Pituitary Cell Growth Research Articles

FRI291 Musashi1 And Musashi2 Mark Distinct Pituitary Stem Cell Populations

Abstract Disclosure: Y. Rahmatallah: None. J. Banik: None. K. Bronson: None. M.C. MacNicol: None. The cell plastic state of the pituitary is required to meet the changing demands upon pituitary function and may also result in these cells being more vulnerable to malignant transformation. The mechanisms that control pituitary cell plasticity are unknown. The RNA binding protein Musashi (MSI) is expressed in embryonic stem cells, and in cancer stem cells, where MSI functions to promote stem cell plasticity and to oppose premature cell maturation. MSI is also expressed at high levels in adult gonad and adult pituitary. The function of MSI in adult tissues is not fully understood, and the mechanisms that control the pro-proliferative and pro-malignant aspects of MSI function in adult tissues are not known. The two evolutionarily conserved MSI proteins, Musashi1 (MSI1) and Musashi2 (MSI2), are predicted to function redundantly, however, regulatory distinctions have been reported for MSI1 and MSI2. Towards a characterization of MSI1 and MSI2 in pituitary, we analyzed transcriptomics data from public datasets of single-nucleus and single-cell and bulk-tissue gene expression data from physiological and pathophysiological human pituitary tissue. We performed bioinformatics analysis utilizing the single-cell genomics analysis tool, Seurat in R and the pseudotime trajectory analysis tool Monocle and additional genomics and statistics analyses tools in R. We identify the MSI1-expressing pituitary cell population as immature stem cells and the distinct MSI2-expressing pituitary cell population as more mature, committed progenitor cells. Pituitary from human fetal tissue contains multiple distinct MSI1-expressing cell populations while, in contrast, pituitary from human adult tissue contains multiple distinct MSI2-expressing cell populations. Pituitary from human pediatric tissue demonstrates an intermediate state with both distinct MSI1-expressing and distinct MSI2-expressing cell populations. We identify increased MSI1 expression but not increased MSI2 expression in adult pituitary adenoma, and a high level of variation in the MSI1 to MSI2 expression ratio between distinct pituitary adenomas. Based upon this re-analysis of reposited datasets from other pituitary investigators, we hypothesize that the increase that we observe, in MSI1 to MSI2 expression ratio in adult pituitary tumors is consistent with a reversion of mature cells to an earlier immature state. Adult pituitary may contain the mechanisms that regulate MSI2 function but may not be equipped to properly control high levels of re-expressed MSI1. An understanding of the physiological functions and mechanisms of regulation of the distinct MSI proteins in pituitary may provide insight into potential mechanisms that could be recruited to increase physiological pituitary cell growth and differentiation in regenerative therapy and to oppose pathological cell growth in pituitary tumor. Presentation: Friday, June 16, 2023

Ultrastructural and Molecular Evidence of Macroautophagy in Functioning PitNETs and Experimental Pituitary Tumors

Introduction: Macroautophagy is a lysosome-mediated degradation process that controls the quality of cytoplasmic components and organelles, with its regulation depending on autophagy-related proteins (Atg) and with Beclin1/Atg6 and microtubule-associated protein light chain 3 (LC3/Atg8) being key players in the mammalian autophagy. As reports on this mechanism in the field of pituitary neuropathology and neuroendocrinology are scarce, our study analyzed the ultrastructural signs of macroautophagy and the expression of Beclin1 and LC3 proteins in human functioning PitNETs and in experimental pituitary tumors. Methods: A group of humans functioning PitNETs and an experimental lactotroph model in rats of the F344 strain stimulated with estradiol benzoate (BE) were used. Ultrastructural and molecular evidence of the macroautophagic process was evaluated using different techniques. Results: In functioning PitNETs cohort, 60% exhibited evidence of macroautophagy, with a significant difference found for Beclin1 and LC3 between macro- and micro-PitNETs (p < 0.05). In the experimental model, the expression of both Beclin1 and LC3 proteins was immunopositive in normal and tumoral glands when analyzed by immunofluorescence, Western blot, and immunohistochemistry. In the experimental model, protein expression was associated with increased glandular size and weight. Conclusions: Our study revealed evidence of macroautophagy at the pituitary level and the important role of Beclin1 and LC3 in the progression of functioning PitNETs, implying that this mechanism participate in regulating pituitary cell growth.

