Download cancer/testis antigens, gametogenesis and cancer

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CANCER/TESTIS ANTIGENS,
GAMETOGENESIS AND CANCER
Andrew J. G. Simpson*, Otavia L. Caballero*, Achim Jungbluth*, Yao-Tseng Chen‡
and Lloyd J. Old*
Abstract | Cancer/testis (CT) antigens, of which more than 40 have now been identified, are
encoded by genes that are normally expressed only in the human germ line, but are also
expressed in various tumour types, including melanoma, and carcinomas of the bladder, lung
and liver. These immunogenic proteins are being vigorously pursued as targets for therapeutic
cancer vaccines. CT antigens are also being evaluated for their role in oncogenesis —
recapitulation of portions of the germline gene-expression programme might contribute
characteristic features to the neoplastic phenotype, including immortality, invasiveness,
immune evasion, hypomethylation and metastatic capacity.
TROPHOBLASTS
The outermost layer of cells of
the blastocyst that attaches the
fertilized ovum to the uterine
wall and serves as a nutritive
pathway for the embryo.
Trophoblasts invade and
penetrate, permiting the
blastocyst to burrow into the
central layer of endometrium.
Early blastocyst trophoblasts
differentiate into all the other
cell types found in the human
placenta.
*Ludwig Institute for
Cancer Research, New York
Branch at Memorial SloanKettering Cancer Center,
1275 York Avenue,
New York, New York 10021,
USA.
‡
Weill Medical College of
Cornell University,
New York, New York 10158,
USA.
Correspondence to A.J.G.S.
e-mail: [email protected]
doi:10.1038/nrc1669
Published online 20 July 2005
It has long been noted that processes of germ-cell
development and tumour development share important similarities. Indeed, as much as 100 years ago, the
similarity of the biological features of TROPHOBLASTS and
cancer cells prompted John Beard to propose a ‘trophoblastic theory of cancer’, which envisaged cancers
as arising from germ cells that fail to complete their
embryonic migration to the gonads1. Evidence for an
association between germ-cell development and cancer has steadily emerged. For example, a much studied facet of a range of human cancers that attracted
considerable attention over the past 25 years is their
frequent production of CHORIONIC GONADOTROPIN and
other trophoblastic hormones2. Such hormones are
now used as prognostic indicator for a range of epithelial tumours3. A further step has been the discovery of a growing number of proteins that appear to be
present only in germ cells, trophoblasts and tumours
— the cancer/testis (CT) antigens4. This discovery
led to the theory that aberrant expression of germline
genes in cancer reflects the activation of the silenced
gametogenic programme in somatic cells, and that this
programmatic acquisition is one of the driving forces
of tumorigenesis5 (FIG. 1).
Extensive data have been assembled concerning
the extent, frequency and control of expression of
CT antigens in tumours 4. Crucially, insights into
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the function of a number of the most frequently
expressed CT antigens are now also beginning to
emerge. These data are all consistent with the concept
that genetic alterations in cancer can result in the
reactivation of normally silent germline expression
programmes, and that these programmes might confer some of the central characteristics of malignancy
to the tumour.
The discovery of CT antigens
In the past 100 years, there has been a continuing
search for tumour antigens, which could be used to
direct the potent cytolytic capacities of the human
immune system against cancer6,7. An ideal cancer
antigen for immunotherapy would be specifically
and stably expressed by the tumour, absent from
normal tissues, and crucial for the survival of the
cancer cell. A crucial turning point in this search
was the adoption of an approach called AUTOLOGOUS
TYPING, in which cultured tumour and normal cells
from a patient were used as targets for specific
immune recognition by the patient’s own antibodies
and T cells6,8. It is noteworthy that due to the immune
system’s capacity to recognize and distinguish among
antigens, several key cancer-related molecules were
initially identified as tumour antigens, including the
viral T antigens and p53 REFS 9,10.
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Summary
• Cancer/testis (CT) antigens are normally expressed by gametes and trophoblasts, and are aberrantly expressed in a
range of human cancers.
• So far, 44 distinct CT-antigen families, some of which have multiple members, have been identified.
• CT antigens are immunogenic and, as a result, have the potential to be used as tumour vaccines.
• CT antigens can be divided between those that are encoded on the X chromosome (CT-X antigens) and those that
are not (non-X CT antigens).
• CT-X antigens tend to form recently expanded gene families that are usually highly expressed in the
spermatogonia — mitotically proliferating germ cells. The CT-X genes are frequently co-expressed in cancer cells,
which tend to express several CT antigens.
• The genes for the Non-X CT antigens are distributed throughout the genome. In the testis, they are usually
expressed in the spermatocytes and many have roles in meiosis. Their aberrant expression in cancer cells might
cause abnormal chromosome segregation and aneuploidy.
• Methylated CpG islands associated with the CT-X genes in normal somatic cells become demethylated in cancer
cells, indicating activation of their expression. Although global hypomethylation is frequently put forward as the
basis of CT-antigen expression in cancer cells, it will be important to define the events leading to the
hypomethylated state.
• MAGEA1 has been shown to be a transcriptional co-repressor that interacts with SKI interacting protein and
autonomously represses transcription. Other CT-X antigens also control gene expression and directly influence
the sensitivity of cancer cell lines to cytotoxic assault as well as cell proliferation.
• As germline stem cells and their trophoblastic derivatives share many characteristics with tumour cells, the
activation of normally silent germline-specific genes in cancer stem cells (gametic recapitulation) could mediate
the malignant phenotype in the absence of mutations in known oncogenes and tumour-suppressor genes.
CHORIONIC GONADOTROPIN
A hormone produced by the
placenta that maintains the
corpus luteum during
pregnancy.
