The role of signaling pathways in melanocyte malignant transformation and components of signaling cascades as targets for melanoma therapy

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Introduction

Melanoma is a malignant tumor that originates from melanocytes and causes high mortality rates. It is characterized by rapid growth, active metastasis, and resistance to many сhemotherapeutic drugs, posing a considerable challenge for researchers and physicians developing effective therapeutic options [1]. One such option is targeting the components of signaling pathways [1].

A signaling pathway is a system that activates second messengers, which causes changes in processes such as gene transcription, proliferation, and cell metabolism. One of the most prevalent signaling pathways is the MAPK cascade, which follows the conventional structure of receptor-mitogen-associated protein kinase kinase (MAPKK—MAPK kinase—MAPK—cellular response). A signaling pathway is initiated by a ligand-receptor interaction, which activates a receptor associated with a G protein or a tyrosine kinase. The receptors activate GTPases of the retrovirus-associated DNA sequences (RAS) and Rho families. GTPases phosphorylate MAPK kinase kinase, before sequential phosphorylation of MAPK kinase, MAPK, and OH groups of serine, threonine, or tyrosine of target proteins [2–4].

ERK (EGFR–RAF–MEK–ERK) signaling pathway

The extracellular signal-regulated kinase (ERK) signaling pathway was named after the key MAPK, which includes two proteins with similar structures (ERK1 and ERK2). The ERK pathway is activated by receptors with tyrosine kinase activity. In response to a signal, the receptor’s cytoplasmic portion assembles a protein complex that activates RAS GTPase. RAS binds and activates ERK kinase kinase (MEKK), which comprises the RAF proteins RAF1, ARAF, and BRAF [5]. When phosphorylated by MEK assumes an active conformation identified as two proteins (MEK1 and MEK2). The key MAPK ERK1/2 is activated by attachment of a phosphate group via MEK1/2 [5].

After phosphorylation, ERK1/2 diffuses from the receptor’s cytoplasmic portion into the cytoplasm, where it phosphorylates specific proteins, such as ELK1, c-JUN, cyclin D1, and c-Myc, which are involved in cell proliferation, survival, and differentiation [2, 6] (see Fig. 1, a).

 

Fig. 1. Functioning of the extracellular signal-regulated kinase (ERK) signaling pathway under normal conditions and in the presence of activating mutations: a, ERK signaling pathway in a normal cell; b, ERK signaling pathway in a tumor cell with an RAS mutation; c, ERK signaling pathway in a tumor cell with a RAF mutation; and d, ERK signaling pathway in a tumor cell with a MEK mutation. EGF, epidermal growth factor; SHC, Src homology 2 domain-containing protein; GRB2, growth factor receptor-bound protein 2; SOS, Son-of-Sevenless guanine nucleotide exchange factor; GAP, GTPase-activating protein; RAS, small GTPase; T599, threonine at position 599; S602, serine at position 602; S218, serine at position 218; S222, serine at position 222; T202, threonine at position 202; Y204, tyrosine at position 204; RAF, mitogen-activated protein kinase kinase kinase; MEK1, mitogen-activated protein kinase kinase; ERK1, mitogen-activated protein kinase; ATP, adenosine triphosphate; ADP, adenosine diphosphate; P, phosphate residue; GTP, guanosine triphosphate; GDP, guanosine diphosphate; G12D mutation, substitution of glycine at position 12 with aspartic acid; V600E mutation, substitution of valine at position 600 with glutamic acid; S222D mutation, substitution of serine at position 222 with aspartic acid. Created in BioRender: K. Vorobev (2025). Available at: https://BioRender.com/ki3ajnh.

 

The activity of protein kinases in the signaling pathway is “balanced” by that of phosphatases at practically every stage of signaling. This balance is a fundamental mechanism that regulates key cellular processes [7].

Molecular genetic defects in any component of this cascade can lead to permanent activation of the signaling pathway, resulting in malignant transformation and cancer [6, 7].

Hyperactivation of the ERK signaling pathway is reported in 90% of cases of cutaneous melanoma, with activating mutations in the genes BRAF and NRAS in more than 50% and 15%–20% of all cutaneous melanomas, respectively [8]. Mutations in the gene ERBB4, which encodes the EGFR receptor tyrosine kinase, are observed in 19% of melanomas without BRAF and NRAS mutations (pan-negative) [8]. These mutations activate the ERK and PI3K-mTOR signaling pathways, promoting proliferation [8].

Excess of growth factors

Impaired signaling may result in permanent receptor activation caused by excessive growth factor production. This process is associated with autocrine and paracrine regulation [7]. In autocrine regulation, the tumor cell synthesizes growth factors with subsequent receptor autoactivation, meaning that the source and target of the growth factor are the same cell, which independently maintains its growth and proliferative activity [7]. In paracrine regulation, the source and target of the growth factor are different cells [7]. In addition to promoting proliferation, paracrine signaling regulates other processes in melanoma, such as invasion and metastasis [9, 10]. Furthermore, the fibroblast growth factor receptor 3 (FGFR-3), which is highly expressed in malignant melanoma, correlates with high Breslow thickness and lymph node involvement [11]. FGFR3 promotes melanoma growth, metastasis, and epithelial–mesenchymal transition, most likely by modulating the phosphorylation of ERK, protein kinase B (AKT), and the EGFR [11].

