BMC Molecular andCell Biology

Malakoutikhah et al.
BMC Molecular and Cell Biology(2023) 24:5
https://doi.org/10.1186/s12860-023-00466-5

Open Access

RESEARCH

The dual role of Nrf2 in melanoma:a systematic review
Zahra Malakoutikhah*, Zahra Mohajeri, Nasim Dana and Shaghayegh Haghjooy Javanmard*

Abstract
Melanoma is the most lethal type of skin cancer that originates from the malignant transformation of melanocytes.
Although novel treatments have improved patient survival in melanoma, the overall prognosis remains poor. To
improve current therapies and patients outcome, it is necessary to identify the influential elements in the development and progression of melanoma.
Due to UV exposure and melanin synthesis, the melanocytic lineage seems to have a higher rate of ROS (reactive
oxygen species) formation. Melanoma has been linked to an increased oxidative state, and all facets of melanoma
pathophysiology rely on redox biology. Several redox-modulating pathways have arisen to resist oxidative stress. One
of which, the Nrf2 (nuclear factor erythroid 2-related factor 2), has been recognized as a master regulator of cellularresponse to oxidative or electrophilic challenges. The activation of Nrf2 signaling causes a wide range of antioxidant
and detoxification enzyme genes to be expressed. As a result, this transcription factor has lately received a lot of interest as a possible cancer treatment target.
On the other hand, Nrf2 has been found to have a variety of activities in addition to its antioxidant abilities, constantNrf2 activation in malignant cells may accelerate metastasis and chemoresistance. Hence, based on the cell type andcontext, Nrf2 has different roles in either preventing or promoting cancer. In this study, we aimed to systematicallyreview all the studies discussing the function of Nrf2 in melanoma and the factors determining its alteration.
Keywords Melanoma, Nrf2, Chemoresistance, Metastasis, Oxidative stress, Systematic review
Background
Melanoma is a potentially fatal type of skin cancer causedby the uncontrolled proliferation of melanocytes [1]. Melanoma is responsible for more than 80% of skin cancer
mortality. Its incidence has increased dramatically over
the world in the last 50 years in all age groups, and basedon incidence curves, the incidence will grow sharply inthe coming years [2]. Although novel treatments such as*Correspondence:
Zahra Malakoutikhah
malakoot213@yahoo.com
Shaghayegh Haghjooy Javanmard
shaghayegh.haghjoo@gmail.com
Applied Physiology Research Center, Cardiovascular Research Institute,Isfahan University of Medical Sciences, Isfahan, Irantargeted and immune therapies have improved patient
survival in melanoma, chemoresistance prevents improving patients’ survival. The prognosis is still unsatisfactory, with most patients relapsing soon after their initial
response [3]. As a result, new therapeutic targets must be
identified to improve existing therapies and the prognosisof refractory melanomas.
Melanin synthesis comprises oxidation reactions
and the generation of ROS (reactive oxygen species) bymelanocytes, such as superoxide anions and hydrogen
peroxide. These reactive oxygen species have a high tendency to react with DNA, lipids, and proteins, causing
cell death. Even while oxidative species are synthesized,
an advanced antioxidant system, paracrine agents, andgene regulatory networks keep them at a low level toprevent oxidative species-induced cellular damage. If© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5

antioxidants do not detoxify ROS, the cell will experiencea rise in oxidative stress, altering the structure and function of critical cellular macromolecules like DNA, leading
to excessive cell proliferation, mutation, and/or chromosome instability, and eventually the development of neoplasms. Oxidative stress and the consequent oxidative
damage have emerged as key determinants involved in all
stages of melanoma development. Redox signaling is disrupted by oxidative imbalance, and metabolic remodelingoccurs in addition to mitochondrial and nucleus genetic
instability. These events play an important role in tumor
growth, metastasis, and the emergence of chemoresistance [4–9]. One of the most essential proteins involved incontrolling antioxidant response is the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), also
known as the master regulator of antioxidant and cytoprotective genes. Nrf2 is located in the cytosol in physiological states, coupled to its inhibitor Keap1 (Kelch-like
ECH-associated protein), resulting in deactivation via
ubiquitination and proteasomal destruction. When
Keap1 is subjected to oxidative stress, its cysteine residues oxidize, causing it to alter shape. Nrf2 is released,translocated into the nucleus, binding to ARE (antioxidant response element) sequences in the promoter ofgenes coding for antioxidant enzymes such as HO-1
(heme-oxygenase-1), SOD (superoxide dismutase), CAT(catalase), and GPX (glutathione peroxidase) [10, 11].
Thus, through the up-regulation of downstream genes,
Nrf2 inhibits carcinogenesis by assuring rapid enzymaticchange and neutralization of various carcinogens, as wellas suppressing ROS or repairing oxidative damage [12, 13].
On the other hand, the endogenous antioxidant system
adjusts with elevated oxidative stress following cancer
initiation, leading to altered redox homeostasis. According to emerging data, the Nrf2 signaling pathway has
been shown to enhance tumor cell proliferation, migration, invasion, angiogenesis, as well as radio/chemotherapy
resistance.
So, depending on the cell type and circumstance,
Nrf2 appears to be a double-edged sword [14]. The
involvement of Nrf2 and its downstream target genes inmelanoma will be discussed in this systematic review,
indicating a dual role of Nrf2 as both oncogene/oncosuppressive protein.

Materials & method

Page 2 of 18“Nuclear Respiratory Factor 2’ OR ‘Nuclear Factor Erythroid 2 Like 2’ OR ‘NFE2L2’ OR ‘Nrf2” and other related
MeSHs in title, abstract, or keywords. We modified thesearch keywords to get the optimal results using the Scopus
database, searching only ‘melanoma’ AND “NFE2L2’ OR‘Nrf2”. All titles and abstracts were screened to find relative
articles that met the inclusion criteria for full-text screening.
Study selection

We used the EndNote reference manager to collectsearch results for initial assessment and exclusion of
duplicate articles. Articles selected for review included
the following inclusion criteria; (I) English language, (II)the title/abstract indicating the relationship between
melanoma and Nrf2 or related genes. In addition, exclusion criteria are books, documents, and reviews. Afterexcluding irrelevant articles, the remaining articles’ fulltext was retrieved for further assessment.
Results analysis

Two researchers screened the search results independently. Study selection was performed based on the relevancy, inclusion, and exclusion criteria. The critical data
extracted from each article included (I) first author name,(II) publication year, (III) location of study, (IV) study
subject, (V) preliminary results, and (VI) conclusion(Table 1). Studies on the correlation of Nrf2 and melanoma cells were the primary outcome of interest. Regarding many heterogeneous study subjects and different
study designs and protocols, conducting a meta-analysis
was not deemed to be possible.

Results
Article selection

According to the search strategy we applied, the literature search provided 594 records, including 151 articles
from WOS, 100 from PubMed, and 343 from Scopus.
Removing duplicate reports resulted in 337 articles available for the initial assessment. We assessed the titles andabstracts and removed irrelevant articles, limiting ourdata to 73 articles for full-text review. Out of 73, 32 were
excluded based on inaccessibility and exclusion criteria.
Eventually, data were collected from 41 articles presenting the role of Nrf2 in melanoma initiation, progression,and metastasis (Fig. 1).

Literature search strategy

Tabulation

The current systematic review followed PRISMA principles. Two researchers evaluated the search results
independently to minimize the bias. Searches were conducted until May 2022, using three databases, including
Web of Science (WOS), PubMed, and Scopus. On these
databases, we searched the keywords ‘melanoma’ AND

All data extracted from the final articles are summarizedin Table 1. All of the articles chosen for this study were
published after 2014. Studies are mainly performed onhuman cancer cell lines, whereas others have focusedon murine melanoma cell lines, mice, or human tissue
samples.

