Human endogenous retrovirus rec interferes with germ cell ... - Nature

Oncogene (2005) 24, 3223–3228

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Human endogenous retrovirus rec interferes with germ cell development in mice and may cause carcinoma in situ, the predecessor lesion of germ cell tumors Uwe M Galli1, Marlies Sauter1, Bernd Lecher2, Simone Maurer2, Hermann Herbst3, Klaus Roemer1 and Nikolaus Mueller-Lantzsch*,1 1 Department of Virology, Bldg. 47, University of Saarland Medical School, 66421 Homburg/Saar, Germany; 2M-F-D Diagnostics Laboratory GdbR, Resources Bldg. of the Johannes-Gutenberg University, Obere Zahlbacher Str. 63, 55131 Mainz, Germany; 3 Institute of Pathology, University Hospital, 48149 Muenster, Germany

Germ cell tumors (GCTs) are among the most common malignancies in young men. We have previously documented that patients with GCT frequently produce serum antibodies directed against proteins encoded by human endogenous retrovirus (HERV) type K sequences. Transcripts originating from the env gene of HERV-K, including the rec-relative of human immunodeficiency virus rev, are highly expressed in GCTs. We report here that mice that inducibly express HERV-K rec show a disturbed germ cell development and may exhibit, by 19 months of age, changes reminiscent of carcinoma in situ, the predecessor lesion of classic seminoma in humans. This provides the first direct evidence that the expression of a human endogenous retroviral gene previously established as a marker in human germ cell tumors may contribute to organ-specific tumorigenesis in a transgenic mouse model. Oncogene (2005) 24, 3223–3228. doi:10.1038/sj.onc.1208543 Published online 28 February 2005 Keywords: endogenous retrovirus; Rec; Rev; Rex; germ cell tumor; PLZF

All humans carry and inherit endogenous retroviral sequences (HERVs) as an integral part of their genomes. Analyses of the recently completed human genome sequence have revealed that retroviral elements account for approximately 8% of the human genome, corresponding to approximately 2000 proviruses (the DNA copies of viral RNA genomes) and perhaps more than 30 000 solitary sequences derived from retroviral longterminal repeats (LTRs). HERVs are relics of ancient, horizontally (via infection) transmitted proviruses that integrated into the genomes of cells of the germ line of primate/human predecessors 35–40 million years ago

*Correspondence: N Mueller-Lantzsch, Institute of Virology, Bdlg. 47, University of Saarland Medical School, Germany; E-mail: [email protected] Received 11 August 2004; revised 5 January 2005; accepted 14 January 2005; published online 28 February 2005

(Barbulescu et al., 1999). Many of these sequences have since then been amplified through retrotransposition, that is, reintegration of reversely transcribed mRNA, and perhaps through re-infection, and have been extensively altered by mutation and recombination (Leib-Mosch and Seifarth, 1996; Lower et al., 1996; Patience et al., 1997; Jurka, 1998). In fact, the great majority of the HERV sequences is dysfunctional due to the accumulation of mutations. Nonetheless, several members of the HERV-K family of endogenous retroviral sequences still contain intact open reading frames for the viral Gag, Prt, Pol and Env proteins, with an almost intact member, HERV-K (HML-2.HOM), being located on chromosome 7 (Mayer et al., 1999). HERVK transcripts have been detected in many human tissues; however, germ cell tumors (GCTs) and GCT cell lines express complex splice variants reminiscent of the variants observed upon the infection of cells with exogenous lentiviruses (the family of viruses that contains HIV and the human T-cell leukemia viruses HTLV-1 and -2) (Lower et al., 1993). rec transcipt, produced from the HERV-K env gene by alternative splicing, is translated into the 14.7 kDa Rec protein, a functional homolog of the regulatory Rev/Rex proteins of the human immunodeficiency and T-cell leukemia viruses (Lower et al., 1995; Magin et al., 1999; Yang et al., 1999). The incidence of GCTs, the most common solid tumors in young men, has more than doubled in the past 50 years (Moller, 1993). GCTs are morphologically distinct neoplasms that originate from primordial germ cells and can be grouped into two entities: seminomatous and nonseminomatous (mixed seminomatous and nonseminomatous areas in one tumor are occasionally observed) (Mostofi, 1980). Approximately half of all GCTs are seminomas, which are either of the ‘classic’ type with uniform medium-sized cells showing clear cytoplasm and defined borders (Skakkebaek et al., 1987), or belong to the rare spermatocytic seminomas which characteristically contain large cells of the spermatocyte or spermatogonia B type (Looijenga and Oosterhuis, 1999). Classic seminomas originate from developmentally early arising, immature and

