Testicular cancer in mice: interplay between stem cells and endocrine insults

Background Incidence of type II germ cell tumors (T2GCT) has increased in young men possibly due to fetal/perinatal exposure to estrogenic compounds. Three-fold increased incidence of T2GCT was reported in men exposed in utero to diethylstilbestrol (DES). T2GCT is a development-related disease arising due to blocked differentiation of gonocytes into spermatogonia in fetal testes which survive as germ cell neoplasia in situ (GCNIS) and initiate T2GCT. In our earlier study, T2GCT-like features were observed in 9 out of 10 adult, 100-day-old mice testes upon neonatal exposure to DES (2 μg/pup/day on days 1–5). Neonatal DES exposure affected testicular very small embryonic-like stem cells (VSELs) and spermatogonial stem cells and resulted in infertility, reduced sperm counts and tumor-like changes leading to our postulate that testicular dysgenesis syndrome possibly has a stem cell basis. The present study was undertaken to further characterize testicular tumor in mice testes. Methods DES-exposed mice pups (n = 70) were studied on D100 and after 12 months to understand how T2GCT progresses. Besides histological studies, a carefully selected panel of markers were studied by immuno-fluorescence and qRT-PCR. Results DES resulted in either atrophied or highly vascularized, big-sized testes and extra-testicular growth was also observed. GCNIS-like cells with big, vacuolated cytoplasm and increased expression of OCT-4, SSEA-1, SCA-1 and CD166 (cancer stem cells marker) along with reduced c-KIT, MVH and PTEN were evident. Global hypomethylation was found associated with altered expression of Dnmts, Igf2-H19 and Dlk-Meg3 imprinted genes along with reduced expression of Ezh2, cell cycle regulator p57KIP2 and Meg3; however, Pten remained unaltered. Increased expression of PCNA and Ki67 was observed in concert with complete lack of SOX-9 suggesting Sertoli cells independent proliferation. Conclusions Mouse model for T2GCT is described which will have immense potential to understand cancer initiation, cancer stem cells and also to develop effective therapies in future. T2GCT initiates from tissue-resident, pluripotent VSELs due to their altered epigenome. Neonatal exposure to DES blocks differentiation (spermatogenesis) and VSELs get transformed into CD166 positive cancer stem cells that undergo excessive self-renewal and initiate cancer in adult life challenging existing concept of fetal origin of T2GCT. Supplementary Information The online version contains supplementary material available at 10.1186/s13287-022-02784-5.

Hypomethylation confers a tumor-suppressing phenotype IGF2-H19 H19 and Igf2 genes are part of a cluster of imprinted genes.
VSELs show erasure or hypomethylation of imprints in paternally methylated and hypermethylation of imprints in maternally methylated ones. These epigenetic characteristics result in upregulation of H19 and repression of Igf2 in VSELs and thus they remain quiescent [2].
This balance may be affected by endocrine disruption and explain increased numbers of VSELs after neonatal exposure to DES.
Pathak et al [4] have reported reduced DNA methylation at Igf2-H19 ICR in sperm upon 60 days of tamoxifen (0.4 mg/kg body weight/day orally, 5 days a week) treatment to 75 days old rats which upon mating resulted in embryo loss.
Doshi et al [5] reported aberrant DNA methylation at Igf2-H19 imprinting control region in sperm upon neonatal exposure to bisphenol A and its association with post implantation loss.
Khambata et al [6] reported similar altered methylation status of several imprinted genes in male partners from couples experiencing recurrent pregnancy loss.
MEG3 encodes a lncRNA which is suggested to function as a tumor suppressor and has been involved in a variety of cancers. Zheng et al [8] demonstrated that MEG3 acts as a tumor suppressor Increased expression of DLK1 is found in several types of cancers. Overexpression of Dlk1 enhances tumor cell stemness & invasiveness in-vitro [9,10].

