Recent Advances in Molecular and Cell Biology of Testicular Germ-Cell Tumors

CHAPTER THREE

Recent Advances in Molecular and Cell Biology of Testicular Germ-Cell Tumors Paolo Chieffi1 Department of Psychology II, University of Naples, Caserta, Italy 1 Corresponding author: e-mail address: [email protected]

Contents 1. 2. 3. 4.

Introduction Epidemiology and Risk Factors Histopathology Prognostic and Diagnostic Markers 4.1 Serum tumor markers in TGCTs 4.2 Newly discovered biomarkers detected by immunohistochemistry in TGCT subtypes 5. Therapy 5.1 Aurora-kinase inhibitors 5.2 Tyrosine-kinase inhibitors 5.3 Angiogenesis inhibitors 6. MicroRNAs in TGCTs 7. Conclusions and Perspectives References

80 80 81 84 84 85 90 90 91 94 95 96 96

Abstract Testicular germ-cell tumors (TGCTs) are the most frequent solid malignant tumors in men 20–40 years of age and the most frequent cause of death from solid tumors in this age group. TGCTs comprise two major histologic groups: seminomas and nonseminomas germ-cell tumors (NSGCTs). NSGCTs can be further divided into embryonal, carcinoma, Teratoma, yolk sac tumor, and choriocarcinoma. Seminomas and NSGCTs present significant differences in clinical features, therapy, and prognosis, and both show characteristics of the primordial germ cells. Many discovered biomarkers including OCT3/4, SOX2, SOX17, HMGA1, Nek2, GPR30, Aurora-B, estrogen receptor β, and others have given further advantages to discriminate between histological subgroups and could represent useful novel molecular targets for antineoplastic strategies. More insight into the pathogenesis of TGCTs is likely to improve disease management not only to better treatment of these tumors but also to a better understanding of stem cells and oncogenesis.

International Review of Cell and Molecular Biology, Volume 312 ISSN 1937-6448 http://dx.doi.org/10.1016/B978-0-12-800178-3.00003-8

#

2014 Elsevier Inc. All rights reserved.

79

80

Paolo Chieffi

1. INTRODUCTION Testicular germ-cell tumors (TGCTs), the most common malignant tumors in males among adolescent and young adults, represent a major cause of death attributable to cancer in this age group (Chaganti and Houldsworth, 2000; Chieffi et al., 2009, 2012; Oosterhuis and Looijenga, 2005; Ulbright, 2005). TGCTs are histologically classified as seminomas and nonseminomas according to the international classification of oncological diseases. Both these tumors display an invasive phenotype and are believed to be derived from a common ancestor, carcinoma in situ (CIS), where the generation and expansion of tumor cells are limited to within the seminiferous tubules (Chaganti and Houldsworth, 2000; Chieffi et al., 2009, 2012; Oosterhuis and Looijenga, 2005; Ulbright, 2005). Nonseminomas, including embryonal carcinoma and teratoma, contain stem cells as well as cells that have differentiated toward somatic lineages to various degrees, thus giving rise to a morphologically pleiotropic appearance (Oosterhuis and Looijenga, 2005). In contrast, seminomas have a rather uniform appearance, at least at the histological level. Due to this apparently homogenous cell composition, seminomas are particularly suitable for investigations of tumor-associated alterations in gene expression. In addition, the cells that constitute seminomas resemble the primordial germ cells (PGCs) and/or the cells in the CIS. Thus, the gene expression profile in seminomas is interesting not only with regard to understanding their oncogenesis, but it also may be useful for research into PGCs (Ulbright, 2005).

2. EPIDEMIOLOGY AND RISK FACTORS The incidence of TGCT varies between different countries and races, being greater in Scandinavia and Switzerland than Asia and Latin America, and in Caucasian Americans compared to African Americans. The incidence in western countries has been increasing over the last decades, probably because of an increased exposure to etiologic factors ( Jones and Vasey, 2003). Remarkably, differences in incidence between adjacent countries such as Sweden and Finland are still largely unexplained, calling for further studies. Both clinical and epidemiological evidence strongly suggest that genetic and environmental factors play an important role in the genesis and development of TGCT. Several genes are implicated in the pathogenesis of TGCT, but the involvement of other genetic factors remains unknown

Biomarkers for TGCTs

81

(Rajpert-De Meyts and Skakkebaek, 2011; Skakkebaek, 1972). Susceptibility genes and environmental factors may deregulate normal differentiation processes of PGCs. In fact, TGCT have an invasive phenotype and are believed to be derived from a common ancestor, CIS, where the generation and expansion of tumor cells are limited to within the seminiferous tubules (Skakkebaek et al., 1987). A number of environmental factors have been investigated to explain the possible links. Some evidence suggests association of increased TGCTs risk and maternal smoking during pregnancy, adult height, body mass index, diet rich in cheese, and others (Bonner et al., 2002; Dieckmann and Pichlmeier, 2002; Dieckmann et al., 2009). However, the biological mechanisms remain to be elucidated. Hypothesized environmental agents involved in the development of TGCTs include pesticides (McGlynn et al., 2008) and nonsteroidal estrogens, such as diethylstilbestrol (DES; Martin et al., 2008). It has been proposed that increased levels of estrogen exposure in utero increase the risk of TGCTs (Garner et al., 2008) and the exposure of women to the nonsteroidal estrogen DES during pregnancy increases the risk of TGCTs (Strohsnitter et al., 2001). However, other studies have not confirmed a role for estrogen in TGCT development (Dieckmann et al., 2001). Familial predisposition to TGCTs, ethnic variations in incidence, and an association with certain chromosome abnormality syndromes strongly suggest that inherited factors also play a role in disease development. The familial predisposition is one of the strongest for any tumor type, since the increased relative risk of TGCT development associated with fathers and sons of TGCT patients is fourfold (Forman et al., 1992). However, gene(s) involved in familial TGCTs have not been identified so far (Oosterhuis and Looijenga, 2005). Genome-wide linkage analysis of affected families has provided evidence for two susceptibility loci, one at Xq27 locus for undescended testis probably playing an indirect role, and another at 12q which results in hyperexpression of the product of the CCND2 gene (Lutke Holzik et al., 2004). It is probable that both genetic and environmental factors produce the high familial risk seen in TGCTs and that the interplay between these two factors, along with genetic heterogeneity, may make familial associated susceptibility loci difficult to determine.