ERK activation is primarily driven by EGFR ligands, such as the epidermal growth factor (EGF), transforming growth factor α (TGFα), and epiregulin [1, 12]. These ligands have conserved sequences, ensuring structural homology. Therefore, overproduction of any of them may cause pathological activation of the ERK signaling pathway [7].

One potential solution is the use of EGFR inhibitors (see Fig. 2). Recent research into melanoma highlight the use of a combination of EGFR inhibitors (crizotinib and foretinib) and a hepatocyte growth factor receptor inhibitor (lapatinib to reduce the proliferation and invasiveness of mucosal melanoma cells [13].

 

Fig. 2. Components of the extracellular signal-regulated kinase (ERK) signaling pathway as potential targets for melanoma therapy. Notes: LCAT, epidermal growth factor receptor ligand conjugated with an antitumor molecule; ISL, isoliquiritigenin; LRIG1, leucine-rich repeats and immunoglobulin-like domains protein 1; miR-301B, microRNA 301b; EGFR, epidermal growth factor receptor; SOS, Son-of-Sevenless guanine nucleotide exchange factor; RAS, small GTPase; H95, histidine at position 95; BRAF, mitogen-activated protein kinase kinase kinase; MEK1, mitogen-activated protein kinase kinase; ERK1, mitogen-activated protein kinase; ATP, adenosine triphosphate; BI-3406, nucleotide exchange factor inhibitor; DARPin, designed ankyrin repeat protein; iBRAF, BRAF inhibitor; iMEK, MEK inhibitor; PP2A, protein phosphatase 2A; EPE, peptide inhibitor of ERK1/2 nuclear translocation. 1, isoliquiritigenin reduces EGFR levels by suppressing miR-301B, a negative regulator of LRIG1 [16]; 2, LRIG1 decreases EGFR expression through ubiquitination of its ligand EGF [14]; 3, EGFR ligand conjugated with an antitumor compound [21]; 4, anti-EGFR antibody [22]; 5, BI-3406 inhibits SOS [34]; 6, DARPin allosterically modulates RAS activity by binding to H95 [35]; 7, BRAF kinase inhibitors [40]; 8, MEK kinase inhibitors [51–53]; 9, PP2A dephosphorylates ERK1, rendering it inactive [57]; 10, EPE peptide prevents ERK1 nuclear translocation [58]; 11, SCH722984, an ATP-competitive ERK1/2 inhibitor with additional allosteric properties that suppress ERK phosphorylation [59]; 12, antibody fragment blocking RAS-effector protein–protein interactions [36]. Created in BioRender: K. Vorobev (2025). Available at: https://BioRender.com/k7wlb9i.

 

Excess EGFR receptors on cell membranes

The EGF receptor is a tyrosine kinase consisting of four domains: an extracellular ligand-binding domain, one hydrophobic transmembrane region, a highly conserved cytoplasmic domain with tyrosine kinase activity, and a C-terminus. When a ligand binds, EGFR dimerizes and changes its conformation in the transmembrane and cytoplasmic domains, resulting in autophosphorylation of tyrosine residues at the C-terminus. The phosphorylated terminus contains interaction sites for proteins with the src homology 2 domain or phosphotyrosine binding domain, which activate the ERK and AKT signaling pathways, increasing the involvement of EGFR in oncogenesis [1].

Hyperexpression of both ligands and receptors can lead to pathological activation of a signaling pathway [14, 15]. The number of EGFRs expressed on the cell surface is regulated by the leucine-rich repeats and the protein immunoglobulin-like domains 1 (LRIG1) protein. LRIG1 antagonizes EGFR by reducing its levels in the cell membrane by ubiquitination of the receptor’s extracellular domain and subsequent degradation [14, 15]. Billing et al. [14] found that LRIG1 production is inhibited at the malignant transformation stage, with simultaneous EGFR activation. They reported an association between high LRIG1 expression and improved survival in patients with metastatic melanoma with high EGFR expression and triple-subtype wild-type melanoma without mutations that can activate the signaling pathway downstream of EGFR. Additionally, an in vitro experiment recorded a correlation between low LRIG1 expression and resistance to BRAF inhibitors [14]. Treatment of resistant melanoma cells with recombinant LRIG1 decreases AKT activity, which inhibit the proliferation of melanoma cells. Thus, recombinant LRIG1 is a potential therapeutic option in melanoma resistant to BRAF inhibitors [14].