Ezh2
Ezh2 has an important role during histone modifications which are crucial to ensure epigenetic changes. It adds trimethylation group to histone H3 at lysine 27 and this leads to gene inhibition.
Stem cell self-renewal is not affected in Ezh2 deficient mice but their differentiation is affected and cells show elevated levels of Oct-4 and Nanog [11].
Interestingly, in Ezh2 plays an essential role in the maintenance of both the proliferative and selfrenewal capacity of stem/progenitor cells and the full execution of their differentiation. DNA methylation enzymes DNA methylation, catalyzed by the DNMTs, plays an important role in maintaining genome stability.
Altered expression of DNMTs and disruption of DNA methylation patterns are closely associated with many forms of cancer.
Dnmt 1 is involved in the maintenance of DNA methylation, Dnmt 3 a & b for de novo methylation, Dnmt 3L shares homology with Dnmt 3 family & essential for DNA methylation.
BPA treated testis showed increased levels of Dnmt3a and b [5].

PTEN
PTEN is a tumor suppressor gene that is frequently mutated or deleted in sporadic human tumors.
PTEN negatively regulates cell growth, migration and survival via the phosphatidylinositol 30kinase/AKT signaling pathway.
PTEN is expressed in germ cells and is virtually absent from 56% of seminomas, 86% of embryonal carcinomas and virtually all teratomas [12].
Loss of PTEN marks the transition from intra-tubular germ cell neoplasia (ITGCN) to invasive germ cell tumors [12]. p57kip2 Cyclin-dependent kinase inhibitor 1C (p57(KIP2) is an imprinted gene that regulates cell cycle and is frequently down-regulated in malignancies denoting its anti-oncogenic function. OCT-4A OCT-4 VSELs are pluripotent stem cells and express nuclear OCT-4 whereas the immediate descendants where differentiation is initiated express cytoplasmic OCT-4. VSELs with nuclear OCT-4A and SSCs with cytoplasmic OCT-4 are expressed in both human [13] and mice testes [14][15][16].
OCT4 is a sensitive and specific biomarker for intratubular germ cell neoplasia of the testis [17][18].

SCA-1
Stem cell antigen 1 (SCA-1) is expressed on stem/progenitor cells in multiple organs and is also reported in testes [16].
c-KIT Epigenetic status of spermatogonia changes dramatically during transition from c-Kit negative to positive state and this epigenetic switch decides whether the cells undergo self-renewal or cross the point of no return (lose stemness) and initiate differentiation [20].
C-kit is a proto-oncogene and KIT mutations are common in testicular seminomas [21].

Dmrt1a
Dmrt1 is a regulator of testis development and is implicated in testicular germ cell tumors of mouse and human. Loss of Dmrt1 in 129Sv strain mice results in a >90% incidence of testicular teratomas [22].
DMRT1 is a transcription factor, belonging to DNA binding gene family and is expressed in TGCTs in addition to Oct-4 and NANOG [23].

PCNA
Proliferating cell nuclear antigen (PCNA) is well established marker specific to DNA synthesis in dividing cells.

Ki67
Ki67 is a marker for proliferation. Serves as a molecular target in the diagnosis of cancer [24].

CD166
It is a cell surface marker for stem/progenitor cells in several cancers including testicular cancer in both mice and men [19,25,26].
Suppl Table 2 List of antibodies used in the study  and Stem Cell Antigen (SCA-1, b) upon DES treatment. DES treated testes showed increased PCNA (d) expression compared with normal testicular section (c). Global hypo-methylation was observed after DES endocrine disruption (f-h) while control testicular section has normal methylation (e). Tumor suppressor PTEN was abundantly expressed by normal testicular germ cells (i) and DES testis showed lost of PTEN expression (k) however (j) in atrophied testis few germ cells expressed PTEN was observed. (l) shows minimal expression of Ki-67 staining in normal control testis section.
Suppl Fig 9. Immuno-localization of MVH, c-KIT, SOX-9, PTEN, PCNA, Ki-67, 5-mC and CD166 in E2 treated testicular paraffin section on D100. MVH (a), PTEN (d) and SOX-9 (c) were not affected. c-KIT positive cells were observed (b) but to a lesser extent from normal age-matched control (Suppl Fig 2, h) Global methylation pattern was normal as evident by expression of 5-mC (g). PCNA was increased (e) but Ki67 was not increased (f). It is noteworthy that CD166 was not expressed (h). We did not observe tumor-lie changes upon E2 exposure at D100.