Cell Oncol. DOI 10.1007/s13402-014-0168-6

REVIEW

The chemosensitivity of testicular germ cell tumors Ioannis A. Voutsadakis

Accepted: 27 February 2014 # International Society for Cellular Oncology 2014

Abstract Although rare cancers overall, testicular germ cell tumors (TGCTs) are the most common type of cancer in young males below 40 years of age. Both subtypes of TGCTs, i.e., seminomas and non-seminomas, are highly curable and the majority of even metastatic patients may expect to be cured. These high cure rates are not due to the indolent nature of these cancers, but rather to their sensitivity to chemotherapy (and for seminomas to radiotherapy). The delineation of the cause of chemosensitivity at the molecular level is of paramount importance, because it may provide insights into the minority of TGCTs that are chemo-resistant and, thereby, provide opportunities for specific therapeutic interventions aimed at reverting them to chemosensitivity. In addition, delineation of the molecular basis of TGCT chemosensitivity may be informative for the cause of chemoresistance of other more common types of cancer and, thus, may create new therapeutic leads. p53, a frequently mutated tumor suppressor in cancers in general, is not mutated in TGCTs, a fact that has implications for their chemosensitivity. Oct4, an embryonic transcription factor, is uniformly expressed in the seminoma and embryonic carcinoma components of non-seminomas, and its interplay with p53 may be important in the chemotherapy response of these tumors. This interplay, together with other features of TGCTs such as the gain of genetic material from the short arm of chromosome 12 and the association with disorders of testicular development, will be discussed in this paper and integrated

I. A. Voutsadakis (*) Division of Medical Oncology, Department of Internal Medicine, Sault Area Hospital, 750 Great Northern Road, Sault Ste. Marie, ON P6B 0A8, Canada e-mail: [email protected] I. A. Voutsadakis e-mail: [email protected]

in a unifying hypothesis that may explain their chemosensitivity. Keywords Testicular germ cell tumors . Seminoma . Non-seminoma . Chemotherapy . Sensitivity . Resistance . p53 . Oct4

1 Background Testicular germ cell tumors (TGCTs) are the most common tumors in the young male population, with a peak incidence in the third and fourth decades of life. Two main sub-types of TGCTs, seminomas and non-seminomas exist, the former of which has a peak incidence in the fourth decade of life, the latter a decade earlier. These two sub-types share several risk factors, including cryptorchidism, infertility and the testicular dysgenesis syndrome and their precursor lesions, testicular carcinoma in situ (also called intratubular germ cell neoplasia unclassified) and gonadoblastoma, but they also exhibit distinct clinical characteristics and treatment modalities [1, 2]. Despite differences in their specific cell of origin, they share the fortunate characteristic of being highly curable. This is mostly due to the treatment responsiveness of these cancers rather than their early detection. Although about one quarter of all seminomas and over half of all non-seminomas are metastatic at diagnosis, the survival rates far exceed those of other cancers and consistently exceed 90 % for the good prognostic sub-groups [1]. Based on this high cure rate, seminomas, the sub-type having the best prognosis of the two, are divided into intermediate and good risk groups in the widely used IGCCC (International Germ Cell Consensus Classification) classification system, whereas no high risk sub-group is considered [3]. Systemic chemotherapy, including cisplatin and etoposide with or without bleomycin, leads to complete remissions and high cure rates even in the worse prognosis sub-groups. The

I.A. Voutsadakis

question of why TGCTs are chemosensitive is of major interest, not only because it may provide information on the small fraction that is intrinsically treatment-resistant or acquires resistance during their evolution, but also because it may provide clues on how to chemosensitize other cancers. This latter question has so far not been addressed in a fully satisfactory manner. Studies comparing chemosensitive TGCTs with chemoresistant counterparts have yielded information on specific single factors correlating with these outcomes, but the ultimate answer most probably lies in the integration of several simultaneous molecular events [4]. In order to formulate a plausible integrated theory one would, therefore, have to take into account the vast amount of data available on embryology and development, molecular biology, cytogenetics as well as epidemiology, linking the testicular dysgenesis syndrome to TGCTs. Here, I will put forward such a critical hypothesis based on existing published data and on emerging insights into the embryonic circuitries present in these tumors.

2 p53 is wild type in TGCTs A characteristic of TGCTs is that the tumor suppressor p53 is retained in its wild type configuration [5, 6] and is activated following exposure to chemotherapeutic agents [7]. p53, a transcription factor that is activated by DNA damage or cellular stress in general and that has the mission of safe-guarding the integrity of the genome, is mutated in about half of all human cancers and in the remaining malignancies it almost invariably becomes functionally inactivated through nongenomic mechanisms [8]. This dichotomy is well illustrated in for example head and neck carcinomas, where two pathogenic sub-types have been revealed, i.e., carcinomas not related to HPV viruses that mostly carry mutant p53 and HPVassociated carcinomas that mostly carry wild type p53, which is inactivated through viral protein E6-promoted proteasomal degradation [9, 10]. The importance of p53 in tumor suppression, which is underlined by these observations of universal neutralization, is further strengthened by recent data associating p53 with differentiation promotion, EMT (Epithelial to Mesenchymal Transition) inhibition and the promotion of asymmetric cell division [11–13]. These actions counteract the maintenance, propagation and metastatic potential of cancer stem cells. Additional parameters that have to be taken into consideration when discussing p53 and cancer are the positive pro-carcinogenic actions of mutant p53 that actively participate in the processes beyond the absence of the wild type transcript, and the fact that normal p53 activates two main distinct and mutually exclusive cellular programs, one leading to apoptosis and the other one leading to cell cycle arrest [14, 15].

The absence of mutant p53 in TGCTs deprives the respective cancer cells from several cancer promoting properties otherwise facilitated by the mutant protein. The presence of mutant p53 in other cancers, on the other hand, attributes functions to cells that are important for the initiation and/or establishment of the carcinogenic process [16, 17]. A significant proportion of p53 point mutants retains the ability to induce transcription from high affinity promoters, such as the p21 promoter, but loses the ability to induce transcription from lower affinity promoters, such as those of the Bax and PIG3 (p53-indicible gene 3) genes [18]. Retention of such selective transcriptional abilities may skew the outcome towards cell cycle arrest over apoptosis. Cell cycle arrest is sufficient to suppress cellular growth in vitro, but not to suppress cellular transformation in vivo [19]. The presence of mutant p53 has been proposed to enhance reprogramming of normal cells to pluripotent stem cells in the presence of Oct4 and Sox2 while, in the absence of the mutant version, the mere absence of wild type p53 requires the presence of Klf4 for efficient reprogramming [20]. Thus, in TGCTs, the absence of p53 mutations may be compatible with, or even imposed by, the (re)expression of Oct4 in order to support carcinogenesis by establishing the pro-survival embryonic network, which is now becoming a characteristic of these tumors as will be discussed in a following section. This (re)expression may also be facilitated by the fact that Oct4 is normally expressed in germ cell progenitors.