AUTOLOGOUS TYPING
An approach developed to test
whether patients with cancer
develop specific antibodies or
T cells to tumour-restricted
antigens. Analysis was restricted
to assays including normal
(fibroblasts) and malignant cells
from the same (autologous)
patient. The specificity was
further confirmed by
absorption analysis with
autologous normal cells.
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The first CT antigen was discovered through autologous typing and the application of a newly developed
DNA-cloning methodology for defining the targets of
T-cell recognition11. A patient with melanoma who had
an unusually favourable clinical course was identified
as having cytotoxic T cells that recognized autologous
tumour cells12. Using this system, the gene encoding
the tumour antigen MZ2-E was cloned13,14. The gene
was termed MAGE1 (melanoma antigen; see BOX 1 for
nomenclature) and closely related genes MAGE2 and
MAGE3 were subsequently identified in the same cell
line15,16 BOX 1. Expression of the MAGEA1 gene, as
MAGE1 is now known, was detected in melanomas,
some breast carcinomas and other tumour types, but
not in any normal tissues except testis. Further analysis
of the MAGEA family revealed 12 closely related genes
clustered at Xq28 REF. 16. A search for the gene responsible for the sex-reversal phenotype revealed a second
cluster of MAGE genes, which encoded the MAGEB
genes and was located at Xp21.3 REF. 17. Subsequently,
a third cluster, encoding the MAGEC genes, was identified at Xq26-27 REFS 1821. The genes in all three
of these families were found to exhibit expression
restricted to the testis or cancers. By contrast, more distantly related clusters of genes (MAGED to MAGEL)16,22
were found to be expressed in many normal tissues.
A range of other tumour-antigen genes, including B melanoma antigen (BAGE)23 and G antigen 1
(GAGE1)24 (which bear no structural similarities with
the MAGE genes), were also discovered using cytotoxic T cells isolated from the same patient in which
MAGEA1 was discovered. Similarly, the BAGE and
GAGE1 genes were also found to be expressed only in
the testis and malignant tumours.
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The next major step in the search for tumour antigens came from the screening of cDNA expression
libraries with antibodies, rather than T cells25,26. This
technology, termed SEREX (serological analysis of
cDNA expression libraries) led to the identification
of several categories of tumour antigens, including
products of both mutated and overexpressed forms of
genes commonly found in adult tissues, as well as genes
that are normally silent in the adult soma but which
are activated in tumours. The latter included several
testis-restricted antigens, including SSX25,27, synaptonemal complex protein 1 (SCP1) REF. 28 and the highly
immunogenic cancer antigen New York oesophageal
squamous cell carcinoma 1 (NY-ESO-1)29. In view of
the growing list of testis genes expressed in cancer, a
nomenclature for this category of tumour antigens was
needed, so the term cancer/testis (CT) antigen was
adopted to encompass all these molecules26. Subsequent
work showed that CT antigens are restricted to germ
cells within the testis30, and are also expressed in trophoblasts31,32 along with the immature germ cells in the
fetal ovary33 (FIG. 2).
MAGEA and NY-ESO-1 are being studied as potential cancer vaccines engineered to stimulate T-cell
responses against tumours BOX 2. A range of vaccine
formulations containing one or other of these antigens
have been demonstrated to induce cellular and antibody responses in clinical trials34, and a small subset
of treated patients have shown clinical benefit following immunization34–37. The importance of CT antigens
as vaccine targets has led to detailed studies of their
expression in a range of human cancers. To identify
additional potential vaccine targets, there has been a
strategic quest to enlarge the known set of germline
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Germ cell
Cancer cell
Zygote
1
Blastula
2
Normal cell
Transformed
cell
2
Embryo
Trophoblast
4
7
3
8
1
4
7
8
3
9
9
5
Primordial
germ cell
1
2
6
8
Testis
Malignant
cell
Tumour
6
Metastasis
Spermatogonia
5
Spermatazoa
Shared phenotypes
Corresponding phenotypes
1
Global hypomethylation
Immortalization
2
Transformation
3
Angiogenesis
Implantation
4
Invasion
7
Immune evasion
Meiosis
5
Aneuploidy
8
CT antigen expression
Migration
6
Metastasis
9
Chorionic gonadotropin expression
Gametogenesis
Tumorigenesis
Figure 1 | Shared characteristics of germ cells and cancer cells. Activation of the
gametogenic programme (shown by brown cells) might contribute to properties of tumour
formation and progression (shown by blue cells). Corresponding features between cancer
cells and those in the germ cell/gamete/trophoblast differentiation pathways include:
immortalization (involved in transformation), invasion, induction of meiosis (can lead to
aneuploidy) and migration (contributes to metastasis). Shared phenotypes between germ
cells and cancer cells include demethylation, angiogenesis induction, downregulation of
the major histocompatibility complex (immune evasion), and expression of chorionic
gonadotropin. The numbers (1–9) indicate gametogenesis- and tumorigenesis-related
phenotypic traits and the stages at which these events occur.
genes that are re-expressed in cancer. This has mostly
been done by using published expressed sequence tag
(EST) data followed up by confirmatory PCR after
reverse transcription of RNA (RT-PCR)16,38,39. Although
transcriptional data do not establish the genes as encoding tumour antigens, for consistency and in recognition
of the origin of this field, the products of all genes identified as having cancer and germline expression are now
referred to as CT antigens.
