Genes, chromosomes and the development of testicular germ cell tumors of adolescents and adults

REVIEWARTICLE

Genes, Chromosomes and the Developmentof Testicular Germ Cell Tumors of Adolescentsand Adults

Alan McIntyre,1 Duncan Gilbert,1 Neil Goddard,1 Leendart Looijenga,2 and Janet Shipley1*

1Molecular Cytogenetics,Section of Molecular Carcinogenesis,The Institute of Cancer Research,Sutton,Surrey,SM2 5NG,UK2Departmentof Pathology,Erasmus MC-University Medical Center Rotterdam,Daniel den Hoed Cancer Center,Josephine Nefkens Institute, 3000 DRRotterdam,The Netherlands

Testicular germ cell tumors (TGCTs) of adults and adolescents are thought to be derived from primordial germ cells or gono-

cytes. TGCTs develop postpuberty from precursor lesions known as intratubular germ cell neoplasia undifferentiated. The

tumors can be divided into two groups based on their histology and clinical behavior; seminomas resemble primordial germ

cells or gonocytes and nonseminomas resemble embryonic or extraembryonic tissues at various stages of differentiation. The

most undifferentiated form of nonseminoma, embryonal carcinoma, resembles embryonic stem cells in terms of morphology

and expression profiling, both mRNAs and microRNAs. Evidence supports both environmental factors and genetic predisposi-

tion underlying the development of TGCTs. Various models of development have been proposed and are discussed. In TGCTs,

gain of material from the short arm of chromosome 12 is invariable: genes from this region include the proto-oncogene KRAS,

which has activating mutations in �10% of tumors or is frequently overexpressed. A number of different approaches to

increase the understanding of the development and progression of TGCTs have highlighted the involvement of KIT, RAS/RAF/

MAPK, STAT, and PI3K/AKT signaling. We review the role of these signaling pathways in this process and the potential influ-

ence of environmental factors in the development of TGCTs. VVC 2008 Wiley-Liss, Inc.

INTRODUCTION

Testicular germ cell tumors of adults and adoles-

cents (TGCTs) are the most common tumor in

male Caucasian patients aged 15–34 years (Hor-

wich et al., 1991) and account for 60% of all male

malignancies between the ages 20–40 years

(Ulbright, 1993). It is a major cause of death in

these age groups, despite its overall curability.

TGCTs can be classified into two main histological

subtypes, seminoma (SE) and nonseminoma (NS).

SE resembles primordial germ cells (PGC) or early

gonocytes, the cells from which all TGCTs are

thought to be derived. NS exhibits various stages

of embryonic differentiation ranging from undiffer-

entiated cells (embryonal carcinoma, EC), which

resemble embryonic stem cells to the highly differ-

entiated cells of somatic tissue types in teratomas

(Mostofi, 1973). NS can also resemble extraem-

bryonic tissues namely yolk sac tumors and chorio-

carcinomas. In addition, TGCTs may present with

a mixture of seminomatous and nonseminomatous

elements. SE and the EC component of NS

express markers of pluripotency including OCT3/

4, STELLAR, and NANOG (Oosterhuis and Looi-

jenga, 2005). Expression profiling data has pro-

vided evidence for similarities between embryonic

stem cells and TGCTs, in particular the EC type

(Sperger et al., 2003). Recently, a similar observa-

tion was also found based on microRNAs expres-

sion profiling (Gillis et al., 2007; Looijenga et al.,

2007).

The most widely accepted model of TGCTs de-

velopment proposes an initial tumorigenic event in

utero and the development of a precursor lesion

known as intratubular germ cell neoplasia undiffer-

entiated (ITGCNU), also known as carcinoma in

situ (Skakkebaek, 1972). This is followed by a

period of dormancy until after puberty when

TGCTs emerge. This prepubertal dormancy sug-

gests that the TGCTs development is hormone

dependant, a factor which may differentiate the

pediatric and adult cases. However, recently

evidence has emerged to suggest potential mecha-

nisms for transformation of later cell types of the

spermatogenic lineage (discussed in detail later).

