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Endocrinology. 2008 July; 149(7): 3339–3345. Published online 2008 March 20. doi: 10.1210/en.2007-1696. | PMCID: PMC2453100 |
Activation of Phosphatidylinositol 3-Kinase Signaling Promotes Aberrant Pituitary Growth in a Mouse Model of Thyroid-Stimulating Hormone-Secreting Pituitary Tumors Changxue Lu, Mark C. Willingham, Fumihiko Furuya, and Sheue-yann Cheng Laboratory of Molecular Biology (C.L., F.F., S.-Y.C.), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4264; and Department of Pathology (M.C.W.), Wake Forest University, Winston-Salem, North Carolina 27157-1072 Received December 10, 2007; Accepted March 13, 2008. TSH-secreting pituitary tumors (TSHomas) are pituitary tumors that constitutively secrete TSH. Molecular mechanisms underlying this abnormality are largely undefined. We recently created a knock-in mutant mouse harboring a mutation (denoted as PV) in the thyroid hormone receptor-β gene (TRβPV/PV mouse). As these mice age, they spontaneously develop TSHomas. Using this mouse model, we investigated the role of the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway in the pathogenesis of TSHomas. Concurrent with aberrant growth of pituitaries, AKT and its downstream effectors, mammalian target rapamycin and p70S6K, were activated to contribute to increased cell proliferation and pituitary growth. In addition, activation of AKT led to decreased apoptosis by inhibiting proapoptotic activity of Bcl-2-associated death promoter, further contributing to the aberrant cell proliferation. These results suggest an activated PI3K-AKT pathway could underscore tumorigenesis, raising the possibility that this pathway could be a potential therapeutic target in TSHomas. Indeed, TRβPV/PV mice treated with a PI3K-specific inhibitor, LY294002, showed a significant decrease in pituitary growth. The progrowth signaling via AKT-mammalian target rapamycin-p70S6K and cyclin D1/cyclin-dependent kinase were inhibited, and proapoptotic activity of Bcl-2-associated death promoter was increased by LY294002 treatment. Thus, activation of the PI3K-AKT pathway mediates, at least in part, the aberrant pituitary growth, and the intervention of this signaling pathway presents a novel therapeutic opportunity for TSHomas. TSH-SECRETING PITUITARY adenomas (TSHomas) are tumors that constitutively secrete TSH. They represent about 2% of all pituitary tumors in humans ( 1). TSHomas are usually large at diagnosis with significant headaches, visual field disturbances, and central hyperthyroidism ( 2). Pituitary surgery and irradiation are effective in improving the symptoms in two-thirds of patients ( 3). However, due to its local invasiveness, one of three patients may lack surgical opportunity and requires chemotherapy to control the tumor size and hyperthyroidism. Treatment with somatostatin analogs, such as octreotide or lanreotide, leads to the restoration of the euthyroid state with or without pituitary tumor shrinkage ( 4, 5). The molecular genetics underlying the development of TSHomas is not well understood. TSHomas could conceivably arise from activation or gain-of-function genetic mutations in the stimulatory pathways or from changes in the inhibitory pathways due to repression or loss-of-function mutations. Efforts to identify potential tumor promoter and suppressor genes involved in the pathogenesis of TSHomas have been hampered by a limitation of available pituitary tumor samples. However, the creation of a knock-in mouse that spontaneously develops TSHomas (TRβ PV/PV mouse) has presented an unusual opportunity to elucidate the molecular basis leading to the development and progression of TSHomas ( 6). The TRβ PV/PV mouse harbors a knock-in mutation (denoted PV) in the thyroid hormone receptor (TR)-β gene. The PV mutation was identified in a patient with thyroid hormone resistance. It has a C insertion in codon 448 that leads to a frame-shift mutation in the C-terminal 14 amino acids of TRβ ( 7). As a result of this mutation, PV has completely lost thyroid hormone (T 3) binding and transcription activity ( 8). TRβ PV/PV mice exhibit severe dysregulation of the hypothalamic-pituitary-thyroid axis with 9- to 15-fold increased thyroid hormone associated with 400- to 500-fold elevated circulating serum TSH levels ( 9). As TRβ PV/PV mice age, they spontaneously develop TSHomas with highly vascular and enlarged pituitaries, containing characteristic TSH-stained large thyroprival cells and adenomas ( 6). Using this mouse model of TSHomas, we identified cyclin D1 as one of the oncogenes that mediate the tumorigenesis of TSHomas ( 6). However, because tumorigenesis frequently results from multigenic alterations, we sought to identify other altered signaling pathways that could also contribute to the pathogenesis of TSHomas. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway regulates a vast array of fundamental cellular responses and plays a critical role in controlling the balance between cell survival and apoptosis ( 10, 11). AKT is the primary mediator of PI3K-initiated signaling by phosphorylation of a number of downstream substrates, such as mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (S6K), the forkhead family of transcription factors (FKHR/FOX), glycogen synthase kinase (GSK)-3, and proapoptotic Bcl-2-associated death promoter (BAD) protein ( 12). That overactivation of the PI3K/AKT signaling pathway has been found in numerous tumors ( 12, 13, 14) prompted us to investigate whether this pathway could be activated during the development of TSHomas in TRβ PV/PV mice. In the present study, we found that indeed the PI3K/AKT signaling pathway was activated via increased phosphorylation of its downstream effectors that promote cell growth and decrease cell survival. Importantly, we demonstrated that the activation of this pathway was attenuated by treating TRβ PV/PV mice with LY294002 (LY), a potent inhibitor of PI3K ( 15), leading to significant reduction in pituitary growth. These in vivo findings suggested that the PI3K/AKT pathway represents novel potential therapeutic targets for TSHomas. Experimental animals and treatment TRβ PV/PV mice were prepared and genotyped as described previously ( 16). All animal experiments were performed according to protocols approved by the National Cancer Institute Animal Care and Use Committee. The reagent LY, a specific PI3K inhibitor, was a gift from Dr. J. Starling (Eli Lilly and Co., Indianapolis, IN). Beginning at the age of 2 months, TRβ PV/PV mice and their wild-type siblings were given either 25 mg/kg LY or vehicle [dimethylsulfoxide (DMSO)] ip twice weekly for more than 10 months. Pituitaries were collected from euthanized mice and kept frozen at −80 C until use. Western blot analysis For Western blot analysis, pituitaries were dissected and frozen immediately until ready to be analyzed. Pituitary extracts were prepared in a similar manner as previously described ( 6). In short, frozen pituitary was homogenized in cell lysis buffer containing 20 m m Tris-HCl (pH 7.5), 125 m m NaCl, 2 m m EDTA, 0.5% Triton X-100, 0.2 μ m okadaic acid, 2 m m Na 3VO 4, 100 m m NaF, and protease inhibitors (Complete mini-EDTA-free; Roche Diagnostics, Basel, Switzerland). The protein concentration was determined by the method of Bradford (Pierce Chemical Co., Rockford, IL). Approximately 40–50 μg of extract proteins were separated by SDS-PAGE, and the Western blot analysis was carried out as described by Furumoto et al. ( 6). Two sets of representative results from three to six independent experiments are shown in each figure. Primary antibodies for phosphorylated S473 AKT (no. 9271, 1:500 dilution), total AKT (no. 9272, 1:1000), phosphorylated S2448 mTOR (no. 2971, 1:500), total mTOR (no. 2972, 1:1000), phosphorylated Thr421/Ser424 p70 S6K (no. 9204, 1:1000), total p70 S6K (no. 9202, 1:500), phosphorylated Ser9/21 GSK3β/α (no. 9331, 1:1000), total GSK-3β (no. 9315, 1:500), phosphorylated Ser253 forkhead box (Fox)-O3a (no. 9466, 1:500), total FoxO3a (no. 9467, 1:500), phosphorylated Ser136 BAD (no. 9295, 1:500), and total BAD (no. 9292, 1:500) were from Cell Signaling Technology (Danvers, MA). Cyclin D1 (sc-450) and CDK4 (sc-260) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a 1:200 dilution concentration. The Bcl-2 interacting mediator of cell death (Bim) antibody (OPAI-01021) from Affinity BioReagents (Golden, CO) was used at a 1:500 dilution concentration. Anti-dynamitin (dynactin 2.p50) antibody (AB5869P) from Chemicon (Temecula, CA) was used at 1:1000 dilution concentration. Histopathological analysis of TSHomas in LY-treated TRβPV/PV mice The percentage of anterior pituitary mass occupied by neoplastic cells was quantified using morphometry as follows. Multiple-step sections were prepared to include the entire mouse pituitary in five equally spaced separate sections for each mouse. Two mice were PV/PV animals untreated, and two were LY treated. Digital images of hematoxylin and eosin (H&E)-stained sections were captured using a ×4 objective. Each field had a total area of 4.3 × 106 m2. Neoplastic cells were easily identified as cells having foci of homogenous morphology with enlarged nuclei and increased cytoplasm that was often eosinophilic or clear. These foci occurred either as large solitary masses or small multifocal lesions. Normal areas of anterior pituitary showed the expected glandular arrangements of cells with small nuclei and variable tinctorial properties. The