3. HISTOPATHOLOGY It has been suggested that the initiating event in the pathogenesis of TGCT occurs during embryonal development (Chieffi and Chieffi,

82

Paolo Chieffi

2013a,b). The most widely accepted model of postpuberal TGCT development proposes an initial tumorigenic event in utero and the development of a precursor lesion known as intratubular germ cell neoplasia undifferentiated (ITGCNU), also known as CIS (Skakkebaek, 1972). This is followed by a period of dormancy until after puberty when postpuberal TGCTs emerge. This prepubertal dormancy suggests that the TGCT development is hormone dependent. Recently, it has been proposed that tumors originate from neoplastic cells that retain stem cell properties such as self-renewal (Wicha et al., 2006), and this novel hypothesis has fundamental implications for the pathogenesis of TGCTs. According to the stem cell hypothesis, tumors originate from tissue stem cells or from their immediate progeny. This cellular subcomponent drives tumorigenesis and aberrant differentiation, contributing to cellular heterogeneity of the tumor and also to the resistance to antineoplastic treatments. ITGCNU cells are generally accepted as the common preinvasive precursor cells that give rise to postpuberal TGCT (Oosterhuis and Looijenga, 2005). ITGCNU are almost invariably found in the periphery of overt postpuberal TGCTs and it is estimated that it is present in approximately 5% of the contralateral testis of patients with postpuberal TGCTs (Berthelsen et al., 1982). Preinvasive ITGCNU cells are supposed to be able to develop in different germinal and somatic tissues and are regarded as pluripotent or totipotent cells and therefore can be considered as TGCT stem cells. ITGCNU cells share morphological similarities with gonocytes and it has been proposed that ITGCNU cells could be remnants of undifferentiated embryonic/fetal cells (Nielsen et al., 1974; Skakkebaek et al., 1987). Their fetal origin is also supported by immunohistochemical studies of proteins present in ITGCNU, also shown to be present in PGCs and gonocytes. The identification of ITGCNU cells in prepubertal patients, who later developed TGCTs, indicated that the cells had originated prior to puberty (Muller et al., 1984). Therefore, the ITGCNU cell represents an interesting variant of cancer stem cell since it originates before the tissue that it propagates in is fully differentiated and functional. The observation that two transcription factors, POU5F1 (OCT3/4) and NANOG, known to be associated with pluripotency in ES cells are expressed in ITGCNU has further contributed to assess the embryonic origin of these cells. A link between ITGCNU cells and embryonic cells has been further supported by a substantial overlap between human ES cells and ITGCNU cell gene expression profiles, as

Biomarkers for TGCTs

83

shown by Almstrup et al. (2004). All histotypes could be present in postpuberal TGCTs, because of its totipontent profile, even seminoma can switch to nonseminoma histotype through reprogramming phenomenon (Fig. 3.1; Hoei-Hansen et al., 2005). The role of these factors will be discussed in more detail in the next sections. Seminoma consists of transformed germ cells, which closely resemble the PGC/gonocyte, apparently blocked in their differentiation. Nonseminoma could be constituted by cells with typical pluripotency of PGC/gonocyte. In particular, embryonal carcinoma reflect undifferentiated stem cells, Teratoma represent somatic differentiation, while choriocarcinoma and YST extraembryonal differentiation. Genetic studies have shown that postpubertal testis tumors are often aneuploid with a consistent chromosomal abnormality composed of a gain of short arm of chromosome 12, usually in the form of an isochromosome, i(12p). In contrast tumors arising in prepubertal gonads are typically unassociated with 12p amplification and tend to be diploid. The most consistent structural chromosomal abnormality is an isochromosome 12p. Tumors lacking i(12p) have other structural abnormalities of 12p, among them the amplification of 12p11.2–p12.1. Gain of 12p

Figure 3.1 Differentiative relations between TGCT histotypes.

84

Paolo Chieffi

sequences may be related to invasive growth (Chaganti and Houldsworth, 2000; Chieffi and Chieffi, 2014) suggesting that cyclin D2 (mapped to 12p13) is the most likely candidate gene of pathogenetic relevance.

4. PROGNOSTIC AND DIAGNOSTIC MARKERS 4.1. Serum tumor markers in TGCTs In recent years, although the implementation to identify novel biomarkers for progression has improved our ability to counsel our patients with regard to likely outcomes and risk and relapse, large gaps remain in our ability to reliably exclude patients from unnecessary treatment or implement lifesaving therapies in those destined to fail. Three serum tumor markers, S-alpha fetoprotein (S-AFP) concentration, S-human-chorionic gonadotropin (S-hCG), and serum lactate dehydrogenase (S-LD) catalytic concentration, are currently used for prognostic markers (von Eyben, 2003). In the fetus, AFP is a major serum-binding protein produced by the fetal yolk sac, liver, and gastrointestinal tract. The highest concentrations approach during the 12th to 14th weeks of gestation and decline 1 year after birth (von Eyben, 2003). AFP is secreted by embryonal cell carcinoma and yolk sac tumor, but not by pure choriocarcinoma or pure seminoma. Falsely elevated AFP values can be seen after treatment in patients with liver disease, hereditary persistence of AFP, and several malignancies including hepatocellular carcinoma, lung, pancreatic, colon, and gastric cancers (von Eyben, 2003). During pregnancy, hCG is produced primarily by the syncytiotrophoblastic cells of the placenta and serves to maintain the corpus luteum. Similarly, in TGCTs, syncytiotrophoblastic cells are responsible for the production of hCG. All the patients with choriocarcinoma and 40–60% of patients with embryonal cell carcinoma have elevated serum levels of hCG. Approximately 10–20% of patients with pure seminoma have elevated serum hCG. S-LD is a cytoplasmic enzyme found in all living cells. S-LD catalyzes the reduction of pyruvate to lactate TGCTs patients typically express high levels of S-LD isoenzyme 1 (S-LD-1). However, S-DL represent a nonspecific marker for the burden of disease and can be elevated in non-TGCT malignancies and conditions of chronic disease, such as liver and congestive heart failure, pancreatitis, hemolytic anemia, collagen vascular disorders (von Eyben, 2003).

