Functional PPAR- receptor is a novel therapeutic target for ACTH-secreting pituitary adenomas

Adrenocorticotrophic hormone (ACTH)-secreting pituitary tumors are associated with high morbidity due to excess glucocorticoid production. No suitable drug therapies are currently available, and surgical excision is not invariably curative. Here we demonstrate immunoreactive expression of the nuclear hormone receptor peroxisome proliferator-activated receptor- (PPAR-) exclusively in normal ACTH-secreting human anterior pituitary cells: PPAR- was abundantly expressed in all of six human ACTH-secreting pituitary tumors studied. PPAR- activators induced G₀/G₁ cell-cycle arrest and apoptosis and suppressed ACTH secretion in human and murine corticotroph tumor cells. Development of murine corticotroph tumors, generated by subcutaneous injection of ACTH-secreting AtT20 cells, was prevented in four of five mice treated with the thiazolidinedione compound rosiglitazone, and ACTH and corticosterone secretion was suppressed in all treated mice. Based on these findings, thiazolidinediones may be an effective therapy for Cushing disease

Peroxisome proliferator-activated receptor- (PPAR-), a member of the nuclear receptor superfamily^{1, 2}, functions as a transcription factor mediating ligand-dependent transcriptional regulation^{3, 4, 5}. High-affinity PPAR- ligands include the insulin-sensitizing thiazolidinedione compounds (TZDs)^{6, 7, 8}. PPAR- activation leads to adipocyte differentiation⁶, glucose regulation⁹, inhibition of macrophage and monocyte activation^{10, 11} and inhibition of angiogenesis¹². PPAR- is expressed in breast, prostate and colon epithelium, and administration of synthetic PPAR- ligands inhibits tumor cell growth in prostate and colon^{13, 14, 15, 16}. Rosiglitazone, a potent thiazolinedione oral antidiabetic agent, recently approved in the United States, differs structurally from pioglitazone and troglitazone, with greater PPAR--binding affinity and antihyperglycemic potency.

Here we report that PPAR- expression is restricted to ACTH-secreting cells of the normal human anterior pituitary, and is abundantly expressed in human ACTH-secreting pituitary tumors. PPAR- ligands potently inhibit ACTH-secreting pituitary-tumor proliferation, and they inhibit pituitary tumor growth and secretion of ACTH and corticosterone. These results support the role for PPAR- as a novel target for treating patients with Cushing syndrome.

Selective PPAR- expression in pituitary corticotroph cells

Using the avidin-biotin-peroxidase method in post mortem−derived normal human pituitary tissue, we carried out immunocytochemical analysis of ACTH and PPAR- expression. This demonstrated characteristic diffuse ACTH immunoreactive cells throughout the anterior pituitary in approximately 15% of cells; however, cells were concentrated in the intermediate region between the anterior and posterior pituitary (Fig. 1a−c). PPAR- immunoreactivity was evident throughout the anterior pituitary in large basophilic cells containing inclusion bodies characteristic of corticotrophs. Immunoreactivity was particularly apparent in ACTH-immunoreactive basophilic cells, which typically invaded the posterior pituitary region (Fig. 1d−f). In rat pituitary tissue, ACTH immunoreactivity was evident in the anterior and intermediate pituitary (Fig. 1g and inset). In support of our findings in human pituitary tissues, PPAR- immunoreactivity occurred throughout the rat anterior pituitary in large basophilic ACTH-immunoreactive cells (Fig. 1h and inset). In the human pituitary, double immunofluorescence studies using tetra-methyl rhodamine isothiocyanate (TRITC)-labeled ACTH (Fig. 1i) and fluorescein isothiocyanate (FITC)-labeled PPAR- (Fig. 1j) antibodies revealed that PPAR- immunoreactivity colocalized with ACTH-immunoreactive cells (Fig. 1k). Co-immunostaining showed that some corticotrophs exhibited both PPAR- and -melanocyte stimulating hormone (-MSH) expression. However, other corticotrophs selectively exhibited only PPAR- or -MSH immunoreactivity respectively (data not shown).

