Sarcom a (1998) 2, 3± 17
RE VIEW
Progress in the m olecular biology of Ewing tum ors
HEIN RICH K OVAR
Children’ s Cancer R esearch Institute, Vienna, Austria
A bstract
Purpose/results/discussion . Rearrangem ent of the E W S gene with an ET S oncogene by chromosomal translocation is a
hallmark of the Ewing family of tumors (EFT). Detectability, incidence, tumor speci® city and variability of this aberration
have been m atters of intense investigation in recent years. A number of related alterations have also been found in other
m alignancies. The common consequence of these gene rearrangements is the generation of an aberrant transcription
factor. In EFT, the ETS partner is responsible for target recognition. However, synergistic and possibly tissue-restricted
transcription factors interacting with either the EWS or the ETS portion m ay in¯ uence target selection. M inimal domains
of both fusion partners were de® ned that have proved necessary for the in vitro transformation of murine ® broblasts. These
functional studies suggest a role for aberrant transcriptional regulation of transforming target genes by the chimeric
transcription factors. Also, fusion of the two unrelated protein domains may affect overall protein conformation and
consequently DNA binding speci® city. Recent evidence suggests that EWS, when fused to a transcription factor, interacts
with differen t partners than germ -line EWS. Variability in EWS± ETS gene fusions has recently been demonstrated to
correlate with clinical outcome. This ® nding may re¯ ect functional differences of the individual chimeric transcription
factors. Alternatively, type and availability of speci® c recom binases at different time-points of stem cell developm ent or in
differen t stem cell lineages may determine fusion type. Studies on EFT cell lines using EWS± ETS antagonists do suggest
a rate-limiting essential role for the gene rearrangement in the self-renewal capacity of EFT cells. The presence of
additional aberrations varying in number and type that may account for immortalization and full transform ation is
postulated. Knowledge about such secondary alterations may provide valuable prognostic m arkers that could be used for
treatment strati® cation.
K ey w ords: EWS , ETS , IGF1 , tum or suppressor, prognosis.
Introduction
Ew ing’ s sarcom a (ES), being a rare m alignant disease affe cting bone and soft tissue in children and
young adults, was hardly known to people other
than pediatric oncologists until the characterization
of a chim eric gene product presum ed to be causally
involved in the generation of this neoplasm . It dram atically gained attention w hen, from investigating
other m alignancies, it becam e apparent that the
ES-derived oncoprotein constitutes the prototype of
a whole class of aberrant proteins speci® cally asso ciated with certain tum or types. C onsequently, ES
m ay be considered a m odel system to study m alignant conversion on a subclinical level. T he discovery
of the ES-asso ciated gene rearrangement transiently
halted a controversy among pathologists about the
existence of distinct categories of ES (i.e. osseous
ES, extra-sk eletal ES, Askin tum or, peripheral
prim itive neuroectoderm al tum or) because it was
found to be expressed in all of them . Clinically,
however, there is a need for diversi® cation.
Although m ore than half of the patients can be
cured by m ultimodal therapy, one third of cases
with localized disease and about 80% of patients
presenting with m etastases succum b to the disease
(for a recent review, see K ovar et al. 1 ). Current
treatment protocols have largely com pensated for
classical prognostic m arkers such as tumor volum e
and localization of the prim ary except for the rather
unfavo rable presence of metastases at diagnosis. It is
likely, therefore, that biological differences exist
between so far incurable aggressive disease and clinically m anageable localized disease inexplicable by
the m ere presence of the ES-assoc iated gene
rearrangement. W hile Ewing’ s tum or research has
focused on the clinical exploitability and the function of the ES-spe ci® c gene rearrangem ent since its
discovery in 1992, this review will also consider
extensively the role of additional m olecular aberrations in the search for useful prognostic m arkers.
N eoplastic transform ation and m etastatic spread is
com m only believed to result from a m ulti-step pro-
Correspondence to: H. Kovar, Children’ s Cancer Research Institute (C CRI), St Anna Kinderspital, Kinderspitalgasse 6, A-1090 Vienna,
Austria. T el: 1 43 1 40470 ext, 409; Fax: 1 43 1 4087230; E-m ail: [email protected].
1357-714 X/98/040003± 15
Ó
1998 Carfax Publishing Ltd
4
H. Kovar
cess. In this context, the ES-spe ci® c gene
rearrangement obviously constitutes a rate-lim iting
event. According to Knudson’ s legendary tw o-hit
hypothesis, at least one additional aberration
should be present in a Ewing tum or. It is possible
that this second hit is less speci® c and affects different genes at different times during developm ent
of the enigmatic Ewing tumor stem cell, thus
de® ning distinct subcategories of the disease.
C onsequently, Ew ing tum or research is slowly
m oving towards m olecular subclassi® cation and
staging.