Biomarkers for TGCTs

85

4.2. Newly discovered biomarkers detected by immunohistochemistry in TGCT subtypes Many novel biomarkers have been described in literature that can help to discriminate the different TGCTs, and they represent new potential molecular therapeutic targets. These biomarkers which are helpful for immunohistochemistry analysis are summarized in Table 3.1 in order to clearly define each TGCT histotypes. HMGA1 and HMGA2 represent a useful diagnostic markers (Chieffi et al., 2002a,b; Franco et al., 2008). In fact, it has been demonstrated that the two isoforms are differently expressed with respect to the state of differentiation of TGCTs (Fig. 3.2; Chieffi et al., 2002a; Franco et al., 2008). Indeed, HMGA1 is able to bind proteins involved in transcriptional regulation, such as RNF4 (Pero et al., 2001, 2003) and PATZ1, which have been shown to be delocalized and overexpressed in human testicular seminomas (Fedele et al., 2008). More recently, we have shown that PATZ1 interacts with ERβ in normal germ cells, while downregulation of ERβ associates with transcriptional coregulator PATZ1 delocalization in human testicular seminomas (Fig. 3.3; Esposito et al. 2011, 2012). NEK2 is a serine/threonine kinase that promotes centrosome splitting and ensures correct chromosome segregation during the G2/M phase of the cell cycle, through phosphorylation of specific substrates. Aberrant expression and activity of NEK2 is present in neoplastic cells of seminomas. In addition, nuclear localization and the upregulation of Nek2 protein was also observed in the TCam-2 seminoma cell line, and correlates with expression of the stemness markers PLZF and OCT4 (Barbagallo et al., 2009; Di Agostino et al., 2004). Recently, it has been shown that NEK2 acts as a novel splicing factor kinase and suggest that part of its oncogenic activity may be ascribed to its ability to modulate alternative splicing, a key step in gene expression regulation that is frequently altered in cancer cells (Naro et al., 2014). OCT3/4 is a well-characterized marker for PGCs, and of CIS, seminoma, and embryonal carcinoma (Ledford, 2007). It has been demonstrated that OCT3/4 is also expressed in normal adult stem cells and nongerm cellderived cancers (Atlasi et al., 2008; Ledford, 2007). OCT3/4 is a transcription factor of the family of octamer-binding proteins (also known as the POU homeodomain proteins) and is regarded as one of the key regulators of pluripotency (Atlasi et al., 2008). In addition to OCT3/4, several other embryonic stem-cell-specific proteins are important for maintaining the

Table 3.1 Immunohistochemical markers identified in TGCT subtypes OCT3/4 SOX2 SOX17 HMGA1

Seminoma

+

Embryonal carcinoma

+

Teratoma

+ + +/

Yolk sac Notes: +, expressed; +©, cytoplasmic localization;

HMGA2

+ +

+

+/ +/

+

, not expressed; +/ , variable expression.

PATZ1

GPR30

+©

CCDC6

Nek2

Aurora-B

+

+

+

+©

+

+/

+

+©

+/

+©

+

Biomarkers for TGCTs

87

Figure 3.2 Immunohistochemical analysis of HMGA1 and HMGA2 expression in TGCTs: (A and B) classic seminoma, (C and D) Embryonal carcinoma, (E and F) a yolk sac tumor, and (G and H) Teratoma. All magnifications 40 .

so-called “stemness” of pluripotent cells, such as NANOG and SOX2 (Avilion et al., 2003; de Jong and Looijenga, 2006; Yamaguchi et al., 2005). LIN28 is an RNA-binding protein involved in maintaining the pluripotency of embryonic stem cells, and its expression is decreased upon

88

Paolo Chieffi

Figure 3.3 Immunohistochemistry and immunofluorescence analyses of ERβ and PATZ1 protein expression in human testicular seminomas. Seminoma in which an absence of immunopositivity of ERβ (A) was observed in association with an intense and diffuse PATZ1 cytoplasmic immunosignal (B) by immunohistochemistry (magnification 40 ); confocal microscopic images displaying an absence of immunopositivity of ERβ (C) and PATZ1 cytoplasmic (D) immunoreactivities in the same seminoma case; Scale bar: 10 μm.

differentiation. In mouse embryonic stem cells, Lin28 mediates the posttranslational expression of OCT4 by directly binding to its messenger RNA (mRNA). LIN28 is important in reprogramming somatic cells to pluripotent stem cells. Recently, it has been shown that LIN28 can be used as a diagnostic marker for testicular CIS, classical seminomas, embryonal carcinomas, yolk sac tumors (Cao et al., 2011). The major advantage of LIN28 over OCT4 is in diagnosing yolk sac tumors (yolk sac tumor negative for OCT4). NANOG protein was detected in gonocytes within the developing testis. In addition, NANOG is highly and specifically expressed in CIS, embryonal carcinoma, and seminomas, but not in teratoma, and YSTs revealing a molecular and developmental link between TGCTs and the embryonic cells from which they arise (Hart et al., 2005). SOX2 is a member of the SOX protein family, transcription factors that regulate development from the early embryonal stage to differentiated lineages of specialized cells. SOX proteins are known to cooperate with POU proteins; in particular, the interaction between SOX2 and OCT3/4 has

Biomarkers for TGCTs

89

been well demonstrated. SOX2 is not detected in human germ cells regardless of their developmental age, in contrast to data in mouse embryos (de Jong et al., 2008). SOX2 is expressed in embryonal carcinoma, but it is not present in seminomas, YSTs, and normal spermatogenesis (de Jong et al., 2008). SOX17 maps to the chromosomal region 8p23, which is gained in seminoma. This indicates that SOX17 is a candidate SOX protein for cooperation with OCT3/4 in CIS and seminoma. These data also demonstrate that SOX17 is a good marker to discriminate CIS and seminoma from embryonal carcinoma. Of interest is that SOX17 distinguishes embryonic from adult hematopoietic stem cells (Kim et al., 2007). Current research focuses on the processes that may regulate the differential expression of SOX2 versus SOX17 and on the role of these SOX proteins in the different histologies of the TGCT subtypes involved. Although the physiologic responses to estrogens are mainly mediated by the ERα and ERβ (Chieffi et al., 2000, 2002b; Vicini et al., 2006), in the last few years, GPR30 has been shown to mediate estrogen signaling in a wide variety of cell types. GPR30 is an intracellular seven-transmembrane G protein-coupled estrogen receptor (GPR30) that functions alongside the traditional estrogen receptors (ERα and ERβ) to regulate physiological responsiveness to estrogen. It has been shown that GPR30 is overexpressed in seminomas and in the derived human seminoma TCam-2 cell line. The design of specific GPR30 inhibitors could be a useful molecular target to block neoplastic germ cells with a high proliferative rate, suggesting its potential therapeutic role for the treatment of TGCTs (Chieffi, 2007; Chieffi and Chieffi, 2013a,b; Franco et al., 2011). Sariola and coworkers have shown that targeted overexpression of glial cell line-derived neurotrophic factor (GDNF) in undifferentiated spermatogonia promotes malignant testicular tumors, which express germ-cell markers. The tumors are invasive and contain aneuploid cells, but no distant metastases have been found. By several histological, molecular, and histochemical characteristics, the GDNF-induced tumors mimic classic seminomas in men, representing a useful experimental model for TGCT (Meng et al., 2001). In addition, recently it has been shown that GDNF promotes invasive behavior, an effect dependent on pericellular protease activity, possibly through the activity of matrix metalloproteinases. GFRA1 overexpression in CIS and seminoma cells, along with the functional analyses in TCam-2 cells, suggests an involvement of the GDNF pathway in the progression of testicular germ cell cancer (Ferranti et al., 2012).

