[Advances in Pharmacology] A New Era of Catecholamines in the Laboratory and Clinic Volume 68 ||...

CHAPTER FOURTEEN

Role of Hypoxia and HIF2ain Development of theSympathoadrenal Cell Lineageand Chromaffin Cell Tumors withDistinct CatecholaminePhenotypic FeaturesSusan Richter*,1, Nan Qin*, Karel Pacak†, Graeme Eisenhofer**Department of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus Dresden,Dresden University of Technology, Dresden, Germany†Program in Reproductive and Adult Endocrinology, Eunice Kennedy ShriverNational Institute of Child Healthand Human Development (NICHD), National Institutes of Health, Bethesda, Maryland, USA1Corresponding author: e-mail address: [email protected]

Contents

1.

AdvISShttp

Introduction

ances in Pharmacology, Volume 68 # 2013 Elsevier Inc.N 1054-3589 All rights reserved.://dx.doi.org/10.1016/B978-0-12-411512-5.00014-2

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2. Regulation of Catecholamine Synthesis and Secretion by Hypoxia 287 3. The Role of HIF2a in Chromaffin Cell Development 290 4. HIF2a Signaling in Tumorigenesis 294 5. Genotype–Phenotype Relationships of Chromaffin Cell Tumors 297 6. What Role Does HIF2a Play in the Development of Chromaffin Cell Tumors? 299 7. HIF2a and Metastatic PHEO/PGL 302 8. Mutations of HIF2a as a Cause of Chromaffin Cell Tumors 305 9. Conclusion 306 Conflict of Interest 306 Acknowledgments 306 References 306
Abstract

Hypoxia has wide-ranging impact in normal physiology and disease processes. This stim-ulus evokes changes in gene expression mediated by transcription factors termedhypoxia-inducible factors (HIFs) that affect numerous processes: angiogenesis, cell sur-vival, cellular metabolism, stem cell self-renewal and multipotency, migration, invasive-ness, and metastatic progression in tumor cells. Over the past decade, increasingnumbers of reports have emerged documenting differential roles of HIF1a and HIF2ain these processes. In cells of the sympathoadrenal lineage, both HIFs differentially medi-ate influences of hypoxia on catecholamine synthesis and secretion, but HIF2a signaling

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286 Susan Richter et al.

has particularly prominent functions in regulating developmental processes of growthand differentiation. This chapter discusses the role of HIF2a and HIF1a in the contextof the development, phenotypic features, and functions of chromaffin cells. Moreover,current knowledge about tumor formation in cells of the sympathoadrenal lineage, lead-ing to catecholamine-producing pheochromocytomas and paragangliomas, is analyzedin the light of the HIF2a signaling network.

ABBREVIATIONSAMPK AMP-activated kinase

ANG angiopoietin

DDC dopa decarboxylase

DBH dopamine b hydroxylase

DLL4 delta-like ligand 4

HES1 hairy and enhancer of split-1

HIF hypoxia-inducible factors

HRE hypoxia-responsive element

MEN2 multiple endocrine neoplasia type 2

mTOR mammalian target of rapamycin

NADPH nicotinamide adenine dinucleotide phosphate

NGF nerve growth factor

OCT4 octamer-binding transcription factor 4

PGL paraganglioma

PHD prolyl hydroxylase

PHEO pheochromocytoma

PI3K phosphoinositide 3-kinase

PNMT phenylethanolamine N-methyltransferase

RCC renal cell carcinoma

SDH succinate dehydrogenase

TAMs tumor-associated macrophages

TH tyrosine hydroxylase

VEGF(R) vascular endothelial growth factor (receptor)

VHL Von Hippel–Lindau

1. INTRODUCTION

Oxygen tension differs widely across tissues and is much lower than

under ambient conditions (21%O2). The termmild hypoxia, the lack of suf-

ficient oxygen supply, is generally used for oxygen concentrations of 1–5%;

in contrast, severe hypoxia is defined as below 1% (Koh & Powis, 2012).

Oxygen shortage leads to stabilization of a class of transcription factors, ter-

med hypoxia-inducible factors (HIFs). HIFs are comprised of a stable

b-subunit and an oxygen-sensitive a-subunit. Protein stability of the latter

287Role of HIF2a in Chromaffin Cells

is regulated by several processes, including modification by prolyl hydrox-

ylases (PHDs) with subsequent normoxic proteasomal degradation; the latter

is partly mediated by the von Hippel–Lindau (VHL) tumor suppressor. The

molecular mechanisms for these processes are well described in several

reviews (Kaelin & Ratcliffe, 2008; Koh & Powis, 2012).

There are two main HIFa isoforms with partly overlapping, though

mostly complementary, functions (Carroll & Ashcroft, 2006; Hu, Wang,

Chodosh, Keith, & Simon, 2003; Rankin et al., 2007). HIF1a is activated

during short periods of severe hypoxia, whereas HIF2a (also referred to as

EPAS1, endothelial PAS protein 1) is active under mild hypoxia for

prolonged periods of time (Holmquist-Mengelbier et al., 2006). This

differential effect is mediated by hypoxia-associated factor, which marks

HIF1a for degradation, but transactivates HIF2a by binding to a different

protein site than in HIF1a (Koh, Darnay, & Powis, 2008; Koh, Lemos,

Liu, & Powis, 2011). This differential regulation leads to distinct cellular

functions, reflected in the expression patterns of the two transcription

factors. HIF1a is ubiquitously present in most cell types, whereas HIF2adisplays a more restricted expression pattern. HIF2a was first identified in

endothelial cells but has since been shown to be expressed in several other

cell types, specifically in the retina, lungs, heart, glial, and neural crest cells.

Both HIF1a and HIF2a employ at least two mechanisms for regulating

gene expression. In addition to their well-known interaction with HIFb,followed by C-terminal transactivation of genes possessing hypoxia-

responsive elements (HREs), both HIFa subunits also functionally interact

with other signal transduction and transcriptional systems. These non-

HRE-mediated mechanisms include NOTCH, WNT, and MYC pathway

interactions (Kaelin & Ratcliffe, 2008). Some evidence suggests that HIF1aand HIF2a can regulate the interaction of MYC and MAX, resulting in

opposing functional effects on MYC-dependent cell proliferation, apopto-

sis, differentiation, and stemness (Dang, Kim, Gao, & Yustein, 2008;

Gordan, Bertout, Hu, Diehl, & Simon, 2007).

The chapter focuses on the roles of HIF2a and HIF1a in cells of the

sympathoadrenal lineage and in particular their influences on catecholamine

synthesis and secretion, developmental processes, and tumorigenesis.

2. REGULATION OF CATECHOLAMINE SYNTHESISAND SECRETION BY HYPOXIA

Hypoxia is a well-established potent stimulus for secretion of cate-

cholamines both in vivo and in vitro in isolated cell systems (Cheung,


1989; Donnelly & Doyle, 1994; Kumar et al., 1998). Direct effects of

hypoxia on chromaffin cell catecholamine release are vital for maintaining

physiological homeostasis of fetuses before sympathetic innervation is

fully developed (Phillippe, 1983; Ream et al., 2008). Increased release of cat-

echolamines at birth facilitates appropriate hemodynamic adjustments and

stimulation of surfactant production by the lungs (Padbury, 1989;

Paulick, Kastendieck, & Wernze, 1985). Thereafter, responses of catechol-

amine systems to hypoxic stress, such as those associated with high altitude,

remain important for maintenance of cardiorespiratory homeostasis

(Gamboa et al., 2006; Kanstrup et al., 1999). On the other hand, chronic

hypoxic stress-associated catecholamine release can also lead to pathological

complications, such as hypertension associated with increased sympathetic

activity in patients with sleep apnea (Dimsdale, Coy, Ziegler, Ancoli-

Israel, & Clausen, 1995; Donnelly, 2005; Prabhakar & Kumar, 2010).

Intermittent hypoxia (5% O2 in the gas phase) increased the efflux of

both norepinephrine and epinephrine from ex vivo adrenal medullae of rats

10 days after beginning of treatment, indicating that catecholamine secre-

tion is upregulated under low oxygen tension (Kumar et al., 2006). Further

studies demonstrated that hypoxia increases cellular calcium influx, leading

to elevated exocytosis (Bournaud, Hidalgo, Yu, Girard, & Shimahara,

2007; Carpenter, Hatton, & Peers, 2000; Mojet, Mills, & Duchen,

1997; Taylor, Batten, & Peers, 1999). More recently, the involvement

of NADPH oxidase and reactive oxygen species signaling in hypoxia-

evoked catecholamine secretion has been established (Souvannakitti

et al., 2010).

Besides stimulating catecholamine secretion, hypoxia induces expression

of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine

synthesis, in numerous catecholamine-producing cells both in vivo and

in vitro (Czyzyk-Krzeska, Bayliss, Lawson, & Millhorn, 1992; Czyzyk-

Krzeska, Furnari, Lawson, & Millhorn, 1994; Schmitt, Garcia, Soulier,

Pujol, & Pequignot, 1992; Schmitt, Pequignot, Hanchin, Pujol, &

Pequignot, 1993). This induction is explained by the presence of a func-

tional HRE on the TH promoter; both HIF isoforms are able to activate this

promoter in a reporter construct assay (Schnell et al., 2003). It has also been

shown that levels of both TH and dopamine b-hydroxylase (DBH) protein

are increased after intermittent and sustained hypoxia (10% O2 in the gas

phase) in the rat carotid body and to lesser extents in the superior cervical

ganglia and adrenal glands; in the carotid bodies, this resulted in an increase

in contents of dopamine and norepinephrine (Hui et al., 2003). In this study,


increased TH activity was shown to result not only from increased levels of

TH protein but also from posttranslation activation of the enzyme by phos-

phorylation at serines 19, 31, and 40. This effect is most likely mediated

by AMP-activated kinase (AMPK), since AMPK inhibition by AICAR

(5-aminoimidazole-4-carboxamide 1-b-D-ribofuranoside) in PC12

cells prevents TH phosphorylation on relevant serine residues (Fukuda

et al., 2007).

Surprisingly, THmRNAwas not downregulated by RNAi knockdown

of Hif2a in immortalized rat chromaffin-cell-derived MAH cells; instead,

HIF2a was shown to directly regulate dopa decarboxylase (DDC) by bind-

ing to an HRE within its promoter (Brown, Kelly, Daniel, & Nurse, 2009).

The authors demonstrated that besides DDC, also DBHmRNA is decreased

by RNAi knockdown of Hif2a. Although no HRE was found in the Dbh

promoter region, the authors speculated that HIF2a regulation is due to

either the presence of an HRE within the gene or a mediating factor.

The same group also showed that HIF2a directly affects adenosine A2A

receptor expression in MAH cells (Brown, Reyes, & Nurse, 2011). Recep-

tor activation induces an increase in intracellular calcium in an HIF2a-dependent manner, leading to increased catecholamine release.

The findings in the preceding text are in tune with an earlier observation

that Hif2a�/� mouse embryos at 12.5 days have dramatically reduced nor-

epinephrine levels compared to wild types (Tian, Hammer, Matsumoto,

Russell, & McKnight, 1998). These Hif2a�/� embryos die in midgestation

similarly to TH- or DBH-deficient mice (Kobayashi et al., 1995; Thomas,

Matsumoto,& Palmiter, 1995; Zhou,Quaife, & Palmiter, 1995), emphasizing

the importance of catecholamines during mammalian development. This cru-

cial requirement is reinforced by findings that maternal oxygen (inspired O2

33% or 63%) prevents midgestational lethality of TH-deficient embryos,

indicating that catecholamines mediate fetal survival by maintaining oxygen

homeostasis (Ream et al., 2008).

As reviewed in detail by Wong and coworkers (Wong et al., 2010),

HIF1a also appears important in regulating adrenergic responses to stress

by activating phenylethanolamine N-methyltransferase (PNMT), the

enzyme that converts norepinephrine to epinephrine. This effect appears

to be indirectly mediated by HIF1a stimulation of EGR-1 and SP-1 tran-

scription factors and is in agreement with other findings that hypoxia

increases expression of PNMT,HIF1a, and EGR-1 in mouse pheochromo-

cytoma (PHEO) cells (Evinger, Cikos, Nwafor-Anene, Powers, &

Tischler, 2002).