Human tissue sample;
Cell lines: COLO-800, SK-MEL-1 with BRAF mutation, SK-MEL-30, and IPC-298 with NRAS mutation

Hintsala et al., 2016[15]

References

The oxidative stress level is directly related to anadvanced stage of melanoma and metastasis.
Redox therapeutic agents inhibit cell growth andsurvival by disrupting PI3K/AKT/mTOR pathway.

Intracellular ROS levels are significantly higher
in Nrf2 knockout cells than in wild-type cells,
sensitizing the melanoma cells to cisplatin or
dacarbazine.

Redox imbalance following UVR favors the condition for melanoma formation. Co-activation ofoxidant and antioxidant responses leads to tumor
growth and progression.

[20]

[19]

[18]

[17]

Nrf2 expression is suppressed in some types of[16]
cancers. However, there are no significant differences between normal and melanoma cells in the
NFE2L2 expression. Down-regulation of NFE2L2 isassociated with poor prognosis in melanoma.

The Nrf2/Keap1 pathway dysregulation relates toskin cancers, including melanoma.

Conclusion

In melanoma cells with BRAF\NRAS mutations,Nrf2 is an influential factor in determining theadministration of their inhibitors would decrease
prognosis of melanoma patients.
the expression of Nrf2.
Nuclear expression of Nrf2 in melanoma correlateswith higher Clark level, deeper Breslow index,
nodular growth, and poor survival.

Human melanoma cell lines: MeWo, WM793B, and Nrf2 protein level was higher in all melanoma
1205Lu, human epidermal neonatal melanocytes cell lines than HEMn. NQO1 was meaningfully(HEMn)
increased in WM793B cell lines, and HMOX1transcript was higher in MeWo and 1205Lu than
HEMn.
Basal oxidative stress in melanoma cells is higher
than in normal melanocytes, consequently upregulating the Keap1/Nrf2 signaling pathway toadapt to this oxidative stress. The elevated oxidative stress inhibits the dependency of melanoma
cell survival on PI3K/AKT/mTOR pathway.

Hambright et al., 2015

Constitutive activation of Nrf2 has a significant
role in emerging melanoma-resistant cells.

The intracellular level of ROS elevates during
human cell transformation, which accompanied
by Nrf2 repression through stimulation of RAS/
RAF/ERK signaling pathway.
Interestingly, regeneration of Nrf2 level in transformed cells elicits antioxidant responses andinhibit tumor growth.

Shinpei et al., 2014

Human mesenchymal stem cells, and humanmammary epithelial cells

Funes et al., 2014

Although Nrf2 expresses in normal skin cells, it isdownregulated in skin cancers, including malignant melanoma. However, the Keap-1 expression
varies in normal cells and skin cancers, suggesting that Nrf2 translocation to nucleus may occur
through Keap-1 independent pathways in humanskin cancers.

NOX activity has a crucial role in UVA-induced
oxidative stress.
UV radiation leads to Nrf2 protein accumulation
as well as Nox1 protein expression and NOX activity. Overexpression of Nrf2 and Nox1 has been
observed in all melanoma cell lines and tissue
samples.

The human melanoma cell line G361

Choi et al., 2014

Primary results

Smith et al., 2014

SampleStudy

Table 1 The summary of the final selected studies

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5
Page 3 of 18

Nrf2 has a protective role against melanogenesis
under UVA irradiation.

Conclusion

Human melanoma cell lines: SKMEL 28 and SKMEL
94
Murine melanoma B16
human glioma cell lines: U138MG and U87MG

Female nu/nu nude mice, Human epidermal melanocytes: HEMa-LP, pancreatic adenocarcinoma:
ASPC-1 and BxPC-3, Human A2058, MeWo, andMelJuso melanoma cells, murine B16 melanoma
F1, mouse pituitary corticotroph tumor cells:
AtT-20Melanoma patient (case report)

Rocha et al., 2016

Benlloch et al., 2016

Cai et al., 2017

A variation of NFE2L2 (NFE2L2 p.Leu266Phe) is
identified in a patient with stage III metastaticmelanoma.

Pterostilbene indirectly downregulates Nrf2 andthe downstream antioxidant defense system
by inhibiting ACTH production, and reducing
plasma levels of corticosterone, thus restricting
melanoma tumor growth. In contrast, geneticallyinduced Nrf2 overexpression or administration ofexogenous corticosterone hindered tumor growth
suppression of melanoma cells.

Temozolomide (TMZ) increases the intracellular
ROS level and triggers Nrf2 and its downstream
GCLM and GSTπ genes. Consequently, theelevated level of GSH mediates temozolomide
resistance. In addition, GSH depletion increases
melanoma cells’ sensitivity to temozolomide. Nrf2
silencing results in temozolomide-induced cell
death in vitro and in vivo.

Nrf2 deficiency results in tumor growth and ahigher risk of lung metastasis in B16-F10 melanoma cells.

Nrf2-null C57BL/6 mice, and male Nrf2-null andwild-type mice
Cell line: B16-F10-luc-G5

[22]

[21]

References

NFE2L2 mutation may play a role in melanogenesis and cancer progression.

Pterostilbene prevents human melanoma growthin vivo. However, it does not affect melanoma
growth in vitro at levels tested within the tumors.
The anti-tumor effect of pterostilbene is dependent on the Nrf2 signaling pathway.

Nrf2 is a key factor in developing TMZ drug resistance in melanoma.

The exact role of the Nrf2 pathway in carcinogenesis needs to be investigated. However, diverse
factors such as type of carcinogens, cancer type,and host immunity appear to contribute to theoncosuppressor/oncogene role of Nrf2.

[27]

[26]

[25]

[24]

Activation of the Nrf2-IL17D pathway in primary[23]
tumorigenesis leads to NK cell-dependent tumor
inhibition. However, the role of the Nrf2-IL17D axis
is context-dependent and may not always associate with NK infiltration or better survival.
Nrf2 stimulates both internal and externalanti-tumor immune responses, prevents tumor
progression, and improves survival.

A high proportion of NQO1-high melanomas
17-AAG is a potential therapeutic option forpresent Nrf2 activation independent of KEA1
NQO1-high melanoma.
mutation.
Melanoma cell lines presenting NQO1 overexpression show sensitivity to 17-AAG compared to celllines with low expression of NQO1.

Zhu et al., 2016

Cell lines; A7, C32, GAK, G-361, HMY-I, HMV-II,
MM-AN, MeWo, SK-MEL-2, SK-MEL-28 and SKMEL-31, nine non-small cell lung cancer cell lines,
normal human dermal fibroblasts, and neonatalskin fibroblasts

Kasai et al., 2016

Knockdown of the Nrf2 gene in B16-F10 and
HEMn cells results in melanogenesis enhancement in response to UVA exposure.
UVA irradiation affects Nrf2 nuclear accumulation,
and Nrf2 target antioxidants time-dependently.

Tert-butylhydroquinone (tBHQ) induced activationof Nrf2 results in elevated expression of il17d
transcript in the B16 cell line.
IL-17D induction leads to NK cell-dependent
tumor inhibition.

