respiration and melanogenesis [37,38,39]. These sources of oxidative stress can then contribute towards the regulation of nuclear factor erythroid 2-like 2 (NFE2L2 encoding NRF2) related signaling pathways [40].

Notably, UV irradiation of melanocytes in vitro has been shown to induce the activation of NRF2 and the subsequent expression of antioxidant target genes including GCLM, GCLC, NQO1, and HMOX1 [41]. Furthermore, in vivo human skin UV irradiation has been shown to induce the expression of another NRF2 target, thioredoxin reductase 1, in melanocytic nevi [42]. We have also shown, using a tissue-specific knockout mouse model, that melanocytic thioredoxin reductase 1 contributes to both melanocyte development and photobiology, as we observed pigmentation defects and elevated UV-mediated 8-oxo-2′-deoxyguanosine DNA damage in its absence [43]. Overall, NRF2 and its antioxidant target genes are important to normal melanocyte physiology and the response to UVR and act as tumor suppressors in this context.

As with many cancers, melanomas exhibit elevated oxidative stress both intracellularly as well as in the tumor microenvironment [44,45]. This elevation is due, in part, to altered metabolic pathways that cancer cells engage to sustain their dysregulated proliferation, which NRF2 contributes to [46,47,48]. Classically, increased oxidative stress has been held to simply contribute to melanoma initiation and progression by inducing a microenvironment conducive to tumor heterogeneity and metastasis. This is due to the increased mutational burden, as well as the activated signaling pathways associated with heightened tissue damage, such as inflammation [49]. It would then follow that exogenous antioxidant therapies should be highly efficacious in reducing proliferation and progression. However, it is currently understood that the role played by oxidative stress in melanomas is highly complex, as cancer cells establish a reprogrammed redox homeostasis that enables the tumor-promoting aspects of oxidative stress while avoiding its associated toxicity [50]. This is often seen by the activation of NRF2 in melanomas, particularly in those harboring KEAP1 mutations, during progression with the upregulation of key downstream redox regulatory factors including elements of the glutathione (GSH) system, thioredoxin (TRX) system, cysteine cycling, nicotinamide adenine dinucleotide phosphate (NADPH): quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), heat shock protein 70 (HSP70), and peroxiredoxins [51,52,53,54,55,56,57,58,59,60,61].

Furthermore, the complexity of this reprogrammed redox homeostasis also explains the lack in efficacy often exhibited by exogenous antioxidant treatments thus far [62]. Altogether, it is certain that further research into how oxidative stress contributes towards melanocyte physiology and melanoma pathophysiology is needed for potential anti- or pro-oxidant therapies to come to fruition. This review primarily focuses on the NRF2 pathway and potential transcriptional targets controlling redox signaling in melanoma.

Conclusion and Future Directions

In this review we have highlighted how protective endogenous antioxidant systems are coopted during melanoma initiation and progression. The net effect of this cooption is an increased antioxidant capacity within melanoma cells which allows for their unrestrained proliferation, metastasis, and drug resistance [84]. Fortunately, this reliance on endogenous antioxidant systems for progression and metastasis reveals potential targets for novel therapies. This is seen in the glutathione antioxidant system where there is upregulation of GSH synthesis genes, increased recycling of GSH mediated by GSR, and increased expression of antioxidant genes associated with drug-resistance like GPX2, GPX4, and GSTs[54]. Furthermore, NRF2 contributes to the elevated expression of G6PD which is an important source for NADPH necessary for the activity of both glutathione and thioredoxin antioxidant systems[54]. With the thioredoxin antioxidant system, there is an upregulation of both TXN and TXNRD1 expression that contributes to PRDX function and the clearance of cellular free radicals[54]. Both of these systems are dependent on the amino acid cysteine which is imported through the cystine/glutamate antiporter xCT (system x_c^-) comprised in part by the NRF2 transcriptional target SLC7A11 that also has upregulated expression in melanoma[54].

Three potential anti-melanoma targets within these antioxidant systems could include (but are not limited to) glutathione synthesis, thioredoxin reductase enzymatic activity, and the cystine/cysteine cycle. A combination of these redox targets would then be combined with conventional melanoma therapies. For example, it has been previously shown that combined inhibition of GSH synthesis and cystine/cysteine cycling was the more effective in killing melanoma cells than either treatment alone[52]. This blockade of coopted antioxidant function could then be combined with MAPK inhibition or immunotherapy. Knockdown of thioredoxin reductase 1 alone in melanoma alone does not prevent metastasis, but does induce a dependency on glycolysis and a complete knockout of TXNRD1 in melanocytes increases nuclear localization of NRF2 and synthesis of GSH post-UVB irradiation[43,53]. Interestingly, acquired resistance to BRAF inhibition induces a shift to oxidative phosphorylation [242]. So, it would be interesting to determine the effect of combinatorial inhibition of MAPK signaling and thioredoxin reductase 1 would have on melanoma cells.

Another avenue to treatment through intracellular redox manipulation is through the targeting of mitochondria. We recently performed a high-throughput screen to identify compounds effective against drug-resistant melanoma and found that compounds that target the electron transport chain were the most potent and others have studied different compounds that act similarly[81, 243]. Inhibition of the electron transport chain leads to the accumulation of oxidative stress making it a pro-oxidant treatment[244]. Also promising are compounds capable of disrupting mitochondrial membrane potential as exhibited by natural product polyphenols acting as proton shuttles that interfere with the electrochemical gradient across the inner mitochondrial membrane[245,246]. Additionally, polyphenols can also inhibit the electron transport chain and thereby further increase the oxidative stress present in cancer cells.

It is also important to examine NRF2 and its transcriptional targets in the context of the immune response. Melanoma cell NRF2 has an indirect connection with an immune response through its negative regulation of the transcription factor RXRα where we have previously shown that knockout of RXRα and RXRβ in melanocytes alters the expression of cytokines/chemokines that recruit IFN-γ secreting immune cells[101]. However, targeting of NRF2 directly in the attempt to improve an antimelanoma immune response is not likely to be effective as it has been previously shown that NRF2-null mice have increased susceptibility to xenograft of B16-F10 melanoma cells possibly due in part to an impaired immune system[247]. These data indicate any intervention that might also compromise NRF2 function in nonmelanoma cancer-adjacent cells could be detrimental, and therefore, endogenous antioxidants downstream of NRF2/KEAP1/ARE signaling could be better targets to benefit immunotherapy. These targets could include thioredoxin secretion, heme oxygenase 1, and cell surface expression of heat shock protein 70 previously shown to play a role in the immune response to melanoma as discussed above.

It should be noted that there has been some efficacy seen in clinical studies investigating the role of exogenous antioxidants in the prevention of melanoma but its utility in treatment requires further detailed studies on intervention at different stages of melanoma progression and metastasis to better understand and subsequently avoid any unintended detrimental effects.