Inhibition of GH3 and HEK293 pituitary adenoma cell growth by Trigonella foenum-graecum extract (TFGE) via mTORC1 down-regulation

The present study investigated the effect of Trigonella foenum-graecum extract (TFGE) on GH3 and HEK293 pituitary adenoma cell growth in vitro in mice model in vivo and evaluated the underlying mechanism. TFGE exposure in dose-dependent manner suppressed the viability of GH3 and HP75 cells at 5–30 µM concentrations. Treatment of GH3 and HP75 cells with 30 µM TFGE inhibited colony formation significantly (P < .05) compared to the un-exposed cells. In GH3 and HP75 cells, TFGE exposure suppressed invasion potential significantly (P < .05) at 5 and 30 µM concentrations. TFGE treatment at 5 and 30 µM for 72 h significantly (P < .05) elevated induction of apoptosis. The H19 expression was significantly (P < .05) inhibited by TFGE-treatment in GH3 and HEK293 cells. TFGE treatment at 2.5 and 10 mg/kg doses led to a significantly (P < .05) suppression in the tumor growth in HP75 cell-implanted mice. TFGE treatment of GH3 and HP75 cells at 5 and 30 µM down-regulated 4E-BP1 activation. TFGE treatment inhibits GH3 and HEK293 cell viability and inhibits tumor development. It suppresses H19 expression and upregulates 4E-BP1 phosphorylation in GH3 and HEK293 cells. Therefore, TFGE may be used for the treatment of pituitary adenoma.

Inhibition of lncRNA-UCA1 suppresses pituitary cancer cell growth and prolactin (PRL) secretion via attenuating glycolysis pathway.

Pituitary tumor is one type of endocrine tumor with high incidence and mortality rates. Long non-coding RNAs (lncRNAs) are a family of non-coding RNAs with longer than 200 nucleotides. Among them, lncRNA-UCA1 is highly expressed in multiple cancers and plays critically oncogenic roles in tumor progressions. However, the potential roles of UCA1 in human pituitary tumor have not been elucidated. In this study, the expressions of lncRNA-UCA1 were analyzed in thirty pituitary tumor samples and thirty normal pituitary tissues. Cancer cell glycolysis rate was examined by glucose uptake and lactate production. The lncRNA-UCA1 expression was detected by qRT-PCR. Glycolysis enzyme expressions were measured by Western blot and qRT-PCR. Consistent with other cancers, lncRNA-UCA1 was highly expressed in pituitary tumors. Meanwhile, we found glycolysis of pituitary tumors was higher than normal pituitary tissues. Overexpression of lncRNA-UCA1 in rat pituitary cancer cell lines, GH3 and MMQ, significantly promoted glucose uptake and lactate production. In addition, expressions of the glycolysis key enzymes, HK2 and LDHA, were significantly upregulated by exogenous overexpression of lncRNA-UCA1. Importantly, silencing lncRNA-UCA1 obviously inhibited pituitary cancer cells growth and prolactin (PRL) secretion. We report higher lncRNA-UCA1 expression is associated with higher serum PRL level in pituitary patients. Finally, by blocking the lncRNA-UCA1-promoted glycolysis of pituitary cancer cells by glycolysis inhibitor, 2-DG, we obtained recovery of cell growth rate and PRL secretion from an in vitro model. Taken together, our investigation revealed an oncogenic role of lncRNA-UCA1 through upregulating glycolysis of pituitary tumors. This study contributes to underlying molecular mechanisms of the tumorigenesis of pituitary tumors.