Primary human epidermal melanocytes (HEMn),
B16-F10 mouse melanoma cells

Chaiprasongsuk et al., 2016

Primary results

Saddawi-Konefka et al., 2016 Mice cell lines: MCA-induced sarcoma, Ramos,
B16, and LLC

SampleStudy

Table 1 (continued)

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5
Page 4 of 18

WT/Sesn2 KO mouse embryonic fibroblast, A375,
iMC23 melanocytes, and normal human epidermal melanocytes

B16-F10 melanoma cells

Tumor specimens, A375 melanoma cell line

C57BL/6 J male mice and melanoma B16-F10 cells Mouse lung tissue with melanoma metastasis
revealed a high expression of Nrf2 and its downstream antioxidant enzymes and inflammatory
factors, including NF-κB P65, IL-6, and TNF-α.

Human Skin Samples, A375 melanoma cell line,
and WT/KO mouse embryonic fibroblasts

Female C57BL/6 J mice,
Cell lines: B16-BL6, HPK, and HPM

Zhao et al., 2017

Gao et al., 2018

Khamari et al., 2018

Li et al., 2018

Sample et al., 2018

Zhu et al., 2018

Nrf2 signaling pathway exerts a dual role inmelanoma. The effect of UV on the Nrf2 signaling
pathway is dose-dependent.

Sesn2 seems to be the upstream suppressor of
Nrf2 in response to UVA exposure.

Nuclear expression of Nrf2 is associated with
distant metastasis and poor prognosis.

Conclusion

Nrf2 and PD-L1 are overexpressed in melanomas
and metastatic melanomas.
UVA-induced PD-L1 upregulation is positively
associated with Nrf2 activation.

UVA induces P62 up-regulation in an Nrf2dependent manner.
Knockdown of Nrf2 prevents p62 up-regulation.
Conversely, knockdown of P62 inhibits UVAinduced Nrf2 up-regulation.
P62 overexpresses in melanoma and malignant
melanoma independent of BRAF/NRAS mutation.

[32]

[31]

[30]

[29]

[28]

References

UVR induces PD-L1 in an Nrf2-dependent manner, [34]facilitating melanoma progression.

Nrf2 and P62 interact with each other in a positive [33]
feedback loop. The P62 overexpression contributes to tumor growth and metastasis.

The antioxidant response following oxidative
stress plays a significant role in melanoma lungmetastasis.

A375 melanoma cells present Nrf2 nuclear accuMAPK-resistant melanomas are OXPHOS dependmulation, notably higher in BRAFi-resistant cells.
ent, expressing Nrf2 downstream antioxidant
Knockout of Nrf2 in BRAF-resistant melanoma cells response.
reversed vemurafenib resistance partially.
Nrf2-dependent glutathione metabolism plays acrucial role in the emergence of resistant cells.

UV radiation induces Nrf2 and caspase-3 expression in B16-F10 melanoma cells, decreasing cell
viability. However, Inhibition of Nrf2 by siRNA also
leads to a decrease in cell viability, invasion, andmigration of irradiated melanoma cells and anincrease in caspase 3 expression.

UV irradiation induces Sesn2 expression inmelanoma cells. Sesn2 silencing stimulates UVAinduced Nrf2 activation, inhibiting UVA-induced
ROS generation.
Nrf2 silencing does not affect UVA-induced
expression of Sesn2 in A375 cells.

The nuclear expression of Nrf2 is significantly
higher in metastatic melanoma than in primarymelanoma, whereas the level of cytoplasmic
Keap1 in metastatic melanoma is lower than in
primary melanoma.

Human tissue sample

Hintsala et al., 2017

Primary results

SampleStudy

Table 1 (continued)

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5
Page 5 of 18

SampleHuman melanoma cell lines: CRL-1585 (C32), GAK,
G-361, HMV-II, HMY-1, MeWo, MM-AN, SK-MEL-2,
SK-MEL-31, and SK-MEL-28 murine melanoma
B16BL6 cell line, and human non-small cell lungcancer H460 cell line

Melanoma B16-bearing mice

Neonatal human skin melanocyte

Human melanoma cell lines: A2058, A375, C8161,
CHL-1, SK-Mel 5, SK-Mel 24, and MeWo

Human tissue samples,
Cell lines: human primary melanoma
IPC-298 (ACC 251), adult primary epidermalmelanocytes (PSC-200-013), and metastatic melanoma SK-MEL-30

Mouse epidermal JB6 P+ cell line, and humanhepatocellular HepG2-C8

StudyArakawa et al., 2018

Aksenenko et al., 2019

Chhabra et al., 2019

Gagliardi et al., 2019

Hämälaïnen et al., 2019

Kuo et al., 2019

Table 1 (continued)

The NRF2/NQO1 axis can play a decisive role in
improving the clinical response rate of NQO1dependent anticancer therapies in malignant
melanomas.

Conclusion[35]

References

Nrf2 knockout mice developed multiple skintumors following exposure to the carcinogens.
Delphinidin prevents JB6 P+ cell transformation
by epigenetic activation of the Nrf2-ARE pathway.

[37]

Redox imbalance predisposes cells to melanoma [40]formation.
Phytochemicals can play a chemopreventive roleby inhibiting the neoplastic transformation of skincells.

[39]

Melanoma cancer cells resistant to ferroptosis cell [38]death efficiently activate Nrf2 and its downstream
enzymes HO1.

Nrf2-Keap1 pathway plays a key role in protecting
skin cells against UVA and UVB induced oxidative
damage.

The Nrf2 mRNA decreases from benign lesions to The miRNAs are associated positively with Nrf2
primary melanomas. Moreover, the Nrf2 mRNA
mRNA but negatively with its protein.
level varies in different stages of melanoma. High
nuclear expression of Nrf2 protein can play a prognostic role in melanoma patients prior to nodal
or distant metastasis. MiR-144-3p, miR-212-3p,
miR-23b-3p, miR-340, and miR-93-5p appear to berelated with Nrf2.

Activation of Nrf2 leads to a constant decrease inerastin-mediated AKR1C1÷3 expression. Moreover, inhibition of Nrf2 causes complete abolishment of ERA-stimulated CHAC1 expression.
Nrf2 expression in tumor cells is lower than innormal tissue samples, without any importantfluctuation during the stages of tumor progression. However, there is no association between
Nrf2 expression and patient overall survival.

UVA and/or UVB exposure increase ROS anddownregulate Nrf2 expression in melanocytes.

The pro-oncogenic miR-155 is significantly eleA reduction in NFE2L2 gene expression in the[36]
vated in the target organ of melanoma metastasis, premetastatic organs may be associated withdownregulating NFE2L2 gene expression.
metastatic niche formation and stromal progression, results in premetastatic tissue reconstruction.

β-lapachone cytotoxicity in melanoma cell lines
is dependent on the Nrf2/NQO1 axis activity.
Carnosic acid induces NQO1 through further
stabilization of Nrf2. Co-treatment of β-lapachone
and carnosic acid increases the melanoma cellsensitivity to β-lapachone by elevating the expression of NQO1.

Primary results

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5
Page 6 of 18

SampleNude (nu/nu) male mice,
Human A2058, COLO-679 and SK-Mel-28 melanoma cell lines

C32, and SK-MEL-28 melanoma cell lines

Normal human embryonic melanocytes (NHEM),
SK-Mel-2, SK-Mel-5, SK-Mel-28, and HEK293FT
melanoma cell lines

A375, A-549, SK-MEL-2, SK-MEL-3, SK-MEL-28,
UACC-62, UACC-257, M14, and LOXIMVI cell lines

The A375 human melanoma cell line

A375, LOXIMVI, M14, UACC-257, UACC-62, SKMEL-2, and SK-MEL-28 cell lines

Skin melanoma tissue sample, the A375 humanmelanoma cell line

Study

Estrela et al., 2019

Chipurupalli et al., 2020

Jasmer et al., 2020

Jessen et al., 2020

De Cicco et al., 2021

Kreß et al., 2021

Li et al., 2021

Table 1 (continued)
References

There is a positive feedback loop between Nrf2
and EGFR signaling pathway. The activation ofthat results in the survival and invasiveness ofmelanoma cancer cells.