*Correspondence to: Janet Shipley, Molecular Cytogenetics,MUCRC, The Institute of Cancer Research, 15 Cotswold Rd,Sutton, Surrey, SM2 5NG, UK. E-mail: [email protected]

Received 19 September 2007; Accepted 18 February 2008

DOI 10.1002/gcc.20562

Published online 31 March 2008 inWiley InterScience (www.interscience.wiley.com).

VVC 2008 Wiley-Liss, Inc.

GENES, CHROMOSOMES & CANCER 47:547–557 (2008)

Genetic aberrations in addition to the tetraploidy

found in ITGCNU are associated with progression

out of the seminiferous tubules and development

of invasive tumor.

Here, we review the impact that environmental

factors and genetic predisposition may have on

TGCTs development. We also consider the gene

products and pathways known to play a role in the

development of TGCTs and their progression from

ITGCNU. In particular, we draw comparisons

between the molecular biology and associated cel-

lular behavior of TGCTs and normal germ cell

development.

ENVIRONMENTAL FACTORS

Evidence of a role for environmental factors in

the etiology of TGCTs comes predominantly from

population migration studies. Sweden has an inci-

dence of TGCTs roughly twice that of Finland

(Parkin and Iscovich, 1997), and although first gen-

eration migrants from Finland to Sweden show no

increased risk (Ekbom et al., 2003), second genera-

tion males born to the migrant parents in Sweden

have a tendency to an increased frequency of

developing TGCTs (Montgomery et al., 2005). A

number of environmental factors have been inves-

tigated to explain the possible links with TGCTs-

development. Some evidence suggests association

of increased TGCTs risk and maternal smoking

during pregnancy, adult height, diet rich in cheese,

dizygotic twins, birth order, and sibship size

(Bonner et al., 2002; Dieckmann and Pichlmeier,

2002; Garner et al., 2003; Kaijser et al., 2003; Pet-

tersson et al., 2004; Richiardi et al., 2004a; Swer-

dlow et al., 1997), but underlying biological mecha-

nisms are unclear.

The strongest association of TGCTs exists with

disorders of genitourinary development including

cryptochidism or undescended testis (UDT), poor

sperm quality, and/or hypospodias (Mostofi, 1973;

Giwercman et al., 1988; Ondrus et al., 1997; Jacob-

sen et al., 2000). Collectively these disorders com-

prise a spectrum with TGCTs termed testicular

dysgenesis syndrome (TDS). This is increasingly

common and is proposed to be caused by genetic

and/or environmental influences (Skakkebaek

et al., 2001; Skakkebaek, 2002; Skakkebaek, 2003;

Skakkebaek et al., 2003). Hypothesized environ-

mental agents include pesticides (Garcia-Rodri-

guez et al., 1996) and nonsteroidal estrogens such

as diethylstilbestrol (DES) (Strohsnitter et al.,

2001). Increased levels of estrogen exposure in

utero have been proposed to increase the risk of

TDS and TGCTs (Weir et al., 2000; English et al.,

2003; Sharpe, 2003), and exposure of women to the

nonsteroidal estrogen DES during pregnancy

increases the risk of TGCTs (Strohsnitter et al.,

2001). In rats, administration of estradiol or ethinyl

estradiol (steroidal estrogens) during pregnancy

increases the rate of cryptorchidism and possibly

increases the risk of testicular teratoma (Lassur-

guere et al., 2003). Other studies however find no

role for high levels of natural estrogen increasing

TGCTs incidence, including two studies where

higher levels of estrogen were found in ethnic

groups with lower incidences of TGCT (Die-

ckmann et al., 2001; Hsieh et al., 2002; Zhang

et al., 2005).

GENETIC PREDISPOSITION

Familial predisposition to TGCTs, ethnic varia-

tions in incidence, and an association with certain

chromosome abnormality syndromes suggest that

inherited factors also play a role in disease develop-

ment. The familial predisposition seen in TGCTs

is one of the strongest for any tumor type. The

increased relative risk of TGCTs development

associated with fathers and sons of TGCTs patients

is fourfold, while between brothers it is higher at 8-

to 10-fold (Forman et al., 1992). Genome-wide

linkage analysis of affected families has thus far

provided evidence for two susceptibility loci, one

at Xq27 (which is possibly an indirect effect,

explained by being a susceptibility locus for unde-

scended testis (UDT which is part of TDS) and

another at 12q (Rapley et al., 2000; Rapley et al.,

2003). It is hypothesized that a genetic polymor-

phism at the Xq27 locus is responsible for the

genetic predisposition, although this remains to be

identified (Rapley et al., 2003). It is probable that

both genetic and shared environmental factors pro-

duce the high familial risk seen in TGCTs and that

the interplay between these two factors, along with

genetic heterogeneity, may make familial associ-

ated susceptibility loci difficult to determine.