D iagnostic tools
In 1988, the cytogenetic translocation t(11;22)
(q24;q12) was described as speci® cally asso ciated
w ith histopathologically diagnosed ES and peripheral prim itive neuroectoderm al tum or (pPN ET). 2
T he presence of this aberration in a largely undifferentiated small round cell tum or of childhood
turned out to be a form idable diagnostic m arker. 3,4
H ow ever, cytogenetic analysis was restricted to
tum or cells with at least lim ited in vitro proliferation potential. T he generation of an antibody,
H BA71, 5 speci® cally reacting with the surface
glycoprotein encoded by the M IC2 gene, 6 which
w as found to be abundantly present in tum or cells
carrying a chrom osom e 22q12 aberration, 7,8
enlarged the spectrum of diagnostic tools. H owever, em bryonal rhabdomyosarcom as, asterocytom as, neuroendocrine tum ors and carcinom as
occasionally stained positive with HBA71 9 and,
w hen using a m ore sensitive antibody (12E7 10 ),
high level expression of this antigen was also noted
in early hem atopoietic precursor cells 11 and several
lymphom as. 12 The characterization of the ES
break-po int regions on chrom osom es 22 and 11 13
and the subsequent cloning of a chim eric cD N A
resulting from a gene fusion between a novel gene,
designated E W S, and the E TS transcription factor
gene FLI1 14 allowed for sensitive detection of
tum or cells carrying a 11;22 translocation even
in sm all sam ples of fresh, frozen or paraf® nem bedded m aterial by m eans of reverse transcriptase polym erase chain reaction (RT -PC R). 15± 18
Subsequently, several alternative fusion partners for
E W S from the ETS oncogene fam ily were
identi® ed in ES and pPN ET cases 19± 24 U sing the
RT -PC R method, combined with genom ic analysis
of the EW S break-point region, on a large series cf
osseous and extra-ske letal ES (including Askin
tum ors and pP N ET, designated Ewing fam ily of
tum ors (EFT )) as opposed to several unrelated
sm all round cell tum ors, the speci® city of the
E W S± ETS gene rearrangement and the correlation
w ith high M IC2 expression w as con® rm ed. 16± 25
Recently, however, the lim itation of this aberration
to typical EF T m em bers has been questioned since
RT -PC R am pli® able EW S± FLI1 fusion transcripts
have been reported in childhood soft tissue sarcom as with m ixed phenotype, 26± 27 in som e olfactory
neuroblastom as 28 which have previously been
show n not to express MIC2, 29 and in two cases of
classical M IC2-negative neuroblastom a. 30 In the
absence of any cytogenetic evidence for a t(11;22)
in neuroblastoma the latter ® nding needs to
be independently con® rm ed. O n the other hand,
RT -PCR failed to dem onstrate the presence of
chim eric EW S transcripts in roughly 5% of histologically classi® ed EFT . Sceptics m ight raise their
® ngers and recall all the potential pitfalls of using
RT -PCR including the m ethod’ s inherent susceptibility to cross-contam ination as a single tool in
the diagnosis of histopathologically am biguous
cases of sm all round cell tum ors. Intriguing questions, i.e. if EW S± ETS gene rearrangements can
occur outside the EF T and if `atypical’ ES exist,
can only be assessed by the use of com plem entary
techniques allowing for the visualization of the
EW S gene rearrangem ent on a single cell level. It
has already been demonstrated that ¯ uorescent in
situ hyb ridization (FISH) using cosm ids ¯ anking
the EF T break-po int regions is not restricted to
m etaphase chrom osom es, but is also feasible to
detect the gene rearrangement ef® ciently on interphase nuclei 31,32 (H attinger et al., unpublished).
Alternatively, antibodies to unique dom ains of the
chim eric gene product could allow the routine
pathologist to screen for the EW S rearran gem ent
by standard im m unohistochem ical m ethods. T he
author and others 125, 12 6 have recently obtained
prelim inary indirect evidence from protein interaction studies that an am ino terminal E W S dom ain,
w hich appears to be inaccessible in germ -line
E W S, m ight be speci® cally exposed on the surface
of the chim eric product (see below). O ne could
endeavor, therefore, to generate an agent that distinctly recognizes the altered conform ation of the
E W S portion present in EW S fusion proteins. T he
hinge region of E W S± E T S chim eric proteins displays a high d egree of variab ility du e to variable
break-p oint locations in the genes contributing to
the translocation. So far, ® ve altern ative ETS fam ily m em bers have been found in E W S gene rearrangem ents. T herefore, antibodies to the lin ker
dom ain of fused partners would be of only lim ited
use in routine diagnosis. For the an alysis of RT PC R negative `atypical’ E FT and for sm all round
cell tum ors with a diagnosis other than EF T but
RT -PC R positive for an EW S chim eric transcript,
it is strongly recom m ended to con® rm the m olecular diagnosis by the dem onstration of an EW S
aberration on either D N A level (FISH or Southern
blot) or on RN A level by N orthern blotting.
Presently, it cannot be excluded that using these
approaches, follow ed by re® ned cloning procedures, fu rther E TS fam ily m em bers will be
identi® ed as alternative fusion partners for E W S in
E FT or non-E FT .
M olecular biology of E wing tum ors
5
F ig. 1. G enera tion of chim eric oncoproteins inv olving an EW S fam ily m em ber and a transcription factor. Protein dom ains presum ably
inv olved in RN A binding (RG G boxes and RN P m otif) are replaced by the DNA binding portion of the transcription factor. The m inim al
dom ains of the fusion partners present in all chim eras are the carboxy term ina l dom ain (CTD) of the E W S family m em ber and the
D NA binding dom ain (D BD) of the transcription factor. The carboxy term inal transactivation dom ain (C TA) of the transcription
factor is lost in fusions of E TS family mem bers but not of CH O P .
The EW S± ETS gene rearra ngem ent
The EW S gene family
E W S is the prototype of a grow ing fam ily of putative RN A-binding proteins including T LS (translocated in liposarcom a)/FU S, 33± 35 hT AF II68 (TAT A
box binding protein asso ciated factor), 36 the sm all
nu clear ribon uclear protein(snRN P)-asso ciated 69kD a protein, 37 the bovin e Pigpen protein 38 an d
D rosophila cabeza/SARF H (sarcom a associated
R N A bindin g ¯ y hom ologue), 39,40 that share distinct structural characteristics such as a conserved
R N A binding m otif ¯ anked by arginine± glycine±
glycine (RG G ) boxes 41 and a putative zinc-® nger
dom ain in the carboxy terminus. T his portion is
replaced by the D N A binding dom ain of a transcription factor in the oncogenic E W S and T L S
fu sion protein s. T he am ino term inus is rich in
glutam ine and prolin e residues. A s such, it resem bles the activation dom ain of certain transcription
fa ctors such as SP-1. 42 In EW S, this N -term inal
dom ain (N T D ), w hich is encod ed by the ® rst seven
exons, 43 is com prised of 30 copies of a repeated
degenerate peptide of 7± 12 residues rich in
tyrosine, serine, threonine, glycine and glutam in e 44
(Fig. 1). T LS w as identi® ed as a heterogenous
nu clear ribon ucleoprotein (hnRN P) in nonspliceosom al com plexes on m R N A continuously
shuttling betw een the nucleus and the cytoplasm 45
and SA RFH w as found to be asso ciated w ith
regions of the D rosophila chrom atin transcribed by
R N A polym erase II. C onsistent w ith a role of E W S
fam ily m em bers in gene transcription, hT A F II 68,
T L S and E W S have been identi® ed in subpopu lations of the general transcription factor T F IID . 126
H owever, recent evidence suggests that the oncogenic derivatives of T L S an d E W S are not stably
associated w ith the RN A polym erase II com plex
and T F IID . 12 6 Accum ulation in nuclear inclusions
such as the coiled bod y and the nucleolus have
been reported for Pigpen, 46 the 69-kD a snRN Passociated protein, 37 and, after transcriptional inhibition, for T L S. 47 Such nuclear subcom partm ents
m ight either constitute the site of norm al function
of these EW S-related proteins or serve as their
reservoir. Interestingly, on coproteins that contain
the am ino terminal dom ain of EW S or T LS are also
targeted to the sam e structure 47 . So far, the functional relevance of this ® nding is com pletely
unknown.