Xiang et al. reported that LRIG1 demonstrated the most significant changes in expression in the treatment of melanoma [16]. The LRIG1 gene has a binding site for the miR-301b microRNA in the three prime untranslated region (3ʹ-UTR). Isoliquiritigenin suppresses the proliferation of melanoma cells by inhibiting miR-301b and inducing the synthesis of its target protein LRIG1. A miR-301b mimic decreases LRIG1 protein and mRNA levels, inhibiting isoliquiritigenin-induced apoptosis [16]. Furthermore, LRIG1 overexpression in melanoma cells under hypoxic conditions significantly suppresses invasion, migration, and vasculogenic mimicry, which increased after LRIG1 inhibition [17]. The mechanisms behind these phenomena suggest that elevated LRIG1 levels inhibit the hypoxia-induced epithelial–mesenchymal transition, which decreases E-cadherin expression and increases N-cadherin expression [17]. Another study suggests that LRIG1 has an oncogenic function during carcinogenesis in epidermal tissues in mice, as well as (potentially) in human keratinocytes and melanoma cells [15].

The nuclear factor erythroid 2 (NRF2) transcription factors constitute another EGFR regulator in melanoma cells [18]. NRF2 regulates oxidative stress and activates EGFR in melanoma, leading to high levels of EGFR and its ligands (EGF and TGFα). Sequencing shows that NRF2 directly binds to the EGF promoter. Therefore, oxidative stress induces EGF production, which increases in tumors with mutation-activated NRF2 [18]. NRF2 indirectly regulates EGFR and TGFα by inhibiting the melanocyte-inducing transcription factor (MITF). MITF effectively inhibits the expression of EGFR and TGFα, linking the NRF2 and EGFR pathways. NRF2 is necessary for complete activation of the EGFR pathway; NRF2 knockout cells show lower AKT activation in response to EGF stimulation than control cells. EGF induces nuclear localization and activation of NRF2, demonstrating that NRF2 and EGFR are linked in a positive feedback loop in melanoma [18].

EGFR overexpression in distant melanoma metastasis is associated with poor survival in male patients and patients with primary cutaneous melanoma without ulceration or Breslow thickness ≤4.0 mm [19]. Additionally, EGFR modulates ferroptosis, making BRAF V600E-mutated melanoma cells resistant to targeted therapy [20].

Treatment options for tumors with ERK hyperactivation caused by EGFR overexpression include anti-EGFR nanocarriers, which are EGFR ligands conjugated to nanoparticles with antitumor components [21], and anti-EGFR antibodies [22]. Both options have sufficient selectivity because of the preferential binding of drugs to cells overexpressing EGFR [21, 22].

Impaired EGFR function

The ligand, receptor, and subsequent links in the signaling cascade must all work together to ensure normal receptor function [7]. Oncogenesis is associated with mutations in EGFR regions that control the structure of the receptor’s tyrosinase domain. The most prevalent mutations are microdeletions in exon 19 (44%) and a point mutation in exon 21, which replaces leucine with arginine at position 858 (41%) [7]. These mutations cause autophosphorylation of the receptor regardless of interaction with the ligand, resulting in signal transmission to the intracellular cascade elements [7].

Mutations play a role in regions that encode the receptor’s extracellular domain [12]. For example, alterations in the extracellular portion of the EGFR receptor interfere with the recognition of the corresponding ligands in glioblastoma multiforme. The dimeric structure of EGFR is a product of ligand-receptor interactions. However, EGF induces the formation of “strong” homodimers, promoting proliferation. In contrast, epiregulin (EREG) promotes the formation of “weaker” heterodimers that regulate cell differentiation. Extracellular domain mutations decrease EGFR’s ability to discriminate between EREG and EGF, allowing it to form EGF-like dimers in response to interactions with EREG and other low-affinity ligands [12].

Mutations in EGFR have a role in tumor progression; however, they can also serve as therapeutic targets [7]. Affinity is one of the most significant properties of receptors with an altered amino acid sequence. High affinity in mutant receptors of tumor cell clones ensures the selectivity of antineoplastic agents [7].

Mutations in RAS proteins

RAS proteins (small GTPases) are the next functional link in the signaling cascade after the receptor [7]. RAS proteins have hydrolytic activity toward GTP, acting as a signaling switch owing to significant conformational differences between the GTP/GDP-bound forms of RAS. After ligand-mediated EGFR activation, GTP-bound RAS adopts an active conformation and interacts with effector molecules and regulatory proteins, whereas GDP-bound RAS becomes inactive and inhibits further signaling along the cascade axis [6, 23]. Amino acids at positions 12, 13, and 61 of the RAS protein are involved in hydrolysis [7]. The duration of GTP hydrolysis limits the cellular response, namely the rate of proliferation. Longer hydrolysis results in longer cellular responses, and vice versa [24]. Mutations in RAS and/or proteins involved in the GTP/GDP cycle in RAS proteins may influence the rate of their hydrolysis. These include GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). GAP activates GTP hydrolysis, promoting the formation of the inactive RAS form bound to GDP, whereas GEF promotes the formation of the active RAS form bound to GTP. Each RAS subfamily has unique GEF and GAP proteins [6, 25]. Their role is attributed to the high affinity of RAS for both GTP and GDP, which complicates small GTPase transition between the “on” and “off” states [6, 25].

