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Bellazzo et al. Cell Communication and Signaling (2025) 23:177
https://doi.org/10.1186/s12964-025-02173-4
Introduction
Oxygen is crucial for cell survival and function. ere-
fore, a prolonged reduction of oxygen availability
(hypoxia) usually represents a lethal condition for cells
and tissues. On the contrary, about 50–60% of solid
tumors are commonly affected by hypoxia [1]. In fact, the
rapid and uncontrolled growth of tumor cells commonly
outstrips the oxygen supply from the preexisting blood
vessels [2].
e adaptive response to hypoxia is mainly mediated
by the hypoxia-inducible factors 1 (HIF-1), 2 (HIF-2), and
3 (HIF-3). HIF proteins are heterodimers composed of an
oxygen sensitive α subunit (HIF-1α, -2α or -3α) and the
Cell Communication
and Signaling
†Arianna Bellazzo and Barbara Montico Contributed equally as rst
authors.
*Correspondence:
Barbara Montico
bmontico@cro.it
Elisabetta Fratta
efratta@cro.it
1Immunopathology and Cancer Biomarkers, Centro di Riferimento
Oncologico di Aviano (CRO), IRCCS, via Franco Gallini, 2, Aviano 33081,
PN, Italy
2Unit of Cancer Epidemiology, Centro di Riferimento Oncologico di
Aviano (CRO), IRCCS, via Franco Gallini, 2, Aviano 33081, PN, Italy
Abstract
Hypoxia is a common feature of solid malignancies, including cutaneous melanoma (CM). Hypoxia-inducible
factor (HIF)-1α and HIF-2α orchestrate cellular responses to hypoxia and coordinate a transcriptional program that
promote several aggressive features in CM, such as angiogenesis, epithelial-mesenchymal transition, metastasis
formation, metabolic rewiring, and immune escape. BRAFV600E, which is the most frequent mutation observed
in CM patients, usually increases HIF-α signaling not only in hypoxia, but also in normoxic CM cells, enabling
HIF-1α and HIF-2α to continuously activate downstream molecular pathways. In this review, we aim to provide
a comprehensive overview of the intricate role and regulation of HIF-1α and HIF-2α in CM, with a brief focus on
the complex interactions between HIF-α subunits and non-coding RNAs. We also discuss HIF-α-mediated cellular
responses in normoxia along with the mechanisms that allow HIF-α subunits to maintain their stability under
normal oxygen conditions. Finally, we resume available evidence on potential therapeutic approaches aimed at
targeting HIF-1α and/or HIF-2α.
Keywords Cutaneous melanoma, Hypoxia-inducible factors, Hypoxia, Normoxia, Non-coding RNAs
Unraveling the role of hypoxia-inducible
factors in cutaneous melanoma:
from mechanisms to therapeutic
opportunities
AriannaBellazzo1†, BarbaraMontico1*†, RobertoGuerrieri1, FrancescaColizzi1, AgostinoStean1, JerryPolesel2 and
ElisabettaFratta1*
Page 2 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
HIF-1β subunit, which is constitutively expressed, inde-
pendently from oxygen levels. In contrast, in normoxia,
cytoplasmic α subunits show a very short half-life, since
they are continuously degraded via the ubiquitin-prote-
asome system, whereas under hypoxic conditions, α sub-
units are stabilized via post-translational modifications
and translocated into the nucleus, where they dimerize
with HIF-1β [3].
HIF-1 was initially discovered through a study on the
erythropoietin gene [4]. Subsequently, the structural
analysis of the HIF-1α protein revealed four distinct
domains: a basic helix-loop-helix (bHLH) domain for
DNA binding and dimerization, two Per-ARNT-Sim
(PAS) domains for dimerization and target gene speci-
ficity, an oxygen-dependent degradation (ODD) domain,
and two transactivation domains (N-TAD and C-TAD)
[5]. HIF-2α is closely related to HIF-1α, whereas HIF-3α
misses the C-TAD domain, which is required for HIF-1α
and HIF-2α transcriptional activity. In fact, the C-TAD
was found to interact with CREB-binding protein (CBP)
and p300 co-activators to activate gene transcription [6].
As shown in Fig.1, α subunits are distinct from HIF-1β
since they all possess the ODD domain, which is crucial
for preventing their degradation in hypoxic condition [7].
e existence of three different α subunits evokes ques-
tions about target genes exclusively induced by each of
them, as well as the specificity of their regulated pheno-
types. Unlike HIF-1α and HIF-2α, that are widely studied
and clearly linked to development and carcinogenesis,
HIF-3α functions are still largely unknown, especially
in the context of malignancies [8–11]. e presence of
multiple transcript variants of HIF-3α, showing tissue-
specific expression and potential divergent roles in the
regulation of gene expression, makes harder to determine
its molecular functions [12]. Differently, HIF-1α and
HIF-2α are well characterized, with a list of both com-
mon and unique target genes, which reflect their similar
as well as independent roles in different tumor types (for
Fig. 1 Schematic illustration of HIF-1α, HIF-2α, HIF-3α isoforms and HIF-1β protein. As shown, HIF-α proteins possess a bHLH and two PAS domains, re-
sponsible for the heterodimerization. Instead of having the C-TAD domain, HIF-3α has a LZIP domain that allows the protein interaction. The constitutively
expressed HIF-1β does not contain the ODD, N-TAD and ID domains. Abbreviations are listed in the legend section. Created in BioRender ( h t t p s : / / B i o R e
n d e r . c o m / s 3 8 c 4 1 7)
Page 3 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
review see [13]). Importantly, N-TAD domain, that differs
between the two isoforms, has been shown to contribute
to HIF-1α and HIF-2α target genes specificity, whereas
the C-TAD domain is homologous and promotes the
expression of HIF-1/HIF-2 common target genes [14].
Knockout mice have showed that both HIF-1α and
HIF-2α are not-redundant and essential in develop-
ment, since the targeted independent disruption of both
subunits resulted in embryonic lethality. Nevertheless,
embryos died of different causes and at different age. For
instance, HIF-1α -/- embryos did not survive beyond car-
diac and vascular defects, whereas HIF-2α -/- had normal
systemic vasculature but suffered of bradycardia, and did
not exhibit completed lung maturation [15]. Accordingly,
HIF-1α and HIF-2α pattern of expression vary among
tissues. Specifically, HIF-1α is detected ubiquitously,
whereas HIF-2α exhibits a more tissue-specific expres-
sion pattern [16]. In response to reduced oxygenation,
HIF-1α primarily mediates acute responses, whereas
the chronic hypoxic response involves HIF-2α activa-
tion [17]. Although both subunits have been delineated
as key factors in promoting tumor progression, how-
ever, the specificity of their functions remains to be fully
elucidated.
Epidemiological characteristics and genetic
alterations in cutaneous melanoma
Cutaneous melanoma (CM) represents a very aggressive
neoplasm originating from the malignant transformation
of melanocytes, accounting for about 20% of all cutane-
ous cancers [18]. Although it is the most lethal form of
skin cancer, the 5-year relative survival is above 80%, with
worse prognosis for nodular CM compared to lentigo
maligna or superficial spreading CM [19]. CM incidence
has dramatically increased in recent decades among
light-skinned populations. Approximately 85% of CM
occurring annually affect populations from North Amer-
ica, Europe and Oceania, with Australia and New Zea-
land showing the highest CM age-standardized incidence
rates (40–50 cases/100000 per year) [18]. In Europe, the
average incidence of CM is 9 cases/100,000 per year,
with higher prevalence in Nordic populations (about 20
cases/100000 per year) [18].
Genetic alterations are common in both familial and
sporadic CM, with frequently alteration of the MAPK/
ERK signaling. In particular, the constitutive activation
of the MAPK/ERK signaling caused by mutations within
BRAF gene appears as a major driver of CM tumorigenic
potential and survival [20, 21]. BRAF is a serine/threo-
nine protein kinase encoded by the BRAF gene located on
chromosome 7. At present, it is well known that over 90%
of BRAF mutations occur in exon 15 and include substi-
tutions of valine at position 600 (V600) with another ami-
noacid. In particular, the substitution of glutamic acid for
valine (V600E, codon GTG > GAG), accounts for 70–80%
of the BRAFV600 mutations. e second most common
mutation is BRAFV600K substituting lysine for valine
(GTG > AAG), followed by BRAFV600R (GTG > AGG),
and by an infrequent two-nucleotide variation of the
predominant mutation that includes the BRAFV600 ′E2′
(GTG > GAA) and the BRAFV600D (GTG > GAT) [22].