A role for EW S and its partner genes in determining the
EFT phenotyp e
Figure 2 sum m arizes all known gene fusions involving either EW S or TLS in human malignancies. In
an N IH 3T3 transform ation study, the type of transcription factor contributing to the chim eric gene
product determined cell m orphology. 35 T his observation m ight in part explain w hy only m em bers of
the ET S transcription factor fam ily are found in
gene fusions with EW S associated w ith an EFT
phenotype. H ow ever, w hile EW S and TLS am ino
termini appear to be functionally interchangable
6
H. Kovar
w hen fused to the transcription factor CHO P in the
in vitro m odel,35 as well as in m yxoid chondrosarcom a, 33,34,48 T LS has never been found to replace
EW S in EF T. In contrast, fusion of T LS to the ET S
fam ily m ember ERG, which is involved in 10% of
EF T, has been reported for poor prognosis, t(l6;21)
positive, acute m yeloid leukem ia. 49± 51 Rearrangem ent of E W S with other transcription factor genes
such as ATF1, the W ilm s’ tum or gene W T1 and the
nuclear receptor C HN /TEC have been shown to be
asso ciated w ith m alignant m elanom a of soft parts,
desm oplastic sm all round cell tumor and myxoid
chondrosarcom a, respectively. 52± 55 T hus, it is the
speci® c com bination of EW S w ith a subset of ETS
transcription factor genes and/or a particular stem
cell in which these genes are sensitive to illegitimate
recom bination that determ ine the EFT phenotype.
Accessibility to rearrangement by an as yet
unde® ned recom binase m ight also determine the
incidence of EFT . Z ucm an et al. reported recently
that sequence analysis of the entire EW S intron 6
region close to the m ajor break-point region in EFT
from Caucasian origin revealed a very high density
of Alu elements resulting from repeated retroposition during evolution. 56 T he Alu fam ily of short
interspersed repetitive D N A elements has previously
been dem onstrated to be frequently involved in
hum an gene rearrangements. 57 T his region was
found to be reduced by 50% due to deletion in the
African population. This inter-ethnic polymorphism
in the EW S gene is accom panied by a striking
difference in the incidence of EF T between populations of European and African origin. 58,59 It should
be noted that the m ajority of EW S genomic breakpoints occur in intron 7 and that intron 6 is, in fact,
never directly rearranged in EF T. So far, only three
E W S genom ic break-po ints have been sequenced,
two in EF T and one in a desm oplastic sm all round
cell tum or, 60± 62 , none of which contained Alu elem ents in the im mediate vicinity of the rearrangem ent sites. T hus, direct proof for the involvem ent of
Alu elements in E W S translocation is not availab le.
In the published cases, the lack of uniform ity of
sequences affe cted by the gene rearrangem ent does
not allow the identi® cation of a speci® c recom binase
responsible for the translocation. Chromosom e 22
alteration m ay occur as the only cytogenetically
visible aberration in EF T suggesting that the EW S±
E TS gene rearrangem ent is not the consequence of
a general genomic destabilization. However, the frequent involvem ent of m ore than two chrom osom es
in complex chrom osom e 22 aberrations and evidence for deletion of considerable am ounts of
sequences from the directly involved genes on the
untranscribed counterpart of the derivative chrom osom e 22 60 im ply a complex m echanism for gene
rearrangement in EFT. In addition, w hile EW S and
FLI1 are equally oriented on the long arm s of chrom osom es 22 and 11 from the centrom ere to the
telom ere allow ing for sim ple reciprocal transloca-
tion, the E RG gene is oriented in the opposite
direction. Consequently, EW S± ERG gene fusions
m ay result from either interstitial deletion/insertion
m echanism s 63 or from com plex genom ic rearrangem ents
involving
additional
chrom osom es. 24
Although a high variability in EW S fusion partners
and genom ic break-point locations has been noted,
rearrangement of EW S intron 7 w ith intron 5 or 4 of
the ETS fam ily gene FLI1 predom inates (about
80% of EF T cases). 16,18,24 Interestingly, the only
known three cases of fusion between EW S and the
ETS transcription factor gene FE V involve E W S
intron 10 which is otherw ise affe cted in only 9% of
EFT . Since, as outlined later, the m inim al portions
of EW S and its fusion partners contained in all
EFT -derived oncoproteins and required for full in
vitro transfo rm ation and transcription activation
function are signi® cantly sm aller than the portions
present in the m ost frequently observed EW S
fusions, it is unlikely that in EFT rearrangement
sites are determined by functional constraints only.
Rather, genom ic structure and accessibility m ight
direct illegitimate recom bination to speci® c regions
in the involved genes. G enom ic accessibility and
availability of recom binases m ight vary during the
developm ent of speci® c stem cell lineages. Therefore, it cannot be excluded that the different E W S
rearrangements de® ne different histogenetic starting
points of EF T developm ent. This m odel would
provide an intriguing explanation for our recent
observation of prognostic differences in EFT correlating with different EW S fusion types. 18 Alternatively, the various chim eric oncoproteins m ight
display functional differences. Since the com plete
EW S and FLI1 genes have been cloned and
sequence inform ation is readily availab le,64± 65 the
description of m ore genom ic rearrangement points
in EFT will hopefully throw m ore light on the
m echanism of gene rearrangem ent in this disease.