In the human body, the RAS family includes more than 100 proteins; however, there is still no agreement on their classification. The RAS (coincides with the family name), Rho, Rab, Ran, and Arf subfamilies are currently recognized [6]. The classification of more recently discovered subfamilies, such as Miro, Roc, and Rag, requires clarification. The RAS subfamily is the most well-studied. Some of its proteins, such as H-RAS, K-RAS, and N-RAS, have been identified as classical proto-oncogenes, whereas other members (D-RAS, Noey2, and Rerg) can be classified as tumor suppressors [6].

Missense mutations that replace an amino acid at position 10, 12, 13, or 61 are the most common cause of a low rate of GTP hydrolysis. Glycine, the only amino acid without a side chain, is normally found at positions 12 and 13. Any mutation that substitutes glycine with another proteinogenic amino acid (except for proline) disrupts the interaction between GAP and KRAS through steric clashes with GAP’s arginine residue (see Fig. 1, b). Mutations cause the KRAS-GTP complex to accumulate, overactivating signal transduction pathways [26]. Additionally, regardless of EGFR status, RAS’s persistent “on” state causes constitutive activation of the signaling cascade [7, 24].

Mutations in NRAS are reported in approximately 20% of cutaneous melanomas, 10% of acral melanomas, and approximately 20% of conjunctival melanomas; however, they have not been found in uveal melanomas [27]. The NRAS mutation in codons 12, 13, or 61 is associated with transformation [28]. However, codon 61 plays a key role in melanoma. When accompanied by p16INK4a inactivation, KRASG12D or NRASQ61R expression promotes melanoma in vivo, whereas NRASG12D expression does not [28]. Furthermore, the NRASQ61R mutation, combined with the loss of Lkb1/Stk11, causes active metastasis [28]. The NRASQ61R mutation plays a more significant role in melanoma than that of NRASG12D because it increases nucleotide binding and reduces intrinsic GTPase activity, leading to the accumulation of the active RAS form bound to GTP [28]. Patients with non-uveal melanoma with NRAS mutations have a median survival of 8.2 months after stage IV diagnosis, which is shorter than the median survival of patients with wild-type melanoma (15.1 months) [29].

KRAS mutations are less common in melanoma; they are detected in 2% of cases, with the G12V mutation being detected in 77% cases [6]. Melanoma with this mutation occur in the female reproductive tract [30].

HRAS alterations are reported in 1.5% of melanoma cases [31]. These mutations are not typical of skin melanoma; however, they are found in Spitz nevus [32].

Additionally, melanoma has been linked to missense and truncating mutations in PREX2, RAS1’s GEF. Truncating mutations in PREX2 boost the activity of the GEFs Rac1, and tumors carrying these mutations have higher PI3K/AKT activation, leading to elevated cell proliferation [33].

RAS is a promising target for antineoplastic therapy; however, no drugs are currently approved for therapeutic use [23]. Difficulties in developing drugs that target RAS proteins stem from the need to identify drug-binding sites on the molecule. This protein seems to have no potential drug-binding pockets except for the nucleotide-binding site, which is difficult to target due to RAS’s high affinity for both GDP and GTP [25]. Molecular fragments suitable for targeting have been identified in the RAS molecule, and strategies have been proposed accordingly. These include disrupting the formation of the RAF/GEF complex to reduce the exchange of GDP for GTP [34, 35], and blocking the sites responsible for interaction with the effector molecules RAF and/or PI3K to inhibit further signaling [35, 36].

BRAF mutations

RAF kinases are a family of three specific serine/threonine protein kinases (ARAF, BRAF, and RAF1 (CRAF)) that are associated with retroviral oncogenes. RAF kinases are activated by RAS small GTPases [37].

BRAF plays the most significant role in melanoma. It includes three conserved regions (CRs): CR1 is the regulatory domain; CR2 is the hinge region between CR1 and CR3; and CR3 is the catalytic domain that mediates substrate phosphorylation. CR3 has the most complex structure, with a small N-terminal lobe responsible for ATP binding, and a large C-terminal lobe that binds substrate proteins. Additionally СR3 has the following subregions: the P-loop, the nucleotide-binding pocket, the catalytic loop, the DFG motif, and the activation loop [37, 38].

BRAF activation occurs as described below. Activated RAS interacts with BRAF (CR1 binding terminates the autoinhibitory effect on the CR3 catalytic domain) and recruits it to the plasma membrane, where conformational changes in the BRAF structure and its dimerization occur (both homodimers and heterodimers with other RAF proteins are possible). BRAF is then autophosphorylated at critical amino acid residues in the activation loop, primarily T599 and S602. The negative charge of phosphorylated amino acids destabilizes the interaction between the activation loop and the P-loop; leading to F595, a member of the DFG motif, releasing the nucleotide-binding pocket, making it accessible to ATP. BRAF gains catalytic activity; it phosphorylates and activates MEK1/2 [38, 39].