TGCTs incidence varies among different racial

groups with the highest rates among Caucasian of

the USA and Western Europe and much lower

rates among Black Americans, Asians, Puerto

Ricans, and Africans (Waterhouse, 1985; Moul

et al., 1994; McGlynn et al., 2003). Variation in

incidence exists between countries in Europe

although this may either be due to racial or envi-

ronmental factors. Denmark has the highest inci-

dence of TGCTs, at 15.4 per 100,000, followed by

Norway and Sweden, whereas Lithuania has a

much lower incidence of 2.1 per 100,000 (Richiardi

et al., 2004b).

Genes, Chromosomes & Cancer DOI 10.1002/gcc

548 MCINTYRE ETAL.

Down and Klinefelter syndromes are associated

with an extra chromosome 21 and X, respectively

and have been identified as predisposing factors to

SE and NS, although tumors in Klinefelter syn-

drome patients are not of the testis, but of the an-

terior mediastinum (Hasle et al., 1995; Satge et al.,

1997). Down syndrome males also have increased

incidence of UDT (Chew and Hutson, 2004). It is

likely that the increased risk of TGCTs may be

due to gene dosage effects of the chromosome 21

trisomy and/or hormonal disturbance (Satge et al.,

1997). In a study of Chinese patients with testicu-

lar dysgenesis and spermatocytic arrest, chromo-

some 21 aberrations were commonly found (Guo

et al., 2002), suggesting that genes within this chro-

mosome may be required for normal testicular and

gonocyte development and that overexpression of

these genes may result in testis malformation and/

or ITGCNU development. The incidence of

mediastinal germ cell tumors in males with Kline-

felter syndrome, which is associated with TDS, is

50 times greater than in the normal population

(Lanfranco et al., 2004; Aguirre et al., 2006).

MODELS OF TGCTs DEVELOPMENT

The generally accepted model of TGCTs devel-

opment proposes that PGC or gonocytes form the

precursor lesion ITGCNU in utero, which post-

puberty develops into either SE or NS (Fig. 1).

Evidence to support the development of ITGCNU

from PGC includes similarities in gene expression

profiles (Almstrup et al., 2004), telomerase activity

(Albanell et al., 1999), and patterns of genomic

imprinting (van Gurp et al., 1994; Kawakami et al.,

2006). The presence of ITGCNU has been

reported in aborted trisomy 21 fetuses in two case

reports, one at Week 18 and the second at Week 22

of gestation (Jacobsen and Henriques, 1992; Satge

et al., 1997). In addition, extragonadal tumors

(which represent 5% SE and NS) develop at sites

along the midline of the body namely the pineal

gland, mediastinum, and retroperitoneum (Hor-

Figure 1. Differential gene expression in the germ cell lineage. ESC, embryonal stem cell; PGC, primor-dial germ cell; GC, gonocyte; SSC, spermatogonial stem cell; 1SC, primary spermatocyte; 2SC, secondaryspermatocyte; CIS, carcinoma in situ (ITGCNU); SE, seminoma; EC, embryonal carcinoma; MT, mature ter-atoma; CC, choriocarcinoma; YS, yolk sac tumor. Bars indicate gene expressed documented, ‘‘?’’ denotesgene expression status unknown.


549TESTICULAR GERM CELL TUMOR DEVELOPMENT

wich et al., 1991). ITGCNU has also been pro-

posed to develop from later stages of gonocyte de-

velopment at the zygotene-pachytene spermato-

cyte (Chaganti and Houldsworth, 2000). In addi-

tion, the recent discovery of a population of

pluripotent cells within mouse adult testis display-

ing features suggestive of a stem cell phenotype

(maGSCs) (Guan et al., 2006) raises the possibility

that TGCTs could arise from malignant transfor-

mation affecting this lineage.