3 p53 decides between cell cycle arrest and apoptosis The outcome of p53 activation differs in different tissues, which is part of the normal tissue physiology [21]. In cancers such as TGCTs that retain wild type p53, any residual p53 activity that remains after non-genomic neutralization could become associated with drug resistance when the cell cycle arrest program is activated, or with treatment sensitivity when this residual activity is directed towards the apoptotic program. In the first scenario, a cancer cell is kept alive with a functional p53 that may promote differentiation which, in turn, may lead to drug resistance by allowing the accumulation of additional cancer promoting lesions. In the second scenario, triggering of p53 may lead to the elimination of a cancer cell in a definitive manner. The apoptotic program is executed through the increased transcription of genes such as PUMA, NOXA and p53AIP1, whereas the cell cycle arrest program is promoted through increased transcription of genes such as p21, GADD45 (Growth Arrest and DNA Damage 45) and TIGAR (TP53 Induced Glycolysis and Apoptosis Regulator) [22]. In TGCTs, p21 expression, despite being a p53 target gene, does not correlate with p53 expression, arguing for a dissociation of its regulation from p53 [23]. Following cisplatin treatment, TGCT cells up-regulate p21 much less

The chemosensitivity of testicular germ cell tumors

than e.g. ovarian carcinoma cells [24]. This means that either p53 is regulated distinct from the cycle arrest program or that other factors counter-balance any effect that it exerts on the p21 promoter. Obviously, factors determining which p53 program will be activated within a cellular context are of paramount importance for the therapeutic responses of these cancers with wild type p53. Cisplatin-resistant TGCT cell lines retain wild type p53 but, in contrast to sensitive TGCT cell lines, activate p21 and HDM2 expression after drug treatment [25]. In TGCTs which retain wild type p53, its triggering after drug-induced stress would be predicted to lead to activation of the apoptotic program in accordance with the drug sensitive phenotype usually observed [26]. At the same time p53induced differentiation, which is usually associated with drug resistance, should be prevented. How are these two conditions contributing to treatment sensitivity fulfilled in TGCT cells? Whether a cell will undergo apoptosis or cell cycle arrest following p53 activation is determined by the nature of the activation signal, the presence of concomitant signals and p53 post-translation modifications (which are partly determined by the activation signal and other inputs to the system). Multiple post-translation modifications, such as phosphorylation, ubiquitination, SUMOylation and acetylation, often working in concert, regulate p53 function [27] and their discussion is appropriate in the context of exploring the role of p53 in TGCT chemosensitivity. A major modification of p53 regulation is exerted through its ubiquitination by the E3 ligase HDM2, the human homologue of mouse mdm2 (mouse double minute 2). This modification leads to p53 proteasomal degradation and keeps p53 under control at baseline

conditions in the absence of stress [28]. HDM2 is a target of p53, allowing the fine-tuning of p53 activity via a feed-back mechanism (Fig. 1). Auto-ubiquitination of HDM2 promotes its own proteasomal degradation [29]. The HDM2 homologous protein HDMX (also called HDM4) is induced in TGCT cells by p53, and this protein participates in p53 regulation by inhibiting it without targeting it for ubiquitin-mediated proteasomal degradation [30]. The de-ubiquitinase HAUSP (Herpes virus-Associated Ubiquitin Specific Protease, also called USP7) reverses the ubiquitination of both p53 and HDM2. Under different stress conditions, p53 degradation is inhibited either through its phosphorylation, which prevents interaction with HDM2, or through inhibition of HDM2 activity via interaction with p14ARF (Alternative Reading Frame, a name that this protein takes due to the fact that it is transcribed from the same DNA sequence but with a different reading frame with the CDK inhibitor p16INK4A at chromosome 9p). p53 degradation is also prevented through deubiquitination by the enzyme HAUSP. At baseline conditions HAUSP binds with higher affinity to HDM2, whose degradation is prevented while p53 degradation is ensued. Under stress conditions phosphorylation of HDM2 decreases its affinity for HAUSP, whereas auto-ubiquitination leads to its degradation and concomitant p53 stabilization (Fig. 1). Mono-ubiquitination of nuclear p53 by HDM2 promotes its export to the cytoplasm where it is transcriptionally inactive [31]. Cytoplasmic translocation may be followed by poly-ubiquitination and degradation, or de-ubiquitination by HAUSP or an alternative de-ubiquitinase, USP10, that would facilitate re-entry [32] or even cytoplasmic sequestration by binding to cytoskeletal proteins such as vimentin,

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Fig. 1 Regulation of p53 network by ubiquitination. a After DNA damage, activation of ATM kinase leads to phosphorylation of p53 and HDM2. These phosphorylations decrease the binding affinity of deubiquitinase USP7 for HDM2 and increase its binding affinity for p53. p53 de-ubiquitination stabilizes it while HDM2 auto-ubiquitinates itself and is degraded by the proteasome. b Stabilization of p53 leads to HDM2

degradation

transcription. If DNA damage persists, HDM2 levels remain low because of its continued degradation. Low levels of activity lead to p53 monoubiquitination that promotes p53 nuclear export where it has non-transcription actions. If the DNA damage signal ceases, HDM2 is stabilized and its activity leads to p53 poly-ubiquitination and proteasomal degradation