The present catalogue of CT antigens4 contains 44
distinct CT-antigen families, of which several have
multiple members, such as MAGEA and GAGE1, as
well as splice variants, such as XAGE1a and XAGE1b,
resulting in a total of 89 transcripts (TABLES 1,2 and
BOX 1). In order for a protein to be designated a CT
antigen, it must be expressed in tumours as well as
in testis and/or the placenta, but not be expressed in
more than two non-germline normal tissues. When
non-germline normal tissue expression is detected,
this is usually at only a fraction of the level detected
in the testis4. Many other genes with an important role
in the development and differentiation of germ cells
are also expressed in somatic tissues — recognition of
these genes that are activated in concert with known
CT antigens is an important element of future research
into the relevance of germline gene expression in
cancer. However, in this article, we will focus on CT
antigens defined as above.
CT antigens can be divided between those that
are encoded on the X chromosome (CT-X antigens;
TABLE 1 ) and those that are not (non-X CT antigens; TABLE 2). Twenty-two of the 44 listed CT antigens,
including the principal CT-antigen cancer-vaccine candidates, are CT-X antigens. In normal testes the CT-X
genes are generally expressed in the SPERMATOGONIA,
which are proliferating germ cells30 (FIG. 2). The genes
tend to form recently expanded gene families associated
with inverted DNA repeats. Remarkably, it is estimated
that 10% of the genes on the X-chromosome belong to
CT-X families40.
The genes for the non-X CTs, on the other hand,
are distributed throughout the genome and do not
generally form gene families or reside within genomic
repeats. In the testis, their expression appears more
dominant in later stages of germ-cell differentiation,
such as in SPERMATOCYTES28,41–43 TABLE 2.
Box 1 | Nomenclature of the cancer/testis antigens
SPERMATOGONIA
The germ cells of
spermatogenesis. Unlike
mammalian oogonia, they
continue to divide throughout
life and give rise to
spermatocytes
Over recent years, a growing list of genes for cancer/testis (CT) antigens has been compiled. In order to create a
coherent picture, a CT-antigen nomenclature has been established as a supplement to the names of the genes. Owing
to a general lack of information regarding the function of CT gene products, this nomenclature is based on the
chronological order of their discovery — for example, MAGEA is CT1; BAGE is CT2 etc. Many CT antigens on the
X chromosome form recently expanded gene families within tandemly repeated DNA domains. As shown in
TABLE 1, 22 CT antigens map to chromosome X, with twelve of them clustering to the telomeric end of the q arm
between q24–28. The family members typically encode proteins with very high sequence similarity. Individual
family members are designated numerically; for example, SSX1 is CT5.1; SSX2 is CT5.2; SSX3 is CT5.3, and so on.
In cases where there are multiple isoforms of a single gene, individual splice variants are designated alphabetically;
for example, XAGE-1a is CT12.1a; XAGE-1b is CT12.1b; and XAGE-3a is CT12.3a. Finally, in deference to the
original discovery, the nomenclature includes the gene identifier (for example, SCP1/CT8).
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a
b
c
d
Figure 2 | Cancer/testis antigen expression in normal and tumour tissues.
a | Immunohistochemical staining of cancer/testis (CT) antigens shows that testicular germ
cells express MAGEA4 (determined by immunoreactivity with antibody 57B). b | Analysis of
CT7/MAGEC1 expression (determined by immunoreactivity with antibody CT7-33) in fetal
ovary is indicated by streaks of immunopositive germ cells (oocytes of the primordial follicle).
c | Expression of MAGEA in trophoblastic epithelia of placental villi (determined by
immunoreactivity with antibody M3H67). d | Expression of NY-ESO-1 (determined by
immunoreactivity with antibody E978) in urinary bladder carcinoma. In all cases antibody
binding is indicated by brown staining.
CT antigen gene expression in tumours
SPERMATOCYTES
Diploid cells that undergo
meiosis to form four
spermatids. A primary
spermatocyte divides into two
secondary spermatocytes,
which in turn divide to form the
spermatids.
The expression of CT-X antigens varies greatly between
tumour types. According to RT-PCR analyses, bladder
cancer, lung cancer, ovarian cancer, hepatocellular
carcinoma and melanoma frequently express CT-X
antigens. By contrast, CT-X antigen expression is
rarely observed in renal cancer, colon cancer, gastric
cancer and leukaemia/lymphoma cells. The CT-X
mRNAs can be a dominant feature of the transcriptome in tumour cells. For example, in a microarray
study of lung cancer, 20 genes were identified as being
highly overexpressed compared with normal tissues44.
Box 2 | Cancer vaccines targeted against cancer/testis antigens
Cancer/testis (CT) antigens are ideal targets for cancer vaccines because of their
highly restricted expression patterns in normal tissues and their expression in a wide
range of human tumour types. In addition, CT antigens such as NY-ESO-1 can have
strong spontaneous immunogenicity in humans, inducing an integrated response
involving both cellular and humoral arms of the immune system. To construct
maximally immunogenic vaccines targeting CT antigens, a range of different vaccine
strategies are being explored and compared in ongoing clinical trials, including the
use of peptide, protein, DNA and RNA as well as viral and bacterial vectors. The
objective is to develop immunization protocols that consistently induce effector and
memory CD8+ and CD4+ T cells with high affinity for naturally processed CT
antigens and the capacity to home to tumours. Because of recent advances in
immunological monitoring technologies, the immune responses to cancer vaccines
can be dissected in great detail and the relation between vaccine induced immune
responses and therapeutic benefit can now be realistically assessed.
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Of these 20 genes, six were known CT antigens: five
members of the MAGEA gene family and NY-ESO-1.
Interestingly, analysis of the other overexpressed genes
revealed one that appeared to encode a novel CT antigen — Na+/K+-transporting ATPase, α-3-polypeptide.
Analysis of publicly available databases reveal that this
gene is expressed in placenta and testis and is absent
from all other normal adult tissues with the possible
exception of brain.
