Song-Guang Ren

EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas

Cushing disease is a condition in which the pituitary gland releases excessive adrenocorticotropic hormone (ACTH) as a result of an adenoma arising from the ACTH-secreting cells in the anterior pituitary. ACTH-secreting pituitary adenomas lead to hypercortisolemia and cause significant morbidity and mortality. Pituitary-directed medications are mostly ineffective, and new treatment options are needed. As these tumors express EGFR, we tested whether EGFR might provide a therapeutic target for Cushing disease. Here, we show that in surgically resected human and canine corticotroph cultured tumors, blocking EGFR suppressed expression of proopiomelanocortin (POMC), the ACTH precursor. In mouse corticotroph EGFR transfectants, ACTH secretion was enhanced, and EGF increased Pomc promoter activity, an effect that was dependent on MAPK. Blocking EGFR activity with gefitinib, an EGFR tyrosine kinase inhibitor, attenuated Pomc expression, inhibited corticotroph tumor cell proliferation, and induced apoptosis. As predominantly nuclear EGFR expression was observed in canine and human corticotroph tumors, we preferentially targeted EGFR to mouse corticotroph cell nuclei, which resulted in higher Pomc expression and ACTH secretion, both of which were inhibited by gefitinib. In athymic nude mice, EGFR overexpression enhanced the growth of explanted ACTH-secreting tumors and further elevated serum corticosterone levels. Gefitinib treatment decreased both tumor size and corticosterone levels; it also reversed signs of hypercortisolemia, including elevated glucose levels and excess omental fat. These results indicate that inhibiting EGFR signaling may be a novel strategy for treating Cushing disease.

Central and peripheral actions of somatostatin on the growth hormone–IGF-I axis

Somatostatin (SRIF) analogs provide safe and effective therapy for acromegaly. In a proportion of patients, however, SRIF analogs may lead to discordant growth hormone (GH) and IGF-I suppression, which suggests a more complex mechanism than attributable to inhibition of GH release alone. To elucidate whether SRIF acts peripherally on the GH-IGF-I axis, we showed that rat hepatocytes express somatostatin receptor subtypes-2 and -3 and that IGF-I mRNA and protein levels were suppressed in a dose-dependent manner by administration of octreotide. The inhibitory effect of SRIF was not apparent without added GH and in the presence of GH was specific for IGF-I induction and did not inhibit GH-induced c-myc or extracellular signal regulated kinase (ERK) phosphorylation. Pertussis toxin treatment of hepatocytes incubated with GH and SRIF, or with GH and octreotide, abrogated the inhibitory effect on GH-induced IGF-I, which confirms the requirement for the inhibitory G-protein. Treatment with SRIF and GH increased protein tyrosine phosphatase (PTP) activity and inhibited signal transducer and activator of transcription-5b (STAT5b) phosphorylation and nuclear localization. Octreotide also inhibited GH-stimulated IGF-I protein content of ex vivo-perfused rat livers. The results demonstrate that SRIF acts both centrally and peripherally to control the GH-IGF-I axis, providing a mechanistic explanation for SRIF analog action in treating patients with GH-secreting pituitary adenomas.

Rat Prolactinoma cell growth regulation by Epidermal Growth Factor receptor ligands

Epidermal growth factor (EGF) regulates pituitary development, hormone synthesis, and cell proliferation. Although ErbB receptor family members are expressed in pituitary tumors, the effects of EGF signaling on pituitary tumors are not known. Immunoprecipitation and Western blot confirmed EGF receptor (EGFR) and p185(c-neu) protein expression in GH3 lacto-somatotroph but not in adrenocorticotropic hormone-secreting AtT20 pituitary tumor cells. EGF (5 nmol/L) selectively enhanced baseline ( approximately 4-fold) and serum-induced (>6-fold) prolactin (PRL) mRNA levels, whereas gefitinib, an EGFR antagonist, suppressed serum-induced cell proliferation and Pttg1 expression, blocked PRL gene expression, and reversed EGF-mediated somatotroph-lactotroph phenotype switching. Downstream EGFR signaling by ERK, but not phosphoinositide-3-kinase or protein kinase C, mediated the gefitinib response. Tumors in athymic mice implanted s.c. with GH3 cells resulted in weight gain accompanied by increased serum PRL, growth hormone, and insulin growth factor 1. Gefitinib decreased tumor volumes and peripheral hormone levels by approximately 30% and restored normal mouse body weight patterns. Mice treated with gefitinib exhibited decreased tumor tissue ERK1/2 phosphorylation and down-regulated tumor PRL and Pttg1 mRNA abundance. These results show that EGFR inhibition controls tumor growth and PRL secretion in experimental lacto-somatotroph tumors. EGFR inhibitors could therefore be useful for the control of PRL secretion and tumor load in prolactinomas resistant to dopaminergic treatment, or for those prolactinomas undergoing rare malignant transformation.

