Department of Gynecology and Obstetrics (G.H.G.M.-B., T.B.H.), Andrology Laboratory, Rikshospitalet, University Hospital, 0027 Oslo, Norway; Department of Public Health and Cell Biology (G.H.G.M.-B., F.G.K., M.D.F.), Section of Histology and Embryology, University of Rome "Tor Vergata," 00133, Rome, Italy; Department of Biochemistry (W.E.), University of Oslo, 0316 Oslo, Norway; and Institute of Population-Based Cancer Research (T.G.), Cancer Registry of Norway, 0310 Oslo, Norway
Address all correspondence and requests for reprints to: Prof. Massimo De Felici, Dipartimento di Sanità Pubblica e Biologia Cellulare, Università di Roma "Tor Vergata," Via Montpellier 1, 00133 Roma, Italy. E-mail: defelici{at}uniroma2.it.
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ABSTRACT |
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Based on these results, we present a novel experimental in vitro model for tumorigenic germ cell transformation and identify molecular pathways likely involved in development of germ cell tumors after estrogen exposure.
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INTRODUCTION |
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In the present work, we aimed to directly verify whether estrogens affect the development of mouse PGCs and eventually cause their transformation into tumorigenic cells. This paper shows that estrogens stimulate Steel gene transcription in gonadal somatic cells increasing the production of the c-Kit ligand (KL) and consequently inducing PGC growth most likely via an Akt/PTEN-dependent pathway. In addition, we found that prolonged exposure of PGCs to estrogens in culture associated with down-regulation of the lipid phosphatase PTEN, a major inhibitor of phosphoinositol 3-OH kinase (PI3-K) activity (6) and with stimulation by leukemia inhibitor factor (LIF), resulted in a high frequency of PGC transformation into tumorigenic cells.
These results highlight the risk that in certain genetic background, exposure of the embryo to estrogens may favor tumors formation from PGCs in the testis and perhaps in other extragonadal sites where PGCs can mislocalize during migration toward the gonadal ridges.
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RESULTS |
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Using RT-PCR and immunoblotting, we found that 11.5 dpc gonadal somatic cells, but not PGCs, express estrogen receptor (ER) and estrogen-related receptors (ERR)
(Fig. 2
, A and B), whereas ERß and ERRß were not expressed by either cell types (not shown), thus confirming that PGCs are not the direct target of estrogens.
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Estrogens Stimulate KL Production in Gonadal Somatic Cells
In vitro studies have identified several factors that can regulate PGC growth (for a review, see Ref. 7). The product of the Steel gene, c-Kit ligand (KL), also known as stem cell factor, is a major positive regulator of PGC growth. PGCs express high levels of the KL receptor c-Kit and gonadal somatic cells synthesize both soluble and membrane-bound forms of KL (8, 9, 10). We found that the addition of E2 and ZEA to the culture medium caused, on average, a significant 4-fold (E2) and 2-fold (ZEA) increase of soluble KL production in gonadal somatic cells in culture, whereas no consistent increase of the membrane bound KL was found (Fig. 3, A and B). Further evidence that the growth promoting action of E2 and ZEA on PGCs is mostly due to the stimulation of the KL/c-Kit system activity, was from the finding that 10 µg/ml anti-KL antibody abolished such action (1 d of culture, control = 62 ± 7%, 10 µM ZEA = 120 ± 9%, ZEA + anti-KL antibody = 55 ± 5%). In addition, we found that other factors known to favor PGC growth (for a review, see Ref. 7), such as the leukemia inhibitory factor (LIF), the basic fibroblast growth factor (bFGF) and increased intracellular levels of cAMP in PGCs, were not involved in the estrogen-dependent PGC growth (Fig. 3C
, and data not shown).
