IN MEMORIAM PR D. CHOPIN
Progrès en Urologie (2005), 15, Supp. N°1, 1303-1313
Cancer-testis antigen expression in bladder cancer
Yves Fradet1, V. Picard 2, A. Bergeron 2, H. LaRue
2
1 Laboratoire d’Uro-Oncologie Expérimentale. Centre de recherche en cancérologie de l’Université Laval.
CHUQ-L’Hôtel-Dieu de Québec. Canada.
2 Centre de recherche en cancérologie de l’Université Laval, * Laboratoire d’Uro-Oncologie Expérimentale
Centre de recherche en cancérologie de l’Université Laval CHUQ-L’Hôtel-Dieu de Québec
RÉSUMÉ
But: Afin d’évaluer le potentiel des antigènes testiculaires du cancer (CTA) comme cibles immunothérapeutiques dans le cancer de la vessie, nous
avons analysé l’expression de 9 des ces antigènes ou familles d’antigènes dans des tissus
humains de vessie. Comme leur expression est
contrôlée par des mécanismes épigénétiques,
nous avons aussi analysé l’effet d’inhibiteurs des
méthylases de l’ADN et des désacétylases des
histones sur l’expression des CTA dans des
lignées de cancer de vessie.
Matériel et méthodes : L’expression de NY-ESO1/LAGE-1, MAGE-A, MAGE-C1, BAGE, HOMTES-85, SCP-1, SSX-1, SSX-2 et SSX-4 a été
analysée par RT-PCR semi-quantitativef dans 10
urothéliums normaux, 24 tumeurs superficielles,
22 tumeurs infiltrantes et dans 10 lignées vésicales traitées par 5-aza-2’-désoxycytidine (5-AZADC) et/ou Trichostatin A.
Résultats: L’expression de tous les gènes des
CTA a été observée dans au moins une tumeur à
l’exception de HOM-TES-85 qui n’a jamais été
détecté. Les ARNm de MAGE-A, BAGE et NYESO-1/LAGE-1 ont été les plus fréquemment
détectés, dans respectivement 67%, 21% et 8%
des tumeurs superficielles et dans 64%, 41% et
27% des tumeurs infiltrantes. À l’exception de
MAGE-A, les transcrits CTA ont rarement été
détectés dans les lignées vésicales. Cependant,
l’expression de tous les gènes des CTA sauf SCP1, a été variablement induite par les drogues, 5AZA-DC étant un inducteur beaucoup plus puissant que le TSA.
Conclusion: Ces données suggèrent que les
CTA pourraient être utilisés comme cibles dans
l’immunothérapie du cancer de la vessie, surtout
ceux qui sont souvent exprimés comme MAGE-A,
BAGE et NY-ESO-1/LAGE-1. De plus, l’induction
de ces antigènes par des agents de chimiothérapie tel le 5-AZA-DC, offre la possibilité d’un prétraitement augmentant l’immunogénicité des
tumeurs.
Keywords: Bladder cancer, Cancer-testis antigens, MAGE, NY-ESO-1, Immunotherapy
ABSTRACT
Purpose: To evaluate the potential of cCancert/Testis antigens (CTAs) as targets for immunotherapy of bladder cancer, we evaluated the expression of 9 CTA genes or families of genes in normal
urothelia, bladder tumours and bladder cancer
human bladder tissuescell lines. As expression of
most CTAs is controlled by epigenetic mechanisms, we also evaluated the effect of the DNA
methylase inhibitor 5-aza-2’-deoxycytidine (5AZA-DC), and/or theand histone deacetylase inhibitors Trichostatin A (TSA) on their expression in
bladder cancer cell lines.
Material and Methods: Expression of NY-ESO1/LAGE-1, MAGE-A, MAGE-C1, BAGE, HOMTES-85, SCP-1, SSX-1, SSX-2 and SSX-4 was
analyzed by semi-quantitative RT-PCR and Western blotting on 10 normal urothelia, 23 24 superficial and 223 invasive tumours and on 10 cell
lines treated with 5-aza-2’-deoxycytidine (5-AZADC) and/or Trichostatin A (TSA).
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Results: Expression of all CTA genes could be
observed in at least 1 tumour except for HOMTES-85 for which mRNA was never detected.