Endogenous retroviruses and cancer UM Galli et al

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differentiation-defective carcinoma in situ (CIS) cells. Since patients with GCT produce antibodies against HERV proteins and since GCT cells express splice variants of the HERV env transcript, of which one is translated into the Rec protein (Lower et al., 1995; Herbst et al., 1996; Sauter et al., 1996; Magin et al., 1999; Yang et al., 1999), we asked whether Rec, inducibly expressed in all tissues of transgenic mice, can induce organ-specific changes indicative of a disturbance of germ cell maturation or the development of CIS. rec is produced in humans from HERV-K env transcripts by alternative splicing (Figure 1a). To detect Rec protein in Raji cells stably transfected with a recexpressing plasmid or in insect cells infected with recbaculovirus, we developed a rabbit anti-Rec polyclonal antibody (#3086). The antibody produced a specific signal at 14.5 kDa only with extracts from Raji-Rec cells (formerly Raji-cORF; Boese et al., 2000) and infected insect cells, but not with extracts from control-transfected Raji cells or after pre-incubation of the antibody with bacterially expressed TrpE–Rec fusion protein (Figure 1b), documenting that the rec gene employed for the following studies can give rise to a protein of the predicted size. Next, transgenic mice that conditionally express rec under the control of a Tet-inducible promoter were generated. For this purpose, the rec gene was inserted into the PvuII site of the pBI-enhanced green fluorescence protein (EGFP) vector (Clontech), which allowed bidirectional expression of the transgene and the EGFP gene in the presence of a Tet transactivator and tetracycline or doxycycline. Purified, vector-free DNA was injected into mouse pronuclei using standard techniques. Tet transgenic mice expressing the Tet transactivator were kindly provided by H Bujard, ZMBH, Heidelberg, Germany. rec/Tet

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transgenic mice were generated by mating male hemizygous rec transgenics with female hemizygous Tet transgenics. PCR genotyping of rec transgenic mice was achieved with the following primers: 50 -ATG AAC CCA TCA GAG ATG CAA-30 ; 50 -CAA TGG TTG TCA ACA GAG TAG-30 . The Tet transgenic mice were identified with the primers: 50 -AAT GAG GTC GGA ATC GAA GG-30 and 50 -TAG CTT GTC GTA ATA ATG GCG G-30 . Animals were either mock-treated or exposed throughout their lifetime to doxycycline hydrochloride (200 mg/ml) dissolved in 5% sucrose and supplied with the drinking water, which was changed every 3 days. To test for inducible transgene expression, skin fibroblasts were cultured by incubating tail tips in RPMI 1640 with 10% FCS and collagenase (1 mg/ml), then grown in the presence or absence of doxycycline, and finally examined by fluorescence microscopy for expression of the EGFP transgene. Figure 2a shows that the transgene was inducible by doxycycline. Inducible expression of the rec transcript was studied by RT–PCR. For this purpose, total RNA from tissues was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer´ s protocol. To remove residual DNA, DNase I treatment was carried out. The RT was performed on 5 mg RNA and 25 pmol of random primers using Superscript II (Life Technologies, Inc.). The results summarized in Figure 2b show that doxycycline treatment induced rec transcript production in various organs including the testes of rec/Tet mice. However, when compared, for instance, with the levels of rec transcript that were detectable in the endogenous rec-expressing human teratocarcinoma cell line Tera-1 (Figure 2c), the levels obtainable in the rec/Tet mice were not very high, and were indeed very low in the testes. Reflecting these differences, Western immunoblot analyses of protein extracts prepared from rec-negative

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Rec (14.5 kDa) Figure 1 Splicing and expression of rec. (a) Schematic outline of the human chromosome 7-located, almost intact member of the HERV-K family, HML-2.HOM, and positioning of the proviral gag, prt, pol and env reading frames. rec is generated from the env reading frame by alternative splicing. The numbers denote the beginning and end of the reading frames relative to the proviral sequence. (b) Western immunoblot analysis of extracts (15 mg) from Raji cells stably transfected with the rec expression plasmid pCEP4-cORF (lanes 1, 5, 9), the same cells stimulated with TPA (lanes 2, 6, 10), Raji cells stably transfected with a similar plasmid harboring rec in inverse orientation (lanes 3, 7, 11), and insect cells infected with rec-baculovirus (lanes 4, 8, 12). The blots were incubated with anti-Rec polyclonal antibody 3086 at 1 : 100 dilution and a secondary peroxidase-conjugated goat anti-rabbit antibody diluted 1 : 2000, and the signals were produced by enhanced chemiluminescence. Specificity was tested by pre-incubation of the antibody with 2 mg of bacterially produced TrpE protein (lanes 5–8) or TrpE–Rec fusion protein (lanes 9–12) Oncogene


