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A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...

Johns Hopkins University School of Medicine

Multikinase inhibitors (MKIs) targeting VEGF receptors and other receptor tyrosine kinases have shown considerable activity in clinical trials of thyroid cancer. Thyroid cancer frequently exhibits activation of the RAS/RAF/MEK/ERK pathway. In other types of cancer, paradoxical ERK activation has emerged as a potential resistance mechanism to RAF-inhibiting drugs including MKIs such as sorafenib and pazopanib. We therefore queried whether the MEK inhibitor trametinib, could augment the activity of pazopanib in thyroid cancer cell lines. Trametinib potently inhibited growth in vitro (GI50 1.1-4.8 nM), whereas pazopanib had more limited in vitro activity, as anticipated (GI50 1.4-7.1 µM). We observed progressive upregulation of ERK activity with pazopanib treatment, an effect abrogated by trametinib. For xenografts (bearing either KRASG12R or BRAFV600E mutations), the combination of trametinib and pazopanib led to sustained shrinkage in tumor volume by 50% or more, compared to pre-treatment baseline. Trametinib also was highly effective as a single agent, compared to pazopanib alone. These preclinical findings support the evaluation of trametinib, alone or in combination with pazopanib or other kinase inhibitors, in thyroid cancer clinical trials. We highlight the importance of pharmacodynamic assessment of the ERK pathway for patients enrolled in trials involving MKIs. 

Trametinib with and without pazopanib has potent preclinical activity in thyroid cancer.

Androgen and its receptor (AR) play a critical role in reproductive function under both physiological and pathophysiological conditions. Female AR global knockout mice are subfertile due to both neuroendocrine and ovarian defects. Female offspring from prenatally androgenized heterozygous AR pregnant mice showed rescued estrous cyclicity and fertility. Ar is expressed in granulosa cells, theca interstitial cells, and oocytes in the ovary. We created mice with theca-specific deletion of Ar (ThARKO) by crossing Cyp17-iCre mice that express Cre recombinase under cytochrome P450 17A1 (Cyp17) promoter with Arfl/fl mice. ThARKO mice exhibited no significant differences in pubertal onset or fertility compared with control littermates, and neither estrogen or testosterone levels were different between these groups. Therefore, Ar expression in theca cells likely does not influence fertility nor androgen levels in female mice. We then tested the role of AR in theca cells under hyperandrogenemic condition. After treatment with a pathophysiological level of dihydrotestosterone (DHT), control mice (control-DHT) showed acyclicity and infertility. However, estrous cycles and fertility were altered to a significantly less degree in ThARKO-DHT mice than in control-DHT mice. Messenger RNA (mRNA) levels of Lhcgr (luteinizing hormone receptor) and Timp1 (tissue inhibitor of metalloproteinase 1, and inhibitor of matrix metalloproteinase) were significantly lower in control-DHT ovary compared with control-no DHT ovaries, whereas mRNA levels of Fshr (follicle-stimulating hormone receptor) were significantly higher. Timp1 gene expression was comparable in the ThARKO-DHT and the control-no DHT ovary. We speculate that the preserved level of Timp1 in ThARKO-DHT mice contributes to retained reproductive function.

Androgen Receptor in the Ovary Theca Cells Plays a Critical Role in Androgen-Induced Reproductive Dysfunction.

Androgen excess in women is associated with metabolic dysfunction (e.g., obesity, hyperinsulinemia, insulin resistance, and increased risk of type 2 diabetes) and reproductive dysfunction (e.g., polycystic ovaries, amenorrhea, dysregulated gonadotropin release, and infertility). We sought to identify the effects of androgen excess on glucose metabolic dysfunction and the specific mechanisms of action by which androgens are inducing pathology. We developed a mouse model that displayed pathophysiological serum androgen levels with normal body mass/composition to ensure that the phenotypes were directly from androgens and not an indirect consequence of obesity. We performed reproductive tests, metabolic tests, and hormonal assays. Livers were isolated and examined via molecular, biochemical, and histological analysis. Additionally, a low-dose dihydrotestosterone (DHT) cell model using H2.35 mouse hepatocytes was developed to study androgen effects on hepatic insulin signaling. DHT mice demonstrated impaired estrous cyclicity; few corpora lutea in the ovaries; glucose, insulin, and pyruvate intolerance; and lowered hepatic insulin action. Mechanistically, DHT increased hepatic androgen-receptor binding to phosphoinositide-3-kinase (PI3K)-p85, resulting in dissociation of PI3K-p85 from PI3K-p110, leading to reduced PI3K activity and decreased p-AKT and, thus, lowered insulin action. DHT increased gluconeogenesis via direct transcriptional regulation of gluconeogenic enzymes and coactivators. The hepatocyte model recapitulated the in vivo findings. The DHT-induced hepatocyte insulin resistance was reversed by the androgen-receptor antagonist, flutamide. These findings present a phenotype (i.e., impaired glucose tolerance and disrupted glucose metabolism) in a lean hyperandrogenemia model (low-dose DHT) and data to support 2 molecular mechanisms that help drive androgen-induced impaired glucose metabolism.

