Human Pituitary Tumors Research Articles

Abstract LB-202: STAT3 induces pituitary tumor transforming gene (PTTG) expression

Abstract PTTG is overexpressed in multiple human tumors, but mechanisms regulating tumor PTTG abundance remain elusive. As EGF induces pituitary cell PTTG expression, and as STAT3 is required for pituitary cytokine responses, we investigated STAT3 regulation of PTTG. STAT3 and PTTG were both concordantly overexpressed in 79 human pituitary tumor specimens (62% and 56% respectively; p<0.01) as assessed by confocal immunofluorescence. We therefore treated ACTH-secreting AtT20 corticotroph tumor cells with IL-6 which dose-dependently increased STAT3 phosphorylation, PTTG and POMC expression. STAT3 also specifically bound to the human PTTG promoter (−2000 to −3000 bp) and induced PTTG transcriptional activity (2.2-fold) as assessed by chromatin immunoprecipitation and luciferase reporter assay, implying that PTTG acts as an intracellular STAT3 target. To elucidate the function of this pathway, we constructed WT STAT3, constitutively activated STAT3 (STAT3-C) and dominant negative STAT3 (STAT3-DN) plasmids, and selected respective stable transfectants. STAT3 transfection doubled PTTG mRNA and protein abundance, while PTTG induction was enhanced 3-fold in STAT3-C, and totally abrogated in STAT3-DN transfectants (0.7-fold). Stable STAT3 overexpression also enabled in vitro colony formation which was strongly attenuated by PTTG RNAi (∼50%, p<0.01). Elevated STAT3 expression, or STAT3 activation, both increased HCT116 cell migration and invasion 5-fold as measured by in vitro cell assays, whereas cell mobility was abolished by STAT3-DN (>90%). Knockdown of PTTG expression by RNAi strongly suppressed STAT3-induced cell migration and invasion by up to 80%. Attenuating PTTG expression by RNAi in STAT3-C stable cells constrained downstream myc, cyclin D3, K-Ras and MMP expression. Moreover, STAT3 inhibitor VI dose-dependently (10-150 M) suppressed STAT3 phosphorylation and subsequently inhibited PTTG, cyclin D3, POMC expression and ACTH secretion as treated for 24 hours. We also generated PTTG RNAi stable transfectants on STAT3-C stable cells and injected these compound transfectants s. c. to nude mice. PTTG RNAi decreased STAT3-C-induced in vivo tumor volume (32%, p<0.01), compared to injected scrambled control transfectants which readily formed tumors. These results elucidate a mechanism for tumor PTTG abundance, whereby STAT3 activation induces PTTG expression to facilitate tumor formation. These findings further elucidate PTTG actions in tumor progression and support the rationale for targeting PTTG to abrogate endocrine tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-202.

Immunoexpression of endoglin and CD‐34 in various human pituitary adenoma types

Angiogenesis is a crucial event in tumor growth, invasion and development of metastasis. Endoglin (CD‐105), a disulfide‐linked homodimeric cell membrane glycoprotein of 180 kDa, located on chromosome 9q and binds to several components of the TGF beta family. In contrast to other endothelial cell markers, endoglin can be demonstrated only in proliferating endothelial cells. We investigated endoglin immunoexpression in various surgically removed human pituitary tumor types and compared the results with CD‐34 immunoexpression. For immunohistochemistry, the streptavidin‐biotin‐peroxidase complex method was used and the number of vessels were counted in 10 low power fields. Results show that endoglin immunostains fewer vessels than CD‐34 in every pituitary adenoma type. The number of endoglin immunopositive vessels is highest in untreated prolactinomas and subtype 3 silent pituitary adenomas and lowest in subtype 1 silent corticotroph adenomas and GH adenomas exposed to somatostatin analogs and prolactinomas exposed to dopamine agonists. Our findings are consistent with the view that endoglin is upregulated in proliferating endothelial cells and its expression can be used as an indicator of vascular neoformation in pituitary tumors. The low levels of endoglin expression in pituitary tumors exposed to somatostatin or dopamine agonists suggests that these therapeutic drugs can inhibit angiogenesis.