From the findings outlined in the preceding text, it appears that PNMT is

predominantly responsive to HIF1a, whereas TH is responsive to both HIFs

and DDC and DBH are regulated mainly by HIF2a. This might suggest dif-

ferential effects on expression of catecholamine biosynthetic enzymes

dependent on the nature of the hypoxic stimulus. In support of this, rat

embryos exposed to long-term hypoxia, a state of predominant HIF2a sig-

naling, develop adrenal medullae with decreased epinephrine and increased

norepinephrine content, and a decreased percentage of chromaffin cells

expressing PNMT (Mamet et al., 2002). Similar findings of reduced num-

bers of PNMT positive adrenal medullary cells were observed after long-

term hypoxia in fetal sheep (Ducsay et al., 2007). In contrast, but still in line

with differential effects on catecholamine biosynthetic machinery, acute

short-term hypoxia in fetal sheep increased expression of PNMT but

decreased that of TH (Adams & McMillen, 2000). Other studies have char-

acterized differential effects of long-term and intermittent hypoxia on

expression of TH and HIF isoforms in catecholamine-producing cells of

the carotid body and brain (Gozal et al., 2005; Lam, Tipoe, Liong, &

Fung, 2008; Raghuraman, Prabhakar, & Kumar, 2012). As outlined in

the succeeding text, at least some of the differential effects of hypoxia on

expression of catecholaminergic biosynthetic enzyme may also partly reflect

influences of HIF2a on chromaffin cell growth and differentiation rather

than direct actions on expression of biosynthetic enzymes.

3. THE ROLE OF HIF2a IN CHROMAFFIN CELLDEVELOPMENT

Initial investigations concerning the role of HIF2a in sympatho-

adrenal development assessed expression patterns during embryogenesis.

A study with chicken embryos demonstrated strong expression in endothe-

lial and vascular smooth muscle cells, liver, kidney, and cellular progenitors

of the sympathetic nervous system characterized by TH expression (Favier,

Kempf, Corvol, & Gasc, 1999). HIF2a distribution in the sympathetic lin-

eage was investigatedmore closely inmouse embryos whereHIF2a-positivecells were observed in the sympathetic chain at embryonic day E11.5 (Tian

et al., 1998). This expression was lost soon after but followed by a strong

immunohistochemical staining signal in forming paraganglia; this signal

was maintained until E15.5. Lower levels of expression were also found

in the adrenal. Death of Hif2a-null embryos coincided with the time of

expression in sympathoadrenal cells. Furthermore, HIF2a colocalized with


TH protein in paraganglia of a human fetus at week 8.5, corresponding to

E15 in mice (Nilsson et al., 2005).

The findings in the preceding text provide strong evidence that HIF2a is

important in the regulation of developmental processes of sympathoadrenal

cells. As discussed earlier, HIF2a regulates catecholamine synthesis and

secretion; hence, Hif2a-null embryos contain less norepinephrine than

wild-type mice (Tian et al., 1998). Since PHD3 was shown to preferentially

hydroxylate HIF2a, labeling the latter for degradation (Appelhoff et al.,

2004), Phd3�/� mice should contain higher HIF2a levels than their

wild-type littermates. However, contrary to what one would expect, these

mice display a hypofunctional sympathoadrenal system with reduced tissue

innervation, lower plasma levels of epinephrine and norepinephrine, and

decreased systolic and diastolic blood pressures (Bishop et al., 2008).

The authors also demonstrated that nerve growth factor (NGF)-stimulated

neuronal survival was increased in Phd3�/� mice in an HIF2a-dependentmanner. Moreover, increased numbers of TH-positive cells were measured

in the adrenal medulla, carotid body, and superior cervical ganglion.

These results suggest that HIF2a renders cells more responsive to neurite

growth-promoting effects but at the same time has dedifferentiating effects

leading to the occurrence of a hypofunctional sympathoadrenal system in

Phd3�/� mice.

During the development of multicellular organisms, the balance

between cell proliferation, differentiation, and death is constantly changing,

leading to processes such as organ morphogenesis. The balance is maintained

by the presence of different factors at certain developmental stages. One such

factor, with extreme importance for the sympathetic nervous system, is NGF

(Fig. 14.1). NGF inhibits both basal and hypoxia-induced Hif2a but not

Hif1a expression in PC12 cells (Naranjo-Suarez et al., 2003). Depriving

sympathetic neurons of NGF results in reduced glucose uptake, elevated

levels of reactive oxygen species, and, hence, increased cell death (Lomb,

Desouza, Franklin, & Freeman, 2009). This process is diminished by

PHD inhibitors and, in concordance with this, knockdown of Hif2a by

shRNA in mouse neurons decreases survival in the presence of NGF com-

pared to control.

In keeping with the concepts in the preceding text, PC12 cells over-

expressing Phd3 (also referred to as EGLN3 or SM-20) display increased

cytochrome c- and caspase-dependent apoptosis (Straub, Lipscomb,

Yoshida, & Freeman, 2003). Similar observations were made in sympathetic

neurons, where the authors also demonstrated increased expression of Phd3

RAS

RAF

MEK

MAPK

PHD3

PI3K

AKT

mTOR

TRK

A

NGF

TRKA

HIF2HIF2α

PHD3

HIF2HIF2α

HIF2HIF2α

OCT4

HES1

ic-NOTCH

ic-N

OTC

H

TIE

2

NO

TCH

HEY1

HEY2

c-JUN

GLUT1VEGFASOD1/2DLL4ANG2

Succinate

KIF1KIF1Bβ

?

Apoptosis

DLL4

AN

G2

AN

G1

HIF2HIF2α

Figure 14.1 HIF2a signaling network in chromaffin cells. Nerve growth factor (NGF)binding to its receptor tyrosine kinase TRKA induces PI3K/AKT signaling, which in turnactivates the global translational regulator mTOR. One of its targets is hypoxia-induciblefactor 2a (HIF2a), a transcription factor able to induce a number of different genes, suchas the NOTCH ligand delta-like ligand 4 (DLL4). HIF2a and intracellular NOTCH(ic-NOTCH) jointly activate genes, such as stem cell marker HES1. The HIF2a proteinis labeled for degradation by prolyl hydroxylase 3 (PHD3) and processed by theproteasome complex, part of which is the von Hippel–Lindau (VHL) protein. PHD3 isinhibited by high concentrations of succinate, which can be achieved by inactivationof the enzyme succinate dehydrogenase. When TRKA is not activated by NGF, theRAS/MAPK pathway is activated instead leading to induction of the transcription factorc-JUN, which stimulates increased transcription of PHD3, and then kinesin KIF1Bbinduces apoptosis. An alternative way of PI3K activation is signaling via the TIE2 recep-tor, which is normally activated by its ligand angiopoietin-1 (ANG1). Whenangiopoietin-2 (ANG2) is present, ANG1 effects can be inhibited, but when ANG1 isabsent, ANG2 is also able to induce TIE2 signaling.


after NGF withdrawal (Lipscomb, Sarmiere, Crowder, & Freeman, 1999;

Lipscomb, Sarmiere, & Freeman, 2001). It is well established that NGF

deprivation causes apoptosis by activating the transcription factor c-JUN

(Estus et al., 1994; Ham et al., 1995; Schlingensiepen et al., 1994; Xia,

Dickens, Raingeaud, Davis, & Greenberg, 1995). More recently, Lee and

coworkers showed that PHD3 but not PHD1 or PHD2 is required for

c-JUN-dependent apoptosis by a mechanism in which c-JUN directly binds


to the PHD3 promoter (Lee et al., 2005). PHD3 in turn induces kinesin

KIF1Bb, a motor protein that contributes to induction of neuronal apoptosis

(Schlisio et al., 2008). The exact mechanism of this induction has yet to

be clarified.

Besides regulation of cell death, HIF2a is also involved in differentiation

processes; thus, similar to chromaffin progenitors,Hif2a is expressed in pan-creatic progenitor cells, but not in differentiated endocrine or exocrine cells

(Chen et al., 2010). In the same study, Hif2a-null embryos were found to

have less HES1 (hairy and enhancer of split-1)-positive cells than wild type,

indicating a less differentiated state. This effect appears to be independent of

the canonical hypoxia pathway, since Hif1b deletion did not impair normal

development. The authors also demonstrated that HIF2a binds to the

NOTCH intracellular domain (ic-NOTCH), explaining the activation of

the classical NOTCH signaling effector HES1.

Knockdown of HIF2a in neuroblastoma tumor-initiating stem cells

resulted in decreased expression of NOTCH target genes and increased

expression of neural differentiation markers (Pietras et al., 2009).

A similar effect was seen with rapamycin, indicating that HIF2a translation

is dependent on the mammalian target of rapamycin (mTOR) pathway. In

keeping with this, hypoxia was shown to induce neural crest genes, includ-

ing NOTCH-1 and HES1, in neuroblastoma cell lines and the embryonic

carcinoma cell line P19 (Gustafsson et al., 2005; Jogi et al., 2002; Nilsson

et al., 2005). These effects are however partly dependent on HIF1a, sinceit was shown that HIF1a is also able to associate with ic-NOTCH

(Gustafsson et al., 2005; Pietras, von Stedingk, Lindgren, Pahlman, &

Axelson, 2011).

There is some evidence that neural crest stem cells cultured under mild

hypoxia undergo sympathoadrenal differentiation to cells expressing TH and

DBH with measurable release of dopamine and norepinephrine (Morrison

et al., 2000). In a similar way, PC12 cells treated with a PHD inhibitor or

shRNA against Phd1 or Phd2 show increased TH activity and dopamine

release (Johansen et al., 2010); however, it is not clear if these effects are

HIF1a- or HIF2a-mediated. On the other hand, HIF2a was shown to

act directly upstream of OCT4 (octamer-binding transcription factor 4), a

transcription factor known to be essential for maintenance of pluripotency

(Covello et al., 2006; Koh et al., 2011).

The results in the preceding text strongly indicate that HIF2a is respon-

sible for maintaining a balance between stemness and differentiation in the

sympathoadrenal lineage. Interestingly, HIF2a was found to be repressed in


murine embryonic stem cells (Hu et al., 2006), suggesting this transcription

factor is needed in later developmental stages. This is in agreement with the

observation thatHif2a expression is only induced at E11.5 in chromaffin cell

progenitors (Tian et al., 1998). On the other hand, HIF1a signaling is fully

functional in embryonic stem cells, where it activates the Wnt/b-cateninpathway, which regulates neural stem cell proliferation and differentiation,

for example, in the subgranular zone of the hippocampus (Mazumdar,

O’Brien, et al., 2010).

In summary, HIF1a and HIF2a have distinct roles in development of

cells of the mammalian sympathoadrenal lineage, wherein HIF2a is a centralplayer in regulating chromaffin cell phenotypic features.

4. HIF2a SIGNALING IN TUMORIGENESIS

HIF2a expression has been linked to malignant progression and poor

prognosis in a number of tumors, including astrocytoma, glioma, neuroblas-

toma, head and neck cancers, melanoma, and others (Keith, Johnson, &

Simon, 2012). In most cases, both overexpression of HIF1a andHIF2a havenegative effects on outcome; however, in neuroblastoma, HIF1a staining in

tissue sections correlated with favorable prognosis, whereas HIF2a staining

indicated poor outcome (Noguera et al., 2009).

Similar observations have been reported in renal cell carcinoma (RCC)

in which VHLmutations lead to decreased HIFa degradation. It was shown

that VHL wild-type cells expressing a stable HIF1a mutant are not able to

reproduce the tumorigenic phenotype (Maranchie et al., 2002; Raval et al.,

2005); in contrast, HIF2a suppression abrogated tumor formation in mice

(Kondo, Kim, Lechpammer, & Kaelin, 2003). Raval et al. also demonstrated

that in RCC lines, proapoptotic genes, such as BNIP3, are predominantly

regulated by HIF1a, whereas protumorigenic genes, such as vascular endo-

thelial growth factor (VEGF), transforming growth factor alpha, and cyclin

D, are more dependent on HIF2a.In a Kras-driven lung tumor mouse model,Hif2a deletion but notHif1a

deletion contributed to tumor growth and progression by direct down-

regulation of the tumor suppressor secretoglobin 3A1, an inhibitor of

AKT signaling (Mazumdar, Hickey, et al., 2010). Interestingly, over-

expression of a stable mutant of Hif2a also increased tumor formation in

the same Kras mouse model; this was shown to be dependent on increased

angiogenesis by induction of Vegf and increased invasiveness, demonstrated

by increased markers of epithelial–mesenchymal transition (EMT), such as


Snail (Kim et al., 2009). These results indicate that Hif2a expression has to

strike a certain balance and that both upregulation and downregulation can

promote tumorigenesis.

