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Molecular Oncology, Markers, Clinical Correlates |
Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and platelet-derived growth factor (PDGF)] by semiquantitative
RT-PCR in 37 NB primary tumors and in 22 NB cell lines. We also
analyzed the relationship between angiogenic factor expression and
clinicopathological factors as well as patient survival. All eight
angiogenic factors examined were expressed at various levels in NB cell
lines and tumors, suggesting their involvement in NB angiogenesis. The
expression levels of most angiogenic factors were correlated with each
other, suggesting their synergy in regulating the angiogenic process.
Significantly higher expression levels of VEGF, VEGF-B, VEGF-C, basic
fibroblast growth factor, Ang-2, transforming growth factor , and
PDGF-A (P < 0.0001–0.026) were found in
advanced-stage tumors (stages 3 and 4) compared with low-stage tumors
(stages 1, 2, and 4S). Expression of PDGF-A was significantly
associated with patient survival (P = 0.04). The
redundancy in angiogenic factor expression suggests that inhibition of
VEGF bioactivity alone might not be a sufficient approach for
antiangiogenic therapy of human NB. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, PDGF, Ang-1, and Ang-2 have been shown to induce angiogenesis
in a variety of experimental models (4, 5, 6, 7, 8, 9, 10, 11)
. VEGF, also known as vascular permeability factor, is an important angiogenic agent and endothelial-specific mitogen, which has been implicated in the neovascularization of a wide variety of tumors (12, 13, 14, 15, 16) . VEGF acts via a paracrine mechanism mainly through two specific receptors on the surface of endothelial cells: Flt-1 and KDR (17 , 18) . Although encoded by a single gene, VEGF has several isoforms generated by alternative splicing (19 , 20) . Of these, the main isoforms, VEGF121 and VEGF165, are secreted soluble glycoproteins, whereas VEGF189 and VEGF206 remain bound to heparan sulfate proteoglycans at the cell surface (21) . The importance of VEGF as a potential target for antineoplastic therapy has been demonstrated in several studies in which neutralizing antibodies to VEGF inhibited tumor growth and vascularization in vivo (22 , 23) .
VEGF-B and VEGF-C are two recently discovered members of the VEGF family (24, 25, 26) , which are expressed in many tissues and have mitogenic and/or chemotactic actions on endothelial cells, indicating that they may also contribute to the induction or maintenance of angiogenesis. VEGF-C was discovered as the ligand for the third member of the VEGF receptor family (Flt-4, or VEGF receptor 3), which is expressed mainly on lymphatic endothelium of adult tissues (26 , 27) . VEGF-C is also angiogenic in vivo (11) . Recently, expression of VEGF-B and VEGF-C has been detected in a variety of human tumors (28) .
Ang-1, the ligand for TIE-2, a receptor-like tyrosine kinase expressed almost exclusively in endothelial cells, seems to be important to maintain vessel integrity by mediating interactions between the endothelium and surrounding matrix (10 , 29) . Because it stabilizes the structure of newly formed vessels, it has a later role in angiogenesis than VEGF. Its naturally occurring antagonist, Ang-2, binds with similar affinity to TIE-2, but does not activate the receptor (7) . Inhibition of Ang-1 by Ang-2 has been suggested to drive angiogenesis in the presence of angiogenic inducers like VEGF by loosening contacts between endothelial and periendothelial cells, thus rendering endothelial cells accessible to angiogenic inducers (6) .
bFGF is a mitogenic, angiogenic, and neurotrophic factor expressed by
many tumor cells (30, 31, 32, 33)
. PDGF consists of two related
polypeptides (A- and B-chain) (34
, 35)
. It originally was
known to be involved in the regulation of cell migration and
proliferation, but it has more recently been found to possess an
angiogenic capability both in vitro and in vivo
(8)
. TGF- has been shown to induce VEGF expression
(36)
and has also an angiogenic role in vivo
(37
, 38)
.
