Targeting zebrafish and murine pituitary corticotroph tumors with a cyclin-dependent kinase (CDK) inhibitor

Abstract

Cushing disease caused by adrenocorticotropin (ACTH)-secreting pituitary adenomas leads to hypercortisolemia predisposing to diabetes, hypertension, osteoporosis, central obesity, cardiovascular morbidity, and increased mortality. There is no effective pituitary targeted pharmacotherapy for Cushing disease. Here, we generated germline transgenic zebrafish with overexpression of pituitary tumor transforming gene (PTTG/securin) targeted to the adenohypophyseal proopiomelanocortin (POMC) lineage, which recapitulated early features pathognomonic of corticotroph adenomas, including corticotroph expansion and partial glucocorticoid resistance. Adult Tg:Pomc-Pttg fish develop neoplastic coticotrophs and pituitary cyclin E up-regulation, as well as metabolic disturbances mimicking hypercortisolism caused by Cushing disease. Early development of corticotroph pathologies in Tg:Pomc-Pttg embryos facilitated drug testing in vivo. We identified a pharmacologic CDK2/cyclin E inhibitor, R-roscovitine (seliciclib; CYC202), which specifically reversed corticotroph expansion in live Tg:Pomc-Pttg embryos. We further validated that orally administered R-roscovitine suppresses ACTH and corticosterone levels, and also restrained tumor growth in a mouse model of ACTH-secreting pituitary adenomas. Molecular analyses in vitro and in vivo showed that R-roscovitine suppresses ACTH expression, induces corticotroph tumor cell senescence and cell cycle exit by up-regulating p27, p21 and p57, and downregulates cyclin E expression. The results suggest that use of selective CDK inhibitors could effectively target corticotroph tumor growth and hormone secretion.

Despite being small (<2 mm) and often undetectable by MRI, pituitary corticotroph tumors are associated with significant morbidities and mortality as a result of adrenal glucocorticoid (Gc) hypersecretion in response to autonomous tumor adrenocorticotropin (ACTH) production (1). The standard of care for Cushing disease consists of transsphenoidal pituitary tumor resection, pituitary-directed radiation, adrenalectomy, and/or medical suppression of adrenal gland cortisol production. Although transsphenoidal ACTH-secreting tumor resection yields 30% to 70% surgical cure rates, adenoma recurrence rate is high (2). Efficacies of other therapeutic modalities are limited by factors such as slow therapeutic response, development of pituitary insufficiency, and uncontrolled pituitary tumor growth in the setting of adrenal gland resection or inhibition (2, 3). Effective pharmacotherapy directly targeting corticotroph tumor growth and/or ACTH production remains a major challenge (4).

The pituitary is highly sensitive to cell cycle disruptions (5, 6). Pituitary tumors acquire oncogene and tumor suppressor genetic and epigenetic alterations, which result in unrestrained proliferation, aberrant neuroendocrine regulatory signals, and disrupted humoral milieu, mediated directly or indirectly by dysregulated cyclin-dependent kinases (CDKs) (5, 7). Although CDK gene mutations have not readily been identified in human pituitary tumors, overexpression of cyclins and dysregulation of CDK inhibitors are encountered in pituitary adenomas, indicating the importance of CDK activation for potential therapeutic targeting (8, 9). However, preclinical studies of CDK inhibitors are hampered by the requirement for large drug quantities and prolonged duration of administration to observe potential efficacy. Furthermore, although the genetic spectrum of tumor-associated mutations and/or their cellular context may dictate specific CDK dependence, it is difficult to predict which CDK inhibitor(s) may be effective against particular tumor types in vivo (10, 11). Therefore, animal models faithfully recapitulating human pituitary tumors, which enable rapid and efficient in vivo testing, are needed to identify small molecule CDK inhibitors with optimal potency.

Regardless of cell lineage origin, pituitary tumors almost invariably overexpress pituitary tumor transforming gene (PTTG), which encodes a securin that binds separase in the APC complex, and governs faithful chromosome segregation during mitosis (12). PTTG was originally isolated from rat pituitary tumor cells (13). Dysequilibrium of intracellular PTTG abundance leads to cell cycle disruption and neoplastic formation, causing chromosomal instability and aneuploidy, and also aberrant G1/S and G2/M transition by transcriptional dysregulation of cyclin expression (12, 14–19). On the contrary, PTTG overexpression also triggers irreversible cell cycle arrest in pituitary growth hormone (GH)- and gonadotropin (e.g., luteinizing hormone, follicle-stimulating hormone)-expressing tumors by activating lineage-specific senescence pathways, contributing to the benign propensity of pituitary tumors (12, 20).