BRAF mutations are able to produce an activation of the
kinase domain of the protein, leading to an uncontrolled
stimulation of cell proliferation [23]. NRAS is the second
most frequent mutation in CM (15–20%), after BRAF
[24]. In the majority of cases (> 80%), NRAS gene pres-
ents a missense mutation within codon 61, which dis-
rupts the GTPase activity of NRAS, locking it in its active
conformation, independent of upstream RTK activation
[25]. Besides BRAF and NRAS, a small subset of CM
presents NF1 (10–15%) and c-Kit (1–3%) mutations [25].
Finally, a high frequency of PTEN mutations have been
reported in highly metastatic CM, where they frequently
coexist with BRAF mutations, but not with NRAS [25].
Constitutive expression of HIF-1α and HIF-2α has
been frequently detected in CM [26]. BRAF-mutant
CM, in particular, usually exhibits dramatic activation
of HIF-1α signaling, often uncoupled from hypoxia [27–
29]. Along this line, microarray profiling on 30 cell lines
derived from various stages of CM progression and 5
melanocytes showed that HIF-1α expression was signifi-
cantly higher in BRAF-mutant CM cell lines respect to
cells with wild-type BRAF or melanocytes. Accordingly,
suppression of BRAFV600E decreased HIF-1α expression
and impaired CM cell survival and proliferation, whereas
its overexpression increased hypoxic tolerance through
HIF-1α up-regulation, suggesting that the oncogenic
activity of BRAFV600E might be partially mediated directly
through the HIF-α signaling pathway [27].
In the last years, a better understanding of the CM
biology has resulted in the development of different
FDA approved therapeutic strategies relying on the
use of small molecule inhibitors of BRAF (BRAFi) and
MEK (MEKi) and blockers of the immune checkpoint
molecules. e advent of targeted and immune-based
therapies has not only expanded the treatment options
for CM but also proven clinical benefits in patients with
unresectable or metastatic CM. However, primary and
acquired resistance often limit the clinical effectiveness
of these therapies. Importantly, aberrant expression of
HIF-1α and HIF-2α might influence response to therapy
and impact on drug resistance in CM.
Hence, this review aims to provide an overview of the
activity and the regulation of HIF-1α and HIF-2α in CM,
not only under hypoxia, which occurs when oxygen (O2)
levels fall below 1 kPa, but also in normoxic environ-
ments. With the term “normoxia”, we refer to the atmo-
spheric oxygen (O2) levels of 18kPa at which the majority
Page 4 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
of pre-clinical studies with cells are conducted. is O2
tension is clearly higher respect to that measured in
physiological tissues (i.e. 3–8kPa in the skin), and termed
“physoxia” [30, 31]. Based on these considerations, a list
of papers with the terms “CM”, “hypoxia”, “normoxia”
and/or “HIF” were retrieved and selected, mainly focus-
ing on those published in the last two decades. e
potential therapeutic targeting of HIFs to improve treat-
ment efficacy in CM will be also discussed.
HIF-1α-related targets in hypoxia and normoxia in
CM
In CM, HIF-1α mediates the transcription of genes
involved in several processes, including angiogenesis and
vascular remodeling, epithelial to mesenchymal transi-
tion (EMT), metastasis, and cellular metabolism, in both
hypoxic and normoxic conditions. A selection of the
most frequently HIF-1α-target genes in CM is given in
Table1.
Angiogenesis and vasculogenic mimicry. It has become
increasingly clear that the interaction of endothelial cells
(EC) and cancer cells affects tumor growth and vascular-
ization. Hypoxia and HIF-α signature are master regu-
lators of microcirculation acquisition, thus supporting
malignant growth and hematogenous dissemination.
e delineation of molecular mechanisms of neo-angio-
genesis in cancer has revealed a critical role for HIF-1α
in positively regulating pro-angiogenic factors, including
vascular endothelial growth factor (VEGF) and its recep-
tors (i.e. FLK-1 and FTL-1), angiopoietins and platelet-
derived growth factor beta [32]. To date, although the
hypoxia-induced new vessel formation is a common fea-
ture of CM, the number of studies directly focused on
HIF-1α and angiogenesis in CM progression and treat-
ment are still limited. However, it has become clear that
HIF-1α fosters pro-angiogenic factors expression in CM
[33]. Indeed, Trisciuoglio et al. reported that Bcl-2, via
BH4 domain, increased HIF-1α half-life by counteracting
its ubiquitination, in order to enhance HIF-1α-mediated
VEGF expression and secretion in CM cells exposed
to hypoxia [34]. Of interest, vasculogenic mimicry, a
process that cooperates with neo-angiogenesis for the
recruitment of new vessels, was firstly identified in CM
and uveal melanoma through in vitro and in vivo stud-
ies [35, 36]. In particular, vasculogenic mimicry describes
the ability of CM cells to express endothelium-associated
genes and ECM-remodeling proteins to mimic the pres-
ence and function of EC and to support a capillary-like
structure formation [35, 36]. Mouse CM cells injected
in mouse ischemic limbs increased the expression of
HIF-1α and VEGF to support the formation of vascu-
logenic mimicry channels [37]. In a study by Comito et
al., once stabilized upon hypoxia-induced accumulation
of mitochondrial reactive oxygen species (ROS), HIF-1α
activated MET expression in CM cells to promote cap-
illary-like structures formation and vasculogenic mim-
icry [38]. In a subsequent study by Spinella et al., hypoxia
induced production of endothelin-1 (ET-1) that, in
turn, sustained HIF-1α/HIF-2α-mediated VEGF-A and
VEGF-C expression in CM cells and EC, suggesting HIFs
involvement in promoting endothelia remodeling and
vasculogenic mimicry. Consistent with this hypothesis,
silencing of HIF-1α/HIF-2α completely abrogated cell
migration and hypoxia-induced capillary-like structure
acquisition, in both CM and EC exposed to CM cells sec-
retome. Furthermore, ET-1 capacity to increase VEGF-A
and VEGF-C mRNA and protein expression in CM and
EC was reduced [39].
EMT and metastasis. e expression of HIF tran-
scription factors has been suggested to correlate with
the increased malignant potential of melanocytes. In
fact, several genes that act as key players in melanogen-
esis and melanocyte behavior contain putative func-
tional hypoxia response elements (HREs) and, therefore,
might be directly targeted by HIF transcription factors
[26]. In another study, transcriptomic analysis of murine
immortalized melanocyte cells exposed to hypoxia iden-
tified a signature that included thirteen EMT-associated
genes, of which only two, Plod1 and Plod2, exhibited
HRE motifs within their promoters [40], supporting that
HIF-1α regulation might occur through both direct and
indirect mechanisms.
In hypoxic conditions, HIF-1α has been demonstrated
to contribute to CM heterogeneity, and to trigger meta-
static progression by reprogramming gene expression
patterns in proliferative CM cells, making them more
invasive in in vitro assays. e incubation of a set of
human CM cells in hypoxia chamber allowed to identify
eighteen HIF1α-dependent genes that were differentially
regulated in the switch from a proliferative to an inva-
sive phenotype in CM cell cultures. Interestingly, the
microphthalmia-associated transcription factor (MITF)
and several of its target (i.e. TRP1, TNFRSF14, CAPN3,
GPM6B, and DAPK1) were among repressed genes [41],
indicating that HIF1α-regulated MITF might act as a key
regulator of the process of hypoxia-induced phenotype
switching in CM. In accordance, a study by Cheli et al.
revealed that, in hypoxic condition, HIF-1α positively
regulated the expression of the transcription factor basic
helix-loop-helix protein 2 (BHLHB2) to inhibit MITF,
thus increasing the tumorigenic and metastatic prop-
erties of CM cells [42]. e interplay between HIF-1α
and the receptor tyrosine kinase-like orphan recep-
tor (ROR)-1 and ROR-2 has been intimately involved in
the switch from a proliferative to an invasive phenotype
as well. More specifically, following hypoxia exposure,
Wnt5A stabilized HIF-1α, resulting in ROR-2 up-reg-
ulation and decreased expression of MITF and ROR-1.