Cynaropicrin plays a chemopreventive role byinducing Nrf2 activation in melanoma cells.

[47]

[46]

[45]

Activation of Nrf2 by intrinsic and extrinsic trig[44]gers present in the melanoma tumor niche resultsin developing a dedifferentiated phenotype andoverproduction of COX2 and PGE2. This phenomenon creates an immune-cold tumor environment. Melanoma benefits from activating of theNrf2 pathway.

The amount of mGPDH protein in melanoma
mGPDH deficient cells are able to activate thecells is significantly lower compared to normal
Nrf2 signaling pathway, leading to melanoma
skin cells, and metastatic melanoma compared to progression and metastasis.
primary melanoma.
Furthermore, the loss of mGPDH triggers Nrf2
expression without changing the NRF2 protein
degradation, promoting melanoma migration andinvasion.

AKT activity was strongly reduced in NRF2-knockout cells in response to EGF induction comparedto the control group, indicating the essential roleof Nrf2 for full activation of EGFR. Nrf2 facilitates
the expression of EGFR and its ligands, attributed
to features such as invasion, tumor metastasis, andBRAFi resistance.

Cynaropicrin has an antioxidant effect; treatedmelanoma cells are less aggressive with lower
clonogenic ability.

Various internal and external signals activate Nrf2
in melanoma cells. siRNAs-dependent downregulation of Nrf2 decreases the proliferation rate,
promoting cell differentiation. Moreover, Nrf2
knockout prevents melanoma occurrence in vivo.

Inhibiting Nrf2 or its downstream enzyme, HMOX1 Melanosphere formation has an HMOX-depend[43]reduces melanosphere formation in BRAF-driven
ent mechanism correlating with tumorigenic
melanoma cell lines.
capacity.
HMOX1 contributes to drug resistance, melanoma
invasion, and migration.

[42]

The cross-talk among GR, p53, and Nrf2 may be[41]
critical in BRAFV600E-mutated melanoma cells
survival and metastasis. Combination of GR antagonists (mifepristone, RU486) with BRAF-related
therapy could be a novel approach for improving the outcome, and preventing resistance totreatment in BRAFV600E-mutated metastaticmelanoma.

Conclusion

Increased activity of the Nrf2 in melanoma cancer
STING activation downregulates the Nrf2 downcells may be due to a defect in STING activation.
stream antioxidant enzymes.
diABZI induced STING-activation reduces proliferation, accelerates cell death, and prevents migration of melanoma cells by restricting the Nrf2
signaling pathway.

Pharmacologic silencing of glucocorticoid
receptor (GR) reduces the expression of Nrf2
and antioxidant responses in metastatic B16-F10
melanoma cells, causing a drastic reduction of cell
survival and tumor growth. The induction of Nrf2
overexpression reversed the anticancer effect of
GR inhibition.

Primary results

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5
Page 7 of 18

SampleNormal skin cell line PIG1, melanoma cell lines:
A375, G-361, HS1-CLS, MEL-CLS1-4, IGR-1, MEWO,NIS-G, WS1-CLS, and MML1

The HEK293, and A375 cell lines

Metastatic MCM1DLN, and Non-metastatic
MCM1G lines from a patient with melanoma,metastatic WM1205Lu and Non-metastatic
WM793b cell lines from ATCC.

The A375, and A2058 cell lines

B16-F10 murine melanoma cell line

SK-MEL-28 human melanoma cell line

A375 human melanoma cell line

Study

Liao et al., 2021

Schmidlin et al., 2021

Weitzenböck et al., 2022

Wang et al., 2022

Sanches et al., 2022

Feng et al., 2022

Li et al., 2022

Table 1 (continued)

Dimethyl fumarate (DMF) suppresses the Nrf2
antioxidant pathway as well as AKT/mTOR/ERK
signaling pathways, causing ROS accumulation.
The combination of DMF with vemurafenib
significantly reduced melanoma cell growth andincreased tumor cell death in vitro and in vivo.

GSK3 expression in melanoma cells is enhanced
by nobiletin, which is usually low in cutaneous
melanoma. GSK3β inhibits the Keap1/Nrf2/HO-1
Signaling Pathway. The buildup of iron and ROS
and the GSH depletion causes lipid peroxidation,
thereby inducing ferroptosis.

References

[50]

[49]

A combination of DMF and vemurafenib could be [54]a novel therapeutic method against melanoma

Nobiletin stimulates ferroptosis in melanoma cells [53]by GSK3β-mediated suppression of the Nrf2/HO-1
axis.

[52]

CAMKK2 regulates the AMPK–Nrf2 signaling[51]
pathway, leading to lower ferroptosis sensitivity ofmelanoma cancer cells.

Nrf2 silencing increases melanoma cell viability,
invasiveness, and the risk of metastasis.

Constitutive activation of Nrf2 induced by
FAM129B potentiates BRAF mutant melanomas
for metastasis.

The expression of miR-130b-3p reinforces activa- [48]tion of the Nrf2/HO-1 pathway followed by DKK1
suppression. The overexpression of Nrf2 correlateswith the resistance of melanoma cells to ferroptosis.

Conclusion

DTIC resistant cells showed a decreased level ofMetformin plays a dual role, inhibiting or proNrf2. However, the addition of metformin in DTIC moting a DTIC-resistant phenotype in B16-F10
resistance leads to increased Nrf2 and less resistmelanoma cells.
ance to DTIC. Moreover, the exclusive administration of metformin also reduces Nrf2 compared tocontrol group, inducing cellular resistance to DTIC.

Following treatment with erastin and RSL3, thelevel of CAMKK2 mRNA was significantly elevated.
Phosphorylation of CAMKK2 reduces during ferroptosis. Moreover, the phosphorylation of AMPK(known as a canonical substrate of CAMKK2)
increases during ferroptosis. CAMKK2 prevents
ferroptosis by repressing the production of lipid
peroxidation in an Nrf2-dependent manner.

Suppression of Nrf2 leads to a loss of ROS protection and promotes the epithelial-mesenchymaltransition (EMT) phenotype, which is hallmarked
by the expression of CD44. Cells devoid of NRF2
exhibit higher survival rates after a BRAF or an
MYC inhibitor treatment.

FAM129B up-regulates Nrf2 by binding to Keap1
protein in A375 melanoma cells. Knockdown of
FAM129B plays a protective role against melanoma metastasis.

miR-130b-3p activates the Nrf2/HO-1 signaling pathway by inhibiting DKK1 expression inmelanoma, leading to reduced melanoma cellferroptosis.

Primary results

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5
Page 8 of 18

SampleM14 melanoma cell line

Study

Argenziano et al., 2022

Table 1 (continued)
The Nrf2 protein level was downregulated
after the treatment of M14 cells with siNrf2-NB.
Subsequently, the treated cells showed significant
downregulation of the viability cells.

Primary results

Nrf2 contributes to maintaining the chemoresistance in melanomas. siRNA-mediated inhibition of
Nrf2 could be a promising strategy to overcome
chemoresistance.