Here, we report the generation of a stable transgenic zebrafish with zPttg overexpression targeted to pituitary proopiomelanocortin (POMC) lineages (corticotrophs and melanotrophs). Tg:Pomc-Pttg larvae develop early pathologic processes reflective of corticotroph tumors including neoplastic corticotrophs with partial Gc resistance, and hypercortisolemia-induced metabolic disturbances in adult transgenic fish. Taking advantage of the early-observed corticotroph pathology, combined with pituitary POMC lineage-specific expression of a fluorescent reporter in live transparent larvae, we tested small-molecule CDK inhibitors, which lead to identification of R-roscovitine against PTTG-overexpressing corticotrophs. Inhibitory effects of R-roscovitine on corticotroph tumor cells were subsequently validated in an in vivo and in vitro mouse model, supporting use of selective CDK inhibitors as effective therapy for Cushing disease.

Results

Stable Transgenic PTTG Overexpression Targeted to Pituitary POMC Cells Rapidly Induces Early Pathologies of Cushing Disease.

As an initial step toward identification of novel targets for Cushing disease therapy, we created a zebrafish model of pituitary corticotroph tumors. Given the highly conserved zebrafish PTTG protein sequence (Fig. S1), we hypothesized that zebrafish PTTG exhibits conserved properties involving cell cycle dysregulation in pituitary tumor formation (21). To test this hypothesis, we targeted PTTG overexpression to pituitary POMC lineages under the control of the zPomc promoter. One- to two-cell stage embryos were coinjected with transposase mRNA and a Tol2 transposon cassette flanking a zPomc proximal promoter fused to a full-length zPttg cDNA. Whole-mount in situ RNA analysis in F2 generation embryos confirmed zPttg overexpression, which temporally and spatially coincided with pituitary POMC cell ontogeny (Fig. 1A) (22).

Fig. 1.

Pituitary pathology of zPttg transgenic zebrafish, Tg:Pomc-zPttg. (A) Top: Schematic representation of Pomc-zPttg transgene. Bottom: Pituitary expression of zPttg in Tg:Pomc-zPttg zebrafish at 72 hpf. F1 Tg:Pomc-zPttg transgenic zebrafish were crossed with WT zebrafish, resulting in F2 embryos with 50% of the progeny positive (Left, Tg), and 50% negative (Right, WT) for pituitary zPttg expression assessed by whole-mount in situ analysis (ventral view, with anterior aspect to the left). (B) Tg:Pomc-Pttg embryos showed increased Tpit/Tbx19 expression, whereas no significant change of Pit-1 expression by whole-mount in situ analysis at 48 hpf. Antisense mRNA probes are indicated at right lower corner of each panel. Top, Lateral view; Middle and Bottom, ventral view, anterior aspect to the left. (C) Tg:Pomc-Pttg; POMC-eGFP embryos exhibited increased pituitary eGFP signal, and are more resistant to Gc-negative feedback than Tg:Pomc-Pttg–negative siblings. Double transgenic embryos (Tg:Pomc-Pttg; POMC-eGFP) were generated by breeding Tg:Pomc-Pttg fish with an established transgenic line POMC-eGFP, wherein eGFP expression is driven by the same zPomc promoter. Fluorescence intensity of POMC-GFP–positive cells was measured in live embryos after dexamethasone treatment at 4 dpf. (D) Pituitary hematoxylin/eosin stain (Top) and ACTH immunohistochemistry (Bottom) of sections derived from WT and Tg:Pomc-Pttg transgenic fish (Tg) at 20 mo. Red arrows indicate neoplastic mitotic ACTH-expressing cells. (E) Tg:pomc-pttg pituitary exhibited increased number of PCNA and ACTH coexpressing cells. Representative confocal pituitary images of fluorescence immunohistochemistry detecting PCNA (red) and ACTH (green) expression in Tg:Pomc-Pttg (a–c) and WT (d–f) zebrafish. Paraffin slides were counterstained with DAPI (blue). Arrow-heads indicate ACTH-producing cells coexpressing intranuclear PCNA. PCNA index was calculated in WT and Tg:Pomc-Pttg pituitary (g) (mean ± SE, n = 500 cells counted per pituitary, two pituitaries per group; *P = 0.05). AP, anterior pituitary; IP, pars intermedia. P, pituitary. (Scale bar, 50 μm.)