MAGE-A, BAGE and NY-ESO-1/LAGE-1 mRNAs
were the most frequently detected, respectively in
5677%, 212% and 89% of superficial and in
6461%, 4139% and 276% of invasive tumours.
With the exception of MAGE-A, CTA transcripts
were rarely detected in the cell lines. However,
expression of all CTA genes, except SCP-1, could
be induced at various levels by the drugs and 5AZA-DC was a much more potent inducer than
TSA.
Conclusion: These data suggest that immunotherapy of bladder cancer could target CTAs,
especially those expressed at higher frequency
such as MAGE-A, BAGE and NY-ESO-1/LAGE-1.
Moreover, their induction by chemotherapeutic
agents such as 5-AZA-DC, provides a potential
pretreatment aimed at inducing the immunogenicity of the tumours.
INTRODUCTION
Superficial transitional cell carcinomas (TCC)
account for about 70% of all bladder cancers in
North America and Europe [1]. Following surgery,
these tumours recur in more than 60% of cases
and up to 25% of recurrences become invasive.
For more than 20 years, intravesical bacillus Calmette-Guérin (BCG) instillation has been used
successfully to prevent recurrence and progression of superficial TCC after surgery, or for the
treatment of carcinoma in situ. Nearly 50% of
patients at high risk of recurrence remain free of
disease after receiving BCG immunotherapy following surgery.
This suggests that immunotherapeutic treatments
targeting specific tumour-associated antigens
might be more effective and associated with less
morbidity than BCG. Bladder cancer thus offers a
unique opportunity to develop cancer vaccines in
an ideal setting of minimal tumour burden. However, the development of specific immunotherapy
depends on the identification of relevant target
antigens.
Cancer-/Ttestis antigens (CTAs) constitute one of
the most interesting class of tumour-associated
antigens [16,19]. They are immunogenic proteins
expressed in various types of cancers. Their
expression in normal tissues is restricted to germ
cells in the testis and is occasional in non-gametogenic tissues. As testis tissues are immunoprivileged by their lack or low expression of HLA molecules, CTAs appear as ideal targets for immunotherapy. Moreover, the identification of CTA epitopes recognized by CD4+ and CD8+ T-cells and
the high-titer antibody responses observed
against some CTAs show their relevance in the
immune response against cancer.
However, a major drawback to their use as targets
is their heterogeneous expression [10]. Successful CTA-based immunotherapy would thus likely
be achieved by use of multiantigenic vaccines.
Expression of CTA genes appears to be mostly
regulated by DNA methylation and their expression in neoplastic tissues seems to correlate with
the frequent cancer-associated hypomethylation
[19]. Previous studies tested new classes of
chemotherapeutic agents inhibiting DNA methylation or histone deacetylation (HDAC) to induce
CTAs expression in tumour cells. The methylation
inhibitor 5-aza-2’-deoxycytidine (5-AZA-DC), [4],
alone or in combination with a histone deacetylaseHDAC inhibitor such as Trichostatin A (TSA),
[14], was shown to efficiently induce expression of
several CTAs in cancer cell lines [5,8]. Thus, these
drugs used in association with an appropriate vaccination strategy targeting CTAs could increase
the number of tumours eligible for immunotherapy
and help to improve the immune response and
increase the number of tumours eligible for immunotherapy.
Previous studies on CTAs expression in bladder
tumours focused on the MAGE-A or NY-ESO-1
families but none reported simultaneous analysis
of many CTAs in one panel of tumours
[2,11,12,15,17] or NY-ESO-1 families but none
reported simultaneous analysis of many CTAs in
one panel of tumours. In this the present study,
we examined the expression of 9 CTA genes or
families of genes in superficial and invasive TCCs.
Moreover to establish a strategy for chemoimmunotherapy, we assessed the potential of 5-AZADC and TSA to induce the expression of CTAs in
bladder cancer cell lines.
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MATERIAL AND METHODS
1. Clinical specimens
This study was approved by the Institutional
Review Board of CHUQ-L’Hôtel-Dieu de Québec.
Bladder tumour specimens were collected between
1984 and 1990, frozen in liquid nitrogen and stored
at –80°C. Normal urothelial mucosa were isolated
from the bladder of organ donors. Normal testis
specimens were also obtained from organ donors.