Conclusion[55]

References

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5
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Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5

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Fig. 1 This flow diagram presents the process of study selection included in the qualitative systematic review

Expression and activation of Nrf2 in melanoma

Due to UV radiation and melanin production, melanocytes create more ROS than surrounding keratinocytes inthe skin. Evidence shows that oxidative stress, increased
ROS level, and redox imbalance are critical parameters
in melanogenesis. Indeed, the entire process of melanoma formation relies on oxidative stress and antioxidant
response. Because of their more significant metabolic
needs, melanoma cells exhibit even higher ROS andantioxidant responses than normal melanocytes [21,56]. Melanocytes use adaptive strategies to withstandoxidative stress by expressing antioxidant scavenging
activity. As a result, it has been proposed that targetingmelanoma cells’ addiction to greater redox capability
could be a suitable and effective treatment method. The
master stress response transcription factor Nrf2, which
regulates the expression of antioxidant enzymes such as
HO-1, SOD, CAT, and GPX, is primarily responsible forredox homeostasis in melanoma [57]. Investigations on
the expression of Nrf2 and its target genes have produced
conflicting results. Even some studies reported no significant differences in Nrf2 expression between normal andmelanoma cells [16].
Taken together, it seems that lower Nrf2 expression
favor melanogenesis, and is appeared in early stages ofmelanoma. Nrf2 mRNA and protein levels are diminished during the early phases of melanoma formation.
Even though Nrf2 is expressed in normal skin cells, it isconsiderably reduced in skin malignancies such as malignant melanoma, indicating that Nrf2 expression is linked
to the incidence of skin cancers. However, higher Nrf2
expression in cancerous cells is linked to more aggressive and resistant melanomas, and poor prognosis [15,39]. It is hypothesized that Nrf2 contributes in melanoma phenotypic switch between a proliferative and aninvasive phenotype. The expression of genes linked tothe melanoma progression change subsequent of Nrf2
expression. For example, Nrf2 inhibits the MITF (microphthalmia-associated transcription factor), the primarytranscriptional regulator of the phenotypic transition
in melanoma, and its expression changes through themelanoma progression. MITF down regulation causes
invasive dedifferentiated melanoma, while overexpression causes a proliferative phenotype. It is plausible that
Nrf2 expression is required during periods of fast development but is repressed once an invasive phenotype has
emerged [11].
When melanoma advances, Nrf2 expression rises.
Melanoma redox capacity is assumed to represent a spectrum ranging from low in normal cells to moderate inMalakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5

drug-sensitive melanomas and to high in drug-resistantmelanomas [57]. Consequently, the expression of Nrf2
changes in benign lesions and progresses to primary andmetastatic melanomas [39]. Although Nrf2 expression ishigher in metastatic lesions than in primary sites, cytoplasmic Keap1 expression is lower, indicating that Nrf2
accumulates in the nucleus, leading to up-regulation of
Nrf2 target genes [28]. For example, HO-1 expression has
been found to be increased in B16-F10 murine melanoma
cells as well as in-vivo melanoma tumor models, which
has been linked to the higher proliferation rate [57].
BRAF (B-rapidly accelerated fibrosarcoma) and NRAS
(N-Rat sarcoma virus) mutations, which are found in 50
and 20% of cases, respectively, are commonly responsible for dysregulation of the MAPK (mitogen-activatedprotein kinase)-proliferation signaling pathway in melanomas [58]. A direct link between Nrf2 activation and
MAPK signaling has recently been discovered. The activation of the MAPK pathway causes Nrf2 to be expressed.
BRAF/NRAS mutant melanoma cells activate the Nrf2
signaling pathway to protect cells against oxidative stress[20, 49]. BRAF inhibitors (BRAFi), like vemurafenib, suppress Nrf2 expression in BRAF mutant melanoma cells,
although the effect is not significant, suggesting that
other oncogenes also trigger the Nrf2 signaling pathway [20]. This is worth noting that BRAFi-resistant cells
have higher Nrf2 expression than non-resistant cells [57].
However, not all Nrf2-dependent genes react the same
way to BRAFV600E expression [43].
UV irradiation is also a determining factor in theexpression of Nrf2 and subsequent antioxidant target
genes. UV exposure enhances the Nrf2 expression andnuclear accumulation in a dose-dependent manner [17,30, 59]. However, the effect of UV radiation on Nrf2
expression is still a matter of contention; some studies
have reported no effect or even a negative effect [29].
GSH (glutathione) depletion and ROS accumulation
caused by UVA (ultraviolet A) irradiation may trigger
melanogenesis in people with defective Nrf2. Although
Nrf2 silencing did not affect melanogenesis in theabsence of UVA exposure, Nrf2-knockout cells exhibit
higher tyrosinase activity and melanin content [21].
Nrf2 in melanoma progression

Nrf2, a crucial activator of antioxidant and phase two
defenses in cells, has a dual role in melanoma. Although
regular Nrf2 expression has a cytoprotective role against
carcinogenesis, Nrf2 overexpression boosts antioxidant responses and changes the cellular redox state. In
this aspect, antioxidants have been demonstrated to aid
melanoma cell survival, proliferation, and tumor growth[44]. Nrf2 activity in transformed cells lowers ROS andinhibits tumor growth in vivo by mechanisms such

Page 11 of 18

as apoptosis sensitization and a reduced angiogenic/
hypoxic response via HIF-1(hypoxia-inducible factor-1)
instability and VEGFA (vascular endothelial growth factor A) suppression [16]. Nrf2 activators like cynaropicrin
can reduce the clonogenic ability of metastatic melanomas in a dose- and time-dependent manner while notaffecting normal melanocytes. Anti-apoptotic proteinssuch as XIAP (X-linked inhibitor of apoptosis protein) and Bcl-2 (B cell lymphoma-2) are also reduced in
response to cynaropicrin treatment [45]. Another study
discovered that delphinidin, an Nrf2 epigenetic enhancer,
suppresses ­JB6P+ cell transformation, implying that delphinidin could be employed as a chemopreventive medication [40]. However, Nrf2 activators or inhibitors should
be utilized carefully and in line with the stage of the disease in order to be effective.
UV-induced aggregation of Nrf2 in B16-F10 cells wasfound to diminish cell viability and increase caspase-3
expression, a key indicator of apoptosis. Surprisingly,
ionizing radiation in Nrf2 silenced cells synergisticallyaffected melanoma cell viability, elevating apoptosis [30].
This dual behavior may be due to the complex web ofinteractions around Nrf2. As Arslanbaeva et al. indicated,in vitro and in vivo, the combination of brusatol, a potent
Nrf2 inhibitor from the plant Brucea javanica, and UVA
irradiation could reduce the melanoma cell proliferationand induce apoptosis in melanoma cells [57].
In melanoma, the activity of receptor tyrosine kinases(RTKs) is normally restricted. Some RTK family members like EGFR may be expressed in MITF-low melanoma cells, and under this circumstance, the EGFR
expression is linked to a pro-invasive and pro-metastaticmelanoma phenotype with resistance to BRAF/MEK
inhibitors. Stress-induced Nrf2 mediates the expression
and activity of EGFR by increasing the EGFR levels andits ligands like EGF and TGFα. A canonical ARE is foundin the EGF promoter, and Nrf2 binds to it directly, leading to a higher level of EGF for AKT activation. Furthermore, Nrf2 blocks the MITF activity, the melanocytic
lineage factor responsible for suppression of EGFR and
TGFA. Hence, Nrf2 indirectly derepresses the EGFR,
contributing in the maintenance of EGFR-expressing
melanomas [46]. Reversely, EGFR stimulates Nrf2, providing a positive feedback loop. In vivo Nrf2 silencing
has been shown the low EGFR expression and proliferation rate, preventing melanoma growth, and increasing
tumor-free survival. However, Nrf2-induced suppression of MITF activity also contributes to developmentof dedifferentiated, invasive phenotype in another ways.
The antigenicity of melanoma cells is significantly influenced by the proteins expressed by MITF target genes.
Nrf2-mediated down regulation of pigmentation markers(melanoma antigens) due to MITF inhibition promoteMalakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5