To investigate the effect of zPttg overexpression on embryonic pituitary POMC lineage development, we analyzed highly conserved pituitary transcription factors as markers for both non-POMC (Pit-1) and POMC (Tpit/Tbx-19) pituitary lineages (23–25). At 2 d post fertilization (dpf), Tg:Pomc-Pttg larva demonstrated increased pituitary Tpit/Tbx-19 expression, but Pit-1 expression was not altered (Fig. 1B), indicating early POMC lineage-specific expansion. We also generated double transgenic embryos (Tg:Pomc-Pttg; POMC-eGFP) by breeding Tg:Pomc-Pttg zebrafish into a previously established transgenic line, POMC-GFP, wherein eGFP expression was targeted to pituitary POMC cells by the same zPomc promoter, thus representing a POMC lineage-specific marker (22). Live double transgenic (Tg:Pomc-Pttg; POMC-eGFP) larvae exhibited POMC lineage expansion as evidenced by increased pituitary eGFP expression (Fig. 1C).

Pituitary corticotrophs are a critical component of the hypothalamic-pituitary-adrenal axis that mediates the stress response via hypothalamic corticotropin-releasing hormone (CRH)-stimulated, and subsequently pituitary ACTH-stimulated, adrenal gland Gc production. Gcs exert negative feedback on CRH and POMC-derived ACTH expression and secretion to restore hypothalamic-pituitary-adrenal homeostasis following stress. In human corticotroph tumors, ACTH hypersecretion is partially resistant to Gc-negative feedback regulation, further exacerbating uncontrolled hypercortisolism (26). To investigate the integrity of the Gc-negative feedback pathway in Tg:Pomc-Pttg corticotrophs, we exposed live zebrafish embryos to dexamethasone-containing culture medium starting from 10 h post fertilization (hpf). Pituitary eGFP expression was suppressed in POMC-GFP larvae exposed to 10⁻⁷ M dexamethasone by 4 dpf but not in double-transgenic (Tg:Pomc-Pttg; POMC-eGFP) larvae, which only exhibited inhibition of pituitary eGFP expression in response to 10 times higher dexamethasone concentrations (10⁻⁶ M; Fig. 1C), suggesting decreased Gc sensitivity of Tg:Pomc-Pttg corticotrophs. Thus, Tg:Pomc-Pttg corticotrophs rapidly develop the hallmark pathology of ACTH-dependent Cushing disease within 4 d of embryonic development, i.e., partial Gc resistance.

In adult Tg:Pomc-Pttg fish (20 mo of age), immunohistochemistry revealed overt neoplastic-appearing pituitary cells with a high nuclear/cytoplasmic ratio, distinct nucleoli, and basophilic cytoplasm that stained strongly for ACTH in two of six Tg:Pomc-Pttg pituitary glands analyzed, morphologically resembling human pituitary ACTH-secreting adenomas, whereas none of six WT pituitary glands showed a similar phenotype (Fig. 1D). WT zebrafish pituitary glands exhibited an overall PCNA index of 2.3 ± 0.9% vs. 3.1 ± 1.3% in Tg:Pomc-Pttg (mean ± SE; P = 0.6), whereas ACTH-producing cells in the Tg:Pomc-Pttg pituitary exhibited increased PCNA index compared with WT (2.8 ± 0.1% vs. 1.8 ± 0.2%, mean ± SE; P = 0.05; Fig. 1E), suggesting altered G1/S in neoplastic corticotrophs as a result of zPttg overexpression.

Hypercortisolism and Metabolic Disturbance in Tg:Pomc-Pttg Zebrafish.

We tested whether the observed neoplastic corticotroph cell changes in Tg:Pomc-Pttg zebrafish lead to autonomous ACTH secretion and subsequent hypercortisolism. Because we are technically hampered from measuring plasma ACTH or serum cortisol levels by the very limited amount of blood obtainable from each adult zebrafish (∼5 μL), we measured total cortisol content in age- and weight-matched Tg:Pomc-Pttg zebrafish and their transgene-negative siblings. At 3 mo of age, adult Tg:Pomc-Pttg fish showed 40% increased cortisol content versus WT siblings (1.4 ± 0.2 vs. 1.0 ± 0.2 μg/L/mg, n = 12 for each group, mean ± SE; P < 0.01). We then performed histological sections of zebrafish kidney to identify zebrafish Gc steroidogenic cells (27). Tg:Pomc-Pttg fish demonstrated increased intrarenal epithelial cell layers surrounding the posterior cardinal vein compared with WT, consistent with ACTH-stimulated adrenal hyperplasia (Fig. 2A).

