Introduction
Pheochromocytomas and paragangliomas (PPGLs) are rare neural crest-derived tumors
(2–8 cases per million per year) that respectively originate from chromaffin cells
of the adrenal medulla or the extra-adrenal sympathetic paraganglia [1 ]. Paragangliomas of the head and neck on the other hand are derived mainly from non-chromaffin
cells associated with parasympathetic ganglia. Most PPGLs do not metastasize, but
up to 20% of patients with PPGLs present with distant metastases at sites where chromaffin
cells are normally absent, such as lymph nodes, lung, liver, and bones [2 ]
[3 ]
[4 ]. Metastatic disease may be identified at either initial diagnosis (synchronous disease)
or only become apparent at follow-up (metachronous disease). This article reviews
the role of hypoxia-inducible factor 2α (HIF2α), also referred to as endothelial PAS
protein 1 (EPAS1), in tumorigenesis and metastasis of PPGLs. In this context, we also
highlight the influence of HIF2α on catecholamine phenotype and its effects on different
steps of the invasion-metastasis cascade to clarify the therapeutic relevance of stabilizing
an altered HIF2α for treatment of patients with metastatic PPGLs.
Whether the metastatic disease is synchronous or metachronous, treatment options are
limited and the prognosis is poor, though highly variable. An early diagnosis or even
prediction of metastatic disease in metachronous cases or patients screened due to
hereditary risk may nevertheless be useful for earlier intervention and an improved
outcome for any affected patient; to this end, several risk factors have been well
established to be associated with metastatic disease. These include germline mutations
in succinate dehydrogenase (SDH) subunit B (SDHB), large tumor size, extra-adrenal
location, and elevated plasma methoxytyramine concentrations [2 ]
[5 ]
[6 ]. Telomerase activation and ATRX chromatin remodeler (ATRX) mutations are also described
as independent risk factors for metastatic PPGLs [7 ]. Our recent findings further demonstrate that patients with PPGLs characterized
by activation of pseudohypoxic pathways due to an increased expression and stabilization
of HIF2α are also at high risk of metastatic disease [8 ]. This association also appears to relate to some other established risk factors.
Hypoxia signaling has a far-reaching impact on cellular differentiation and tumorigenesis
mainly driven by the two main HIFα isoforms: HIF1α and HIF2α, which are structurally
comparable and mostly have complementary functions [9 ]
[10 ]. In presence of oxygen, proline residues within HIFα subunits are hydroxylated by
α-ketoglutarate-and oxygen-dependent prolyl hydroxylases (PHDs); this then allows
for von Hippel-Lindau (VHL) tumor suppressor-mediated proteasomal degradation of the
HIF proteins ([Fig. 1a ]). In absence of oxygen (hypoxia), HIFα subunits form transcriptionally active complexes
with aryl hydrocarbon receptor nuclear translocator (ARNT, also known as HIFβ) and
co-factors (e. g., CREB-binding protein and p300) followed by C-terminal transactivation
of genes possessing hypoxia-responsive elements (HREs) ([Fig. 1b ]). In addition to HRE-mediated mechanisms, both HIFα subunits show interactions with
Notch, Wnt, and MYC pathways, thereby regulating gene expression through these additional
pathways [11 ]. During cellular adaptation to hypoxia, the activity of HIFα differs temporally;
while HIF1α primarily mediates the acute response to severe hypoxia, HIF2α mediates
the response to chronic, even mild hypoxia [12 ]. This differential regulation is mediated by different hypoxia-associated factors
and involves distinct cellular functions [13 ]. Moreover, several studies have indicated an oncogenic activity of HIF2α, and tumor
suppressor role of HIF1α [14 ]
[15 ]. HIF1α is ubiquitously expressed in many cell types, while HIF2α expression is restricted
to specific cellular populations, including endothelial cells, neural crest cells,
and glial cells [16 ].