Ultrastructural differences in the endoplasmic reticulum among various pituitary tumor types

Our aim was to reveal ultrastructural differences in the endoplasmic reticulum (ER) among surgically removed human pituitary tumors. More than 2000 pituitary tumors were studied by electron microscopy. In growth hormone (GH) adenomas, the ER is well developed and consists mainly of rough surfaced endoplasmic reticulum (RER) and few ER membranes without ribosomes, called smooth surfaced endoplasmic reticulum (SER). In the sparsely granulated GH adenomas, the ER is less prominent and is seen in fibrous bodies containing keratin immunopositive microfilaments. In prolactin (PRL) adenomas, the RER is conspicuous, forming concentric whorls. In adenocorticotropin (ACTH) and thyroid stimulating hormone (TSH) adenomas, the ER and consists of RER and to a lesser extent, SER. In gonadotroph adenomas and null cell adenomas, the ER is poorly developed. In GH adenomas exposed to somatostatin analogs and in prolactin adenomas exposed to dopamine agonists, regression of ER is noted. In pituitary carcinomas, the ER is recognizable but it is less extensive than in the benign neoplasms. In conclusion, the ultrastructural investigation of ER provides valuable information on the cytogenesis and cellular composition of pituitary tumors. There is also a mild correlation between endocrine activity and extent of ER. However, no correlation exists between invasive and non‐invasive tumors and growth potential. Pituitary carcinomas cannot be distinguished from benign adenomas based on the ultrastructural features of ER.

Cyclins and their related proteins in pituitary tumourigenesis

Pituitary tumours are benign neoplasms that may cause major endocrine dysfunction. Transgenic disruption of the cell cycle machinery frequently leads to pituitary adenoma formation in animal models. The molecular analysis of human pituitary tumours has found various alterations in the expression of cell cycle regulators: cyclins, cyclin-dependent kinases and their inhibitors. There are also different mechanisms (e.g. hypermethylation, frameshift mutations, increased proteasome degradation) responsible for changed expression in cyclin mRNA and protein. It is probable that the primary initiating events lie beyond the cell cycle and may be related to co-activation of Akt, MAP-kinase and β-catenin pathways. Nevertheless, molecular CDK inhibitors may play a role in pituitary tumour treatment in the future.

Inhibitory Effect of Prepro‐Thyrotrophin‐Releasing Hormone (178–199) on Adrenocorticotrophic Hormone Secretion by Human Corticotroph Tumours

Prepro-thyrotrophin-releasing hormone (TRH) (178-199), a 22-amino acid cleavage product of the TRH prohormone, has been postulated to act as an adrenocorticotrophin hormone (ACTH)-release inhibitor. Indeed, although in vitro evidence indicates that this peptide may inhibit basal and stimulated ACTH secretion in rodent anterior pituitary primary cultures and cell lines, not all studies concur and no study has as yet evaluated the effect of this peptide in Cushing's disease. The present study aimed to test the effect of preproTRH(178-199) in human tumoural corticotrophs. Twenty-four human ACTH-secreting pituitary tumours (13 macroadenomas, 11 microadenomas) were collected during surgery and incubated with 10 or 100 nm preproTRH(178-199). ACTH secretion was assessed after 4 and 24 h of incubation by immunometric assay and expressed relative to levels observed in control, unchallenged wells (= 100%). Parallel experiments were performed in rat anterior pituitary primary cultures. A clear inhibition of ACTH secretion at 4 and 24 h was observed in 12 specimens (for 10 nm ppTRH: 70 +/- 4% control at 4 h and 83 +/- 5% control at 24 h; for 100 nm ppTRH: 70 +/- 4% control at 4 h and 85 +/- 5% control at 24 h), whereas a mild and short-lasting stimulatory effect was observed in three tumours and no changes in ACTH secretion in the remaining nine tumoural specimens. The inhibitory effect of preproTRH(178-199) was more evident in macroadenomas and significantly correlated with sensitivity to dexamethasone inhibition. Significant inhibition of ACTH secretion by preproTRH(178-199) in rat pituitary cultures was observed after 24 h of incubation. The present study conducted in a large series of human corticotroph tumours shows that preproTRH(178-199) inhibits tumoural ACTH secretion in a sizable proportion of specimens, in close relation to the size of the tumour and its sensitivity to glucocorticoid negative feedback. This appears a promising avenue of research and further studies are warranted to explore the full scope of preproTRH(178-199) as a regulator of ACTH secretion.