Somatostatin Receptor Type 5 Modulates Somatostatin Receptor Type 2 Regulation of Adrenocorticotropin Secretion

Somatostatin inhibits adrenocorticotropin (ACTH) secretion from pituitary tumor cells. To assess the contribution of somatostatin receptor subtype 5 (SST5) to somatostatin receptor subtype 2 (SST2) action in these cells, we assessed multipathway responses to novel highly monoreceptor-selective peptide agonists and multireceptor agonists, including octreotide and somatostatin-28. Octreotide and somatostatin-28 cell membrane binding affinities correlated with their respective SST2-selective peptide ligand. Although octreotide had similar inhibiting potency (picomolar) for cAMP accumulation and ACTH secretion as an SST2-selective agonist, somatostatin-28 exhibited a higher potency (femtomolar). Baseline spontaneous calcium oscillations assessed by fluorescent confocal microscopy revealed two distinct effects: SST2 activation reduced oscillations at femtomolar concentrations reflected by high inhibiting potency of averaged normalized oscillation amplitude, whereas SST5 activation induces brief oscillation pauses and increased oscillation amplitude. Octreotide exhibits an integrated effect of both receptors; however, somatostatin-28 exhibited a complex response with two separate inhibitory potencies. SST2 internalization was visualized with SST2-selective agonist at lower concentrations than for octreotide or somatostatin-28, whereas SST5 did not internalize. Using monoreceptor-selective peptide agonists, the results indicate that, in AtT-20 cells, SST5 regulates the dominant SST2 action, attenuating SST2 effects on intracellular calcium oscillation and internalization. This may explain superior somatostatin-28 potency and provides a rationale for somatostatin ligand design to treat ACTH-secreting pituitary tumors.

Growth hormone is a cellular senescence target in pituitary and nonpituitary cells

Significance Growth hormone (GH) is secreted to the circulation by anterior pituitary somatotroph cells and is also expressed locally in peripheral tissues. This study identifies GH as a target for p53-induced senescence in both pituitary and nonpituitary cells. We show marked induction of intracellular GH expression and secretion in response to activation of the p53 DNA damage pathway. GH is shown to be a direct transcriptional target for p53, and up-regulated GH protects pituitary tumor cells from apoptosis. The results demonstrate a novel mechanism determining the pathophysiology of GH-secreting pituitary tumors and elucidate a basis for the invariably benign nature of these adenomas. Our findings show that induced GH can serve as a readily measurable marker for p53-induced senescence. Premature proliferative arrest in benign or early-stage tumors induced by oncoproteins, chromosomal instability, or DNA damage is associated with p53/p21 activation, culminating in either senescence or apoptosis, depending on cell context. Growth hormone (GH) elicits direct peripheral metabolic actions as well as growth effects mediated by insulin-like growth factor 1 (IGF1). Locally produced peripheral tissue GH, in contrast to circulating pituitary-derived endocrine GH, has been proposed to be both proapoptotic and prooncogenic. Pituitary adenomas expressing and secreting GH are invariably benign and exhibit DNA damage and a senescent phenotype. We therefore tested effects of nutlin-induced p53-mediated senescence in rat and human pituitary cells. We show that DNA damage senescence induced by nutlin triggers the p53/p21 senescent pathway, with subsequent marked induction of intracellular pituitary GH in vitro. In contrast, GH is not induced in cells devoid of p53. Furthermore we show that p53 binds specific GH promoter motifs and enhances GH transcription and secretion in senescent pituitary adenoma cells and also in nonpituitary (human breast and colon) cells. In vivo, treatment with nutlin results in up-regulation of both p53 and GH in the pituitary gland, as well as increased GH expression in nonpituitary tissues (lung and liver). Intracrine GH acts in pituitary cells as an apoptosis switch for p53-mediated senescence, likely protecting the pituitary adenoma from progression to malignancy. Unlike in the pituitary, in nonpituitary cells GH exerts antiapoptotic properties. Thus, the results show that GH is a direct p53 transcriptional target and fulfills criteria as a p53 target gene. Induced GH is a readily measurable cell marker for p53-mediated cellular senescence.