Fig. 1 Oxygen-dependent and independent regulation of HIFα. (a ) Under normoxic conditions, HIFα subunits are hydroxylated by α-ketoglutarate-and
O2-dependent prolyl hydroxylases (PHDs) enabling von Hippel-Lindau (VHL)-mediated
degradation of the HIFα protein. (b ) Under hypoxic conditions, HIFα subunits form transcriptionally active complexes
with ARNT (also known as HIFβ) and co-factors followed by C-terminal transactivation
of genes possessing hypoxia-responsive elements (HREs). (c ) Pseudohypoxic conditions are characterized by increased stabilization of HIFα, especially
HIF2α, although oxygen is present. In PPGLs, pseudohypoxia can be caused by mutations
in hypoxia-related genes encoding PHDs, VHL, and HIF2a themselves, or by mutations
in tricarboxylic acid (TCA) cycle-related genes leading to an alteration in TCA cycle
metabolites, leading to an inhibition of α-ketoglutarate-dependent PHDs. Genes reported
as altered in PPGLs are highlighted by stars (yellow: loss-of-function mutation, red:
gain-of-function mutation). CBP: CREB-binding protein; FH: fumarate hydratase; MDH2:
mitochondrial malate dehydrogenase; CS: citrate synthase; ACO2: mitochondrial aconitase;
IDH2: isocitrate dehydrogenase 2; IDH3A/IDH3B/IDH3G: subunits of isocitrate dehydrogenase
3; OGDH/DLD/DLST: subunits of the alpha-ketoglutarate dehydrogenase complex; SUCLx:
subunits of succinyl-CoA synthetase; SDHx: subunits of the succinate dehydrogenase
complex.
HIF2α-dependent genotype-phenotype relationships in PPGLs
Currently, germline or somatic driver mutations can be identified in approximately
70% of catecholamine-producing PPGLs [17 ]
[18 ]. Close links between genotype and phenotype including the risk of metastatic disease,
age of onset, syndromic presentation, and a predominant anatomic site have been recognized
[19 ]
[20 ]. PPGLs are most broadly classified into two main clusters in accordance with their
transcriptional profiles [21 ]
[22 ]. Cluster 1 PPGLs comprise those with mutations encoding two groups of genes; these
are either directly involved in the stabilization of HIFs or encode components of
the tricarboxylic acid (TCA) cycle. The former include VHL , EGLN1/2 (encoding PHD1/2), and HIF2α , whereas the latter include SDH subunits (SDHA, SDHB, SDHC, SDHD ), fumarate hydratase (FH ), malate dehydrogenase 2 (MDH2 ), mitochondrial 2-oxoglutarate/malate carrier (SLC25A11 ), isocitrate dehydrogenases (IDH1/IDH2/IDH3B ), glutamic-oxaloacetic transaminase 2 (GOT2 ), and dihydrolipoamide S-succinyltransferase (DLST ) ([Fig. 1c ]) [18 ].
All cluster 1 mutations result in the stabilization of HIFα and lead to activation
of hypoxia pathways even in the presence of oxygen (pseudohypoxia) [23 ]. Cluster 1 PPGLs are more prone to metastasize (18.6% excluding patients with SDHB mutation) compared to cluster 2 PPGLs (4.3%) caused by mutations of genes including
(Ret protooncogene (RET ), neurofibromin 1 (NF1 ), transmembrane protein 127 (TMEM127 ), H-Ras (HRAS ), fibroblast growth factor receptor 1 (FGFR1 ), MYC associated factor X (MAX ) that lead to activation of kinase signaling pathways [8 ]. However, due to mutations in SDHB , cluster 1 PPGLs are particularly prone to metastasize (75.6% in [8 ]) [24 ]. Both higher expression and stabilization of HIF2α are characteristics of cluster
1 compared to cluster 2 PPGLs [21 ]
[25 ]
[26 ] and this seems to contribute to the pro-metastatic behavior of cluster 1 tumors
[8 ]
[27 ].
HIF2α blocks glucocorticoid-mediated induction of phenylethanolamine N-methyltransferase
(PNMT), the enzyme that converts norepinephrine to epinephrine, providing a direct
link between genotype and biochemical phenotype [26 ]. This, therefore, explains the expression of PNMT and epinephrine production by
cluster 2 PPGLs, but not by cluster 1 PPGLs. HIF2α also influences phosphorylation
of tyrosine hydroxylase (TH), the rate-limiting step in catecholamine synthesis, thereby
affecting cellular dopamine and norepinephrine production [28 ]. Cluster 1 PPGLs that produce predominantly norepinephrine with varying amounts
of dopamine, have lower total tissue catecholamine content than cluster 2 PPGLs; nevertheless,
rates of catecholamine secretion and urinary excretion are higher in cluster 1 than
cluster 2 PPGLs [29 ]. This may reflect a more completely developed secretory system in cluster 2 than
in cluster 1 PPGLs that acts to restrain otherwise continuous or constitutive secretion
[30 ]. Thus, although cluster 1 tumors tend to secrete catecholamines more actively than
cluster 2 tumors, they have lower catecholamine contents per unit of tissue and produce
lower amounts of O-methylated metabolites than cluster 2 tumors. The role of HIF2α
in these differences remains unclear.