a dedifferentiated melanoma cell that can escape from
immune recognition [44]. Another study has demonstrated that inhibiting Nrf2 by pharmacologic activationof the Nrf2’s upstream gene reduces proliferation, accelerates cell death, and slows melanoma progression [42].
Inhibiting either Nrf2 or its target gene, HMOX-1 (hemeoxygenase 1 gene) prevents melanosphere formation, an
indicator of cell tumorigenic potential, in melanoma celllines. Moreover, HMOX-1 overexpression in A375 melanoma cells increases tumor volume, whereas silencing
HMOX-1 reduces tumor growth in mouse models [43].
The cellular redox status has been shown to alter the
innate immune responses. Albeit this has yet to be determined, Nrf2 may cooperatively modulate the innate
immune response, repressing the pro-inflammatory
mediators’ expression by mediating the transactivation of antioxidant and other cytoprotective genes [60].
Nrf2 depletion can increase the susceptibility to infection and inflammatory diseases due to impaired cellular stress response and exacerbated immune-mediatedhypersensitivity and autoimmunity [61]. Nrf2 can also aid
anti-tumor immunity by inducing immune-dependentcascades such as IL-17D-dependent NK (natural killer)
cell recruitment early in carcinogenesis before the protumor action of Nrf2 manifests. However, induction of
IL-17D at advanced stages is not always associated with agood prognosis [23].
There is evidence that Nrf2 has a significantly greater
tendency to suppress immunity, mostly in advanced
stages. BRAF wild-type melanomas have been treatedwith monoclonal antibodies that target immune checkpoint proteins such as anti-PD-1 (programmed cell death
protein-1) (like pembrolizumab or nivolumab). Nrf2
inhibits the recruitment of interferon-secreting immune
cells, reducing PD-L1 (programmed death-ligand 1)
expression in the process. Given that increased PD-L1
expression has been linked to enhanced anti-PD-1 therapeutic effectiveness, this might explain why Nrf2 overexpression is linked to immunotherapy resistance [11]. Nrf2
has also been found to have a direct impact on PD-L1
expression. Zhu et al. found that Nrf2 stimulates PD-L1
transcription, and targeted Nrf2 inhibition is an alternative way of suppressing PD-1/PD-L1 to trigger tumor
infiltration by ­CD4+ and ­CD8+ T lymphocytes and consequentially restrict melanoma growth [34].
Cytosolic DNA induced from nuclear/mitochondrial DNA damage in cancer triggers the activation ofcGAS-STING (cyclic GMP-AMP synthase-stimulatorof interferon genes) pathway. The recruitment of cGASSTING pathway plays an important role in tumor immunogenicity, and Its activation has been demonstrated
to reduce mice’s resistance to PD-1 blockage [62, 63].
Evidence exists to show the suppressive role of Nfr2

Page 12 of 18

on cGAS-STING pathway, providing an immune-cold
microenvironment [44, 64]. Moreover, Nrf2 is demonstrated to be a potent inducer of COX2, the enzymethat facilitate the production of PGE2. Following this,the PGE2 contributes to the suppression of the innate
immune response and the attenuation of T cell receptors, establishing an immune-evasive tumor microenvironment [64, 65]. Nrf2 inhibitors are thus expected tofunction in conjunction with checkpoint inhibitors in thetreatment of melanoma. Such a combination is presently
not possible due to the lack of potent Nrf2 inhibitors.
Nevertheless, preclinical studies have shown that COX
inhibitors and STING agonists work in conjunction with
anti-PD-1 immunological therapy, indicating that targeting Nrf2 downstream indicators may be beneficial [42,44].
Overall, it can be concluded that the onco-promoter/onco-suppressor function of Nrf2 varies by cell stage
and is context-dependent. Furthermore, whereas transient Nrf2 activity protects against cancer, constitutive
Nrf2 activation caused by Keap1 or Nrf2 genetic variations may promote cancer progression and tumor growth(Fig. 2).
Nrf2, and melanoma metastasis

There is evidence that Nrf2 activation enhances cancer
cell metastasis in melanoma, suggesting that Nrf2 has anonco-promoter role [34, 47, 59]. In fact, as long as the cellis intact, Nrf2 and its lower genes try to save the cell frombecoming cancerous by reducing oxidative stress. However, if the situation progresses in such a way that celltransformation occurs, the cancer cell uses Nrf2 in favorof itself, and higher expression of Nrf2 makes melanoma
cells resistant to high levels of ROS and oxidative stressproduced from cell metabolism. In this regard, Nrf2 isassociated with invasiveness and pro-metastatic features.
Studies have shown that the ROS or ROS-enhancing
chemicals at this phase might be effective in cancer treatment, whereas antioxidants provide a more desirable
environment for melanoma progression and metastasis[32, 45, 57, 66].
HMOX1 is one of the driving forces in melanoma invasion and migration. Silencing Nrf2, which causes HO-1
to be down-regulated, prevents melanoma cells frominvasion and migration [43]. UV irradiation also has asignificant role in regulating the apoptosis, migration,and invasion of melanoma cells in the Nrf2-dependent
manner. Gao et al. found that downregulation of Nrf2
reduces the ability of radiation-exposed B16-F10 cells
to invade and migrate. Surprisingly, however, overexpression of Nrf2 exhibited similar effects on cell survival, invasion, and migration, demonstrating Nrf2’s
dual function in melanoma [30]. It has been discovered

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5

Page 13 of 18

Fig. 2 Nrf2 plays a dual function in melanoma, depending on the cell stage. The standard expression of Nrf2 has a protective role against oxidative
stress and cell damage, maintaining normal melanocytes. Nrf2 depletion and ROS accumulation make cells susceptible to transformation andcancer initiation. Evidence exists to show that reduced Nrf2 expression is associated with the incidence of melanoma. On the contrary, aberrant
overexpression or constitutive activation of Nrf2 leads to invasive, metastatic, and radio/chemoresistant melanomas with poor prognosis. Indeed,
after cell transformation has occurred, Nrf2 can become a pathway for cancer progressionthat inhibiting the function of Nrf2 by pharmacologically
activating the STING pathway accelerates cell death anddecreases melanoma cell migration, and has a synergistic
effect with BRARi in cell death [42].
It has been shown that FAM129B, an Nrf2 inducer,
also makes melanoma cells more susceptible to metastasis. Intriguingly, knocking down FAM129B or reducing its phosphorylation reduces melanoma cell invasion
and migration in an Nrf2-dependent way [49]. In metastatic melanomas, mGPDH (mitochondrial glycerol3-phosphate dehydrogenase) activity is reduced. Li et al.
showed that in A375 melanoma cells, knocking downmGPDH activates the Nrf2 pathway, causing melanoma
cell metastasis, whereas co-inhibiting Nrf2 prevents
melanoma cell metastasis in vivo and in vitro. Thesefindings show that via inhibiting the Nrf2 signaling pathway, mGDPH suppresses melanoma cell motility andinvasion [47]. P62, an autophagy-degrading factor, isoverexpressed in malignant and metastatic melanomas
regardless of BRAF/NRAS mutant status. Overexpression of P62 has been shown to promote tumor growthand metastasis in various malignancies, including melanoma. P62 accumulates in melanocytes and melanoma
cells due to different stimuli, including UVA [33, 49]. P62
interacts with Keap1, preventing it from inhibiting Nrf2
and boosting Nrf2. Surprisingly, p62 is a direct target of
Nrf2, and the two are linked in a positive feedback loop.
UVA-induced p62 upregulation is inhibited when Nrf2
is silenced. P62 knockdown, on the other hand, hinders