CT-X genes are frequently co-expressed, and
tumours that express them tend to express several CT-X
antigens. In a study of nine CT-X antigens expressed
by breast tumours and melanomas, it was found that
whereas 47% and 26% of breast tumours and melanomas, respectively, expressed none of the antigens
tested, 40% of breast tumours and 65% of melanomas
expressed three or more of these antigens45. Similarly,
in a study of lung tumours for expression of nine CT-X
antigens, 33% were reported to totally lack CT-antigen
expression, 10% expressed a single CT antigen, 57%
expressed two or more and 37% three or more of the
antigens analysed46. Moreover, the permutations of
antigens expressed are non-random — MAGEA3 is
almost always expressed in tumours that are positive
for CT-X antigen, irrespective of the presence of any
other CT antigens. On the other hand, NY-ESO-1 is
very rarely found in the absence of MAGEA3. All these
observations are consistent with CT-antigen expression
being the result of the activation of part, or parts, of a
coordinated gene-expression programme, rather than
being independent events.
A key element in the induction of CT-X gene
expression appears to be promoter demethylation.
Methylation of CpG islands within gene promoters is responsible for gene silencing, due to both its
effect on chromatin structure and to transcriptionfactor binding47. ‘Epigenetic reprogramming’, consisting of both alterations in DNA methylation and
chromatin re-structuring, occur during two phases
in the human life cycle: gametogenesis and early
embryogenesis48. So far, all CT-X genes studied have
methylated CpG islands in normal somatic tissues
and are activated by demethylation during spermatogenesis49–52. It has been shown that experimental
demethylation of CT-X gene promoters induces
antigen expression in cells that do not normally produce them49,50,52,53. Global DNA hypomethylation,
gene-specific hypomethylation and regional hypermethylation occur during tumorigenesis, and global
hypomethylation in tumours has been correlated
with CT-X expression. A recent study indicated that
in tumours, global genome hypomethylation, repetitive DNA hypomethylation and the hypomethylation
of the promoters of MAGE genes (leading to their
expression) are all associated with, but are independent of, the hypermethylation of tumour-suppressor
genes54. Nevertheless, hypomethylation alone is not
sufficient for the induction of CT-X gene expression,
as DNA in colon cancer cells, for example, is universally hypomethylated55, even though CT-X antigen
gene expression is rare in this tumour type.
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Table 1 | Characteristics of CT-X antigens
CT antigen
gene family
CT identifier
Number of genes
in family
Chromosome
location
Expression
during germline
maturation
Function
References
MAGEA
CT1
12
Xq28
Spermatogonia
Translational co-repressor
14
MAGEB
CT3
4
Xp21–p22
Migrating PGCs
Unknown
83
GAGE1
CT4
8
Xp11.4–p11.2
ND
Unknown
84
SSX
CT5
5
Xp11.23–p11.22
ND
Transcriptional repressor
27
NY-ESO-1
CT6
3
Xq28
Spermatogonia
Unknown
29
MAGEC1
CT7
2
Xq26, Xq27.2
ND
Unknown
18
MAGEC2
CT10
1
Xq27
ND
Unknown
19
CTp11/SPANX
CT11
4
Xq27.1
Spermatids
Unknown
85
XAGE1/GAGED
CT12
8
Xp11.22–p11.21
ND
Unknown
86
SAGE1
CT14
1
Xq26
ND
Unknown
87
PAGE5
CT16
2
Xp11.22
ND
Unknown
38
NA88
CT18
1
Xp22.12
ND
Unknown
88
IL13RA1
CT19
1
Xq24
ND
Receptor for interleukin-13
89
CSAGE
CT24
2
Xq28
ND
Unknown
90
CAGE
CT26
1
Xp22.13
Spermatids,
spermatozoa
Possible helicase
91
HOM-TES-85
CT28
1
Xq23
ND
Possible transcriptional
regulatory protein
92
E2F-like/HCA661
CT30
1
Xq26.2
ND
Transcription factor
93
NY-SAR-35
CT37
1
Xq28
ND
Unknown
94
FTHL17
CT38
1
Xp21
Spermatogonia
Possible ferritin heavy
polypeptide-like protein
95
NXF2
CT39
1
Xq22.1
Spermatogonia
mRNA export to the
cytoplasm
95
TAF7L
CT40
1
Xq22.1
Spermatogonia
Possible TATA box binding
protein-associated factor
71
FATE1
CT43
1
Xq28
ND
Unknown
96
CAGE, cancer antigen 1; CSAGE , chondrosarcoma-associated gene 1; FATE1, fetal and adult testis expressed 1; FTHL17, ferritin, heavy polypeptide-like 17;
GAGE1, G antigen 1; IL13RA1, interleukin-13 receptor-α1; MAGE, melanoma antigen; ND, not determined; NXF2, nuclear RNA export factor 2; NY-ESO-1, New York
oesophageal squamous cell carcinoma 1; NY-SAR-35, New York sarcoma 35; PAGE5, P antigen family, member 5; PGCs, primordial germ cells; SAGE1, sarcoma
antigen 1; SPANX, sperm associated with the nucleus, X chromosome; SSX, synovial sarcoma; TAF7L, TAF7-like RNA polymerase II, TATA box binding protein
associated factor; XAGE1, X antigen family, member 1
Immunohistochemical analysis has revealed that
CT antigens are rarely homogeneously expressed in
tumours30 (FIG. 2). They are frequently found in only a
relatively small proportion of the cells in a tumour —
a phenomenon that has yet to be fully understood but
which can be viewed in the light of growing awareness of tumours as ‘quasi-tissues’ that contain both
stem cells and differentiated cells. As CT-X antigens
are expressed in the spermatogonia, which include
the spermatogonial stem cells, it could be that CT
antigens serve as markers for cells with stem-cell-like
properties (cancer stem cells) within the tumour. The
absence of any immunohistochemical evidence for
focal CT-X antigen expression in normal somatic tissues indicates that normal somatic tissues stem cells
do not express CT antigens.