90

Paolo Chieffi

DNA damage response has been clearly described as an anticancer barrier in early human tumorigenesis. Moreover, interestingly, TGCTs have been reported to lack the DNA damage response pathway activation. CCDC6 is a proapoptotic phosphoprotein substrate of the ataxia telangectasia mutated able to sustain DNA damage checkpoint in response to genotoxic stress and is commonly rearranged in malignancies upon fusion with different partners. Recently, it has been shown that the loss of CCDC6 expression is the most consistent feature among the TGCTs and in the TCam-2 seminoma cell line (Staibano et al., 2013). Cancer/testis (CT) antigens are cancer antigens normally expressed in adult testicular germ cell. The expression of chromosome X-encoded CT (CT-X) antigens were initially identified during the search for immunogenic tumor antigens capable of eliciting spontaneous immune responses in patients with cancer. A study of MAGE, BAGE, and GAGE antigens, the first group of tumor antigens shown to elicit cell-mediated immune responses in melanoma patients, shown mRNA expression limited to testis and no expression in any other normal adult tissue (Chen et al., 2011). Subsequent serological cloning of antigens that elicited antibody responses in patients with cancer identified SSX, NY-ESO, and CT7, all of which also shared this distinctive characteristic of testis-restricted expression and aberrant activation in various types of human cancer. This unique feature led us to designate this group of antigens as CT antigens, recognizing them as attractive targets for immunotherapy, particularly for therapeutic cancer vaccines (Chen et al., 2011). Recently, it has been published that CT-X antigens are not expressed in the fetal precursor cells for germ-cell tumors, and their expression likely reflects germ cell differentiation of the neoplastic cells (in seminomas) or aberrant gene activation as cancer antigens (in nonseminomatous tumors) (Chen et al., 2013).

5. THERAPY 5.1. Aurora-kinase inhibitors Tumorigenesis is associated with genomic instability due to errors in mitosis. Many mitotic regulators are aberrantly expressed in tumor cells. Aurora-B is a chromosomal passenger protein. During mitosis, Aurora-B is required for phosphorylation of histone H3 on serine 10, and this might be important for chromosome condensation. Aurora-B clearly regulates kinetochore function, as it is required for correct chromosome alignment and segregation.

Biomarkers for TGCTs

91

Aurora-B is also required for spindle checkpoint function and cytokinesis (Carmena and Earnshaw, 2003). The kinase Aurora-B represents another marker that could be used to discriminate the different tumor histotype is Aurora-B expression; in fact, it was detected in all CIS, seminomas and embryonal carcinomas analyzed, but not in teratomas and yolk sac carcinomas (Chieffi et al. 2004; Esposito et al. 2009; Portella et al. 2011). The increase of Aurora-B expression in TGCTs has been confirmed by using Ki67 and PCNA as molecular markers (Chieffi et al. 2004; Esposito et al. 2009; Portella et al. 2011). Different Aurora-kinase inhibitors have recently been described targeting the enzymatic activity of the Aurora kinase and in particular blocking Aurora-B activity. AZD1152, ZM447439, Hesperadin 8, and VX-680 (Fig. 3.4; Harrington et al. 2004; Hauf et al. 2003; Keen and Taylor, 2004). AZD1152 is a reversible ATP-competitive Aurora inhibitor and it is 1000fold more selective for Aurora kinase B than for Aurora kinase A (Fig. 3.4). AZD1152 has shown highly significant tumor growth inhibition in a diverse panel of solid human cancer tumor xenograft models, including lung and colorectal cancers and its good solubility makes it suitable for clinical use. AZD1152 and other Aurora inhibitors (ZM2, ZM3, GSK1070916) are currently in early clinical evaluation, showing reversible neutropenia as major side effect (Figs. 3.4, 3.5; Carmena and Earnshaw, 2003). All these molecules act by inhibiting phosphorylation of histone H3 on serine 10 (Ota et al., 2002; Terada et al. 1998), and consequently blocking cell division, as shown by using GC1 and TCam-2 germ cell lines, respectively, derived from immortalized type B murine spermatogonia and human seminoma (Esposito et al., 2009; Portella et al., 2011). Although germinal cell tumors are highly responsive to commonly used chemotherapeutic treatment, cases of acute toxicity and chronic collateral effects, such as sterility, are recorded. Therefore, the availability of novel drugs such as Aurora-B inhibitors could represent an escape from chemotherapy early and late effects.

5.2. Tyrosine-kinase inhibitors Protein phosphorylation plays key roles in many physiological processes and is often deregulated in neoplastic lesions. Current understanding of how protein kinases and phosphatases orchestrate the phosphorylation changes that control cellular functions, has made these enzymes potential drug targets for the treatment of different types of cancer. Recently, receptor and

Figure 3.4 Aurora-kinase inhibitors. Dual inhibitors of Aurora kinases. Chemical structures of (A) Hesperadin, (B) ZM447439, (C) ZM2, (D) ZM3, (E) VX-680.

Biomarkers for TGCTs

93

Figure 3.5 Selective Aurora-B inhibitors. Chemical structures of (A) AZD1152, (B) GSK1070916.

nonreceptor tyrosine kinases (TKs) have emerged as clinically useful drug target molecules for treating cancer (Krause and Van Etten, 2005). Imatinib mesilate (STI-571) was primarily designed to inhibit bcr-abl TK activity and to treat chronic myeloid leukemia. STI-571 is also an inhibitor of c-Kit receptor TK, and is currently the drug of choice for the therapy of metastatic gastrointestinal stromal tumors (GISTs), which frequently express constitutively activated forms of the c-Kit-receptor (Krause and Van Etten, 2005). It has been described case with disseminated testicular seminoma that was refractory to salvage chemotherapy, and who had complete remission after imatinib administration suggesting that imatinib could be a potential therapeutic strategy for selected patients with refractory seminoma (Kemmer et al., 2004). Platelet-derived growth factor receptor-a (PDGFRa), and c-Kit are expressed at high levels in TGCTs (Kemmer et al., 2004; Palumbo et al., 2002). The c-Kit/stem cell factor system is a signaling pathway for migration and survival of PGCs (Manova et al., 1990). c-Kit is a TK receptor for the stem cell factor (SCF), ligand binding leads to the c-Kit receptor heterodimerization and TK activity and the downstream signal involves both apoptosis and cell cycle progression (Manova et al., 1990;