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KL Increases Akt and PTEN Phosphorylation in PGCs
To identify molecular pathways downstream of c-Kit activation in PGCs and to verify their involvement in the increased proliferation of PGCs caused by estrogens, we first studied whether KL stimulation of PGCs resulted in phosphorylation of the serine/threonine kinase Akt, a well-known effector of c-Kit in other cell type (for a review, see Ref. 14). Western blot analysis demonstrated that stimulation of purified PGCs, cultured in suspension in the absence of cell monolayers, for 5 min with 100 ng/ml KL, increased Akt phoshorylation (Fig. 4A). The presence in the assay of 20 µM SU5416, a potent inhibitor of c-Kit-dependent Akt activation (15), or of 10 µM LY294002, a highly specific inhibitor of the lipid kinase PI3-K, resulted in complete inhibition of Akt phosphorylation (Fig. 4A
). This indicated that c-Kit and PI3-K activation in 11.5 dpc PGCs, as in other cell type, are upstream events of Akt phosphorylation. Interestingly, we also found that the lipid phosphatase PTEN, known as a major inhibitor of PI3-K activity (6), was also phosphorylated in PGCs subjected to KL stimulation (Fig. 4B
). Because PTEN phosphorylation is believed to restrict PTEN activity (16), a parallel activation of Akt and inactivation of PTEN seem to occur in PGCs after KL stimulation that should contribute to sustain their proliferation. Moreover, we found that SU5416 also inhibited PTEN phosphorylation induced in PGC by KL stimulation (Fig. 4B
), confirming a novel upstream c-Kit-dependent regulation of PTEN. We did not further investigate the molecular pathway(s) of the c-Kit-dependent PTEN phosphorylation. It is unlikely, however, that they involve PI3-K activation because it is known that the PTEN tail contains phosphorylation sites for GSK3, PKA, CK1, and CK2 kinases but not for PI3-K (16).
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We also tested if estrogen stimulation and PTEN inactivation affected the embryonal germ (EG)-forming efficiency of PGCs in vitro. It is known that PGCs cultured in continuous presence of a combination of growth factors or compounds [for example KL, LIF, and bFGF or forskolin (FRSK)] for 710 d give rise to pluripotent tumorigenic cells called EG cells (17, 18, 19). We found that in cultures of no-AS-treated PGCs, estrogens can substitute for exogenous KL and partly FRSK in the cocktail of compounds necessary for EG cell formation and that the efficiency of EG cell colony formation was markedly increased in PTEN AS-treated PGC cultures. Moreover, in this latter condition ZEA or E2 plus LIF produced the most favorable compound combination for EG formation (Fig. 6). Control replicates indicated that antisense oligonucleotide treatment in the absence of growth factors and estrogens was not sufficient to induce PGC transformation in EG cells and that cell treatment with sense oligonucleotide did not produce any significant increase of EG cell formation with any compound combination in comparison to untreated cultures (data not shown). Moreover, PGC transformation into EG cells with or without estrogen stimulation required c-Kit-dependent Akt activation because no EG colonies were formed when PGC culture was carried out in the presence of 20 µM SU5416 or 10 µg/ml anti-Kl antibody (Fig. 6
).
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DISCUSSION |
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In the present paper, however, we report that relatively high concentrations of estrogens increase PGC proliferation in culture in a dose-dependent manner. In addition, we demonstrated that estrogens do not act directly on PGCs but stimulate gonadal somatic cells to synthesize PGC proliferation factor(s). In support of these observations, we found that these early stage PGCs do not express estrogen receptors, whereas gonadal somatic cells express mRNA for ER. The immunohistochemistry for the ER
on tissue sections of 11.5 dpc gonads gave no conclusive results, which might indicate a low expression of this receptor at protein level. It is known, however, that certain tissues (e.g. bone), which are highly responsive to estrogens, express a low level of ERs. In such tissues, the effect of estrogens is explained by the simultaneous expression of ERR
or ERRß, which, acting via a cross talk to the ER
or ERß, amplify estrogen signaling (11). This might also occur in 11.5 dpc gonadal ridges because we found that somatic cells express, in addition to ER
, ERR
mRNA. At later fetal stages, both male and female mouse gonadal somatic cells and germ cells can express ERs, but their functions at these stages are unknown (24, 25).
Among several compounds that can stimulate PGC proliferation (7), we obtained evidence that estrogens increase the synthesis of KL, mainly the soluble form, in gonadal somatic cells, most likely by directly increasing Steel promoter activity. In support of the possibility that increased levels of KL produced by gonadal somatic cells is responsible for the estrogen-dependent growth of PGC in culture, we found that this growth induction was abolished or significantly reduced when anti-KL antibodies were added to the culture medium.