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Rec (14.5 kDa) Figure 2 Inducible expression of rec in transgenic mice. (a) Immunofluorescence documenting the expression of EGFP in rec/Tet mouse fibroblasts from a bidirectional promoter upon induction with doxycycline (Dox). DAPI stains cell nuclei. (b) RT–PCR analysis of RNA from the indicated organs of transactivator (Tet)-only transgenics and rec/Tet double transgenics upon treatment with doxycycline. Two testes from rec/Tet mice are shown. No transcript could be detected in lung and spleen tissue. þ denotes a positive control. (c) RT–PCR (left) and Western immunoblot analysis (right) of rec transcript and Rec protein expression in the naturally rec-overexpressing Tera-1 cells as compared to the rec-negative B95-8 cells and testes from doxycycline-treated rec/Tet and Tet-control mice. Western blot conditions were as outlined in the legend to Figure 1b

B95-8 cells, highly rec-expressing Tera-1 cells, and testes from docycyline-treated rec/Tet and Tet control mice, produced a specific 14.5 kDa signal upon incubation with our anti-Rec polyclonal antibody only with the Tera-1 cell extracts (Figure 2c). In general, even transiently overproduced Rec protein was extremely difficult to detect, biochemically or by immunofluorescence, in all cell types tested (e.g., diverse breast carcinoma, lymphoma and ovarian carcinoma cell lines) apart from the stably transfected Raji cells (see Figure 1b) and the naturally Rec-producing teratocarcinoma cells. Male rec/Tet transgenics, produced from two independent founders, and male control mice, expressing only the Tet transactivator, were constantly fed with doxycycline and were subjected to an in-depth histopathological examination in a blinded study. Testes were

freshly dissected and frozen in liquid nitrogen or fixed in either Bouin or 4% paraformaldehyde for 2–12 h, depending on the size of the sample. Paraffin-embedded samples were sectioned at 5 mm and stained with H&E. At 4 months of age, Rec-expressing mice characteristically showed a reduced lumen diameter (LD) of the seminiferous tubuli in their testes when compared to control mice but had no other abnormalities (Tet control mice: tubulus diameter (TD) ¼ 170.6716.6 mm, LD ¼ 103.6716.0 mm, TD/LD ¼ 1.67; rec/Tet double transgenics: TD ¼ 194.7579.9 mm, LD ¼ 73.977.1 mm, TD/LD ¼ 2.65; see also Figure 3a, top panel). At 12 months of age, four rec and four control mice were subjected to a detailed histopathological examination in a blinded study. All rec-expressing mice (100%), but none of the control mice, exhibited characteristic changes in their testes. Most prominent was a focal Oncogene


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Figure 3 Disturbed germ cell development in mice expressing Rec. (a) Seminiferous tubules of 4-, 12- or 19-months-old transgenic mice treated with doxycycline and producing either the Tet transactivator only (controls) or the Rec protein under the control of Tet. Note that by 4 months of age, the rec/Tet transgenics showed a reduced tubular diameter. By 12 months of age, nests of tubules with atypic spermatogonia and spermatocytes emerged (arrows). rec mice, 19 months old, exhibited tubules with the lumen filled with pleomorphic cells (arrow), and the occasional presence of multinucleated cells (inset). (b) RT–PCR analysis of the testes of 12- or 19months-old, doxycycline-treated rec/Tet double transgenic mice and Tet control mice. Only the testes of the rec/Tet mice produced rec transcript. ( þ ): reactions with or without polymerase

irregular hyperplasia of cells of early spermatogenesis, including spermatogonia and spermatids, suggesting that rec expression can disturb germ cell development (Figure 3a). By 19 months of age, all pathologically examined rec mice (100%; n ¼ 4), but none of the control animals (n ¼ 2) harbored in their testes – in the presence of intact tubular walls – extended nests of atypical germ cells reminiscent of spermatogonia and spermatocytes (Figure 3a). Despite the striking pathology, the rec mice displayed a focally intact spermatogenesis and spermiogenesis. Lymphocytic infiltration was rare or absent. Oncogene