The ETS partner in the E W S fusion gene
T he ET S transcription factor fam ily currently
counts m ore than 30 mem bers. It is characterized by
the presence of a unique D N A binding domain
which is highly conserved from ¯ ies to hum ans and
has been ® rst described for the viral oncogene v-ets
of avian erythroblastosis virus E 26 (E twentysix
speci® c). Several E TS subfam ilies can be de® ned on
the basis of evolutionary sequence conservation. In
ET , 95% of cases show EW S fusion to the FLI1
(Friend leukemia virus integration site 1)/ERG
(ET S related gene) subfam ily of transcriptional activators. 16,18 These two gene products share, in
addition to alm ost identical D N A binding dom ains,
a 16-am ino acid stretch im m ediately upstream of
the ETS dom ain which is 100% conserved betw een
X enopus and humans, suggesting an im portant but
as yet unidenti® ed functional role. 66 T his portion is
retained in alm ost all EW S± FLI1 and EW S± ERG
M olecular biology of E wing tum ors
F ig. 2. Tum or-speci® c rearrangem ents betw een an EWS
family gene and a transcription factor gene .
fusions, w hile the genuine F LI1 and ERG transactivation dom ain is alw ays replaced by the EW S am ino
term inus resulting in a potentiation of transcriptional activation properties. 67± 69 The 85-am ino acid
D N A binding domain folds into three helices and
a four-stranded û sheet (w inged helix-turn-helix
m otif), 70± 72 m ost frequently refered to as `the ET S
dom ain’ . In som e ETS fam ily m embers, this
dom ain is ¯ anked by auto-inhibitory a helical structures that fold back and interact with the ET S
dom ain. 73 Structural studies on m urine ET S1 suggest that upon speci® c binding to D N A a conform ational change takes place that m ight expose distinct
portions of the ET S dom ain and its ¯ anking regions
for interactions with other proteins. 74 D N A bindingdependent complex formation with other transcription factors m ediated by the ET S dom ain and
additional residues has been reported for several
ET S fam ily m embers including GABP ~ , ELK1,
SAP1, Pu1, ETS1 and ETS2. 75 As dem onstrated
for ET S1, w hen binding to the speci® c recognition
sequence, intercalation of a tryptophan into the
m inor groove induces a sharp kink and a w idening
of D N A that m ight facilitate synergistic binding of
other regulatory proteins. 76 Since alm ost all ET S
proteins bind to a (G /C)(A/C )GG A(A/T)T consensus m otif, 75,77 synergy with other transcription factors m ight determ ine target speci® city of the
individual ET S fam ily mem bers. Interestingly,
7
ET S1, G ABP a , and F LI1 have recently been
demonstrated to bind to the transcription factor
PAX 5 in vitro. 78 A ¯ anking PAX5 binding site
allowed for ET S1 binding to an im perfect ET S
recognition m otif indicating that cooperativity m ight
change ET S binding speci® city to som e extent.
Som e ET S proteins, including ETS1, ETS2, F LI1,
ERG , G ABP a and TEL, share a further conserved
region ¯ anking the am ino terminal transactivation
dom ain, refered to as the `pointed or B-dom ain’ .
T his structure has been show n to de® ne a speci® c
oligom erization interface. For T EL, it governs
hom otypic aggregation. 79 N o interaction partner has
been de® ned for FLI1 and ERG so far. The conserved fold of the am ino terminal dom ain of ET S
proteins is, however, likely to underlie a conserved
function. T hus, replacem ent of this portion by the
EW S am ino term inus may alter not only quantitatively but also qualitatively the transcriptional activation properties of these two ETS fam ily m em bers
by placing them into a different protein context. In
addition, several ET S fam ily m embers have been
demonstrated to be a target for the RAS/RAF M AP
kinase signaling pathway. For ET S1, ras regulation
involves phospho rylation of residues within the
am ino terminal `pointed’ dom ain. So far, no link
between this signalling pathw ay and FLI1 or ERG
has been reported. Consequently, it rem ains unclear
if fusion to EW S w ill uncouple FLI1/ERG target
gene regulation from extracellular signaling.
In about 1% of ET cases, EW S is rearranged with
FEV (® fth Ew ing’ s tum or varian t) on chrom osom e
2. 21 T his ETS fam ily m ember displays 90% identity
to FLI1 in the D N A binding dom ain but the F LI1and E RG -sp eci® c ¯ anking 16 am ino acid sequence
is m issing in this protein. Interestingly, F EV also
lacks an am ino terminal transactivation dom ain present in m ost other ETS fam ily m embers. Instead, it
carries a long C-term inus w hich, because of the
presence of abundant alanine residues, m ight serve
as a putative repressor dom ain. H owever, experim ental proof for such an activity is not yet available. Since this portion is retained in the EW S
fusion, it rem ains to be established if the gene
rearrangement w ould result in a functional conversion to an activator. Interestingly, germ -line FE V
cD N A has been cloned from an E W S± FLI1 expressing Ew ing tum or cell line, indicating coexpression of
the two genes w ithin the sam e cell. If E W S± FLI1
and FEV target the sam e genes and FEV operates as
a repressor, it is possible that the EFT gene
rearrangement results in the release of these genes
from transcriptional inhibition by com petitive binding.
So far, three cases of suspected EF T have been
reported in w hich EW S w as fused to ETS fam ily
genes of a different subclass on chromosom es 7p22
and 17q21, ETV1 (ETS translocation varian t 1) and
E1AF (Adeno virus E1A enhancer binding factor),
the putative hum an hom ologues of m ouse ER81 and
F ig. 3. Com parison of DNA -binding domains of ETS proteins rearranged or coexpressed in E wing’ s tum ors. *: detectable only by RT-PCR;
underlined: positions hom ologous to am ino acids making contact to DNA in the PU .1 E TS-fam ily m em ber .
·
:identical am ino acids;
8
H. Kovar
M olecular biology of E wing tum ors
19,20,23
M ost notably, the D N A
PEA3, respectively.
binding dom ains of these tw o transcription factors
diverge from F LI1, ERG and FEV by 38% including the third a -helix that contacts the central core of
ET S binding sequences. D o EW S± ET V1 and
EW S± E1AF chim eric transcription factors target the
sam e genes as EW S± FLI1, EW S± ERG and EW S±
F EV? By a subtractive cloning strategy for genes
differentially expressed in EW S± FLI1-transfo rm ed
and
FLI1-transfected
untransfo rmed
m urine
® broblasts (NIH3T 3), several potential EW S± FLI1speci® c target genes were identi® ed. 80 Am ong them
w ere the m urine hom ologue of cytochrom e P-450
F 1, cytokeratine 15, a novel SH 2 dom ain containing
protein, EAT 2 (EW S± FLI1 activated transcript 2) 81
and the strom elysin gene. Strom elysin is a m atrix
m etalloproteinase involved in m etastatic invasio n.