Page 5 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
Pathway HIF-α Target gene (s) Hypoxia/Normoxia Cell line(s) BRAF/NRAS
mutational
status
Ref-
er-
ence
Angiogenesis and vas-
culogenic mimicry
HIF-1α MET Hypoxia Hs29-4T BRAFV600E [38]
HIF-2α sVEGF Hypoxia/normoxia B16F10
A375
wt
BRAFV600E [64]
HIF-1α/HIF-2α VEGF-A
VEGF-C
Hypoxia/normoxia 1007
M10
M14
SK-Mel-28
wt
*n.a.
BRAFV600E
BRAFV600E
[39]
EMT and metastasis HIF-1α Genes downregulated
in the switch from a
proliferative to an
invasive phenotype
CAPN3
DAPK1
GALNT3
GPM6B
MITF
MYO1D
TNFRSF14
TYRP1
Genes upregulated in
the switch from a pro-
liferative to an invasive
phenotype
ADAM12
AXL
BIRC3
COL13A1
CRIM1
FGF2
FLNB
HS3ST3A1
KCNMA1
ITGA3
Hypoxia M000921
M010817
M080307
M080423
BRAFV600E
NRASQ61R
n.a.
n.a.
[41]
HIF-1α HIF1α repressed MITF
promoter through the
transcription factor
BHLHB2
Hypoxia B16F10
SK-Mel-28
wt
BRAFV600E [42]
HIF-1α HIF1α induced a switch
from ROR-1 to ROR-2 to
activate Wnt5A signal-
ling pathway
Hypoxia G361
M93-047
UACC1273EV
UACC647
UACC903
WM35
WM793
WM983B
451Lu
1205Lu
BRAFV600E
NRASQ61R
n.a.
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
[43]
HIF1α directly bound
to CD147 promoter
to induce MMP-2
activation
Hypoxia A375
G361
BRAFV600E
BRAFV600E [44]
HIF-1α uPAR Hypoxia R-18 n.a. [48]
HIF-1α TB-4 Hypoxia B16F10 wt [49]
HIF-1α MET Hypoxia Hs29-4T BRAFV600E [38]
HIF-1α BIRC-7 Hypoxia/normoxia A875
M14
wt
BRAFV600E [52]
HIF-1α Rab27 Normoxia B16-F10 wt [53]
Table 1 HIF-1α and HIF-2α targets in CM
Page 6 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
ROR2 expression associated with a more aggressive phe-
notype and was critical for Wnt5A-mediated invasion
and metastasis in CM [43].
HIF-1α was also shown to orchestrate EMT and metas-
tasis in CM by increasing the expression of proteins
and enzymes directly implicated in extracellular-matrix
(ECM) remodeling, including matrix metalloprotein-
ase-2 (MMP-2) and urokinase-type plasminogen activa-
tor receptor (uPAR). Once activated by hypoxia, HIF-1α
enhanced the invasion and metastatic potential of CM
Pathway HIF-α Target gene (s) Hypoxia/Normoxia Cell line(s) BRAF/NRAS
mutational
status
Ref-
er-
ence
HIF-2α Snail Hypoxia 1205Lu WM35
WM793
WM115A
WM3523A
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
[65]
HIF-2α AP2a Hypoxia 501-mel BRAFV600E [66]
HIF-1α/HIF-2α CTGF Hypoxia K457 BRAFV600E [67]
HIF-1α SFK via PDGFRA
MT1-MMP
Hypoxia A375SM
WM266-4
BRAFV600E [68]
HIF-2α SFK via FAK
MMP-2
Metabolic
reprogramming
HIF-1α GLUT1
HK2
LDHA
Hypoxia SbCl2
WM35 WM239A
WM1158
WM1232
WM1366
WM3211
451Lu
1205Lu
Mel-Im
Mel-Ju
NRASQ61R
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
NRASQ61R
wt
BRAFV600E
BRAFV600E
n.a.
n.a.
[56]
HIF-1α PDK1 Normoxia Mel 272
Mel 593
Mel 611
BRAFV600E
BRAFV600E
BRAFV600E
HIF-1α GLUT1 and LDHA
overexpression
ATP5ME, NDUFA6,
NDUFB5, NDUFB6
and NDUFS8
downregulation
Hypoxia A2058
HT-144
SK-Mel-5
SK-Mel-28
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
[57]
HIF-1α PKM2 Hypoxia RPMI 8322
SK-Mel-23
SK-Mel-103
SK-Mel-187
VMM39 WM2664
501-mel
526-mel
624-mel
wt
wt
NRASQ61R
wt
NRASQ61R
BRAFV600E
BRAFV600E
BRAFV600E
BRAFV600E
[58]
HIF-1α PDK1 Normoxia A375
MEWO
Sk-mel-28 WM35
BRAFV600E
wt
BRAFV600E
BRAFV600E
[59]
HIF-1α GM3S Hypoxia B16F10
G361
wt
BRAFV600E [61]
Inammation and
immunomodulation
HIF-1α ACKR2 Hypoxia B16F10 wt [63]
HIF-1α /HIF-2α Treg Hypoxia/normoxia B16F10 wt [70]
HIF-1α /HIF-2α CD8 + T cells Hypoxia B16 wt [71]
* Data not available
Table 1 (continued)
Page 7 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
cells by fostering MMP-2 expression through the involve-
ment of CD147 [44], a key inducer of MMPs [45]. Chro-
mosome immunoprecipitation assay supported that
HIF-1α directly bound to CD147 promoter. Indeed, point
mutations within CD147 promoter attenuated MMP-2
response to hypoxia both in vitro and in vivo [44]. e
interaction between urokinase-type plasminogen acti-
vator receptor (uPAR) and its ligand, which is mainly
involved in pericellular proteolysis, is a central step in
mesenchymal motility. By binding to the HRE within
uPAR promoter, HIF-1α mediated uPAR transcription in
various cancers under hypoxia [46, 47]. In CM, in partic-
ular, uPAR overexpression promoted spontaneous lymph
nodes metastasis in in vivo models [48]. In addition to
MMP-2 and uPAR, hypoxia might induce CM metastasis
through a putative cooperation between HIF-1α and thy-
mosin beta-4 (Tβ4) [49], an actin-sequestering molecule
involved in cytoskeletal reorganization [50]. At present,
the molecular mechanism by which the HIF-1α/Tβ4 axis
contributes to CM metastasis is still largely unknown.
Nevertheless, in a study by Makowiecka et al., Tβ4 was
not only a component of focal adhesions, but also directly
regulated their formation, thus altering the adhesion abil-
ities of CM cells [51].
As described above, the ROS/HIF-1α/MET signal-
ing axis has been strongly involved in hypoxia medi-
ated vasculogenic mimicry in CM. In the same study,
MET expression and activation were also correlated with
enhanced CM cells migration in vitro and lung metasta-
sis formation in vivo [38], thereby suggesting that HIF-1α
and MET might cooperate in eliciting an escape strategy
to avoid the hypoxic CM environment.
More recently, Xu et al. unveiled baculoviral IAP
repeat-containing protein 7 (BIRC7) as a downstream
HIF-1α target gene. Although BIRC7 is known to act as
an apoptosis inhibitor, its knockdown inhibited the inva-
sion of A875 and M14 CM cells in both hypoxia and
normoxia [52], indicating that even in normoxic condi-
tion HIF-1α could sustain invasiveness. Under lactate
stimulation, HIF-1α was found to drive lung metastasis
in B16F10 transplanted mice, specifically by regulating
Rab27 expression and extracellular vesicles secretion
[53].
Metabolic reprogramming. HIF-1α represents the main
transcription factor for the induction of genes encod-
ing glucose transporters (GLUT 1/4) and glycolytic
enzymes, such as aldolase, enolase, lactate dehydroge-
nase (LDHA) and phosphoglycerate kinase-1, allowing
hypoxic tumors cells to take up glucose more efficiently.