The phthalate DEHP modulates the estrogen receptors α and β increasing lactotroph cell population in female pituitary glands

Humans are exposed to numerous endocrine disruptors on a daily basis, which may interfere with endogenous estrogens, with Di-(2-ethylhexyl) phthalate (DEHP) being one of the most employed. The anterior pituitary gland is a target of 17β-estradiol (E2) through the specific estrogen receptors (ERs) α and β, whose expression levels fluctuate in the gland under different contexts, and the ERα/β index is responsible for the final E2 effect. The aim of the present study was to evaluate in vivo and in vitro the DEHP effects on ERα and β expression in the pituitary cell population, and also its impact on lactotroph and somatotroph cell growth.Our results revealed that perinatal exposure to DEHP altered the ERα and β expression pattern in pituitary glands from prepubertal and adult female rats and increased the percentage of lactotroph cells in adulthood. In the in vitro system, DEHP down-regulated ERα and β expression, and as a result increased the ERα/β ratio and decreased the percentages of lactotrophs and somatotrophs expressing ERα and β. In addition, DEHP increased the S + G2M phases, Ki67 index and cyclin D1 in vitro, leading to a rise in the lactotroph and somatotroph cell populations. These results showed that DEHP modified the pituitary ERα and β expression in lactotrophs and somatotrophs from female rats and had an impact on the pituitary cell growth. These changes in ER expression may be a mechanism underlying DEHP exposure in the pituitary gland, leading to cell growth deregulation.

The ERα membrane pool modulates the proliferation of pituitary tumours.

The molecular mechanisms underlying the ERα nuclear/cytoplasmic pool that modulates pituitary cell proliferation have been widely described, but it is still not clear how ERα is targeted to the plasma membrane. The aim of this study was to analyse ERα palmitoylation and the plasma membrane ERα (mERα) pool, and their participation in E2-triggered membrane-initiated signalling in normal and pituitary tumour cell growth. Cell cultures were prepared from anterior pituitaries of female Wistar rats and tumour GH3 cells, and treated with 10 nM of oestradiol (E2). The basal expression of ERα was higher in tumour GH3 than in normal pituitary cells. Full-length palmitoylated ERα was observed in normal and pituitary tumour cells, demonstrating that E2 stimulation increased both, ERα in plasma membrane and ERα and caveolin-1 interaction after short-term treatment. In addition, the Dhhc7 and Dhhc21 palmitoylases were negatively regulated after sustained stimulation of E2 for 3 h. Although the uptake of BrdU into the nucleus in normal pituitary cells was not modified by E2, a significant increase in the GH3 tumoural cell, as well as ERK1/2 activation, with this effect being mimicked by PPT, a selective antagonist of ERα. These proliferative effects were blocked by ICI 182780 and the global inhibitor of palmitoylation. These findings indicate that ERα palmitoylation modulated the mERα pool and consequently the ERK1/2 pathway, thereby contributing to pituitary tumour cell proliferation. These results suggest that the plasma membrane ERα pool might be related to the proliferative behaviour of prolactinoma and may be a marker of pituitary tumour growth.

Effects of metformin on cell growth and AMPK activity in pituitary adenoma cell cultures, focusing on the interaction with adenylyl cyclase activating signals.

For a few years we have been investigating AMP-activated protein kinase (AMPK) as a target for drug therapy of GH-secreting pituitary adenomas. Aim of this study was to investigate the direct effects of metformin, which causes AMPK activation in different cell types, on rat pituitary adenoma cell growth and on related cell signalling pathways. Our results suggest that metformin can exert a growth-inhibitory activity in rat pituitary tumor cells mediated by AMPK activation, although multiple mechanisms are most likely involved. Membrane proteins, including growth factor receptors, are valuable targets of AMPK. The inhibition of the mTOR-p70S6 kinase signalling pathway plays a role in the suppressive effect of metformin on pituitary tumor cell growth. Metformin did not affect the MTT reduction activity in energetic stress conditions. Finally, metformin was still able to induce AMPK activation and to inhibit cell growth in cells treated with forskolin and in transfected cells overexpressing GHRH-receptor and treated with GHRH. Hence, adenylyl cyclase over-activation does not account for the lack of response of some human pituitary tumors to AMPK-activating compounds invitro.