94

Paolo Chieffi

Sorrentino et al., 1991). The SCF c-Kit signaling pathway is important for proper germ cell development. This was first illustrated by mice carrying naturally occurring mutations in the Steel (SI) and White-spotting (W) loci that involved the ligand-receptor pair SCF and c-KIT, respectively (Loveland and Schlatt, 1997; McCoshen and McCallion, 1975). Activating mutations of c-Kit have recently been found in 93% of bilateral TGCTs, albeit in less of 2% of unilateral TGCTs (Looijenga et al., 2003). These mutations affect codon 816 of c-Kit gene resulting in a constitutional kinase active, in a manner similar to other receptorial TK activating mutations (Looijenga et al., 2003). However, the mutation in exon 17 is not inhibited by the TK inhibitor imatinib mesylate (Heinrich et al., 2002). In addition, c-kit activate a number of signaling molecules, including PI3-kinase (PI3K). PI3K is activated by a number of proteins, such as AKT3, which is generally overexpressed in the majority of nonseminoma and seminoma. PTEN, which is a tumor suppressor gene, inhibits PI3K activity. Di Vizio et al. (2005) reported that the loss of PTEN is implicated in the progression from ITGCNU to invasive tumors. The success of the TK inhibitors in the treatment of some cancers has invigorated the development of kinase inhibitors as anticancer drugs and a large number of these compounds are currently undergoing clinical trials and it is likely that molecules capable to inhibit exon 17 c-Kit activating mutations will be identified contributing to the development of molecular targeted therapies.

5.3. Angiogenesis inhibitors Tumors require access to blood vessels for the supply of oxygen and to maintain growth. The development and the growth of new vessels (angiogenesis) are essential for tumor growth and progression. Judah Folkman in the early 1970s proposed the inhibition of tumor blood vessel as a therapeutic approach for treating cancer patients (Folkman, 1996). The blood vessel growth in normal tissues is regulated through a balance between the action of proangiogenic factors, such as vascular endothelial growth factor (i.e., VEGF; Ferrara, 2004) and the action of angiogenic inhibitors (i.e., thrombospondin-1; Gasparini et al., 2005). In neoplastic lesions the angiogenic balance is shifted toward the proangiogenic factors, and irregular and uncoordinated tumor vessel growth is the result. VEGFR tyrosine kinase, p53, cyclooxygenase-2 (COX-2), and matrix metallo proteinases (MMPs) all directly and/or indirectly influence the proangiogenic switch (Ferrara, 2004). More than five inhibitors of the

Biomarkers for TGCTs

95

VEGF pathway have entered clinical phases I–III trials. Bevacizumab (Avastin™), an antibody against VEGF, was shown to prolong survival in a phase III clinical trial in renal cell cancer and was efficient in two randomized clinical trials investigating the treatment of metastatic colorectal cancer (Ranieri et al., 2006). ZD6474 is an orally bioavailable inhibitor of VEGF receptor-2 tyrosine kinase activity that in preclinical studies has been shown to inhibit both VEGF-induced signaling in endothelial cells and tumor-induced angiogenesis. ZD6474 produced significant broad-spectrum antitumor activity in a panel of human tumor xenografts (Lee, 2005; Zakarija and Soff, 2005). The results obtained so far with inhibitors of angiogenesis suggest that these are novel molecules, currently in development could be useful for the treatment of chemotherapeutic resistant TGCTs and to increase patients survival.

6. MicroRNAs IN TGCTs In recent years, the role of miRNAs in carcinogenesis of human testicular cancer and germ cell development has emerged (Bernstein et al., 2003). It was demonstrated that knockout mice for Dicer suffered from an early decrease in germ cell number and an impaired ability to differentiate, indicating that Dicer1 and miRNAs are important for both survival and proper differentiation of male germ cells (Maatouk et al., 2008). Subsequently, it was demonstrated that miRNAs 372 and 373 can overcome cell cycle arrest mediated by p53 (Voorhoeve et al., 2006). In contrast, TGCT cell lines with mutated p53 or expressing low levels of p53 were shown to be negative for these miRNAs and it can be assumed that miRNAs 372 and 373 can bypass the p53 checkpoint allowing the growth of TGCT. Another interesting link to the importance of miRNAs for germ cells and GCTs came from research on the dead end gene (DND1). Until recently, DND1 was known to regulate germ-cell viability and to suppress the formation of germ-cell tumors. Recently Kedde et al. (2007) demonstrated that DND1 counteracts miRNA-mediated destabilization of mRNAs by binding to mRNAs and prohibiting the association of miRNAs with their target sites. This underlines the important role of miRNAs and regulation of miRNA expression in germ cell development. Linger et al. (2008) focused on the role of DND1 in humans and analyzed the presence of DND1 mutations in 263 human TGCTs. Further research into the functional mechanisms of miRNAs and the role of DND1 in TGCT are likely to give more interesting clues.

96

Paolo Chieffi

7. CONCLUSIONS AND PERSPECTIVES Both environmental and genetic factors play an important role in the development of human testicular seminomas. These factors cause the deregulation of the normal differentiation processes of PGC. The incidence of seminomas has been increasing over the last decades. Remarkably, differences in incidence between adjacent countries such as Sweden and Finland are still largely unexplained, calling for further studies. Diagnosis is usually based on identification of histological subgroups. In recent years, immunohistochemistry with a panel of suitable markers, including OCT3/4, SOX2, SOX17, HMGA1, and HMGA 2, Aurora-B, PATZ1, GPR30 and others has given further advantages to discriminate between subgroups. A unique characteristic of seminoma is their sensitivity to treatment. Although the better responses of seminomas versus nonseminomas is well reported, as the frequent recurrence of mature teratomas in residual treatment-resistant tumors highlighting the need for more effective therapies in these resistant forms. A deeper understanding of the molecular mechanisms underlying the development of TGCTs may provide new tools to specifically target neoplastic cells and could contribute to overcome acquired and intrinsic chemotherapy resistance. Promising molecules capable to selectively target neoplastic cells, that are, the Aurora-B serine–threonine kinases, TKs, HMGAs, GPR30 antagonist, and proangiogenic factors inhibitors, already under clinical evaluation will open a new scenario for seminomas treatment.