It is well known that HIF2a is a potent inducer of angiogenesis. In 2000,

Peng and coworkers showed that mouse embryos originating from Hif2a-deficient embryonic stem cells display severe vascular defects in the embryo

itself and the yolk sac, where vessels are formed but fail to connect and estab-

lish the correct network (Peng, Zhang, Drysdale, & Fong, 2000). Over-

expression of Hif2a in rat glioma tumors increases Vegf mRNA and

vascular tumor area, whereas the expression of the dominant negative form

of Hif2a resulted in the opposite (Acker et al., 2005).

HIF2a expression has also been shown to be associated with increased

vascular density in breast tumors (Giatromanolaki, Sivridis, Fiska, &

Koukourakis, 2006) and increased VEGF expression and advanced clinical

stage in neuroblastoma (Holmquist-Mengelbier et al., 2006). In VHL-

deficient mouse livers, Vegf expression and the development of hemangi-

omas were demonstrated to be dependent on Hif2a and not Hif1aexpression (Rankin et al., 2008). Patients with VHL germline mutations

are at higher risk to develop hemangioblastomas. These highly vascularized

but nonmalignant tumors mainly originate from stromal cells in the central

nervous system and retina, but can also occur in other organs. Further

tumors associated with the VHL syndrome are clear cell RCC and PHEO.

Another area related to angiogenesis and where HIF2a plays a critical

role is hematopoiesis. Mice lacking Hif2a have pancytopenia but intact

multilineage maturation processes in the bone marrow, which led the

authors to suggest a mechanism related to a disturbed microenvironment

(Scortegagna, Morris, Oktay, Bennett, & Garcia, 2003). Later, it was

established that HIF2a directly regulates erythropoietin expression

(Rankin et al., 2007; Scortegagna et al., 2005; Warnecke et al., 2004) and

is a crucial regulator of iron absorption, a process essential for the normal

functionality of erythrocytes (Mastrogiannaki et al., 2009).

Other processes important for both normal development of the vascula-

ture and tumor progression are cell migration and matrix vascular remo-

deling. Epidermal growth factor receptor activation in hypoxic foci in

head and neck squamous cell carcinoma leads to enhanced cell migration,

but not proliferation, a process shown to be dependent on the expression

ofHIF2a (Wang & Schneider, 2010). This pathway is proposed to promote

a more aggressive phenotype in this type of cancer. An HIF2a target gene

possibly mediating this effect is plasminogen activator inhibitor-1 (PAI1)


(Sato et al., 2004), a serine protease inhibitor shown to induce cancer inva-

sion and vascularization by facilitating attachment and migration of cancer

cells (Bajou et al., 1998; Chazaud et al., 2002).

Another HIF2a target identified in RCC cells lacking the VHL gene is

type 1 matrix metalloproteinase, a protein capable of extracellular matrix

degradation (Petrella, Lohi, & Brinckerhoff, 2005). In the context of carti-

lage destruction, a number of other matrix metalloproteinases and matrix

catabolic factors were identified to be HIF2a targets (S. Yang et al.,

2010). In RCC cells deficient for VHL, HIF2a was shown to induce genes

that drive metastasis, including chemokine (C-X-C motif ) receptor 4 and

cytohesin 1 interacting protein; they are involved in chemotactic cell inva-

sion and protection from death cytokine signaling, respectively (Vanharanta

et al., 2013). These effects were dependent on the induction of epigenetic

changes, such as histone H3 and DNA methylation. HIF2a also activates

expression of the adenosine A2A receptor; activation affects not only cate-

cholamine release (Brown et al., 2011) but also other processes, including

cell proliferation, cell migration, and tube formation in primary cultures

of human lung endothelial cells (Ahmad et al., 2009).

Interestingly, in murine endothelial cells, HIF2a appears to have an

inhibitory effect on tumor cell migration and metastasis mediated by reduc-

tion of nitric oxide (NO) synthesis; in contrast, HIF1a has an opposing effect(Branco-Price et al., 2012; Takeda et al., 2010). This is consistent with the

finding of Skuli et al., where Hif2a deletion resulted in increased migration

and invasion, and dysfunctional arteriogenesis (Skuli et al., 2012), which was

associated with decreased expression of delta-like ligand 4 (DLL4, NOTCH

ligand),Hes1, and other NOTCH target genes and the proangiogenic factor

angiopoietin-2 (ANG2). Hif2a expression in host endothelial cells was also

shown to be crucial for tumor neovascularization in a xenograft model of

melanoma, where this process was identified to be dependent on the

HIF2a-driven expression of ephrin A1 (Yamashita et al., 2008).

The preceding examples emphasize that not only tumor cells need to be

considered in the process of tumorigenesis but also cells of the stromal

microenvironment. High HIF2a levels are found in tumor-associated mac-

rophages (TAMs) (Talks et al., 2000), and their number has been associated

with poor clinical outcome in different cancers (Tang, Mo, Wang, Wei, &

Xiao, 2013). HIF2a is crucial for TAM infiltration into tumor lesions and,

through this mechanism, promotes tumor progression in mouse models

(Imtiyaz et al., 2010). On the other hand, in a melanoma mouse model,

it was shown that HIF2a stabilization by a PHD3 inhibitor decreases tumor


growth; this was attributed to TAMs secreting an increased amount of sol-

uble vascular endothelial growth factor receptor (VEGFR) that inhibits

VEGF function (Roda et al., 2012).

TAMs appear to reside close to an HIF2a expressing immature neural

crest-like cell population in the perivascular niche of neuroblastomas

(Pietras et al., 2008). These cells resemble a population termed neural

crest-like neuroblastoma tumor-initiating stem cells, which have been iso-

lated from patient bone marrow (Pietras et al., 2009). Similarly, tumor-

associated stem cells with high HIF2a expression have been identified in

glioblastomas (Li et al., 2009).

In summary, the role of HIF2a in tumorigenesis appears to be complex

and context-dependent in that both increased and decreased expression can

result in tumor development. Hence, targeting hypoxic signaling pathways

as cancer therapy requires careful evaluation of tumor type and phenotypic

features.

5. GENOTYPE–PHENOTYPE RELATIONSHIPSOF CHROMAFFIN CELL TUMORS

PHEOs and paragangliomas (PGLs) are catecholamine-producing

tumors derived, respectively, from chromaffin cells of the adrenal medulla

and extra-adrenal paraganglia. Most catecholamine-producing PGLs occur

in abdominal and thoracic regions. Other PGLs are found in the head and

neck region, but these usually produce negligible or low amounts of cate-

cholamines. Occurrence of PHEOs/PGLs in the general population is rare,

but a substantial portion of these tumors have a hereditary basis, where about

one-third are caused by germline mutations in one of the following genes:

neurofibromatosis type 1 (NF1); rearranged during transfection (RET )

proto-oncogene; transmembrane protein 127 (TMEM127); myc-associated

factor (MAX);VHL; or one of the genes for succinate dehydrogenase (SDH)

subunits (SDHA, B, C, D, and AF2) (Astuti et al., 2003; Baysal et al., 2000;

Burnichon et al., 2010; Comino-Mendez et al., 2011; Hao et al., 2009; Latif

et al., 1993; Mulligan et al., 1993; Niemann & Muller, 2000; Qin et al.,

2010; Viskochil et al., 1990).

The diverse genotypic backgrounds of PHEOs/PGLs are associated with

distinct differences in clinical presentation (Eisenhofer, Pacak, et al., 2011).

This includes tumor location, propensity to malignancy, tumor tissue

catecholamine contents, the dominant type of catecholamine (dopamine,

norepinephrine, or epinephrine) produced, and catecholamine secretory

Dopamine & mixed

dopamine/noradrenaline

producing tumors

• Immature phenotype• Extra-adrenal locations• Early presentation age• Malignant tendency

Noradrenaline-producing

tumors

• Immature secretory phenotype• Adrenal & extra-adrenal locations

• Early presentation age• Occasionally malignant

Adrenaline-producing

tumors

• Mature secretory phenotype• Adrenal locations

• Late presentation age• Rarely malignant

Cluster 1

Cluster 2

Sporadic

Sporadic

SDHBSDHD

VHL

RETNF1

Figure 14.2 Unsupervised hierarchical clustering based on measured values forepinephrine-related analytes in plasma and urine. Increasing analyte levels are illus-trated in gray scale by progression from lighter to darker heat map areas. Patients withVHL or SDHx mutations are confined to cluster 1, whereas patients with RET and NF1mutations are confined to cluster 2. Image modified from Eisenhofer, Pacak, et al., 2011.


characteristics (Fig. 14.2). Tumors due to mutations of SDH subunit genes

predominantly occur at extra-adrenal locations, whereas those due to RET,

NF1, TMEM127, and MAX mutations predominantly occur at adrenal

locations. Tumors due to VHL mutations can occur at both locations but

predominate at adrenal locations. PGLs due to SDHB mutations are partic-

ularly prone to metastasis (Blank et al., 2010; Gimenez-Roqueplo et al.,

2003; King et al., 2011).

Hereditary tumors associated withRETmutations in multiple endocrine

neoplasia type 2 (MEN2) produce epinephrine, whereas VHL tumors lack

expression of PNMT leading to termination of catecholamine synthesis at

the level of norepinephrine (Eisenhofer et al., 2001). Similarly, tumors

due to NF1 mutations express PNMT and, thus, produce epinephrine,


whereas those due to mutations of SDH subunit genes do not and tend

towards immature phenotypic features of low tissue catecholamine contents

with significant production of dopamine and its O-methylated metabolite

methoxytyramine (Eisenhofer et al., 2012; Eisenhofer, Pacak, et al., 2011).

Interestingly, while total tissue catecholamine contents are highest in

epinephrine-producing hereditary and sporadic tumors and lowest in those

that do not produce epinephrine, rates of catecholamine secretion and

excretion into urine are highest in dopamine- and norepinephrine-

producing tumors (Eisenhofer, Pacak, et al., 2011). This difference in cat-

echolamine secretory characteristics has been linked to a more fully devel-

oped regulatory secretory pathway in tumors that produce epinephrine than

in those that do not (Eisenhofer et al., 2008). Lack of regulatory controls in

the more immature norepinephrine- and dopamine-producing tumors leads

to more continuous or constitutive catecholamine-secretory activity than in

the more fully differentiated tumors that produce epinephrine.

6. WHAT ROLE DOES HIF2a PLAY IN THE DEVELOPMENTOF CHROMAFFIN CELL TUMORS?

The differences in genetic backgrounds of PHEOs/PGLs are reflected

by distinct differences in gene expression profiles, with consistent differences

among hereditary groups observed in several studies (Burnichon et al., 2011;

Dahia et al., 2005; Eisenhofer et al., 2004; Lopez-Jimenez et al., 2010). In par-

ticular, these various gene expression profiling studies have all described two

cluster groups with epinephrine-producing tumors due to RET, NF1, and

TMEM127 mutations in one cluster group (cluster 2) and norepinephrine-

or dopamine-producing tumors due to VHL and SDHx mutations in the

other (cluster 1).

The first gene expression profiling study noted distinct differences in

gene expression between both hereditary and sporadic norepinephrine-

and epinephrine-producing tumors with overexpression of genes involved

in hypoxia–angiogenic pathways in the former tumors (Eisenhofer et al.,

2004). The most important upregulated gene in both hereditary and spo-

radic norepinephrine-producing tumors was HIF2a. Taking into account

the role of HIF2a in maintaining stem cell-like traits in chromaffin cells,

these findings were interpreted to suggest a key role of HIF2a in develop-

ment of PHEOs/PGLs with an immature catecholamine phenotype.


Expression of HIF2a in developing chromaffin progenitors was consid-

ered to confer susceptibility of these cells to mutations of genes, such asVHL

and SDHx. Mutations of these genes lead to impaired degradation of HIFs at

the protein level. Thus, for such mutations to have effect, the appropriate

HIF genes must be expressed at the transcriptional level, which for HIF2ahas a restricted tissue distribution that includes, during embryogenesis, neu-

ral crest-derived cells of the sympathoadrenal lineage. This restricted tissue

distribution thereby confers susceptibility of sympathoadrenal progenitors

for amplification of HIF2a at the protein level. This in turn enhances hyp-

oxia signaling and creates an environment favoring survival of cells and their

further susceptibility to the effects of second-hit deletions or point muta-

tions. The end result is cluster 1 tumors maintained in an HIF2a-overexpressed undifferentiated state characterized by lack of epinephrine

production and underexpression of numerous other genes that otherwise

contribute to the fully differentiated chromaffin cell phenotype.