Figure 1. Pituitary PPAR- expression is restricted to normal human corticotroph cells. a−k, Immunocytochemical analysis of autopsy- derived normal human and rat pituitary tissue, using immunoperoxidase (a−c and g) and TRITC-labeled ACTH (i) and immunoperoxidase (d−f and h) and FITC-labeled (j) PPAR- antibodies. In human pituitary tissue, ACTH− and PPAR-−DAB immunoreactivity (brown) was visible in corticotroph cells throughout the anterior pituitary and concentrated in the basophilic cells, which invade the posterior pituitary. In the rat pituitary, ACTH immunoreactivity was seen in the anterior and intermediate pituitary (g and inset), whereas PPAR- immunoreactivity was confined to the anterior pituitary corticotrophs (h and inset). Double immunofluorescence using TRITC-labeled ACTH (i) and FITC-labeled PPAR- (j) antibodies demonstrated colocalization of PPAR- and ACTH immunoreactivity (k). AP, anterior pituitary; IP, intermediate pituitary; PP, posterior pituitary; BI, basophil invasion. Arrows indicate inclusion bodies, a characteristic feature of corticotroph cells. Magnification: 200 (a and d); 400 (b, e and i−k); 100 (c, f and insets in g and h). Full Figure and legend (80K)

PPAR- expression in human ACTH-secreting pituitary tumors

Given the observed colocalization of PPAR- and ACTH in normal human anterior pituitary corticotroph cells, PPAR- expression was assessed in six surgically resected ACTH-secreting pituitary tumor specimens. Western blotting of pituitary tumor−derived protein extracts revealed abundant PPAR- expression in all six ACTH-secreting pituitary tumors examined, in comparison with modest PPAR- expression in two normal pituitary-derived extracts (Fig. 2a). Longer film exposures confirmed low-level PPAR- expression in normal pituitary tissue extracts (data not shown). Immunocytochemical analysis demonstrated abundant PPAR- immunoreactivity localized to those pituitary adenoma cells that also expressed ACTH (Fig. 2b).

Figure 2. PPAR- is abundantly expressed in human ACTH-secreting pituitary tumors. a−b, Western-blot (a) and immunocytochemical (b) analysis depicting abundant PPAR- expression in 6 surgically resected ACTH-secreting pituitary tumors (T1-6), in comparison with PPAR- expression in 2 normal pituitary tissue-derived extracts (NP). -actin immunoblotting confirmed equivalent total protein loading. MCF-7 breast-cancer cells served as a positive control. M, protein marker. High magnification confirmed cytoplasmic granular PPAR- immunoreactivity; magnification: 400 (b) and 800 (inset). Full Figure and legend (44K)

Functional role for pituitary PPAR- receptor in vitro

To determine the functional significance of pituitary corticotroph PPAR- expression, we tested the effects of PPAR- ligands on ACTH-secreting pituitary tumor cells in vitro. Troglitazone or rosiglitazone treatment (1 10^-6 to 10^-4 M) for up to 48 hours in serum-free conditions, or up to 96 hours for cells maintained in serum-replete conditions, induced G₀/G₁ cell-cycle arrest, and led to decreased number of cells in S-phase (Fig. 3a). Western-blot analysis of protein extracts derived from TZD-treated murine and human corticotroph cells showed an approximately 60% decrease in Ser⁷⁹⁵ phosphorylated retinoblastoma (Rb) protein expression (Fig. 3b and c), indicating an Rb-mediated mechanism for the G₀/G₁ cell-cycle arrest.