Fifty percent of patients with ITGCNU develop

invasive lesions within 5 years of diagnosis (Ooster-

huis and Looijenga, 2005). Furthermore, it is pro-

posed that most if not all diagnosed ITGCNU pro-

gress to invasive tumor as ITGCNU frequency in

the population is equal to the frequency of TGCTs

(Linke et al., 2005). Molecular evidence support-

ing the development of invasive tumor from

ITGCNU includes conserved genetic lesions and

expression of histological markers. Shared genetic

aberrations include activating mutations of KIT(Looijenga et al., 2003) and gain of material from

12p, invariably found in the invasive component of

tumor and found in the adjacent ITGCNU from 8

of 29 samples examined (Looijenga et al., 2000;

Rosenberg et al., 2000; Summersgill et al., 2001;

Ottesen et al., 2003). Other genomic imbalances

frequently found in both the ITCGN and the inva-

sive tumor component using metaphase CGH

include gain of material from chromosomes 1, 5, 7,

and X, and loss of material from chromosome 18

(Summersgill et al., 2001). Histological markers

that can be expressed in both ITGCNU and the

invasive components include OCT3/4, placental

alkaline phosphatase, and KIT (Izquierdo et al.,

1995; Honecker et al., 2004).

Around 10% of TGCTs contain both SE and NS

(Mostofi, 1973) suggesting that ITGCNU may not

initially be subtype-specific. However, the associa-

tion of immunohistochemical markers in ITGCNU

with a particular histology suggests that the

ITGCNU may develop along pathways associated

more with a single subtype. Immunohistochemical

staining for a number of markers found differences

in the expression of antigens, particularly in

ITGCNU adjacent to combined tumors (SE and

NS elements) (Meyts et al., 1996). This study con-

cluded that ITGCNU is a phenotypically hetero-

geneous lesion and even adjacent cells may be at

different developmental stages (Meyts et al.,

1996). This might also be due to the plasticity of

the ITGCNU cells along the physiological germ

cell lineage. Other evidence that there is subtype-

specific development within ITGCNU includes

the association of chromosomal imbalances in the

ITGCNU and particular histologies. These in-

clude loss of material from chromosome 15 (NS

and ITGCNU adjacent to NS) and loss of hetero-

zygosity of 3q27–q28 (only ITGCNU adjacent to

EC) (Faulkner et al., 2000; Looijenga et al., 2000;

Summersgill et al., 2001; Ottesen et al., 2003).

It has also been suggested that the development

of TGCTs might follow a single pathway with pro-

gression from ITGCNU through SE to NS (Oliver,

1987; Oosterhuis et al., 1989). This might explain

the presence of NS elements reported in the me-

tastasis of pure SE primary tumors (Oliver, 1987)

but is inconsistent with the average age at develop-

ment of these tumors, with SE presenting 10 years

later than NS. Therefore, this linear progression

model is unlikely to apply to all NS (Oosterhuis

and Looijenga, 2005).

SE and NS form two distinct types of TGCTs.

EC represents NS at its most pluripotent stage,

with yolk sac, choriocarcinoma, or malignant tera-

tomatous elements arising from varying degrees

and paths of differentiation. These differences are

demonstrable at the level of gene expression (Juric

et al., 2005; Skotheim et al., 2005) and also micro-

RNA expression (Looijenga et al., 2007; Gillis

et al., 2007). MicroRNAs are short RNA molecules

that regulate gene expression and are associated

with processes including differentiation (Houbaviy

et al., 2003). SE express a number of pluripotency

genes (Skotheim et al., 2005) and EC are notable

for their similarity with embryonic stem cells

(Sperger et al., 2003). A cassette of these genes

located on chromosome 12 (NANOG, CD9, EDR1,SCNN1A, GDF3, Glut3, Stella) plus OCT3/4 on

chromosome 6 are repressed by retinoic acid,

resulting in loss of pluripotency (Giuliano et al.,

2005) and activation of homeobox genes (Mavilio

et al., 1988). OCT3/4 is an important controller of

this pluripotency, as demonstrated using siRNA

knockdown (Giuliano et al., 2005) and has been

linked with differentiation patterns early in

embryological development (Niwa et al., 2005).