2. Cell lines
Mycoplasma-free bladder cancer cell lines (MGHU3, SW780, RT4, 5637, 639V, T24, VMCUB-3,
J82, JON and SW1710) were cultured in Minimal
Essential Medium (MEM, Gibco/BRL, Burlington,
ON) containing 10% foetal calf serum. 5-AZA-DC
(Sigma Chemical Company, St-Louis, MO) and
TSA (Sigma) treatments were carried out in T25
tissue culture flasks on 25-50% confluent cells,
twenty-four hours after plating. Cells were treated
for 48 hours with 1 µM 5-AZA-DC and/or 0.5 µM
TSA. Alternatively, after 48 hours of 5-AZA-DC
treatment, cells were treated for an additional 48
hours with 5-AZA-DC alone or with TSA.
3. RNA and protein extractions
RNA and proteins were isolated from frozen
tumour specimens or cultured cells using TRIzol
(Invitrogen, Burlington, ON). Proteins were quantified using the Bradford method following the
manufacturer’s recommendations. RNA was
quantified by the optical density at 260 nm and its
quality assessed on formaldehyde-agarose gels.
The presence of normal or near normal proportions of 28S and 18S ribosomal RNAs attested to
the quality of the specimens. DNA contamination
was eliminated using DNAse I (2 units/10 µg RNA,
Ambion, Austin, TX). The enzyme was inactivated
with the DNAse Inactivating Reagent (Ambion).
4. Reverse-transcriptase polymerase chain
reaction (RT-PCR)
RT reactions were performed at 37°C for 1 h using
1 µg of DNA-free RNA and 200 units of M-MLV
reverse transcriptase in 50 mM Tris-HCl pH 8.3,
75 mM KCl, 3 mM MgCl2, 0.5 mM dNTP, 10 mM
DTT, containing 100 ng of hexanucleotide primers
and 25 units of RNA Guard (Amersham BioSciences, Baie d’Urfé, QC) in a final volume of 20 µl.
PCR primers for the human ß-ACTIN and CTA
genes were selected using the Prime function of
GCG (Wisconsin Package Version 10.2, Genetics
Computer Group, Madison, Wisc.) (Table 1). Specificity of MAGE-A primers was confirmed using
plasmids containing full-length cDNAs provided by
Table I: List of primers and amplification conditions used to analyze the expression of ß-ACTIN and 9
CTA genes or families of genesPROGCOMP.
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Dr. De Plaen (Brussels, Belgium) (MAGE-A1, A2,
A3, A4, A6, A12) or purchased from Invitrogen
(MAGE-A8, A9, A10, A11). PCR amplification of ßACTIN transcripts was first performed to select the
cDNA quantity limiting the amplification to the
exponential phase.
CTA gene expression was measured using 300
times more cDNA than for ß-ACTIN. PCR reactions were performed using up to 5 µl of cDNA and
0.5 units of Platinum Taq DNA polymerase (Invitrogen) in PCR buffer containing MgCl2, 0.2 µM of
both primers, 0.2 mM dNTP, 0.1 mM dATP and 2.5
mCi of dATP-[a32P] (3000 Ci/mmole, Mandel
Scientific Co. Ltd., Guelph, ON). PCR reactions
were conducted in a Perkin Elmer 9600 thermocycler (Norwalk, CT) as follows: 95°C for 5 min followed by 28 (for ß-ACTIN) or 32 (for CTA genes)
cycles of 95°C (15 sec) - 52-57°C (30 sec) - 72°C
(30 sec) with final extension at 72°C for 10 min.
MgCl2 concentrations and annealing temperatures are indicated in Table I. After drying, electrophoresis gels were exposed to a phosphor screen
and analyzed with a PhosphorImager Storm 860
(Molecular Dynamics, Sunnyvale, CA). Band
intensity was evaluated using the ImageQuant
software (Molecular Dynamics) to determine
CTA/ß-ACTIN ratios. Ratios between 0.13 and 0.9
were considered to represent low expression,
those between 0.9 and 2.5, medium expression
and those ≥ 2.5, high expression. Ratios below
0.13 were considered as background and not relevant.