The BRAF V600E mutation, which replaces valine with glutamic acid at position 600, is especially important. It mimics constitutive phosphorylation of T599 and S602, which activating BRAF independently of RAS signaling [6, 38] (see Fig. 1, c).

BRAF inhibitors, such as vemurafenib, dabrafenib, and encorafenib, are currently the most well-studied. The Food and Drug Administration and the European Medicines Agency have approved them for the treatment of patients with progressive melanoma with the BRAF V600 mutation [6, 40]. Their advantages include the possibility of oral administration, a low molecular weight, and high selectivity for BRAF, particularly BRAF with the V600 mutation. This specificity can be explained by the preferential inhibition of the active conformation of BRAF by competitive displacement of the ATP-binding pocket, which stabilizes the kinase in its active conformation [41].

Preclinical studies found that vemurafenib and dabrafenib selectively inhibit BRAF kinase in V600-mutated melanoma cell lines by suppressing ERK phosphorylation and cell proliferation while inducing G1 cell-cycle arrest and apoptosis. The efficacy of drugs is determined by the type or presence/absence of mutation (V600E, V600D, V600R, or V600K) [40]. For example, encorafenib, which targets the V600E and V600K mutations, also inhibits wild-type BRAF to some extent [42].

Additionally, BRAF inhibitors influence the immune system by increasing the expression of tumor antigens and promoting tumor infiltration by T cells [40]. This indicate the potential efficacy of combination therapy with immune checkpoint inhibitors such as PD-1/PD-L1 (programmed death-ligand 1) and CDK4/6 (cyclin-dependent kinase 4/6) inhibitors [43].

Although therapy is initially effective, tumors frequently become resistant to it [44]. The primary resistance mechanisms include the following:

• Reactivation of the MAPK/ERK pathway by the NRAS mutation or enhanced CRAF expression, which bypasses BRAF inhibition;
• BRAF alternative splicing to produce truncated forms capable of the pathway dimerization and activation without an inhibitor; and
• Activation of parallel pathways (e.g., PI3K/AKT) that promote cell survival independent of the MAPK/ERK pathway [44].

MEK1/2 mutations

The MEK1 and MEK2 mutations are reported in 8% of melanoma cases [6].

There is an association between MEK molecular structure and activity, and changes in molecular structure during MEK activation have been investigated [36, 45]. The MEK molecule has the following key sequences: the N-terminal regulatory domain, the D domain, the kinase domain, the activation loop, the DVD domain, and the C-terminus [36, 45].

To induce further signaling, BRAF interacts with MEK via specialized regions, such as the D and DVD domains, ensuring highly specific binding and proper orientation within the BRAF catalytic pocket. In this complex, BRAF phosphorylates two conserved serine residues in the activation loop of MEK1 (S218 and S222) or MEK2 (S222 and S226), initiating a sequence of intramolecular conformational changes necessary for MEK activation [2, 45].

Unlike other protein kinases with similar structure, МЕК1/2 has modest basal activity [5]. MEK, like other classical kinases, has two subunits, each with its own regulatory sequence: an αA helix for the small subunit and an activation loop for the large subunit [46].

Phosphorylation of residues S218 and S222 stabilizes the activation loop via electrostatic interactions with neighboring amino acid residues (e.g., arginines). The DFG motif (D208–F209–G210) at the beginning of the activation loop shifts from the DFG-out position to the DFG-in position, in which D208 is directed into the active center and coordinates the Mg²⁺ ion, which is necessary for ATP binding and orientation. In the DFG-out position, phenylalanine blocks the ATP-binding pocket, whereas aspartate is exposed and interacts with H119 of the αC helix [5, 45]. The αC helix conformation associated with D208 at the DFG-out position is known as αC-out. Phosphorylation of the activation loop stabilizes the αC helix, which rotates to form an ion pair between K97 (β3-sheet) and E114 (αC helix). This position, known as αC-in, is typical of active kinases [5, 47]. Phosphorylation of the activation loop seems to induce a combined conformational change to form DFG-IN and αC-IN, making MEK1 completely active.

MEK basal activity is regulated by the αA helix, which, like the αC helix, is located in the N-terminal lobe [5, 45, 48]. The αA helix is the backbone of the small lobe, ensuring its compact structure. This is crucial for correct positioning of the αC helix. The αA helix indirectly influences E114, the catalytically important residue of the αC helix, which forms an ion pair with K97, a catalytically significant salt bridge located in the β3-sheet adjacent to the αA helix [5, 45, 48]. For E114 to be correctly oriented, αA and its interactions with adjacent structural elements must provide stable support. Small shifts in the αA helix position can disrupt or enhance the αC helix rotation, altering the enzyme’s active state [3, 45]. Nakae et al. found that H119 of the αC helix is oriented toward the αA helix and interacts with K57 in the DFG-in position, potentially affecting MEK activity [45]. Additionally, the αA helix has an allosteric effect on the αC helix after inhibitor binding. Allosteric MEK inhibitors bind near the αA/αC helices and can either stabilize or disrupt their mutual orientation [49].