NB is the most common extracranial malignant solid tumor of childhood and arises from the sympathetic nervous system. A number of different biological and genetic factors are known to influence the heterogeneous biological and clinical behavior of NBs (39) . The invasive, metastatic, and hypervascular nature of high-stage NB may be one of the key obstacles to the cure of this disease. Evidence suggests that higher vascularity in NB correlates with metastasis, MYCN amplification, unfavorable histology, and poor outcome (40) . Hence, it is likely that NBs elaborate angiogenic peptides. It has been reported previously that VEGF is expressed in human NB specimens (41 , 42) , but to our knowledge, no analysis of other important angiogenic factors has been performed thus far in NB. However, it is unlikely that a single angiogenic factor regulates the angiogenic process in any tumor system. Therefore, the purpose of this study was to determine the angiogenic profile of NB. Differential mRNA expression of a panel of angiogenic factors was examined in 37 primary tumors and 22 NB cell lines and compared with age, stage, histology, MYCN amplification, TrkA expression, and outcome.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RNA Extraction and First-Strand cDNA Synthesis.
Total RNA was extracted either by using the RNeasy Kit (Qiagen,
Valencia, CA) or according to the method of Chomczynski and Sacchi
(43)
. Reactions were carried out using 1.0 µg of total
RNA in a total volume of 20 µl containing 150 ng of random hexamers
(Life Technologies, Inc., Gaithersburg, MD), 0.5 mM dNTPs
(Life Technologies, Inc.), 10 mM DTT, and 200 units of
SuperScriptII reverse transcriptase (Life Technologies, Inc.) in the
reaction buffer [20 mM Tris-HCl (pH 8.4), 50
mM KCl, 2.5 mM MgCl2].
Initially, the total RNA was denatured at 70°C for 10 min and
immediately chilled on ice. First-strand cDNAs were obtained after 10
min at 23°C and 50 min at 42°C. The reaction was terminated at
70°C for 15 min. RNase H (2 units; Life Technologies, Inc.) was added
to each RT reaction followed by incubation at 37°C for 20 min.
Semiquantitative RT-PCR.
PCR was carried out in a final volume of 10 µl containing 0.5 units
of Taq Gold Polymerase, 200 µM
dNTPs, and 0.4 µM of each primer in a buffer
[50 mM KCl, 10 mM Tris-HCl
(pH 8.3), 2.0 mM MgCl2, and
1 µl of the RT product (reverse-transcribed total RNA)]. Specific
PCR primers for VEGF, PDGF-A, TGF-,
and bFGF have been described previously (44)
and were designed to bracket cDNA sequences that cross an intron-exon
boundary in genomic DNA. Primer sequences for VEGF were able to detect
two of four different molecular species produced by alternative
splicing: mRNA VEGF165 and
VEGF121. The expected PCR product size was 576
bp and 444 bp, respectively. Primers for VEGF-B were able to
detect VEGF-B167 with an expected PCR product
size of 299 bp. Specific primer sequences for VEGF-B,
PDGF-A (PCR product size 228 bp), TGF- (PCR
product size 241 bp), bFGF (PCR product size 238 bp),
Ang-1 (PCR product size 263 bp), Ang-2 (PCR
product size 202 bp), VEGF-C (PCR product size 228 bp), and
glyceraldehyde-3-phosphate dehydrogenase (PCR product size,
160 bp) are available upon request. All PCR primers were biotinylated
at their 5' ends. PCR samples were overlayed with mineral oil, and
amplification was performed on a PTC-100 Programmable Thermal
Controller (MJ Research). The samples were denatured initially at
95°C for 12 min, followed by 20 cycles with denaturation at 95°C
for 30 s, annealing at 55°C for 30 s, and extension at
72°C for 90 s. The final cycle was followed by a 5-min extension
step at 72°C. The absence of contaminants was routinely checked by
RT-PCR assays of negative control samples (H2O
control or no reverse transcriptase added). The housekeeping gene
GAPD was coamplified as an internal standard control as
described previously.4
Analysis and Quantification of Amplified Products.
Each PCR sample (10 µl + 2 µl of Ficoll dye reagent) was analyzed
in parallel with a biotinylated molecular weight marker (Amersham,
Arlington Heights, IL) on a nondenaturing 6% polyacrylamide gel. DNA
was electrotransferred to a nylon membrane (Hybond N+; Amersham) and
immobilized by UV cross-linking. Detection of biotin-labeled DNA was
performed according to the "Southern-Light-Protocol" (Tropix,
Bedford, MA). Quantification of RNA transcript expression was performed
by densitometric analysis on X-ray films using Scion Image 1.55
software. A modification of the GAPD primers
(biotinylated:nonbiotinylated at a ratio of 1:49) allowed accurate
quantification within the linear range of X-ray detection of both the
target transcript and GAPD.4
The
expression of the target transcript was normalized by taking the ratio
of the densitometric unit of the transcript:densitometric unit of the
internal control, GAPD.4
MYCN Gene Analysis and Histology.