5. Western blots
Thirty µg of tumour proteins or 15 µg of cultured
cells proteins were separated on SDS-PAGE and
transferred onto Hybond C nitrocellulose membranes (Amersham BioSciences). After blocking, filters were incubated at room temperature for 1-2 h
with monoclonal antibodies (mAb) 57Bb to MAGEA or D8.38 to NY-ESO-1 (dilution 1:100), provided
by Dr Spagnoli (Basel, Switzerland), or with a
mouse polyclonal against human ß-ACTIN
(Sigma) [10]. Membranes were washed and incubated for 1 h with horseradish-labelled goat antimouse secondary antibody (Jackson Immunoresearch Laboratories, West Grove, PA). Bound
antibodies were revealed using enhanced chemiluminescence (Perkin Elmer).
RESULTS
To maximize mRNA detection from clinical samples with various levels of integrity, random primers were used rather than oligo-dT for RT reactions and primers for 80-140 bp amplicons were
selected. Primers for non-familial CTA genes such
as SCP-1 and HOM-TES-85, for the first member
of the MAGE-C family, MAGE-C1 (CT7), for members of the SSX gene family, SSX-1, SSX-2, SSX4, for BAGE-1, -4 and -5 but not for BAGE-2 and 3, for NY-ESO-1 and LAGE-1a but not LAGE-1b
and finally for multiple members of the MAGE-A
family i.e. MAGE-A1, A2, A3, A4, A6, A8, A9, A12
but not A10 and A11 were used as described in
Table I.
1. CTA expression in clinical specimens
No significant expression of any CTA genes was
detected in 10 normal urothelia except for MAGEA which gave a very weak signal in 7 out of the 10
samples (results not shown). Exceptionally, the
threshold of low reactivity for MAGE-A genes was
increased from 0.13 to 0.30 to show the overexpression of these genes in the tumours compared
to the normal tissues. Figure 1 shows an example of the signals obtained for ß-ACTIN, NY-ESO1/LAGE-1 and MAGE-A the results of CTA gene
expression analysis in the 23 24 superficial (7 6 Ta
and 16 18 T1), 23 22 invasive bladder tumours (10
9 T2, 10 T3 and 3 T4) and one testis specimen.
Expression of all CTA genes or families of genes
was detected in at least one bladder tumour sample, with the exception of HOM-TES-85 (Figure
2). Eighteen Sixteen (359%) tumours did not express any of the CTA genes analyzed. Twelve Thirteen (286%) tumours expressed only 1 CTA gene
while the remaining 16 17 (3437%) expressed up
to 6 CTA genes. The expression of CTA genes
was generally 2-3 times more frequent in invasive
than in superficial tumours, except for MAGE-A
genes, which were also the most frequently
expressed, in 5767% of superficial and 6164% of
invasive tumours. About 8070% of MAGE-A-positive tumours showed a medium to high expression. MAGE-A expression was not associated with
grade as 1/2 G1 (50%), 1115/20 21 G2 (5571%)
and 1514/24 G3 (6258%) tumours were positive.
The second CTA transcripts most frequently
expressed were BAGE in 2221% of superficial
and 3941% of invasive tumours. However, more
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Figure 1: Expression of CTA genes in superficial (Ta-T1) and invasive (≥ T2) bladder tumour samples.
Semi-quantitative RT-PCR results are expressed as ratios between CTA and ß-ACTIN gene expressions.
White cases : No expression; Yellow cases : Low expression; Blue cases : Medium expression; Red cases
: High expression . NY-ESO-1*(NY-ESO-1/LAGE-1)
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Figure 2: Western blot analysis of the expression of NY-ESO-1 and MAGE-A proteins. Proteins were extracted from the same bladder tumour samples used for RT-PCR analyses presented in Figure 1, separated on
SDS-PAGE, transferred to nitrocellulose membranes and tested with mAbs 57B (anti-MAGE-A) and D8.38
(anti-NY-ESO-1).
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than 50% of the positive tumours showed only a
weak expression. Expression of BAGE correlated
with grade as 0/2 G1, 4/20 G2 (15%) and 10/24
G3 (42%) (p=0.035, chi-square) tumours were
positive. The third most often detected CTA were
NY-ESO-1/LAGE-1, expressed at high or medium
level in 98% of superficial and 2627% of invasive
tumours. Three tumours expressed NY-ESO1/LAGE-1 mRNA more strongly than the testis. As
for BAGE, NY-ESO-1/LAGE-1 expression correlated increased with tumour grade as it was found in
0/2 G1, 12/20 G2 (510%) and 76/24 (2925%) G3
tumours. (p=0.05, Fisher). This association was
however not statistically significant. The other CTA
genes analyzed were less frequently expressed,
in at most 9% of bladder tumours.