Sca1⁺ murine pituitary adenoma cells show tumor-growth advantage.

The role of tumor stem cells in benign tumors such as pituitary adenomas remains unclear. In this study, we investigated whether the cells within pituitary adenomas that spontaneously develop in Rb+/- mice are hierarchically distributed with a subset being responsible for tumor growth. Cells derived directly from such tumors grew as spheres in serum-free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor. Some cells within growing pituitary tumor spheres (PTS) expressed common stem cell markers (Sca1, Sox2, Nestin, and CD133), but were devoid of hormone-positive differentiated cells. Under subsequent differentiating conditions (matrigel-coated growth surface), PTS expressed all six pituitary hormones. We next searched for specific markers of the stem cell population and isolated a Sca1(+) cell population that showed increased sphere formation potential, lower mRNA hormone expression, higher expression of stem cell markers (Notch1, Sox2, and Nestin), and increased proliferation rates. When transplanted into non-obese diabetic-severe combined immunodeficiency gamma mice brains, Sca1(+) pituitary tumor cells exhibited higher rates of tumor formation (brain tumors observed in 11/11 (100%) vs 7/12 (54%) of mice transplanted with Sca1(+) and Sca1(-) cells respectively). Magnetic resonance imaging and histological analysis of brain tumors showed that tumors derived from Sca1(+) pituitary tumor cells were also larger and plurihormonal. Our findings show that Sca1(+) cells derived from benign pituitary tumors exhibit an undifferentiated expression profile and tumor-proliferative advantages, and we propose that they could represent putative pituitary tumor stem/progenitor cells.

Diminished Pancreatic β-Cell Mass in Securin-Null Mice Is Caused by β-Cell Apoptosis and Senescence

Pituitary tumor transforming gene (PTTG) encodes a securin protein critical in regulating chromosome separation. PTTG-null (PTTG(-/-)) mice exhibit pancreatic beta-cell hypoplasia and insulinopenic diabetes. We tested whether PTTG deletion causes beta-cell senescence, resulting in diminished beta-cell mass. We examined beta-cell mass, proliferation, apoptosis, neogenesis, cell size, and senescence in PTTG(-/-) and WT mice from embryo to young adulthood before diabetes is evident. The roles of cyclin-dependent kinase inhibitors and DNA damage in the pathogenesis of diabetes in PTTG(-/-) mice were also addressed. Relative beta-cell mass in PTTG(-/-) mice began to decrease at 2-3 wk, whereas beta-cell proliferation rate was initially normal but decreased in PTTG(-/-) mice beginning at 2 months. Apoptosis was also much more evident in PTTG(-/-) mice. At 1 month, beta-cell neogenesis was robust in wild-type mice but was absent in PTTG(-/-) mice. In addition, the size of beta-cells became larger and macronuclei were prominent in PTTG(-/-) animals. Senescence-associated beta-galactosidase was also active in PTTG(-/-) beta-cells at 1 month. Cyclin-dependent kinase inhibitor p21 was progressively up-regulated in PTTG(-/-) islets, and p21 deletion partially rescued PTTG(-/-) mice from development of diabetes. mRNA array showed that DNA damage-associated genes were activated in PTTG(-/-) islets. We conclude that beta-cell apoptosis and senescence contribute to the diminished beta-cell mass in PTTG(-/-) mice, likely secondary to DNA damage. Our results also suggest that ductal progenitor beta-cells are exhausted by excessive neogenesis induced by apoptosis in PTTG(-/-) mice.