DNA was extracted from primary NB tumor tissue, and the MYCN
gene copy number was determined by Southern blot analysis as previously
described (45)
. Tumor tissue was classified histologically
according to the criteria described by Shimada et al.
(46)
. Tumor staging was based on International
Neuroblastoma Staging System criteria (47)
.
Statistical Analysis.
A two-sample t test was used to examine possible
associations between clinical stages and the expression of the genes of
interest. The Pearson correlation coefficient (r) and
P for each gene pair examined were calculated. Cox
regression models were used to explore associations between angiogenic
factor expression and survival, as well as other prognostic variables,
such as age, stage, and MYCN amplification. Statistical
analysis was performed using STATA version 5.0 (State Corp.,
College Station, TX).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Biological Features.
From the NB specimens in our study, 20 tumors had a favorable histology
and 17 had an unfavorable histology. MYCN amplification was
detected in 6 of 37 tumors (all of them stage 4 tumors), and 31 tumors
had a single copy of the MYCN proto-oncogene. Sixteen of 22
NB cell lines were MYCN-amplified, and six cell lines had a
single copy of MYCN.
Angiogenic Factors Are Expressed at Various Levels in Primary
NBs and NB Cell Lines.
We used semiquantitative RT-PCR analysis to examine the
expression of eight angiogenic factors in NB. Fig. 1
demonstrates the expression of three
angiogenic factors in six different NB cell lines as a
representative example. Transcripts of
VEGF165 and VEGF121 were
detected in 36 and 35, respectively, of 37 tumors. All 37 tumors
expressed various levels of VEGF-B, VEGF-C,
Ang-2, and PDGF-A. Transcripts for
bFGF were detected in 36 tumors, and transcripts for
Ang-1 and TGF- were detected in 35 tumors.
From the 22 NB cell lines examined, all 22 expressed various levels of
VEGF165, VEGF121,
VEGF-B, and bFGF (Fig. 2)
. Nineteen cell lines expressed
PDGF, 18 cell lines expressed Ang1 and
TGF-, and 16 cell lines expressed transcripts of
VEGF-C and Ang-2 (Fig. 2)
. Data obtained by RT-PCR were
confirmed by a specific ELISA for VEGF and bFGF protein in supernatants
of NB cell lines. Expression on mRNA and protein levels correlated well
with each other (data not shown). However, no paraffin-embedded
material for immunohistochemistry or protein material for Western
blotting was available for the primary tumor specimens.
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). This association
was statistically significant for VEGF165
(P < 0.0001), VEGF121
(P = 0.0001), VEGF-B (P =
0.004), VEGF-C (P = 0.003), bFGF
(P = 0.002), Ang-2 (P =
0.0001), TGF- (P = 0.0007), and
PDGF-A (P = 0.026; Table 1
).
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and PDGF-A.
The expression of VEGF121 was also correlated
with VEGF-B, VEGF-C, bFGF,
Ang-1, and Ang-2 (r = 0.4–0.65;
P < 0.0001–0.05). In addition, VEGF-Btranscript levels were positively correlated with
VEGF-C, bFGF, Ang-2, and
TGF- (r = 0.4–0.7; P <
0.0001–0.028), but not with Ang-1 and PDGF-A. VEGF-Cexpression was also correlated with Ang-2 and
bFGF (r = 0.65; P <
0.0001), and bFGF was correlated with Ang-1
(r = 0.4; P = 0.014) and
Ang-2 (r = 0.82; P <
0.0001). Ang-1 and Ang-2 were positively
correlated with each other (r = 0.53; P = 0.0006). There was no correlation of PDGF-A expression
with any other angiogenic factor examined. In NB cell lines, we found a
significant correlation in the expression of
VEGF165, VEGF121,
VEGF-B, and bFGF (r = 0.45–0.92;
P < 0.0001–0.05). TGF- expression was
positively correlated with Ang-1 (r =
0.55; P = 0.008) in NB cell lines. All other
correlations were not significant in NB cell lines.
Correlation with TrkA Expression.
Because the expression of angiogenic factors was found to be associated
with advanced-stage tumors, we examined whether their expression was
inversely associated with TrkA expression, a
well-established prognostic marker of favorable NB
(48, 49, 50)
. The expression of TrkA in our study
cohort of NB examined with the same semiquantitative RT-PCR also served
as a control for sampling bias (see "Discussion"). High
TrkA expression was positively associated with better
outcome and survival, younger age, lower stage, and favorable
histology. We showed an inverse correlation of TrkA
expression and expression of TGF- in NB tumors
(r = -0.37; P = 0.02) and cell
lines (r = -0.42; P = 0.05).
The correlation in expression of all other angiogenic factors with
TrkA was not significant.
Correlation of TrkA and Angiogenic Factor Expression with Age,
Histology, and MYCN Amplification.
We detected an inverse correlation of TrkA expression and
age (r = -0.33; P = 0.046),
which has been published before (48)
. The expression
levels of PDGF-A (r = 0.34;
P = 0.038) and VEGF-B (r =
0.32; P = 0.05) were positively related with age, but
no statistically significant correlation could be found for the other
angiogenic factors (Table 1)
.
A two-sample t test revealed a significant association
between high TrkA expression and favorable histology
(P = 0.037) and between higher age and unfavorable
histology (P = 0.006). The mean expression levels of
VEGF121 (P = 0.044) and
Ang-2 (P = 0.012) were significantly higher
in tumors with unfavorable histology, whereas no other
angiogenic factor showed any significant association with
histology or age (Table 1)
.
In NB tumors, high TrkA expression (P =
0.009) and younger age (P = 0.037) were associated with
single-copy MYCN. From the angiogenic factors, high
expression of PDGF-A correlated significantly with
MYCN amplification, but no other correlation of angiogenic
factor expression and MYCN status was found in primary
tumors. In NB cell lines, high expression of TrkA was
associated with single-copy MYCN (P =
0.023). The mean expression level of VEGF-B was also higher
in NB cell lines with single-copy MYCN (P =
0.017), whereas the mean expression levels of VEGF-C were
higher in NB cell lines with MYCN amplification
(P = 0.0046; Fig. 4
). No
other correlations with MYCN status were found to be
significant in cell lines.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our results indicate that all eight angiogenic factors examined are
present at various levels in NB cell lines and tumors, suggesting their
involvement in NB angiogenesis. We found that high expression
levels of seven angiogenic factors
(VEGF165/VEGF121,
VEGF-B, VEGF-C, bFGF,
Ang-2, TGF-, and PDGF-A) correlated
strongly with the advanced stage of NB. In the study by Roessler
et al. (41)
, VEGF immunoreactivity in 10 NB
tumor samples did not correlate with clinical stage. This might be
attributable to the lower number of tumor specimens in that study, but
might also suggest that RT-PCR is a more sensitive method than
immunohistochemistry for the quantification of angiogenic factor
expression in NB.
We also examined whether the expression of angiogenic factors was associated with well-established favorable and unfavorable prognostic markers of NB, i.e., TrkA expression and MYCN amplification, respectively. The examination of TrkA expression by the same RT-PCR method also served as a control for method reliability and sampling bias because TrkA expression has been investigated in large study cohorts, and its expression pattern in NB has been well established (48, 49, 50) . In fact, our data on TrkA expression were consistent with the previous findings that high TrkA expression was associated with low-stage, favorable NB (P = 0.0002; data not shown). These and other data suggest that our study cohort is generally representative of an unselected population of these patients. Thus, the expression pattern of the angiogenic factors examined is likely to reflect that in the general NB population.
We demonstrated in our study that the expression of PDGF-A correlates significantly with overall survival of the patients, but no other angiogenic factor was found to be associated with outcome or survival. However, future studies in larger study cohorts will be required to determine whether the expression of angiogenic factors is predictive of NB outcome and might serve as an independent prognostic factor.
The fact that the expression level of most angiogenic factors is not
associated with MYCN amplification (except for an inverse
correlation with PDGF-A) in NB tumors and that only TGF-
expression is inversely correlated with TrkA expression might also be
attributable to the low number of patients in our study. However, it is
interesting that VEGF-B is correlated with single-copy
MYCN in NB cell lines, whereas VEGF-C is highly
associated with MYCN amplification. One might speculate that
up-regulation of VEGF-C is a mechanism used by
MYCN-amplified, aggressive NB tumors to attract not only
vascular endothelial cells, but also lymphatic endothelial cells.
Expression of VEGF-C is associated with the development of
lymphatic vessels. Although angiogenesis refers to an increase in blood
vessel formation, others have found that angiogenesis is also
associated with increased lymph node metastasis (52
, 53) .