Similar information on the extent and control of
expression of non-X CT antigens is not available, but
examination of their mutual expression, as well as the
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relationship of their expression with the CT-X, will be
of significant interest.
Functions of CT-X antigens
Although CT-X antigens have taken centre stage in
the development and clinical testing of experimental
cancer vaccines56, their biological function in both
the germ line and tumours has remained poorly
understood. A central question is whether their
expression contributes to tumorigenesis or is a functionally irrelevant by-product of the process of cellular transformation, possibly due to global-chromatin
changes. Clues have emerged, however, to indicate
that expression of CT antigens such as MAGE could
have a fundamental role in human tumorigenesis.
The extensive MAGE family of CT antigens comprises more than 25 genes that are characterized by the
presence of a large central region termed the MAGE
homology domain (MHD)16. Genes containing MHDs
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are present in many multicellular organisms, including Drosophila melanogaster and Aspergillus nidulans,
but have not been found in Caenorhabditis elegans or
unicellular organisms. The MHD does not contain
any regions of significant homology with other known
proteins, but detailed analysis of a number of type II
MAGE proteins (non-CT) shows that this domain is an
important site of protein–protein interaction57.
So far, the only CT-X gene product for which
protein binding partners have been actively sought,
through a yeast two-hybrid assay, has been MAGEA1.
Using proteins expressed from a testis cDNA library,
Table 2 | Characteristics of non-X CT antigens
CT antigen
gene family
CT identifier
Number of genes
in family
Chromosome
location
Expression
during germline
maturation
Function
References
BAGE
CT2
5
13
Spermatogonia
Unknown
23
SCP1
CT8
1
1p13–p12
Spermatocyte
Structural component of
synaptonemal complexes
28
BRDT
CT9
1
1p22.1
ND
Possible transcriptional regulatory
protein
97
HAGE
CT13
1
6q12–q13
ND
ATP-dependent RNA helicase
87
ADAM2
CT15
1
8p11.2
Spermatocyte,
spermatids
Sperm–egg membrane binding
38
LIP1
CT17
1
21q11.2
ND
Membrane-associated
phospholipase
38
TSP50
CT20
1
3p14–p12
Spermatocyte
Protease
98
CTAGE1
CT21
2
18p11.2
ND
Unknown
99
SPA17
CT22
1
11q24.2
Spermatocyte,
round spermatids
Binds sperm to the zona pellucida
and other cell–cell adhesion
functions
100
OY-TES-1
CT23
1
12p13.31
Spermatocytes,
spermatids
Binds to proacrosin to mediate
packaging and condensation of the
acrosin zymogen in the acrosomal
matrix
101
DSCR8 (also
known as
MMA1)
CT25
2
21q22.2
ND
Unknown
102
BORIS
CT27
1
20q13.31
Spermatocyte
Contains 11 zinc fingers, possible
transcriptional regulatory protein
103
AF15q14
(also known
as D40)
CT29
1
15q14
ND
Possible suppressor of cell
proliferation
104
PLU-1
CT31
1
1q32.1
Spermatogonia
Transcriptional repression
78
LDHC
CT32
1
11p15.5–p15.3
Spermatocyte
Catalyses the conversion of
L-lactate and NAD to pyruvate and
NADH in the final step of anaerobic
glycolysis
39
MORC
CT33
1
3q13
Spermatogonia
Role in spermatogenesis, possibly
by affecting entry into apoptosis
105
SGY1
CT34
1
19q13.33
ND
Role in signal transduction
106
SPO11
CT35
1
20q13.2–q13.3
Spermatocyte
Formation of double-strand breaks
in paired chromosome homologues
74
TPX1
CT36
1
6p21–qter
Spermatocyte
Mediates the binding of
spermatogenic cells to Sertoli cells
107
TDRD1
CT41
2
10q26.11
Spermatogonia
RNA-binding protein
95
TEX15
CT42
1
8p12
Spermatogonia
Unknown
95
TPTE
CT44
1
21p11
Secondary
spermatocyte and/
or prespermatids
PTEN-related tyrosine phosphatase
96
ADAM2, a disintegrin and metalloproteinase domain 2; BAGE, B melanoma antigen; BORIS, brother of the regulator of imprinted sites; BRDT, Bromodomain, testis
specific; CTAGE1, cutaneous T-cell lymphoma-associated antigen 1; DSCR8, Down syndrome critical region gene 8; LDCH, lactate dehydrogenase C; LIP1, lipase,
member 1; MMA1, malignant melanoma associated protein 1; MORC, microchordia homologue (mouse); ND, not determined; SCP1, synaptonemal complex protein 1;
SPA17, sperm autoantigenic protein 17; SPO11, Sporulation protein, meiosis specific; TDRD1, tudor domain containing 1; TPX1, testis specific protein 1; TEX15, testis
expressed sequence 15; TPTE, transmembrane phosphatase with tensin homology; TSP50, testes-specific protease 50.
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PACLITAXEL
A naturally occurring
compound, originally purified
from the pacific yew tree, that
stabilizes microtubules and has
antitumour activity.
DOXORUBICIN
A chemotherapeutic drug that
induces breaks in DNA strands,
which initiates apoptosis.