Additionally, HIF-1α can suppress oxidative phosphory-
lation (OXPHOS) by reducing metabolites entry into
the tricarboxylic acid cycle (TCA), via the direct induc-
tion of the key glycolytic enzyme pyruvate dehydroge-
nase kinase (PDK) 1 that, in turn, phosphorylates the
pyruvate dehydrogenase (PDH) complex, thus preventing
the conversion of pyruvate into acetyl-CoA [54]. Respect
to melanocytes, malignant CM cells, particularly those
harboring the BRAFV600E mutation, exhibit the Warburg
effect with high glucose consumption and more lactate
production even in the presence of oxygen and fully
functioning mitochondria [55]. e BRAFV600E mutation
might transactivate a panel of transcriptional regulators
of glycolysis, including HIF-1α [28] and its target genes
GLUT1, hexokinase 2 (HK2), LDHA [56], and PDK1 [29].
Under hypoxia, HIF-1α-dependent upregulation was
accompanied by a decreased expression of several com-
ponents of the OXPHOS, including ATP5ME, NDUFA6,
NDUFB5, NDUFB6 and NDUFS8 [57]. HIF-1α activa-
tion could also up-regulate pyruvate kinase M2 (PKM2),
a rate-limiting enzyme of glycolysis, further minimizing
OXPHOS [58].
HIF-1α-dependent glycolytic program offers metabolic
plasticity but also growth advantage for CM cells. For
example, Liu et al. demonstrated that HIF-1α enhanced
PDK1 transcription by recruiting the Ku80 protein to
PDK1 promoter. Consistently, treatment with melato-
nin, which affects the stability of HIF-1α at the protein
level, suppressed the binding of Ku80 to the PDK1 pro-
moter and inhibited CM growth [59]. By reprogramming
the glucose metabolic pathway and ensuring scaveng-
ing of mitochondrial free radicals, HIF-1α enhances the
antioxidant capacity of tumor cells, fostering radioresis-
tance [60]. In this context, it has been demonstrated that
HIF-1α could indirectly decrease the expression of the
ganglioside GM3 synthase, thus reducing synthesis of
glycosphingolipids and increasing resistance of CM cells
to oxidative stress and radiation therapy [61].
Inammation. In silico analysis suggested that inflam-
matory response, together with increased recruitment of
T-cells, B-cells, NK-cells, monocytes and macrophages,
were strongly associated with CM [62]. Intriguingly,
HIF-1α has emerged as one the main pathways impli-
cated in the regulation of the inflammatory response.
Atypical chemokine receptor 2 (ACKR2), which regulates
a number of pro-inflammatory chemokines reported to
drive cytotoxic cells in tumor microenvironment (TME),
has been recently identified as a direct transcriptional
target of HIF-1α. In a study by Benoit et al., the decreased
ACKR2 expression observed following HIF-1α deple-
tion was associated with an augment of Chemokine (C-C
motif) ligand 5 levels in hypoxic B16F10 CM cells, pro-
viding novel evidence on how hypoxia might impair pro-
inflammatory TME and hamper immune infiltration [63].
Page 8 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
HIF-2α-related phenotypes in hypoxia and
normoxia in CM
While HIF-2α has been largely characterized in renal
cell carcinoma, where it has been shown to represent a
key driver of this disease, studies focused on its function
in other human malignancies, including CM, are still in
their infancy. At present, however, it is known that, simi-
larly to HIF-1α, HIF-2α can regulate several pathways,
thus contributing to the malignant behaviors of CM cells
(Table1).
Angiogenesis and vasculogenic mimicry. Unlike HIF-1α,
the role of HIF-2α in regulating angiogenesis is still
ambiguous and not complete clarified. As stated above,
ET-1 production sustained HIF-1α/HIF-2α-mediated
VEGF-A and VEGF-C expression in CM and EC, thus
promoting neo-vascularization [39]. Surprisingly, and
in contrast with the study by Spinella et al., a puta-
tive tumor-suppressor role for HIF-2α has been also
described. In fact, stabilization of HIF-2α with the pro-
line hydroxylases (PHD) 3 inhibitor AKB-6899 decreased
angiogenesis in B16F10 CM-bearing mice and in A375
CM cell-line derived xenograft, by promoting secretion
in the tumor environment of a soluble form of the VEGF
receptor (VEGFR), which usually inhibits VEGF activity.
On the contrary, HIF-1α accumulation and VEGF pro-
duction were not affected by AKB-6899 treatment [64].
EMT, metastasis and stemness. In the last years, a
number of evidence has confirmed that aberrant HIF-2α
activation contributes to CM metastasis. Under hypoxia,
HIF-2α promotes EMT and cell migration by enhanc-
ing the expression of specific target genes, which can
be, or not, commonly regulated by HIF-1α. For instance,
HIF-2α, but not HIF-1α, directly enhanced Snail expres-
sion in CM cells, fostering features associated with stem-
ness in vitro and increasing metastatic capacity in vivo
[65]. A mass spectrometry analysis of HIF-2α interac-
tome identified specific HIF-2α interactors involved in
CM development, including MITF, SRY-box transcrip-
tion factor (SOX) 10, and adaptor protein-2α (AP2α).
Although MITF and SOX10 were confirmed as HIF-1α
partners as well, the transcription factor AP2α was not.
Furthermore, HIF-2α and AP2α interaction had func-
tional consequences for the invasive properties of CM
cells since HIF-2α overexpression in AP2α-expressing
cells could reduce their invasion potential [66]. On the
other hand, an independent silencing of HIF-1α and
HIF-2α performed in metastatic CM cells K457 by using
short hairpin RNA (shRNA) identified connective tis-
sue growth factor (CTGF), an invasion positive MMPs
regulator, as a specific target of both isoforms. Inhibition
of CTGF decreased invasion and migration of CM cells
along with reduced MMP-9 expression [67]. shRNA con-
structs against HIF1α and HIF2α did not affect tumor ini-
tiation and progression in PTEN-deficient, BRAF-mutant
genetically engineered mouse model of CM, but clearly
abrogated metastasis at lymph node. Mechanistically,
HIF-1α and HIF-2α drove CM invasion through Platelet
Derived Growth Factor Receptor Alpha (PDGFRα) and
Focal Adhesion Kinase (FAK)-dependent SRC activa-
tion, and by directly regulating the expression of MMPs
involved in invadopodia-associated ECM degradation.
Notably, HIF-1α and HIF-2α acted independently since
HIF-1α positively regulated PDGFRα-dependent SRC
activation and Metallothionein 1-MMP expression,
whereas HIF-2α specifically fostered FAK signaling and
MMP-2 expression in CM cells cultured in hypoxic con-
ditions [68].
Immunomodulation. Regulatory T (Treg) cells usu-
ally prevent effective anti-tumor immune response and
promote immune evasion, thus playing a crucial role
in CM initiation and progression [69]). However, some
Treg cells can be converted into effector T cells, which
lose suppressive capacity and release pro-inflammatory
cytokines to promote inflammation [70]. In this con-
text, Treg-selective HIF-2α deletion or treatment with
the HIF-2α PT2385 inhibitor reduced Treg capacity to
impair the activities of effector T cells, and suppressed
cells dissemination in immunogenic B16F10 CM mouse
models, apparently via increasing HIF-1α expression
[70]. In contrast, in a study by Doedens et al., HIF-2α,
along with HIF-1α maintains the functions of CD8+ cyto-
toxic T lymphocytes (CTL) [71], which are the front-line
defense in the elimination of infected or tumor cells [72].
In fact, in CTL, HIF-1α and HIF-2α sustained the expres-
sion of effector molecules, co-stimulatory receptors and
key transcriptional regulators, thus hampering tumor
growth in CM mouse models [71].