T3 receptors in human pituitary tumors

Objective: The purpose of this work was to investigate the synthesis of T3 receptors in human tumors of the anterior pituitary gland, its relationship with the hormone synthesized and/or secreted by the tumor and the post-surgical evolution of the patient.Methods: Patients were evaluated clinically and by magnetic nuclear resonance to classify the adenoma according to their size. Hormonal concentrations in sera were determined by radioimmunoassay. Immunohistochemistry of the pituitary hormones was performed in the tumors. Tumors were obtained at surgery and immediately frozen in ice, transported to the laboratory and stored at −70°C. Reverse transcription was performed with purified RNA from the tumors.Results: Out of 33 pituitary tumors, 29 had RNA for T3 receptors synthesis (88%). They were present in different histological specimens, the tumors were grades 1–4 according to their size, and there was no relationship between the size of the tumor and the presence of T3 receptor RNAs. The post-surgical evolution of the patient was mostly dependent on the size and not on the presence of T3 receptors.Discussion: The presence of thyroid hormone receptors in pituitary tumors is in line with two important characteristics of these tumors: they are histologically benign and well differentiated.

E2F1 Induces Pituitary Tumor Transforming Gene (PTTG1) Expression in Human Pituitary Tumors

Rb/E2F is dysregulated in murine and human pituitary tumors. Pituitary tumor transforming gene (PTTG1), a securin protein, is required for pituitary tumorigenesis, and PTTG1 deletion attenuates pituitary tumor development in Rb(+/-) mice. E2F1 and PTTG1 were concordantly overexpressed in 29 of 46 Rb(+/-) murine pituitary tissues and also in 45 of 80 human pituitary tumors (P < 0.05). E2F1 specifically bound the hPTTG1 promoter as assessed by chromatin immunoprecipitation and biotin-streptavidin pull-down assay, indicating that hPTTG1 may act as a direct E2F1 target. Transfection of E2F1 and its partner DP1 dose-dependently activated hPTTG1 transcription up to 3-fold in p53-devoid H1299 cells but not in p53-replete HCT116 cells. E2F1 overexpression enhanced endogenous hPTTG1 mRNA and protein levels up to 3-fold in H1299 cells. The presence of endogenous p53/p21 constrained the induction, whereas knocking down either p53 or p21 in HCT116 cells restored E2F1-induced hPTTG1 transactivation and expression. Moreover, suppressing Rb by small interfering RNA concordantly elevated E2F1 and hPTTG1 protein levels. In contrast, transfection of E2F1 small interfering RNA lowered hPTTG1 levels 24 h later in HCT116 than in H1299 cells, indicating that p53 delays E2F1 action on hPTTG1. These results elucidate a mechanism for abundant tumor hPTTG1 expression, whereby Rb inactivation releases E2F1 to induce hPTTG1. This signaling pathway may underlie the requirement of PTTG1 for pituitary tumorigenesis.

Proliferative and anti-proliferative effects of dietary levels of phytoestrogens in rat pituitary GH3/B6/F10cells - the involvement of rapidly activated kinases and caspases

BackgroundPhytoestogens are a group of lipophillic plant compounds that can have estrogenic effects in animals; both tumorigenic and anti-tumorigenic effects have been reported. Prolactin-secreting adenomas are the most prevalent form of pituitary tumors in humans and have been linked to estrogen exposures. We examined the proliferative effects of phytoestrogens on a rat pituitary tumor cell line, GH3/B6/F10, originally subcloned from GH3 cells based on its ability to express high levels of the membrane estrogen receptor-α.MethodsWe measured the proliferative effects of these phytoestrogens using crystal violet staining, the activation of several mitogen-activated protein kinases (MAPKs) and their downstream targets via a quantitative plate immunoassay, and caspase enzymatic activities.ResultsFour phytoestrogens (coumestrol, daidzein, genistein, and trans-resveratrol) were studied over wide concentration ranges. Except trans-resveratrol, all phytoestrogens increased GH3/B6/F10 cell proliferation at some concentration relevant to dietary levels. All four phytoestrogens attenuated the proliferative effects of estradiol when administered simultaneously. All phytoestrogens elicited MAPK and downstream target activations, but with time course patterns that often differed from that of estradiol and each other. Using selective antagonists, we determined that MAPKs play a role in the ability of these phytoestrogens to elicit these responses. In addition, except for trans-resveratrol, a serum removal-induced extrinsic apoptotic pathway was blocked by these phytoestrogens.ConclusionPhytoestrogens can block physiological estrogen-induced tumor cell growth in vitro and can also stimulate growth at high dietary concentrations in the absence of endogenous estrogens; these actions are correlated with slightly different signaling response patterns. Consumption of these compounds should be considered in strategies to control endocrine tumor cell growth, such as in the pituitary.