The second gene profiling study also clustered PHEOs/PGLs in two

groups, one containing tumors with VHL, SDHB, and SDHD mutations

and another with RET and NF1 mutations (Dahia et al., 2005). Notably,

almost all extra-adrenal PGLs analyzed in this cohort were confined to clus-

ter group 1, which was comprised of both adrenal and extra-adrenal tumors

in equal proportions. In agreement with the earlier study, cluster 1 displayed

a gene signature of activated hypoxia pathways and enhanced angiogenesis

and extracellular matrix processes. Furthermore, the cluster 1 gene profile

showed a suppressed mitochondrial function, an observation confirmed

by decreased SDHB protein in the majority of all cluster 1 tumors, including

VHL and sporadic cases. These features, according to gain-of-function and

loss-of-function experiments in cell line models, were described as depen-

dent on HIF1a signaling.

Subsequent gene expression profiling studies confirmed the strong

HIF2a expression in VHL and SDHx tumors compared to cluster 2 tumors

(Burnichon et al., 2011; Lopez-Jimenez et al., 2010). This was also further

confirmed by immunohistochemical analyses of HIF2a in tumor sections

(Favier et al., 2009). HIF1a expression on the other hand was not different

between clusters.

In 2005, Lee et al. proposed that the PHEO/PGL mutations of NF1,

c-RET, VHL, and SDHx all act on the same signaling network, resulting

in decreased apoptosis during chromaffin cell development and, hence,

tumor formation (Lee et al., 2005). NF1 is a negative regulator of RAS sig-

naling induced by stimulation of the NGF receptor TrkA. c-RET is the


receptor for GDNF (glial cell line-derived neurotrophic factor) that cross

talks with TrkA (Dechant, 2002; Peterson & Bogenmann, 2004; Tsui-

Pierchala, Milbrandt, & Johnson, 2002) and induces JUNB, an antagonist

of c-JUN, leading to decreased apoptosis (Lee et al., 2005). In the same

study, it was demonstrated that loss of VHL, similar to c-RET activation,

results in JUNB induction. The authors went on to show that succinate

inhibits PHD3. The former accumulates in the cell when SDH is inhibited

(Selak et al., 2005; Smith, Janknecht, & Maher, 2007). NF1 mutations and

the more recently identified TMEM127 mutations were shown to hyper-

phosphorylate mTOR (Dasgupta, Yi, Chen, Weber, & Gutmann, 2005;

Qin et al., 2010), potentially inducing HIF2a translation. The fundamental

differences observed between clusters could potentially be due to a more

severe activation of HIF2a in cluster 1 due to the impairment of the deg-

radation machinery (Fig. 14.1).

Given the highly divergent phenotypic features and gene expression pro-

files observed between different groups of hereditary PHEOs/PGLs, it

seems counterintuitive that all these tumors might develop through a single

signaling network as proposed by Lee et al. (Lee et al., 2005). Nevertheless,

this remains plausible should components of this network be differentially

susceptible to the mutations of the various tumor-susceptibility genes in

chromaffin progenitors or mature cells at different locations and stages of

development. In this way, expression of HIF2a in chromaffin cell types at

specific locations or in sympathoadrenal progenitors at a particular stage

in chromaffin cell development might make only these cells vulnerable to

mutations impacting hypoxia pathways. As originally proposed

(Eisenhofer et al., 2004), the expected result is development of tumors with

the same immature catecholamine phenotypic features characteristic of the

sympathoadrenal progenitor cells from which the tumors derive.

Support for the concept that chromaffin cell tumors develop from differ-

ent progenitors has been derived from analysis of a large clinical dataset

documenting highly significant differences in age of diagnosis of sporadic

and hereditary PHEOs/PGLs according to catecholamine phenotypic fea-

tures (Eisenhofer, Timmers, et al., 2011). Specifically, patients with

epinephrine-producing cluster 2-type tumors are diagnosed on average a

decade later than patients with norepinephrine- or dopamine-producing

cluster 1 tumors. While hereditary PHEOs/PGLs also present on average

10 to 15 years earlier than sporadic tumors, the differences in ages of presen-

tation of tumors with different catecholamine phenotypes occur indepen-

dently of this additional influence (Fig. 14.3). Thus, patients with

RET NF1 VHL SDHB SDHD Sporadic

Age

(ye

ars)

20

25

30

35

40

45

50

55

Figure 14.3 Age at diagnosis of hereditary and sporadic PHEO/PGLs. Summed data(mean and standard deviation) from nine studies (Amar et al., 2005; Casanova et al.,1993; Cascon et al., 2009; Eisenhofer, Timmers, et al., 2011; Mannelli et al., 2009;Neumann et al., 2002; Pomares et al., 1998; Ricketts et al., 2010; Walther, Herring,Enquist, Keiser, & Linehan, 1999) weighted according to the number of subjects ineach study.


dopamine- or norepinephrine-producing hereditary tumors showed the

youngest ages of disease presentation. Importantly, among this group, pre-

sentation of disease was much earlier in patients with multifocal extra-

adrenal tumors than in those with solitary PHEOs/PGLs.

The observations in the preceding text not only support origins of

PHEOs/PGLs from different chromaffin progenitor cells with variable

developmental susceptibility to disease-causing mutations but also suggest

development of multifocal disease from HIF2a-overexpressing tumor stem

cells that have suffered a second hit to inactivate gene function before migra-

tion to different sites. Later age of presentation of tumors that produce epi-

nephrine than norepinephrine is expected, since in those patients, any

genetic abnormalities leading to tumorigenesis can only be expected to have

impact after chromaffin progenitors have migrated and differentiated into

adrenaline-producing chromaffin cells within the adrenals.

7. HIF2a AND METASTATIC PHEO/PGL

In addition to phenotypic differences between tumors of the twomain

gene expression cluster groups, there are also differences between tumors of

the same cluster; SDHB-related tumors are often extra-adrenal and have an


enhanced tendency to metastatic progression, whereas VHL tumors are

mostly adrenal and have a low risk of malignancy (Brouwers, Eisenhofer,

et al., 2006; Burnichon et al., 2011; Gimenez-Roqueplo et al., 2003). Gene

expression profiling also clearly distinguishes between those two hereditary

groups of tumors (Favier et al., 2009; Lopez-Jimenez et al., 2010). VHL

tumors have a more HIF1a-driven signature than SDHx-related tumors,

which manifests in upregulation of glycolytic and suppression of mitochon-

drial genes, the so calledWarburg effect. These differences were additionally

associated with increased expression of apoptotic target genes such as

BNIP3.

Overexpression of HIF2a in a VHL-deficient RCC line increased

mitochondrial and decreased glycolytic metabolism compared to controls,

also suggesting that SDHx-related tumors are more dependent on HIF2a(Biswas et al., 2010). Pollard and colleagues not only confirmed higher

levels of BNIP3 in VHL tumors by immunohistochemistry but also found

somewhat increased VEGF, cyclin D1, and HIF2a levels (Pollard et al.,

2006). The authors, however, concluded that VHL tumors are more

HIF2a-driven than those with SDHx mutations; nevertheless, based on

their observations that both target genes of HIF1a (BNIP3) and HIF2a(cyclin D1) are increased and that VEGF expression is known to be

induced by both transcription factors (Keith et al., 2012), the question

of whether one signaling pathway is more prominent than the other

may not have such a simple answer. Interestingly, PHD3 is more highly

expressed in VHL tumors, which is however not reflected in the protein

level analyzed by immunohistochemistry, indicating some form of post-

transcriptional mechanism (Eisenhofer et al., 2004; Lopez-Jimenez

et al., 2010).

More than 50% of childhood PHEO/PGL cases have metastatic disease,

and about 70% of these carry SDHBmutations (King et al., 2011). This again

not only is in line with concepts that phenotypically immature tumors

develop earlier in life than more fully differentiated tumors but also indicates

an additional link to aggressiveness of tumors. Nevertheless, SDHB muta-

tions carry a poor prognosis in both children and adults (Amar et al.,

2007; Blank et al., 2010; King et al., 2011). Gene profiling studies

attempting to shed light on the distinctive biology between SDHx-mutated

and VHL tumors have so far failed to show a definitive lead (Brouwers,

Elkahloun, et al., 2006; Waldmann et al., 2010). Both studies however

established downregulation of JUNB in malignant PHEOs/PGLs, pointing

towards a potential involvement of the HIF2a signaling network.


Recently, Favier and colleagues established increased vascularization

coupled with increased VEGF and ANG2 expression in cluster 1 tumors

(Favier, Igaz, et al., 2012). Interestingly, malignant tumors of both clusters

showed strongly reduced ANG1 expression. In the presence of ANG1,

ANG2 is known to inhibit TIE2 receptor activation, but if ANG1 is absent,

ANG2 is able to activate TIE2 and downstream signaling of pho-

sphoinositide 3-kinase (PI3K) and AKT (Yuan, Khankin, Karumanchi, &

Parikh, 2009) (Fig. 14.1). This evidence points to a possible activation of

the mTOR pathway in malignant PHEOs/PGLs; however, the sample size

of this study was too low for any definite conclusions (Favier, Igaz, et al.,

2012). Preliminary investigations concerning mTOR inhibition in the

mouse PHEO cell lines indicate a concentration-dependent decrease in cell

survival (Nolting & Grossman, 2012).

The question remains, why are SDHB-related tumors so much more

malignant than VHL and other cluster 1 tumors, including those due to

SDHD? They are more immature, have dramatically decreased total cate-

cholamine contents with high proportions of dopamine, and they are larger

at diagnosis compared to most other PHEOs/PGLs (Eisenhofer et al., 2012).

Is this solely mediated by HIF2a signaling or are there other factors

involved? The study by Guzy et al. would support the first hypothesis, since

SDHB knockdown in a cell line model led to increased tumor xenograft

growth, which could be attenuated by inhibiting both HIF1a or HIF2aor both (Guzy, Sharma, Bell, Chandel, & Schumacker, 2008). On the other

hand, it needs to be considered that SDHB mutations alter the balance of

energy metabolites in these cells dramatically by strongly elevating succinate

concentrations (Selak et al., 2005; Smith et al., 2007). Succinate not only

broadly inhibits a-ketoglutarate-dependent enzymes, such as PHDs, but also

affects the TET family of DNA hydroxylases and inhibits other enzymes,

such as histone demethylases, leading to increased histone H3 methylation

(Cervera, Bayley, Devilee, & McCreath, 2009; Smith et al., 2007; Xiao

et al., 2012). The same effects have been demonstrated for fumarate accu-

mulation caused by fumarate hydratase knockdown (Xiao et al., 2012).

Clearly changes in metabolite levels can lead to profound epigenetic

alterations and hence changes in gene expression that may differ between

SDHx-mutated PHEO/PGL and other tumors. Possibly, therefore, the

aggressive nature of SDHB-related PHEOs/PGLs might reflect downstream

actions of succinate on methylation-related epigenetic mechanisms of gene

regulation. Strong support for this concept has been provided by Favier and

colleagues (Letouze et al., 2013), who showed that hypermethylation is


responsible for many of the differences in gene expression between tumors

due to SDHx mutations compared to other mutations. Interestingly,

hypermethylation was more extensive in tumors due to SDHB than SDHD

mutations, which may contribute to the more aggressive nature of SDHB-

related PHEOs/PGLs than other tumors, including those due to

SDHD mutations.

8. MUTATIONS OF HIF2a AS A CAUSE OF CHROMAFFINCELL TUMORS

A central role of HIF2a signaling in PHEO/PGL development has

been substantiated by findings of somaticHIF2a gain-of-function mutations

in two patients with multiple PGLs and somatostatinomas (Zhuang et al.,

2012). Shortly after this initial report, there followed several further publi-

cations from different groups describing either somatic or germline HIF2amutations in patients characterized with mostly multiple PGLs and polycy-

themia (Comino-Mendez et al., 2013; Favier, Buffet, & Gimenez-

Roqueplo, 2012; Lorenzo et al., 2012; Pacak et al., 2013; C. Yang et al.,

2013). These gain-of-function mutations protect HIF2a from degradation

processes mediated by PHDs, but whether they predispose or rather are a

direct cause of tumorigenesis is currently unclear.