The predisposing role of HIF2α expression may also explain the reason for tumorigenesis
of certain neural crest derivatives according to mutations that involve stabilization
of HIF2α protein. As mentioned earlier, while HIF1α is ubiquitously expressed, HIF2α
exhibits a much more restricted expression pattern [16 ]. In particular, HIF2α is expressed transiently during the migration of trunk neural
crest cells to sympathetic paraganglia and the adrenal medulla, and before further
differentiation [31 ]. This expression impacts migration and proliferation and has also been associated
with the development of neural-crest derived sympathoblast precursors to neuroblastoma
subtypes with more aggressive and less differentiated features than other subtypes
[32 ]. Furthermore, using single-cell transcriptomics, human embryos were shown to have
different neural crest cell derivatives that populate both the sympathetic paraganglia
and adrenal medulla and may be responsible for the heterogeneity of associated tumors
[33 ]. It, therefore, seems possible that cluster 1 tumors may be derived from more primitive
neural crest derivatives compared to cluster 2 tumors and that this is associated
with expression of HIF2α and different phenotypic features including the propensity
for metastasis. Similarly, the younger age of patients with cluster 1 noradrenergic
than cluster 2 adrenergic tumors, along with findings of higher proportions of multifocal,
extra-adrenal, and metastatic tumors in younger than older patients with PPGLs, has
also been proposed to reflect the development of cluster 1 tumors from different populations
of chromaffin cell precursors [34 ]
[35 ].
In line with the above considerations and as outlined by Fliedner et al. [19 ], the cellular origins and the location of tumors can also influence tumor behavior
in terms of catecholamine phenotype and metastatic behavior. The majority of chromaffin
cell tumors arise from the adrenal medulla [35 ]
[36 ]. About half of these produce norepinephrine exclusively and reflect cluster 1 tumors
and the other half, the cluster 2 tumors, present with variable amounts of both norepinephrine
and epinephrine [29 ]
[37 ]. Sympathetic paragangliomas usually exclusively produce norepinephrine, in some
cases additional variable amounts of dopamine, and in isolated cases exclusively dopamine
[29 ]. Head and neck paragangliomas, which are derived from parasympathetic ganglia, show
only limited expression of TH [38 ]
[39 ]; thus, most are biochemically non-functional, though about 30% produce dopamine
as manifested by the increase in the dopamine metabolite, methoxytyramine [40 ].
A recent study focusing on Sino-European differences in the genetic landscape of patients
with PPGLs initiated a paradigm shift in the understanding of genotype-phenotype relationships
of these genetically heterogeneous tumors [41 ]. Epinephrine production in the adrenal medulla depends on the expression of PNMT,
which is regulated by glucocorticoids produced in the surrounding adrenal cortex [42 ]. According to previous understanding, mainly based on findings in Caucasian populations,
this explains why epinephrine production is confined to adrenal pheochromocytoma [37 ]
[43 ]
[44 ]. Among the Chinese population, a substantial proportion of extra-adrenal PGLs produce
epinephrine mainly associated with somatic mutations in HRAS and FGFR1 . These findings further clarify that the adrenergic phenotype of PPGLs primarily
depends on the underlying genetic mutation rather than tumor location [41 ].
Nevertheless, the aforementioned revised understanding does not mean that glucocorticoids
are not relevant to induction of PNMT, but only that they may act on chromaffin cells
to impact phenotypic features more distantly than previously believed. As outlined
earlier, failure to induce PNMT, whether at adrenal or extra-adrenal locations, is
related to the activity of HIF2α to block glucocorticoid-mediated induction of the
enzyme. As also clarified by Qin et al. [26 ], these activities possibly involve interactions of HIF2α with the MYC/MAX complex,
actions that are independent of binding of HIF2α to ARNT/HIFβ.
HIF2α mutations in PPGL tumorigenesis
Gain-of-function mutations of HIF2α are associated with pheochromocytomas and extra-adrenal paragangliomas and are often
multifocal and recurrent. HIF2α -mutant PPGLs are more frequent in females than in males and are characterized by
the production of norepinephrine [23 ]. Mutations in HIF2α are predominantly somatic and postzygotic, and are closely located on the oxygen-dependent
degradation domain of HIF2α; this hinders hydroxylation by PHDs and degradation by
VHL [45 ]
[46 ]. Germline mutations (germline variant F374Y) are rare, but mosaicism is more common
[47 ]
[48 ]. Such patients present with PPGL–somatostatinoma–polycythemia syndrome (Pacak–Zhuang
syndrome), which occurs exclusively in females [46 ]
[49 ]
[50 ].
