Oncogene (2003) 22, 3269–3278 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc High levels of luteinizing hormone analog stimulate gonadal and adrenal tumorigenesis in mice transgenic for the mouse inhibin-a-subunit promoter/ Simian virus 40 T-antigen fusion gene Maarit Mikola1, Jukka Kero2, John H Nilson3, Ruth A Keri3, Matti Poutanen1 and Ilpo Huhtaniemi*,1 1Department of Physiology, University of Turku, FIN-20520 Turku, Finland; 2Department of Pediatrics, University of Turku, FIN-20520 Turku, Finland; 3Department of Pharmacology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA Transgenic (TG) mice expressing the Simian virus 40 are involved in the appearance and/or maintenance of T-antigen under the control of the murine inhibin-a gonadal tumors. Among human gonadal malignancies, promoter (Inha/Tag) develop granulosa and Leydig cell ovarian tumors are the commonest, with the poorest tumors at the age of 5–6 months, with 100% penetrance. prognosis. In attempts to improve the diagnostics and When these mice are gonadectomized, they develop treatment of ovarian cancers, it is essential to learn more adrenocortical tumors. Suppression of gonadotropin about their pathogenesis, including the regulation of secretion inhibits the tumorigenesis in the gonads of intact their aggressive growth and invasion, and to develop animals and in the adrenals after gonadectomy. To study better predictive markers. It has often been suggested further the role of luteinizing hormone (LH) in gonadal that LH could promote the development of certain types and adrenal tumorigenesis, a double TG mouse model was of ovarian and testicular cancer. This is supported by generated by crossing the Inha/Tag mice with mice epidemiological findings showing an increased incidence producing constitutively elevated levels of LH (bLHb- of ovarian cancer after menopause, when circulating CTP mice). Our results show that in double TG mice gonadotropin levels are elevated (Wise et al., 1996). (bLHb-CTP/Inha/Tag), gonadal tumorigenesis starts There are also data showing that ovarian stimulation earlier and progresses faster than in Inha/Tag mice. Both with gonadotropins is associated with an increased ovarian and testicular tumors were histologically compar- incidence of granulosa cell tumors (Willemsen et al., able with the tumors found in Inha/Tag mice. In addition, 1993). In addition, certain activating mutations of the adrenal tumorigenesis was found in intact double TG LH receptor (LHR)gene have been shown to cause females, but not in Inha/Tag females. Inhibin-a and LH Leydig cell tumors in humans (Liu et al., 1999), while receptor (LHR) were highly expressed in tumorigenic the results of in vitro studies have demonstrated that LH gonadal tissues, and the elevated LH levels were shown to and FSH can stimulate the growth of human ovarian be associated with ectopic LHR and high inhibin-a carcinoma cells (Simon et al., 1983; Wimalasena et al., expression in the female adrenals. We conclude that in 1992). In line with these results, a modest clinical the Inha/Tag tumor mouse model, elevated LH levels act response has been observed in the treatment of certain as a tumor promoter, advancing gonadal and adrenal types of ovarian cancer with gonadotropin-releasing tumorigenesis. hormone (GnRH)analogs (Adelson and Reece, 1993). Oncogene (2003) 22, 3269–3278. doi:10.1038/sj.onc.1206518 Studies on experimental animals have also supported the gonadotropin theory. In inhibin-a-deficient mice, gona- Keywords: LH; adrenal and gonadal tumorigenesis; dotropins are essential for gonadal and adrenal tumor- inhibin igenesis (Kumar et al., 1996), and chronically elevated circulating levels of LH or human chorionic gonado- tropin (hCG)can cause ovarian tumors in certain strains Introduction of mice (Risma et al., 1995; Rulli et al., 2002). Furthermore, in male rats, chronic treatment with LH The gonadotropins, luteinizing hormone (LH)and produces Leydig cell hyperplasia (Neumann, 1991). follicle-stimulating hormone (FSH), are the main We have studied gonadal and adrenal tumorigenesis regulators of gonadal differentiation, growth and in transgenic (TG)mice expressing the Simian virus 40 steroidogenesis. It is therefore to be expected that they T-antigen under the control of the murine inhibin-a- subunit promoter (Inha/Tag). As shown previously *Correspondence: Ilpo Huhtaniemi, Institute of Reproductive and (Kananen et al., 1995, 1996a), these mice develop Developmental Biology, Imperial College, Faculty of Medicine, Du gonadal tumors originating from Leydig and granulosa Cane Road, London W12 ONN, UK; E-mail: [email protected] cells, with 100% penetrance, at the age of 5–6 months. Received 16 August 2002; revised 18 February 2003; accepted 