Nrf2 overexpression. As a result, p62’s oncogenic activity
may be influenced by Nrf2 antioxidant responses [49, 67].
Nrf2 has also been linked to establishing the metastatic
niche and stromal progression in various studies. In the
pre-metastatic liver of melanoma-bearing mice, a reduction in Nrf2 expression was discovered, leading to oxidative stress and acidosis. This state is consistent with thetumor microenvironment, enhances melanoma cell survival, and facilitates metastasis [36]. These findings imply
that redox imbalance changes the environment in favorof metastasis and that the Nrf2 antioxidant response iscritical in this process.
There is also evidence of the preventive role of Nrf2 inmetastasis. The phytochemical cynaropicrin enhances
the Nrf2 activity in melanoma cells. Cicco et al. provedthat cynaropicrin reduces the capacity of human melanoma cells to migrate, invade, and proliferate in vitro.
However, this phytochemical can also suppress MAPK/ERK (extracellular signal-regulated kinase) and NFκB
(nuclear factor kappa-light-chain-enhancer of activated
B cells) pathways in human melanoma cells. Thus wecannot consider the anticancer effects of cynaropicrin
to be attributed only to Nrf2 stimulation [45]. Zhu et al.
reported that B16-F10 melanoma cells lacking Nrf2 are
more prone to tumor development and lung metastasis [24]. Weitzenböck et al. found that Nrf2 suppression
leads to the expression of CD44 and the development ofepithelial-mesenchymal transition phenotype. Of note,treatment of Nrf2 depleted cells with BRAF or MYC
inhibitor results in a higher survival rate of melanoma

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5

cells [50]. This shows that antioxidant intervention could
be a game-changing strategy for improving melanoma
patient survival.
Taken together, it can be concluded that despite thecontradictory findings regarding the role of Nrf2 inmetastasis, most studies indicate the facilitative role ofNrf2 in this subject. Further investigation is necessary tofind a clear conclusion as to whether or not nrf2 activity
is metastatic.
Clinical role of Nrf2 in melanoma

Melanoma is a poorly responsive malignancy with a
5-year survival of about 15% based on conventional
chemotherapy [68]. In recent years, novel targeted treatments and immunotherapy have drastically altered thegeneral approach to melanoma treatment. BRAF and
NRAS mutations in the MAPK signaling pathway arefound in about half of all melanoma cases [58], resulting
in constitutive activation of the MAPK pathway.
In patients with advanced mutant BRAF melanoma
diseases, BRAFi like as vemurafenib or dabrafenib
have been used successfully in conjunction with MEK(mitogen-activated protein kinase) inhibitors such as
trametinib. Moreover, for BRAF wild-type melanomas,
current recommendations advocate using monoclonal
antibodies (i.e., ipilimumab) that target immunologicalcheckpoint proteins such as anti-PD-1 or CTLA-4 (cytotoxic T-lymphocyte antigen 4) in combination with antiPD-1 therapy [69].
Despite the fact that new agents for melanoma have
improved survival compared with conventional chemotherapy, the response rate of metastatic melanoma is still
low and has little influence on prognosis. Malignant melanoma cells’ extensive resistance typically limits modern
treatments’ efficacy. Adaptive processes of melanoma
cells allow them to offset the effects of medicines, resulting in a multidrug-resistant phenotype [70]. Melanoma
cells rewire their proliferation and survival pathways,
putting their inherent resistance to apoptosis and ferroptosis to work, making them ‘bulletproof ’ against a wide
range of chemotherapeutic medicines [71]. Identifying
signaling pathways involved in the regulation and execution of apoptosis and altering them to promote melanoma cell sensitivity has offered a unique method forbattling melanoma chemoresistance in recent years. In
this context, a mountain of evidence suggests that metabolic modification plays a significant role in melanoma
cells’ chemo- and radio-resistance. During melanoma
progression, metabolic reprogramming, including redox
status, results in constitutive activation of Nrf2 and its
downstream enzymes. As a result, intracellular ROS levels drop considerably, conferring chemoresistance [18,72]. Therefore, Nrf2 is critical for controlling the redox

Page 14 of 18

system and fostering melanoma resistance cells throughmediating downstream antioxidant enzymes, detoxifying and drug-metabolizing enzymes, drug transporters, and multidrug resistance proteins [73]. Althoughmelanoma cells often create reactive oxygen species, theco-evolution of adaptive systems, specifically Nrf2 as amaster regulator, makes them more sensitive to emerging
chemoresistant cells’ ROS tolerance. In vitro studies haveshown that suppressing Nrf2 can make melanoma cells
more sensitive to anticancer medicines. As expected,
Nrf2 devoid cells have increased ROS levels, making
them more susceptible to anticancer drugs such as dacarbazine (DTIC) or cisplatin [18, 55]. Thus targeting Nrf2
may prevent melanoma from further progression andchemoresistance. Nevertheless, whether targeting Nrf2
alone is more beneficial or detrimental is still debated.
If Nrf2 is targeted as a single therapy, melanoma phenotypic change will likely occur. Thus A combinatorial strategy would very certainly be required [11].
Drugs that reduce the corticosterone production or its
effect on the receptor indirectly downregulate Nrf2, preventing tumor growth. In contrast, the induction of Nrf2
overexpression reversed this anti-tumor effect [26, 41].
According to the Rocha study, TMZ (temozolomide) promotes Nrf2 activation, which leads to the expression of
the GCLM (glutamate-cysteine ligase modifier) and GST(glutathione S-transferase) genes. Melanoma cells eventually develop TMZ resistance due to high levels of GSH.
GSH depletion, on the other hand, boosted TMZ sensitivity in cells [25]. As a result, using TMZ in conjunction
with Nrf2/GSH inhibitors enhances therapy outcomes.
According to multiple studies, RAF/RAS isoforms
activate the Nrf2 pathway, and BRAF/NRAS mutant
melanomas express more Nrf2 [57, 74]. Melanoma cells
also accumulate more Nrf2 in their nuclei, much higher
in MAPKi-resistant cells. Moreover, MAPKi-resistantmelanomas exhibit higher oxidative phosphorylation andoxygen consumption in their mitochondria. As a resultof the robust activation of the Nrf2 signaling pathway,these cells can modify mitochondrial metabolism, allowing MAPKi-resistant cells to survive. In BRAFi-resistantcells, Nrf2 silencing improves the efficacy of treatments
like vemurafenib or PLX4032 [31, 43, 54, 75]. These findings imply that resistant melanoma cells rely on the mitochondrial oxidative phosphorylation system (OXPHOS)
and that Nrf2-dependent glutathione metabolism isessential for the development of refractory melanomas.
Thus, the activation of the Nrf2 pathway is closely linked
to the development of resistant melanomas [31].
However, the effect of Nrf2 inhibitors on tumor growth
suppression cannot yet be conclusively theorized. Studies have revealed that adding metformin besides themain chemotherapeutic agents has a promising effect inMalakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5