REFERENCES Almstrup, K., Hoei-Hansen, C.E., Wirkner, U., Blake, J., Schwager, C., Ansorge, W., et al., 2004. Embryonic stem cell-like features of testicular carcinoma in situ revealed by genome-wide gene expression profiling. Cancer Res. 64, 4736–4743. Atlasi, Y., Mowla, S.J., Ziaee, S.A., Gokhale, P.J., Andrews, P.W., 2008. OCT4 spliced variants are differentially expressed in human pluripotent and non-pluripotent cells. Stem Cells 26, 3068–3074. Avilion, A.A., Nicolis, S.K., Pevny, L.H., Perez, L., Vivian, N., Lovell Badge, R., 2003. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140. Barbagallo, F., Paronetto, M.P., Franco, R., Chieffi, P., Dolci, S., Fry, A.M., et al., 2009. Increased expression and nuclear localization of the centrosomal kinase Nek2 in human testicular seminomas. J. Pathol. 217, 431–441. Bernstein, E., Kim, S.Y., Carmell, M.A., Murchison, E.P., Alcorn, H., Li, M.Z., et al., 2003. Dicer is essential for mouse development. Nat. Genet. 35, 215–217. Berthelsen, J.G., Skakkebaek, N.E., von der Maase, H., Sorensen, B.L., Mogensen, P., 1982. Screening for carcinoma in situ of the contralateral testis in patients with germinal testicular cancer. Br. Med. J. 285, 1683–1686.

Biomarkers for TGCTs

97

Bonner, M.R., McCann, S.E., Moysich, K.B., 2002. Dietary factors and the risk of testicular cancer. Nutr. Cancer 44, 35–43. Cao, D., Allan, R.W., Cheng, L., Peng, Y., Guo, C.C., Dahiya, N., et al., 2011. RNAbinding protein LIN28 is a marker for testicular germ cell tumors. Hum. Pathol. 42, 710–718. Carmena, M., Earnshaw, W.C., 2003. The cellular geography of aurora kinases. Nat. Rev. Mol. Cell Biol. 4, 842–854. Chaganti, R.S., Houldsworth, J., 2000. Genetics and biology of adult human male germ cell tumors. Cancer Res. 60, 1474–1482. Chen, Y.T., Chiu, R., Lee, P., Beneck, D., Jin, B., Old, L.J., 2011. Chromosome X-encoded cancer/testis antigens show distinctive expression patterns in developing gonads and in testicular seminoma. Hum. Reprod. 26, 3232–3243. Chen, Y.T., Cao, D., Chiu, R., Lee, P., 2013. Chromosome X-encoded cancer/testis antigens are less frequently expressed in non-seminomatous germ cell tumors than in seminomas. Cancer Immun. 13, 1–10. Chieffi, P., 2007. Molecular targets for the treatment of testicular germ cell tumors. Mini Rev. Med. Chem. 7, 755–759. Chieffi, P., Chieffi, S., 2013a. Molecular biomarkers as potential targets for therapeutic strategies in human testicular germ cell tumours: an overview. J. Cell. Physiol. 228, 1641–1646. Chieffi, P., Chieffi, S., 2013b. An up-date on the molecular biomarkers as potential therapeutic targets in human testicular germ cell tumours. Open Androl. J. 5, 6–9. Chieffi, P., Chieffi, S., 2014. An up-date on newly discovered immunohistochemical biomarkers for the diagnosis of human testicular germ cell tumors. Histol. Histopathol 29, 999–1006. Chieffi, P., Colucci D’Amato, G.L., Staibano, S., Franco, R., Tramontano, D., 2000. Estradiol-induced mitogen-activated protein kinase (extracellular signal-regulated kinase 1 and 2) activity in the frog (Rana esculenta) testis. J. Endocrinol. 167, 77–84. Chieffi, P., Battista, S., Barchi, M., Di Agostino, S., Pierantoni, G., Fedele, M., Chiariotti, L., Tramontano, D., Fusco, A., 2002a. HMGA1 and HMGA2 protein expression in mouse spermatogenesis. Oncogene 21, 3644–3650. Chieffi, P., Colucci-D’Amato, G.L., Guarino, F., Salvatore, G., Angelini, F., 2002b. 17βestradiol induces spermatogonial proliferation through mitogen-activated protein kinase (extracellular signal-regulated kinase 1) activity In the lizard (Podarcis s. sicula). Mol. Reprod. Dev. 61, 218–225. Chieffi, P., Troncone, G., Caleo, A., Libertini, S., Linardopoulos, S., Tramontano, D., Portella, G., 2004. Aurora B expression in normal testis and seminomas. J. Endocrinol. 181, 263–270. Chieffi, P., Franco, R., Portella, G., 2009. Molecular and cell biology of testicular germ cell tumors. Int. Rev. Cell Mol. Biol. 278, 277–308. Chieffi, P., Chieffi, S., Franco, R., Sinisi, A., 2012. Recent advances in the biology of germ cell tumors: implications for the diagnosis and treatment. J. Endocrinol. Invest. 35, 1015–1020. de Jong, J., Looijenga, L.H., 2006. Stem cell marker OCT3/4 in tumor biology and germ cell tumor diagnostics: history and future. Crit. Rev. Oncog. 12, 171–203. de Jong, J., Stoop, H., Gillis, A.J., van Gurp, R.J., van de Geijn, G.J., Boer, M., et al., 2008. Differential expression of SOX17 and SOX2 in germ cells and stem cells has biological and clinical implications. J. Pathol. 215, 21–30. Di Agostino, S., Fedele, M., Chieffi, P., Fusco, A., Rossi, P., Geremia, R., et al., 2004. Phosphorylation of high mobility group protein A2 by Nek kinase during the first meiotic division in mouse spermatocytes. Mol. Biol. Cell 15, 1224–1232. Dieckmann, K.P., Pichlmeier, U., 2002. Is risk of testicular cancer related to body size? Eur. Urol. 42, 564–569.

