Truncated somatostatin receptor variant sst5TMD4 confers aggressive features (proliferation, invasion and reduced octreotide response) to somatotropinomas.
ABSTRACT: The GH/IGF1 response of somatotropinomas to somatostatin analogues (SSA) is associated with their pattern of somatostatin receptor (sst1-sst5) expression. Recently, we demonstrated that expression of a truncated sst5-variant (sst5TMD4) can influence the secretory response of somatotropinomas to SSA-therapy; however, its potential relationship with aggressive features (e.g. invasion/proliferation) is still unknown. Here, we show that sst5TMD4 is present in 50% of non-functioning pituitary-adenomas (NFPA) (n?=?30) and 89% of somatotropinomas (n?=?36), its expression levels being highest in somatotropinomas?>?>?NFPAs?>?>?>?normal pituitaries (negligible expression; n?=?8). In somatotropinomas, sst5TMD4 mRNA and protein levels correlated positively, and its expression was directly associated with tumor invasiveness (cavernous/sphenoid sinus), and inversely correlated with age and GH/IGF1 reduction after 3-6?months with octreotide-LAR therapy. GNAS+ somatotropinomas expressed lower sst5TMD4 levels. ROC analysis revealed sst5TMD4 expression as the only marker, within all sst-subtypes, capable to predict tumor invasiveness in somatotropinomas. sst5TMD4 overexpression increased cell viability in cultured somatotropinoma (n?=?5). Hence, presence of sst5TMD4 associates with increased aggressive features and worse prognosis in somatotropinomas, thereby providing a potentially useful tool to refine somatotropinoma diagnosis, predict outcome of clinical response to SSA-therapy and develop new therapeutic targets.
Project description:Filamin-A (FLNA) plays a crucial role in somatostatin receptor (sst) subtype-2 signaling in somatotropinomas. Our objective was to investigate the in vivo association between FLNA and sst2 expression, sst5 expression, dopamine receptor subtype-2 (D2) expression, somatostatin receptor ligand (SRL) responsiveness and tumor invasiveness in somatotropinomas. Quantitative real-time PCR was used to evaluate the absolute mRNA copy numbers of FLNA/sst2/sst5/D2 in 96 somatotropinomas. FLNA, sst2 and sst5 protein expression levels were also evaluated using immunohistochemistry. The Knosp-Steiner criteria were used to evaluate tumor invasiveness. Median FLNA, sst2, sst5 and D2 copy numbers were 4,244, 731, 156 and 3,989, respectively. Thirty-one of the 35 available tumors (89%) were immune positive for FLNA in the cytoplasm and membrane but not in the nucleus. FLNA and sst5 expression were positively correlated at the mRNA and protein levels (p < 0.001 and p = 0.033, respectively). FLNA was positively correlated with sst2 mRNA in patients who were responsive to SRL (p = 0.014, R = 0.659). No association was found between FLNA and tumor invasiveness. Our findings show that in somatotropinomas FLNA expression positively correlated with in vivo sst5 and D2 expression. Notably, FLNA was only correlated with sst2 in patients who were controlled with SRL. FLNA was not associated with tumor invasiveness.
Project description:?-arrestins seem to have a role in endocytosis and desensitization of somatostatin receptor subtype 2 (sst2) and could be associated with the responsiveness to somatostatin receptor ligands (SRL) in patients with acromegaly. To investigate the in vivo correlation between ?-arrestins 1 and 2 with sst2, sst5 and dopamine receptor subtype 2 (D2) expressions, and the association of ?-arrestins with response to first-generation SRL and invasiveness in somatotropinomas. ?-arrestins 1 and 2, sst2, sst5 and D2 mRNA expressions were evaluated by quantitative real-time RT-PCR on tumoral tissue of 96 patients. Moreover, sst2 and sst5 protein expressions were also evaluated in 40 somatotropinomas by immunohistochemistry. Response to SRL, defined as GH <1 ?g/l and normal IGF-I levels, was assessed in 40 patients. The Knosp-Steiner criteria were used to define invasiveness. Median ?-arrestin 1, ?-arrestin 2, sst2, sst5 and D2 mRNA copy numbers were 478; 9375; 731; 156; and 3989, respectively. There was a positive correlation between ?-arrestins 1 and 2 (R = 0.444, P < 0.001). However, no correlation between ?-arrestins and sst2, sst5 (mRNA and protein levels) or D2 was found. No association was found between ?-arrestins expression and SRL responsiveness or tumour invasiveness. Although previous data suggest a putative correlation between ?-arrestins and sst2, our data clearly indicated that no association existed between ?-arrestins and sst2, sst5 or D2 expression, nor with response to SRL or tumour invasiveness. Therefore, further studies are required to clarify whether ?-arrestins have a role in the response to treatment with SRL in acromegaly.