Furthermore, phenotypic similarities between the

behavior of differentiating embryonic stem cells

and EC cells in culture correspond to karyotypic

changes common to both (Andrews et al., 2005).

A stage in tumor progression between ITGCNU

and overt TGCTs has been described, termed as

microinvasive germ cell tumor (MGCT). MGCT is

identified as a series of small groups or single

malignant germ cells in the peritubular interstitial

tissue and has a histological appearance similar to

that of SE whether associated with SE or NS.


550 MCINTYRE ETAL.

MGCT is found in a smaller proportion of cases

than ITGCNU (9/106 SE and 32/149 NS samples)

(von Eyben et al., 2004). There are few studies

examining this stage and little is known about its

molecular biology, although it does stain positively

for PLAP and KIT even if associated with embry-

onal carcinoma (von Eyben et al., 2004).

MOLECULAR BIOLOGYOF ESTROGENS IN

ITGCNU FORMATION

Given the evidence that TGCTs develop from

ITGCNU it can be assumed that factors that

increase the likelihood of developing TGCTs must

assert this effect by increasing the likelihood of

developing ITGCNU. Estrogens are a proposed

etiological factor in TGCTs development.

Research in the mouse has determined that estro-

gens stimulate PGC proliferation. This occurs

through the somatic cells of the testis, which in

response to the estrogen stimulation upregulate

stem cell factor (SCF) expression and secretion

leading to activation of the AKTsignaling pathway

in the PGC. This research also showed that expo-

sure to high levels of estrogen in conjunction with

factors that inhibit PGC differentiation (such as

leukemia inhibitory factor) can result in oncogenic

transformation of PGC; however, no ITGCNU or

SE was observed (Moe-Behrens et al., 2003). This

provides evidence of a molecular mechanism

whereby increased levels of estrogen during testic-

ular development can lead to TGCTs. This work

also determined that estrogen acts through

increased activation of KIT signaling through AKT

(Moe-Behrens et al., 2003). In addition, activating

mutations of KIT have been determined in

ITGCNU (Looijenga et al., 2003). This evidence

suggests that KIT activation may play a role in

ITGCNU development, but also requires other

factors in transformation of the PGC. Research has

determined activation of KIT, along with other fac-

tors, to be important in both proliferation and sur-

vival of PGC (De Miguel et al., 2002). Physiologi-

cally, KIT plays a key role in PGC proliferation (Li

et al., 2003), survival (De Felici, 2000), and migra-

tion where SCF, the ligand for KIT, is localized to

the membranes of somatic cells associated with the

PGC migratory pathway (Kierszenbaum and Tres,

2001). Only a few studies have examined the rela-

tionship between estrogen stimulation, KIT signal-

ing pathway activation, and the phenotypic effects

of this in PGC. Further work is required to fully

understand any role for these in transformation of

PGC. Investigations have also highlighted the im-

portance of AKTactivation in maintaining pluripo-

tency (Armstrong et al., 2006; Watanabe et al.,

2006), suggesting this signaling may have a role in

inhibiting the differentiation process of normal

PGC development. Further evidence for this

comes from in vivo and in vitro experiments with

mouse PGCs which lacked PTEN, a negative reg-

ulator of AKT activation. The PTEN-null PGCs

exhibited significant effects on the differentiation

state of germ cell lineage and the authors con-

cluded that PTEN appeared to be essential for

germ cell differentiation (Kimura et al., 2003).

KITAND RAS SIGNALING IN TGCTs

A number of investigations in PGC have high-

lighted the importance of signaling molecules

downstream of KIT and RAS in the phenotype of

these cells. In addition, some of these have been

investigated in cell lines providing further evi-

dence that these signaling pathways are of impor-

tance in TGCTs development. An overview of the

signaling interactions of these proteins within the

KIT and RAS signaling pathways can be seen in

Figure 2.