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a process facilitated by the RBR (RING between RING) type E3 ligase Parc [33]. Cytoplasmic sequestration of p53 by PARC has been linked to treatment resistance in neuroblastoma [34]. Other E3 ligases have been found to contribute to p53 ubiquitination in diverse cellular environments and circumstances. In papilloma virus-infected cells, the HECT domain E3 ligase E6-AP (E6-Associated Protein) binds to the viral protein E6 and promotes p53 degradation, an event that, together with inhibition of the tumor suppressor Rb by viral protein E7, contributes to viral oncogenesis [9]. Additional E3 ligases with p53 as a substrate include PIRH2 (p53-induced RING H2) [35], ARF-BP1/Mule (ARF-Binding Protein 1/Mcl1 ubiquitin ligase E3) [36] and COP1 (Constitutively Photomorphogenic 1) [37]. ARF-BP1/Mule represents an interesting example of how ubiquitination networks may contribute to the decision between apoptosis or cell cycle arrest after p53 activation. This protein is a HECT domain ubiquitin ligase that, as its name implies, can be bound and inactivated by p14ARF in a manner analogous to HDM2 [36]. In addition, ARF-BP1/Mule may be regulated by an isoform of the deubiquitinase USP7 that prevents its auto-ubiquitination [38]. ARF-BP1/Mule inactivation leads to promotion of apoptosis in both p53-dependent and p53-independent ways, implying that the ligase may have other apoptosis promoting substrates besides p53 (Fig. 2). In addition, it ubiquitinates and promotes the degradation of an anti-apoptotic protein, the Bcl2 family member Mcl1 [39]. ARF-BP1/Mule possesses a BH3 (Bcl2 homology 3) domain through which it can interact with Mcl1. As a result of having both p53 and Mcl1 as substrates, ARFBP1/Mule can promote or impede apoptosis under different conditions [40]. An alternative hypothesis could be that ARFBP1/Mule may favour apoptosis by facilitating any residual p53 activity towards apoptosis, given that even a low PUMA or NOXA transcription would be sufficient to induce apoptosis in a cell with a decreased Mcl-1 level. This process could be ARF-BP/Mule USP7

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Fig. 2 Interactions of the E3 ligase ARF-BP/Mule with p53. ARF-BP/ Mule may have anti-apoptotic actions by directly inhibiting p53 by ubiquitination and, thus, its apoptotic target genes and pro-apoptotic actions by inhibiting base excision repair (BER) and promoting degradation of the anti-apoptotic Bcl-2 family member Mcl1

further enhanced by ARF-BP1/Mule-mediated suppression of BER (Base Excision Repair) after DNA damage [38]. The existence of multiple pathways regulating p53 stability and degradation by the Ubiquitin Proteasome System (UPS) allows both a strict control of its function and a versatility of its activation and inhibition. Thus, the UPS constitutes a vital component and central node of p53 regulation. The expression levels of the diverse E3 ligases playing a role in p53 regulation beyond HDM2 have, however, not yet been studied in TGCTs. In TGCTs p53 is not only functional but also increased in amount, which implies that either its production is increased or its degradation is decreased [6]. This modified level may have ramifications on the choice of the transcriptional p53 program which is triggered following activation, for example, if increased stability would favour a particular posttranslational modification due to slower kinetics of this modification. Indeed there are specific p53 modifications that have been associated with the execution of apoptosis versus cell cycle arrest and vice versa (Fig. 3), i.e., phosphorylation of serine at position 46 of p53 by the kinases DYRK2 (Dual Specificity Tyrosine Regulated Kinase 2) and HIPK2 (Homeodomain-Interacting Protein Kinase 2) promotes apoptosis over cell cycle arrest [41, 42]. Acetylation of two lysine residues K120 and K320 located in the DNA binding domain and the tetramerization domain of p53, respectively, also plays a role in the decision between apoptosis or cell cycle arrest. K120 is acetylated by the acetyltransferases TIP60 (Tat Interacting Protein of 60 kDa, also called KAT5, Lysine Acetyl-Transferase 5) and hMOF (human Male absent On the First, also called KAT8) and promotes apoptosis through transcription-dependent and -independent mechanisms [43–45], and acetylation at K320 by PCAF (p300/CBP-Associated Factor, also called KAT2B) promotes cell cycle arrest over apoptosis [46]. Several other lysine residues at the carboxy-terminus of p53 serve as common targets for acetylation and ubiquitination, and their acetylation antagonizes the ubiquitination and degradation, thereby promoting p53 stability [27]. De-acetylation of p53 offers an additional level of regulation. The de-acetylase SIRT1, for example, deacetylates p53 at K382 and suppresses apoptosis [47]. SIRT1 may, however, act as a tumor suppressor in other contexts [48]. This latter activity may relate to promotion of cytoplasmic retention of p53 when it is in the de-acetylated state and to induction of transcription-independent apoptosis secondary to interaction with mitochondrial proteins [49]. It is conceivable that, in other contexts, cytoplasmic retention does not result in apoptosis because of, for example, PARC-facilitated vimentin entrapment [34]. TGCTs express vimentin only in a minority of cells [50], which may not be sufficient for inhibition of p53induced transcription-independent apoptosis. An explanatory hypothesis for the role of p53 in TGCT chemosensitivity would be that enzymes serving apoptosispromoting p53 post-translation modifications are over-

The chemosensitivity of testicular germ cell tumors Fig. 3 Examples of posttranslational modifications of p53 that affect the decision between cell cycle arrest or apoptosis

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expressed or up-regulated or, alternatively, that enzymes that are involved in p53 modifications promoting cell cycle arrest are suppressed or down-regulated. However, no data supporting such occurrence in TGCTs specifically exist yet. More complex regulations and loops of interactions are operational in distinct contexts and result in imbalances of the p53 decision with up-regulation or down-regulation of just one protein involved. For example, kinase HIPK2 stability is regulated by the E3 ligase HDM2 and another RING type E3 ligase, SIAH2 (Seven in Absentia Homolog 2), which are both p53-induced [51]. Thus, auto-regulatory loops may operate in this case too, akin to the p53-HDM2 loop. Another layer of regulation in the choice of the transcriptional program initiated by p53 is provided by co-regulators that influence the occupation of target gene promoters by p53 and modulate the final result in the expressed set of these genes in a given cellular context. An example is the protein JMY (Junction Mediating and regulatory protein) that cooperates with p53 in transcription initiation from apoptotic gene promoters [52, 53]. ASPP (Apoptosis Stimulating Protein of p53) proteins also act as co-factors that specifically favour transcription of p53 apoptotic genes [54, 55]. ASPP cooperates with the acetyltransferase p300 and p53 to induce the apoptotic target PIG3, but not p21 [56]. In contrast, APAK (ATM and p53-associated KZNF protein, also called ZNF420) is a repressing co-factor that specifically suppresses transcription of p53-associated apoptosis genes, but not cell cycle arrest genes [57]. In addition, APAK promotes interaction of p53 with the de-acetylase HDAC1, reversing p53 acetylation and interaction with apoptotic genes promoters [58]. These are examples of how p53 post-translational modifications and co-regulators interact to determine a final program output. Again, although over-expression or suppression of these co-regulatory proteins in TGCTs could contribute to promotion of an apoptotic p53 response program over a cell cycle arrest outcome, this option remains speculative and no data are available yet. Obviously, this would be an area interesting to investigate.