We attempted to identify downstream effectors of KL in PGCs, with the aim of studying their involvement in the stimulation of PGC proliferation by estrogens and the consequence of an exposure of PGCs to estrogens in culture conditions in which the action of these effectors was experimentally altered. Several gain-of-function mutations have been recently identified in the c-Kit receptor or its dependent signaling to be associated with highly malignant tumors including germ cell testicular tumors in humans (14). It is noteworthy that the c-Kit receptor is expressed at high levels in carcinoma in situ and seminoma testicular tumors (26). The lipid kinase phosphoinositol 3-OH kinase (PI3-K) and its downstream target, the protein-serine/threonine kinase Akt, are crucial effectors of tumorigenic protein-tyrosine kinases including c-Kit. Interestingly, the tumor suppressor gene PTEN encodes a phosphatase that inhibits PI3-K activity. Deletion or inactivation of PTEN results in constitutive Akt activation and is implicated in the pathogenesis of tumors of various histological origin (6).
It has been suggested that PTEN may be inactivated upon growth factor stimulation and estrogens have been reported to inhibit PTEN-mediated growth suppression in MCF-7 breast cancer cells (6, 27). The stability and activity of PTEN depend on the phosphorylation of three residues (S380, T382, and T383) within its tail. In particular, PTEN tail phosphorylation is believed to restrict PTEN activity (16). Recently, Akt activation by estrogen in estrogen receptor-negative breast cancer cells has also been reported (28). Based on these reports, we decided to verify whether stimulation of PGCs by KL or estrogens resulted in Akt-phosphorylation (activation) and PTEN-phosphorylation (inactivation). Western blot analysis demonstrated that stimulation of purified PGCs in suspension with 100 ng/ml KL increased both Akt and PTEN phoshorylation, thus suggesting a parallel KL-dependent activation of Akt and inactivation of PTEN. The presence in the assay of SU5416, an inhibitor of c-Kit-dependent Akt activation (15) or of LY294002, an inhibitor of PI3-K, one of the major effectors of Akt, resulted in complete inhibition of Akt phosphorylation. This indicates that c-Kit and PI3-K activation in 11.5 dpc PGCs, as in other cell types, are upstream events of Akt phosphorylation. Two studies using transgenic and knockout mice (29, 30) and one by De Miguel et al. (31), have recently reported that Akt activation in PGCs may occur independently of PI3-K action suggesting alternative downstream effectors of c-Kit in PGCs. Although we were unable to demonstrate that in our culture system estrogen action resulted in increased Akt and PTEN phosphorylation in PGCs, the findings that the estrogen-dependent increase of PGC growth in culture was abolished or significantly decreased by anti-KL antibodies and by SU5416 or LY294002 presence in the culture medium, suggest that such effect depends on c-Kit activation and Akt/PI3-K downstream pathways. Most importantly, we show that the tumor suppressor phosphatase PTEN, a major regulator of PI3-K activity (6), plays a crucial role in regulating PGC growth and their response to prolonged estrogen stimulation. We demonstrated that the reduction of PTEN activity in PGCs markedly increased their growth in culture and that PGCs with low PTEN activity were much more sensitive to tumorigenic transformation induced by the addition to the culture medium of KL, LIF, and FRSK or of estrogens and LIF, thus suggesting that PGCs in which PTEN is inactive are more prone to tumorigenic transformation by any proliferative stimulus. Estrogen might be one of many factors that can act as such a proliferative stimulus. Finally, we found that under the culture conditions used in the present paper, PGC transformation into EG cells required a functional KL/c-Kit system. In support, our results regarding the crucial role of PTEN in germ cell tumor formation, while we were preparing the present paper, Kimura et al. (32) have reported that PGCs from PTEN-null mice exhibited an increased proliferation capacity, Akt hyper-phosphorylation and enhanced EG cell colony-formation. They have also demonstrated that the PTEN-null mice developed bilateral testicular teratomas.