Fig. 2.

Tg:Pomc-Pttg transgenic zebrafish develop hypercortisolism, exacerbated insulin resistance, glucose intolerance, hepatic steatosis, and cardiomyopathy. (A) Tg:pomc-pttg (Top, Tg) fish showed steroidogenic cell expansion as depicted by H&E stain of kidney head paraffin slides. Zebrafish Gc steroidogenic cells are arranged as epithelial cell layers (arrowheads) in association with the renal head posterior cardinal vein (27). Tg:pomc-pttg fish also showed blood cell accumulation within the posterior cardinal vein, which was not observed in WT (Bottom). (B) Glucose tolerance tests were performed in 72 Tg:pomc-pttg and WT fish. Zebrafish were given ad libitum feeding of regular diet for 1 h (gray column) after 16 h of fasting. Glucose levels are presented as mean ± SE (area under the curve; P < 0.0001). (C) After 20 h of fasting, zebrafish were intraperitoneally injected with insulin (0.1 U/100 mg), and blood glucose levels were measured at 30 and 60 min after insulin injection (n = 24, mean ± SE; *P < 0.01). (D) Oil red O staining of liver sections reveal hepatic lipid accumulation in Tg:Pomc-pttg transgenic fish. (E) Area distribution and intensity of hepatic oil red O staining were scored as described in SI Methods (mean ± SE; *P < 0.01). (F) Top: Tg:Pomc-pttg zebrafish (Tg) with gross pericardial fluid accumulation (arrow) compared with WT at 24 mo. Middle: H&E stain of midbody cross section. Bottom: High magnification (20×) showing ventricular hypertrophy of the heart. Tg, Tg:Pomc-Pttg; Pc, pericardial space; h, heart. (Scale bar, 50 μM.)

To determine the metabolic impact of hypercortisolism in Tg:Pomc-Pttg zebrafish, we subjected adult Tg:Pomc-Pttg and WT fish to 16 h fasting followed by ad libitum feeding of regular diet for 1 h. Tg:Pomc-Pttg zebrafish exhibited consistently higher levels of fasting and postprandial blood glucose levels than WT zebrafish (96 ± 9 vs. 65 ± 10 mg/dL, mean ± SD; P < 0.0001; Fig. 2B), demonstrating both attenuated fasting and postprandial glucose tolerance. Because teleost fish are glucose intolerant as a result of blunted peripheral responses to insulin (28), and Gcs induce insulin resistance in mammals, we assessed insulin sensitivity by testing blood glucose responses to intraperitoneally administered insulin. Whereas WT fish exhibited a brisk hypoglycemic response 30 min after injection of a relatively high insulin dose (0.1 U/100 mg; P < 0.01), Tg:Pomc-Pttg fish showed no significant change of blood glucose levels for up to 90 min after insulin injection (Fig. 2C). Hepatic lipid content as detected by oil red O staining was increased in Tg:Pomc-Pttg fish (Fig. 2 D and E), suggesting visceral adiposity resulting from increased insulin resistance. Finally, chronic hypercortisolism exerts specific myocardial effects leading to increased ventricular wall thickness, with subsequent systolic and diastolic dysfunction contributing to high risk of heart failure in patients with Cushing disease (1, 29). Reflective of the chronic hypercortisolemic status caused by corticotroph PTTG overexpression, a spectrum of cardiac hypertrophy was observed in late-stage (24 mo) Tg:Pomc-Pttg fish, with increased heart wall thickness involving trabecular and compact zones of the single ventricular chamber (Fig. 2F). Four of 18 Tg:Pomc-Pttg transgenic fish also exhibited coexisting overt pericardial effusion (Fig. 2F, Top). Taken together, corticotroph targeted PTTG overexpression in Tg:pomc-pttg zebrafish results in ACTH-dependent hypercortisolism and metabolic disruptions mimicking features of mammalian Cushing disease.