Specific amplification, overexpression, and mu-

tation of KIT have been reported in SE. These

changes are predominantly associated with SE,

however a small number of KIT-activating muta-

tions have been found in NS generally associated

with bilateral tumors (Looijenga et al., 2003;

Kemmer et al., 2004; McIntyre et al., 2004; Rapley

et al., 2004; McIntyre et al., 2005a; Willmore-Payne

et al., 2005). KIT expression is also predominantly

associated with SE but is also found in �30% of

NS where the staining is restricted to the cyto-

plasm unlike the SE which also show membranous

staining (Izquierdo et al., 1995). Significantly

experiments inhibiting KIT expression in the SE

cell line TCam-2 using siRNA resulted in a reduc-

tion in the number of viable cells over a time

course (Goddard et al., 2007). Activating mutation,

amplification, and overexpression of KRAS, whichsignals downstream of KIT, are also found in

TGCTs (Olie et al., 1995; McIntyre et al., 2005b).

Interestingly, activated KRAS resulted in and

increased in vitro survival of SE cells, which was

also found for the presence of a restricted 12p-

amplification (Roelofs et al., 2000). Both RAS and

KIT activate a number of signaling molecules

including PI3-kinase (PI3K). Activated RAS binds

directly to and activates the p110 catalytic subunit

of PI3K (Marte and Downward, 1997; Cox and

Der, 2003; Downward, 2003; Campbell and Der,

2004). Similarly upon stimulation, KIT activates

the p85 subunit of PI3kinase either through direct



interaction or through the signaling mediator Src

(Hong et al., 2004).

The tumor suppressor PTEN inhibits PI3K ac-

tivity. In PGC, upregulation of PTEN phosphoryl-

ation (resulting in deactivation of the protein) was

determined upon stimulation with SCF or estro-

gen. This work also demonstrated that reduction in

PTEN activity increased growth and sensitivity to

transformation of PGC upon the addition of growth

factors including SCF (Moe-Behrens et al., 2003).

Male mice with an engineered PGC-specific dele-

tion of PTEN develop bilateral testicular teratoma

(Kimura et al., 2003). That this occurred with early

onset implies it is unlikely that any additional

genetic alterations were required for the tumor for-

mation. This study also determined that PTEN

was important in differentiation of PGC to form

mature germ cells (Kimura et al., 2003). A study of

PTEN in five TGCTs cell lines revealed three with

LOH and one harboring a PTEN missense muta-

tion (Teng et al., 1997). PTEN loss has also been

implicated in the progression from ITGCNU to

invasive disease, with investigators finding loss/

decreased expression of PTEN in 56% SE and

86% NS. LOH and inactivating mutations of

PTEN were found at frequencies of 36 and 9%,

respectively (Di Vizio et al., 2005). In addition in-

hibition of PI3K, using wortmannin, reduced inva-

sive migration in an embryonal carcinoma cell line

(Diez-Torre et al., 2004).

Activation of PI3K results in its translocation to

the membrane and activation through secondary

messengers of a number of proteins including

AKT. AKT is therefore activated upon activation

of RAS and/or KIT (Marte and Downward, 1997;

Cox and Der, 2003; Downward, 2003; Campbell

and Der, 2004). Moe-Behrens et al. (2003) deter-

mined upregulation of AKT phosphorylation upon

stimulation with SCF or estrogens (which induced

upregulation of SCF secretion by the somatic cells

Figure 2. A model of RAS signaling in TGCTs. Striped moleculesdenote proteins for which aberrant expression or aberrant expressioncopy number and/or mutation of the gene that encodes the protein hasbeen observed in studies of TGCTs. This diagram was constructed withinformation from: Campbell and Der, 2004; Cox and Der, 2003; Di Vizioet al., 2005; Downward, 2003; Downward, 2004; Han et al., 2001; Hong

et al., 2004; Houldsworth et al., 1997; Houldsworth et al., 1998; Kamaiet al., 2002; Kemmer et al., 2004; Leaman et al., 1996; Li et al., 2003;Marte and Downward, 1997; McIntyre et al., 2005a; Miyagi et al., 2004;Murty and Chaganti, 1998; Ronnstrand, 2004; Sommerer et al., 2005;Teng et al., 1997; Wennerberg et al., 2005; Ye et al., 1993; Yu et al.,2005.