Curcumin acts as anti-tumorigenic and hormone-suppressive agent in murine and human pituitary tumour cells in vitro and in vivo

Curcumin (diferuloylmethane) is the active ingredient of the spice plant Curcuma longa and has been shown to act anti-tumorigenic in different types of tumours. Therefore, we have studied its effect in pituitary tumour cell lines and adenomas. Proliferation of lactosomatotroph GH3 and somatotroph MtT/S rat pituitary cells as well as of corticotroph AtT20 mouse pituitary cells was inhibited by curcumin in monolayer cell culture and in colony formation assay in soft agar. Fluorescence-activated cell sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G2/M. Analysis of cell cycle proteins by immunoblotting showed reduction in cyclin D(1), cyclin-dependent kinase 4 and no change in p27(kip). FACS analysis with Annexin V-FITC/7-aminoactinomycin D staining demonstrated curcumin-induced early apoptosis after 3, 6, 12 and 24 h treatment and nearly no necrosis. Induction of DNA fragmentation, reduction of Bcl-2 and enhancement of cleaved caspase-3 further confirmed induction of apoptosis by curcumin. Growth of GH3 tumours in athymic nude mice was suppressed by curcumin in vivo. In endocrine pituitary tumour cell lines, GH, ACTH and prolactin production were inhibited by curcumin. Studies in 25 human pituitary adenoma cell cultures have confirmed the anti-tumorigenic and hormone-suppressive effects of curcumin. Altogether, the results described in this report suggest this natural compound as a good candidate for therapeutic use on pituitary tumours.

Targeting MAPK (Ras/ERK) and PI3K/Akt pathways in pituitary tumorigenesis

Background: Pituitary adenomas are common intracranial neoplasms, comprising 10 – 15% of all brain tumors. Data from autopsy studies suggest that pituitary adenomas develop in 17 – 25% of the population. Nevertheless, the pathogenesis of sporadic pituitary tumors still remains obscure. Objective: In this review, the roles of MAPK (mainly Ras/extracellular signal-regulated protein kinase (ERK)) and PI3K/Akt signaling pathways in pituitary tumorigenesis are summarised. Methods: A full data search was performed through PubMed over the years 2000 – 2009 with key words ‘pituitary, pituitary tumor, molecular biology, Akt, MAPK, PI3K, ERK’, and all relevant publications have been included, together with selected publications prior to that date. Growth factor receptor mutations and overexpression, G protein mutations, other signaling pathway abnormalities or genetic syndromes associated with pituitary tumors are not discussed as these topics are behind the scope of this review. Conclusions: There are preclinical data and human pituitary tumor studies that are compatible with increased Ras/ERK and/or PI3K/Akt pathway activity in pituitary tumors. Future research focusing on scaffold proteins and signaling modulators regulating these pathways may help identify the initiating transforming events and accordingly new strategies may be developed targeting these pathways in pituitary tumors.

The dopamine–somatostatin chimeric compound BIM-23A760 exerts antiproliferative and cytotoxic effects in human non-functioning pituitary tumors by activating ERK1/2 and p38 pathways

The study investigated the effects of the dopamine-somatostatin chimeric compound BIM-23A760 on cell proliferation and apoptosis in cultured cells from human non-functioning pituitary tumors (NFPTs). Both BIM-23A760 and the dopaminergic agonist BIM-53097 induced a significant inhibition of cell proliferation associated with increased p27 expression, together with a significant increase in caspase-3 activity. Conversely, null or marginal effects were elicited by somatostatin analogs. Moreover, BIM-23A760 and BIM-53097 induced ERK1/2 and p38 phosphorylation and the blockade of these pathways prevented both the antiproliferative and the pro-apoptotic effects of these drugs. In conclusions the chimeric compound BIM-23A760 is able to exert cytostatic and cytotoxic effects in NFPTs, these phenomena being mainly mediated by DR2D and involving ERK1/2 and p38 pathways activation.