In order to correlate the expression of CTA
mRNAs measured in tumour samples with that of
proteins, we performed Western blot analyses on
total proteins, isolated from these same tumours.
These analyses could be performed only for
MAGE-A and NY-ESO-1 antigens as antibodies
for the other CTA were not available to us. Figure
2 shows Western blots obtained with mAbs 57b
57B to multiple MAGE-A antigens and D8.38 to
NY-ESO-1 antigen. NY-ESO-1 antigen expression
detected with mAb D8.38 paralleled mRNA
expression. However, only 1316/27 30 (53%)
MAGE-A mRNA-positive tumours were positive
with mAb 57Bb.
2. Cell lines
Nine TCC and one adenocarcinoma (JON) cell
lines were tested for expression of CTA genes. To
assess a possible induction of expression of the
various CTAsantigens, cells were treated with 5AZA-DC (inhibitor of methylation) and/or with TSA
(inhibitor of histone deacetylationHDAC) for 48 or
96 hours, as described. For some cell lines, no
data could be obtained after 96 hours because of
drug toxicity or because RNA was lost.
Four cell lines expressed none of the CTA mRNAs
whereas 5 others expressed only MAGE-A (Figure 3). In addition to MAGE-A, the JON cells also
expressed BAGE and SSX-4 mRNAs. Treatment
with 5-AZA-DC and/or TSA induced the expression of all CTA genes or families of genes with the
exception of SCP-1. For each cell line, expression
of at least 6 and up to 8 CTA genes was induced.
In general, expression was more efficiently indu-
Figure 3: Expression of CTA genes in bladder cancer
cell lines treated or not with methylase and/or histone desacetylase inhibitors. Semi-quantitative RTPCR results are expressed as ratios between CTA
and ß-ACTIN gene expressions. Treatment conditions were: 1, Control (untreated cells); 2, Cells treated with 1µM 5-AZA-DC for 48 h; 3, Cells treated with
0.5 µM TSA for 48 h; 4, Cells treated with 1µM 5-AZADC and 0.5 µM TSA for 48 h; 5, Cells treated with 1µM
5-AZA-DC for 96 h; 6, Cells treated with 1µM 5-AZADC for 96 h and with 0.5 µM TSA for the last 48 h.
White cases: No expression; Yellow cases: Low
expression; Blue cases: Medium expression; Red
cases: High expression. NY-ESO-1*(NY-ESO1/LAGE-1). N.D.: Not determined.
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ced by 5-AZA-DC than by TSA. The strongest
inductions were observed for MAGE-A, MAGEC1, SSX-1 and SSX-2 genes. However, in most
cases, the level of expression observed in testis
could not be attained.
Western blotting with mAb D8.38 showed a weak
induction of the NY-ESO-1 antigen only in the
639V, VMCUB-3 and SW1710 (Figure 4). A strong
induction of the MAGE-A antigens, as detected by
mAb 57b 57B could be observed following treatment with 5-AZA-DC and TSA. It paralleled perfectly mRNA expression at the exception of the
RT4 cell line which showed no reactivity with mAb
57b 57B despite medium mRNA expression.
DISCUSSION
We evaluated the expression of 9 CTA genes or
families of genes in bladder tumours and normal
urothelia to evaluate their potential as targets for
specific immunotherapy. MAGE-A genes were the
most frequently expressed with overall 5965% of
both superficial and invasive tumours expressing
any combination of MAGE-A1, A2, A3, A4, A6, A8,
A9 and A12. Patard et al. analysed the expression
of MAGE-A1, -A2, -A3 and -A4 genes in 29 super-
Figure 4: Western blot analysis of the expression of NY-ESO-1 and MAGE-A proteins. Proteins were extracted from the
same cells used for RT-PCR analyses presented in Figure 3, separated on SDS-PAGE, transferred to nitrocellulose
membranes and tested with mAbs 57B (anti-MAGE-A) and D8.38 (anti-NY-ESO-1). Treatment conditions were as described in Figure 3.
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ficial and 28 invasive bladder tumours, using RTPCR [15]. They found that 61% of invasive
tumours expressed at least one of these MAGE-A
genes, a result comparable to our own 5964%.