4 Oct4 and its role in chemo-sensitivity A relevant observation that has been described in the literature in recent years is that seminomas and embryonal carcinomas (in contrast to choriocarcinomas, teratomas and yolk sac components of non-seminomas) universally express the octamer transcription factor Oct4 (also called OTF3 or POU5F1) [59]. This is a transcription factor that binds to the octamer DNA sequence ATGC(A/T)AAT through its POU domain (named after the founder proteins of the family, PIT-1, Oct1, Oct2 and UNC86) [60]. Oct4 is expressed in embryonic stem cells where it ensures their survival [61] and maintains their pluripotency in co-operation with other transcription factors such as Sox2 [62]. Leydig cell-associated signaling through the IGF-1 (Insulin-like Growth Factor 1) and PI3K/Akt pathways induces Oct4 expression in germ cells (Fig. 4) [63]. Phosphorylation of Oct4 by Akt facilitates its interaction with Sox2 on promoters of target genes such as Oct4 itself and Nanog [64]. In addition, this phosphorylation inhibits ubiquitination of Oct4, which leads to degradation by the proteasome and promotes dissociation of unphosphorylated Oct4 from the Akt promoter where it acts as transcription suppressor [64]. Oct4 is down-regulated by promoter methylation and its expression is suppressed in differentiated adult tissues and organs, such as the normal testis [65]. In TGCT cells, Oct4 may become down-regulated by hypoxia, an event that leads to cisplatin and bleomycin resistance [66]. Hypoxia promotes SUMOylation (association with the ubiquitin-like protein SUMO) of Oct4 at lysine K123 and, consequently, Oct4 down-regulation. In contrast, peptidase SENP1 can deSUMOylate Oct4 and, hence, enhance chemosensitivity of TGCT cells [66]. Re-expression of Oct4 in seminomas and the embryonal component of non-seminomas may contribute to both prevention of differentiation and induction of apoptosis in these cancers following p53 activation [67]. In other words, Oct4 re-expression may prevent p53-induced cell cycle arrest and differentiation. Thus, Oct4 may have a triple role in (i) survival of TGCT cells at baseline conditions similar to that in normal embryonal cells, (ii) apoptosis following stress

I.A. Voutsadakis Activation through Leydig cell-derived IGF-1 signaling in normal germ cells Signaling-independent activation in TGCTs

Fig. 4 Regulation of Oct4 expression in normal testis and TGCTs that leads to the establishment of embryonic networks

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and p53 activation and (iii) prevention of differentiation as witnessed by loss of expression in teratomas, choriocarcinomas or yolk sac components of non-seminomatous TGCTs. Mechanistically, these effects may be related to the effect of Oct4 on transcription of the CDK inhibitor p21 and Nanog, which are co-regulated but in opposite directions by p53 [68]. The cell cycle arrest inducer p21 is induced by p53 but suppressed by Oct4, while the embryonic stem cell maintenance factor Nanog is suppressed by p53 but induced by Oct4 (Fig. 5). Several lines of evidence support the idea that these reciprocal regulatory mechanisms in TGCT cells are critical for determining chemotherapy responsiveness: the expression of these proteins correlates with sensitivity or resistance to therapy, i.e., in contrast to teratomas which are the a priori chemo-resistant tumors [23, 69, 70], p21 is not expressed in seminomas and embryonal carcinomas which are the a priori chemo-sensitive tumors [71]. Cisplatin-resistant embryonal carcinoma cells show a high p21 expression, whereas Oct4 has the ability to down-regulate this expression and to sensitize these cells to cisplatin [72]. In addition, primary embryonal carcinomas from patients that were cisplatin-sensitive showed high Oct4 and no p21 expression, whereas patients with cisplatin-resistant mature teratomas were Oct4 negative miR-371-3

LATS2

miR-302 miR-17-92 Oct4

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Fig. 5 Representation of the p53/Oct4 network in TGCTs with important targets that are mutually regulated

and strongly p21 positive [72]. Thus, lack of Oct4 expression in embryonal carcinoma cells and primary tumors correlates with cisplatin resistance [73]. How does a stemness factor such as Oct4, which is a prosurvival factor for normal germ cell progenitors, become a factor promoting cell death following chemotherapy? Indeed, stemness in general is associated with chemo-resistance in cancer [74, 75]. In other cancer types, such as oral squamous carcinomas, increased expression of Oct4 (and other stem cell factors) is associated with cisplatin resistance [76]. Thus, it is the particular cellular context of TCGCs rather than the presence Oct4 per se that favors chemosensitivity in these tumors. In this respect it is worth mentioning here that oral squamous cancers have a functionally debilitated p53 as a result either of gene mutations or of the presence of the HPV protein E6 that promotes its E3 ligase E6AP-mediated ubiquitination and proteasomal degradation [77].

5 p53-Oct4 interplay: p21, microRNAs and the pluripotency network From the above discussion it can be concluded that it is not the individual expression of p53 or Oct4 that leads to chemosensitivity of TGCTs, but rather the combined expression and function of both. The balance between them negates effects that both have (i.e., promotion of differentiation, cell cycle arrest and autophagy for p53, establishment of stemness and promotion of survival for Oct4) and creates a unique stable inbetween state that, despite having acquired all other neoplastic properties, retains the sensitivity to apoptotic stimuli (Fig. 6). Conversely, both mutations in p53 and loss of Oct4 expression lead to cisplatin resistance [78, 79]. Oct4 is one of the core factors of stemness and an integral component of the recently proposed reprogramming of