In conclusion, whereas studies in mice lacking ER and ERß suggest that estrogens are not required for normal PGC development, our results indicate that exposure to estrogens during embryonic life may have a profound effect on PGC growth and differentiation. Moreover, although it remains controversial if prenatal estrogen excess exposure in itself increases the risk of testicular germ cell cancer (5, 33), in the present paper, using an in vitro culture system we show that estrogens stimulate PGC proliferation and that this can result in their tumorigenic transformation when associated with other conditions affecting the control of their proliferation and differentiation. We provide evidence that together with genetic background and conditions (i.e. KL/c-Kit mutations, PTEN inactivation) favoring increased and/or prolonged PGC proliferation and the action of factors that inhibit PGC differentiation (most importantly LIF, or members of the LIF family), exposure to high levels of estrogens, altering the normal supply of growth factors provided by estrogen-responsive somatic cells surrounding PGCs, might constitute a high risk for PGC transformation into pluripotent tumorigenic cells. These conditions may give rise to germ cell tumor formation in the fetal testis and perhaps in other extragonadal sites containing estrogen-responsive cells where PGCs can mislocalize during migration toward the gonadal ridges and find conditions favorable for their tumorigenic transformation.
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MATERIALS AND METHODS |
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Mice and Cell Culture
129 SvSl/+ random-bred with 129 Sv+/+ mice (Jackson Laboratories, Bar Harbor, ME), and random-bred CD1 mice (Charles River, Como, MI) were used. The morning of vaginal plug was defined as 0.5 dpc. Gonadal ridges were obtained from 11.5 dpc embryos. PGC plus gonadal somatic cells and MiniMACS (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) purified PGCs and gonadal somatic cells were prepared using MiniMACS magnetic separation system as previously described (34). Culture of PGCs on mitomycin C-treated STO (ATCC, Manassas, VA) or Sl4m220 (a kind gift from Dr. Peter Donovan, T. Jefferson University, Philadelphia, PA) cell feeder layers was carried out as previously described (34) using phenol red-free DMEM with high glucose, supplemented with 15% FCS, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine and penicillin-streptomycin. PGCs were identified by alkaline phosphatase (APase) staining (34). In some experiments, PGC identity was confirmed by TG-1 (SSEA-1-like) antibody as described (34). The number of adherent PGCs 4 h after seeding was defined as the number of seeded PGCs. Mouse recombinant KL and anti-KL antibody (MABG56) were purchased from R & D Systems (Minneapolis, MN) and mouse LIF ESGRO and human bFGF were from Life Technologies (Tåstrup, Denmark). E2, ZEA, and FRSK were from Sigma (Oslo, Norway). SU5416 was a kind gift from SUGEN, Inc. (South San Francisco, CA) and LY294002 was from ALEXIS Biochemicals (Vinci, Italy).
BrdU labeling was carried out using the Amersham Pharmacia Biotech (Oslo, Norway) cell proliferation kit as reported in (35).
Tumors in Nude Mice
EG cells were subcultured onto STO cell feeder layers in the presence of 1000 UI LIF as described (18). Approximately 2 x 104 cells were injected sc or under the testis capsule of CD-1 nude mice (Charles River). After 3 wk, tumors were fixed in Bouins fixative, processed for histology and sections stained with hematoxylin and eosin.
RT-PCR
Total RNA was isolated from whole 11.5 dpc gonadal ridge or purified 11.5 dpc PGCs and somatic cells. RT-PCR was performed as described (36). Adult uterus and 16.5 dpc heart served as positive controls for ER and ERR
, respectively. PCR primers were designed from the murine sequence (GenBank accession no. M38651 for ER
and U85259 for ERR
) and purchased from DNA Technology (Aarhus, Denmark). The sequence for ER
of the sense primer was 5'-ATTGACAAGAACCGGAG-3' and that of the antisense was 5'-ATAGATCATGGGCGGTTCAG-3'. For ERR
, the sense primer was 5'-GAAAGTGAATGCCCAGGTGT-3' and for antisense 5'-GGAGATCGGATTAAGCAGCA-3' was used. For ERß the sense primer was 5'-GAA GCT GGCTGACAAGGAAC-3' and the antisense was 5'-GTGTCAGCTTCCGGCTACTC-3'. For ERRß the sense primer was 5'-GATGCCCTCAGCCACCAC-3' and the antisense was 5'-CAG CCG TCGCTTGTACTTCT-3'. Identity of the PCR products was confirmed by direct sequencing by BMR Bio Molecular Research (Padova, Italy).