However, they only found 28% of superficial
tumours expressing any of these genes, in
contrast to the 5767% we detected. This difference suggests that about about half of our positive
superficial tumours expressed MAGE-A genes
other than MAGE-A1, -A2, -A3, -A4, in agreement
with the reactivity of only 69/13 16 (56%) mRNApositive tumours with mAb 57bB. This mAb was
shown to preferentially recognize MAGE-A4-positive tumours although it was shown to efficiently
react with MAGE-A1, -A2, -A3, -A4, -A6, and -A12
antigens in specificity analyses using transfected
cells [13]. A higher level of expression of MAGEA4 compared to other MAGE-A antigens has been
proposed to explain the preferential reactivity of
57bB. More recently, Bar-Haim et al. reported the
overexpression of MAGE-A8 mRNA in 70% of
bladder tumours [2]. Their higher sensitivity of
detection compared to ours may be explained by
their amplification parameters, which included
more cDNA and a higher number of amplification
cycles. These results suggest that MAGE-A8
could be the MAGE-A mRNA observed in our
57bB-negative tumours. However we obtained
evidences that other specific MAGE-A transcripts
are also expressed in these tumours (unpublished
data). A comprehensive analysis of the expression
of MAGE-A antigens in bladder tumours will require the use of specific primers for each MAGE-A
family member.
The second CTAs most frequently expressed in
our panel of tumours were BAGE-1, -4 and -5.
Their mRNAs were detected in 22 21 % of superficial and 3941% of invasive bladder tumours.
These data agree with those of Boel et al. who
found that 30% of invasive bladder carcinomas
expressed BAGE [3]. However in more than 50%
of positive tumours the level of expression was
low compared to that observed in testis.
NY-ESO-1 is one of the most immunogenic known
antigens. Nearly 50% of patients with NY-ESO-1
positive tumours have detectable humoral immune response and CD8+ T-cell reactivity against
this antigen [9,18]. We found NY-ESO-1/LAGE-1
to be the third most frequently expressed CTAs, in
98% of superficial and 2627% of invasive
tumours.
Expression of NY-ESO-1/LAGE-1 was also correlated with the differentiation status as 7/8 positive
tumours were G3 tumours. Kurashige et al. [12]
reported NY-ESO-1 gene expression in 28% of
superficial and 38% of invasive tumours while
Sharma et al. [17] found NY-ESO-1 and/or LAGE1 expression in 14% of low-grade and 48% of
high-grade bladder tumours. The higher rates of
expression observed by these investigators may
again be explained by their amplification parameters. In our study, we chose to avoid saturation of
signals to limit amplification of weak, less significant signals. We feel that the perfect match we
observed between our RT-PCR and Western blot
data attested to the validity of this strategy. Other
CTAs were expressed in less than 10% tumours
with SCP-1 and HOM-TES-85 being the less frequently expressed with respectively only 1 or no
positive tumour.
The results presented in this study suggest that
NY-ESO-1/LAGE-1, BAGE but mostly MAGE-A
antigens could be relevant targets for bladder cancer immunotherapy, especially for superficial
tumours. Several clinical trials assessing the efficacy of immunotherapy targeting CTAs (mostly
MAGE-A3) in patients with various types of cancers, including bladder cancer, have already been
reported. Generally speaking, their success has
been limited, with less than 20% of patients showing regression after vaccination [7]. The failure to
obtain clinical responses may have two major causes: failure to induce a significant T-cell response
or resistance of the tumour to immunological
attack. While the first cause may be eventually
resolved by more efficient vaccine formulations,
the second one will need better characterization of
the patients’ tumour and immune system status.
One mechanism developed by tumours to escape
immune attack is outgrowth of antigen-loss or antigen-negative variants. Heterogeneous expression
of CTAs in tumour tissues is a condition favouring
tumour escape [10]. In this study we evaluated
the potential of 5-AZA-DC alone or in combination
with TSA to induce the expression of CTA genes in
bladder cancer cell lines. We showed that all
CTAs analysed except SCP-1, could be induced
by 5-AZA-DC. However, this induction raises
questions about the ability of tumour cells to properly present induced tumour epitopes by HLA
class I. Coral et al. [5] showed that de novo
expression of NY-ESO-1 in 5-AZA-treated renal
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carcinoma cells could lead to efficient lysis by NYESO-1-specific cytotoxic T lymphocytes and that
such treatment may also lead to increased
expression of HLA class I and co-stimulatory
molecules [6]. Thus, the use of 5-AZA-DC, also
known as Decitabine, with an immunisation strategy aimed at inducing an efficient immune response against CTA antigens could provide an opportunity for to 1) increaseing the number of tumours
potentially treated by chemoimmunotherapy, 2)
improveing antigen presentation by HLA class I
antigens and 3) possibly providing a mean to
counter tumour escape mechanisms by reexpressinginducing antigens expression in antigen-negative loss or antigen-negative tumour cell
variants.