Angiogenesis of lymphatic vessels might be a process similar to
vascular angiogenesis, providing a subset of tumors with the
opportunity of metastatic spread via the lymphatics. VEGF-C
could be an important factor regulating paracrine relationships between
tumor cells and lymphatic endothelial cells (28)
. At
present, it is unclear if high levels of MYCN expression
regulate this process at least in part by up-regulation of
VEGF-C. Whether VEGF-C expression is also
associated with MYCN amplification in primary NB tumors has
to be determined in a larger study cohort with more
MYCN-amplified tumors. A possible relationship between
MYCN amplification and VEGF-C up-regulation is in
agreement with previous studies demonstrating that NB angiogenesis
correlates with MYCN amplification and metastatic disease
(40)
and that enhanced MYCN expression induces
angiogenesis of experimental human NBs (54)
.
Most angiogenic factors were expressed at relatively low levels in the four 4S tumors in our study. In contrast, in the report by Meitar et al. (40) , three 4S tumors were highly vascular, which might be expected because of the metastatic disease that 4S patients demonstrate at diagnosis. However, unlike stage 4 tumors, it is well known that the outcome for this special stage of disseminated NB is generally favorable because these 4S tumors have a propensity to undergo spontaneous remission. Although stage 4S tumors might be widely metastatic because they initially have an angiogenic phenotype, the unique biological features of this special subset of NB appear to alter this phenotype. Thus, the time point of diagnosis (before or after initiation of regression) might be important for the determination of angiogenic factor expression.
Expression levels of VEGF, VEGF-B,
VEGF-C, bFGF, Ang-1, and
Ang-2 are correlated with each other in NB specimens. This
suggests that several angiogenic peptides act in concert in the
regulation of neovascularization. Up-regulation of VEGF family members
might be mediated by up-regulation of common transcription factors, or
some angiogenic factors may act through a second messenger system by
inducing the expression of other angiogenic factors. VEGF-related
factors may interact with the VEGF system in a number of ways,
e.g., VEGF-B is known to form heterodimers with VEGF
(24)
. The existence of a gene family consisting of several
related growth factors suggests that these family members have
overlapping but distinct functions. A synergistic effect of bFGF and
VEGF has also been reported before (55)
. TGF- was found
to function as a potent inducer of VEGF synthesis by transcription of
the VEGF gene promoter via AP2 transcription factors
(36)
. However, we did not find a statistically significant
correlation between expression of TGF- and VEGF in our study,
suggesting that interaction of VEGF with other angiogenic factors may
be more important.
Taken together, our results suggest that several angiogenic factors have a biological role in NB angiogenesis. They might contribute synergistically to a more aggressive unfavorable tumor biology. The ubiquitous expression of several angiogenesis stimulators in NB suggests that antiangiogenesis therapy may provide a novel strategy that may be particularly useful for highly vascularized, advanced-stage tumors. However, the redundant expression of other angiogenic factors also suggests that molecules targeting only VEGF and inhibiting its bioactivity selectively might not be sufficient as antiangiogenic agents in NB. More general antiangiogenic approaches may be necessary, like therapy with the angiogenesis inhibitor TNP-470, a synthetic angiostatic agent derived from Aspergillus fumigatus. This agent specifically inhibits endothelial proliferation independent of angiogenic factor expression, and so it might be more promising (56) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Dr. Mildred
Scheel-Stiftung (to A. E.), the NIH Grant NS 34514 (to
G. M. B.), the Audrey E. Evans Endowed Chair (to G. M. B), and the
W. W. Smith Charitable Trust (to B. P. H.) 
2 To whom requests for reprints should be
addressed, at Children’s Hospital of Philadelphia, Division of
Oncology, ARC Room 902, 3516 Civic Center Boulevard, Philadelphia, PA
19104. Phone: (215) 590-4855; Fax: (215) 590-3770; E-mail: eggert{at}email.chop.edu
3 VEGF, vascular endothelial growth factor;
bFGF, basic fibroblast factor; TGF-, transforming growth factor ;
PDGF, platelet-derived growth factor; Ang, angiopoietin; NB,
neuroblastoma; RT, reverse transcription.
4 Eggert, A., Brodeur, G. M., Ikegaki, N. A
relative quantitative RT-PCR protocol for TrkB expression in
neuroblastoma using GAPD as an internal control. Biotechniques,
28: in press, 2000.
Received 11/22/99; revised 2/15/00; accepted 2/24/00.
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