Western Blot Analysis
Western blotting analyses were performed according to standard methods. Briefly, after 10% SDS-PAGE electrophoresis, proteins were transferred to nitrocellulose, blocked with TBST/5% nonfat milk or 5%BSA and incubated with specific antibodies: anti-ER (kindly provided by National Hormone & Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA), anti-KL (R&D Systems, Abingdon, UK), anti-LIF (Santa Cruz Biotechnology, Santa Cruz, CA), antiactin (Sigma), anti-P-PTEN-Ser380 and PTEN (Cell Signaling, Beverly, MA) or anti-P-Akt-Ser473 and anti-Akt (Cell Signaling). After exposure to the secondary antibodies (Amersham Pharmacia Biotech), blots were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech). The band intensity was quantified using actin as internal quantitative control and SigmaGel software (Jandel Scientific, Chicago, IL). For each determination, at least two experiments were performed.
Immunohistochemistry
Immunohistochemistry was performed on paraffin or cryostat sections of 11.5 dpc gonadal ridges using the antibody against ER (National Hormone & Pituitary Program, Harbor-UCLA Medical Center, Torrance, CA) and a protocol as reported (25).
For PTEN immunocytochemistry, PGCs in culture were fixed with methanol for 10 min at 4 C, washed extensively with PBS + 10% BSA and incubated overnight with 1:300 TG-1 and 1:150 PTEN (Cell Signaling, Beverly, MA) primary antibodies in PBS + BSA at 4 C. Finally, cells were labeled with secondary antibody (TRITC conjugated antimouse IgM and fluorescein isothiocyanate-conjugated antirabbit IgG, respectively) for 30 min at room temperature, washed and mounted in 90% glycerol/PBS.
Reporter Gene Assay
TM4 cells were transfected with 2 µg ß-galactosidase plasmid and 3.5 µg of the pTAL-Luc (CLONTECH, Palo Alto, CA) reporter plasmid that contained a luciferase gene under transcriptional control of the thymidine kinase promoter and a Steel gene promoter AP-1 response element as described (37). Briefly, the construct 5'T TTA ATC CTG AGT CAC TTG TTT TC3' (893916) from Mus musculus mast cell KL gene 5' flanking region U44724 (gi 1172214) was inserted in its normal orientation into a unique XhoI site of the pTAL-Luc reporter plasmid (CLONTECH). The transfected TM4 cells were treated in charcoal stripped and phenol red-free medium with 4 nM E2 or 10 µM ZEA. After 48 h, cells were harvested. Luciferase activity was assayed according to the manufacturer (Promega, Madison, WI), ß-galactosidase activity was assayed as described (38). Luciferase activity was normalized to ß-galactosidase activity, and the difference relative to the empty vector was calculated.
Antisense Treatment
Two PTEN AS were constructed according to the sequence obtained from the GenBank accession no. NM 008960.
AS1: (5'-GCTCAACTCTCAAACTTCCAT-3'; 43% GC; corresponds to nucleotides 153173 and AS2: (5'-GCCGCCGCCGTCTCTCATCTC-3'; 71% GC; corresponds to nucleotides 269289). A sense oligonucleotide (S) control was (5'-GAGATGAGAGACGGCGGCGGC-3'; 71% GC). The oligonucleotides were obtained from GIBCO Life Technologies. PGCs were subjected to transfection with oligonucleotides after the procedures as described (39, 40). A sense oligonucleotide control was performed for all treatments. The expression of PTEN in cultured PGCs subjected to oligo treatment was analyzed as reported above.
Statistical Analysis
Data are expressed as mean ± SEM of at least three independent experiments with three replicates per experimental group. Comparisons were made by one-way ANOVA, and significance was accepted at the 0.05 level of probability. P < 0.05 was considered to be significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The first two departments listed contributed equally to this work.
Abbreviations: AP, Activator protein; APase, alkaline phosphatase; AS, antisense PTEN oligo nucleotides; bFGF, basic fibroblast growth factor; BrdU, 5-bromo-2'-deoxyuridine; dpc, days post coitum; E2, 17ß-estradiol; EG, embryonal germ; ER, estrogen receptor; ERR, estrogen-related receptor; FRSK, forskolin; KL, c-Kit ligand; LIF, leukemia inhibitor factor; PGC, primordial germ cells; PI3-K, phosphoinositol 3-OH kinase; ZEA, zearalenone.
Received for publication January 8, 2003. Accepted for publication September 10, 2003.
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REFERENCES |
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