Previously, E1AF has been demonstrated to regulate the stromelysin gene. In transfection experim ents, E1AF was suf® cient to confer an invasive
phenotype to non-m etastatic hum an breast cancer
cells (M CF7) 82 and antisense RN A to E1AF was
able to revert it in a squam ous cell carcinom a cell
line.83 Strom elysin has also been demonstrated to be
activated by ETS2, a m ember of another ET S subfam ily. 84 However, coexpression of ERG, w hich also
strongly binds to the same prom oter but is by itself
unable to activate it, resulted in inhibition of ET S2m ediated strom elysin gene activation. 85 T hus, different ET S fam ily m embers, irrespective of their
subclass, app ear to com pete for binding to speci® c
target genes. It is likely, therefore, that target selectivity and the speci® c m ode of gene regulation by
the EW S chim eric ETS transcription factors
strongly depends on the cellular background. A
spectrum of ET S-related gene products coexpressed
w ith EW S± FLI1 in EFT cell lines by am pli® cation
of ETS D N A binding dom ain encoding cD N As has
recently been de® ned using degenerate prim ers. 86
Am ong them, E TS2, E4TF1-60, ELK , ELF1, the
putative hum an hom ologue of ER71, and a novel
gene product ELF R w ere identi® ed. 127 N one of the
ET S fam ily m embers involved in EFT -spe ci® c gene
rearrangements w ere found to be expressed in their
germ -line con® guration. T his assay, however, m ay
have m issed low level expression of som e ET S fam ily m embers (i.e. FEV). As show n in Fig. 3 com paring the D N A binding dom ains of the ET S gene
products alternatively fused to EW S in EFT with
those found to be coexpressed with the chim eric
transcription factors, ELK is the m ost likely ET Srelated gene product that m ight interfere with the
EF T fusion proteins in target site selection because
the D N A-contacting third a -h elix of the ET S
dom ain is identical to that of FLI1, ERG and FEV.
EL K is one of several alternative ternary com plex
factors regulating a num ber of growth factor
inducible genes. 87 In fact, EW S± F LI1 can replace
EL K within the ternary com plex form ed on the
serum response elem ent of the cfos and the EGR1
9
promoters.88,89 In contrast to germ -line FLI1, binding of EW S± FLI1 to the serum response element
did not require interaction with the serum response
factor SRF. T ernary com plex form ation by FLI1
and EW S± FLI1 was m ediated by a dom ain preceding the D N A binding dom ain and present in the
m ajority of EFT -derived EW S± FLI1 fusions that
show lim ited sim ilarity to the ELK1± SRF interaction dom ain. H ow ever, no hom ologous structure
can be identi® ed in ERG and evidence for EW S±
ERG involvem ent in ternary com plex form ation is
not available.
In sum m ary, current know ledge about norm al
and aberrant ET S proteins suggest a num ber of
interesting candidate target genes for the EF Tspeci® c chim eric transcription factors, potentially
involved in the regulation of cell growth, signaling
and m etastasis, as revealed by the study of heterologous cellular system s such as m urine
® broblasts. H owever, am ple evidence exists that
ET S transcription factor action is largely context
speci® c. F or EFT , the cell of origin rem ains a
m atter of speculation. Because of lim ited neural
differentiation potential, EFT is considered as derived from the neuroectoderm . 90 Interestingly,
X enopus FLI1 has been shown to be expressed in a
restrictive pattern during embryogenesis evocative of
neural crest cell invasio n. 66 It is therefore speculated
that FLI1 m ight be involved in neural differentiation
in the context of these early stem cells. If so,
unscheduled activation of F LI-responsive genes by
the EFT -sp eci® c EW S fusion proteins in an undifferentiated cell possibly unrelated to the neural crest
m ight result in lim ited neural differentiation of the
EFT stem cell depending on the degree of determ ination achieved at the time of gene rearrangement. 1
T his m odel could explain the variable degree of
neuroectoderm al m arker expression in ES and
pP N ET as well as the occurrence of biphenotypic
tum ors. 26
The rate-lim iting (® rst) hit in EFT pathogenesis
EW S± ETS gene rearrangements are the only genetic
aberrations that have so far been identi® ed as highly
associated w ith histologically diagnosed EFT. This
association is the only available compelling argum ent that EW S± ETS gene rearrangements can be
rate-limiting for tum origenesis. Although com plem entary experim ental evidence supports this
assu m ption, the m echanism of m alignant transfo rm ation by these chimeric oncoproteins rem ains elusive. The best studied biological m odel for the
pathogenic role of inappropriately activated FLI1 is
Friend m urine leukemia virus(F -M uL V)-induced
erythroleukem ia. Insertional activation of the FLI1
gene appears to be the ® rst detectable genetic
change asso ciated w ith this disease. T he association
between the detection of FLI1 rearrangement and
clonal outgrowth of erythroleukem ia cells suggests
10
H. Kovar
that the activation of this transcription factor m ay be
affe cting the self-renewal potential of the infected
erythroid progenitors. However, leukem ogenesis
proceeds in m ultiple steps and additional aberrations affe cting viab ility of cells (e.g. inactivation of
the tum or suppressor gene p53) can be observed in
F -M uLV induced erythroleukem ia. 91,92 In contrast
to ERG , 93 norm al FLI1 was reported to be unable
to transfo rm m urine ® broblasts (NIH3T 3) while
expression of an EW S fusion protein resulted in
pronounced anchorage-independent clonogenicity
of N IH 3T3 cells. 94,95 H owever, rat embryo
® broblasts and som e m urine ® broblast subclones
w ere resistant to EW S± FLI1-mediated transform ation. These ® ndings again suggest that the oncogenic potential of norm al and aberrant FLI/ERG
ET S subfam ily m embers m ay depend on a cell
type-sp eci® c availability of relevant synergistic factors and possib ly on the presence of additional
aberrations. N IH3T 3 transfection studies with various recom binant EW S± F LI1 deletion m utants
revealed a dependence of transfo rm ation on both
the EW S portion and the ETS dom ain. 95 However,
optim al transactivation potential m ediated by the 30
EW S am ino term inal degenerate repeats included in
almost all EFT -derived fusion proteins was dispensable for m axim al focus form ation of transfected
N IH3T 3 in soft agar. T he m inim al EW S dom ain
required to transform m urine ® broblasts was delineated to the ® rst 82 am ino acids. 94 Recently, evidence obtained shows that w ithin the EW S± FLI1
fusion protein, but not within germ -line EW S, this
peptide directly contacts a com ponent of the RN A
polym erase II complex, RP B7, and that this interaction is suf® cient to drive EW S± FLI1-m ediated
reporter gene transactivation.125 Interactions of fulllength EW S with the general transcription factor
T FIID , an essential com ponent of the transcriptional preinitiation com plex, w ere absent from EW S
fusion proteins.126 It is therefore possible that fusion
of the EW S amino terminus to the FLI1 D N A
binding dom ain alters the protein conform ation and
directly recruits RN A polym erase II to F LI1 target
genes. Since RPB7 displays sim ilarities to prokaryotic sigm a factors, it m ight be involved in EFTspeci® c target site selection. Further protein± protein
interactions presum ably occurring downstream of
the 82 am ino acids m ight be required for ef® cient
gene regulation w ithin the EFT context. In
addition, using the yeast two-hybrid protein interaction trap, further candidate proteins not directly
related to transcription regulation were identi® ed
that interacted with the 82 am ino acids long m inim al transfo rm ation dom ain. T hese interactions
aw ait detailed characterization.