Recent advances in the molecular biology of growth-hormone secreting human pituitary tumours.

The application of DNA technology has led to significant progress in our understanding of the aetiology of GH-secreting pituitary tumours. X-chromosome inactivation and RFLP analysis has revealed that GH-secreting tumours are monoclonal in origin and thus arise as a consequence of a somatic mutation within a single cell. Using PCR and sequence analysis, such a mutation has been identified in 30-40% of tumours. This is the so-called gsp mutation in which the gene for the alpha s subunit of the Gs protein is converted to an oncogene the expression of which results in constitutive adenyl cyclase activity and thus excessive cAMP production. In our own studies, we demonstrate that it is unlikely that a defect within the promoter region of the GH gene will prove to be a cause of excessive GH secretion in acromegaly. In contrast, we have shown that the GH gene is hypomethylated in tumour derived DNA and this may account, at least in part, for abnormal GH gene expression.

Lanreotide promotes apoptosis and is not radioprotective in GH3 cells

Somatostatin analogs are a mainstay of medical therapy in patients with GH producing human pituitary tumors, and it has been suggested that somatostatin analogs may be radioprotective. We utilized GH secreting rat GH3 cells to investigate whether a somatostatin analog may limit the effects of radiation on proliferation and apoptosis in vitro and on tumor growth in vivo. Treatment with lanreotide alone at doses of either 100 or 1000 nM for 48 h reduced clonogenic survival by 5-10%. Radiation alone produced a dose-dependent survival curve with a SF2 of 48-55%, and lanreotide had no effect on this curve. The addition of lanreotide resulted in a 23% increase in the proportion of apoptotic sub-G1 cells following irradiation (P<0.01). In a mouse GH3 tumor xenograft model, lanreotide 10 mg/kg moderately inhibited the growth of GH3 tumors, with a 4x tumor growth delay (TGD) time that ranged from 4.5 to 8.3 days. Fractionated local tumor radiation alone significantly inhibited tumor growth and produced a TGD of 35.1+/-5.7 days for 250 cGy fractions. The combination of lanreotide, either antecedent to or concurrent, with radiation of 250, 200 or 150 cGy/fraction for 5 days inhibited tumor growth and produced the TGD times that were similar to radiation alone (P>0.05). Pretreatment with lanreotide had the most significant radiosensitizing effect. These studies demonstrate that the somatostatin analog lanreotide is not radioprotective in GH3 cells, and further studies are necessary to determine the impact of lanreotide on apoptosis.

Histone acetyltransferase p300 regulates the expression of human pituitary tumor transforming gene (hPTTG)

The human pituitary tumor transforming gene (hPTTG) serves as a marker for malignancy grading in several cancers. hPTTG is involved in multiple cellular pathways including cell transformation, apoptosis, DNA repair, genomic instability, mitotic control and angiogenesis induction. However, the molecular mechanisms underlying hPTTG regulation have not been fully explored. In this study, we found that overexpression of histone acetyltransferase (HAT) p300 upregulated hPTTG at the levels of promoter activity, mRNA and protein expression. Moreover, the HAT activity of p300 was critical for its regulatory function. Chromatin immunoprecipitation (ChIP) analysis revealed that overexpression of p300 elevated the level of histone H3 acetylation on the hPTTG promoter. Additionally, the NF-Y sites at the hPTTG promoter exhibited a synergistic effect on upregulation of hPTTG through interacting with p300. We also found that treatment of 293T cells with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) increased hPTTG promoter activity. Meanwhile, we provided evidence that HDAC3 decreased hPTTG promoter activity. These data implicate an important role of the histone acetylation modification in the regulation of hPTTG.