Estrogen receptor β regulates the tumoral suppressor PTEN to modulate pituitary cell growth.

In this study, we focused on ERβ regulation in the adenohypophysis under different estrogenic milieu, by analyzing whether ER modulates the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression and its subcellular localization on anterior pituitary glands from Wistar rats and GH3 lactosomatotroph cells that over-expressed ERβ. ERβ was regulated in a cyclic manner, and underwent dynamic changes throughout the estrous cycle, with decreased ERβ+ cells in estrus and under E2 treatment, but increased in ovariectomized rats. In addition, the ERα/β ratio increased in estrus and under E2 stimulation, but decreased in ovariectomized rats. Double immunofluorescence revealed that lactotroph and somatotroph ERβ+ were significantly decreased in estrus. Also, variations in the PTEN expression was observed, which was diminished with high E2 conditions but augmented with low E2 milieu. The subcellular localization of this phosphatase was cell cycle-dependent, with remarkable changes in the immunostaining pattern: nuclear in arrested pituitary cells but cytoplasmic in stimulated cells, and responding differently to ER agonists, with only DPN being able to increase PTEN expression and retaining it in the nucleus. Finally, ERβ over-expression increased PTEN with a noticeable subcellular redistribution, and with a significant nuclear signal increase in correlation with an increase of cells in G0/G1 phase. These results showed that E2 is able to inhibit ERβ expression and suggests that the tumoral suppressor PTEN might be one of the signaling proteins by which E2, through ERβ, acts to modulate pituitary cell proliferation, thereby adapting endocrine populations in relation with hormonal necessities.

Programmed cell senescence: role of IL-6 in the pituitary.

IL-6 is a pleiotropic cytokine with multiple pathophysiological functions. As a key factor of the senescence secretome, it can not only promote tumorigenesis and cell proliferation but also exert tumor suppressive functions, depending on the cellular context. IL-6, as do other cytokines, plays important roles in the function, growth and neuroendocrine responses of the anterior pituitary gland. The multiple actions of IL-6 on normal and adenomatous pituitary function, cell proliferation, angiogenesis and extracellular matrix remodeling indicate its importance in the regulation of the anterior pituitary. Pituitary tumors are mostly benign adenomas with low mitotic index and rarely became malignant. Premature senescence occurs in slow-growing benign tumors, like pituitary adenomas. The dual role of IL-6 in senescence and tumorigenesis is well represented in pituitary tumor development, as it has been demonstrated that effects of paracrine IL-6 may allow initial pituitary cell growth, whereas autocrine IL-6 in the same tumor triggers senescence and restrains aggressive growth and malignant transformation. IL-6 is instrumental in promotion and maintenance of the senescence program in pituitary adenomas.

FGFR4 polymorphic alleles modulate mitochondrial respiration: A novel target for somatostatin analog action in pituitary tumors

We reported that a single nucleotide polymorphism (SNP) at codon 388 of the fibroblast growth factor receptor 4 (FGFR4-Gly388Arg) can result in distinct proteins that alter pituitary cell growth and function. Here, we examined the differential properties of the available therapeutic somatostatin analogs, octreotide and pasireotide, in pituitary tumor cells expressing the different FGFR4 isoforms. Consistent with their enhanced growth properties, FGFR4-R388-expressing cells show higher mitochondrial STAT3 serine phosphorylation driving basal and maximal oxygen consumption rate (OCR) than pituitary cells expressing the more common FGFR4-G388 isoform. While both somatostatin analogs reduce the OCR in FGFR4-G388 cells, pasireotide was more effective in decreasing OCR in cells expressing the variant FGFR4-R388 isoform. Down-regulation of somatostatin receptor 5 (SSTR5) abrogated the effect of pasireotide, demonstrating its involvement in mediating this action. The effects on OCR were recapitulated by introducing a constitutively active serine STAT3 but not by a tyrosine-active mutant. Moreover, pharmacologic inhibition demonstrated the role for the phosphatase PP2A in mediating the dephosphorylation of STAT3-S727 by pasireotide. Our data indicate that FGFR4 polymorphic isoforms mediate signaling that yields mitochondrial therapeutic targets of relevance to the actions of different somatostatin analogs.