Project description:Chimeric somatostatin/dopamine compounds such as BIM-23A760, an sst2/sst5/D2 receptors-agonist, have emerged as promising new approaches to treat pituitary adenomas. However, information on direct in vitro effects of BIM-23A760 in normal and tumoral pituitaries remains incomplete. The objective of this study was to analyze BIM-23A760 effects on functional parameters (Ca2+ signaling, hormone expression/secretion, cell viability and apoptosis) in pituitary adenomas (n?=?74), and to compare with the responses of normal primate and human pituitaries (n?=?3-5). Primate and human normal pituitaries exhibited similar sst2/sst5/D2 expression patterns, wherein BIM-23A760 inhibited the expression/secretion of several pituitary hormones (specially GH/PRL), which was accompanied by increased sst2/sst5/D2 expression in primates and decreased Ca2+ concentration in human cells. In tumoral pituitaries, BIM-23A760 also inhibited Ca2+ concentration, hormone secretion/expression and proliferation. However, BIM-23A760 elicited stimulatory effects in a subset of GHomas, ACTHomas and NFPAs in terms of Ca2+ signaling and/or hormone secretion, which was associated with the relative somatostatin/dopamine-receptors levels, especially sst5 and sst5TMD4. The chimeric sst2/sst5/D2 compound BIM-23A760 affects multiple, clinically relevant parameters on pituitary adenomas and may represent a valuable therapeutic tool. The relative ssts/D2 expression profile, particularly sst5 and/or sst5TMD4 levels, might represent useful molecular markers to predict the ultimate response of pituitary adenomas to BIM-23A760.
Project description:<b>Background:</b> Imatinib, a tyrosine kinase inhibitor, causes growth failure in children with chronic myeloid leukemia probably by targeting the growth hormone (GH)/insulin like growth factor-1 (IGF-1) axis. We aim to explore the imatinib targets expression in pituitary adenomas and study the effect of imatinib on GH secretion in somatotropinoma cells and GH3 cell line. <b>Materials and Methods:</b> The expression pattern of imatinib's targets (c-kit, VEGF, and PDGFR-?/?) was studied using immunohistochemistry and immunoblotting 157 giant (?4 cm) pituitary adenomas (121 non-functioning pituitary adenomas, 32 somatotropinomas, and four prolactinomas) and compared to normal pituitary (<i>n</i> = 4) obtained at autopsy. The effect imatinib on GH secretion, cell viability, immunohistochemistry, electron microscopy, and apoptosis was studied in primary culture of human somatotropinomas (<i>n</i> = 20) and in rat somato-mammotroph GH3 cell-line. A receptor tyrosine kinase array was applied to human samples to identify altered pathways. <b>Results:</b> Somatotropinomas showed significantly higher immunopositivity for c-kit and platelet-derived growth factor receptor-? (PDGFR-?; <i>P</i> < 0.009 and <i>P</i> < 0.001, respectively), while staining for platelet-derived growth factor receptor-? (PDGFR-?) and vascular endothelial growth factor (VEGF) revealed a weaker expression (<i>P</i> < 0.001) compared to normal pituitary. Imatinib inhibited GH secretion from both primary culture (<i>P</i> < 0.01) and GH3 cells (<i>P</i> < 0.001), while it did not affect cell viability and apoptosis. The receptor tyrosine kinase array showed that imatinib inhibits GH signaling via PDGFR-? pathway. <b>Conclusion:</b> Imatinib inhibits GH secretion in somatotropinoma cells without affecting cell viability and may be used as an adjunct therapy for treating GH secreting pituitary adenomas.