PACHYTENE
The third stage of the prophase
of meiosis. In this phase the
homologous chromosomes
become short and thick and
divide into four distinct
chromatids.
the assay identified the transcriptional regulator SKIinteracting protein (SKIP) as a binding partner for
MAGEA158. Binding of MAGEA1 to SKIP depends
on the extreme carboxyl terminus of the MAGE protein, a domain shared with MAGEA4, which was also
shown to be able to bind to SKIP. SKIP connects DNAbinding proteins to other proteins that either activate
or repress transcription, and participates in a range
of signalling pathways, including those involving
vitamin D, retinoic acid, oestrogens, glucocorticoids,
NOTCH1 and trasforming growth factor-β.
In the NOTCH1 pathway, MAGEA1 was found
to disrupt SKIP-mediated NOTCH1 signal transduction by binding to SKIP and recruiting histone
deacetylase. Therefore, at least in this signalling pathway, MAGEA1 can act as transcriptional repressor58.
The function of MAGEA1 in the germ line has not
been elucidated, but it is clear that pathways acting
through SKIP are involved. It is likely that MAGEA1
represses the expression of genes required for differentiation, as it is expressed in spermatogonia but
not during later developmental stages. A similar
function in the inhibition of cellular differentiation
in cancer cells could make an important contribution
to tumorigenesis.
Yeast two-hybrid studies using other cancer-related
genes as bait have twice pulled out MAGE proteins:
MAGEA11 and MAGEA4. MAGEA11 was found to
have a role in the regulation of androgen-receptor
function by modulating its internal domain interactions59. MAGEA11-stabilized ligand-free androgen
receptor in the cytoplasm, leading to its accumulation
and, in the presence of agonist, increasing the exposure
of the amino-terminal ligand-binding domain of the
receptor to the recruitment of SRC/p160 co-activators.
So, MAGE was found to have a dual amplifying effect
on androgen signalling.
MAGEA4 was identified60 in a search for binding
partners of the oncoprotein gankyrin. Both MAGEA4
and gankyrin are frequently overexpressed in hepatocellular carcinomas61. Gankyrin destabilizes the
retinoblastoma tumour suppressor, contributing to
unscheduled entry into the cell cycle and escape from
cell-cycle arrest and/or apoptosis62. MAGEA4 suppresses the oncogenic activity of gankyrin through
the action of a peptide that is naturally cleaved from
the carboxyl terminus of MAGEA4, which induces
p53-dependent and p53-independent apoptosis. The
fact that this action is dependent on a cleavage product
indicates that MAGEA4 has additional functions. It
is also worth noting that the same carboxy-terminal
region of MAGEA4 can bind to SKIP.
So it appears that MAGE genes encode multifunctional regulator molecules that exert a range
of effects. This is consistent with the more clearly
defined functions of those MAGE genes that are
widely expressed in somatic cells. To take a single example, the MAGE family member necdin
is a potent suppressor of cell proliferation that is
expressed predominantly in post-mitotic cells,
such as neurons and skeletal muscle cells. Necdin
NATURE REVIEWS | C ANCER
interacts through its MHD with several oncoproteins, tumour-suppressor proteins, the SV40 large
antigen, adenovirus E1A, E2F1, E2F4, p53, NEFA,
heterogeneous nuclear ribonucleoprotein U and with
the p75 neutrophin receptor63–65. It will be important
to determine whether other MAGE family members
that are CT antigens interact, through their MHD,
with any of these gene products. Moreover, necdin’s
range of molecular interactions has been extended
to the D1x/Msx homeodomain proteins, which are
key transcription factors for cellular differentiation.
Necdin interacts with the Msx homeodomain proteins, by first interacting with another MAGE protein
(MAGED1) through the MHD domains of the two
MAGE proteins. MAGED1 then interacts directly
with the Dlx/Msx homeodomain proteins, resulting
in their functional modulation.
Importantly, recent data indicate that expression of
MAGE genes in cancer cells contributes directly to the
malignant phenotype and response to therapy. In one
study, for example, the relationship between sensitivity to tumour-necrosis factor (TNF) and MAGEA1,
MAGEA2 or MAGEA3 expression was examined in
a range of human cell lines66. It was found that cell
lines that express at least one of the three MAGE
genes were more resistant to TNF-mediated cytotoxicity. In addition, transfection and stable expression
of MAGEA1 in ME-180 cervical carcinoma cells
resulted in a diminished sensitivity to TNF-mediated
cytotoxicity. Other experiments have indicated that
MAGE expression might contribute to the malignant
phenotype, in that overexpression of MAGEA2 or
MAGEA6 genes leads to the acquisition of resistance
to the widely used chemotherapeutic drugs PACLIT
AXEL and DOXORUBICIN in human cell lines. Resistance
to drugs such as these is typical of aggressive cancer
phenotypes67. Transfection of cells with MAGEA2 or
MAGEA6 genes also confers a proliferative advantage, although there has been no insight as to what
the molecular mechanism of these effects might be68.
Other studies have provided independent evidence
for an association between MAGEA3 expression and
doxorubicin resistance69.
Outside the MAGE-gene family, there is one report
that indicates a CT antigen contributes directly to a
crucial element of malignant transformation — the
inhibition of apoptosis. In this study, it was found
that members of the GAGE family, GAGE7C or
GAGE7B, conferred resistance to apoptosis induced
by either interferon-γ or by the death receptor FAS
(also known as CD95/APO-1) when the GAGE genes
were transfected into HeLa cells70.