CONCLUSIONS
The BAGE, the NY-ESO-1/LAGE1 but mostly the
MAGE-A families of antigens are the most frequently CTA expressed in bladder tumours and
thus constitute targets of choice for bladder cancer immunotherapy. A more detailed analysis is
needed to identify which specific MAGE-A antigens are expressed in bladder tumours. We showed that in bladder cancer cell lines the expression of these CTA can be induced by 5-AZA-DC.
Thus a treatment combining immunotherapy and
5-AZA-DC chemotherapy could increase the number of tumours eligible for immunotherapy and
help to improve the immune response.
ACKNOWLEDGEMENTS
We are grateful to Dr Etienne De Plaen for his
gift of plasmids, Dr Spagnoli for his gift of
monoclonal antibodies and to Mrs Claire
Ménard for technical assistance. This work
was partially supported by the Canadian National Cancer Institute (Grant # 10333) with funds
from the Canadian Cancer Society.
BAGE: a new gene encoding an antigen recognized on
human melanomas by cytolytic T lymphocytes. Immunity.,
1995 ; 2 ; 167-175.
4. CHRISTMAN, J.K.: 5-Azacytidine and 5-aza-2’-deoxycytidine as inhibitors of DNA methylation: mechanistic studies
and their implications for cancer therapy. Oncogene, 2002
; 21 ; 5483-5495.
5. CORAL, S., SIGALOTTI, L., ALTOMONTE, M., ENGELSBERG, A., COLIZZI, F., CATTAROSSI, I., MARASKOVSKY, E., JAGER, E., SELIGER, B., MAIO, M.: 5-aza-2’deoxycytidine-induced expression of functional cancer
testis antigens in human renal cell carcinoma: immunotherapeutic implications. Clin.Cancer Res., 2002 ; 8 ;
2690-2695.
6. CORAL, S., SIGALOTTI, L., GASPAROLLO, A., CATTAROSSI, I., VISINTIN, A., CATTELAN, A., ALTOMONTE,
M., MAIO, M.: Prolonged upregulation of the expression
of HLA class I antigens and costimulatory molecules on
melanoma cells treated with 5-aza-2’-deoxycytidine (5AZA-CdR). J.Immunother., 1999 ; 22 ; 16-24.
7. COULIE, P.G., KARANIKAS, V., LURQUIN, C., COLAU,
D., CONNEROTTE, T., HANAGIRI, T., VAN PEL, A.,
LUCAS, S., GODELAINE, D., LONCHAY, C., MARCHAND, M., VAN BAREN, N., BOON, T.: Cytolytic T-cell
responses of cancer patients vaccinated with a MAGE
antigen. Immunol.Rev., 2002 ; 188 ; 33-42.
8. GURE, A.O., WEI, I.J., OLD, L.J., CHEN, Y.T.: The SSX
gene family: characterization of 9 complete genes.
Int.J.Cancer, 2002 ; 101 ; 448-453.
9. JAGER, E., NAGATA, Y., GNJATIC, S., WADA, H., STOCKERT, E., KARBACH, J., DUNBAR, P.R., LEE, S.Y.,
JUNGBLUTH, A., JAGER, D., ARAND, M., RITTER, G.,
CERUNDOLO, V., DUPONT, B., CHEN, Y.T., OLD, L.J.,
KNUTH, A.: Monitoring CD8 T cell responses to NY-ESO1: correlation of humoral and cellular immune responses.
Proc.Natl.Acad.Sci.U.S.A, 2000 ; 97 ; 4760-4765.
10. JURETIC, A., SPAGNOLI, G.C., SCHULTZ-THATER, E.,
SARCEVIC, B.: Cancer/testis tumour-associated antigens: immunohistochemical detection with monoclonal
antibodies. Lancet Oncol., 2003 ; 4 ; 104-109.