Leukemia inhibitory factor (LIF) modulates pro-opiomelanocortin (POMC) gene regulation in stably transfected AtT-20 cells overexpressing LIF

Leukemia inhibitory factor (LIF) levels are elevated in sepsis and correlate with shock and poor prognosis. We have previously shown that lipopolysaccharide (LPS) administration induces hypothalamic and pituitary LIF expression in vivo, which is associated with the acute rise in circulating adrenocorticotrophic hormone (ACTH) levels. As AtT-20 cells respond to LIF, we established murine LIF (mLIF) stably transfected AtT-20 cell lines to study LIF regulation of pro-opiomelanocortin (POMC) expression and ACTH secretion. Our results show that mLIF transfectants accumulated mLIF (up to 15.6±3.2 ng/mL after 24h) as well as increased ACTH secretion (up to 2.4-fold above control cells) in conditioned medium. The magnitude of ACTH induction correlated with mLIF concentrations in different transfectants (r=0.75–0.88,p<0.05). Moreover, mLIF transfectants showed a higher sensitivity to CRH stimulation with an increased ACTH production within 8 h (p<0.05), whereas control cells were responsive to CRH at 24h. Additionally, mLIF transfectants exhibited a maximum threefold ACTH induction, compared to 1.7-fold in control cells. Furthermore, mLIF transfectants have a blunted dexamethasone-mediated inhibition of ACTH (35% inhibition in control cells vs no inhibition in mLIF-transfected cells at 24h). These findings support and extend the previous observations of LIF acting at the pituitary level, and indicate that mLIF stably-transfected AtT-20 cells are a useful model for studying mLIF-mediated gene regulation in pituicytes.

STAT3 upregulation in pituitary somatotroph adenomas induces growth hormone hypersecretion

Pituitary somatotroph adenomas result in dysregulated growth hormone (GH) hypersecretion and acromegaly; however, regulatory mechanisms that promote GH hypersecretion remain elusive. Here, we provide evidence that STAT3 directly induces somatotroph tumor cell GH. Evaluation of pituitary tumors revealed that STAT3 expression was enhanced in human GH-secreting adenomas compared with that in nonsecreting pituitary tumors. Moreover, STAT3 and GH expression were concordant in a somatotroph adenoma tissue array. Promoter and expression analysis in a GH-secreting rat cell line (GH3) revealed that STAT3 specifically binds the Gh promoter and induces transcription. Stable expression of STAT3 in GH3 cells induced expression of endogenous GH, and expression of a constitutively active STAT3 further enhanced GH production. Conversely, expression of dominant-negative STAT3 abrogated GH expression. In primary human somatotroph adenoma-derived cell cultures, STAT3 suppression with the specific inhibitor S3I-201 attenuated GH transcription and reduced GH secretion in the majority of derivative cultures. In addition, S3I-201 attenuated somatotroph tumor growth and GH secretion in a rat xenograft model. GH induced STAT3 phosphorylation and nuclear translocation, indicating a positive feedback loop between STAT3 and GH in somatotroph tumor cells. Together, these results indicate that adenoma GH hypersecretion is the result of STAT3-dependent GH induction, which in turn promotes STAT3 expression, and suggest STAT3 as a potential therapeutic target for pituitary somatotroph adenomas.

Constitutive Somatostatin Receptor Subtype-3 Signaling Suppresses Growth Hormone Synthesis

Somatostatin signals through somatostatin receptor subtypes (SSTR) 2 and 5 to attenuate GH secretion. Although expressed in normal pituitary glands and in GH-secreting pituitary tumors, SSTR3 function was unclear, and we have now determined the role of SSTR3 in somatotroph function. Stable rat pituitary tumor cell (GC) transfectants of human SSTR3 (GpSSTR3WT) showed suppression of rat (r) GH promoter activity, GH mRNA expression, and secreted GH concordant with suppressed cAMP/protein kinase A (PKA) signaling. In contrast, cAMP levels and GH expression were unchanged in cells expressing a mutant SSTR3 DRY motif (GpSSTR3R141A). GH expression was rescued by treatment of GpSSTR3WT with forskolin and 8-bromo-cAMP. GpSSTR3WT exhibited activation of glycogen synthase kinase3-β (GSK3-β), a PKA substrate, which was also reversed by 8-Bromo-cAMP treatment. Moreover, SSTR3-dependent GH transcriptional inhibition was rescued by inhibition of GSK3-β. GpSSTR3WT exhibited elevated Pit-1 serine phosphorylation and decreased Pit-1 occupancy of the rGH promoter with sustained Pit-1 expression. GSK3-β and Pit-1 physically interacted with each other, indicating that Pit-1 may be a GSK3-β phosphorylation substrate. In conclusion, constitutive SSTR3 activity mediates transcriptional repression of GH through cAMP/PKA, leading to subsequent activation of GSK3-β and increased Pit-1 phosphorylation and ultimately attenuating Pit-1 binding to the rGH promoter.