Corticotroph PTTG Overexpression Induces Cyclin E.

Our previous studies indicated that PTTG facilitates G1/S transition by acting coordinately with Sp1 to up-regulate cyclin D expression in human choriocarcinoma cells (16). To understand the mechanism for zebrafish corticotroph PTTG overexpression inducing altered G1/S transition (Fig. 1), we analyzed expression of key G1/S cell cycle regulators by real-time PCR in adult Tg:Pomc-Pttg and WT pituitary glands. Whereas expression of pituitary cyclin D, p21, and p27 were not different between WT and Tg:Pomc-Pttg, cyclin E mRNA levels were more than doubled in the Tg:Pomc-Pttg pituitary (Fig. 3A).

Fig. 3.

In vivo drug testing in Tg:Pomc-Pttg zebrafish. (A) Pttg overexpression directed by zebrafish Pomc promoter induced cyclin E up-regulation in Tg:Pomc-Pttg transgenic pituitary at 3 mo. mRNA levels were assayed by quantitative real-time PCR (mean ± SE of relative expression; n = 30 pituitaries for each group). (B) Western blot of mouse corticotroph tumor AtT20 cells transfected with a control or PTTG siRNA. (C) In vivo treatment of Tg:Pomc-Pttg;Pomc-eGFP embryos with small-molecule CDK inhibitors (50 μM) or 0.2% DMSO as control from 18 to 40 hpf. One hundred to one hundred fifty embryos were treated with each compound. Representative images of live embryos are shown with gross morphology (Right) and pituitary Pomc-GFP–positive cells at higher magnification (Left) at 40 hpf. Embryos exposed to flavopiridol developed early developmental defect before pituitary POMC cell ontogeny occurs. (D) Relative expression of pituitary Pomc-eGFP fluorescence analyzed using Volocity 5.2 software (Improvision; mean ± SE of relative expression, n = 7). (E) R-roscovitine specifically suppresses expansion of pituitary POMC cells overexpressing zPttg from 18 to 48 hpf. Double transgenic Tg:Pomc-Pttg;Prl-RFP embryos were generated by breeding Tg:Pomc-Pttg fish with a previously generated PRL-RFP transgenic line, in which RFP was targeted to pituitary lactotrophs by a zebrafish Prolactin promoter (34). Representative fluorescent microscopy of pituitary POMC-eGFP (a and b) and PRL-RFP (c and d) expression in live Tg:Pomc-Pttg;Pomc-eGFP and Tg:Pomc-Pttg;Prl-RFP embryos treated with 0.2% DMSO (a and c) or 50 μM R-roscovitine (b and d). (F) Relative expression of pituitary POMC-eGFP or PRL-RFP fluorescence were analyzed (mean ± SE of relative expression; n = 10). Results represent one of three similar experiments; *P < 0.02 and **P < 0.000005. (Scale bar, 50 μm.)

Cyclin E up-regulation has been associated with poor clinical outcomes in human malignancies (30). In the adult pituitary, cyclin E is undetectable in normal cells but preferentially up-regulated in tumors of corticotroph, but not other, lineage(s) (31). In murine pituitary POMC cells, cyclin E overexpression collaborates with p27^kip1-null mutation to increase cell proliferation, centrosome instability, and tumor formation (9). Up-regulated cyclin E is also associated with loss of Brg1 observed in approximately one third of human corticotroph adenomas (9). Enhanced pituitary cyclin E mRNA levels observed in Tg:Pomc-Pttg fish may not represent protein expression, but the minute adult zebrafish pituitary size (<1 mm) technically hampered analysis of protein expression by Western blot. We therefore determined whether PTTG regulates cyclin E expression in mammalian (murine) AtT20 corticotroph tumor cells that express abundant endogenous PTTG and cyclin E proteins. Suppression of endogenous PTTG expression with a PTTG-specific siRNA resulted in decreased cyclin E expression and enhanced p27^kip1 levels (Fig. 3B), whereas p21 expression was not changed (Fig. 3B). These observations suggest that PTTG up-regulation of cyclin E and down-regulation of p27^kip1 in pituitary corticotroph tumor cells occurs independently of p21.

In Vivo Testing of CDK/Cyclin Inhibitors in Tg:Pomc-Pttg Zebrafish.