preventing chemoresistance in an Nrf2-dependent manner. Sanches et al. investigated how metformin affectsoxidative stress and how it affects DTIC resistance in
B16-F10 murine melanoma cells. They found that whenmelanoma cells were first pre-treated with metformin
before and during the induction of DTIC resistance,
metformin synergized with DTIC and the cells became
sensitive to the therapy. This effect does not occur in the
exclusive treatment with metformin or DTIC, and bothof these conditions lead to the chemoresistance. According to findings, sensitive cells pre-treated with metformin
and DTIC had an increased level of Nrf2 compared to the
low Nrf2 levels of resistant cells [52]. Moreover, the cytotoxicity of some therapeutics depends on the activationof the Nrf2/NQO1 (NAD(P)H Quinone Dehydrogenase
1) axis. Co-treatment of melanoma cell lines with an Nrf2
inducer and β-lapachone increase the cytotoxic effect of
β-lapachone on the melanoma cells [35].
Ferroptosis is a kind of programmed cell death accompanied by lipid peroxidation and iron accumulation, vitalfor tumor growth control. It has been shown that activating the Nrf2 signaling pathway can prevent ferroptosis
cell death, boosting tumor development and resistance tochemotherapy. Thus, melanomas with a high Nrf2/HO-1
activity are resistant to ferroptosis cell death. The Nrf2/
HO-1 signaling pathway impresses melanoma cell chemosensitivity by modulating microRNAs and ferroptosisrelated proteins. For instance, activation of AKR1C1-3(Aldo-keto reductase family 1-member C1-3) inhibit
ferroptosis by lowering lipid ROS levels in melanoma
cells. Nrf2 enhances elastin-mediated AKR1C1-3 production, whereas Nrf2 inhibition prevents erastin-stimulated CHAC1 (cation transport regulator-like protein
1) expression during ferroptosis induction and inhibits ferroptosis-induced AKRs (Aldo-keto reductases)
expression [30, 38, 66]. Liao et al. reported that the miR130b-3p activates Nrf2/HO-1 pathway by targeting Dickkopf1, inhibiting ferroptosis cell death in melanoma cells[48]. Therefore, inducing ferroptosis following Nrf2 suppression has the potential to overcome tumor cell apoptosis resistance. Nobiletin, a natural product extracted
from citrus peel, can induce ferroptosis by GSK3βmediated downregulation of the Nrf2/HO-1 axis [53].
The prognostic role of Nrf2 in melanoma

Constant Nrf2 activation, as previously stated, promotes
tumor development and metastasis, resulting in a weak
prognosis [59]. Many studies have linked greater Nrf2
expression in melanoma cells to a more aggressive phenotype and a worse prognosis [20, 27, 57, 64]. A greater
Clark level, a deeper Breslow index, and nodular development are linked to increased Nrf2 expression. Furthermore, higher Nrf2 expression can predict patient survival

Page 15 of 18

before nodal or distant metastases [39]. Hintsala et al.
reported that nuclear NFE2L2 expression is also linked tomelanoma cell distant metastasis, nodular histology, anda deeper invasion, indicating that Nrf2 has a predictivefunction in original melanomas [20, 28]. Patients withmelanoma cells expressing nuclear Nrf2 have a worse
survival rate [57]. Hence, genetic manipulation or pharmacologic inhibition of Nrf2 may enhance melanoma
patient survival in advanced stages. These findings support the pharmacological use of Nrf2 inhibitors such as
ML385 and brusatol to avoid distant melanoma spreadand treat metastatic melanomas. Although these drugs
have drawbacks, they are effective [26, 41, 47].
Unlike other studies, one study found that lower Nrf2
expression is associated with a worse outcome in cutaneous melanomas. Oncogene-induced decrease of Nrf2 isthought to be an adaptive mechanism for establishing a
pro-inflammatory state that promotes cell survival andproliferation, according to researchers [16].

Conclusion
Nrf2, the master regulator of the antioxidant defense system, is expressed in melanocytes and malignant melanoma cells. The Nrf2 expression level is associated with
the incidence of melanoma and co-opted during cancer
progression. Indeed, the dual function of Nrf2 is context-dependent and varies by cell stage. Nrf2’s activity asa cellular defender at an early stage of melanogenesis is
likely to be transformed into a cancer driver by oncogenic
alterations that promote cell survival and provide protection against oxidative stress. Nrf2 addicted cancer cells
exhibit more aggressive disease and poor prognosis. Thusa potential treatment option might target the disturbed
redox homeostasis present in melanoma. However, it is
becoming clear that treating melanomas by reversing
increased oxidative stress is a misreading of the tumors’complex altered redox homeostasis. Targeting Nrf2
combined with conventional anti-melanoma treatments
like MAPK inhibition or immunotherapy may be more
beneficial.
Nevertheless, there are conflicting data on the role ofNrf2 in melanoma; Lack of sufficient knowledge makes
an emerging goal in melanoma research to elucidate theunderlying mechanism of Nrf2 activity and its functionin this cancer.
Abbreviations
ARE	Antioxidant response element
AKR	Aldo-keto reductase
AKR1C1-3	Aldo-keto reductase family 1-member C1-3
BRAF	B-rapidly accelerated fibrosarcoma
Bcl-2	B cell lymphoma-2
DTIC	Dacarbazine
CAT​	Catalase

Malakoutikhah et al. BMC Molecular and Cell Biology(2023) 24:5

cGAS	Cyclic GMP-AMP synthase
CHAC1	Cation transport regulator-like protein 1
COX2	Cyclooxygenase 2
CTLA-4	Cytotoxic T-lymphocyte antigen 4
EGFR	Epithelial growth factor receptor
GCLM	Glutamate-cysteine ligase modifier
GPX	Glutathione peroxidase
GSH	Glutathione
GST	Glutathione S-transferase
HIF-1	Hypoxia-Inducible Factor-1
HMOX-1	Heme oxygenase 1 gene
HO-1	Heme-oxygenase-1
Keap1	Kelch-like ECH-associated protein
MAPK	Mitogen-activated protein kinase
MEK	Mitogen-activated protein kinase
MITF	Microphthalmia-associated transcription factor
NRAS	N-Rat sarcoma virus
NFE2L2	Nuclear Factor Erythroid 2 Like 2
Nrf2	Nuclear factor erythroid2-related factor
OXPHOS	Oxidative phosphorylation system
PD-1	Programmed cell death protein 1
PD-L1	Programmed death ligand 1
PGE2	Prostaglandin E2
ROS	Reactive oxygen species
RTK	Receptor tyrosine kinase
SOD	Superoxide dismutase
STING	Stimulator of interferon genes
TMZ	Temozolomide
UV	Ultraviolet
VEGFA	Vascular endothelial growth factor A
WOS	Web of science
XIAP	X-linked inhibitor of apoptosis protein
Acknowledgments
None.

Page 16 of 18

2.
3.
4.
5.
6.
7.

8.

9.

10.
11.
12.
13.
14.

Authors’ contributions
Zahra Malakoutikhah: Conceptualization, Methodology, Investigation, Data
curation, Writing - Original Draft, Visualization, Writing - Review & Editing.
Zahra Mohajeri: Investigation, Data curation, Writing - Original Draft. Nasim
Dana: Writing - Review & Editing. Shaghayegh Haghjooy Javanmard: Supervision. The author(s) read and approved the final manuscript.

15.

FundingNone.

17.

Availability of data and materials
The datasets used in this investigation can be found in the paper.

18.

16.

Declarations

19.

Ethics approval and consent to participate
Not applicable.

20.

Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 11 September 2022 Accepted: 1 February 2023

21.

22.

23.
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