1. AMLING, C.L.: Diagnosis and management of superficial
bladder cancer. Curr.Probl.Cancer, 2001 ; 25 ; 219-278.
11. KOCHER, T., ZHENG, M., BOLLI, M., SIMON, R., FORSTER, T., SCHULTZ-THATER, E., REMMEL, E., NOPPEN,
C., SCHMID, U., ACKERMANN, D., MIHATSCH, M.J.,
GASSER, T., HEBERER, M., SAUTER, G., SPAGNOLI,
G.C.: Prognostic relevance of MAGE-A4 tumor antigen
expression in transitional cell carcinoma of the urinary
bladder: a tissue microarray study. Int.J.Cancer, 2002 ;
100 ; 702-705.
3. BOEL, P., WILDMANN, C., SENSI, M.L., BRASSEUR, R.,
RENAULD, J.C., COULIE, P., BOON, T., VAN DER, B.P.:
13. LANDRY, C., BRASSEUR, F., SPAGNOLI, G.C., MARBAIX, E., BOON, T., COULIE, P., GODELAINE, D.: Monoclonal antibody 57B stains tumor tissues that express
REFERENCES
2. BAR-HAIM, E., PAZ, A., MACHLENKIN, A., HAZZAN, D.,
TIROSH, B., CARMON, L., BRENNER, B., VADAI, E.,
MOR, O., STEIN, A., LEMONNIER, F.A., TZEHOVAL, E.,
EISENBACH, L.: MAGE-A8 overexpression in transitional
cell carcinoma of the bladder: identification of two tumourassociated antigen peptides. Br.J.Cancer, 2004 ; 91 ; 398407.
12. KURASHIGE, T., NOGUCHI, Y., SAIKA, T., ONO, T.,
NAGATA, Y., JUNGBLUTH, A., RITTER, G., CHEN, Y.T.,
STOCKERT, E., TSUSHIMA, T., KUMON, H., OLD, L.J.,
NAKAYAMA, E.: NY-ESO-1 expression and immunogenicity associated with transitional cell carcinoma: correlation
with tumor grade. Cancer Res., 2001 ; 61 ; 4671-4674.
1312
gene MAGE-A4. Int.J.Cancer, 2000 ; 86 ; 835-841.
14. MARKS, P.A., RICHON, V.M., BRESLOW, R., RIFKIND,
R.A.: Histone deacetylase inhibitors as new cancer drugs.
Curr.Opin.Oncol., 2001 ; 13 ; 477-483.
15. PATARD, J.J., BRASSEUR, F., GIL-DIEZ, S., RADVANYI,
F., MARCHAND, M., FRANCOIS, P., ABI-AAD, A., VAN
CANGH, P., ABBOU, C.C., CHOPIN, D., .: Expression of
MAGE genes in transitional-cell carcinomas of the urinary
bladder. Int.J.Cancer, 1995 ; 64 ; 60-64.
16. SCANLAN, M.J., SIMPSON, A.J., OLD, L.J.: The cancer/testis genes: review, standardization, and commentary. Cancer Immun., 2004 ; 4 ; 1-
18. STOCKERT, E., JAGER, E., CHEN, Y.T., SCALAN, M.J.,
GOUT, I., KARBACH, J., ARAND, M., KNUTH, A., OLD,
L.J.: A survey of the humoral immune response of cancer
patients to a panel of human tumor antigens. J.Exp.Med.,
1998 ; %20;187 ; 1349-1354.
19. ZENDMAN, A.J., RUITER, D.J., VAN MUIJEN,G.N. :
Cancer/testis-associated genes: identification, expression
profile, and putative function. J.Cell Physiol, 2003 ; 194 ;
272-288.
17. SHARMA, P., GNJATIC, S., JUNGBLUTH, A.A.,
WILLIAMSON, B., HERR, H., STOCKERT, E., DALBAGNI, G., DONAT, S.M., REUTER, V.E., SANTIAGO, D.,
CHEN, Y.T., BAJORIN, D.F., OLD, L.J.: Frequency of NYESO-1 and LAGE-1 expression in bladder cancer and evidence of a new NY-ESO-1 T-cell epitope in a patient with
bladder cancer. Cancer Immun., 2003 ; 3 ; 19-
1313