98

Paolo Chieffi

Dieckmann, K.P., Endsin, G., Pichlmeier, U., 2001. How valid is the prenatal estrogen excess hypothesis of testicular germ cell cancer? A case control study on hormone-related factors. Eur. Urol. 40, 677–683. Dieckmann, K.P., Hartmann, J.T., Classen, J., Diederichs, M., Pichlmeier, U., 2009. Is increased body mass index associated with the incidence of testicular germ cell cancer? J. Cancer Res. Clin. Oncol. 135, 731–738. Di Vizio, D., Cito, L., Boccia, A., Chieffi, P., Insabato, L., Pettinato, G., et al., 2005. Loss of tumour suppressor gene PTEN marks the transition from intratubular germ cell neoplasias (ITGCN) to invasive germ cell tumors. Oncogene 24, 1882–1894. Esposito, F., Libertini, S., Franco, R., Abagnale, A., Marra, L., Portella, G., et al., 2009. Aurora B expression in post-puberal testicular germ cell tumours. J. Cell. Physiol. 221, 435–439. Esposito, F., Boscia, F., Franco, R., Tornincasa, M., Fusco, A., Kitazawa, S., et al., 2011. Down-regulation of estrogen receptor-β associates with transcriptional coregulator PATZ1 delocalization in human testicular seminomas. J. Pathol. 224, 110–120. Esposito, F., Boscia, F., Gigantino, V., Tornincasa, M., Fusco, A., Franco, R., et al., 2012. The high mobility group A1-oestrogen receptor β nuclear interaction is impaired in human testicular seminomas. J. Cell. Physiol. 227, 3749–3755. Fedele, M., Franco, R., Salvatore, G., Paronetto, M.P., Barbagallo, F., Pero, R., et al., 2008. PATZ1 gene has a critical role in the spermatogenesis and testicular tumours. J. Pathol. 215, 39–47. Ferranti, F., Muciaccia, B., Ricci, G., Dovere, L., Canipari, R., Magliocca, F., et al., 2012. Glial cell line-derived neurotrophic factor promotes invasive behaviour in testicular seminoma cells. Int. J. Androl. 35, 758–768. Ferrara, N., 2004. Vascular endothelial growth factor: basic science and clinical progress. Endocr. Rev. 25, 581–611. Folkman, J., 1996. New perspectives in clinical oncology from angiogenesis research. Eur. J. Cancer 32, 2534–2539. Forman, D., Oliver, R.T., Brett, A.R., Marsh, S.G., Moses, J.H., Bodmer, J.G., et al., 1992. Familial testicular cancer: a report of the UK family register, estimation of risk and an HLA class 1 sib-pair analysis. Br. J. Cancer 65, 255–262. Franco, R., Esposito, F., Fedele, M., Liguori, G., Pierantoni, G., Botti, G., et al., 2008. Detection of high mobility group proteins A1 and A2 represents a valid diagnostic marker in post-puberal testicular germ cell tumours. J. Pathol. 214, 58–64. Franco, R., Boscia, F., Gigantino, V., Marra, L., Esposito, F., Ferrara, D., et al., 2011. GPR30 is over-expressed in post puberal testicular germ cell tumors. Cancer Biol. Ther. 11, 609–613. Garner, M., Turner, M.C., Ghadirian, P., Krewski, D., Wade, M., 2008. Testicular cancer and hormonally active agents. J. Toxicol. Environ. Health B Crit. Rev. 11, 260–275. Gasparini, G., Longo, R., Toi, M., Ferrara, N., 2005. Angiogenic inhibitors: a new therapeutic strategy in oncology. Nat. Clin. Pract. Oncol. 2, 562–577. Harrington, E.A., Bebbington, D., Moore, J., Rasmussen, R.K., Ajose-Adeogun, A.O., Nakayama, T., et al., 2004. VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat. Med. 10, 262–267. Hart, A.H., Hartley, L., Parker, K., Ibrahim, M., Looijenga, L.H., Pauchnik, M., et al., 2005. The pluripotency homeobox gene NANOG is expressed in human germ cell tumors. Cancer 104, 2092–2098. Hauf, S., Cole, R.W., LaTerra, S., Zimmer, C., Schnapp, G., Walter, L., et al., 2003. The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore microtubule attachment and in maintaining the spindle assembly checkpoint. J. Cell Biol. 161, 281–294. Heinrich, M.C., Blanke, C.D., Druker, B.J., Corless, C.L., 2002. Inhibition of KIT tyrosine kinase activity: a novel molecular approach to the treatment of KIT-positive malignancies. J. Clin. Oncol. 20, 1692–1703. Hoei-Hansen, C.E., Almstrup, K., Nielsen, J.E., Brask Sonne, S., Graem, N., Skakkebaek, N.E., et al., 2005. Stem cell pluripotency factor NANOG is expressed

Biomarkers for TGCTs

99

inhuman fetal gonocytes, testicular carcinoma in situ and germ cell tumours. Histopathology 47, 48–56. Jones, R.H., Vasey, P.A., 2003. Part II: testicular cancer-management of advanced disease. Lancet Oncol. 4, 738–747. Kedde, M., Strasser, M.J., Boldajipour, B., Oude Vrielink, J.A., Slanchev, K., le Sage, C., et al., 2007. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Cell 131, 1273–1286. Keen, N., Taylor, S., 2004. Aurora-kinase inhibitors as anticancer agents. Nat. Rev. Cancer 4, 927–936. Kemmer, K., Corless, C.L., Fletcher, J.A., McGreevey, L., Haley, A., Griffith, D., et al., 2004. KIT mutations are common in testicular seminomas. Am. J. Pathol. 164, 305–313. Kim, I., Saunders, T.L., Morrison, S.J., 2007. Sox17 dependence distinguishes the transcriptional regulation of fetal from adult hematopoietic stem cells. Cell 130, 470–483. Krause, D.S., Van Etten, R.A., 2005. Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med. 353, 172–187. Ledford, H., 2007. Doubts raised over stem-cell marker. Nature 449, 647. Lee, D., 2005. Phase II data with ZD6474, a small-molecule kinase inhibitor of epidermal growth factor receptor and vascular endothelial growth factor receptor, in previously treated advanced non-small-cell lung cancer. Clin. Lung Cancer 7, 89–91. Linger, R., Dudakia, D., Huddart, R., Tucker, K., Friedlander, M., Phillips, K.A., et al., 2008. Analysis of the DND1 gene in men with sporadic and familial testicular germ cell tumors. Genes Chromosomes Cancer 47, 247–252. Looijenga, L.H., de Leeuw, H., van Oorschot, M., van Gurp, R.J., Stoop, H., Gillis, A.J., et al., 2003. Stem cell factor receptor (c-KIT) codon 816 mutations predict development of bilateral testicular germ-cell tumors. Cancer Res. 63, 7674–7678. Loveland, K.L., Schlatt, S., 1997. Stem cell factor and c-kit in the mammalian testis: lessons originating from Mother Nature’s gene knockouts. J. Endocrinol. 153, 337–344. Lutke Holzik, M.F., Rapley, E.A., Hoekstra, H.J., Sleijfer, D.T., Nolte, I.M., Sijmons, R.H., 2004. Genetic predisposition to testicular germ-cell tumours. Lancet Oncol. 5, 363–371. Maatouk, D.M., Loveland, K.L., McManus, M.T., Moore, K., Harfe, B.D., 2008. Dicer1 is required for differentiation of the mouse male germline. Biol. Reprod. 79, 696–703. Manova, K., Nocka, K., Besmer, P., Bachvarova, R.F., 1990. Gonadal expression of c-kit encoded at the W locus of the mouse. Development 110, 1057–1069. Martin, O.V., Shialis, T., Lester, J.N., Scrimshaw, M.D., Boobis, A.R., Voulvoulis, N., 2008. Testicular dysgenesis syndrome and the estrogen hypothesis: a quantitative metaanalysis. Environ. Health Perspect. 116, 149–157. McCoshen, J.A., McCallion, D.J., 1975. A study of the primordial germ cells during their migratory phase in Steel mutant mice. Experientia 31, 589–590. McGlynn, K.A., Quraishi, S.M., Graubard, B.I., Weber, J.P., Rubertone, M.V., Erickson, R.L., 2008. Persistent organochlorine pesticides and risk of testicular germ cell tumors. J. Natl. Cancer Inst. 100, 663–671. Meng, X., de Rooij, D.G., Westerdahl, K., Saarma, M., Sariola, H., 2001. Promotion of seminomatous tumors by targeted overexpression of glial cell line-derived neurotrophic factor in mouse testis. Cancer Res. 61, 3267–3271. Muller, J., Skakkebaek, N.E., Nielsen, O.H., Graem, N., 1984. Cryptorchidism and testis cancer. A typical infantile germ cells followed by carcinoma in situ and invasive carcinoma in adulthood. Cancer 54, 629–634. Naro, C., Barbagallo, F., Chieffi, P., Bourgeois, C., Paronetto, M.P., Sette, C., 2014. The centrosomal kinase NEK2 is a novel splicing factor kinase involved in cell survival. Nucleic Acids Res. 42, 3218–3227. Nielsen, H., Nielsen, M., Skakkebaek, N.E., 1974. The fine structure of possible carcinoma in-situ in the seminiferous tubules in the testis of four infertile men. Acta Pathol. Microbiol. Scand. A 82, 235–248.