HIF-α regulation in CM
Canonical-oxygen dependent negative regulation of
HIF-α signaling is commonly driven by the PHD 1–3
proteins. PHDs hydroxylate two conserved proline resi-
dues in the ODD motif of HIF-α subunits, in order to
promote the binding of HIF-α with the von Hippel-
Lindau protein (VHL) that leads to its subsequent pro-
teasome-dependent degradation [2]. Surprisingly, Liu et
al. found that psychological stress enhanced EMT and
vasculogenic mimicry in CM by reducing the ubiquitin-
mediated degradation of HIF-1α through the activity of
the D2 dopamine receptor (DRD2), which expression
was upregulated in tumor tissues under stress condi-
tion. More precisely, DRD2 could interact with VHL
and competitively inhibit the HIF-1α/VHL interaction,
resulting in increased HIF-1α stability [73]. In addition,
the transcriptional activity of both HIF-1α and HIF-2α
is also negatively regulated through the hydroxylation
of an asparagine residue in the C-TAD domain, medi-
ated by the factor-inhibiting HIF, that blocks the HIF-α
Page 9 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
binding to CBP/p300 co-transcription factors [74]. ese
processes are hampered under hypoxic conditions, since
HIF-α subunits accumulate and heterodimerize with
HIF-1β to form a transcriptional complex that translo-
cates into the nucleus [75].
In addition to the canonical regulation, supplementary
factors can influence HIF-1α transcription, protein sta-
bility and activity in response to hypoxia in CM (Fig.2).
Among them, poly (ADP-ribose) polymerase 1 (PARP-1)
has recently emerged as a novel HIF-1α regulator in CM.
Mechanistically, following incubation in a hypoxia cham-
ber, Martí JM et al. observed an early ROS induction that
led to PARP-1 activation in C8161 metastatic CM cells.
Once activated, PARP-1 stabilized HIF-1α by PARyla-
tion at specific lysine and arginine residues within the
C-terminus domain. Consistently, PARP-1 and HIF-1α
expression were strongly associated in tissues from CM
patients, thus suggesting that PARP inhibitors might
be considered as a potential therapeutic approach in
CM displaying HIF-1α overactivation [76]. Intriguingly,
Lakhter et al. unveiled that HIF-1α could localize at Golgi
compartment in CM cells cultured in hypoxia cham-
ber. Treatment with brefeldin A, an ER-Golgi transport
inhibitor, not only induced HIF-1α protein accumulation
in the nucleus, but also reduced its transcriptional activ-
ity, indicating that ER-Golgi transport is essential to fine-
tuning the transcriptional activity of HIF-1α [77].
In cancer, hypoxia is only one factor among several
HIF-α-activating mechanisms. Interestingly, normoxic
activation of HIF-1α has been detected in several cancer
Fig. 2 Overview of the multiple pathways impacting on HIF-1α in CM. In the upper panel, a graphic representation of the main pathways regulated
by HIF-1α under hypoxic conditions (1 kPa O2 levels) in CM is shown. Conversely, the lower panel resumes pathways that are controlled by HIF-1α in
normoxia (18 kPa O2 levels). Panels are divided into pro-tumorigenic (blue) and anti-tumorigenic (orange). Created in BioRender ( h t t p s : / / B i o R e n d e r . c
o m / p 7 3 u 9 1 5). Abbreviations: HIF: Hypoxia-inducible factor; ROS: Reactive oxygen species; PARP1: Poly (ADP-ribose) polymerase; PAR: Poly ADP-ribose;
HDAC8: Histone deacetylase 8; IL-18R: interleukin-18 receptor; RAC1: Rac Family Small GTPase 1; NF-kB: The Nuclear Factor Kappa B; VEGF: Vascular en-
dothelial growth factor; ER: endoplasmic reticulum; NLGN4X: Neuroligin; VBP1: VHL Binding Protein 1; VHL: Von Hippel-Lindau; DRD2: D2 dopamine
receptor; GPR81: G protein-coupled receptor 81; PKA: protein kinase A; TEV: tumour-derived extracellular vesicles; SOX2: SRY-box transcription factor; TCA:
Tricarboxylic acid; GPX3: Glutathione Peroxidase 3; NOX5: NADPH oxidase 5; EMT: Epithelial to mesenchymal transition; BNIP3: BCL-2 interacting protein
3; NCOA4: Nuclear Receptor Coactivator 4; MITF: Microphthalmia-associated transcription factor; ANXA3: Annexin A3; PDK1: Pyruvate dehydrogenase
kinase; UPR: unfolded protein response
Page 10 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
cell types, including CM, where contributes to the acqui-
sition and maintenance of a malignant phenotype [78, 79]
(Fig.2). Accordingly, Martí-Díaz et al. observed that, in
normoxic conditions, the inhibition of HIF-1α reduced
glucose viability, impaired glucose metabolism, and sup-
pressed CM cell survival [80]. In line with these findings,
in a study by Andreucci et al., the SOX2-driven HIF-1α
inhibition decreased the glycolytic activity and induced
a shift towards oxidative phosphorylation in CM cells in
normoxia [81].
Different non-hypoxic stimuli could enhance HIF-1α
signaling, including lipopolysaccharides [81], thrombin,
angiotensin I and cytokines [82]. As reported above, a
study by Luo et al. showed an interplay between lactate
stimulation and HIF-1α-driven CM metastasis forma-
tion in vivo. In detail, lactate could bind to G protein-
coupled receptor 81 (GPR81) to decrease cAMP levels
and the subsequent protein kinase A (PKA) activation,
finally suppressing the ubiquitin-mediated proteolysis of
HIF-1α [53].
At present, several proteins have been listed as HIF-1α
non-hypoxic activators in CM. In a recent paper by
Schörghofer et al., decreased amount of the adhesion
molecule Neuroligin (NLGN4X) has been correlated
with increased HIF-1α accumulation and migratory
phenotypes in metastatic CM. e same group further
demonstrated that HIF-1α regulation by NLGN4X was
primarily mediated by Von Hippel-Lindau Binding Pro-
tein 1, a member of the canonical prefoldin complex [83].
In hypoxia, MITF expression was found to be suppressed
following HIF-1α activation in CM [41–43]. However, in
response to cAMP, MITF bound to the HIF-1α promoter
to stimulate its transcriptional activity in normoxic mela-
nocytes and CM cells [84]. Mitochondrial ROS have
been involved in HIF-1α regulation in hypoxia in CM
[38]. Besides mitochondria, ROS can be produced in the
cytosol as well, and their accumulation can also occur in
non-hypoxic conditions. For instance, cytosolic NADPH
oxidase 5 (NOX5) contributed to sustain CM cell prolif-
eration, at least in part, by generating high local concen-
tration of ROS that in turn, regulated normoxic HIF-1α
expression [85]. ROS are known to activate NF-κB [86],
and NF-κB activation might enhance HIF-1α transcrip-
tion under hypoxic and normoxic environments [87, 88],
suggesting a potential crosstalk among ROS, NF-κB and
HIF-1α. Consistent with this hypothesis, NF-κB activa-
tion triggered by ROS increased the normoxic HIF-1α
activity, whereas NF-κB inhibition attenuated accumula-
tion in CM cell lines [79]. In another study by Kim J et
al., interleukin (IL)-18 signaling induced HIF-1α expres-
sion through the Rac1/NF-κB pathway in the mouse
CM B16F10 cell line. Of interest, following treatment
with the antioxidants N-acetyl-l-cysteine and pyrro-
lidine dithiocarbamate (PDTC), IL-18 was not able to
up-regulate HIF-1α expression. Furthermore, PDTC
alone, or in combination with an inactive form of Rac1,
markedly reduced NF-κB activation, suggesting that once
activated by IL-8 signaling, Rac1 promoted ROS genera-
tion to mediate NF-κB activation and HIF-1α expression.