Low-Dose Dihydrotestosterone Drives Metabolic Dysfunction via Cytosolic and Nuclear Hepatic Androgen Receptor Mechanisms.

Impaired estrogens action is associated with features of the metabolic syndrome in animal models and humans. We sought to determine whether disruption of hepatic estrogens action in adult male mice could recapitulate aspects of the metabolic syndrome to understand the mechanistic basis for the phenotype. We found 17β-estradiol (E) inhibited hepatic gluconeogenic genes such as phosphoenolpyruvate carboxykinase 1 (Pck-1) and glucose 6-phosphatase (G6Pase) and this effect was absent in mice lacking liver estrogen receptor α (Esr1) (LERKO mice). Male LERKO mice displayed elevated hepatic gluconeogenic activity and fasting hyperglycemia. We also observed increased liver lipid deposits and triglyceride levels in male LERKO mice, resulting from increased hepatic lipogenesis as reflected by increased mRNA levels of fatty acid synthase (Fas) and acetyl-CoA carboxylase (Acc1). ChIP assay demonstrated estradiol (E) induced ESR1 binding to Pck-1, G6Pase, Fas and Acc1 promoters. Metabolic phenotyping demonstrated both basal metabolic rate and feeding were lower for the LERKO mice as compared to Controls. Furthermore, the respiratory exchange rate was significantly lower in LERKO mice than in Controls, suggesting an increase in lipid oxidation. Our data indicate that hepatic E/ESR1 signaling plays a key role in the maintenance of gluconeogenesis and lipid metabolism in males.

Hepatic estrogen receptor α is critical for regulation of gluconeogenesis and lipid metabolism in males.

Female offspring of many species exposed to high doses of androgens in utero experience endocrine dysfunction during adulthood. The phenotype of offspring from females with prepregnancy hyperandrogenemia and impaired ovulation, however, has not been examined. We developed a mouse model of hyperandrogenemia by implanting a low-dose dihydrotestosterone (DHT) pellet 15 days before conception. Female offspring born to dams with hyperandrogenemia (DHT daughters) had delayed puberty (P < 0.05) with first estrus on postnatal day (PND) 41 compared with daughters from dams with physiological levels of DHT (non-DHT daughters, PND37.5). Serum follicle-stimulating hormone (FSH) levels in the DHT daughters were fourfold higher (P < 0.05) on PND21, and anti-Müllerian hormone levels were higher (P < 0.05) on PND26 than in non-DHT daughters (controls). DHT daughters showed an extended time in metestrus/diestrus and a shorter time in the proestrus/estrus phase compared with non-DHT daughters (P < 0.05). To examine ovarian response to gonadotropins, superovulation was induced and in vitro fertilization (IVF) was performed. Fewer numbers of oocytes were retrieved from the DHT daughters compared with non-DHT daughters (P < 0.05). At IVF, there was no difference in rates of fertilization or cleavage of oocytes from either group. There were fewer (P < 0.01) primordial follicles (6.5 ± 0.8 vs 14.5 ± 2.1 per ovary) in the ovaries of DHT daughters compared with non-DHT daughters. Daughters from hyperandrogenemic females exhibited elevated prepubertal FSH levels, diminished ovarian response to superovulation, impaired estrous cyclicity, delayed onset of puberty, and reduced ovarian reserve, suggesting that fetal androgen exposure had lasting effects on female reproductive function.

Ping Xue

Department of Pediatrics

Johns Hopkins University School of Medicine

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome