
N-
myc
oncogene overexpression down-regulates leukemia inhibitory
factor in neuroblastoma
Elissavet Hatzi
1
, Carol Murphy
1,2
, Andreas Zoephel
3
, Horst Ahorn,
3
Ulrike Tontsch
3
, Ana-Maria Bamberger
4
,
Keiko Yamauchi-Takihara
5
, Lothar Schweigerer
6
and Theodore Fotsis
1
1
Laboratory of Biological Chemistry, Medical School, University of Ioannina, Greece;
2
Biomedical Research Institute,
Ioannina, Greece;
3
Boehringer Ingelheim Austria GmbH, Vienna, Austria;
4
Institute of Pathology, Department of Gynecophathology,
University Hospital Hamburg Eppendorf, Hamburg, Germany;
5
Department of Molecular Medicine, Osaka University Graduate
School of Medicine, Suita, Japan;
6
Abt. Ha
¨matologie, Onkologie und Endokrinologie, Universita
¨ts-Kinderklinik Essen, Germany
Amplification of N-myc oncogene is a frequent event in
advanced stages of human neuroblastoma and correlates
with poor prognosis and enhanced neovascularization.
Angiogenesis is an indispensable prerequisite for the pro-
gression and metastasis of solid malignancies, which is
modulated by tumor suppressors and oncogenes. We have
addressed the possibility that N-myc oncogene might regu-
late angiogenesis in neuroblastoma. Here, we report that
experimental N-Myc overexpression results in down-regu-
lation of leukemia inhibitory factor (LIF), a modulator of
endothelial cell proliferation. Reporter assays using the LIF
promoter and a series of N-Myc mutants clearly demon-
strated that down-regulation of the LIF promoter was
independent of Myc/Max interaction and required a
contiguous N-terminal N-Myc domain. STAT3, a down-
stream signal transducer, was essential for LIF activity as
infection with adenoviruses expressing a phosphorylation-
deficient STAT3 mutant rendered endothelial cells insensi-
tive to the antiproliferative action of LIF. LIF did not
influence neuroblastoma cell proliferation suggesting that, at
least in the context of neuroblastoma, LIF is involved in
paracrine rather than autocrine interactions. Our data shed
light on the mechanisms by which N-myc oncogene ampli-
fication enhances the malignant phenotype in neuroblas-
toma.
Keywords:N-myc; LIF; STAT3; endothelial cell; neuro-
blastoma.
The N-myc proto-oncogene encodes a 64-kDa nucleopro-
tein (N-Myc) which associates with a 21- to 22-kDa Max
protein to form N-Myc/Max heterodimers [1]. These dimers
can bind to the E-box consensus sequence (CACGTG) in
the promoter regions of target genes [2], including alpha
prothymosin and ornithine decarboxylase, eventually
inducing their up-regulation [3]. The physiological functions
of N-Myc have remained elusive although there is evidence
for developmentally important activities [4–6]. In the neural
crest, enhanced N-Myc expression may facilitate prolifera-
tion of immature neuronal precursor cells at the expense of
differentiation [7,8].
N-myc is implicated in the pathogenesis of neural crest-
derived tumors including neuroblastoma [9], the most
frequent solid malignancy of infants. Amplification of
N-myc oncogene is a frequent event in advanced stages (III
and IV) of human neuroblastoma [10] and correlates with
poor prognosis [11]. In fact, N-myc amplification is nearly
exclusively observed in neuroblastoma. N-myc amplifica-
tion results in high N-Myc protein levels that could perturb
the finely tuned interplay of N-Myc and Max and eventually
induce abnormal expression patterns of target genes [1].
However, few N-Myc target genes have been identified so
far [1]. A number of data indicate that some of the target
genes might modulate the cardiovascular system. Indeed,
whereas N-myc knockout mice die in utero [12,13], com-
pound heterozygotes suffer from serious heart defects [6].
Moreover, N-myc may also regulate the growth of tumor
vessels as neuroblastoma with N-myc amplification exhibit
enhanced neovascularization [14] suggesting that N-Myc
oncogene could stimulate tumor angiogenesis and thereby
enhance neuroblastoma progression. Indeed, stable trans-
fection and 100-fold overexpression of N-Myc in a neuro-
blastoma cell line (SH-EP) resulted in an enhanced
malignant phenotype of the transfectants (WAC2) and the
ability to form well vascularized tumors in nude mice [15].
As tumor angiogenesis derives from an imbalance
between angiogenic factors and inhibitors [16,17], it is
conceivable that the genetic changes of cancer could initiate
angiogenesis by disturbing this balance in the tumor vicinity.
Indeed, normal p53 regulates the expression of the angio-
genesis inhibitors thrombospondin [18] and glioma-derived
angiogenesis inhibitory factor [19]. Also, activation of
oncogenes, such as ras, has been shown to cause both
up-regulation of the expression of angiogenesis stimulators,
such as basic fibroblast growth factor (bFGF) or vascular
endothelial growth factor (VEGF), and down-regulation of
angiogenesis inhibitors such as tissue inhibitor of matrix
metalloproteinases (TIMP) and thrombospondin [20].
Towards the aim of identifying N-myc-regulated mole-
cules modulating neuroblastoma tumor angiogenesis, we
Correspondence to T. Fotsis, Laboratory of Biological Chemistry,
Medical School, University of Ioannina, 45110 Ioannina, Greece.
Tel.: + 30 65197560, Fax: + 30 65197868,
E-mail: thfotsis@cc.uoi.gr
Abbreviations: LIF, leukemia inhibitory factor; bFGF, basic fibroblast
growth factor; VEGF, vascular endothelial growth factor; TIMP,
tissue inhibitor of matrix metalloproteinases.
(Received 20 February 2002, revised 30 May 2002,
accepted 21 June 2002)
Eur. J. Biochem. 269, 3732–3741 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03066.x