captured digital images were manually subdivided and selected using an overlay color based on either normal or neoplastic morphology. Middle lobe and posterior lobe were not included in the analysis. Using Photoshop 7.0 (Adobe, San Jose, CA), the selected areas were quantified by pixel area using the Histogram function. The normal and neoplastic areas were recorded for each section of each mouse, and the percentage of anterior pituitary occupied by neoplasia was calculated using the formula: percent neoplasia = number of pixels neoplastic/number of pixels neoplastic + number of pixels normal. The mean for each mouse was compiled and a sd determined among the five sections for each mouse. Statistical analysis All data are expressed as means ± sd. In statistical analysis, the Student’s t test was performed using GraphPad Prism (version 4.00 for Mac; GraphPad Software, San Diego, CA), and P < 0.05 was considered significant. Activation of AKT-mTOR-p70S6K pathway during development of TSHomas in TRβPV/PV mice We have previously shown that as TRβ PV/PV mice age, they spontaneously develop TSHomas with enlarged pituitaries ( 6). Because activation of the AKT-mTOR-p70 S6K pathway leads to increased organ growth, we ascertained whether this pathway is affected during pituitary tumorigenesis of TRβ PV/PV mice. Western blot analysis indicated that, compared with wild-type siblings, AKT was activated by increased phosphorylation in the pituitaries of TRβ PV/PV mice (compare lanes 3 and 4 with lanes 1 and 2, Fig. 1a) without apparent effects on the respective total AKT protein levels (Fig. 1b). The downstream effectors, mTOR and p70 S6K, were also activated by increased phosphorylation (compare lanes 3 and 4 with lanes 1 and 2, Fig. 1c; compare lanes 3 and 4 with lanes 1 and 2, Fig. 1e, respectively). No significant alterations in the protein levels of total mTOR and p70 S6K were observed (Fig. 1, d and f, respectively). These data indicate that activation of the AKT-mTOR-p70 S6K pathway could contribute to aberrant growth of pituitaries in TRβ PV/PV mice. LY294002 decreases the pituitary tumor weight in TRβPV/PV mice That the AKT-mTOR-p70 S6K pathway was activated provided us with an opportunity to assess whether this pathway could be a potential therapeutic target. We therefore treated TRβ PV/PV mice with LY, a specific inhibitor of its immediate upstream regulator, PI3K, and determined its effects on the spontaneous development of TSHomas. Figure 2 shows that treatment of wild-type mice with LY had no significant effect on the growth of pituitaries (Fig. 2; compare bar 2 with bar 1). Consistent with our previous observations ( 6), the pituitaries of untreated TRβ PV/PV mice were enlarged about 2.6-fold as compared with untreated wild-type siblings (Fig. 2, bar 3 vs. bar 1). This abnormal increase in pituitary growth in TRβ PV/PV mice was blocked by LY treatment (Fig. 2, bar 3 vs. bar 4). These results indicate that inhibition of PI3K activity resulted in the inhibition of aberrant growth of pituitaries in TRβ PV/PV mice. To clearly demonstrate that the decreased pituitary tumor weight was due to reduced tumor growth by LY treatment, the area of pituitaries occupied by neoplastic cells was determined using morphometry analysis of five H&E-stained sequential step-sections to encompass each pituitary (see Materials and Methods). Figure 3A shows a representative H&E-stained micrograph of the untreated pituitary (Fig. 3A, a, c, and e) and treated pituitary (Fig. 3A, b, d, and f). On evaluation, the area of pituitaries occupied by neoplastic cells was marked in red and the area occupied by normal cells was marked in blue (Fig. 3A, c and d). The pathohistologic differences between neoplastic cells and normal cells are illustrated in the high magnification of the transition zone marked with as asterisk (Fig. 3A, e and f, for untreated and treated, respectively). The magnified area of the pituitary in the untreated animal shows homogeneous neoplastic cells that have enlarged eosinophilic or clear cytoplasm with a mild degree of nuclear enlargement and pleomorphism. Adjacent normal areas of anterior pituitary show cells in a glandular pattern with small nuclei and variable cytoplasmic tinctorial properties. In the treated animal, the areas of neoplastic morphology are small and heterogenous in distribution. The areas occupied by the neoplastic cells (e.g. marked in red, Fig. 3A, c and d) and the normal cells (marked in blue) in the anterior lobe were measured from H&E-stained sections, and the data are expressed as the percentage of anterior pituitary occupied by neoplasia. As shown in Fig. 3B, LY treatment led to a marked decrease in the area of the anterior lobe occupied by neoplastic cells. These data