In Caucasian cohorts, frequency of HIF2α mutations in PPGLs varies between 1.6–4.6%
[8 ]
[17 ]
[45 ]
[51 ], which is lower than in a Chinese cohort at 6.2% [41 ]. In our cohort, one-third of patients bearing HIF2α mutations were diagnosed with
metastatic disease [8 ]. This confirmed a previous study in which 29% of HIF2α mutant PPGLs showed metastatic disease [52 ]. The comparatively high metastatic tendency of HIF2α mutant PPGLs together with the generally increased metastatic risk of cluster 1 PPGLs,
characterized by stabilization of HIF2α, further supports the potential role of HIF2α as a pro-metastatic factor in PPGLs.
HIF2α-driven mesenchymal transition promotes a pro-metastatic phenotype in PPGLs
During the invasion-metastasis cascade, tumor cells must pass multiple steps to reach
distant organs. Acquisition of a motile and invasive phenotype (epithelial-mesenchymal
transition, EMT) is the initiation step of this cascade and involves various changes
in gene expression, including genes encoding proteins involved in cell adhesion and
extracellular matrix interactions [53 ]. Due to the non-epithelial origin of chromaffin cells, PPGLs are assumed to undergo
a neuroendocrine-to-mesenchymal transition (neuroendoMT) associated with activation
of specific signaling pathways [54 ]
[55 ]
[56 ]. In pheochromocytoma cells, SDHB mutation-induced neuroendoMT is associated with a pro-metastatic phenotype [54 ]. Our data also confirmed the involvement of HIF2α in this transition. The expression
of HIF2α leads to changes in focal adhesion and extracellular matrix-receptor interaction
pathways [8 ].
In relation to the above-mentioned findings, a study from Morin et al. showed that
the neuroendoMT and pro-metastatic phenotype of SDHB-deficient cells results from
synergistic effects of HIF2α and ten-eleven translocation (TET) dioxygenase-mediated
hypermethylation [27 ]. The 2-oxoglutarate-dependent TET hydroxylates DNA-methylated cytosine to form 5-hydroxymethylocystosine
and is thereby directly involved in epigenetic regulation. Especially oncometabolite-driven
tumors, such as PPGLs of the TCA cycle-related cluster 1, and here in particular tumors
with SDHB mutations show hypermethylation [57 ]
[58 ]. A genome-wide DNA methylation analysis in metastatic PPGLs identified hypermethylation
of negative elongation factor complex member E (RDBP) as a prognostic marker for stratifying
patients according to the risk to develop metastatic disease [59 ]. This, therefore, provides a potential explanation for the much higher metastatic
risk in patients with PPGLs due to mutations of SDHB compared to other cluster 1 genes. Thus, although HIF2α may be pro-metastatic, other
factors clearly play important roles in the development of metastatic disease. These
factors can include secondary mutations, such as those in ATRX , TERT , and p53 , which may occur after initiation of tumorigenesis and lead to an event that further
contributes to the development and progression of metastatic disease [7 ]
[60 ].
In addition to its participation in neuroendoMT and thus the initiation of tumor cell
migration from a primary tumor, HIF2α is also involved in the formation of pseudopodia
in PPGLs [8 ]. This critical step of mesenchymal cell migration enables intravasation; this involves
penetration by tumor cells of the endothelial basement membrane, thereby facilitating
their entry into adjacent blood or lymph vessels [61 ]. The increased ability of HIF2α-expressing cells to attach to extracellular matrixes,
such as laminin, facilitates the adhesion to the endothelial cell layer of the blood
vessel, thereby encouraging subsequent extravasation [8 ]. The expression of genes involved in focal adhesion and the interaction of extracellular
matrix-receptor promotes settlement of tumor cells in the pro-metastatic niche; the
enhanced proliferation ability of HIF2α-expressing cells allows further metastatic
colonization [8 ]. The multifaceted involvement of HIF2α in the invasion-metastasis cascade emphasizes
the importance of this factor for potential therapeutic approaches in metastatic PPGLs.
Targeting HIF2α as a therapeutic option for metastatic PPGLs
For metastatic PPGLs, treatment options are limited and could benefit from personalized
considerations according to the nature of underlying disease-causing mutations [62 ]. The enlarging understanding of the genetic background and associated molecular
alterations in metastatic PPGLs offers for the first time a promising approach for
individualized treatment of these patients. HIF2α appears to play an important role
in tumorigenesis and metastatic spread of PPGLs ([Fig. 2 ]), making it an ideal target for therapeutic approaches. Hypoxia/pseudohypoxia and
derived signaling pathways are also associated with increased resistance to chemotherapy
and radiotherapy [63 ]
[64 ]. Therefore, targeting HIF2α may provide a potential chemo-and/or radiosensitizing
approach to employ alongside other therapies.