The chemosensitivity of testicular germ cell tumors Fig. 6 p53 and Oct4 have both individual effects that may result in cheomoresistance, but their combined action leads to sensitivity for apoptotic stimuli. Mutations in p53 or Oct4 downregulation disrupt this balance and promote chemoresistance

mtp53

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somatic cells to induced pluripotent stem cells [80]. In contrast, p53 is known to act as a blocker of pluripotency induction [81]. In these opposing actions, several targets of the two transcription factors are important as already alluded to in previous sections. The CDK inhibitor p21, induced by p53 and down-regulated by Oct4 has, besides its role in cell cycle arrest, a role in the suppression of pluripotency by applying a brake to the rapid cycling of self-renewing progenitors [82]. Additionally, p21 mRNA is a repression target of a network of microRNAs (miRs) involved in the establishment of pluripotency during the transition from a fertilized oocyte to a zygote during embryogenesis (Fig. 5). This network includes the miR clusters miR-371, miR-302 and miR-17-92 in humans, which are under the positive control of the pluripotency transcription factors Oct4, Sox2 and Nanog [83]. miR-302a is up-regulated in TGCT cells following cisplatin treatment and, subsequently, down-regulates p21 [84]. Another miR, miR-145, is repressed by Oct4, Sox2 and Klf4 and reciprocally represses Oct4 [85]. miR-145 is induced by p53 [86] and, interestingly, serves as a suppression target of the EWS-FLI-1 fusion protein in Ewing sarcoma, a sarcoma of the primitive neuroectodermal family of tumors with the highest prevalence in the second and third decade of life [87]. miR-372/373 (induced by Oct4, Sox2 and Nanog) suppresses the serine/threonine kinase LATS2 (Large Tumor Suppressor homolog 2), an inducer of senescence and suppressor of cyclin E and endoduplication during mitosis [88]. LATS2 is a target gene for induction by p53 and, thus, represents an additional target of reciprocal regulation by the p53/ Oct4 pair. The importance of the miR-372/373 cluster in the Oct4 circuitry is further demonstrated by the fact that it can substitute for transcription factors in the induction of pluripotent stem cells from mature somatic cells [89]. The mechanism of this induction involves suppression of the histone demethylases AOF1 and 2 and the DNA methyltransferase DNMT1, leading to DNA demethylation and, consequently, expression of Oct4, Sox2 and Nanog [90]. By suppressing DNMT1, miR372/372 may relieve the need to have the DNA demethylation machinery in place. This machinery is

Sensitivity to apoptotic stimuli

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comprised of Gadd45, a target of the p53 cell cycle arrest program and of proteins of the excision repair pathways that are down-regulated in TGCTs (see below) [91]. In TGCT patients, miR372 and miR373 are expressed in the majority of both seminomas and non-seminomas (especially the embryonal carcinoma component) where they suppress LATS2, which is induced by wild type p53 [92]. Nanog, another protein reciprocally regulated by Oct4 and p53, is a homeobox-containing transcription factor and a core pluripotency network member itself [93]. At least in some cases, such as in the induction of Xist, an RNA that guides the inactivation of one X chromosome in female embryonic cells, Nanog acts as a pioneering factor for Oct4 [94]. Pioneering factors are proteins bound to DNA interacting with transcription factors and helping them to interact with subsets of target promoters in a particular cellular context where they are expressed. As such, the availability of Nanog in seminoma and embryonal carcinoma components of TGCTs [95], resulting from an inter-play between Oct4 and p53 and a gain of chromosome 12p through isochromosome formation, could have important effects on modulating the Oct4 transcriptome.

6 Gain of chromosome 12p: contributor to sensitivity or bystander? TGCTs most probably arise from primordial germ cells (PGCs) and gonocytes (GCs) entering the gonadal ridge, or at least from cells that have acquired the phenotype of these progenitors. Arguing for this notion are both the imprinting pattern of TGCTs, which is erased (as in PGCs and GCs before being paternally established in normal spermatogonia) [96], and the fact that TGCTs arise as primary tumors all along the route of PGCs along the gonadal ridge. Thus, TGCTs have the unique characteristic among cancers that they can form primaries in locations outside the normal adult location of the neoplastic cell of origin, i.e., the testis. In contrast, spermatocytic seminomas, a sub-type of TGCTs that is usually seen in men older than 50, develop from later cells in

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spermatozoan development, spermatogonia or spermatocytes, and have the paternal imprinting pattern of these cells [97]. Normal PGCs travel along the gonadal ridge guided by SCF (Stem Cell Factor) and SDF-1 (Stromal cell Derived Factor 1, also known as CXCL12), which are secreted by stromal cells of the ridge and bind to their receptors (c-kit and CXCR4 respectively) expressed on PGCs. These receptors activate intracellular K-ras and the down-stream Raf/MEK/ERK and PI3K/Akt pathways that promote cell survival and evasion from apoptosis [98]. During neoplastic transformation PGCs gain the ability to activate these pathways in the absence of external signals without losing their capacity to undergo apoptosis if stress signals or p53 activation ensue. Gain of genetic material from the short arm of chromosome 12 through isochromosome formation in the majority of cases or, less frequently, through more restricted 12p amplifications is a universal feature of TGCTs and takes place during the transition from in situ to invasive tumors [99, 100]. This is a critical moment when neoplastic cells obviate the support of neighboring cells and the micro-environment of the seminiferous tubule in order to detach and become invasive. At this stage it is important that they have acquired their own self-supporting signals for survival. Therefore, the c-kit and CXCR4 receptors and down-stream pathways need to be activated without ligand binding. The increased expression of chromosome 12p genes, including K-ras, may contribute to this. An additional role may be played by the pluripotency genes Nanog and Stella (also called DPPA3, Developmental Pluripotency Associated 3), which also reside on 12p [101]. These genes may contribute to evasion from differentiation that may result from the activity of certain pathways down-stream of K-ras. The benefit endowed to cancer cells by gain of genes on 12p must be specific toTGCT cells, since this karyotypic abnormality is specific to these tumors. In order to derive benefit from the presence of increased dosages of these genes, TGCTs must maintain an active transcription machinery together with a primed epigenetic state in order to be able to transcribe them. These pre-requisites appear to be present in TGCT cells. It has been shown, for example, that the transcriptional regulator DDX1, a DEAD-box ATP-dependent RNA helicase, is present in TGCT cell lines and primary tumors and is critical for the transcription of genes located on 12p such as cyclin D2, CD9 and Nanog [102]. Knock-down of DDX1 by RNA interference in a TGCT cell line resulted in the repression of 12p genes and a reduced ability of this cell line to form tumors in nude mice [102]. Moreover, retinoic acid (RA) is known to induce the differentiation of embryonal carcinoma cells, which is accompanied by loss of tumorigenicity and repression of genes on 12p [103]. As yet, it is unclear whether this RA-mediated repression acts directly or through the repression of Oct4, which is also affected by RA. These data argue for a critical role of 12p-derived genes in TGCTs invasion, but its contribution to the chemo-sensitivity

of these tumors remains to be resolved. Answering this question becomes even more complicated given that, as the above example of RA illustrates, a particular intervention or modulator may have effects not only on genes on 12p, but also on other critical players. Nevertheless, the fact that gain of genetic material on 12p provides a strong benefit in the particular cellular context of TGCTs that is selected for, may imply that genes on 12p such as Nanog participate in generating a cancer stabilizing infrastructure for TGCT cells and, thus, contribute directly or indirectly to chemosensitivity.