Protein Kinase C Delta restrains growth in ACTH-secreting pituitary adenoma cells

Protein Kinase C Delta (PRKCD) has been highlighted among disrupted pathways in corticotroph adenomas. PRKCD is expressed at low level in human corticotroph adenomas and controls cell cycle in vitro. Therefore, PRKCD may play an important role in the development/progression of corticotroph adenomas, warranting further studies to understand the role of PRKCD and related pathways in restraining pituitary cell growth. We evaluated PRKCD role in influencing cell behavior in terms of cell viability, hormone expression and protein expression profile, by silencing PRKCD in AtT-20/D16v-F2 cells. PRKCD silencing increases cell viability, enhances hormone expression and induces morphological changes associated with deregulation of adhesion molecules. PRKCD silencing is associated with an increase in Epithelial Growth Factor Receptor (EGFR) expression, a marker of tumor aggressive behavior, and sensitivity to anti-EGFR molecules. PRKCD might restrain corticotroph adenoma cells from acquiring an aggressive behavior, candidating PRKCD as a possible molecular target for the treatment of corticotroph adenomas.

Abstract LB-303: Identification of a mammalian sterile-20 like kinase 4 (MST4) inhibitor to target pituitary tumorigenesis

Abstract Pituitary tumors of the gonadotrope lineage are often large and invasive, resulting in hypopituitarism. There are no medical treatments currently available. Using a combined genetic and genomic screen of human pituitary tumor samples, we identified the Mammalian sterile-20 like kinase 4 (MST4) as a pro-tumorigenic effector, driving increased pituitary cell proliferation and survival in response to a hypoxic microenvironment. MST4 is a member of the germinal center kinase III subfamily of Ste-20 kinases with limited data available concerning its role in tumorigenesis. To identify selective inhibitors of the MST4 kinase, computational-based virtual library screening was employed to dock the commercially available SelleckChem kinase inhibitor library into the ATP binding site of the MST4 crystal structure. Several candidate MST4 kinase inhibitors were identified with the potential to bind with high affinity. Using a newly developed TRFRET in vitro kinase assay, one of the small molecule compounds exhibited a potent inhibition of the MST4 kinase (IC50 = 6.18 nM +/- 2.2 nM). To determine the ability of this inhibitor to antagonize MST4 actions, a hypoxic LβT2 gonadotrope pituitary cell model was used. Under chronic hypoxia (5% O2), cells stably overexpressing MST4 showed an increased rate of proliferation (at 7 days, 3.16-fold, p = 0.006) compared to vector control cells as assessed by BrdU staining. These effects were completely blocked by the inhibitor at 50nM (-2.61-fold, p = 0.01, NS from controls). Similarly, MST4 protection from hypoxia induced apoptosis under 1% O2 for 17h was also inhibited at the same dose as assessed by TUNEL staining (-2.72-fold, p&amp;lt;0.001, NS from controls). Analysis of transfectants expressing wildtype and Mst4-mutants (K53E-kinase dead, T178A-loss of autophosphorylation, and C-terminus deletion) confirmed the inhibitory effect was dependent on the kinase sequence. Together, these data identify a potent inhibitor of the MST4 kinase with the capacity to selectively block pituitary cell growth under a hypoxic microenvironment and support future studies of its ability to modulate pituitary tumor growth in rodent models. Citation Format: Weipeng Xiong, Chris Matheson, Donald S. Backos, Mei Xu, Katja Kiseljak-Vassiliades, Smita Salian-Mehta, Philip Reigan, Margaret E. Wierman. Identification of a mammalian sterile-20 like kinase 4 (MST4) inhibitor to target pituitary tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2015-LB-303

Evidence of cellular senescence during the development of estrogen-induced pituitary tumors.