With regard to other CT-X genes, detailed functional
information is available in some instances. For example,
the CT-X protein TAF7L is a paralogue of the somatic
transcription factor TFIID subunit TAF7, and is developmentally regulated during male germ-cell differentiation71. Researchers have suggested that TAF7L replaces
its somatic counterpart during the PACHYTENE stage of
germline development, so that TFIID composition
during spermatogenesis is unique. TAF7L is likely to
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REVIEWS
be translated from stored mRNA, as the TAF7L gene is
encoded on the X chromosome, which is not expressed
during the stages at which TAF7L is found. Other
X-linked CT antigens might also have roles in the control of gene expression. For example, there is evidence
that the SSX genes are also transcriptional repressors72,
Box 3 | Schematic summary of spermatogenesis in humans
The germ line derives from the PRIMORDIAL GERM CELLS (PGCs; see figure). These migrate through the dorsal
mesentery of the embryo and enter the developing fetal gonad in the genital ridge. In the genital ridge, in males the
PCGs are enclosed by somatic Sertoli cells and become prospermatogonia, which proliferate for a few days and then
arrest at G0/G1 until birth. At purberty, proliferation is resumed to initiate spermatogenesis. Spermatogonia, the
male germline stem cells, remain proliferative throughout adult life, and are diploid (n = 2 in diagram).
Spermatogonia can either renew themselves to maintain the pool of stem cells or undergo differentiation to produce
spermatozoa. This occurs through two meiotic divisions, in which tetraploid (n = 4 in diagram) primary
spermatocytes undergo meiosis I to form diploid secondary spermatocytes, and then undergo meiosis II to form
haploid spermatids (n = 1 in diagram), which in turn develop into spermatozoa. Many cancer/testis (CT) antigens,
including most CT-X antigens such as MAGE (melanoma antigen) and NY-ESO-1 (New York oesophageal
squamous cell carcinoma 1; red boxes), are expressed in spermatogonia, whereas those associated with meiosis, such
as synaptonemal complex protein 1 (SCP1), are expressed in the spermatocytes. A few of these, such as OY-TES-1/
ACRBP (Acrosin-binding protein), are also expressed by spermatids. CT-X antigens expressed during
spermatogenesis are shown in red boxes, whereas non-X CT-antigen expression is shown in blue boxes.
SPAN-X
MAGEB
Migration
to the
genital ridge
SPANX
MAGEA
NY-ESO-1
FTHL17
NXF2
TAF7L
FATE
Mitosis
Growth
Meiosis I
Meiosis II
Spermiogenesis
PGCs
Spermatogonia
ploidy n = 2
BAGE
PLU-1
MORC
TDRD1
TEX15
Primary spermatocytes
ploidy n = 4
SCP1
ADAM2
TSP50
SPA-117
OY-TES-1
BORIS
LDHC
SPO11
TPX1
TPTE
Secondary spermatocytes
ploidy n = 2
Spermatids
Ploidy n = 1
ADAM2
SPA17
TPTE
OY-TES-1
Spermatogonial stage
Mitotic clonal expansion of germ cells
PRIMORDIAL GERM CELLS
The progenitor cells of
gametogenesis.
622 | AUGUST 2005
Meiotic stage
Chromosome replication,
pairing and recombination
Reduction division
Results in haploid
germ cells
Spermatozoa
ploidy n = 1
Spermiogenesis
Non-dividing,
post-meiotic germ cells
undergo morphological
differentiation
ADAM2, a disintegrin and metalloproteinase domain 2; BAGE, B melanoma antigen; BORIS, brother of the regulator of
imprinted sites; FATE, fetal and adult testis expressed; FTHL17, ferritin, heavy polypeptide-like 17; LDHC, lactate
dehydrogenase C ; MAGE, melanoma antigen; MORC, microchordia homologue (mouse); NXF2, nuclear RNA export factor 2;
NY-ESO-1, New York oesophageal squamous cell carcinoma; SCP1, synaptonemal complex protein 1; SPA17, sperm
autoantigenic protein 17; SPANX, sperm associated with the nucleus, X chromosome; SPO11, Sporulation protein, meiosis
specific; TAF7L, TAF7-like RNA polymerase II, TATA box binding protein associated factor; TDRD1, tudor domain
containing 1; TEX15, testis expressed sequence 15; TPTE, transmembrane phosphatase with tensin homology; TPX1, testis
specific protein 1; TSP50, testis specific protease 50.
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and the nuclear RNA export factor 2 (NXF2) CT protein
is thought to act at the level of translational control as a
transporter of RNA to the cytoplasm73.
to be explored, and most of our knowledge is derived
from the study of adult male germ cells.
Germline genes and tumorigenesis
Functions of non-X CT antigens
SYNAPTONEMAL COMPLEX
A protein structure that forms
between two homologous
chromosomes during meiosis
and that mediates chromosome
pairing, synapsis and
recombination. The
synaptonemal complex is a
tripartite structure, consisting
of two parallel lateral regions
and a central element.
TRANSVERSE FILAMENTS
Proteins that connect the two
lateral elements of the
synaptonemal complex.
ANEUPLOIDY
The state of having an abnormal
number of chromosomes. Most
human epithelial cancers
harbour genomes that are
characterized by gross
aneuploidy.
KARYOTYPE
A complete description of the
chromosomes present in a cell;
characterized by numerical and
structural abnormalities in most
cancers.
DIPLOTENE
The stage of the first meiotic
prophase, following the
pachytene, in which the two
chromosomes in each bivalent
begin to repel one another and a
split occurs between the
chromosomes, which are then
held together by regions where
exchanges have taken place
(chiasmata) during crossing
over.
SPERMATID
Any of the four haploid cells
formed by meiosis in a male
organism that develop into
spermatozoa without further
division.