Figure 3. Functional PPAR- in pituitary corticotroph tumors. a−e, FACS analysis of troglitazone- or rosiglitazone (Ros)-treated serum-replete mouse (a,b,d and f) and human (c and e) ACTH-secreting pituitary tumor cells in vitro demonstrated increased G1 phase, decreased S phase (a) (*, P = 0.009; **, P = 0.001), decreased expression of Ser794 phosphorylated retinoblastoma protein (b and c), and a dose-dependent increase in Annexin-FITC immunoreactive cells in murine (d) and human (e) ACTH-secreting pituitary tumor cells respectively, in keeping with TZD-mediated induction of G1 cell-cycle arrest and apoptosis. M, protein marker (b and c). d, , vehicle; , 1 10-5 M Ros; , 2 10-5 M Ros; , 5 10-5 M Ros; , 10 10-5 M Ros. f, TUNEL analysis demonstrated a 2-fold increase in rosiglitazone-induced apoptosis. *, P = 0.0003. Pretreatment of AtT20 cells with rosiglitazone blocked anti-apoptotic effects of CRH and induced corticotroph cell apoptosis (**, P = 0.0003). , control; , 1 10-6 M Ros; , 1 10-5 M Ros; , 50 nM CRH; , 50 nM CRH + 1 10-5 M Ros. Each bar represents mean s.e.m. of 3 fields in 2 separate experiments. JEG-3 choriocarcinoma cells served as a positive control. Full Figure and legend (24K)

Rosiglitazone (1 10^-5 to 10^-4 M) treatment of mouse corticotroph pituitary tumor cells (AtT20) cells for 48 hours demonstrated a dose-dependent increase in Annexin-FITC immunoreactive AtT20 cells, and enhanced apoptosis (Fig. 3d) (P < 0.001). A similar increase in Annexin-FITC immunoreactive cells occurred after treatment of human pituitary corticotroph tumor cultures with rosiglitazone (1 10^-5 M) (Fig. 3e). TUNEL analysis confirmed that TZD induced cell death, and increased apoptosis about three-fold (Fig. 3f). Corticotrophin-releasing hormone (CRH) is a potent corticotroph proliferative factor³¹. Pretreatment of AtT20 cells with TZD before CRH treatment (50 nM) blocked the growth-promoting effects of CRH (50 nM for 24 h) and induced corticotroph cell apoptosis (Fig. 3f).

Western-blot analysis of protein extracts derived from TZD-treated corticotroph cells showed decreased expression of the anti-apoptotic protein Bcl-2 and increased levels of pro-apoptotic Bax and p53 expression (Fig. 4a and b), indicating a mechanism for PPAR- mediated apoptosis. An approximately four-fold increase in cleaved caspase-3 and a concomitant decrease in total caspase-3 resulted from TZD-treatment, and were consistent with enhanced apoptosis (Fig. 4c).

Figure 4. TZDs inhibit corticotroph cell proliferation, ACTH synthesis and secretion, and induce apoptosis. a and b, Western-blot analysis demonstrated reduced expression of the anti-apoptotic protein Bcl-2, increased pro-apoptotic proteins Bax (a) and p53 (b). c, Dose-dependent increase in TZD-mediated cleaved caspase-3 and a concordant decrease in uncleaved caspase-3 confirms the pro-apoptotic effect of PPAR- ligands. d, Northern-blot analysis of TZD-treated corticotroph cell-derived total RNA extracts demonstrated inhibition of Pomc mRNA levels, and decreased Pttg mRNA expression, confirming decreased proliferative rates in the TZD-treated corticotroph pituitary tumor cells. 18S ribosomal RNA served to normalize RNA loading. Rat testis served as a positive control. e, Rosiglitazone effects on baseline and CRH-induced (50 nM) Pomc transcription. AtT20 cells pretreated for 48 h with 1 10-5 M rosiglitazone, and Pomc-luc transfected using lipofectamine. Results are expressed as fold induction of luciferase activity over control (vehicle), corrected for -galactosidase activity (mean of 3 wells in 3 independent experiments s.e.m.), by ANOVA and Bonferroni multiple-comparison test. *, P< 0.05 compared with control; **, P < 0.05 compared with CRH. JEG-3 choriocarcinoma cells served as a positive control. M represents protein or RNA marker. Full Figure and legend (16K)