HIF2a-mutated tumors have increased levels of HIF2a mRNA, similar

to cases with VHL and SDHx mutations (Comino-Mendez et al., 2013).

However, Favier et al. noted that the activation of hypoxia-inducible genes

is rather mild compared to other cluster 1 tumors (Favier, Buffet, &

Gimenez-Roqueplo, 2012). Interestingly, all subjects described in these

studies who presented with multifocal PGLs and increased red blood cell

mass were first diagnosed at the age 35 or younger, with most being younger

than 20 years; in contrast, patients without polycythemia presented later

with PHEO/PGL and more usually with solitary than multifocal tumors.

Of further relevance to these observations, Comino-Mendez et al. deter-

mined the same HIF2a mutation in different tumors from the same patient,

but not in the germline (Comino-Mendez et al., 2013). This finding of iden-

tical somatic mutations in different tumors from the same patient indicates a

common embryological stem cell origin of the tumors. It also supports the

hypothesis that the early age of presentation of multifocal tumors for patients

with germline mutations of cluster 1 genes (i.e., VHL, SDHB, and SDHD)

might reflect second-hit deletions or point mutations early during develop-

ment before cells have migrated to their final location.


Based on the considerations in the preceding text, it would be of con-

siderable interest to establish whether mice overexpressing Hif2a (e.g., by

introduction of a nondegradable gene variant) have a higher tumor inci-

dence. Phd3 knockout mice exhibit mild hyperplasia in the adrenal, carotid

body, and superior cervical ganglia (Bishop et al., 2008). However, no

PHEOs or PGLs have been found in Sdhd or Sdhd/H19 knockout mice

(Bayley et al., 2009).

9. CONCLUSION

HIF2a signaling appears to be a central pathway in chromaffin cell

development and differentiation, with cells highly expressing HIF2aexhibiting a less differentiated and more stem cell-like phenotype. Recent

evidence suggests that HIF2a signaling in PHEO/PGL patients leads to

tumors with less mature catecholamine phenotypes that occur earlier in life

and are more often multifocal and potentially more aggressive than tumors

that do not display features of upregulated HIF2a signaling. This may indi-

cate a mutational second hit during fetal development, activating HIF2a (by

gene mutation or inhibition of degradation through VHL or SDHx inacti-

vation), which results in maintenance of a more undifferentiated phenotype.

Hence, HIF2a may be a useful biomarker for more aggressive disease.

Moreover, targeting HIF2a by small molecule inhibitors could be a valid

therapeutic strategy for PHEOs/PGLs of the cluster 1 type, especially since

inhibition of HIF2a in a cell line model of VHL-deficient RCC increased

cell death and sensitivity to radiation (Bertout et al., 2009).

CONFLICT OF INTERESTThe authors have no conflicts of interest to declare.

ACKNOWLEDGMENTSThis work has been supported by the Deutsche Forschungsgesellschaft (EI855/1-1).

REFERENCESAcker, T., Diez-Juan, A., Aragones, J., Tjwa, M., Brusselmans, K., Moons, L., et al. (2005).

Genetic evidence for a tumor suppressor role of HIF-2alpha. Cancer Cell, 8(2), 131–141.Adams, M. B., & McMillen, I. C. (2000). Actions of hypoxia on catecholamine synthetic

enzyme mRNA expression before and after development of adrenal innervation inthe sheep fetus. The Journal of Physiology, 529(Pt 3), 519–531.

Ahmad, A., Ahmad, S., Glover, L., Miller, S. M., Shannon, J. M., Guo, X., et al. (2009).Adenosine A2A receptor is a unique angiogenic target of HIF-2alpha in pulmonary

http://refhub.elsevier.com/B978-0-12-411512-5.00014-2/rf0005








endothelial cells. Proceedings of the National Academy of Sciences of the United States ofAmerica, 106(26), 10684–10689.

Amar, L., Baudin, E., Burnichon, N., Peyrard, S., Silvera, S., Bertherat, J., et al. (2007). Suc-cinate dehydrogenase B gene mutations predict survival in patients with malignant pheo-chromocytomas or paragangliomas. Journal of Clinical Endocrinology and Metabolism,92(10), 3822–3828.

Amar, L., Bertherat, J., Baudin, E., Ajzenberg, C., Bressac-de Paillerets, B., Chabre, O., et al.(2005). Genetic testing in pheochromocytoma or functional paraganglioma. Journal ofClinical Oncology, 23(34), 8812–8818.

Appelhoff,R. J., Tian,Y.M.,Raval,R.R.,Turley,H.,Harris, A. L., Pugh,C.W., et al. (2004).Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulationof hypoxia-inducible factor. Journal of Biological Chemistry, 279(37), 38458–38465.

Astuti, D., Hart-Holden, N., Latif, F., Lalloo, F., Black, G. C., Lim, C., et al. (2003). Geneticanalysis of mitochondrial complex II subunits SDHD, SDHB and SDHC in paragangliomaand phaeochromocytoma susceptibility. Clinical Endocrinology, 59(6), 728–733.

Bajou, K., Noel, A., Gerard, R. D., Masson, V., Brunner, N., Holst-Hansen, C., et al.(1998). Absence of host plasminogen activator inhibitor 1 prevents cancer invasionand vascularization. Nature Medicine, 4(8), 923–928.

Bayley, J. P., van Minderhout, I., Hogendoorn, P. C., Cornelisse, C. J., van der Wal, A.,Prins, F. A., et al. (2009). Sdhd and SDHD/H19 knockout mice do not develop para-ganglioma or pheochromocytoma. PLoS One, 4(11), e7987.

Baysal, B. E., Ferrell, R. E., Willett-Brozick, J. E., Lawrence, E. C., Myssiorek, D.,Bosch, A., et al. (2000). Mutations in SDHD, a mitochondrial complex II gene, in hered-itary paraganglioma. Science, 287(5454), 848–851.

Bertout, J. A., Majmundar, A. J., Gordan, J. D., Lam, J. C., Ditsworth, D., Keith, B., et al.(2009). HIF2alpha inhibition promotes p53 pathway activity, tumor cell death, and radi-ation responses. Proceedings of the National Academy of Sciences of the United States of America,106(34), 14391–14396.

Bishop, T., Gallagher, D., Pascual, A., Lygate, C. A., de Bono, J. P., Nicholls, L. G., et al.(2008). Abnormal sympathoadrenal development and systemic hypotension inPHD3�/� mice. Molecular and Cellular Biology, 28(10), 3386–3400.

Biswas, S., Troy, H., Leek, R., Chung, Y. L., Li, J. L., Raval, R. R., et al. (2010). Effects ofHIF-1alpha and HIF2alpha on growth and metabolism of clear-cell renal cell carcinoma786-0 xenografts. Journal of Oncology, 2010, 757908.

Blank, A., Schmitt, A. M., Korpershoek, E., van Nederveen, F., Rudolph, T., Weber, N.,et al. (2010). SDHB loss predicts malignancy in pheochromocytomas/sympathethicparagangliomas, but not through hypoxia signalling. Endocrine-Related Cancer, 17(4),919–928.

Bournaud, R., Hidalgo, J., Yu, H., Girard, E., & Shimahara, T. (2007). Catecholaminesecretion from rat foetal adrenal chromaffin cells and hypoxia sensitivity. Pflugers Archiv,454(1), 83–92.

Branco-Price, C., Zhang, N., Schnelle, M., Evans, C., Katschinski, D. M., Liao, D., et al.(2012). Endothelial cell HIF-1alpha and HIF-2alpha differentially regulate metastaticsuccess. Cancer Cell, 21(1), 52–65.

Brouwers, F. M., Eisenhofer, G., Tao, J. J., Kant, J. A., Adams, K. T., Linehan, W. M., et al.(2006). High frequency of SDHB germline mutations in patients with malignantcatecholamine-producing paragangliomas: Implications for genetic testing. Journal ofClinical Endocrinology and Metabolism, 91(11), 4505–4509.

Brouwers, F. M., Elkahloun, A. G., Munson, P. J., Eisenhofer, G., Barb, J., Linehan, W.M.,et al. (2006). Gene expression profiling of benign and malignant pheochromocytoma.Annals of the New York Academy of Sciences, 1073, 541–556.
























































Brown, S. T., Kelly, K. F., Daniel, J. M., & Nurse, C. A. (2009). Hypoxia inducible factor(HIF)-2 alpha is required for the development of the catecholaminergic phenotype ofsympathoadrenal cells. Journal of Neurochemistry, 110(2), 622–630.

Brown, S. T., Reyes, E. P., & Nurse, C. A. (2011). Chronic hypoxia upregulates adenosine 2areceptor expression in chromaffin cells via hypoxia inducible factor-2alpha: Role in mod-ulating secretion. Biochemical and Biophysical Research Communications, 412(3), 466–472.

Burnichon, N., Briere, J. J., Libe, R., Vescovo, L., Riviere, J., Tissier, F., et al. (2010). SDHAis a tumor suppressor gene causing paraganglioma. Human Molecular Genetics, 19(15),3011–3020.

Burnichon, N., Vescovo, L., Amar, L., Libe, R., de Reynies, A., Venisse, A., et al. (2011).Integrative genomic analysis reveals somatic mutations in pheochromocytoma and para-ganglioma. Human Molecular Genetics, 20(20), 3974–3985.

Carpenter, E., Hatton, C. J., & Peers, C. (2000). Effects of hypoxia and dithionite on cat-echolamine release from isolated type I cells of the rat carotid body. The Journal ofPhysiology, 523(Pt 3), 719–729.

Carroll, V. A., & Ashcroft, M. (2006). Role of hypoxia-inducible factor (HIF)-1alpha versusHIF-2alpha in the regulation of HIF target genes in response to hypoxia, insulin-likegrowth factor-I, or loss of von Hippel-Lindau function: Implications for targeting theHIF pathway. Cancer Research, 66(12), 6264–6270.

Casanova, S., Rosenberg-Bourgin, M., Farkas, D., Calmettes, C., Feingold, N.,Heshmati, H. M., et al. (1993). Phaeochromocytoma in multiple endocrine neoplasiatype 2 A: Survey of 100 cases. Clinical Endocrinology, 38(5), 531–537.

Cascon, A., Pita, G., Burnichon, N., Landa, I., Lopez-Jimenez, E., Montero-Conde, C.,et al. (2009). Genetics of pheochromocytoma and paraganglioma in Spanish patients.Journal of Clinical Endocrinology and Metabolism, 94(5), 1701–1705.

Cervera, A. M., Bayley, J. P., Devilee, P., & McCreath, K. J. (2009). Inhibition ofsuccinate dehydrogenase dysregulates histone modification in mammalian cells.MolecularCancer, 8, 89.

Chazaud, B., Ricoux, R., Christov, C., Plonquet, A., Gherardi, R. K., & Barlovatz-Meimon, G. (2002). Promigratory effect of plasminogen activator inhibitor-1 on inva-sive breast cancer cell populations. American Journal of Pathology, 160(1), 237–246.

Chen, H., Houshmand, G., Mishra, S., Fong, G. H., Gittes, G. K., & Esni, F. (2010).Impaired pancreatic development in Hif2-alpha deficient mice. Biochemical and Biophys-ical Research Communications, 399(3), 440–445.

Cheung, C. Y. (1989). Direct adrenal medullary catecholamine response to hypoxia in fetalsheep. Journal of Neurochemistry, 52(1), 148–153.

Comino-Mendez, I., de Cubas, A. A., Bernal, C., Alvarez-Escola, C., Sanchez-Malo, C.,Ramirez-Tortosa, C. L., et al. (2013). Tumoral EPAS1 (HIF2A) mutations explain spo-radic pheochromocytoma and paraganglioma in the absence of erythrocytosis. HumanMolecular Genetics, 22(11), 2169–2176.

Comino-Mendez, I., Gracia-Aznarez, F. J., Schiavi, F., Landa, I., Leandro-Garcia, L. J.,Leton, R., et al. (2011). Exome sequencing identifies MAX mutations as a cause ofhereditary pheochromocytoma. Nature Genetics, 43(7), 663–667.