Zebrafish pituitary POMC cell differentiation starts at the anterior neural ridge by 20 hpf, and is completed within the mature pituitary by 48 hpf (22). Within the first few days of embryonic development, our transgenic fish shown here recapitulate hallmark features of Cushing disease, i.e., lineage-specific corticotroph expansion with partial Gc resistance (Fig. 1). The observed G1/S alteration, cyclin E up-regulation, and neoplastic corticotroph changes led us to screen small-molecule CDK inhibitors with different spectra of inhibitory selectivity, including flavopiridol (CDK 4/6, 2, 1, 9), R-roscovitine (CDK 2, 1) (32), olomoucine (CDK 2, 1) (32), PD-0332991(CDK 4/6), and CAY10572 (CDK 7) (33). Tg:Pomc-Pttg; POMC-eGFP double transgenic embryos were exposed to each compound added to the embryo culture medium. Although flavopiridol retarded early embryonic development before corticotroph ontogeny occurred, in vivo treatment of zebrafish embryos with R-roscovitine, olomoucine, PD-0332991, and CAY10572 starting at 18 hpf caused no apparent growth defect by 40 hpf (Fig. 3C). Strikingly, R-roscovitine-treated embryos exhibited approximately 40% reduction in pituitary POMC-eGFP expression compared with controls (1.0 ± 0.08 vs. 0.6 ± 0.09, mean ± SE; n = 7 for each group; P < 0.02; Fig. 3 C and D). A modest, approximately 20%, reduction of POMC-eGFP expression was also observed in the olomoucine-treated group (1.0 ± 0.08 vs. 0.8 ± 0.07, mean ± SE; n = 7 for each group; P = 0.07), whereas PD-0332991 and CAY10572 caused no significant change in pituitary POMC-eGFP expression compared with controls (Fig. 3 C and D).

To determine the specificity of R-roscovitine action against zPttg-overexpressing POMC cells, we generated another double transgenic line (Tg:Pomc-Pttg;Prl-RFP) by breeding Tg:Pomc-Pttg fish with a previously generated PRL-RFP transgenic line, in which RFP was targeted to pituitary lactotrophs by a zebrafish Prolactin promoter (34). In vivo treatment between 18 and 48 hpf of Tg:Pomc-Pttg;Prl-RFP and Tg:Pomc-Pttg;POMC-eGFP embryos with R-roscovitine revealed no effect on Prl-RFP expression (1.0 ± 0.08 vs. 1.0 ± 0.09, mean ± SE; n = 9 for each group; P = 0.3), but a greater than 50% reduction of POMC-eGFP expression (1.0 ± 0.07 vs. 0.5 ± 0.05, mean ± SE; n = 10 for each group; P < 0.000005) compared with control groups (Fig. 3 E and F).

R-Roscovitine Action in Mouse Corticotroph Tumor Cells.

Olomoucine and roscovitine are structurally related 2,6,9-trisubstituted purines, which cause G1/S or G2/M arrest by competing for ATP binding sites on CDK1 and CDK2. The R-isomer of roscovitine (R-roscovitine, CYC202) is a more potent and selective inhibitor of CDK2/cyclin E, and murine corticotrophs are highly sensitive to disrupted CDK2/cyclin E-mediated cell cycle pathways (9). Cyclin E up-regulation leads to cell cycle reentry of differentiated POMC cells and also inactivates p27^kip1, further enhancing cell cycle progression (9). In addition, p27^kip1 protects differentiated pituitary POMC cells from reentering the cell cycle, whereas p57^Kip2 is required for cell cycle exit of pituitary precursor cells (35). Given the in vivo potency of R-roscovitine against zebrafish Pttg-overexpressing corticotrophs (Fig. 3), we studied its effect on CDK2/cyclin E-mediated cell cycle pathways in mouse ACTH-secreting pituitary tumor cells (Fig. 4).

Fig. 4.

In vitro inhibition of mouse corticotroph tumor cells by R-roscovitine. (A) Treatment of ACTH-secreting AtT20 cells with R-roscovitine (1–2 × 10⁻⁵ M) led to decreased number of viable cells at 24 and 48 h, as depicted by Wst-1 proliferation assay (mean ± SE; **P < 0.01). (B) Western blot of protein extracts derived from AtT20 cells treated with vehicle or R-roscovitine. (C) R-roscovitine treatment (10 μM) for 48 h induced senescence as indicated by increased β-gal expression. (D) ACTH concentration by radioimmunoassays of culture medium from AtT20 cells treated with vehicle or R-roscovitine (mean ± SE; **P < 0.01 and ***P < 0.001). (E) Western blot of protein extracts derived from AtT20 cells treated with R-roscovitine. Vehicle is 0.2% DMSO.