indicate that LY treatment effectively inhibited the tumor cell proliferation. LY294002 inhibits the PI3K-AKT-mTOR-p70S6K signaling in the pituitary of TRβPV/PV mice Activation of the PI3K downstream AKT-mTOR-p70S6K increases protein synthesis, cell growth, and proliferation, a process whereby cells accumulate mass and increase organ growth. The finding that LY treatment decreased pituitary growth of TRβPV/PV mice (Figs. 2 and 3) prompted us to examine whether this pathway was inhibited by LY treatment. Two representative sets of Western blot analyses from three to five independent experiments are shown in Fig. 4. Compared with untreated mice (Fig. 4a, lanes 1 and 3), TRβPV/PV mice treated with LY show decreased phosphorylated AKT (Fig. 4a, lanes 2 and 4). Compared with untreated mice (Fig. 4, c and e, lanes 1 and 3), concordant decreases in phosphorylated mTOR (Fig. 4c, lanes 2 and 4) and phosphorylated p70S6K (Fig. 4e, lanes 2 and 4) were detected in the treated mice. No significant changes in the total respective protein levels were observed in the pituitaries between treated and untreated mice (Fig. 4, b, d, and f). Dynactin-2, a subunit of the multiprotein complex dynactin, was used as a loading control in all Western blot analyses (Fig. 4g). These results indicate that LY treatment reduced the pituitary tumor growth of TRβPV/PV mice by attenuating AKT-mTOR-p70S6K signaling, mainly via phosphorylation cascades. LY reduces cell proliferation by altering key cell cycle regulators in pituitaries of TRβPV/PV mice To further understand molecular mechanisms by which LY treatment led to the reduction of pituitary tumors in TRβ PV/PV mice, we evaluated several AKT-affected key cell cycle regulators by Western blot analysis (Fig. 5). GSK-3β is a downstream effector of the PI3K-AKT pathway (also see Fig. 7). Phosphorylation of GSK-3β by AKT inhibits its kinase activity to phosphorylate cyclin D1, thereby decreasing the proteasome degradation of cyclin D1 ( 17). Because cyclin D1 is a critical regulator of cell cycle ( 18), we evaluated the effect of LY treatment on the cellular abundance of phosphorylated GSK-3β and cyclin D1. As shown in Fig. 5 from two independent sets of experiments, LY treatment significantly reduced the phosphorylated GSK-3β (Fig. 5a, lane 2 vs. lane 1 and lane 4 vs. lane 3), and there was a concordant decrease in the cellular levels of cyclin D1 (Fig. 5c, lane 2 vs. lane 1 and lane 4 vs. lane 3). Furthermore, cellular levels of cyclin-dependent kinase 4 (CDK4) were also decreased (Fig. 5d, lane 2 vs. lane 1 and lane 4 vs. lane 3). The dynactin 2 loading controls are shown in Fig. 5e. These findings indicate that LY treatment reduced the cellular levels of cyclin D1 and CDK4 to decrease cell proliferation. LY decreases cell survival by activating key proapoptotic regulators Numerous studies indicate that activation of PI3K-AKT pathway promotes cell survival by directly phosphorylating key regulators of the apoptotic cascade. BAD, a member of the Bcl-2 family, is a direct phosphorylation target of AKT (see also Fig. 7). Its phosphorylation by AKT results in cytosolic accumulation, thereby preventing its interaction with and antagonizing the actions of prosurvival members of the family such as Bcl-2 at the mitochondrial membrane ( 19, 20). We therefore examined the effect of LY on cellular protein levels of BAD and its phosphorylation status by Western blot analysis. Two representative sets of comparisons between LY-treated and untreated mice are shown in Fig. 6. Compared with pituitaries of untreated mice (Fig. 6a, lanes 1 and 3), pituitaries of treated mice show decreased phosphorylation of BAD (Fig. 6a, lanes 2 and 4) without apparent alterations of total cellular BAD protein levels in each set (Fig. 6b, lane 1 vs. lane 2 and lane 3 vs. lane 4). The reduction in the phosphorylation of BAD induced by LY treatment led to increased apoptosis (see Fig. 7). Another means by which AKT promotes cell survival is by directly phosphorylating transcription factors that control the expression of pro- and antiapoptotic genes ( 14). The transcriptional factor FoxO family member such as FoxO3a is a direct phosphorylation target of AKT. When phosphorylated, it represses the expression of proapoptotic genes such as Bim ( 21). To understand whether LY treatment could lead to decreased cell survival via this mechanism, we also compared the phosphorylated Fox (p-Fox)-O3a in the pituitaries of untreated and treated TRβ PV/PV mice (Fig. 6). Compared with pituitaries of untreated mice, pituitaries of TRβ PV/PV mice treated with LY show reduced p-FoxO3a (Fig. 6c; compare lane 2 with lane 1 and lane 4 with lane 3). Total cellular FoxO3a protein levels were not significantly affected by LY