Covello, K. L., Kehler, J., Yu, H., Gordan, J. D., Arsham, A. M., Hu, C. J., et al. (2006).HIF-2alpha regulates Oct-4: Effects of hypoxia on stem cell function, embryonic devel-opment, and tumor growth. Genes & Development, 20(5), 557–570.

Czyzyk-Krzeska, M. F., Bayliss, D. A., Lawson, E. E., &Millhorn, D. E. (1992). Regulationof tyrosine hydroxylase gene expression in the rat carotid body by hypoxia. Journal of Neu-rochemistry, 58(4), 1538–1546.

Czyzyk-Krzeska, M. F., Furnari, B. A., Lawson, E. E., & Millhorn, D. E. (1994). Hypoxiaincreases rate of transcription and stability of tyrosine hydroxylase mRNA in pheochro-mocytoma (PC12) cells. Journal of Biological Chemistry, 269(1), 760–764.






















































Dahia, P. L., Ross, K. N.,Wright,M. E., Hayashida, C. Y., Santagata, S., Barontini, M., et al.(2005). A HIF1alpha regulatory loop links hypoxia and mitochondrial signals in pheo-chromocytomas. PLoS Genetics, 1(1), 72–80.

Dang, C. V., Kim, J. W., Gao, P., & Yustein, J. (2008). The interplay between MYC andHIF in cancer. Nature Reviews. Cancer, 8(1), 51–56.

Dasgupta, B., Yi, Y., Chen, D. Y., Weber, J. D., & Gutmann, D. H. (2005). Proteomic anal-ysis reveals hyperactivation of the mammalian target of rapamycin pathway in neurofi-bromatosis 1-associated human and mouse brain tumors. Cancer Research, 65(7),2755–2760.

Dechant, G. (2002). Chat in the trophic web: NGF activates Ret by inter-RTK signaling.Neuron, 33(2), 156–158.

Dimsdale, J. E., Coy, T., Ziegler, M. G., Ancoli-Israel, S., & Clausen, J. (1995). The effect ofsleep apnea on plasma and urinary catecholamines. Sleep, 18(5), 377–381.

Donnelly, D. F. (2005). Development of carotid body/petrosal ganglion response to hypoxia.Respiratory Physiology & Neurobiology, 149(1–3), 191–199.

Donnelly, D. F., & Doyle, T. P. (1994). Developmental changes in hypoxia-induced cate-cholamine release from rat carotid body, in vitro. The Journal of Physiology, 475(2),267–275.

Ducsay, C. A., Hyatt, K., Mlynarczyk, M., Root, B. K., Kaushal, K. M., & Myers, D. A.(2007). Long-term hypoxia modulates expression of key genes regulatingadrenomedullary function in the late gestation ovine fetus. American Journal of Physiology.Regulatory, Integrative and Comparative Physiology, 293(5), R1997–R2005.

Eisenhofer, G., Huynh, T. T., Elkahloun, A., Morris, J. C., Bratslavsky, G., Linehan,W.M.,et al. (2008). Differential expression of the regulated catecholamine secretory pathway indifferent hereditary forms of pheochromocytoma. American Journal of Physiology, Endocri-nology and Metabolism, 295(5), E1223–E1233.

Eisenhofer, G., Huynh, T. T., Pacak, K., Brouwers, F.M.,Walther, M.M., Linehan,W.M.,et al. (2004). Distinct gene expression profiles in norepinephrine- and epinephrine-producing hereditary and sporadic pheochromocytomas: Activation of hypoxia-drivenangiogenic pathways in von Hippel-Lindau syndrome. Endocrine-Related Cancer, 11(4),897–911.

Eisenhofer, G., Lenders, J. W., Siegert, G., Bornstein, S. R., Friberg, P., Milosevic, D., et al.(2012). Plasma methoxytyramine: A novel biomarker of metastatic pheochromocytomaand paraganglioma in relation to established risk factors of tumour size, location andSDHB mutation status. European Journal of Cancer, 48(11), 1739–1749.

Eisenhofer, G., Pacak, K., Huynh, T. T., Qin, N., Bratslavsky, G., Linehan, W. M., et al.(2011). Catecholamine metabolomic and secretory phenotypes in phaeochromocytoma.Endocrine-Related Cancer, 18(1), 97–111.

Eisenhofer, G., Timmers, H. J., Lenders, J. W., Bornstein, S. R., Tiebel, O., Mannelli, M.,et al. (2011). Age at diagnosis of pheochromocytoma differs according to catecholaminephenotype and tumor location. Journal of Clinical Endocrinology and Metabolism, 96(2),375–384.

Eisenhofer, G., Walther, M. M., Huynh, T. T., Li, S. T., Bornstein, S. R., Vortmeyer, A.,et al. (2001). Pheochromocytomas in von Hippel-Lindau syndrome and multiple endo-crine neoplasia type 2 display distinct biochemical and clinical phenotypes. Journal ofClinical Endocrinology and Metabolism, 86(5), 1999–2008.

Estus, S., Zaks, W. J., Freeman, R. S., Gruda, M., Bravo, R., & Johnson, E. M., Jr. (1994).Altered gene expression in neurons during programmed cell death: Identification ofc-jun as necessary for neuronal apoptosis.The Journal ofCell Biology,127(6Pt 1), 1717–1727.

Evinger, M. J., Cikos, S., Nwafor-Anene, V., Powers, J. F., & Tischler, A. S. (2002). Hyp-oxia activates multiple transcriptional pathways in mouse pheochromocytoma cells.Annals of the New York Academy of Sciences, 971, 61–65.






















































Favier, J., Briere, J. J., Burnichon, N., Riviere, J., Vescovo, L., Benit, P., et al. (2009). TheWarburg effect is genetically determined in inherited pheochromocytomas. PLoS One,4(9), e7094.

Favier, J., Buffet, A., & Gimenez-Roqueplo, A. P. (2012). HIF2A mutations in para-ganglioma with polycythemia. The New England Journal of Medicine, 367(22), 2161,author reply 2161–2162.

Favier, J., Igaz, P., Burnichon, N., Amar, L., Libe, R., Badoual, C., et al. (2012). Rationalefor anti-angiogenic therapy in pheochromocytoma and paraganglioma. Endocrine Pathol-ogy, 23(1), 34–42.

Favier, J., Kempf, H., Corvol, P., & Gasc, J. M. (1999). Cloning and expression pattern ofEPAS1 in the chicken embryo. Colocalization with tyrosine hydroxylase. FEBS Letters,462(1–2), 19–24.

Fukuda, T., Ishii, K., Nanmoku, T., Isobe, K., Kawakami, Y., & Takekoshi, K. (2007).5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside stimulates tyrosine hydroxy-lase activity and catecholamine secretion by activation of AMP-activated protein kinase inPC12 cells. Journal of Neuroendocrinology, 19(8), 621–631.

Gamboa, A., Gamboa, J. L., Holmes, C., Sharabi, Y., Leon-Velarde, F., Fischman, G. J., et al.(2006). Plasma catecholamines and blood volume in native Andeans during hypoxia andnormoxia. Clinical Autonomic Research, 16(1), 40–45.

Giatromanolaki, A., Sivridis, E., Fiska, A., & Koukourakis, M. I. (2006). Hypoxia-induciblefactor-2 alpha (HIF-2 alpha) induces angiogenesis in breast carcinomas. Applied Immuno-histochemistry & Molecular Morphology, 14(1), 78–82.

Gimenez-Roqueplo, A. P., Favier, J., Rustin, P., Rieubland, C., Crespin, M., Nau, V., et al.(2003). Mutations in the SDHB gene are associated with extra-adrenal and/or malignantphaeochromocytomas. Cancer Research, 63(17), 5615–5621.

Gordan, J. D., Bertout, J. A., Hu, C. J., Diehl, J. A., & Simon, M. C. (2007). HIF-2alphapromotes hypoxic cell proliferation by enhancing c-myc transcriptional activity. CancerCell, 11(4), 335–347.

Gozal, E., Shah, Z. A., Pequignot, J. M., Pequignot, J., Sachleben, L. R., Czyzyk-Krzeska, M. F., et al. (2005). Tyrosine hydroxylase expression and activity in the ratbrain: Differential regulation after long-term intermittent or sustained hypoxia. Journalof Applied Physiology, 99(2), 642–649.

Gustafsson, M. V., Zheng, X., Pereira, T., Gradin, K., Jin, S., Lundkvist, J., et al. (2005).Hypoxia requires notch signaling to maintain the undifferentiated cell state.Developmen-tal Cell, 9(5), 617–628.

Guzy, R. D., Sharma, B., Bell, E., Chandel, N. S., & Schumacker, P. T. (2008). Loss of theSdhB, but Not the SdhA, subunit of complex II triggers reactive oxygen species-dependent hypoxia-inducible factor activation and tumorigenesis. Molecular and CellularBiology, 28(2), 718–731.

Ham, J., Babij, C.,Whitfield, J., Pfarr, C.M., Lallemand, D., Yaniv,M., et al. (1995). A c-Jundominant negative mutant protects sympathetic neurons against programmed cell death.Neuron, 14(5), 927–939.

Hao, H. X., Khalimonchuk, O., Schraders, M., Dephoure, N., Bayley, J. P., Kunst, H., et al.(2009). SDH5, a gene required for flavination of succinate dehydrogenase, is mutated inparaganglioma. Science, 325(5944), 1139–1142.

Holmquist-Mengelbier, L., Fredlund, E., Lofstedt, T., Noguera, R., Navarro, S.,Nilsson, H., et al. (2006). Recruitment of HIF-1alpha andHIF-2alpha to common targetgenes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressivephenotype. Cancer Cell, 10(5), 413–423.

Hu, C. J., Iyer, S., Sataur, A., Covello, K. L., Chodosh, L. A., & Simon, M. C. (2006). Dif-ferential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha





















































(HIF-1alpha) and HIF-2alpha in stem cells. Molecular and Cellular Biology, 26(9),3514–3526.

Hu, C. J., Wang, L. Y., Chodosh, L. A., Keith, B., & Simon, M. C. (2003). Differential rolesof hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hypoxic gene reg-ulation. Molecular and Cellular Biology, 23(24), 9361–9374.

Hui, A. S., Striet, J. B., Gudelsky, G., Soukhova, G. K., Gozal, E., Beitner-Johnson, D., et al.(2003). Regulation of catecholamines by sustained and intermittent hypoxia in neuro-endocrine cells and sympathetic neurons. Hypertension, 42(6), 1130–1136.

Imtiyaz, H. Z.,Williams, E. P., Hickey,M.M., Patel, S. A., Durham, A. C., Yuan, L. J., et al.(2010). Hypoxia-inducible factor 2alpha regulates macrophage function in mousemodels of acute and tumor inflammation. Journal of Clinical Investigation, 120(8),2699–2714.

Jogi, A., Ora, I., Nilsson, H., Lindeheim, A., Makino, Y., Poellinger, L., et al. (2002). Hyp-oxia alters gene expression in human neuroblastoma cells toward an immature and neuralcrest-like phenotype. Proceedings of the National Academy of Sciences of the United States ofAmerica, 99(10), 7021–7026.

Johansen, J. L., Sager, T. N., Lotharius, J., Witten, L., Mork, A., Egebjerg, J., et al. (2010).HIF prolyl hydroxylase inhibition increases cell viability and potentiates dopaminerelease in dopaminergic cells. Journal of Neurochemistry, 115(1), 209–219.

Kaelin,W. G., Jr., &Ratcliffe, P. J. (2008). Oxygen sensing bymetazoans: The central role ofthe HIF hydroxylase pathway. Molecular Cell, 30(4), 393–402.

Kanstrup, I. L., Poulsen, T. D., Hansen, J. M., Andersen, L. J., Bestle, M. H.,Christensen, N. J., et al. (1999). Blood pressure and plasma catecholamines in acuteand prolonged hypoxia: Effects of local hypothermia. Journal of Applied Physiology,87(6), 2053–2058.

Keith, B., Johnson, R. S., & Simon, M. C. (2012). HIF1alpha and HIF2alpha: Sibling rivalryin hypoxic tumour growth and progression. Nature Reviews. Cancer, 12(1), 9–22.

Kim, W. Y., Perera, S., Zhou, B., Carretero, J., Yeh, J. J., Heathcote, S. A., et al. (2009).HIF2alpha cooperates with RAS to promote lung tumorigenesis in mice. Journal ofClinical Investigation, 119(8), 2160–2170.