Even though the molecular mechanism has been inves-
tigated under normoxic conditions, hypoxic stimulation
dramatically increased IL-18 and HIF-1α mRNA expres-
sion, indicating that IL-18 might perform regulatory
functions in HIF-1α expression under both normoxic
and hypoxic conditions [89]. Although the molecular
mechanism is yet to be defined, the calcium-dependent
phospholipid-binding protein annexin A3 (ANXA3) has
been implicated in HIF-1α aberrant activation in CM
as well. In fact, in a study by Xu et al., ANXA3 overex-
pression increased HIF-1α and VEGF protein expres-
sion levels [90]. In CM cells, under conditions of oxygen
availability, BCL-2 interacting protein 3 (BNIP3) could
act as the upstream regulator of the HIF-1α-mediated
glycolytic program. More specifically, by controlling the
intracellular availability of iron and the mediated autoph-
agic degradation of ferritin, BNIP3 was found to regulate
HIF-1α stability [91]. Since BNIP3 represents one of the
main HIF-1α target genes, results from this study indi-
cated that this bidirectional loop between BNIP3 and
HIF-1α fostered normoxic HIF-1α-linked glycolysis and
pro-tumorigenic program in CM. Finally, the histone
deacetylase 8 (HDAC8) directly bound to HIF-1α to sus-
tain the stabilization of the protein in both hypoxia and
normoxia in CM [92]. On the other hand, the glutathi-
one peroxidase 3, an enzyme involved in protecting cells
from oxidative stress, and often downregulated in CM,
was reported to reduce HIF-1α and HIF-2α stability and
activity by inactivating ROS production [93].
Non-coding RNAs associated to HIF-α signaling
Functional RNAs that are not translated into proteins are
called non-coding RNA (ncRNA) (for review see [94]). In
the last years, a growing number of studies have revealed
their importance as key molecular mediators in several
many critical aspects of tumor biology, including hypoxia
and HIF-α signaling [95, 96].
MicroRNAs involved in HIF-α regulation in CM
MicroRNAs (miRNAs) are small single-stranded ncRNAs
whose length ranges between 18 and 25 nucleotides.
MiRNAs are transcribed as pri-miRNAs by RNA poly-
merase II. Pri-miRNAs are then processed by the com-
plex formed by DROSHA/DGCR8 to produce miRNA
precursors (pre-miRNAs). ereafter, pre-miRNAs are
transported to the cytoplasm where they are processed
by the RNase III enzyme DICER/TRBP2 to form the
mature miRNA duplex. is miRNA duplex includes the
two-strand pair miR-3p/miR-5p, each of which may be
Page 11 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
selected by the ARGONAUTE proteins associated with
the RNA-induced silencing complex. e final single-
stranded miRNA can regulate gene expression at the
post-transcriptional level through fine-tuning of target
protein expression. It has become increasingly clear that
miRNAs deregulation is a hallmark of cancer, where they
might function as oncogenes to promote tumor forma-
tion (oncomiRs), or act as tumor suppressors. Not sur-
prisingly, miRNAs are involved in cancer development
and progression, including the metastatic process and the
TME remodeling (for review see [97]).
Regulation of HIF-1α on miRNAs is common during
cancer initiation and progression (for review see [98]).
However, recent evidence has documented complex
interactions between HIF-1α and miRNAs since, besides
to be directly or indirectly regulated by HIF-1α, miRNAs
can modulate the expression or activity of HIF-1α as well
(Fig.3) [96, 99, 100].
MiRNAs that negatively modulate HIF-1α. HIF-1α was
identified as a target of miR-199a-5p in several tumors,
including CM. In fact, miR-199a-5p mimics impaired
CM cell proliferation by decreasing HIF-1α mRNA and
protein expression both in vitro and in vivo. Consistently,
low expression levels of miR-199a-5p were observed in
CM, particularly in tissue samples from patients with
advanced tumor stage [101]. Zhou et al. reported that
miR-33a expression in metastatic CM cells was also rela-
tively low. Interestingly, when overexpressed, miR-33a
inhibited HIF-1α and, consequently, reduced BRAF-
mutant CM cell proliferation and invasion in vitro, and
tumor growth in vivo [102]. Subsequently, Cao et al. con-
firmed that miR-33a-5p expression was decreased in CM
cells, and that miR-33a-5p negatively regulated HIF-1α,
thereby repressing glycolysis and increasing radiosen-
sitivity of CM cells [103]. In addition to miR-199a-5p
and miR-33a, Mazar et al. found that miR-211-5p indi-
rectly reduced HIF-1α protein levels and decreased CM
cell growth in hypoxia. In detail, when overexpressed
in A375 BRAF-mutant CM cells, miR-211-5p improved
mitochondrial respiration by targeting PDK4. On the
contrary, miR-211 down-regulation led to increased
PDK4 protein expression, which finally resulted in less
oxidative phosphorylation, reduced mitochondrial activ-
ity and HIF-1α stabilization. Accordingly, miR-211-5p
was found to be downregulated during CM progression
[104]. Intriguingly, although miR-211-5p overexpression
was more frequent in primary tumors respect to metas-
tases, in a study by Moritz et al., high miR-211-5p expres-
sion in CM metastases, but not primary tumors, was
associated with worse overall survival [105]. In line with
these findings, miR-211-5p was upregulated in CM cells
resistant to BRAFi, and involved in the early phases of
resistance acquisition to targeted therapy through HIF-
1α-independent mechanisms [106, 107].
HIF-associated oncogenic miRNAs. Among miRNAs
that positively support HIF-α signaling, miR-675-3p was
significantly up-regulated in CM cell lines, tissues and
peripheral blood from CM patients. In vitro analyses
indicated that miR-675-3p might function as a media-
tor of many cellular pathways in CM, since the use of a
miR-675 mimic could activate TGFβ and HIF-1α signal-
ing pathways [108]. In a subsequent study by Hwang et
al., high levels of HIF-1α transcriptional activity were
detected in a panel of TGFβ1 positive CM cell lines
under normoxic conditions, and HIF-1α silencing down-
regulated several miRNAs, including miR-210, miR-218,
miR-224 and miR-452 [109]. MiR-210 is a direct target
of both HIF-1α and HIF-2α, and its expression has been
correlated with metastasis, poor prognosis and early
risk of recurrence in CM [110, 111]. HIF-1α-dependent
miR-210 upregulation impaired the ability of immune
system to target CM cells. Indeed, miR-210 overexpres-
sion promoted immune escape from CTL-mediated lysis
by degrading protein tyrosine phosphatase non-receptor
type 1, homeobox A1, and tumor protein p53 inducible
protein 11 [112]. Besides to impact on immune response,
miR-210 might also influence mitochondrial activity in
CM. In fact, miR-210 silencing accelerated mitochondrial
respiratory, thus improving ROS elimination and label-
ling CM cells for self-destruction, in both normoxic and
hypoxic conditions [113, 114]. erefore, miR-210 might
act as a key factor in HIF-1α-mediated tumorigenesis,
thus representing a good prognostic and therapeutic tar-
get in both normoxic and hypoxic CM.
At present, differently from HIF-1α, only a study by
Hao et al. has focused on HIF-2α-associated miRNA
in CM. More specifically, HIF-2α protein was found to
mediate stemness of CM cells by regulating the miRNA-
363-3p/p21 axis [115]. Nevertheless, the deeper correla-
tion between HIF-2α and miRNA-363-3p remains to be
further explored.
Long non-coding RNAs involved in HIF-α
regulation in CM
Long non-coding RNAs (lncRNAs) are defined as
ncRNAs of more than 200 nucleotides with little or no
coding information. LncRNAs can be located either in
the nucleus or the cytoplasm, or in both compartments
simultaneously, and can be classified into five categories:
sense, antisense, bidirectional, intronic, and intergenic.
LncRNAs have been involved in a wide range of cellular
mechanisms in several tumors, including CM (for review
see [94]).
To our knowledge, LINC00518 is the only lncRNA
that has been characterized for its role in regulat-
ing HIF-1α expression in CM [116]. More precisely,
LINC00518 sponged miR-33a-3p to increase the expres-
sion of HIF-1α, thus accelerating glycolytic metabolism
Page 12 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
and triggering radioresistance in BRAF-mutant CM
cells. Additionally, HIF-1α recruited HDAC2 to the
miR-33a-3p promoter region to decrease its expression.
Hence, the LINC00518/miR-33a-3p/HIF-1α axis might
represent an efficient target to increase the therapeutic
efficiency of radiotherapy in CM [116].