In addition to activating ERK, MEK can interact with AKT via proline-rich sequence recognition domains (PRD), influencing the PI3K/AKT signaling pathway [50].

Similar to BRAF, phosphomimetic mutations (S218D, S222D, S222E) frequently cause MEK hyperactivation (see Fig. 1, d). The E203K mutation can also promote activation by increasing the active conformation stability, whereas the F53S mutation decreases the inactive conformation stability [5, 47, 48].

There are two main categories of MEK inhibitors:

1. Allosteric inhibitors, such as trametinib, which inhibits MEK1/2 and is used in combination with dabrafenib in melanoma with BRAF mutations [51], or binimetinib, which is effective in melanoma with BRAF and NRAS mutations [52];
2. ATP-competitive inhibitors, such as E6201, which is effective against MEK1 mutations resistant to allosteric inhibitors, including the C121S mutation in melanoma [53].

Resistance to MEK inhibitors in melanoma is caused by Q56P mutations in the αA helix or C121S mutations in the kinase domain [5, 47].

ERK1/2 mutations

The proteins ERK1 and ERK2 are 84% identical. They are activated by MEK-mediated phosphorylation of the TEY motif of ERK1 T202/Y204 and ERK2 T185/Y187 by MEK and remain active until dephosphorylation by dual-specificity phosphatases (DUSPs) [54].

Activated ERK moves to the nucleus and phosphorylates various nuclear substrates, influencing cell proliferation, survival, differentiation, motility, and angiogenesis [2]. The proliferative effects of ERK are mediated by the activation of positive cell-cycle regulators cyclin D1 and c-Myc, as well as the downregulation of antiproliferative proteins, such as Tob1, FOXO3a, and p21. ERK promotes cell survival by blocking NF-kB, which stimulates transcription of anti-apoptotic and pro-survival genes, such as Bcl-2 and Mcl-1 [2]. Therefore, ERK plays a significant role in oncogenesis.

ERK mutations are rare in primary tumors, especially melanoma [54]. They primarily arise on treatment with BRAF inhibitors (e.g., vemurafenib) or MEK inhibitors (e.g., trametinib) as a mechanism for “bypassing” the activation of blocked MAPK pathway links [54, 55]. Mutations in E322K, ERK1/2 A206V/A189V, and ERK1/2 S219P/S202P increase the activity of the ERK protein. Mutations that promote resistance to ERK inhibitors include ERK1 C82Y, ERK1 R84H, ERK1 Q90R, ERK1 Y148H, ERK2 Y131F, ERK2 D321G, and ERK2 E322K [54]. However, ERK inhibitors are promising antineoplastic drugs, given their therapeutic efficacy in disorders that occur upstream of ERK in the signaling cascade [4]. Furthermore, ERK inhibitors help overcome resistance to various antineoplastic agents [4].

Overexpression of wild-type ERK1/2 in the mutant BRAF V600E melanoma cell line A-375 leads to growth inhibition [54]. Two other melanoma cell lines with BRAF mutation, SKMEL-19 and WM266.4,also inhibit growth [54]. ERK2 overexpression in BRAF-mutant A-375 cells induced antitumor effects in vitro and in vivo, which could only be eliminated by ERK2 or BRAF knockdown. However, ERK2 overexpression in wild-type BRAF cells has no antitumor effect. Perhaps, ERK2 overexpression in these cells is caused by endoplasmic reticulum stress and DNA damage, as well as pro-apoptotic signals [56]. Studying this feature may lead to a new strategy for treating BRAF-mutant tumors with high ERK expression. There are several other options for the treatment of tumors that lack ERK hyperactivation-related antiproliferative properties, but are resistant to inhibitors of upstream elements of the signaling cascade.

One of these options is to model the activity of protein phosphatase 2A (PP2A), which has many isoforms that model various processes. PP2A in the MAPK pathway initiates both positive and negative regulation [57]. PP2A-B56β/B56γ dephosphorylates ERK1/2, inhibiting its activity and negatively regulating this pathway. PP2A-B55α dephosphorylates the kinase suppressor of RAS-1 (KSR1) and RAF-1, dissociating them from the 14-3-3 complex and activating MEK1, which ultimately leads to positive regulation of the MAPK pathway. Differences in downstream signaling regulation are determined by the therapeutic activation or inhibition of the holoenzyme PP2A [57].