King, K. S., Prodanov, T., Kantorovich, V., Fojo, T., Hewitt, J. K., Zacharin, M., et al.(2011). Metastatic pheochromocytoma/paraganglioma related to primary tumor devel-opment in childhood or adolescence: Significant link to SDHB mutations. Journal ofClinical Oncology, 29(31), 4137–4142.

Kobayashi, K., Morita, S., Sawada, H., Mizuguchi, T., Yamada, K., Nagatsu, I., et al. (1995).Targeted disruption of the tyrosine hydroxylase locus results in severe catecholaminedepletion and perinatal lethality in mice. Journal of Biological Chemistry, 270(45),27235–27243.

Koh, M. Y., Darnay, B. G., & Powis, G. (2008). Hypoxia-associated factor, a novelE3-ubiquitin ligase, binds and ubiquitinates hypoxia-inducible factor 1alpha, leadingto its oxygen-independent degradation. Molecular and Cellular Biology, 28(23),7081–7095.

Koh, M. Y., Lemos, R., Jr., Liu, X., & Powis, G. (2011). The hypoxia-associated factorswitches cells fromHIF-1alpha- to HIF-2alpha-dependent signaling promoting stem cellcharacteristics, aggressive tumor growth and invasion. Cancer Research, 71(11),4015–4027.

Koh, M. Y., & Powis, G. (2012). Passing the baton: The HIF switch. Trends in BiochemicalSciences, 37(9), 364–372.

Kondo, K., Kim, W. Y., Lechpammer, M., & Kaelin, W. G., Jr. (2003). Inhibition ofHIF2alpha is sufficient to suppress pVHL-defective tumor growth. PLoS Biology, 1(3),E83.





















































Kumar, G. K., Overholt, J. L., Bright, G. R., Hui, K. Y., Lu, H., Gratzl, M., et al. (1998).Release of dopamine and norepinephrine by hypoxia from PC-12 cells. American Journalof Physiology, 274(6 Pt 1), C1592–C1600.

Kumar, G. K., Rai, V., Sharma, S. D., Ramakrishnan, D. P., Peng, Y. J., Souvannakitti, D.,et al. (2006). Chronic intermittent hypoxia induces hypoxia-evoked catecholamineefflux in adult rat adrenal medulla via oxidative stress. The Journal of Physiology,575(Pt 1), 229–239.

Lam, S. Y., Tipoe, G. L., Liong, E. C., & Fung, M. L. (2008). Differential expressions androles of hypoxia-inducible factor-1alpha, -2alpha and -3alpha in the rat carotid body dur-ing chronic and intermittent hypoxia. Histology and Histopathology, 23(3), 271–280.

Latif, F., Tory, K., Gnarra, J., Yao,M., Duh, F.M., Orcutt, M. L., et al. (1993). Identificationof the vonHippel-Lindau disease tumor suppressor gene. Science, 260(5112), 1317–1320.

Lee, S., Nakamura, E., Yang, H., Wei, W., Linggi, M. S., Sajan, M. P., et al. (2005). Neu-ronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytomagenes: Developmental culling and cancer. Cancer Cell, 8(2), 155–167.

Letouze, E., Martinelli, C., Loriot, C., Burnichon, N., Abermil, N., Ottolenghi, C., et al.(2013). SDHmutations establish a hypermethylator phenotype in paraganglioma. CancerCell, 23(6), 739–752.

Li, Z., Bao, S., Wu, Q., Wang, H., Eyler, C., Sathornsumetee, S., et al. (2009). Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell, 15(6),501–513.

Lipscomb, E. A., Sarmiere, P. D., Crowder, R. J., & Freeman, R. S. (1999). Expression of theSM-20 gene promotes death in nerve growth factor-dependent sympathetic neurons.Journal of Neurochemistry, 73(1), 429–432.

Lipscomb, E. A., Sarmiere, P. D., & Freeman, R. S. (2001). SM-20 is a novel mitochondrialprotein that causes caspase-dependent cell death in nerve growth factor-dependent neu-rons. Journal of Biological Chemistry, 276(7), 5085–5092.

Lomb, D. J., Desouza, L. A., Franklin, J. L., & Freeman, R. S. (2009). Prolyl hydroxylaseinhibitors depend on extracellular glucose and hypoxia-inducible factor (HIF)-2alphato inhibit cell death caused by nerve growth factor (NGF) deprivation: Evidence thatHIF-2alpha has a role in NGF-promoted survival of sympathetic neurons. MolecularPharmacology, 75(5), 1198–1209.

Lopez-Jimenez, E., Gomez-Lopez, G., Leandro-Garcia, L. J., Munoz, I., Schiavi, F.,Montero-Conde, C., et al. (2010). Research resource: Transcriptional profiling revealsdifferent pseudohypoxic signatures in SDHB and VHL-related pheochromocytomas.Molecular Endocrinology, 24(12), 2382–2391.

Lorenzo, F. R., Yang, C., Ng Tang Fui,M., Vankayalapati, H., Zhuang, Z., Huynh, T., et al.(2012). A novel EPAS1/HIF2A germline mutation in a congenital polycythemia withparaganglioma. J Mol Med (Berl), 91(4), 507–512.

Mamet, J., Peyronnet, J., Roux, J. C., Perrin, D., Cottet-Emard, J. M., Pequignot, J. M.,et al. (2002). Long-term prenatal hypoxia alters maturation of adrenal medulla in rat.Pediatric Research, 51(2), 207–214.

Mannelli, M., Castellano, M., Schiavi, F., Filetti, S., Giacche, M., Mori, L., et al. (2009).Clinically guided genetic screening in a large cohort of italian patients with pheochro-mocytomas and/or functional or nonfunctional paragangliomas. Journal of ClinicalEndocrinology and Metabolism, 94(5), 1541–1547.

Maranchie, J. K., Vasselli, J. R., Riss, J., Bonifacino, J. S., Linehan,W.M., & Klausner, R. D.(2002). The contribution of VHL substrate binding and HIF1-alpha to the phenotype ofVHL loss in renal cell carcinoma. Cancer Cell, 1(3), 247–255.

Mastrogiannaki, M., Matak, P., Keith, B., Simon, M. C., Vaulont, S., & Peyssonnaux, C.(2009). HIF-2alpha, but not HIF-1alpha, promotes iron absorption in mice. Journal ofClinical Investigation, 119(5), 1159–1166.






















































Mazumdar, J., Hickey,M.M., Pant, D. K., Durham, A. C., Sweet-Cordero, A., Vachani, A.,et al. (2010). HIF-2alpha deletion promotes Kras-driven lung tumor development. Pro-ceedings of the National Academy of Sciences of the United States of America, 107(32),14182–14187.

Mazumdar, J., O’Brien, W. T., Johnson, R. S., LaManna, J. C., Chavez, J. C., Klein, P. S.,et al. (2010). O2 regulates stem cells through Wnt/beta-catenin signalling. Nature CellBiology, 12(10), 1007–1013.

Mojet, M. H., Mills, E., & Duchen, M. R. (1997). Hypoxia-induced catecholamine secre-tion in isolated newborn rat adrenal chromaffin cells is mimicked by inhibition of mito-chondrial respiration. The Journal of Physiology, 504(Pt 1), 175–189.

Morrison, S. J., Csete, M., Groves, A. K., Melega, W., Wold, B., & Anderson, D. J. (2000).Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentia-tion by isolated neural crest stem cells. Journal of Neuroscience, 20(19), 7370–7376.

Mulligan, L.M., Kwok, J. B., Healey, C. S., Elsdon,M. J., Eng, C., Gardner, E., et al. (1993).Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type2A. Nature, 363(6428), 458–460.

Naranjo-Suarez, S., Castellanos, M. C., Alvarez-Tejado, M., Vara, A., Landazuri, M. O., &del Peso, L. (2003). Down-regulation of hypoxia-inducible factor-2 in PC12 cells bynerve growth factor stimulation. Journal of Biological Chemistry, 278(34), 31895–31901.

Neumann, H. P., Bausch, B., McWhinney, S. R., Bender, B. U., Gimm, O., Franke, G.,et al. (2002). Germ-line mutations in nonsyndromic pheochromocytoma. The NewEngland Journal of Medicine, 346(19), 1459–1466.

Niemann, S., & Muller, U. (2000). Mutations in SDHC cause autosomal dominant para-ganglioma, type 3. Nature Genetics, 26(3), 268–270.

Nilsson, H., Jogi, A., Beckman, S., Harris, A. L., Poellinger, L., & Pahlman, S. (2005). HIF-2alpha expression in human fetal paraganglia and neuroblastoma: Relation to sympa-thetic differentiation, glucose deficiency, and hypoxia. Experimental Cell Research,303(2), 447–456.

Noguera, R., Fredlund, E., Piqueras, M., Pietras, A., Beckman, S., Navarro, S., et al. (2009).HIF-1alpha and HIF-2alpha are differentially regulated in vivo in neuroblastoma: HighHIF-1alpha correlates negatively to advanced clinical stage and tumor vascularization.Clinical Cancer Research, 15(23), 7130–7136.

Nolting, S., & Grossman, A. B. (2012). Signaling pathways in pheochromocytomas andparagangliomas: Prospects for future therapies. Endocrine Pathology, 23(1), 21–33.

Pacak, K., Jochmanova, I., Prodanov, T., Yang, C., Merino, M., Fojo, T., et al. (2013).A new syndrome of paraganglioma and somatostatinoma associated with polycythemia.Journal of Clinical Oncology, 31(13), 1690–1698.

Padbury, J. F. (1989). Functional maturation of the adrenal medulla and peripheral sympa-thetic nervous system. Bailliere’s Clinical Endocrinology and Metabolism, 3(3), 689–705.

Paulick, R., Kastendieck, E., & Wernze, H. (1985). Catecholamines in arterial and venousumbilical blood: Placental extraction, correlation with fetal hypoxia, and transcutaneouspartial oxygen tension. Journal of Perinatal Medicine, 13(1), 31–42.

Peng, J., Zhang, L., Drysdale, L., & Fong, G. H. (2000). The transcription factor EPAS-1/hypoxia-inducible factor 2alpha plays an important role in vascular remodeling. Proceed-ings of the National Academy of Sciences of the United States of America, 97(15), 8386–8391.

Peterson, S., & Bogenmann, E. (2004). The RET and TRKA pathways collaborate to reg-ulate neuroblastoma differentiation. Oncogene, 23(1), 213–225.

Petrella, B. L., Lohi, J., & Brinckerhoff, C. E. (2005). Identification of membrane type-1matrix metalloproteinase as a target of hypoxia-inducible factor-2 alpha in vonHippel-Lindau renal cell carcinoma. Oncogene, 24(6), 1043–1052.

Phillippe, M. (1983). Fetal catecholamines. American Journal of Obstetrics and Gynecology,146(7), 840–855.






















































Pietras, A., Gisselsson, D., Ora, I., Noguera, R., Beckman, S., Navarro, S., et al. (2008). Highlevels of HIF-2alpha highlight an immature neural crest-like neuroblastoma cell cohortlocated in a perivascular niche. The Journal of Pathology, 214(4), 482–488.

Pietras, A., Hansford, L. M., Johnsson, A. S., Bridges, E., Sjolund, J., Gisselsson, D., et al.(2009). HIF-2alpha maintains an undifferentiated state in neural crest-like humanneuroblastoma tumor-initiating cells. Proceedings of the National Academy of Sciences ofthe United States of America, 106(39), 16805–16810.

Pietras, A., von Stedingk, K., Lindgren, D., Pahlman, S., & Axelson, H. (2011). JAG2 induc-tion in hypoxic tumor cells alters Notch signaling and enhances endothelial cell tube for-mation. Molecular Cancer Research, 9(5), 626–636.

Pollard, P. J., El-Bahrawy, M., Poulsom, R., Elia, G., Killick, P., Kelly, G., et al. (2006).Expression of HIF-1alpha, HIF-2alpha (EPAS1), and their target genes in paragangliomaand pheochromocytoma with VHL and SDH mutations. Journal of Clinical Endocrinologyand Metabolism, 91(11), 4593–4598.

Pomares, F. J., Canas, R., Rodriguez, J. M., Hernandez, A. M., Parrilla, P., & Tebar, F. J.(1998). Differences between sporadic and multiple endocrine neoplasia type 2A pha-eochromocytoma. Clinical Endocrinology, 48(2), 195–200.