7 Increased incidence of germ cell tumors in testicular dysgenesis syndromes Testicular dysgenesis syndromes encompass a spectrum of disorders with an increased risk of TGCT development. Persistence of Oct4 expression beyond its normal developmental presence is seen in dysgenetic gonads, a fact that adds to the argument of the association of Oct4 with testicular carcinogenesis [104]. This persistence may also represent and explain the link of testicular dysgenesis with TGCTs. According to this hypothesis, germ cells of dysgenetic gonads fail to downregulate Oct4 at the point where this normally occurs and these germ cells are, therefore, at an increased risk to become neoplastic. This same molecular event levies the pressure for p53 disabling, and permits its presence in a wild type configuration, making it compatible with germ cell cancer survival but also contributing to chemosensitivity. Dysgenetic gonads are associated with various disorders of sex determination which result from inherited numerical chromosomal anomalies, such as Klinefelter or Turner syndrome, or from insensitivity to sex steroids [105]. These conditions present with various phenotypes, and some of them exhibit an increased risk for germ cell tumors. This increased risk is encountered independent of the phenotype if the Y chromosome is present. Presence of the Y chromosome leads to expression of the Y-associated gene SRY (Sex determining Region in Y) by gonadal stromal cells and the target genes Sox9 (SRY box-containing 9) and FGF9 (Fibroblast Growth Factor 9) that support the differentiation of Sertoli cells [106] (Fig. 7). Sertoli cells, in turn, promote the differentiation of gonocytes towards pre-spermatogonia and spermatogonia, a process during which primordial germ cell-associated proteins such as Oct4, Nanog and c-kit are physiologically downregulated and testis specific proteins such as MAGE4, VASA and TSPY are induced [106]. In dysgenetic gonads of a genetically male individual, defective signaling to gonocytes leads to differentiation delay, thereby prolonging the interval of expression of proteins such as Oct4 and, thus, creating the cellular context described in previous sections. Testicular dysgenesis syndromes are also associated with maternal exposure to environmental estrogenic mimetics

The chemosensitivity of testicular germ cell tumors Fig. 7 Abnormal estrogen signaling in genetic males impact both testis development by interfering with Sertoli cell development and imprinting reestablishment and chemosensitivity by altering retinoic acid (RA) levels

Y chromosome present

Gonadal cells Express SRY

Estrogen signaling

BLIMP1

Imprinting re-establishment during the normal transition from pre-spermatogonia to spermatogonia

Sertoli cells development SOX9

Exogenous estrogen mimetics

De novo methylation

FGF9

Nuclear entry

AP-2γ

DNMT3b

RA degradation (persistence in female embryos)

[107]. This exposure triggers signaling from the Estrogen Receptor (ER). Transcription from ER promoter sites not normally initiated in the male developing testis would result in expression of genes such as AP-2γ (Activator Protein 2γ), which have an ER binding site in their promoter [108]. AP-2γ is a member of the AP-2 family of transcription factors with a role in promotion of proliferation and suppression of differentiation during embryonic development [109]. Another regulator of AP-2γ is the transcription factor BLIMP1 (B lymphocyte-induced maturation protein 1, also named PRDM1, PR-SET domain molecule 1), which is a germ cell specification factor [110]. AP-2γ is normally expressed in gonocytes but becomes down-regulated as gonocytes differentiate towards spermatogonia. In contrast, AP-2γ remains expressed in dysgenetic testes and TGCTs [111]. Target genes of AP-2γ, such as Nanos3, DMRT1 (Doublesex- and mab-3related transcription factor 1) and DNMT3b (DNA methyltransferase 3b), are important regulators of germ cells and, thus, their deregulation may be involved in the pathogenesis of testicular carcinomas. In addition, they represent a link between testicular dysgenesis and carcinogenesis as well as TGCT chemosensitivity. For instance, the cytochrome P250 enzyme CYP26B1 is a target of DMRT1, and upregulation of this enzyme results in the degradation of retinoic acid, RA [112, 113]. In contrast, in female embryos the normally persisting RA plays a role in the development of female gonads and the concomitant induction of meiosis [114]. Down-regulation of RA in TGCTs by AP-2γ as part of normal male physiology, and in dysgenetic gonads as part of abnormal ER signaling, may contribute to the chemosensitivity in TGCTs by preventing differentiation associated with resistance. DNMT3b, a de novo DNA methylation enzyme and also a target of AP-2γ, is important for the re-establishment of

DMRT1

Gonocytes differentiation to pre-spermatogonia and Spermatogonia (downRegulation of Nanog, PLAP, Oct4, c-kit, SALL4, Induction of MAGE4, VASA, TSPY)

CYP26B1

imprinting during the transition from primordial germ cells to pre-spermatogonia [115]. Persistence beyond this stage could lead to inappropriate silencing of additional genes (e.g. tumor suppressors) contributing to carcinogenesis. Additionally, ER signaling interferes with normal testicular development by preventing SOX9 nuclear entry and, thus, normal Sertoli cell establishment in genetic males [116].