PPAR- ligands inhibit Pomc and Pttg expression

PPAR- ligands inhibit in vivo pituitary tumor growth

As the TZDs exhibited anti-proliferative and pro-apoptotic effects in vitro, effects of TZD-treatment were assessed on growth of corticotroph pituitary tumors in vivo. Corticotroph AtT20 tumor cells (2 10⁵ cells) were inoculated subcutaneously in four-week-old female athymic nude mice, and animals randomized to receive either oral rosiglitazone (150 mg/kg/day) or vehicle. Baseline pretreatment ACTH and corticosterone levels in serum were comparable in the control and rosiglitazone-treated groups (data not shown; P = not significant). After four weeks, four of five untreated control mice had developed visible subcutaneous corticotroph tumors (1−2 cm diameter). In contrast, only one of five rosiglitazone-treated animals developed a small subcutaneous corticotroph cell tumor (0.1 cm diameter) (Fig. 5a and b). Plasma ACTH (98 33 pg/ml versus 2085 847 pg/ml, ACTH, mean s.e.m., P 0.05) and serum corticosterone (113 17 ng/ml versus 785 374, corticosterone, mean s.e.m., P 0.05) levels were markedly lower in rosiglitazone-treated mice compared with vehicle-treated tumor-bearing animals (Fig. 5c and d).

Figure 5. Rosiglitazone inhibits corticotroph pituitary tumor growth in vivo. Following subcutaneous inoculation of corticotroph pituitary tumor cells (2000 cells) into 4-wk-old female athymic nude mice, animals were randomized to receive either oral rosiglitazone (150 mg/kg/day; ) or vehicle (). a, Representative photograph of mice treated with vehicle (upper panel) or rosiglitazone (lower panel). b−c, Tumor numbers (b), serum ACTH (c) and corticosterone levels (d) after 4 wk treatment with vehicle or rosiglitazone. n = 5 per group. *, P < 0.05. Full Figure and legend (30K)

Although TZD-treatment prevented experimental pituitary corticotroph tumor development in vivo, patients invariably present with already established and actively growing pituitary tumors. We therefore tested the effects of TZD treatment on growth of already established experimental pituitary corticotroph tumors and steroid hormone levels in vivo. AtT20 were inoculated subcutaneously in athymic nude mice, and tumors were allowed to develop. By three weeks, all mice had developed large visible tumors, and they were then randomized to receive either oral rosiglitazone (150 mg/kg/day) or vehicle. Baseline levels of serum ACTH and corticosterone and tumor volumes did not differ among the mice subsequently randomized as control or rosiglitazone-treated groups (data not shown; P = not significant). In both groups, tumor growth continued, but it was diminished (P < 0.05) in three of five rosiglitazone-treated mice (Fig. 6a and b). The phenotypic appearance of the mice was markedly different, and vehicle-treated mice were wasted, developed skin atrophy and appeared pigmented, with features of hypercortisolism (Fig. 6b). In contrast, rosiglitazone-treated mice continued to thrive and gain weight and did not display skin atrophy or pigmentation. In addition, rosiglitazone treatment led to an approximately 80% reduction in the proliferative marker, Pttg mRNA, and an approximately 60% reduction in Pomc mRNA (Fig. 6c), confirming the in vitro observations. Moreover, there was a 75% reduction in plasma ACTH (Fig. 6d) (controls, 3841 308 versus Ros-treated, 978 452 pg/ml, mean s.e.m. ACTH, P = 0.002) and a 96% reduction in serum corticosterone levels (Fig. 6e) (controls, 4304 2593 versus ros-treated, 181 56 ng/ml, mean s.e.m. corticosterone, P < 0.05).