Prabhakar, N. R., & Kumar, G. K. (2010). Mechanisms of sympathetic activation and bloodpressure elevation by intermittent hypoxia. Respiratory Physiology & Neurobiology,174(1–2), 156–161.

Qin, Y., Yao, L., King, E. E., Buddavarapu, K., Lenci, R. E., Chocron, E. S., et al. (2010).Germline mutations in TMEM127 confer susceptibility to pheochromocytoma. NatureGenetics, 42(3), 229–233.

Raghuraman, G., Prabhakar, N. R., & Kumar, G. K. (2012). Differential regulation of tyro-sine hydroxylase by continuous and intermittent hypoxia. Advances in ExperimentalMedicine and Biology, 758, 381–385.

Rankin, E. B., Biju, M. P., Liu, Q., Unger, T. L., Rha, J., Johnson, R. S., et al. (2007).Hypoxia-inducible factor-2 (HIF-2) regulates hepatic erythropoietin in vivo. Journalof Clinical Investigation, 117(4), 1068–1077.

Rankin, E. B., Rha, J., Unger, T. L., Wu, C. H., Shutt, H. P., Johnson, R. S., et al. (2008).Hypoxia-inducible factor-2 regulates vascular tumorigenesis in mice. Oncogene, 27(40),5354–5358.

Raval, R. R., Lau, K. W., Tran, M. G., Sowter, H. M., Mandriota, S. J., Li, J. L., et al.(2005). Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 invon Hippel-Lindau-associated renal cell carcinoma. Molecular and Cellular Biology,25(13), 5675–5686.

Ream, M. A., Chandra, R., Peavey, M., Ray, A. M., Roffler-Tarlov, S., Kim, H. G., et al.(2008). High oxygen prevents fetal lethality due to lack of catecholamines. American Jour-nal of Physiology. Regulatory, Integrative and Comparative Physiology, 295(3), R942–R953.

Ricketts, C. J., Forman, J. R., Rattenberry, E., Bradshaw, N., Lalloo, F., Izatt, L., et al.(2010). Tumor risks and genotype-phenotype-proteotype analysis in 358 patients withgermline mutations in SDHB and SDHD. Human Mutation, 31(1), 41–51.

Roda, J. M.,Wang, Y., Sumner, L. A., Phillips, G. S., Marsh, C. B., & Eubank, T. D. (2012).Stabilization of HIF-2alpha induces sVEGFR-1 production from tumor-associated mac-rophages and decreases tumor growth in a murine melanoma model. Journal of Immunol-ogy, 189(6), 3168–3177.

Sato, M., Tanaka, T., Maemura, K., Uchiyama, T., Sato, H., Maeno, T., et al. (2004). ThePAI-1 gene as a direct target of endothelial PAS domain protein-1 in adenocarcinomaA549 cells. American Journal of Respiratory Cell and Molecular Biology, 31(2), 209–215.

Schlingensiepen, K. H., Wollnik, F., Kunst, M., Schlingensiepen, R., Herdegen, T., &Brysch, W. (1994). The role of Jun transcription factor expression and phosphorylation





















































in neuronal differentiation, neuronal cell death, and plastic adaptations in vivo. Cellularand Molecular Neurobiology, 14(5), 487–505.

Schlisio, S., Kenchappa, R. S., Vredeveld, L. C., George, R. E., Stewart, R., Greulich, H.,et al. (2008). The kinesin KIF1Bbeta acts downstream from EglN3 to induce apoptosisand is a potential 1p36 tumor suppressor. Genes & Development, 22(7), 884–893.

Schmitt, P., Garcia, C., Soulier, V., Pujol, J. F., & Pequignot, J. M. (1992). Influence of long-term hypoxia on tyrosine hydroxylase in the rat carotid body and adrenal gland. Journal ofthe Autonomic Nervous System, 40(1), 13–19.

Schmitt, P., Pequignot, J., Hanchin, F., Pujol, J. F., & Pequignot, J. M. (1993). Regionalspecificity of long-term regulation of tyrosine hydroxylase in some catecholaminergicrat brainstem areas. II. Effect of a chronic dihydralazine treatment. Brain Research,611(1), 61–66.

Schnell, P. O., Ignacak, M. L., Bauer, A. L., Striet, J. B., Paulding, W. R., & Czyzyk-Krzeska, M. F. (2003). Regulation of tyrosine hydroxylase promoter activity by thevon Hippel-Lindau tumor suppressor protein and hypoxia-inducible transcription fac-tors. Journal of Neurochemistry, 85(2), 483–491.

Scortegagna, M., Ding, K., Zhang, Q., Oktay, Y., Bennett, M. J., Bennett, M., et al. (2005).HIF-2alpha regulates murine hematopoietic development in an erythropoietin-dependent manner. Blood, 105(8), 3133–3140.

Scortegagna, M., Morris, M. A., Oktay, Y., Bennett, M., & Garcia, J. A. (2003). The HIFfamily member EPAS1/HIF-2alpha is required for normal hematopoiesis in mice. Blood,102(5), 1634–1640.

Selak, M. A., Armour, S. M., MacKenzie, E. D., Boulahbel, H., Watson, D. G.,Mansfield, K. D., et al. (2005). Succinate links TCA cycle dysfunction to oncogenesisby inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell, 7(1), 77–85.

Skuli, N., Majmundar, A. J., Krock, B. L., Mesquita, R. C., Mathew, L. K., Quinn, Z. L.,et al. (2012). Endothelial HIF-2alpha regulates murine pathological angiogenesis andrevascularization processes. Journal of Clinical Investigation, 122(4), 1427–1443.

Smith, E. H., Janknecht, R., & Maher, L. J., 3rd. (2007). Succinate inhibition of alpha-ketoglutarate-dependent enzymes in a yeast model of paraganglioma. Human MolecularGenetics, 16(24), 3136–3148.

Souvannakitti, D., Nanduri, J., Yuan, G., Kumar, G. K., Fox, A. P., & Prabhakar, N. R.(2010). NADPH oxidase-dependent regulation of T-type Ca2þ channels and ryanodinereceptors mediate the augmented exocytosis of catecholamines from intermittenthypoxia-treated neonatal rat chromaffin cells. Journal of Neuroscience, 30(32),10763–10772.

Straub, J. A., Lipscomb, E. A., Yoshida, E. S., & Freeman, R. S. (2003). Induction of SM-20in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c inthe cytosol, and caspase-dependent cell death. Journal of Neurochemistry, 85(2), 318–328.

Takeda, N., O’Dea, E. L., Doedens, A., Kim, J. W., Weidemann, A., Stockmann, C., et al.(2010). Differential activation and antagonistic function of HIF-{alpha} isoforms in mac-rophages are essential for NO homeostasis. Genes & Development, 24(5), 491–501.

Talks, K. L., Turley, H., Gatter, K. C., Maxwell, P. H., Pugh, C. W., Ratcliffe, P. J., et al.(2000). The expression and distribution of the hypoxia-inducible factors HIF-1alpha andHIF-2alpha in normal human tissues, cancers, and tumor-associated macrophages. Amer-ican Journal of Pathology, 157(2), 411–421.

Tang, X., Mo, C., Wang, Y., Wei, D., & Xiao, H. (2013). Anti-tumour strategies aiming totarget tumour-associated macrophages. Immunology, 138(2), 93–104.

Taylor, S. C., Batten, T. F., & Peers, C. (1999). Hypoxic enhancement of quantal catechol-amine secretion. Evidence for the involvement of amyloid beta-peptides. Journal ofBiological Chemistry, 274(44), 31217–31222.























































Thomas, S. A., Matsumoto, A. M., & Palmiter, R. D. (1995). Noradrenaline is essential formouse fetal development. Nature, 374(6523), 643–646.

Tian, H., Hammer, R. E., Matsumoto, A. M., Russell, D. W., & McKnight, S. L. (1998).The hypoxia-responsive transcription factor EPAS1 is essential for catecholaminehomeostasis and protection against heart failure during embryonic development.Genes &Development, 12(21), 3320–3324.

Tsui-Pierchala, B. A., Milbrandt, J., & Johnson, E. M., Jr. (2002). NGF utilizes c-Ret via anovel GFL-independent, inter-RTK signaling mechanism to maintain the trophic statusof mature sympathetic neurons. Neuron, 33(2), 261–273.

Vanharanta, S., Shu, W., Brenet, F., Hakimi, A. A., Heguy, A., Viale, A., et al. (2013). Epi-genetic expansion of VHL-HIF signal output drives multiorgan metastasis in renal can-cer. Nature Medicine, 19(1), 50–56.

Viskochil, D., Buchberg, A. M., Xu, G., Cawthon, R. M., Stevens, J., Wolff, R. K., et al.(1990). Deletions and a translocation interrupt a cloned gene at the neurofibromatosistype 1 locus. Cell, 62(1), 187–192.

Waldmann, J., Fendrich, V., Holler, J., Buchholz, M., Heinmoller, E., Langer, P., et al.(2010). Microarray analysis reveals differential expression of benign and malignant pheo-chromocytoma. Endocrine-Related Cancer, 17(3), 743–756.

Walther, M. M., Herring, J., Enquist, E., Keiser, H. R., & Linehan, W. M. (1999). vonRecklinghausen’s disease and pheochromocytomas. Journal of Urology, 162(5),1582–1586.

Wang, X., & Schneider, A. (2010). HIF-2alpha-mediated activation of the epidermal growthfactor receptor potentiates head and neck cancer cell migration in response to hypoxia.Carcinogenesis, 31(7), 1202–1210.

Warnecke, C., Zaborowska, Z., Kurreck, J., Erdmann, V. A., Frei, U., Wiesener, M., et al.(2004). Differentiating the functional role of hypoxia-inducible factor (HIF)-1alpha andHIF-2alpha (EPAS-1) by the use of RNA interference: Erythropoietin is a HIF-2alphatarget gene in Hep3B and Kelly cells. The FASEB Journal, 18(12), 1462–1464.

Wong, D. L., Tai, T. C., Wong-Faull, D. C., Claycomb, R., Siddall, B. J., Bell, R. A., et al.(2010). Stress and adrenergic function: HIF1alpha, a potential regulatory switch. Cellularand Molecular Neurobiology, 30(8), 1451–1457.

Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., & Greenberg, M. E. (1995). Opposingeffects of ERK and JNK-p38 MAP kinases on apoptosis. Science, 270(5240), 1326–1331.

Xiao, M., Yang, H., Xu, W., Ma, S., Lin, H., Zhu, H., et al. (2012). Inhibition of alpha-KG-dependent histone and DNA demethylases by fumarate and succinate that are accu-mulated in mutations of FH and SDH tumor suppressors. Genes & Development, 26(12),1326–1338.

Yamashita, T., Ohneda, K., Nagano, M., Miyoshi, C., Kaneko, N., Miwa, Y., et al. (2008).Hypoxia-inducible transcription factor-2alpha in endothelial cells regulates tumorneovascularization through activation of ephrin A1. Journal of Biological Chemistry,283(27), 18926–18936.

Yang, S., Kim, J., Ryu, J. H., Oh, H., Chun, C. H., Kim, B. J., et al. (2010). Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction.Nature Medicine, 16(6), 687–693.

Yang, C., Sun, M. G., Matro, J., Huynh, T. T., Rahimpour, S., Prchal, J. T., et al. (2013).Novel HIF2A mutations disrupt oxygen sensing leading to polycythemia,paragangliomas and somatostatinomas. Blood, 121(13), 2563–2566.

Yuan, H. T., Khankin, E. V., Karumanchi, S. A., & Parikh, S. M. (2009). Angiopoietin 2 is apartial agonist/antagonist of Tie2 signaling in the endothelium. Molecular and CellularBiology, 29(8), 2011–2022.




















































Zhou, Q. Y., Quaife, C. J., & Palmiter, R. D. (1995). Targeted disruption of the tyrosinehydroxylase gene reveals that catecholamines are required for mouse fetal development.Nature, 374(6523), 640–643.

Zhuang, Z., Yang, C., Lorenzo, F., Merino, M., Fojo, T., Kebebew, E., et al. (2012).Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia. TheNew England Journal of Medicine, 367(10), 922–930.







[Advances in Pharmacology] A New Era of Catecholamines in the Laboratory and Clinic Volume 68 ||...

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