8 DNA damage sensing and response: TGCTs versus common chemo-resistant tumors Attempts have been made to advance unifying theories of the mechanisms and causes underlying chemosensitivity in various tumors including TGCTs, ovarian germ cell tumors, choriocarcinomas, Hodgkin lymphomas and Non-Hodgkin lymp h o m a s [ 11 7 ] . T h e s e t h e o r i e s r e c o g n i z e d t h a t chemosensitivity is an intrinsic state of the tumors and not related to the particular drug or drug combination used. Moreover, a role of complex DNA rearrangements as part of the normal physiologic function of the cells from which all these malignancies derive has been proposed [117]. In TGCTs, these rearrangements include changes that germ cells normally undergo in their preparation for meiosis. The chemosensitivity of a given cancer cell depends on its ability to sense damage, to activate the DNA damage response (DDR) and to respond by undergoing apoptosis instead of cell cycle arrest and DNA damage repair. The activated autophosphorylated form of ATM (Ataxia Telangiectasia Mutated) has been detected in sub-sets of spermatocytes and in TGCTs, especially the embryonic carcinoma part and less in the seminomatous part, confirming that these cells are capable of sensing damage [118]. The phosphorylation target of ATM

I.A. Voutsadakis

and marker of DNA double strand breaks, γH2AX (phosphorylated form of histone H2AX), is constitutively present in normal fetal gonocytes and TGCT cells [118]. In addition, during the early phase of development before meiotic division, male germ cells down-regulate their DNA repair mechanisms and they are, therefore, intrinsically more sensitive to apoptosis [119]. It has been shown that, after DNA damage, developing germ cells exhibit a high level of apoptosis during the meiotic recombination phase. This characteristic of decreased repair ability is retained in TGCTs [120] and may be related to the low DNA repair protein expression levels in TGCTs [121]. Primary tumor specimens from non-seminoma patients, who generally exhibit a higher resistance to chemotherapy, tend to show a higher expression of the Nucleotide Excision Repair (NER) protein ERCC1 (Excision Repair Cross Complementation group 1) and its enzymatic active partner XPF (Xeroderma Pigmentosum protein F) than primary tumor specimens from seminoma patients [122]. ERCC1 and another DNA repair protein, XPA, were confirmed to exhibit increased expression levels in both cisplatin resistant cell lines and primary TGCT patient specimens compared to their respective cisplatin-sensitive counterparts [123]. Intrastrand cross links, the main type of DNA lesions induced by cisplatin, are repaired by the NER system which is dependent on the presence of p53 [124]. This repair activity, however, does not correlate with cisplatin sensitivity in TGCT cell lines [125]. In addition, although low NER protein ERCC1 expression has been found to correlate with cisplatin sensitivity in non-small cell lung cancer, a prototypic chemotherapy insensitive carcinoma [126], the benefit of low ERCC1 expression in this tumor type is much lower than that seen in TGCT patients. The same is true for triple negative breast cancers (clinically more sensitive to cisplatin than Estrogen Receptor positive breast cancers, but hardly so compared to TGCTs), which express significantly lower levels of ERCC1, XPF and the Fanconi Anemia (FA) repair pathway proteins BRCA1, FANCD1 (BRCA2), FANCF and PALB2 than luminal A type breast cancers [127]. Interestingly, in patients with lung cancer not receiving chemotherapy, high ERCC1 expression has been associated with a better prognosis than in patients with a low expression [126, 128]. This high expression may reflect p53 transcriptional activity that induces NER protein expression [129], rather than directly affecting prognosis. Thus, although a degree of down-regulation in DNA repair mechanisms may contribute to chemosensitivity, the way DNA damage is sensed by the cell and the p53 response to it both seem to account for chemotherapy responsiveness. Arguing in the same direction are data from head and neck cancer cell lines where cisplatin sensitivity correlates with levels of drugDNA adducts, but not with mRNA expression of the DNA repair genes ERCC1, ATM, ATR, BRCA1 and BRCA2 [130]. An additional challenge in the interpretation of DNA repair data comes from the realization that all currently available

antibodies for the evaluation of ERCC1 by immunohistochemistry fail to discern between the different isoforms of the protein, only one of which is functionally competent in DNA repair [131]. Despite these issues, which deserve further study, DNA repair defects may affect the way damage is sensed by increasing the burden of simultaneous DNA lesions at a given time in a cell. In TGCT cells treated ex-vivo with cisplatin, no DNA adduct repair was observed [132]. This inability to repair affects, in turn, the program activated down-stream of p53, given that increased damage burden favors apoptosis over cell cycle arrest [133]. In addition, exogenous factors may affect the ability of a cell to repair DNA and to respond to p53 activation. Osteosarcoma cells exogenously expressing p53 and growing in 10 % serum display an increase in DNA repair in response to cisplatin and a decrease to cisplatin sensitivity, while they fail to down-regulate anti-apoptotic Bcl-2 in comparison to their p53-negative counterparts [134]. In contrast, the same cells growing in 1 % serum down-regulate Bcl-2, do not display an increase in DNA repair but show an increase in sensitivity to cisplatin compared to their p53-negative counterparts [134]. Small non-histone chromatin associated proteins of the High Mobility Group (HMG) family play a role in DNA damage recognition by identifying and binding to damaged sites [135]. The binding of HMGB1 to damaged sites leads to shielding and concealing these sites from the repair machinery, resulting in decreased cisplatin adduct repair and drug sensitivity [136, 137]. Others, however, have reported an increased expression of HMGB1 in cisplatin-resistant cells [138, 139]. HMGB1 also plays a role in transcription regulation and is able to directly interact with transcription factors on DNA, or independently from it in colorectal cells [140]. This interaction modulates the outcome of p53 activation, favoring apoptosis or autophagy following DNA damage [141]. In addition, HMGB1 is shed to the extracellular space and serves as a ligand for the cell surface receptors RAGE (Receptor for Advanced Glycation End-products) and TLR4 (Toll-Like Receptor 4) and, as such, can activate several intracellular signaling cascades in cells that express these receptors [142], with ramifications through modulation of the tumor microenvironment. Another HMG family member, HMGB4, is expressed specifically in the testis and inhibits the repair of cisplatin adducts in vitro [143]. HMGA1 and HMGA2, two other HMG proteins that bind to AT-rich DNA sequences through basic domains called AT-hooks, are expressed in TGCTs, especially the embryonal component [144]. Seminomas express only HMGA1, while mature teratomas lose expression of both HMGA proteins. These expression patterns imply an association of these proteins to chemosensitivity, but their actual role in this process remains to be established. HMGA1 interacts with HIPK2 and sequesters it to the cytoplasm, where it interferes with its interaction