Therapeutic opportunity in CM
As mentioned above, HIF-α subunits not only play a
crucial role in the response of CM cells to hypoxia but
also regulate different biological processes under nor-
moxic conditions, thus representing attractive therapeu-
tic molecular targets. In the last years, several anticancer
Fig. 3 Schematic representation of non-coding RNAs involved in HIF-1α pathway regulation in CM. In the upper semicircle, a summary of the current
advances about the bidirectional interactions between HIF-1α and miRNAs in CM (blue: suppressor miRNAs; orange: oncogenic miRNAs). The lower semi-
circle illustrates a graphic representation of the mechanism by which the lncRNA linc00518 modulates HIF-1α expression in CM. Created in BioRender
( h t t p s : / / B i o R e n d e r . c o m / 8 3 3 o 8 2 4). Abbreviations: HIF: Hypoxia-inducible factor; miR: mirna; CM: cutaneous melanoma; ROS: Reactive oxygen species;
MNT: MAX Network Transcriptional Repressor; CTL: cytotoxic T lymphocyte; TCA: Tricarboxylic acid; PDK4: Pyruvate Dehydrogenase Kinase 4; LDHA: Lac-
tate dehydrogenase A; PTPN1:Protein Tyrosine Phosphatase Non-Receptor Type 1; HOXA1: Homeobox A1; TP53I11. Tumor Protein P53 Inducible Protein
11.LDHA: Lactate dehydrogenase A; PTPN1:Protein Tyrosine Phosphatase Non-Receptor Type 1; HOXA1: Homeobox A1; TP53I11. Tumor Protein P53 Induc-
ible Protein 11
Page 13 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
drugs that modulate HIF-1α expression and/or activity
without directly targeting it have been described.
HIF-1α significantly impacts on CM cell metabolism as
it influences the expression of different genes involved in
glycolysis. Among the key effectors of HIF-1α-mediated
metabolic remodeling, the 6-phosphofructo-2-kinase/
fructose-2,6-biphosphatase 3 (PFKFB3) enzyme plays a
crucial role in glycolysis since, once activated, it stimu-
lates allosterically the activity of PFK-1 in BRAF-mutant
CM, fostering the Warburg’s effect. Intriguingly, (2E)-
3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO),
an inhibitor of PFKFB3, was shown to interfere with the
HIF-1α/PFKFB3/PFK-1 axis, and to induce cell cycle
arrest in G1/0, apoptosis and glucose uptake reduction in
BRAF-mutant A375 CM cells [117].
In a study by Furfaro et al., MeOV-1 CM cells cultured
under 18 kPa O2 (normoxia), 5 kPa O2 (physioxia), and
1 kPa O2 (hypoxia) were treated with BRAFi. Of inter-
est, HIF-1α expression was significantly decreased by
the treatment under all O2 tensions tested [118]. Consis-
tently, BRAFi vemurafenib (PLX4032) suppressed HIF-1α
expression at the mRNA and protein levels in a panel of
BRAFV600 CM cell lines cultured under standard condi-
tions. More interestingly, HIF-1α expression was also
clearly decreased early on treatment with BRAFi and
restored after progression in a subset of CM biopsies.
Similarly, the expression of several glycolysis-associated
genes (i.e. HK2, SLC2A1 and SLC2A3) was significantly
decreased following BRAFi treatment and restored in
CM patients that developed drug resistance [28], sug-
gesting a possible involvement of HIF-1α in resistance
to BRAF-targeted therapies. Under hypoxia, HIF-1α lev-
els were decreased in CM cells exposed to PLX4032 in a
cell type-dependent manner [82, 119], further suggesting
that, besides to favor tumor heterogeneity, HIF-1α might
influence the response to the drug treatment. Accord-
ingly, Qui et al. demonstrated that hypoxia, by inducing
MET and HGF via HIF-1α, prompted the acquisition of
vemurafenib resistance in CM BRAF-mutant models
both in vitro and in vivo [120]. Blocking MET signalling
potentiated the antitumor effect of vemurafenib, indicat-
ing that MET inhibition might represent a potential ther-
apeutic strategy for overcoming HIF-1α-driven PLX4032
resistance in CM patients.
In NRAS-driven CM, the growth factor receptor-
binding protein 2-associated protein 2 (GAB2) has been
reported to enhance tumor formation and angiogen-
esis by stabilizing HIF-1α at the post-transcriptional
level. Histological examination of GAB2wt/NRASG12D
xenografts showed longer vessels with markedly dilated
lumina, together with increased expression of CD31,
VEGFR2, VEGF, respect to NRASG12D tumors. Phar-
macological inhibition of MEK significantly suppressed
angiogenesis, providing evidence that HIF-1α mediated
angiogenic response was dependent on RAS-RAF-MEK-
ERK signaling in GAB2/NRAS-driven tumorigenesis
[121].
At present, no study has evaluated the potential con-
tribution of NF1 mutation in HIFs signalling activa-
tion in CM. Conversely, in c-Kit mutated melanocytes,
HIF-1α overexpression was found to promote oncogenic
transformation. Treatment with imatinib, a c-Kit inhibi-
tor, reversed proliferation and anchorage-independent
growth of melanocytes, confirming that HIF-1α induced
melanocytes transformation in a c-Kit-dependent man-
ner [122].
PTEN has been clearly identified as a potent negative
regulator of angiogenesis via inhibition of HIF-1α sig-
naling. In normoxic conditions, PTEN enhances HIF-1α
ubiquitination and degradation, whereas PTEN inactiva-
tion increases the transcriptional activity of HIF-1α in
hypoxia. Hence, to restore PTEN expression or activity
might impair HIF-1-α-induced angiogenesis, with poten-
tial therapeutic benefit in PTEN-deficient CM. In this
context, it has been proposed that the administration of
plant-derived natural bioactive compounds with demeth-
ylating effects, such as phytochemicals, might favor
PTEN upregulation [123].
So far, a broad spectrum of molecules of natural origin
has proven to affect HIF-1α expression. Among them,
isoliquiritigenin was effective in suppressing cell prolif-
eration and in inducing apoptosis via reduced HIF-1α
protein stability and mRNA expression of a number of
glycolysis-related genes in B16F10 transplanted mice
[124]. Vanillin was also reported to possess anti-cancer
and anti-metastatic activities in CM. In particular, van-
illin treatment significantly decreases HIF-1α mRNA
expression by suppressing STAT3 transcriptional activity
in CM cells [125]. A subsequent study by Li et al. indi-
cated that luteolin, a natural flavonoid, exhibited a strong
anti-cancer activity in CM by impairing angiogenesis and
EMT both in vitro and in vivo. is effect was mainly
achieved through the disruption of the HIF-1α/VEGF
signaling pathway [126]. Finally, celastrol, a chemical
compound extracted from the root extract of the Tripte-
rygium Wilfordii, reduced the expression of HIF-1α
mRNA by inhibiting the PI3K/Akt/mTOR signaling,
leading to ROS accumulation, a decrease in that mito-
chondrial membrane potential that promoted cell cycle
arrest and apoptosis [127].
Recent evidence has unveiled that HIF-1α has an
important role in modulating recruitment and functions
of immune cells to create an anti-tumorigenic micro-
environment. Chromatin immunoprecipitation and
luciferase reporter assay revealed that HIF-1α directly
bound to a transcriptionally active HRE within the PD-L1
proximal promoter. Hence, hypoxia could modulate HIF-
1α-transcriptional activity in order to increase PD-L1
Page 14 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
expression on macrophages, dendritic cells and CM
mouse models [128]. Along this line, in a study by Bar-
soum et al., HIF-1α induced PD-L1 expression in human
breast and prostate cancer cells, as well as in mouse CM
and mammary carcinoma cells, leading to resistance to
CTL-mediated lysis [129]. More recently, high-through-
put analyses performed in metastatic CM patients cor-
related hypoxia with non-response to anti-PD-1 and
with immunosuppression and changes in tumor-stromal
communication in TME. Intriguingly, this response was
mainly orchestrated by the expression of HIF-2α in EC
and fibroblasts within CM microenvironment. Indeed,
HIF-2α inhibition with the small molecule PT2399, in
combination with anti-PD1, delayed tumor growth in
mice implanted with B16F10 murine CM cells. How-
ever, enhanced HIF-1α expression was observed in intra-
tumoral EC following the anti-PD-1/PT2399 treatment.
ese findings suggest that the lack of HIF-2α could pro-
voke a compensatory increase of the HIF-1α signaling to
improve tumor growth control [130], thus impairing the
effect of the combined anti-PD-1/HIF-2α inhibition.