Project description:Secretion of GH by pituitary somatotrophs is primarily stimulated by GHRH and ghrelin and inhibited by somatostatin through the activation of specific receptors [GHRH receptor (GHRH-R), GH secretagogue receptor (GHS-R) and somatostatin receptors (sst1-5), respectively]. However, we have shown that somatostatin, at low doses, can also stimulate GH release, directly and specifically, in primary pituitary cultures from a nonhuman primate (baboons, Papio anubis) and pigs. To determine whether somatostatin, GHRH, and ghrelin can also regulate the expression of their receptors in primates, pituitary cultures from baboons were treated for 4 h with GHRH or ghrelin (10(-8) m) or with high (10(-7) m) and low (10(-15) m) doses of somatostatin, and GH release and expression levels of all receptors were measured. GHRH/ghrelin decreased the expression of their respective receptors (GHRH-R and GHS-R). Both peptides increased sst1, only GHRH decreased sst5 expression, whereas sst2 expression remained unchanged. The effects of GHRH/ghrelin were completely mimicked by forskolin (adenylate cyclase activator) and phorbol 12-myristate 13-acetate (protein kinase C activator), respectively, indicating the regulation of receptor subtype levels by GHRH and ghrelin involved distinct signaling pathways. In contrast, high-dose somatostatin did not alter GH release but increased sst1, sst2, and sst5 expression, whereas GHRH-R and GHS-R expression were unaffected. Interestingly, low-dose somatostatin increased GH release and sst1 mRNA but decreased sst5 and GHRH-R expression, similar to that observed for GHRH. Altogether, our data show for the first time in a primate model that the primary regulators of somatotroph function (GHRH/ghrelin/somatostatin) exert both homologous and heterologous regulation of receptor synthesis which is dose and subtype dependent and involves distinct signaling pathways.
Project description:Acromegaly is a rare but severe disease, originated in 95% of cases by a growth hormone-secreting adenoma (somatotropinoma) in the pituitary. Magnetic resonance imaging (MRI) is a non-invasive technique used for the diagnosis and prognosis of pituitary tumours. The aim of this study was to determine whether the use of T2-weighted signal intensity at MRI could help to improve the characterisation of somatotropinomas, by analysing its relationship with clinical/molecular features. An observational study was implemented in a cohort of 22 patients (mean age?=?42.1?±?17.2 years; 59% women; 95% size>10?mm). Suprasellar-extended somatotropinomas presented larger diameters vs. non-extended tumours. T2-imaging revealed that 59% of tumours were hyperintense and 41% isointense adenomas, wherein hyperintense were more invasive (according to Knosp-score) than isointense adenomas. A higher proportion of hyperintense somatotropinomas presented extrasellar-growth, suprasellar-growth and invasion of the cavernous sinus compared to isointense adenomas. Interestingly, somatostatin receptor-3 and dopamine receptor-5 (DRD5) expression levels were associated with extrasellar and/or suprasellar extension. Additionally, DRD5 was also higher in hyperintense adenomas and its expression was directly correlated with Knosp-score and with tumour diameter. Hence, T2-weighted MRI on somatotropinomas represents a potential tool to refine their diagnosis and prognosis, and could support the election of preoperative treatment, when required.
Project description:<b>Objective/purpose: </b>The aryl hydrocarbon receptor (AHR) pathway plays a critical role in the biology of Growth Hormone (GH)-secreting pituitary tumor (somatotropinoma). Germline rs2066853 <i>AHR</i> variant was found to be more frequent among acromegaly patients and associated with a more severe disease with larger invasive somatropinoma, and with resistance to somatostatin analogs treatment in patients living in polluted areas. However, no somatic changes in <i>AHR</i> gene have been reported so far in acromegaly patients. On that basis, the aim of the study was to assess at the somatic level the <i>AHR</i> gene status encompassing exon 10 region, also because of the high rate of variants found in this genomic region.<br><br><b>Methods: </b>A cohort of 13 patients aged 20-76 years with biochemical, clinical and histological diagnosis of somatotropinoma was studied. DNA and RNA from pituitary tumor histological samples have been extracted and analyzed by PCR and direct sequencing for <i>AHR</i> gene variants, and compared with corresponding patients' germline DNA as well as normal pituitary tissue as reference control.<br><br><b>Results: </b>A degenerated letter codes in the region corresponding to <i>AHR</i> exon 10 (c.1239-c.2056) was detected in somatotropinomas-derived DNA but not in that of matched germline and pituitary normal tissue. By multiple PCR and sequencing analysis, we observed amplification only before codon 1246 and after codon 1254, confirming the presence of a tumor-restricted somatic deletion in the 5' upstream region of <i>AHR</i> exon 10. Analysis of PCR-amplified cDNA revealed a wildtype sequence of exon 9 and 10 in normal pituitary tissue, and a wildtype sequence of exon 9 and 10 up to codon 1246 and no sequence after the deletion region (c.1246-c.1254) in 6 out of 9 tumor samples. Patients carrying the germline rs2066853 <i>AHR</i> variant showed no somatic LOH at the corresponding genetic locus.<br><br><b>Conclusion: </b>This is the first demonstration of a recurrent somatic deletion in the exon 10 of the <i>AHR</i> gene in somatotropinomas. The functional impact of this genetic finding needs to be clarified.