The expression of several of the non-X CT-antigen genes
in the testis coincides with meiosis, and some of these
gene products function during this process BOX 3. For
example, the non-X CT antigens SCP1 and SPO11 are
components of the SYNAPTONEMAL COMPLEX. SPO11 causes
double-stranded DNA breaks, possibly functioning to
initiate recombination events during meiosis74. SCP1, on
the other hand, is thought to make up the TRANSVERSE FILA
75
MENTS of the synaptonemal complex . The admixture of
meiotic proteins such as those expressed from non-X CT
genes and mitotic-specific proteins in cancer cells might
be expected to lead to abnormal chromosome segregation and ANEUPLOIDY, a hallmark of human cancer and
a driver of genomic instability, although this has yet to
be directly tested. However, it has been shown that the
expression of SCP1 in COS cells (transformed monkey
fibroblasts) leads to the formation of synaptonemal
complexes, raising the possibility of perturbation of the
mitotic process in cancer cells with aberrant expression
of SCP1. Unfortunately, the KARYOTYPE of COS cells is
aneuploid, precluding accurate assessment of the effect
of the presence of SCP1 on chromosome structure76.
The non-CT-Xs exhibit a spectrum of functions. For
example, PLU-1 is a transcriptional co-repressor that
acts in concert with the transcription factors BF-1 and
PAX9 (paired box gene 9) to regulate gene expression
in the germ line77. All three factors are co-expressed
during early mouse embryogenesis, and PLU-1 and
PAX9 influence gene expression through alteration of
chromatin structure. PLU-1 is most highly expressed
in pre-meiotic spermatogonia, as well as pachytene and
DIPLOTENE spermatocytes, where it is thought to repress
the expression of genes that would otherwise cause the
spermatogonia to remain in the mitotic phase78. PLU1 and PAX9 are also overexpressed in tumours and in
lymph nodes that contain malignant breast cells77.
The product of another non-CT-X gene, BRDT,
causes chromatin to compact following acetylation
of histones79, and is thought to act at the stage of the
elongating SPERMATIDS. And lastly, there are non-CT-X
antigens, such as TPX1 and ADAM2 (a disintegrin
and metalloproteinase domain 2), that are expressed
on the cell surface. TPX1 encodes a protein that serves
to attach spermatogenic cells to the surrounding Sertoli
cells in the testis80 and the metalloproteinase ADAM2
participates in sperm–egg membrane binding 81. In
addition, we recently identified the testis-specific anion
transporter SLCO6A1 as a novel CT antigen82.
Although our knowledge of the function of CT-X
and non-X-CT antigens in both normal and malignant cells is rudimentary and fragmented, it is already
clear that their products influence a range of cellular
processes, including signalling, transcription, translation and chromosomal recombination. The expression profiles of CT antigens in embryonic and fetal
development, and in female gametes have only begun
NATURE REVIEWS | C ANCER
Cancer is a genetic disease that is dependent on the
accumulation of mutations that alter the gene expression
profiles of normal stem-cells, rendering these already
continuously replicating cells malignant. This mechanism involves key mutations in tumour-suppressor genes
and oncogenes that are expressed and functional in normal cells from which the tumours arise. By contrast, CT
antigen expression is aberrant in the sense that proteins
normally restricted to one lineage are now expressed in
another. Such aberrant expression is rare in cancer where
lineage programme fidelity is the rule.
What then is the basis and significance of the coordinated expression in cancer of genes limited to the
gametic differentiation programme? We propose that
the aberrant expression of these genes by cancer cells
confers a range of phenotypic traits that are essential for
the survival and function of gametes and their descendents. These gamete-specific products would be deleterious for the orderly requirements of normal somatic
cells, but highly advantageous for the cancer cell.
In our model, normal stem cells become altered
not in the genes that directly control the cell cycle and
proliferation, but in genes that control germ-cell gene
expression. Candidates for this role are genetic master
switches that control gametogenesis, the activation of
which would lead to the widespread expression of the
normally silent and forbidden germline-specific genes
in somatic cells. These genes remain to be identified,
but important candidates are those that directly control
genome demethylation.
Perspectives
Our immediate goals must be to delineate in greater
detail the gene-expression pathways that operate during
gametogenesis, and to undertake a systematic comparison of the expression of these genes in tumours. Further
examination of the functions of individual molecules,
in the context of both normal germline expression
and in cancer can be performed using a range of
techniques, such as yeast two-hybrid systems, short
interfering RNA knockdowns, whole animal knockout
models, cell-level overexpression and whole animal
knock ins, using both in vivo and in vitro models. The
crucial experiments are those that will test whether CTantigen genes are tumorigenic through their individual or coordinated expression in non-transformed
cell lines, and whether features generally associated
with malignancy are acquired by cells that aberrantly
express CT antigens. This line of enquiry could reveal
that malignant transformation results not only from the
subtle alteration of pre-existing signalling pathways, but
by the activation of entire gene-expression programmes
through as yet unknown mutations. In addition, it will
lead to the identification of a novel set of therapeutic
targets with highly restricted expression in normal
cells. These targets could be susceptible to a range of
treatment modalities beyond cancer vaccines.
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Acknowledgements
We thank C. Pentlow for her diligent literature searches and
preparation of background material used in the preparation of this
review.
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
ADAM2 | BAGE | BRDT | GAGE1 | MAGEA1 | MAGEA11 |
MAGEA2 | MAGEA3 | MAGEA4 | MAGEA6 | MAGED1 | NXF2 |
NY-ESO-1 | PAX9 | PLU-1 | SCP1 | SKIP | SLCO6A1 | SPO11 |
TAF7L | TPX-1
National Cancer Institute: http://www.cancer.gov/
bladder cancer | lung cancer | melanoma | ovarian cancer
FURTHER INFORMATION
Cancer/Testis Gene Database: http://www.cancerimmunity.
org/CTdatabase/
Ludwig Institute for Cancer Research: http://www.licr.org/
Access to this interactive links box is free online.
VOLUME 5 | AUGUST 2005 | 625
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