The atypical cells were reminiscent of neoplasmic type I giant cells and lacked the polarity and layering indicative of normal spermatogenesis. Cellular and nuclear pleomorphisms, multinucleated cells (arrows in Figure 3a, 19 months), and increased mitotic counts with occasional atypical mitoses, strongly indicated transformation, that is, the presence of early stages of multifocal intratubular germ cell neoplasia. The tumors arose bilaterally, and the tumor cells showed a clear cytoplasm and well-defined cell borders, which together is characteristic of CIS, the predecessor lesion of classic seminoma in humans (Skakkebaek et al., 1987);


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furthermore, the presence of immature gonocyte-like germ cells is also compatible with CIS (Mostofi, 1980). Other rec-expressing organs, such as the liver and kidneys (see Figure 2b), were free of histopathological changes. All tumor-containing testes were positive for rec transcript, whereas all control testes were negative (Figure 3b). Endogenous retroviral proteins have been shown to be able to support tumorigenesis in several experimental systems. For instance, Env protein produced from (murine) endogenous retroviral sequences allowed murine MCA205 tumor cells to escape immune rejection (Mangeney and Heidmann, 1998). The results presented here are, however, the first documenting that a human endogenous retrovirus sequence that has previously been established as a marker in human germ cell tumors (Herbst et al., 1996) may disturb germ cell development and thereby ultimately lead to organ-specific neoplastic changes in a mammal. Although HERV expression and anti-HERV protein serum antibodies had been strongly associated with GCT in the past (Sauter et al., 1996), humoral immune responses to HERV antigens have occasionally been observed in patients with other diseases such as breast cancer, HIV- or cytomegalovirus infection; it was therefore unclear whether proteins produced by HERVs can induce changes that may contribute to germ cell tumorigenesis. Spontaneous germ cell tumors in mice are extremely rare and are almost invariably characterized as spermatocytic seminomas (Veeramachaneni and Vandewoude, 1999). A recently characterized transgenic mouse was the first animal model that mimics classic seminoma in humans. In these mice, the glial cell-derived neurotropic factor (GDNF), a distant member of the TGF-b superfamily of proteins which is normally produced in the testis by the spermatogenesis-regulating Sertoli cells, was overexpressed specifically in spermatogonia through regulation via the translation elongation factor 1a promoter (Meng et al., 2000). The result was the accumulation of undifferentiated spermatogonia, of which most were cleared by apoptosis, and eventually the development of malignant testicular tumors in old mice (Meng et al., 2001). In contrast to the rec mice reported here, and in contrast to human patients, the GDNF mice were infertile; rec mice and human GCT

patients have a functional spermatogenesis (Mostofi, 1980; Skakkebaek et al., 1987; Looijenga and Oosterhuis, 1999). A further difference between the animal models is that the GDNF tumors, which were histopathologically very similar to the rec tumors and also arose mostly bilaterally, were invasive while the rec tumors were not. Whether the rec/Tet mouse reported here indeed constitutes a faithful model of human germ cell tumorigenesis, and whether rec expression can affect the Ret receptor tyrosine kinase pathway that is the signalling pathway for GDNF, awaits further study. How might rec induce germ cell tumorigenesis? We have recently been able to identify several cellular Recinteracting proteins via yeast-two-hybrid screens (unpublished results) and have come across a factor that may be of particular interest in this context: the promyelocytic leukemia zinc-finger protein (PLZF) (Boese et al., 2000). Although PLZF was initially identified as a causative agent of acute promyelocytic leukemia when fused to the retinoid acid receptor alpha, it is now established that the intact protein acts as a transcriptional repressor and chromatin remodeller that can suppress cell proliferation, perhaps primarily through the repression of c-myc expression, and thus exhibits the hallmarks of a tumor suppressor (McConnell et al., 2003). Moreover, Barna et al. (2000) have reported that PLZF-deficient mice, unexpectedly, exhibit severe testicular dysfunction, and Buaas et al. (2004) and Costoya et al. (2004) have recently documented that PLZF has a crucial role in the self-renewal of spermatogonial stem cells in the testis. Inactivation of PLZF apparently results in the unrestricted exit of spermatogonia from quiescence. It is thus conceivable that the PLZF-interacting Rec presumably produced in the testes of our rec/Tet transgenic mice can interfere with germ cell maturation and support the development of CIS and, finally, GCT through the modulation of PLZF.

Acknowledgements We thank J Mayer, Institute for Human Genetics, University of Saarland Medical School, for discussion. This work was supported by grant Mu 452/5 from the German Research Foundation (DFG) to NM-L.

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