Expression of p18INK4C is Down-regulated in Human Pituitary Adenomas

Cyclin-dependent kinase inhibitors represented by the INK4 family comprising p16(INK4A), p15(INK4B), p18(INK4C), and p19(INK4D) are regulators of the cell cycle shown to be aberrant in many types of cancer. Mice lacking p18(Ink4c) exhibit a series of phenotypes including the development of widespread organomegaly and pituitary adenomas. The objective of our study is to examine the role of p18(INK4C) in the pathogenesis of human pituitary tumors. The protein and mRNA levels of p18(INK4C) were examined by immunohistochemistry and real-time reverse transcription-polymerase chain reaction, respectively. The methylation status of the p18(INK4C) gene promoter and somatic mutations of the p18(INK4C) gene were also investigated. p18(INK4C) protein expression was lost or significantly reduced in 64% of pituitary adenomas compared with levels in normal pituitary glands. p18(INK4C) mRNA levels were low in all ACTH adenomas and non-functioning (NF)-FSH and in 42%, 70% and 66% of GH, PRL, and subtype 3 adenomas, respectively. p18(INK4C) mRNA levels were significantly associated with p18(INK4C) protein levels. Neither methylated promoters in pituitary adenomas, except in one NF-FSH adenoma, nor somatic mutations of the p18(INK4C) gene in any pituitary adenomas were detected. The down-regulation of p18(INK4C) expression may contribute to the tumorigenesis of pituitary adenomas.

Identification and Characterization of Two Novel Truncated but Functional Isoforms of the Somatostatin Receptor Subtype 5 Differentially Present in Pituitary Tumors

Somatostatin and its related peptide cortistatin exert multiple actions on normal and tumoral tissue targets through a family of receptors termed somatostatin receptor (sst)1-5. Despite the considerable advances in the knowledge on these receptors and their (patho)physiological roles, there is still evidence that additional receptors for these peptides should exist to fully explain their actions. The growing number of spliced variants found in similar receptor families, often present in tumors, and results from our group obtained on sst5 from other species (pig) led us to explore the existence of new human sst5 isoforms. A rapid amplification of cDNA ends PCR approach on samples from a human pituitary tumor and a cell line enabled identification of two novel alternatively spliced sst5 receptor variants. The sequences obtained encode putative proteins that correspond to truncated isoforms of five and four transmembrane domains (TMDs), accordingly named sst5TMD5 and sst5TMD4, respectively. Both novel receptors show a differential expression pattern in normal tissues and are also present in pituitary tumors of diverse etiology including nonfunctioning adenomas, corticotropinomas, somatotropinomas, and a prolactinoma. In contrast to the predominant plasma membrane localization of full-length sst5, both sst5TMD5 and sst5TMD4 show a preferentially intracellular localization. Despite their truncated nature, both receptors are functional, as shown by their ability to mediate selective, ligand-induced rises in free cytosolic calcium concentration. Specifically, whereas sst5TMD5 is selectivity activated by somatostatin compared with cortistatin, cells transfected with sst5TMD4 almost exclusively respond to cortistatin and not to somatostatin. Our results demonstrate the existence of two previously unidentified sst5 spliced variants with distinct distribution in normal tissues and pituitary tumors, unique ligand-selective signaling properties, and subcellular distribution, which could contribute to somatostatin and cortistatin signaling in normal and tumoral cells.

Ultrastructure of mitochondria in various human pituitary tumor types

The mitochondria, the major powerhouse for cellular functions, are predominantly ovoid or rod‐shaped cytoplasmic organelles enclosed by double outer membrane. The cristae, are formed by the infoldings of the inner membranes. Morphologic variations in size, shape, number of cristae exist, attuned to the function of the resident organ. Specific ultrastructural features characteristic of certain tumor types may occur. Such mitochondrial alterations were recorded by EM in more than 5000 various types of surgically removed pituitary adenomas. Changes are chiefly quantitative: A) size and density of internal structures (large/small; active/functionally suppressed prolactinoma); B) increase in number, i.e. oncocytic change. Generally occurring within the endocrine system owing to functional decline. Most prevalent in gonadotroph adenomas lineage. Specific alterations: A) giant mitochondria: specific marker of acidophil stem cell adenoma. With preservation of the double membranes, progressive enlargement and disappearance of the internal compartment and occurrence of unidentified tubular structures take place; B) Mitochondrial enlargement and homogenization of the entire internal compartment evident only in gonadotroph adenoma. C) Close spatial association of mitochondria and RER. It occurs only in silent adenoma subtype 3. The specific alterations may be used as ultrastructural markers.