Figure 6. Rosiglitazone treatment retards growth of established pituitary corticotroph tumors and suppresses steroid hormone levels in vivo (n = 5). Mice were inoculated subcutaneously with corticotroph pituitary tumor cells, and tumors were allowed to develop. By 3 wk, all mice developed large visible tumors and animals and were randomized to receive either rosiglitazone (150 mg/kg/day) (n = 5) or vehicle (n = 5). Baseline serum ACTH and corticosterone levels and tumor volumes were comparable in vehicle- and rosiglitazone-treated groups. a, After 4 wk treatment, tumor volumes (representative growth curve) were diminished in rosiglitazone-treated animals (P < 0.05). , vehicle-treated; , rosiglitazone-treated mice. Arrow indicates time of treatment initiation. b, Amelioration of Cushing phenotypic appearance coincided with tumor reduction. c, Tumor abrogation was associated with reduced Pttg and Pomc mRNA expression. 18s ribosomal RNA served to normalize RNA loading. M, RNA marker. d and e, Tumor abrogation was associated with reduction in serum ACTH (d) and corticosterone (e). , vehicle; , rosiglitazone. *, P = 0.002; **, P < 0.05. Values represent samples drawn 4 wk after starting rosiglitazone or vehicle treatment. f, Pituitary corticotroph responsiveness is preserved following long-term rosiglitazone-treatment in normal mice. Plasma ACTH (at 14:00 hours, i.e. 2pm), unstressed () and following 15 min restraint stress () in normal female athymic nu/nu mice following 6 wk oral treatment with vehicle or rosiglitazone (Ros; 150 mg/kg/day). P = not significant Full Figure and legend (41K)

Given the observed pituitary corticotroph PPAR- expression, the TZD-induced decrease in plasma ACTH and corticosterone levels in vivo, and the potential use of these agents as medical therapy for Cushing disease, we assessed the effects of prolonged rosiglitazone administration on endogenous non-tumorous murine pituitary-adrenal responses in stressed and unstressed conditions. Restraint stress is a potent inducer of ACTH and Corticosterone. Plasma ACTH (Fig. 6f) and serum corticosterone (data not shown) were similar in rosiglitazone-treated (150 mg/kg for 6 wk) and untreated normal athymic nu/nu mice in both unstressed (quiet environment overnight) and stressed conditions (15 min restraint).

PPAR- expression in normal pituitary tissue is restricted and colocalizes with ACTH-secreting cells in the pituitary intermediate region. Immunoreactive PPAR- was not expressed in the rat pituitary intermediate lobe. In contrast, abundant PPAR- expression was seen in all of six corticotroph pituitary tumors examined, compared with minimal expression seen in normal pituitary tissues; the latter finding was confirmed by longer film exposures (data not shown). PPAR- protein immunostaining is localized within the ACTH-secreting tumor cells themselves. TZD treatment of corticotroph pituitary tumor cells in vitro induced G₀/G₁ cell arrest and apoptosis, indicating the functional significance of PPAR- expression in corticotroph tumors. Furthermore, TZD-treatment prevented pituitary corticotroph tumor formation in vivo, and led to marked abrogation of pituitary tumor growth of already established and actively growing pituitary corticotroph tumors. Notably, long-term rosiglitazone treatment (150 mg/kg for 6 wk) did not alter murine endogenous pituitary-adrenal responses. PPAR- expression appears restricted to a subset of corticotrophs, raising the possibility that PPAR- ligand-mediated actions in the corticotroph cell may be selective, allowing their use in treatment of Cushing disease without affecting functions of corticotroph cells that do not express PPAR-.