Treatment with R-roscovitine (1–2 × 10⁻⁵ M) led to decreased cell number by 24 h (Fig. 4A). Western blot analysis of protein extracts derived from R-roscovitine–treated cells revealed evidence for cell cycle arrest, including decreased cyclin E, increased p27^Kip1, p57^Kip2, and p21^Cip1 expression, as well as reduced Thr821 phosphorylation of Rb (Fig. 4B). R-roscovitine treatment also induced senescent features by 48 h as evidenced by increased β-gal expression (Fig. 4C).

Consistent with decreased cell viability, we detected decreased ACTH concentrations in culture medium derived from R-roscovitine–treated AtT20 cells (Fig. 4D). Western blot analysis of protein extracts derived from R-roscovitine–treated AtT20 cells showed suppressed ACTH expression (Fig. 4E). These results indicate that R-roscovitine targets cdk2/cyclin E-mediated cell cycle progression, and also inhibits corticotroph ACTH protein expression.

R-Roscovitine Inhibits in Vivo Corticotroph Tumor Growth and ACTH Expression.

To further establish R-roscovitine action on corticotroph tumors in vivo, we injected athymic nude mice (approximately 6–8 wk old) s.c. with AtT20 corticotroph tumor cells (1 × 10⁵ cells). Three days after tumor cell injection, 29 of 30 mice had developed small (approximately 2–3 mm³) but visible s.c. tumors, and were randomized to receive R-roscovitine (150 mg/kg) or vehicle via oral gavage twice daily for 5 d each week. After 3 wk, R-roscovitine caused approximately 50% weight reduction of dissected tumor xenografts (40.0 ± 4.7 mg vs. 21.0 ± 2.6 mg, mean ± SE; n = 13–14 for each group; P < 0.02; Fig. 5A).

Fig. 5.

In vivo action of R-roscovitine in mouse corticotroph adenomas. Athymic nude mice were s.c. inoculated with corticotroph tumor AtT20 cells (1 × 10⁵ cells). Three days after injection, mice were randomized to receive R-roscovitine (150 mg/kg) or vehicle by oral gavage twice daily, 5 d/wk. After 3 wk, tumor xenografts were dissected and (A) tumor volumes were decreased in R-roscovitine–treated animals. (B) Western blot of representative tumor specimens showed decreased ACTH and PCNA expression in R-roscovitine–treated tumors. (C) R-roscovitine–treated corticotroph tumors exhibited decreased PCNA and ACTH coexpressing cells. Fluorescence microscopy image of immunohistochemistry detecting PCNA (red) and ACTH (green) expression in control (a–c) and R-roscovitine–treated tumors (d–f). Cryosection slides were counterstained with DAPI (blue). (D) Blood was collected from each animal for measurement of plasma ACTH and serum corticosterone levels (mean ± SE; n = 13–14 mice for each group; **P < 0.01).

Consistent with the in vitro observations, Western blot and immunohistochemistry analysis of tumor specimens showed suppressed ACTH and PCNA protein expression by R-roscovitine (Fig. 5 B and C). R-roscovitine–treated mice exhibited more than 50% reduction in plasma ACTH levels (1256 ± 596 pg/mL vs. 596 ± 103 pg/mL, mean ± SE; n = 13–14 for each group; P < 0.01), and approximately 50% reduction in serum corticosterone levels [1,046 ± 109 ng/mL vs. 561 ± 72 ng/mL, mean ± SE; n = 13–14 for each group (P < 0.005); linear regression between ACTH and corticosterone, r = 0.9425 (P < 0.0001); Fig. 5D]. The high baseline plasma ACTH levels may represent tumor secretion as well as stress-induced responses during CO₂ euthanasia.

Discussion

Tumor-targeted drug development for Cushing disease is a major challenge, as the pathogenesis of corticotroph adenomas remains enigmatic. Recently, protein kinases, i.e., epidermal growth factor receptor family (e.g., HER) and CDKs, have been suggested as therapeutic targets for pituitary tumors (8).* Although tumor responses to protein kinase inhibitors is selective, and may be dictated by specific mutations and/or tumor cellular context, preclinical testing is hampered by poor predictabilities with respect to molecular pathophysiology of the tumors being assessed.