Since no tissue of EFT origin has been identi® ed
so far, transfo rm ation studies of authentic EFT stem
cells cannot be perform ed. Alternatively, several
investigators have used EW S± F LI1 antagonists
(antisense RN A expression vectors, antisense
oligonucleotides, dom inant negative proteins) to
m odulate expression of the chim eric oncoprotein in
EFT cell lines. 86,96± 98 T hese studies revealed a
growth inhibitory and anti-tum origenic effect of
these agents. Reduction in cell growth appeared to
result from cell cycle arrest and not from reduced
tum or cell viability. Recently, EW S± FLI1-m ediated
transform ation of m urine ® broblasts was demonstrated to require the presence of a functional
insulin-like growth factor-1 (IGF 1) receptor. 99
Interestingly, consistent expression of IG F1 and its
receptor was previously reported for EFT and IGF1
was demonstrated to act as a potent growth factor
for EFT cells in the absence of serum . l00± l03 This
cytokine appears to regulate negatively several
m echanism s of program m ed cell death at a far
dow nstream step. 104 It has been show n that inhibition of the IG F1 autoregulatory circuit by antiIGF1 receptor antibodies resulted in increased
apoptosis and reduced tumorigenicity of EFT
cells. l03 T aken together, the EW S± ETS gene
rearrangement appears to be involved in the aberrant self-renewal capacity of E FT cells but m ight
not be suf® cient to guarantee survival of initiated
tum or cells. However, as demonstrated recently,
there m ight still be som e role for F LI1, ERG and
their E W S fusions to play in the protection from
stress (i.e. calcium ionophore and serum deprivation)-induced cell death. 105
The second hit
Assum ing that the EW S± ETS gene rearrangement is
able to initiate EFT pathogenesis but is not
suf® cient to generate m alignant transfo rm ation, the
presence of additional m utations m ust be postulated. These aberrations m ight not necessarily be
tum or speci® c but m ay display inter-individual variation that could account for variations in EF T phenotype as w ell as in clinical behavio r. Also, they m ay
determ ine differentiation capacity, invasive potential
and treatm ent resistance. Consequently, while from
a clinical point of view the EW S± ETS gene
rearrangement provides a valuable diagnostic tumor
m arker, knowledge about the nature of additional
aberrations in EFT m ay assist subclassi® cation and
provide prognostic tools. Since studies on facultative
genetic anomalies in EF T have been largely
neglected since the discovery of the E W S± ETS gene
rearrangements, enhanced efforts to de® ne the m ultitude of additional aberrations are warranted for the
bene® t of patients.
C lues from cytogenetics
In three independent reviews of
inform ative EF T cases, non-random
structural chrom osom al aberrations
to occur with variable frequencies in
cytogenetically
num erical and
were reported
addition to the
M olecular biology of E wing tum ors
tum or speci® c t(11;22)(q24;q12) 106± 107 (H attinger et
al., unpublish ed). T hese include trisom y 8 in about
50% of cases frequently coupled with trisom y 12
occurring in roughly 20% , and a derivative chrom osom e 16 as a result of an unbalanced t(1;16) in 18%
of EF T. In rarer cases, other aneuploidies have
been identi® ed. Structural chrom osom e 1 aberrations that either result in gains of chrom osom e
1q21± 22 or relative losses of the short arm of chrom osom e 1, a frequent alteration in neuroblastom a
and other neuroectoderm al tum ors, have also been
observed in EFT (Hattinger et al., unpublished).
Interestingly, this chrom osomal region harbours a
gene encoding a protein (p73) that is structurally
and functionally related to the tum or suppressor
p53, a transcription factor involved in the regulation
of cell growth and apoptosis, and frequently inactivated during the progression of m any tum ors. 108,109
W hile research currently focuses on the role of
p73 for neuroblastom a pathogenesis, its relevance
for a subset of EFT is som ething that has to be
explored.
In general, excluding chrom osom e 22q12 translocations, num erical chrom osome changes are the
m ost frequent cytogenetic ® ndings in EF T. T hese
are likely to affect gene dosage. However, no candidate genes that could prom ote EW S± ETS-initiated
EF T pathogenesis when expressed at aberrant levels
have been identi® ed so far. Also, inform ation on
genes affe cted by the recurrent chrom osom e 16 and
1 structural alterations is not available yet. Frequently, these cytogenetic alterations occur in only a
subpopulation of neoplastic cells within the tum or
suggesting that they m ay be associated with late
stages of tumor progression.
The role of non-speci® c cancer genes
In the absence of recurrent candidate progressionasso ciated genetic alterations identi® able in EFT by
the means of cytogenetics, work has focused on the
analysis of m utations generally associated w ith a
broad range of hum an m alignancies. Genes investigated during the last years include the oncogenes
ras, cmyc and M DM 2, the tumor suppressors p53,
p16, and Rb, the metatasis-asso ciated splice
varian ts of the CD 44 adhesion m olecule, and
the tum or-speci® c m etastasis suppressor gene
110± 113,127
N one of the studied oncogenes
nm 23H1.
w as found to be altered by m utation or in expression
although occasional low level am pli® cation of
M DM 2 has been reported in an independent study
on a sim ilar sized cohort of EFT patients. 111 N either
m utation nor differences in expression levels of the
nucleotide
diphosph ate-kinase
were
nm 23H1
observed irrespective of the disease extension. 112
O nly standard C D44 expression was detectable in
EF T. 127 In contrast, hom ozygous deletions of the
p16 tum or suppressor was identi® ed in about onethird of prim ary EFT sam ples. This ® nding was
11
surprising since chrom osom al aberrations of band
9q21 containing the p16 gene were not reported
before suggesting a high-freq uency of m icrodeletions. Expression studies on EFT cell lines suggested that the frequency of p16 inactivation m ight
be even higher since post-transcriptional gene
silencing w as observed in several cases. 113 p16 acts
as an inhibitor of the cyclin D 1/cyclin-dependent
kinase 4 (CD K 4) com plex that inactivates the cell
cycle inhibitor pRb by phosph orylation. Inactivation
of p16 should com prom ise the G1 cell cycle checkpoint. Over-expression of either cyclin D 1 or
C D K4, or loss of pRb function, is believed to m ediate a sim ilar effect.114 In fact, we observed frequent
cyclin D 1 over-expression as well as variable CD K4
abundancy in EFT cell lines and loss of Rb in one
case. How ever, expression data from prim ary tumor
m aterial are not available yet. In addition, low level
C DK4 am pli® cation w as previously reported for two
of 30 EFT sam ples. 111
In virally induced m alignancy, G1 check-point
control is frequently com promised by concom ittant
inactivation of the pRb and p53 pathways. In
addition, F-M uLV-induced erythroleukem ia involves not only the activation of the FLI1 oncogene
as a rate-lim iting step but also m utation of p53. T he
author and others have, therefore, investigated the
status and expression of p53 and related genes in
EFT . 110,115 T he frequency of p53 m utations in prim ary tumors w as found to be low er than 10% as
opposed to a m ean frequency of 40± 60% in m ost
hum an m alignancies. In contrast, in about half of
EFT cell lines, the p53 gene was mutated and
show ed loss of heterozygosity. Com pariso n of p53
gene status between cell lines and the respective
prim ary tum ors of origin, w hen available, demonstrated that the observed increase in m utation frequency was due to selection and was not acquired
during in vitro expansion of tumor cells. T his result
suggested that p53 m utation m ight release EFT cells
from som e in vivo grow th or survival factor dependency. Transient transfection and over-expression of
wildtype p53 in cell lines with endogenous m utant
or wildtype gene status demonstrated frequent but
variable reduction in apoptotic responsiveness, 116
suggesting the presence of som e as yet unidenti® ed
cell-protective m echanism in EF T cell lines. Prelim inary expression analysis of m embers of the cell
death regulatory Bcl2 gene fam ily did not reveal any
signi® cant variations between individual EFT cell
lines (our unpublished observations). Previously,
high levels and activity of poly(AD P-rib ose) polym erase, a nuclear enzym e that participates in DN A
replication, repair and the triggering of apoptosis
induced by D N A strand breaks, have been reported
for som e EFT cell lines. 117 Sensitivity of EFT cell
lines to D N A dam aging agents (etoposide, actinom ycin D , X-rays) varied considerably in a m anner
independent from p53 responsiveness and endogenous p53 gene status suggesting that com plete
12
H. Kovar
m utational or partial inhibition of the p53 apoptosis pathway in EFT cell lines plays a role different
from radio- and chemosensitivity. However, the
physiolo gical signals that stim ulate p53-dependent
cell death have not been de® ned so far.
M olecular markers of prognosis
As a result of variable break-po int localization in
the involved genes, EW S± ETS gene products vary
considerably in size. M ost fusions include EW S
exons 1 to 7 (89% ) and FLI1 exons 6 to 9 (54% ).
E W S/FLI1 exon 7/6 fusions (type 1) predominate
independent of the disease extension (51% ). In
about one-third of EFT , FLI1 exon 5 is included
into the chim eric gene product, m ost frequently
joined to EW S exon 7 (type 2) (27%). In rare
cases, the chrom osom al translocation results in the
inclusion of EW S exons 9 (1%) or 9 plus 10 (10% )
or FLI1 exon 4 (1% ). In about 3% of cases, FLI1
exon 6 or exon 6 plus 7 are m issing from the gene
fusion. T his variability has prom pted us to investigate a possib le prognostic im pact of the gene fusion
type. The study, perform ed on 55 patients with
localized disease and 30 patients with metastases at
diagnosis, treated according to the European Intergroup Coordinated Ewing’ s Sarcom a Studies
(C ESS 86 and EIC ESS 92), revealed a signi® cantly
better outcom e for patients with localized disease
carrying a type 1 EW S± FLI1 expressing tum or as
com pared to non-type 1 cases. 18 A recent update
1
after a m edian observation time of 3 2 years
con® rm ed this result (Zoubek et al., unpublished).
In addition, an independent Am erican study perform ed on a sim ilar sized cohort of patients after a
m edian follow up of 31 m onths, using a sim ilar
treatment regimen, not only supported our ® ndings
but also identi® ed the EW S± ETS gene fusion type
as a prognostic m arker independent from the presence of m etastases at diagnosis in a m ulti-variate
analysis. 128 About 55% of the `non-type 1’ group in
the two studies w ere com prised of type 2 gene
fusions. Because of the low incidence of the individual `other-gene’ fusion types, no distinction has
been m ade betw een various non-type 1 subgroups
so far. In the absence of a biological explanation
for the observed prognostic differences, large collaborative prospective studies are warranted to
highlight the speci® c chim eric m olecules and the
protein dom ains associated w ith adverse patients’
outcom e.
Still, about 20% of patients with localized
tum ors and m ore than half of the patients with
m etastases succum b from the disease despite the
expression of a type 1 EW S± FLI1 gene fusion, suggesting the existence of additional adverse factors.
Com pariso n of pl6 gene status and clinical
course of 23 EFT patients analyzed so far suggested an adverse prognosis asso ciated w ith this
aberration independent from the extension of the
disease. 113 T hese results, w hich have not been subjected to statistical analysis, m ust be considered as
prelim inary since patients’ num bers in the study
were sm all and the median observation period did
not exceed 2 years. Retrospective im m unohistochem ical analysis of biopsy m aterial from a large
num ber of patients will help to clarify the prognostic relevance of a disrupted pRb cell cycle regulatory pathw ay in EFT . Also, m utation of p53 m ight
be linked to an adverse outcom e, since none of the
three EFT patients from our series carrying such
an aberration survived. H ow ever, because of the
rarity of this alteration it cannot serve as a useful
prognostic m arker.
A prognostic relevance for EFT of the observed
num erical and structural cytogenetic changes has
not been demonstrated with con® dence due to low
sample num bers in the studies perform ed so far.
M ost recently, deletion at 1p36, occurring in 6/22
localized EFT, was discussed as being asso ciated
with unfavo rable outcom e in this group (Hattinger
et al., unpublish ed).
In the absence of reliable m olecular m arkers to
predict outcom e in EFT , the presence of clinically
overt m etastases at diagnosis is com m only considered as the only prognostic criterion that is used
for treatment strati® cation. The EW S± ETS gene
rearrangement as a tum or cell speci® c m arker
detectable by the highly sensitive RT -PCR m ethod
provides a powerful m eans for the detection of
m inute num bers of circulating tum or cells that m ay
be the source of clinically occult m icrom etastases. 118,119 However, except shortly after surgical
intervention, 120 m obilization of PCR detectable
am ounts of tum or cells ( . 1/10 6 ) into the bloodstream has rarely been observed. In contrast, tumor
cells were detected at diagnosis by this m ethod in
the bone m arrow of 30% of patients with localized
disease, 50% of cases with isolated lung m etastases
and all patients w ith bone m etastases. 120,121 In a
prelim inary series of 23 patients lacking clinically
overt dissem ination, RT -PC R screening for bone
m arrow involvem ent did not allow the prediction of
early relapse after a m edian observation time of 30
m onths. It is, however, necessary to recall several
factors that m ay affect tum or cell detection in the
bone m arrow by RT -PC R: (1) tum or cell
in® ltration m ay be focal and bone m arrow aspiration m ay m iss these sites, (2) bone m arrow aspirates m ay contain variable am ounts of diluting
blood resulting in insuf® cient sensitivity, (3) prim ary EFT cells m ay differ in vitality, although
diluted tumor cells from cell lines can be detected
in blood sam ples even after 48 h at 4 C, 119 and
(4) bone m arrow in® ltrating tum or cells m ay be in
a resting state and express lower levels of chim eric
EW S RN A than proliferating tumor cells. RT -PCR
m easures RN A quantity rather than tum or cell
abundance. In a recent study, up to 10-fold variations in the content of chim eric EW S± ETS tran-
°
M olecular biology of E wing tum ors
scripts between individual EFT cell lines have been
reported. 122 M oreover, germ -line EW S expression in
T -cells has been demonstrated to depend on the
proliferative activity. 123 Since the EFT -sp eci® c chrom osom al rearrangement places the chim eric gene
under the control of the EW S regulatory sequences,
it is possible that EW S± ETS gene expression m ay
also vary. Consequently, detectability of EW S± ETS
chim eric transcripts does not necessarily re¯ ect true
tum or cell content. Even if RT-PCR studies fail to
dem onstrate signi® cance of positive blood or bone
m arrow screening results for relapse in patients with
localized disease, the question of prognostic relevance of tum or cell in® ltration rem ains unsolved.
T o assess this problem , im m unohistochem ical studies m ay prove to be superior to RT -PC R. W hile
M IC2 m ay serve as a valuable surface marker in
tum or diagnosis, its exploitability for tum or cell
detection in hem atopoietic tissue is lim ited. 124 T he
recently discovered expression of gastrin-releasing
peptide (GRP) by all EFT cell lines and about half
of the prim ary tumors tested (Lawlor et al., unpublished) m ay provide a m arker that, in conjunction
w ith M IC2, m ay allow the identi® cation of EFT
cells in blood and bone m arrow w ith increased
speci® city. T he study of E W S± FLI1 transcriptional
targets m ay result in the identi® cation of tum or
cell-restricted im m unohistochem ical m arkers. In
order to detect positively staining cells with very low
abundance on a routine basis, automated m icroscopic screening and consequently sophisticated
technical equipm ent is w arranted.
C onclusions
In this review, I have sum m arized evidence for the
im portance of studying EFT-spe ci® c genetic alterations in an authentic cellular background. Since the
histogenesis of EF T is still enigm atic and no experim ental evidence for E W S± FLI-m ediated tum origenesis has been reported from transgenic mouse
m odels so far, EF T cell lines rem ain the only available system for such investigations. In this institution, cell lines could be established from 12 EFT
patients with well docum ented clinical course. If a
cell line could be expanded from the prim ary tumor,
all subsequent tum or sam ples also gave rise to a cell
line. All but one patient died from the disease
suggesting that establishing a cell line selects for
patients with adverse prognosis. In fact, non-type 1
E W S± ETS gene fusions, p16 deletions and p53
m utations were clearly increased in EF T cell lines
from 23 patients investigated. They m ay, therefore,
represent the m ost therapy-resistant subpopulation
of tumor cells despite variable in vitro sensitivity to
cytotoxic agents. T hus, EFT cell lines m ay serve as
a pool for the identi® cation of putative bad prognostic m arkers. However, only large cooperative clinical
studies and m ultivariate statistical analysis will
help to address the question: how far can the
13
identi® cation of such m arkers translate into clinically useful criteria for treatm ent strati® cation?
W hen com paring a wide spectrum of EF T-derived
cell lines for marker expression and response to
either differentiation inducing agents, growth factors
or cytotoxic compounds, an im m ense variability was
observed. C onsequently, in order to sort out the
biological defects com m on to all E FT, a large panel
of genetically well de® ned cell lines w ill have to be
investigated. In the long term , such studies w ill
result in the identi® cation of EW S± ETS-sp eci® c
target genes and in a m ore detailed knowledge of the
m echanism of m alignant conversion of the enigm atic E FT precursor cell. H opefully, for the bene® t
of the patients, this knowledge will potentially provide novel targets for therapeutic intervention.
A cknowledgem ents
I thank D rs Enrique de Alava, Peter Am bros, D ave
Aryee, M arc Ladanyi, Poul Sorensen, Laszlo Tora
and Jeff T oretsky for perm itting m e to refer to their
before publication results.
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