Surveillance of more than 4,500 patients shows that rosiglitazone is a safe, effective monotherapy or combination therapy for patients with type 2 diabetes³⁸, and unlike troglitazone, which has been associated with idiosyncratic hepatotoxicity, rosiglitazone is associated with low incidence of liver abnormalities. Given abundant and selective PPAR- expression in corticotroph pituitary tumors compared with normal pituitary tissue, we propose a role for the use of PPAR- receptor ligands, such as rosiglitazone, in the management of Cushing syndrome due to ACTH-secreting pituitary tumors.

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In accordance with Cedars-Sinai Institutional Animal Care and Use Committee guidelines, mouse pituitary ACTH-secreting AtT20 tumor cells (2 10⁵) were inoculated subcutaneously in 4-wk-old female nu/nu mice, and animals randomized to receive rosiglitazone 150 mg/kg/day or vehicle. Mice were killed by CO₂ inhalation, tumors weighed, and aliquots frozen and stored for subsequent analysis.

Subconfluent AtT20 mouse pituitary (ACTH-secreting) cells (1 10⁶ per well) were cultured in DMEM medium in 6-well plates, supplemented with 10% FBS and antibiotics at 37 °C in 5% CO₂ for 24 h before treatment with troglitazone or rosiglitazone. Medium was replenished with ligands every 2 d, and cells maintained in medium with serum or in serum-free medium for up to 96 h.

AtT20 cells were trypsinized, centrifuged (1,500g for 2 min), washed with PBS, and treated with 20 g/ml Rnase A (Calbiochem, La Jolla, California). DNA was stained with 100 g/ml propidium iodide (PI) for 30 min at 4 °C and protected from light, before analysis with a FACScan (Becton Dickinson, Franklin Lakes, New Jersey).

AtT20 and human corticotroph tumor cells were treated, trypsinized, centrifuged and washed before incubation with a FITC-labeled monoclonal Annexin antibody and PI for 30 min at room temperature according to manufacturer's instructions (Pharmingen, San Diego, California). Approximately 1 10⁶ cells were analyzed by flow cytometry, labeled nuclei (PI) were gated on light scatter to remove debris and the percentage of Annexin-FITC-positive cells was determined. Apoptosis-induced nuclear DNA fragmentation (TUNEL) was detected using the in situ cell-death detection kit, TMR red (Roche Diagnostics, Indianapolis, Indiana). Briefly, TZD-treated AtT20 cells were fixed in 4% paraformaldehyde, permeabilized with a solution of 0.1% sodium citrate and 0.6% Tween 20 (pH 7.4) for 2 min at 4 °C before incubation in a TUNEL reaction mix (TMR red in dNTP mix, TdT and labeling buffer) for 60 min at 37 °C. Annexin-FITC- or TMR red-positive cells were also visualized with an Olympus BH2 immunofluorescent microscope.

Following incubation in serum-replete medium with or without rosiglitazone (1 10^-5 M) for 48 h, a Pomc promoter reporter construct (-706 to +64 rat Pomc-luc; provided by M. Low)³⁹ was transiently transfected into AtT20 cells using Lipofectamine 2000 (Gibco BRL). Cells were then incubated with rosiglitazone (1 10^-5 M) or corticotrophic-releasing hormone (CRH, 50 nm) as described⁴⁰.

Total RNA was extracted from cell cultures (3 10⁷ cells per group) or from excised tissues with TRIzol (Gibco BRL). Rat testis RNA served as a positive control for Pttg expression. Electrophoresed RNA was transferred to Hybond-n nylon membranes (Amersham International, Buckinghamshire, UK) and hybridized as described⁴¹.

All tissues were fixed in 4% paraformaldehyde and processed for paraffin embedment. 10-m sections were immunostained using antibodies to human or mouse ACTH (1:1000) (DAKO, Carpinteria, California) and PPAR- (1:500) (Calbiochem, La Jolla, California), using both the avidin-biotin-peroxidase method and avidin-biotin-FITC and -TRITC for double immunostaining, and then counter-stained with hematoxylin. Negative controls were performed for each slide, using pre-absorbed or non-immune serum.

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