Inhibition of ERK1/2 nuclear translocation can be used as an alternative to negative ERK regulation. This can be achieved using the EPE peptide, which blocks the ERK1/2-importin 7 interaction, inhibits ERK1/2 nuclear translocation, and thus accumulates active ERK1/2 in the cytoplasm. The EPE peptide significantly decreases the viability of melanomas with BRAF, NRAS, and NF1 mutations [58]. Notably, the combination of EPE peptide and trametinib show synergy in reducing the viability of some melanomas with the NRAS mutation [58]. Furthermore, this combination significantly decreases the viability of other melanoma cells, including those resistant to monotherapy with the EPE peptide and ERK cascade inhibitors [58].

The molecule SCH722984 has been tested against BRAF-mutant, NRAS-mutant, and wild-type melanoma [59]. SCH722984 is a potent ATP-competitive ERK 1/2 inhibitor with additional allosteric properties that inhibit ERK phosphorylation. It is effective against BRAF-, NRAS-, BRAF/NRAS-mutant melanomas and wild-type melanomas by arresting the cell cycle in the G1 phase and inducing apoptosis. The combination of vemurafenib and SCH722984 in BRAF-mutant melanoma is synergistic in most cell lines and significantly delays acquired resistance in long-term in vitro experiments [59].

Moreover, short hairpin RNAs (shRNAs) can be used to achieve long-term inhibition. Using A375 melanoma cells with an activating BRAF V600E mutation, it was found that ERK1 or ERK2 inhibition is associated with a decrease in cell proliferation, colony formation on agar, and apoptosis induction [60]. Melanoma cell death caused by ERK1 and/or ERK2 downregulation is caspase-dependent and is associated with high Bak, Bad, and Bim levels, decreased p-Bad levels, detection of activated Bax, and loss of mitochondrial membrane permeability. Moreover, direct targeting of ERK levels results in decreased BRAF, CRAF, and pMEK levels, confirming the significance of ERK inhibition in overcoming drug resistance [60].

AKT and ERK: two sides of the same coin

The EGF/ERK pathway has a parallel with the PI3K/AKT pathway. This section summarizes the data on the relationship between these cascades (see Fig. 3).

 

Fig. 3. Interaction between the AKT and ERK signaling pathways. Notes: EGF, epidermal growth factor; GRB2, growth factor receptor-bound protein 2; GEF, guanine nucleotide exchange factor; RAS, small GTPase; T599, threonine at position 599; S602, serine at position 602; S259, serine at position 259; S218, serine at position 218; S222, serine at position 222; T202, threonine at position 202; Y204, tyrosine at position 204; RAF, mitogen-activated protein kinase kinase kinase; MEK1, mitogen-activated protein kinase kinase; ERK1, mitogen-activated protein kinase; ATP, adenosine triphosphate; ADP, adenosine diphosphate; P, phosphate group; GTP, guanosine triphosphate; GDP, guanosine diphosphate; FoxO1, Forkhead box protein O1; PI3K, phosphoinositide 3-kinase; p85, regulatory subunit of PI3K; p110, catalytic subunit of PI3K; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PTEN, dual-specificity phosphatase; AKT, protein kinase B; S473, serine at position 473; T308, threonine at position 308; TSC1/2, tuberous sclerosis complex 1/2 (hamartin/tuberin); RHEB, RAS homolog enriched in brain; mTORC1, mammalian target of rapamycin complex 1. 1, EGFR can activate both the AKT and ERK signaling pathways [61]; 2, RAS can activate the catalytic subunit of PI3K [28]; 3, AKT inhibits RAF activity by phosphorylating the S259 residue [62]; 4, the MEK–AKT complex activates FoxO1 [61]; 5, ERK exerts a suppressive effect on TSC1/2; 6, ERK-dependent regulation of PTEN expression occurs through c-Jun-mediated transcriptional repression [61]; 7, PTEN controls cell-cycle phases by modulating ERK phosphorylation [61]. Created in BioRender: K. Vorobev (2025). Available at: https://BioRender.com/jazfnn2.

 

The interaction of the MAPK/ERK and PI3K/AKT pathways is a critical aspect of intracellular signal transduction that regulates cell proliferation, survival, migration, and metabolism. These cascades are activated by various receptors, primarily tyrosine kinase receptors, such as the EGFR under normal or abnormal conditions, including malignant transformation. The pathways can function independently, in a coordinated way, or through interaction [50].

The similarities between these cascades are typically determined by a single activator, such as EGFR. The phosphorylated C-terminus of the intracellular domain contains binding sites for proteins with the src homology 2 domain (including the p85 regulatory subunit, which is necessary for PI3K activation, and GRB2, which regulates the GEFs SOS) and the phosphotyrosine binding domain, initiating the PI3K-AKT and MEK–ERK pathways [1]. AKT activity decreases when treating resistant melanoma cells with recombinant LRIG1, a protein that decreases EGFR levels in the cell membrane by ubiquitinylation of the receptor’s extracellular domain [14]. Knockdown of NRF2, a factor that regulates EGFR expression and activation in melanoma, also results in a decrease in AKT activation. NRF2 indirectly regulates EGFR by inhibiting the MITF, which effectively suppresses EGFR expression [18].