100

Paolo Chieffi

Oosterhuis, J.W., Looijenga, L.H., 2005. Testicular germ-cell tumors in a broader perspective. Nat. Rev. Cancer 5, 210–222. Ota, T., Suto, S., Katayama, H., Han, Z.B., Suzuki, F., Maeda, M., et al., 2002. Increased mitotic phosphorylation of histone H3 attributable to AIM-1/Aurora-B overexpression contributes to chromosome number instability. Cancer Res. 62, 5168–5177. Palumbo, C., van Roozendaal, K., Gillis, A.J., van Gurp, R.H., de Munnik, H., Oosterhuis, J.W., et al., 2002. Expression of the PDGF alpha-receptor 1.5 kb transcript, OCT-4, and c-KIT in human normal and malignant tissues Implications for the early diagnosis of testicular germ cell tumours and for our understanding of regulatory mechanisms. J. Pathol. 196, 467–477. Pero, R., Lembo, F., Di Vizio, D., Boccia, A., Chieffi, P., Fedele, M., et al., 2001. RNF4 is a growth inhibitor expressed in germ cells and lost in human testicular tumours. Am. J. Pathol. 159, 1225–1230. Pero, R., Lembo, F., Chieffi, P., Del Pozzo, G., Fedele, M., Fusco, A., et al., 2003. Translational regulation of a novel testis-specific RNF4 transcript. Mol. Reprod. Dev. 66, 1–7. Portella, G., Passaro, C., Chieffi, P., 2011. Aurora B: a new prognostic marker and therapeutic target in cancer. Curr. Med. Chem. 18, 482–496. Rajpert-De Meyts, E., Skakkebaek, N.E., 2011. Pathogenesis of testicular carcinoma in situ and germ cell cancer: still more questions than answers. Int. J. Androl. 34, e2–e6. Ranieri, G., Patruno, R., Ruggirei, E., Montemurro, S., Valerio, P., Ribatti, D., 2006. Vascular endothelial growth factor (VEGF) as a target of bevacizumab in cancer: from the biology to the clinic. Curr. Med. Chem. 1, 1845–1847. Skakkebaek, N.E., 1972. Possible carcinoma-in-situ of the testis. Lancet 2, 2516–2517. Skakkebaek, N.E., Berthelsen, J.G., Giwercman, A., Muller, J., 1987. Carcinoma-in-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma. Int. J. Androl. 10, 19–28. Sorrentino, V., Giorgi, M., Geremia, R., Besmer, P., Rossi, P., 1991. Expression of the c-kit proto-oncogene in the murine male germ cells. Oncogene 6, 149–151. Staibano, S., Ilardi, G., Leone, V., Luise, C., Merolla, F., Esposito, F., et al., 2013. Critical role of CCDC6 in the neoplastic growth of testicular germ cell tumors. BMC Cancer 13, 433. Strohsnitter, W.C., Noller, K.L., Hoover, R.N., Robboy, S.J., Palmer, J.R., TitusErnstoff, L., et al., 2001. Cancer risk in men exposed in utero to diethylstilbestrol. J. Natl. Cancer Inst. 93, 545–551. Terada, Y., Tatsuka, M., Suzuki, F., Yasuda, Y., Fujita, S., Otsu, M.A., 1998. AIM-1: a mammalian midbody-associated protein required forcytokinesis. EMBO J. 17, 667–676. Ulbright, T.M., 2005. Germ cell tumors of the gonads: a selective review emphasizing problems in differential diagnosis, newly appreciated, and controversial issues. Mod. Pathol. 18, S61–S79. Vicini, E., Loiarro, M., Di Agostino, S., Corallini, S., Capolunghi, F., Carsetti, R., et al., 2006. 17-β-estradiol elicits genomic and non-genomic responses in mouse male germ cells. J. Cell. Physiol. 206, 238–245. von Eyben, F.E., 2003. Laboratory markers and germ cell tumors. Crit. Rev. Clin. Lab. Sci. 40, 377–427. Voorhoeve, P.M., le Sage, C., Schrier, M., Gillis, A.J., Stoop, H., Nagel, R., et al., 2006. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124, 1169–1181. Wicha, M.S., Liu, S., Dontu, G., 2006. Cancer stem cells: an old idea-a paradigm shift. Cancer Res. 66, 1883–1890. Yamaguchi, S., Kimura, H., Tada, M., Nakatsuji, N., Tada, T., 2005. Nanog expression in mouse germ cell development. Gene Expr. Patterns 5, 639–646. Zakarija, A., Soff, G., 2005. Update on angiogenesis inhibitors. Curr. Opin. Oncol. 6, 578–583.