Although pituitary adenomas represent 25% of intracranial tumors, they are usually benign, with the mechanisms by which these tumors usually avoid an invasive profile and metastatic growth development still remaining unclear. In this context, cellular senescence might constitute a plausible explanation for the benign nature of pituitary adenomas. In this study, we investigated the emergence of cellular senescence as a growth control mechanism during the progression of estrogen-induced pituitary tumors. The quantification of Ki67-immunopositive cells in the pituitaries of estrogenized male rats after 10, 20, 40, and 60 days revealed that the mitogenic potential rate was not sustained for the whole period analyzed and successively decreased after 10 days of estrogen exposure. In addition, the expression of cellular senescence features, such as the progressive rise in the enzymatic senescence-associated b-galactosidase (SA-b-gal) activity, IL6, IL1b, and TGFb expression, was observed throughout pituitary tumor development. Furthermore, tumoral pituitary cells also displayed nuclear pATM expression, indicating activated DNA damage signaling, with a significant increase in p21 expression also being detected. The associations among DNA damage signaling activation, SA-b-gal expression, and p21 may provide a reliable combination of senescence-associated markers for in vivo pituitary senescence detection. These results suggest a role for this cellular process in the regulation of pituitary cell growth. Thus, cellular senescence should be conceived as a contributing component to the benign nature of pituitary adenomas, thereby influencing the capability of the pituitary gland to avoid unregulated cell proliferation.

Interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK) and the estrogen receptor activities in regulating rat pituitary tumor cell (GH3) growth in vitro

Estrogen receptor α has a role in regulating rat somatolactotroph tumor cell growth (GH3 cells). AMP-activated protein kinase (AMPK) is a metabolic checkpoint which is able to negatively regulate intracellular signaling downstream of growth factors receptors in conditions increasing cellular AMP levels. We have recently reported on the role of AMPK activation in affecting viability and proliferation of GH3 cells. In the present study, we investigated the interplay between ER- and AMPK-pathways. Results can be regarded as relevant to the development of novel multi-targeted pharmacological therapies against pituitary tumors. We confirmed that estradiol (E2) and the ER antagonist fulvestrant exert stimulatory and inhibitory effects, respectively on GH3 cell growth in a competitive manner. The upstream kinase LKB1 is known to phosphorylate and activate AMPK. Here we showed that neither E2 nor fulvestrant caused a downregulation of LKB1 expression and phospho-AMPK levels in GH3 cells. Actually, fulvestrant strongly reduced the phosphorylation of ACC, which is a direct target of AMPK and a known index of AMPK activity. 2-deoxyglucose, a compound reducing glucose utilization, caused an increase in AMPK activity vs baseline and was able to hinder the stimulatory effect of E2 on cell viability, confirming that the exposure of GH3 cells to estrogens does not prevent them from being responsive to the inhibitory activity of compounds activating AMPK. Finally, the AMPK activator AICAR (AMP analog) did not cause further decrease in cell viability in the course of co-treatments with fulvestrant versus fulvestrant alone, in agreement with impaired phospho-AMPK activity in the presence of the anti-estrogen.

The FGFR4-G388R Polymorphism Promotes Mitochondrial STAT3 Serine Phosphorylation to Facilitate Pituitary Growth Hormone Cell Tumorigenesis

Pituitary tumors are common intracranial neoplasms, yet few germline abnormalities have been implicated in their pathogenesis. Here we show that a single nucleotide germline polymorphism (SNP) substituting an arginine (R) for glycine (G) in the FGFR4 transmembrane domain can alter pituitary cell growth and hormone production. Compared with FGFR4-G388 mammosomatotroph cells that support prolactin (PRL) production, FGFR4-R388 cells express predominantly growth hormone (GH). Growth promoting effects of FGFR4-R388 as evidenced by enhanced colony formation was ascribed to Src activation and mitochondrial serine phosphorylation of STAT3 (pS-STAT3). In contrast, diminished pY-STAT3 mediated by FGFR4-R388 relieved GH inhibition leading to hormone excess. Using a knock-in mouse model, we demonstrate the ability of FGFR4-R385 to promote GH pituitary tumorigenesis. In patients with acromegaly, pituitary tumor size correlated with hormone excess in the presence of the FGFR4-R388 but not the FGFR4-G388 allele. Our findings establish a new role for the FGFR4-G388R polymorphism in pituitary oncogenesis, providing a rationale for targeting Src and STAT3 in the personalized treatment of associated disorders.