552 MCINTYRE ETAL.

of the testis) in PGC. This increase in AKTactiva-

tion was inhibited with the PI3K inhibitor

LY294002 (Moe-Behrens et al., 2003) (Fig. 2).

Overexpression of AKT dramatically increased

PGC growth in another study (De Miguel et al.,

2002).

A high level of (activated) phospho-AKT was

found in the majority of both NS and SE. How-

ever, no difference in AKT activation was deter-

mined between samples with or without KIT acti-

vating mutations suggesting that activation may be

achieved through other mechanisms. These could

include the functional loss of PTEN as described

earlier (Kemmer et al., 2004; Sommerer et al.,

2005). Activated AKT inhibits proapoptotic pro-

teins including BAD and, through its phosphoryl-

ating interactions with MDM2, inhibits P53 tran-

scriptional promotion of additional proapoptotic

genes including BAX (Downward, 2004). BAX is

required for both differentiation and apoptosis in

PGC (Stallock et al., 2003) and is upregulated in

PGC undergoing apoptosis in culture. Addition of

SCF inhibited an increase in BAX protein levels in

PGC in response to proapoptotic stimuli (Felici

et al., 1999). In TGCTs, the level of BAX protein

was reduced in a cisplatin-resistant cell line com-

pared to levels in a cisplatin-sensitive cell line

(Houldsworth et al., 1998).

RAS activation also upregulates the MAPK sig-

naling cascade, directly binding the RAF kinases.

Activating mutations of BRAF were found in 9% of

NS where they were restricted to the embryonal

carcinoma components (Sommerer et al., 2005).

However, another study which did not examine

the different components of the NS separately

found no BRAF mutations in 32 SE, 27 NS, and

6 combined tumors (McIntyre et al., 2005b).

Recently, an activating mutation has also been

found in the SE cell line TCam-2 (de Jong et al.,

2008). The interaction with activated RAS induces

the relocation of RAF to the plasma membrane

where it becomes phosphorylated by additional

factors, activating the downstream signaling cas-

cade. Activation of this cascade results in activation

of transcription factors which upregulate transcrip-

tion of genes (such as cyclinD1) promoting migra-

tion and proliferation (Marte and Downward, 1997;

Cox and Der, 2003; Downward, 2003; Campbell

and Der 2004; Yu et al., 2005). Inhibitors of MAPK

signaling reduce PGC numbers suggesting that the

MAPK signaling pathway is involved in promoting

survival and proliferation (De Miguel et al., 2002).

High levels of activated ERK (part of the MAPK

signaling cascade) were found in the majority of

both NS and SE (Kemmer et al., 2004; Sommerer

et al., 2005).

KIT is also known to activate members of the

STAT family of proteins, including STAT-3, -5A,

and -5B (Leaman et al., 1996; Ronnstrand, 2004).

Upon activation, STATs dimerize and translocate

to the nucleus where they regulate gene expres-

sion. STAT signaling is important for both migra-

tion and proliferation in PGC (Li et al., 2003).

High levels of activated STAT3 were found in

most NS and SE (Kemmer et al., 2004; Sommerer

et al., 2005). Experiments in zebrafish have sug-

gested that STAT-3 activates RhoA kinase signal-

ing (Miyagi et al., 2004), also activated by RAS in a

PI3K-dependent manner (Downward 2003; Wen-

nerberg et al., 2005). RhoA and Rho-kinase have

been reported to be overexpressed in TGCTs

(Kamai et al., 2002).

A number of additional growth factor receptors

have been investigated in TGCT. These, like

KIT, also have the ability to activate the down-

stream signaling pathways discussed here. Alterna-

tive splicing and alternative promoter use of

PDGFRA, which encodes a protein structurally

related to KIT, was identified in TGCT. This gives

rise to a 1.5-kb transcript which is a highly selec-

tive marker for TGCT and ITGCNU (Mosselman

et al., 1996). PDGFRA is adjacent to KIT on chro-

mosome 4 but is not part of the minimum region of

amplification of this imbalance (McIntyre et al.,

2005a). EGFR expression was found to correspond

to the b-HCG component of TGCT, associated

with choriocarcinoma, by immunohistochemistry.

However, only 27% of positive cases had activated

phosphorylation of EGFR (Moroni et al., 2001).