19 To find out whether the extragonadal tumors frequently February 2003 found in these mice were primary or because of LH in mouse adrenal and gonadal tumorigenesis M Mikola et al 3270 metastases of gonadal tumors, we gonadectomized To study further the role of LH in this gonadal neonatal mice (Kananen et al., 1996b). To our surprise, tumor model, we produced double TG mice with gonadectomy led to the development of adrenal gland constitutively elevated LH levels and Inha/Tag expres- tumors, with 100% penetrance. It was first hypothesized sion by crossbreeding Inha/Tag mice with bLHb-CTP that these tumors were formed as a result of post- mice (Risma et al., 1995). Under the LH a-subunit gonadectomy elimination of inhibin production, since promoter, bLHb-CTP mice express a modified form of inhibin-a knockout mice have provided evidence that the bovine LH b-subunit gene, containing a 24-amino- this peptide may be a tumor suppressor (Matzuk et al., acid C-terminal peptide (CTP)of hCG b, which prolongs 1992). Moreover, inhibin-a knockout mice also develop its circulatory half-life. Upon coupling with the en- adrenal tumors if they are gonadectomized, to save them dogenous a-subunit it forms dimers with high LH from death caused by gonadal tumors (Matzuk et al., bioactivity. The chronically increased levels of serum 1994). The finding of high LHR expression in adrenal bioactive LH in bLHb-CTP mice induce increased tumors of Inha/Tag mice evoked a question about the steroid levels and a polycystic appearance in the ovaries, role of gonadotropins in adrenal tumorigenesis. We later and cause infertility. In some mouse strains also showed that either pharmacologically or genetically ovarian tumors appear. In males, the only phenotype induced hypogonadotropic hypogonadism prevented found is reduced testis size (Risma et al., 1995). The both gonadal and adrenal tumor development in double TG mice obtained allowed us to determine gonad-intact Inha/Tag mice (Kananen et al., 1997), the extent to which elevated LH levels play a specific thus supporting a role for gonadotropins in tumor role in gonadal and adrenal tumorigenesis, and to development. In addition, the effect of testosterone on analyse the influence of elevated LH levels on the tumorigenesis was excluded (Rilianawati et al., 2000). expression of the inhibin-a gene. Figure 1 (a)Ovarian weights of 3- and 5-month-old control (WT),Inh a/Tag, bLHb-CTP and bLHb-CTP/Inha/Tag mice (***Po0.001 compared with bLHb-CTP mice, tested after logarithmic transformation of the data, n ¼ 5 or 6 mice per group). (b) Histological pictures of ovaries of 3-month-old control, Inha/Tag, bLHb-CTP and bLHb-CTP/Inha/Tag mice (bar ¼ 125 mm). The sections are stained with hematoxylin/eosin. C, cyst; CL, corpus luteum; DF, developing follicle; GCT, granulosa cell tumor; L, luteinized tissue. The bottom right panel is a higher power magnification from the framed area of the adjacent bLHb-CTP/Inha/Tag picture. The arrows indicate late mitotic anaphases (bar ¼ 31 mm) Oncogene LH in mouse adrenal and gonadal tumorigenesis M Mikola et al 3271 Results at an older age and shown to be of granulosa cell origin (Kananen et al., 1995), and they had luteinized follicles, Gonadal tumorigenesis in double TG bLHb-CTP/Inha/ as found in bLHb-CTP mice (Risma et al., 1995) Tag mice (Figure 1b). The granulosa cell origin of these tumors was reconfirmed by aromatase-specific immunostaining To find out whether constitutively elevated LH levels (Figure 3b). Female bLHb-CTP mice never form promote gonadal and adrenal tumorigenesis in Inha/ granulosa cell tumors in the C57Bl background (Keri Tag mice, gonadal and adrenal sizes, as well as et al., 2000), but the effect of the bLHb-CTP transgene histology, were compared among wild-type (WT), has not been studied in a DBA/2J background. To Inha/Tag, bLHb-CTP and double TG bLHb-CTP/ exclude the possibility that the bLHb-CTP transgene Inha/Tag mice at the ages of 3 and 5 months. At these induces tumorigenesis in a DBA/2J background, we ages, no significant differences were seen in the weights always used littermates for comparison between the of the ovaries of WT and Inha/Tag mice, while the groups. As expected, some of the ovaries of the Inha/ female bLHb-CTP mice had multicystic and enlarged Tag mice also showed evidence of granulosa cell ovaries with numerous luteinized follicles (Figure 1a, b), tumorigenesis in its initial stage, which, however, was as reported previously (Risma et al., 1995). The weights slight at the age of 3 months. No overgrowth of luteal of the ovaries of double TG and bLHb-CTP mice tissue was found in these ovaries (data not shown). showed no significant difference at 3 months, but the At the age of 3 months, the weights