Lineage-Specific Restraint of Pituitary Gonadotroph Cell Adenoma Growth

Although pituitary adenomas are usually benign, unique trophic mechanisms restraining cell proliferation are unclear. As GH-secreting adenomas are associated with p53/p21-dependent senescence, we tested mechanisms constraining non-functioning pituitary adenoma growth. Thirty six gonadotroph-derived non-functioning pituitary adenomas all exhibited DNA damage, but undetectable p21 expression. However, these adenomas all expressed p16, and >90% abundantly expressed cytoplasmic clusterin associated with induction of the Cdk inhibitor p15 in 70% of gonadotroph and in 26% of somatotroph lineage adenomas (p = 0.006). Murine LβT2 and αT3 gonadotroph pituitary cells, and αGSU.PTTG transgenic mice with targeted gonadotroph cell adenomas also abundantly expressed clusterin and exhibited features of oncogene-induced senescence as evidenced by C/EBPβ and C/EBPδ induction. In turn, C/EBPs activated the clusterin promoter ∼5 fold, and elevated clusterin subsequently elicited p15 and p16 expression, acting to arrest murine gonadotroph cell proliferation. In contrast, specific clusterin suppression by RNAis enhanced gonadotroph proliferation. FOXL2, a tissue-specific gonadotroph lineage factor, also induced the clusterin promoter ∼3 fold in αT3 pituitary cells. As nine of 12 pituitary carcinomas were devoid of clusterin expression, this protein may limit proliferation of benign adenomatous pituitary cells. These results point to lineage-specific pathways restricting uncontrolled murine and human pituitary gonadotroph adenoma cell growth.

Utilization of cell systems

Macroscopic observations at organism level provide with crucial information about the impact of different hormonal systems in organism homeostasis and wellbeing and how they can be altered during certain experimental manipulations. However they do not readily provide with information about the events taking place inside endocrine cells that lead to the observed hormonal responses. Unravelling the intracellular events underlying the physiology of these systems at molecular and genetic level is of outmost importance in order to understand the pathogenesis of endocrine disorders and to device improved treatments. To this end, cultured endocrine cells provide with a direct and efficient tool that enables to dissect the intracellular pathways and genetic events triggered by and leading to certain hormonal stimuli. During this talk it will be shown how data derived from pituitary cells in primary cell culture or as immortalized cell lines provided with insights in the transcriptional regulation of pituitary (pre)hormone synthesis in response to organism and environmental stimuli; highlighted the importance of certain pathways in the regulation of cell growth and helped to identify dysregulations that may contribute to aberrant pituitary cell growth and underlie pituitary tumorigenesis; illuminated the cell specific mode of action of biologicals and triggered the development of therapeutic schemes able to amplify their therapeutic potential.

Effect of Everolimus on Cell Viability in Nonfunctioning Pituitary Adenomas

Pituitary adenomas can cause specific syndromes due to hormone excess and/or determine sellar mass symptoms. Pituitary cell growth can sometimes be influenced by medical therapy, such as for somatotroph adenomas treated with somatostatin analogs or prolactinomas treated with dopaminergic drugs. However, nonfunctioning pituitary adenomas (NFAs) are still orphans of medical therapy. Everolimus (RAD001), a derivative of rapamycin, is a well-known immunosuppressant drug, which has been recently shown to have antineoplastic activity in several human cancers. The objective of the study was to investigate the possible antiproliferative effects of RAD001 in human NFAs. We collected 40 NFAs that were dispersed in primary cultures, treated without or with 1 nm to 1 microm RAD001, 10 nm cabergoline, 10 nm SOM230 (a somatostatin receptor multiligand), and/or 50 nm IGF-I. Cell viability and apoptosis were evaluated after 48 h, and vascular endothelial growth factor (VEGF) secretion was assessed after an 8-h incubation. Somatostatin and dopamine subtype 2 receptor expression was investigated by quantitative PCR. In 28 cultures (70%), Everolimus significantly reduced cell viability (by approximately 40%; P < 0.05 vs. control), promoted apoptosis (+30%; P < 0.05 vs. control), inhibited p70S6K activity (-20%), and blocked IGF-I proliferative and antiapoptotic effects. In selected tissues cotreatment with SOM230, but not cabergoline, exerted an additive effect. Everolimus did not affect VEGF secretion but blocked the stimulatory effects of IGF-I on this parameter. Everolimus reduced NFA cell viability by inducing apoptosis, with a mechanism likely involving IGF-I signaling but not VEGF secretion, suggesting that it might represent a possible medical treatment of invasive/recurrent NFAs.