Fig. 2 Involvement of HIF2α in tumorigenesis and metastasis of cluster 1 pheochromocytomas
and paragangliomas (PPGLs). During embryogenesis, HIF2α is involved in the migration
of trunk neural crest cells to sympathetic paraganglia or adrenal medulla. Mutations
in HIF2α predispose to the development of pheochromocytomas (PCCs) and paragangliomas
(PGLs). Moreover, mutations in genes related to activation of hypoxia pathways (pseudohypoxic
cluster 1 PPGLs) are characterized by an increased expression and stabilization of
HIF2α. Patients with pseudohypoxic cluster 1 PPGLs bear a higher metastatic risk.
HIF2α supports, among other factors, the pro-metastatic behavior of these tumors by
the acquisition of a motile and invasive phenotype (neuroendocrine-to-mesenchymal
transition, neuroendoMT). HIF2α blocks the glucocorticoid-mediated induction of phenylethanolamine
N-methyltransferase (PNMT) and is thereby directly linked to the immature catecholamine
phenotype of cluster 1 PPGLs. The versatile involvement of HIF2α during tumorigenesis
and metastasis in PPGLs makes it an ideal target for therapeutic interventions. Available
HIF2α inhibitors block dimerization with ARNT, but some tumors do not seem to respond
to these kinds of inhibitors. Addressing ARNT-independent mechanisms of HIF2a, such
as interaction with the MYC/MAX complex, maybe an alternative strategy for these resistant
tumors. Genes reported as altered in PPGLs are highlighted by stars (yellow: loss-of-function
mutation, red: gain-of-function mutation). ARNT: aryl hydrocarbon receptor nuclear
translocator; MAX: MYC associated factor X; PHDs: prolyl hydroxylases; VHL: von Hippel-Lindau
tumor suppressor.
Initially, HIF2α was considered to be undruggable, but the discovery of specific structural
features led to the development of small molecule antagonists that can block the dimerization
of HIF2α with ARNT/HIFβ [65 ]
[66 ]. In clear cell renal cell carcinoma (ccRCC), which is frequently characterized by
inactivation of VHL causing an enhanced stabilization of HIF2α, two specific HIF2α
inhibitors, PT2385 and Belzutifan (PT2977) showed promise in phase I clinical trials
[67 ]
[68 ]. Phase II clinical trials in glioblastoma [69 ] and ccRCC (e. g., NCT03108066, NCT04489771, NCT03401788) are ongoing and a phase
I clinical trial is also in planning stages for patients with PPGLs (personal communication).
However, some VHL-mutant ccRCC cell lines showed resistance towards HIF2α inhibitors
[70 ], which is also in line with recent clinical findings [71 ]. The ccRCC cell lines that were sensitive to HIF2α inhibitors displayed a distinct
HIF2α-dependent gene signature and higher levels of HIF2α than other cell lines [70 ]. Resistance can be induced by prolonged treatment with HIF2α inhibitors [70 ]
[71 ]. A gatekeeper mutation in HIF2α (G323E) that interferes with drug binding further promotes the acquisition of resistance
[71 ]. Our data showed a lack of efficiency of PT2385 in Hif2α-dependent pheochromocytoma
cell models [8 ]. Expression of Hif2α induced a pro-metastatic phenotype in these cells, which could
not be reversed by treatment with PT2385 [8 ]. Similar results were also obtained in HIF2α-dependent neuroblastomas [72 ]. This raises the possibility of an ARNT/HIFβ-independent mechanism in these models,
for example through interactions with the MYC/MAX complex [73 ]
[74 ]. These ARNT/HIFβ-independent mechanisms of HIF2α, already mentioned earlier, may
offer alternative therapeutic approaches for patients who show resistance to HIF2α
inhibitors ([Fig. 2 ]).
Further studies are needed to demonstrate the suitability of HIF2α inhibitors for
the treatment of metastatic disease. Due to the described resistance mechanisms and
the radio-and chemotherapy resistance associated with hypoxia/pseudohypoxia, suitable
combination therapies should also be considered. There is also a need to identify
markers that can predict sensitivity towards HIF2α inhibitors in suitable patients
for such therapy.