In addition to their shared origin, these cascades have points of cross-regulation. For example, missense and truncating mutations of the PREX2 gene, which encodes GEF for RAS1 in melanoma cells, increase the activity of the PI3K/AKT pathway [33]. RAS proteins stimulate BRAF, an ERK pathway kinase, in addition to PI3K activation [37, 28]. NRAS mutations in melanoma cells may influence the extent to which NRAS binds to both PI3K and RAF [28]. Allosteric site-binding KRAS inhibitors inhibit the RAF/MEK/ERK and PI3K/AKT pathways in various melanoma cell lines [36]. Another crossover point is the MAPK kinase MEK, which can activate ERK and directly interact with AKT via the PRD. The specific mechanism of action of the MEK–AKT complex involves phosphorylation of the migration-associated transcription factor FoxO1. Researchers created a peptide that inhibits the interaction between MEK and AKT, regulating cell migration and adhesion [50]. Additionally, ERK can regulate PTEN, a negative PI3K regulator, and influence the transcription of PI3K pathway proteins [61]. AKT, in turn, can inhibit RAF-1 by phosphorylating Ser259, reducing MAPK cascade activation [62].

When one pathway is inhibited, the other can compensate by being enhanced, which is common in drug-resistant tumors [44].

Conclusion

The impaired signaling cascade function, specifically MAPK/ERK and PI3K/AKT, is crucial in the malignant transformation of melanocytes and melanoma. These pathways can be activated by mutations in the genes of individual components (BRAF, NRAS, MEK, and ERK), as well as external mechanisms, such as hyperexpression of ligands and receptors, disruption of their regulation by regulatory proteins (e.g., LRIG1), and activation of transcription factors (NRF2). Of special significance is the cross-regulation between the ERK and AKT cascades, which promotes the survival and drug resistance of tumor cells.

Current therapeutic strategies use targeted inhibitors to suppress the activity of individual links in signaling pathways; however, the efficacy of monotherapy is limited due to the rapid development of resistance to antineoplastic agents. Therefore, there is need to develop combination therapies that target multiple points of regulation and include not only BRAF and MEK inhibitors but also drugs that modulate the activity of AKT and ERK via regulatory proteins, such as PP2A and LRIG1.

A comprehensive approach that considers the molecular characteristics of the tumor, interactions between signaling pathways, and drug resistance mechanisms could facilitate personalized melanoma therapy and improve its efficacy.

Additional information

Author contributions: V.K.P.: conceptualization, visualization, writing—original draft; S.E.A.: writing—review & editing; N.O.L.: conceptualization, writing—review & editing; S.L.V.: writing—review & editing; N.V.M.: writing—review & editing. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding sources: No funding.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: No previously obtained or published material (text, images, or data) was used in this study or article.

Data availability statement: The editorial policy regarding data sharing does not apply to this work, as no new data was collected or created.

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer-review: This paper was submitted unsolicited and reviewed following the standard procedure. The peer review process involved three external reviewers, a member of the Editorial Board, and the in-house science editor.

About the authors

Kirill P. Vorobev

Siberian State Medical University

Author for correspondence.
Email: kirill72v@gmail.com
ORCID iD: 0009-0004-9237-2086
SPIN-code: 4202-9964

Postgraduate

Russian Federation, Tomsk

Elena A. Stepovaya

Siberian State Medical University

Email: stepovaya.ea@ssmu.ru
ORCID iD: 0000-0001-9339-6304
SPIN-code: 5562-4522

MD, Dr. Sci. (Medicine), Professor, Depart. of Biochemistry and Molecular Biology with the Course of Clinical Laboratory Diagnostics

Russian Federation, Tomsk

Olga L. Nosareva

Siberian State Medical University

Email: olnosareva@yandex.ru
ORCID iD: 0000-0002-7441-5554
SPIN-code: 5688-7566

MD, Dr. Sci. (Medicine), Professor, Depart. of Biochemistry and Molecular Biology with a course in clinical laboratory diagnostics

Russian Federation, Tomsk

Ludmila V. Spirina

Siberian State Medical University

Email: spirinalvl@mail.ru
ORCID iD: 0000-0002-5269-736X
SPIN-code: 1336-8363

MD, Dr. Sci. (Medicine), Professor, Head, Depart. of Biochemistry and Molecular Biology with a course in clinical laboratory diagnostics

Russian Federation, Tomsk

Vladimir М. Nagaitsev

Siberian State Medical University

Email: vn71@list.ru
ORCID iD: 0009-0003-3921-3935

MD, Cand. Sci. (Medicine), applicant, Depart. of Biochemistry and Molecular Biology with the Course of Clinical Laboratory Diagnostics

Russian Federation, Tomsk

References

Supplementary files