The chemosensitivity of testicular germ cell tumors

with p53 [145]. This interference could hamper the apoptotic program elicited by HIPK2-induced p53 S46 phosphorylation. In addition, HMGA2 is a transcriptional target of c-Myc, which is over-expressed in various cancers (not always chemosensitive), and is expressed in poorly differentiated aggressive sub-sets of common and commonly chemoresistant cancers such as breast and bladder carcinomas [146, 147]. It seems that a partial concealment of DNA damage from the repair machinery, together with an intrinsic lower activity of this machinery, could contribute to TGCT chemosensitivity. On the other hand, damage recognition by proteins like ATM to activate down-stream p53 is concomitantly required. Data regarding DNA mismatch repair (MMR) in TGCTs argue for the importance of DNA repair in cisplatin resistance [148]. In contrast to repair by NER, where a low activity is associated with chemosensitivity, MMR defects are associated with resistance. Resistant tumors were frequently found to be associated with defects in MMR and to exhibit microsatellite instability (MSI) resulting from a low expression of the MMR proteins MLH1 and MSH6 [148]. These tumors were also found to be associated with the mutant V600E form of the oncogene BRAF, which is recurrently found in subsets of melanomas and leads to constitutive activation of BRAF. Of note, in colorectal cancer where the MMR system is best characterized due to its pathogenic association with Lynch syndrome, MMR defects are associated with right colon tumors that tend to have a better prognosis, but a lower benefit from anti-metabolite fluoro-pyrimidine treatment [149]. When oxaliplatin, an analogue of cisplatin, is added to the regimen, however, the negative predictive association of MSI with chemotherapy response tends to be neutralized [150], as MMR participates in oxaliplatin-DNA adduct repair [151]. In T G C Ts , t h e a s s o c i a t i o n o f M M R f u n c t i o n w i th Fig. 8 Building blocks of DNA damage sensing and response in chemosensitive TGCTs and other chemosensitive cancers (left) and in chemoresistant TGCTs and other chemoresistant cancers such as breast and lung cancer (right)

chemosensitivity may relate to the aforementioned decreased NER activity that creates a pressure on the presence of an alternative repair mechanism for the protection of the genome from excessive damage, especially in damage prone sites such as microsatellites (both drug-induced and spontaneous), that otherwise would produce too many lesions for the cell to be viable. In contrast, if MMR function is lost, additional lesions such as BRAF activating mutations are required for the cells to remain viable, and these may lead to concomitant chemoresistance [148]. This chemoresistance is a characteristic of melanomas often harboring V600E BRAF mutations. Targeted inhibition of mutant BRAF by small kinase inhibitors has produced impressive clinical responses [152]. In summary, following DNA damage by chemotherapy, TGCTs exhibit a low threshold for the activation of DNA damage-associated kinases such as ATM that can activate p53. They additionally are able to conceal damaged sites from the NER repair machinery, which is down-regulated in TGCTs. This, in turn, leads to the accumulation of damage and a further activation of the p53-associated apoptotic program. In TGCTs this program is favored over cell cycle arrest. An intact MMR mechanism contributes to chemosensitivity by protecting the cells from acquiring activating point mutations in oncogenes. An overall model encompassing all aspects discussed in this paper and a comparison of TGCTs with common less sensitive cancers is depicted in Fig. 8.

9 Perspectives: clinical prediction of chemosensitivity and therapy Experimental evidence for the relevance of any step conferring chemosensitivity of cells may be obtained by interfering with them in TGCTs or inducing them in chemoresistant

Chemosensitive TGCTs and other chemosensitive cancers

Chemoresistant TGCTs and other common, chemoresistant cancers

Machinery for DNA recombination or complex re-arrangements present

Machinery for DNA re-arrangements present in (pre-meiotic) resistant TGCTs but absent in other tumors

NER down-regulated

NER not down-regulated

DNA damaged sites concealed from repair machinery

DNA damaged sites accessible to repair machinery

p53 wild type and in the apoptotic module MMR proficient and absence of BRAF mutations

p53 mutated or debilitated and in the cell cycle arrest module MMR deficiency and presence of BRAF mutations in a sub-set

I.A. Voutsadakis

tumors. Therapeutic opportunities to chemosensitize tumors with de novo or acquired resistance lay in every step of this model, but some may be more feasible than others. For example, skewing the p53 response towards apoptosis may be relatively easy attainable by inhibiting an enzyme involved in a post-translational modification step favoring cell cycle arrest, whereas attaining re-expression of Oct4 will have to bypass all the hurdles currently faced by gene delivery technologies in vivo. Nevertheless, even the former approach will not be without problems because many if not all enzymes involved in p53 post-translational modification have other targets besides p53. The E3 ubiquitin ligase HDM2, for example, targets besides itself and p53 the EpithelialMesenchymal Transition (EMT) transcriptional modulator Slug, the degradation of which inhibits EMT [153]. This is of particular concern because it has been suggested (although there are data favoring the reverse in certain cellular contexts [154]) that the pluripotency network promotes EMT [155, 156] and that the absence of HDM2 activity may further exacerbate this transition associated with metastasis and drug resistance. Moreover, the model implies that drug resistance may have different molecular causes in individual cases and, thus, strategies to combat this resistance will need to be tailored to the underlying mechanism. Resistance due to the acquisition of MMR lesions and BRAF V600E mutations may be amenable to treatment with the small kinase inhibitors Vemurafenib or Dabrafenib. Resistance linked to upregulation of p21 due to a molecular lesion causing skewing of the p53 response towards the cell cycle arrest program, e.g. an enzyme that performs a post-translational modification of p53 may be more amenable to targeted inhibition of this enzyme. Resistance associated with mutations of p53 per se also may lead to up-regulation of the cell cycle arrest program and could be addressed therapeutically by interventions inhibiting key proteins of this program. More common and more chemoresistant cancers such as breast cancer express in sub-sets of their cells pluripotency factors such as Oct4 and Nanog [157], but harbor also mutated or functionally debilitated p53. These sub-sets may represent resident stem cell-like cells or cells that have acquired stem cell characteristics. Thus, interventions similar to those proposed for chemoresistant TGCTs could be efficacious in them. This hypothesis implies that an efficacious therapeutic strategy would be a targeted treatment addressing the chemoresistance-inducing lesion(s) followed by chemotherapy. It also implies that the sub-sets of cells with the pluripotency network in place would be the ones to monitor for therapeutic efficacy and to focus on for treatment response marker development.

Conflict of interest The author declares no conflicts of interest regarding this paper.

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