treatment (Fig. 6d). The consequences of de-repression in the expression of its target genes mediated by the reduced p-FoxO3a were made evident by the increased levels of proapoptotic Bim in the pituitaries of treated mice (Fig. 6e; compare lane 2 with lane 1 and lane 4 with lane 3). Figure 6f shows the loading controls using dynactin 2. Taken together, these results indicate that LY treatment increased apoptosis by reduced phosphorylation of FoxO3a and increased proapoptotic regulator Bim (see Fig. 7). TSHomas are believed to derive from the monoclonal expansion of a single transformed thyrotrope that has lost growth regulation ( 21). The availability of a mouse model of TSHomas induced by radiothyroidectomy has helped understand the histopathological changes during the development of TSHomas ( 22). Studies of transplantable tumors have yielded valuable information of the secondary changes caused by the tumors and insights into effects of thyroid hormone in the development of tumors ( 23, 24, 25). However, studies to identify the gene alterations leading to TSHomas are limited. The discovery that the TRβ PV/PV mouse spontaneously develops TSHomas has provided us with an opportunity to elucidate the molecular basis underlying the pathogenesis of TSHomas ( 6). Indeed, we found that overexpression of oncogenic cyclin D1 is one of the genetic changes that results in constitutive activation of the cyclin D1/cyclin-dependent kinase/retinoblastoma protein/E 2F family of DNA-binding transcription factors (E 2F) pathway to promote cell proliferation ( 6). The overexpression of cyclin D1 is mediated at the transcriptional level ( 6). In the present study, we found that AKT was activated and that phosphorylation of GSK-3β by AKT inhibited the kinase activity of GSK-3β to phosphorylate cyclin D1 (Fig. 7). Via the phosphorylation cascade, the degradation of cyclin D1 by the proteasome machinery is prevented ( 17). Thus, we identified an additional posttranslation mechanism by which cyclin D1 protein is stabilized to increase cell proliferation. It is important to point out that in addition to the activation of cyclin D1, the present study shows that activation of AKT-mTOR-p70 S6K signaling also contributes to thyrotrope growth. Thus, the aberrant proliferation is mediated by at least two pathways: via cyclin D1 at cell cycle progression and via AKT-mTOR-p70 S6K signaling to increase protein synthesis and cell growth and proliferation (see Fig. 7). The molecular mechanisms by which PI3K-AKT is activated during the development of TSHomas are currently unknown. We have previously shown that in addition to TSHomas, TRβ PV/PV mice also spontaneously develop follicular thyroid carcinoma similar to human thyroid cancer ( 26, 27). One of the altered signaling pathways identified during thyroid carcinogenesis of TRβ PV/PV mice is overactivation of the PI3K-AKT pathway, which was also reported in human thyroid cancer ( 28, 29, 30). The activation of PI3K is mediated via a novel nongenomic action by physical interaction of PV with the regulatory p85α subunit of PI3K ( 29). It is likely that PV could act in a similar fashion to activate PI3K-AKT in the pituitary. However, at present, the small size of the pituitary in TRβ PV/PV mice has made extensive biochemical and molecular analysis very difficult to confirm this possibility. New methodologies are being developed to overcome this difficulty. The currently available treatment options for TSHomas are surgery, radiotherapy, and somatostatin analog administration ( 2). Whereas each treatment or combinations of these treatments could be beneficial to patients, it would be valuable to explore other options that could improve treatment modalities. The finding that PI3K-AKT was activated in the pituitary of TRβ PV/PV mice suggested that the PI3K-AKT pathway is a potential molecular target for TSHomas. Indeed, treatment of TRβ PV/PV mice with a PI3K inhibitor, LY, led to a significant inhibition in the proliferation of neoplastic cell growth (Fig. 3), resulting in the reduction of pituitary size (Fig. 2). Biochemical analysis indicated that LY treatment attenuated PI3K-AKT-mTOR-p70 S6K signaling (Fig. 4) and cyclin D1/CDK4 activity to reduce cell proliferation (Fig. 5). LY treatment also inhibited PI3K-AKT downstream signaling to decrease cell survival to further reduce cell proliferation (Fig. 6). The present finding that LY, a prototype of PI3K inhibitors, was effective in reducing pituitary tumor growth in vivo suggests that currently available second- or third-generation PI3K inhibitors such as ZSK474, IC187114, TG100–115, and others ( 31, 32) could be tested as potential therapeutic approaches for TSHomas. 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