Activation of phosphatidylinositol 3-kinase signaling promotes aberrant pituitary growth in a mouse model of thyroid-stimulating hormone-secreting pituitary tumors

Abstract

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-beta gene (TRbeta(PV/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 p70(S6K), 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, TRbeta(PV/PV) mice treated with a PI3K-specific inhibitor, LY294002, showed a significant decrease in pituitary growth. The progrowth signaling via AKT-mammalian target rapamycin-p70(S6K) 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.

Figure 1

Activation of the AKT-mTOR-p70^S6K pathway in pituitaries of TRβ^PV/PV mice. Protein levels of phosphorylated AKT (p-AKT) (a), total AKT (b), phosphorylated mTOR (p-mTOR) (c), total mTOR (d), phosphorylated p70^S6K (p-p70^S6K) (e), and total p70^S6K (f) were determined in 40 μg of pituitary extract by Western blot analysis. Representative results from two wild-type (WT) and two TRβ^PV/PV mice are shown. Dynactin 2 (g) was used as a loading control.

Figure 2

Reduced pituitary weight in TRβ^PV/PV mice treated with LY. Bars 1 and 2 represent pituitary weights of wild-type mice (WT) without or with LY treatment, respectively. Bars 3 and 4 represent the weight of the same tissue from TRβ^PV/PV mice without or with LY treatment, respectively. The data are expressed as means ± sd [n = 5 for DMSO treated wild-type mice (bar 1); n = 10 for LY treated wild-type mice (bar 2); n = 23 for DMSO treated TRβ^PV/PV mice (bar 3); and n = 24 for LY treated TRβ^PV/PV mice (bar 4)]. NS, Not significant.

Figure 3

LY treatment inhibited tumor cell growth (A). The area of pituitaries occupied by neoplastic cells was determined using morphometric analysis of five H&E-stained sequential step sections to encompass each pituitary as described in Materials and Methods. Representative examples from sections of untreated mice (panels a, c, and e) and LY-treated mice (panels b, d, and f) are shown. The neoplastic cells are marked in red (panels c and d) and normal cells are marked in blue. The abnormal and normal histologic characteristic details are shown in panels e and f. B, The areas occupied by the neoplastic cells and normal cells in the anterior lobe were quantified as described in Materials and Methods. The percentage of the pituitary mass occupied by neoplasia was dramatically reduced by LY treatment (n = 10).

Figure 4

LY attenuates activation of the AKT-mTOR-p70^S6K pathway in pituitaries of TRβ^PV/PV mice. Pituitary extract (50 μg) from TRβ^PV/PV mice without or with LY treatment were used to determine phosphorylated and total protein levels of AKT (panels a and b), mTOR (panels c and d), and p70^S6K (panels e and f) by Western blot analysis as described in Materials and Methods. Two sets (A and B) of representative results from three to six independent experiments are shown. Dynactin 2 (panel g) was used as a total protein loading control.

Figure 5

LY reduces cell proliferation by altering key cell cycle regulators in TRβ^PV/PV mice. From 50 μg of pituitary extract, protein levels of phosphorylated GSK-3β (a), total GSK-3β (b), cyclin D1 (c), and CDK4 (d) were determined by Western blot analysis as described in Materials and Methods. Two sets (A and B) of representative results from three to six independent experiments are shown. Dynactin 2 (e) was used as a total protein loading control.

Figure 6

LY decreases key proapoptotic regulators in pituitaries of TRβ^PV/PV mice. Phosphorylated proapoptotic protein BAD (p-BAD; a), total BAD (b), p-Fox03a protein (c), total FoxO3a (d), and Bim (extra large isoform; e) protein levels were determined by Western blot analysis in 50 μg of pituitary extract as described in Materials and Methods. Two representative sets of results (A and B) are shown. Dynactin 2 (f) was used as the loading control.

Figure 7

Schematic representation of the activated PI3K-AKT pathway and its downstream effectors that contribute to the tumorigenesis of TSHomas in TRβ^PV/PV mice. Aberrant activation of the PI3K-AKT-mTOR-p70^S6K pathway in pituitaries of TRβ^PV/PV mice increases protein synthesis and cell growth. AKT decreases cyclin D1 degradation by inhibiting GSK-3β activity through phosphorylation (17). AKT also inhibits cell apoptosis through phosphorylation of BAD and FoxO3a. Phosphorylated BAD is incapable of inhibiting the activity of antiapoptotic protein Bcl-2 (33,34). Similar to the phosphorylated BAD, phosphorylated FoxO3a is retained in cytoplasm and thus is incapable of activating the expression of the proapoptotic protein Bim (35). All these processes are reversed by LY294002 in vivo by deactivating the PI3K-AKT signaling pathway. By decreasing the subsequent phosphorylation events after the AKT activation, LY ultimately reduces tumor growth by inhibiting cell proliferation and promoting apoptosis in TSHomas of TRβ^PV/PV mice.