Here, we report generation of germline transgenic zebrafish overexpressing zPttg targeted to pituitary POMC cells, as a small vertebrate animal model of Cushing disease. Although the phenotype of hypercortisolism was observed in adult Tg:Pomc-Pttg zebrafish by 3 mo of age, pituitary corticotroph expansion with partial resistance to Gc-negative feedback was already detected within the first 2 d of embryonic development of stable transgenic zebrafish. Furthermore, the Tg:Pomc-Pttg pituitary demonstrates a characteristic feature of human corticotroph adenomas, i.e., cyclin E up-regulation and G1/S phase disruption. The molecular features and early pathologies of corticotroph tumors in Tg:Pomc-Pttg transgenic fish allowed us to gain insight into mechanisms underlying the disease pathogenesis, and also to test drug efficacy in vivo.

Cyclin E overexpression is associated with disrupted G1/S transition contributing to development and progression of breast carcinomas, leukemia, and lymphomas (30). In the pituitary, cyclin E expression is preferentially up-regulated in corticotroph adenomas compared with tumors arising from other lineages, the mechanisms of which remain to be fully defined (31, 36, 37). In a subgroup of corticotroph adenomas, cyclin E up-regulation was associated with loss of Brg1 expression, suggesting the presence of additional cyclin E regulators in corticotrophs (26). Our results show that corticotroph zPttg overexpression induces cyclin E, whereas PTTG siRNA suppresses cyclin E expression in murine corticotroph tumor cells (Fig. 3). PTTG is overexpressed in more than 90% of pituitary tumors, including corticotroph adenomas (12). In addition to inducing aberrant G1/S and G2/M transition via transcriptional dysregulation of cyclin expression (12, 14–17), causing chromosomal instability and aneuploidy, pituitary PTTG overexpression activates lineage-specific senescence pathways triggering irreversible cell cycle arrest in GH- and gonadotropin-expressing tumors (12, 19, 20). Corticotroph cyclin E up-regulation may represent another pathway for PTTG-induced pituitary lineage-specific effects, although it is yet unclear whether PTTG regulates cyclin E expression directly or indirectly.

Corticotroph cyclin E up-regulation contributes to cell cycle reentry of differentiated corticotrophs and centrosome instability (9). To investigate the clinical significance of cyclin E dysregulation in corticotroph adenomas, we performed in vivo drug testing on Tg:Pomc-Pttg embryos using known small molecule compounds with different spectra of CDK/cyclin inhibitory selectivity. Our results indicated inhibition of PTTG-overexpressing corticotrophs by the 2,6,9-substituted purine analogues, olomoucine and R-roscovitine, with the latter demonstrating a higher efficacy in vivo (Fig. 3). Corticotroph inhibitory effects of R-roscovitine were further validated in mouse corticotroph tumors (Figs. 4 and 5). R-roscovitine arrests G1/S or G2/M phases via CDK1/2 inhibition by competing for ATP binding sites (11), inhibition of RNA polymerase II-dependent transcription, and selective action against CDK2/cyclin E (38, 39). The molecule is currently undergoing clinical trials for several malignancies, and the oral dosing route and relatively mild side effects of R-roscovitine make daily long-term treatment of Cushing disease feasible.

Our results suggest that R-roscovitine inhibits corticotroph tumor cell growth via CDK2/cyclin E and Rb-mediated pathways, independent of p53 (Fig. 4). Both in vitro and in vivo results show that R-roscovitine also suppresses ACTH expression/production (Figs. 4 and 5), suggesting other regulatory mechanisms in addition to CDK2/cyclin E-mediated cell growth. One of the possible mechanisms may involve inhibition of CRH receptor signaling pathways in corticotroph tumor cells, as other 2,6,9-trisubstituted purine analogues have been developed as CRH receptor antagonists exhibiting potential anxiolytic and antidepressant activity (40). Further in vivo screening of small molecule libraries with Tg:Pomc-Pttg transgenic fish may lead to identification of compounds with more potent dual effects targeting both corticotroph tumor growth and ACTH production.

Methods

We used a tol2 transposon cassette to generate the Tg:pomc-pttg transgene, and transgenic founder fish were generated as described previously (22). Further details on experimental procedures can be found in SI Methods.