ERBB2 expression was detected in 24% of NS by

immunohistochemistry where positive staining was

associated with the teratoma and choriocarcinoma

components of the tumors and was significantly

correlated with both stage and adverse clinical out-

come (Mandoky et al., 2004). Another study found

consistent results with increased expression of

ERBB2 in NS of differentiated histology excised

from the lymph nodes of patients postchemother-

apy (McIntyre et al., 2005b).

In addition to receptors, a number of growth fac-

tors have also been investigated, of these possibly

the most interesting is VEGF. VEGF expression is

associated with angiogenesis and metastasis in

TGCTas shown at both the RNA and protein lev-

els (Viglietto et al., 1996, Fukuda et al., 1999).

Analysis of the expression of VEGF identified

predominant expression of the VEGF121 and

VEGF165 isoforms which are highly active in



inducing vascularization as they are more effi-

ciently secreted. Thus it is proposed that VEGF

induces angiogenesis in a paracrine manner in

these tumors (Viglietto et al., 1996). Further to

VEGF, a number of other growth factors have been

identified to have increased expression levels,

these include Pleotrophin and FGF2 which

showed increased levels of roughly 20- and 7-fold,

respectively, in the serum of TGCT patients, both

SE and NS, compared to controls. Similarly EGF

showed increased levels but to a lesser extent

(Aigner et al., 2003). The expression of FGF4

(HST-1) in TGCT conversely to KIT was associ-

ated more with NS where it also correlated with tu-

mor stage (Strohmeyer et al., 1991). FGF4 has

more recently been shown to act as a survival factor

in germ cells reducing apoptotic death in mice tes-

tis when exposed to mild hypothermia where

FGF4 increased MAPK activation (Hirai et al.,

2004). The teratocarcinoma-derived growth factor-

1 (TDGF-1), which is structurally related to the

EGF family of growth factors was overexpressed in

100% of NS and 31% SE compared to normal tes-

tis. The authors hypothesized TDGF-1 may act as

an autocrine growth factor as functional work

showed exogeneous TDGF-1 induced prolifera-

tion of the NT2/D1 TGCT cell line (Baldassarre

et al., 1997).

Taken as a whole, this evidence suggests that in

TGCTs the upregulation of RAS signaling is im-

portant and that in SE, KIT signaling pathways are

important in TGCTs, promoting the survival, inva-

sive migration, and proliferative phenoptype of

these cells. Furthermore, results indicate that this

is achieved through reactivation of signaling impor-

tant in maintaining the same phenotype in the de-

velopment of PGC, the precursor cells for this tu-

mor type. The implication of KIT and RAS signal-

ing in TGCTs and further unraveling of the

downstream pathways involved should provide

potential therapeutic targets (Pero et al., 2002;

Downward, 2003; Pero et al., 2004; Ross et al.,

2004).

CONCLUDING COMMENTS

It is clear that both environmental and genetic

factors play an important role in the development

of TGCTs. Current models propose these factors

cause the deregulation of the normal differentia-

tion processes of PGC and development of the tes-

tis in utero. Little light has been shed on the

development of the tumors at this stage. Aside

from the invariable gain of material from 12p, only

a few consistent copy number imbalances and asso-

ciated genes, such as amplification of KIT (McIn-

tyre et al., 2005a), have been identified and investi-

gated. Recent analysis supports DNA copy number

having a major impact on the gene expression lev-

els in TGCTs (Almstrup et al., 2005; McIntyre

et al., 2007). Evidence indicates that RAS and KIT

signaling play a role in the tumor phenotype in

common with PGC. Recapitulation or maintenance

of signaling molecules and pathways important in

PGC appear to be involved in TGCTs develop-

ment. Therefore, future studies of both PGCs and

TGCTs will complement our understanding of

both. More functional-based studies using RNAi-

based technologies and small molecular inhibitors

of the molecules and pathways discussed will fur-

ther increase our understanding of the role of this

signaling in TGCT. In addition, work using model

organisms further examining deregulation of these

signaling pathways in PGCs will enhance our com-

prehension of their role in tumor initiation.

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Genes, chromosomes and the development of testicular germ cell tumors of adolescents and adults

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