Fibroblast Growth Factor-2 Autofeedback Regulation in Pituitary Folliculostellate TtT/GF Cells

To investigate paracrine regulation of pituitary cell growth, we tested fibroblast growth factor (FGF) regulation of TtT/GF folliculostellate (FS) cells. FGF-2, and FGF-4 markedly induced cell proliferation, evidenced by induction of pituitary tumor transforming gene-1 (Pttg1) mRNA expression and percentage of cells in S phase. Signaling for FGF-2-induced FS cell proliferation was explored by specific pharmacological inhibition. A potent inhibitory effect on FGF-2 action was observed by blocking of Src tyrosine kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (>or=0.1 microm), followed by protein kinase C (PKC) inhibition with GF109203X. Treatment with FGF-2 (30 ng/ml; 10 min) activated phosphorylation of signal transducer and activator of transcription-3, ERK, stress-activated protein kinase/c-Jun N-terminal kinase, Akt, and focal adhesion kinase. Src inhibition with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine suppressed FGF-2-induced Akt and focal adhesion kinase, indicating effects downstream of FGF-2-induced Src activation. FGF-2 also markedly induced its own mRNA expression, peaking at 2-4 h, and this effect was suppressed by Src tyrosine kinase inhibition. The PKC inhibitor GF109203X abolished FGF-2 autoinduction, indicating PKC as the primary pathway involved in FGF-2 autoregulation in these cells. In addition to pituitary FGF-2 paracrine activity on hormonally active cells, these results show an autofeedback mechanism for FGF-2 in non-hormone-secreting pituitary FS cells, inducing cell growth and its own gene expression, and mediated by Src/PKC signaling.

Effects of TGFβ1 on gene expression in the HP75 human pituitary tumor cell line identified by gene expression profiling

The pathogenesis of pituitary adenomas and many of the genes influencing growth of these tumors are unknown. TGFbeta is known to inhibit proliferation of cultured anterior pituitary cells and anterior pituitary tumors, but the signal transduction pathways involved in the inhibition of growth are unclear. We treated the human HP75 pituitary cell line with 10(-9) M TGFbeta1 for 4, 24, and 96 h and performed global gene expression profiling by Affymetrix GeneChip microarray analysis. Quantitative PCR validation of specific genes involved in the TGFbeta1-induced regulation of pituitary cell growth was also done. Of the 15,000 genes queried, there were 37 genes up-regulated and 48 genes down-regulated twofold or more after 4 h of TGFbeta1 treatment. There were 121 genes up-regulated and 109 genes down-regulated twofold or more after 24 h of TGFbeta1 treatment and 112 genes up-regulated and 43 genes down-regulated twofold or more after 96 h of TGFbeta1 treatment. Galectin-3 (Gal-3) protein was decreased by TGFbeta1 treatment and several genes which interacted with Gal-3 including RUNX1 and WNT5B were up-regulated after TGFbeta1 treatment. SOX4 was also up-regulated by TGFbeta1 treatment. SMAD3, which is directly involved in the TGFbeta signal transduction pathway, was down-regulated by TGFbeta1 treatment. These findings highlight the diverse gene networks and pathways through which TGFbeta operates in its effects on pituitary tumor cells.