RUNX1 and RUNX2 upregulate Galectin-3 expression in human pituitary tumors

Galectin-3 is expressed in a cell-type specific manner in human pituitary tumors and may have a role in pituitary tumor development. In this study, we hypothesized that Galectin-3 is regulated by RUNX proteins in pituitary tumors. Transcription factor prediction programs revealed several putative binding sites in the LGALS3 (Galectin-3 gene) promoter region. A human pituitary cell line HP75 was used as a model to study LGALS3 and RUNX interactions using Chromatin immunoprecipitation assay and electrophoresis mobility shift assay. Two binding sites for RUNX1 and one binding site for RUNX2 were identified in the LGALS3 promoter region. LGALS3 promoter was further cloned into a luciferase reporter, and the experiments showed that both RUNX1 and RUNX2 upregulated LGALS3. Knock-down of either RUNX1 or RUNX2 by siRNA resulted in a significant downregulation of Galectin-3 expression and decreased cell proliferation in the HP 75 cell line. Immunohistochemistry showed a close correlation between Galectin-3 expression and RUNX1/RUNX2 level in pituitary tumors. These results demonstrate a novel binding target for RUNX1 and RUNX2 proteins and suggest that Galectin-3 is regulated by RUNX1 and RUNX2 in human pituitary tumor cells by direct binding to the promoter region of LGALS3 and thus may contribute to pituitary tumor progression.

P21 Cip1 restrains pituitary tumor growth

As commonly encountered, pituitary adenomas are invariably benign. We therefore studied protective pituitary proliferative mechanisms. Pituitary tumor transforming gene (Pttg) deletion results in pituitary p21 induction and abrogates tumor development in Rb(+/-)Pttg(-/-) mice. p21 disruption restores attenuated Rb(+/-)Pttg(-/-) pituitary proliferation rates and enables high penetrance of pituitary, but not thyroid, tumor growth in triple mutant animals (88% of Rb(+/-) and 72% of Rb(+/-)Pttg(-/-)p21(-/-) vs. 30% of Rb(+/-)Pttg(-/-) mice developed pituitary tumors, P < 0.001). p21 deletion also accelerated S-phase entry and enhanced transformation rates in triple mutant MEFs. Intranuclear p21 accumulates in Pttg-null aneuploid GH-secreting cells, and GH(3) rat pituitary tumor cells overexpressing PTTG also exhibited increased levels of mRNA for both p21 (18-fold, P < 0.01) and ATM (9-fold, P < 0.01). PTTG is abundantly expressed in human pituitary tumors, and in 23 of 26 GH-producing pituitary adenomas with high PTTG levels, senescence was evidenced by increased p21 and SA-beta-galactosidase. Thus, either deletion or overexpression of Pttg promotes pituitary cell aneuploidy and p53/p21-dependent senescence, particularly in GH-secreting cells. Aneuploid pituitary cell p21 may constrain pituitary tumor growth, thus accounting for the very low incidence of pituitary carcinomas.

Cell cycle control of pituitary development and disease

The pituitary gland regulates diverse physiological functions, including growth, metabolism, reproduction, stress response, and ageing. Early genetic models in the mouse taught us that the pituitary is highly sensitive to genetic alteration of specific cell cycle regulators such as the retinoblastoma protein (pRB) or the cell cycle inhibitor p27(Kip1). The molecular analysis of human pituitary neoplasias has now corroborated that cell cycle deregulation is significantly implicated in pituitary tumorigenesis. In particular, proteins involved in cyclin-dependent kinase regulation or the pRB pathway are altered in nearly all human pituitary tumors. Additional cell cycle regulators such as PTTG1/securin may have critical roles in promoting genomic instability in pituitary neoplasias. Recent experimental data suggest that these cell cycle regulators may have significant implications in the biology of putative progenitor cells and pituitary homeostasis. Understanding how cell cycle regulation controls pituitary biology may provide us with new therapeutic approaches against pituitary diseases.