In an in vivo model proposed by Wei et al., tumors
form the aggressive B16F10 CM line grew significantly
slower in mice lacking HIF-1α in their CD8+ T cells with
respect to those implanted in wild-type mice. Similar
effects were observed in CD8+ T cells treated with the
HIF-1α inhibitor acriflavine alone, whereas the combina-
torial treatment using acriflavine and the Treg depletion
agent cytoxan further suppressed tumor development by
improving CD8+ T cell responses [131]. Consistently, in
a study by Lequez et al., the impairment of the transcrip-
tional activity of HIF-1α significantly reduced B16F10
CM tumor growth by enhancing NK, CD4+, and CD8+
T cell infiltration. Acriflavine ameliorated the therapeu-
tic benefit of anti-PD-1 immune checkpoint- and TRP-2
vaccine-based cancer immunotherapy [132]. Another
HIF-1α inhibitor, namely IDF-11,774, also improved
CD8+ T cell infiltration and impaired invasiveness of
B16F10 CM cells in vivo by reducing the expression of
the EMT markers SNAIL and N-cadherin [133]. On the
other hand, dimethyloxalylglycine, a hypoxia mimetic
agent, induced HIF-1α, HIF-2α and HIF-1β binding to
the genes encoding the costimulatory receptors CD81,
GITR, OX40 and 4-1BB, rising their expression in
CD8+ T cells, and increasing T-cell mediated killing of
CM cells in vivo. Similar effects were observed in CD8+
T-cells treated with the PHDs inhibitor molidustat [134].
Altogether, these results suggest that, although to tar-
get intra-tumoral hypoxia might improve the response
to immunotherapy [135], HIF-α signaling activation in
T-cells might further stimulate anti-tumor immunity in
CM.
Conclusion
HIF-α subunits play an essential role in cancer since they
modulate the transcription of several genes involved
in tumour progression and response to therapies. As
we have discussed in this review, although HIF-1α and
HIF-2α are closely related and share similar domain
structures, it is worth noting that they are mainly non-
redundant and have distinct target genes in CM. ere-
fore, HIF-α subunits are driver of multiple aggressive
features in CM, including angiogenesis, EMT and metas-
tasis, metabolic alterations, but also immune escape.
e pleiotropic effects of HIF-α subunits in CM sug-
gest that inhibiting both HIF-1α and HIF-2α might offer
greater therapeutic benefits respect to target either pro-
tein alone. In this setting, combination therapies that
incorporate HIF-α inhibitors with existing treatments,
such as BRAF-targeted therapy and/or immune check-
point inhibitors, might provide novel therapeutic options
for CM patients with recurrent or metastatic disease. At
present, however, the majority of studies with molecules
that inhibit HIF-α expression or promote its degradation
are mostly restricted to pre-clinical models. In the future,
CM humanized mouse models may allow to accurately
assess the efficacy of combining HIF-α inhibitors and
immunotherapy.
HIF-1α and HIF-2α expression is regulated by different
pathways involving both oxygen-dependent and oxygen-
independent mechanisms. While the canonical oxygen-
dependent HIF-α regulation has been well described,
the mechanisms by which HIF-α subunits maintain their
stability under normoxic conditions in CM are still less
understood, highlighting a critical area for further inves-
tigation. ese observations implicate that future drug
development should focus on defining additional molec-
ular pathways that allow HIF-1α subunits to escape deg-
radation in normoxia and elucidating how this stability
might contribute to CM development and progression.
Abbreviations
ACKR2 Atypical chemokine receptor 2
ADAM12 ADAM Metallopeptidase Domain 12
ADRP Adipose dierentiation-related protein
ANXA3 Annexin A3
AP2α Adaptor protein-2α
AXL AXL Receptor Tyrosine Kinase
ATP5ME ATP Synthase Membrane Subunit E
bHLH Basic helix-loop-helix
BIRC7 Baculoviral IAP repeat-containing protein 7
BNIP3 BCL-2 interacting protein 3
BRAFi BRAF inhibitor
CAPN3 Calpain-3
CBP CREB-binding protein
CM Cutaneous melanoma
COL13A1 Collagen type XIII alpha 1 chain
CRIM1 Cysteine-rich motor neuron 1
CTGF Connective tissue growth factor
CTL Cytotoxic T lymphocyte
DAPK1 Death Associated Protein Kinase 1
DRD2 D2 dopamine receptor
Page 15 of 19Bellazzo et al. Cell Communication and Signaling (2025) 23:177
EC Endothelial cells
ECM Extracellular-matrix
EMT Epithelial to mesenchymal transition
ET-1 Endothelin-1
FABP3 Fatty acid binding protein 3
FABP7 Fatty acid binding protein 7
FAK Focal adhesion kinase
FGF2 Fibroblast Growth Factor 2
FLNB Filamin B
GALNT3 Polypeptide N-Acetylgalactosaminyltransferase 3
GLUT Glucose transporters
GM3S Ganglioside GM3 synthase
GPM6B Glycoprotein M6B
GPR81 G protein-coupled receptor 81
HDAC Histone deacetylase
HIF Hypoxia-inducible factors
HK2 Hexokinase 2
HRE Hypoxia response elements
HS3ST3A1 Heparan Sulfate-Glucosamine 3-Sulfotransferase 3A1
IL Interleukin
ITGA3 Integrin Subunit Alpha 3
KCNMA1 Potassium calcium-activated channel subfamily M alpha 1
LDHA Lactate dehydrogenase
lncRNA Long non-coding RNA
miRNA MicroRNAs
MITF Microphthalmia-associated transcription factor
MMP Matrix metalloproteinase
MT1A Metallothionein 1A
MYO1D Myosin ID
ncRNA Non-coding RNA
NDUFA6 NADH: Ubiquinone Oxidoreductase Subunit
NLGN4X Neuroligin
NOX5 NADPH oxidase 5
ODD Oxygen-dependent degradation
OXPHOS Oxidative phosphorylation
PARP Poly (ADP-ribose) polymerase
PAS Per-ARNT-Sim
PDGFRα Platelet Derived Growth Factor Receptor Alpha
PDH Pyruvate dehydrogenase
PDK Pyruvate dehydrogenase kinase
PDTC Pyrrolidine dithiocarbamate
PFKFB3 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3
PHD Prolyl hidroxylases
PKA Protein kinase A
PKM2 Pyruvate kinase M2
pre-miRNA miRNA precursor
ROR Receptor tyrosine kinase-like orphan receptor
ROS Reactive oxygen species
SFK Src family kinases
shRNA Short hairpin RNA
SOX SRY-box transcription factor
TAD Transactivation domain
Tβ4 Thymosin beta-4
TCA Tricarboxylic acid
TME Tumor microenvironment
TNFRSF14 TNF Receptor Superfamily Member 14
Treg Regulatory T lymphocyte
TYRP1 Tyrosinase related protein 1
uPAR Urokinase-type plasminogen activator receptor
VEGF Vascular endothelial growth factor
VEGFR VEGF receptor
VHL Von Hippel-Lindau
WT Wild type
Supplementary Information
The online version contains supplementary material available at h t t p s : / / d o i . o r
g / 1 0 . 1 1 8 6 / s 1 2 9 6 4 - 0 2 5 - 0 2 1 7 3 - 4.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Acknowledgements
Not applicable.
Author contributions
AB, BM and EF conceptualised the article, performed literature search and
wrote the initial manuscript. AB prepared the table. BM designed gures. RG,
FC and JP contributed to writing the manuscript. EF revised the manuscript
and provided substantial feedback throughout the writing process. All authors
approved the nal version of the manuscript.
Funding
This work was supported by the Italian Ministry of Health (Ricerca Corrente,
grant GR-2018-12366312 to EF) and 5 × 1000 Institutional Grant from CRO
Aviano, National Cancer Institute, Istituto di Ricovero e Cura a Carattere
Scientico (IRCCS) (seed grant to BM and EF; no grant number provided).
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
All the authors agree to publish this paper.
Competing interests
The authors declare no competing interests.
Received: 14 February 2025 / Accepted: 24 March 2025
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