Project description:The purpose of this study was to (1) identify specific miRNAs in growth hormones (GH)-secreting pituitary adenomas; (2) determine the relationship between the expression of these miRNAs and tumor size, somatostatin analogs treatment, and responsiveness to somatostatin analogs (SSA).Fifteen GH-secreting adenomas patients were treated with lanreotide for 4 months before surgery. Patients with 50% reduction of GH secretion by lanreotide were considered as SSA responders, while patients with less than 50% of GH reduction were considered as SSA nonresponders. We analyzed the miRNAs in 21 GH-secreting pituitary adenomas and 6 normal pituitaries by miRCURY™ LNA array and some differentially expressed miRNAs were validated by quantitative real-time PCR.Fifty-two miRNAs were differentially expressed between GH-secreting pituitary adenomas and normal pituitaries. Differential expression of 9 miRNAs was observed between micro- and macro-adenomas. Thirteen miRNAs were differentially expressed between tumor samples from lanreotide-treated patients and those from lanreotide-untreated patients. Seven miRNAs were differentially expressed between SSA responders or GH nonresponders. Several identified miRNAs may be involved in cell proliferation, apoptosis, cancer development and progression.Our results indicate that altered miRNAs expression is involved in GH-secreting pituitary adenomas transformation, which will shed light on the mechanisms for the treatment of acromegaly by SSA. Identification and characterization of the targets of altered miRNAs genes may elucidate molecular mechanisms involved in the pathogenesis of pituitary adenoma.
Project description:<h4>Context</h4>Dopamine receptor (DR) and somatostatin receptor subtype expression in pituitary adenomas may predict the response to postsurgical therapies.<h4>Objectives</h4>Our objectives were to assess and compare the mRNA levels of DR1-5 and somatostatin receptors 1-5 in normal pituitaries (NPs), nonfunctioning pituitary adenomas (NFPAs), and somatotropinomas. In addition, we determined whether the level of DR expression correlates with the in vivo response to octreotide-LAR in acromegalic patients.<h4>Design and patients</h4>Eight NPs, 30 NFPAs, and 39 somatotropinomas were analyzed for receptor mRNA levels by real-time RT-PCR. The DR2 short variant was estimated as the DR2 long/DR2 total (DR2T). The relationship between DR expression and the postsurgical response to octreotide-LAR was assessed in 19 of the acromegalic patients.<h4>Results</h4>DR3 was not detected. The relationship between expression levels of DR subtypes in NPs and somatotropinomas was DR2T>>>DR4>>DR5>DR1, whereas in NFPAs, DR2T>>>DR4>>DR1>DR5. The DR2 short variant was the predominant DR2 variant in the majority of samples. In acromegalics treated with octreotide-LAR, DR1 was negatively correlated with percent GH reduction (3 months: r = -0.67, P = 0.002; and 6 months: r = -0.58, P = 0.009), and DR5 was positively correlated with percent IGF-I reduction (3 months: r = 0.55, P = 0.01; and 6 months: r = 0.47, P = 0.04).<h4>Conclusions</h4>DR2 is the predominant DR subtype in NPs, NFPAs, and somatotropinomas. The fact that DR1, DR4, and DR5 are also expressed in many adenomas tested suggests that these receptors might also play a role in the therapeutic impact of postsurgical medical therapies in patients with NFPA and acromegaly. This was supported by the finding that the in vivo response to octreotide-LAR was negatively associated with DR1 and positively associated with DR5.
Project description:Acromegaly is a disease mainly caused by pituitary neuroendocrine tumor (PitNET) overproducing growth hormone. First-line medication for this condition is the use of somatostatin analogs (SSAs), that decrease tumor mass and induce antiproliferative effects on PitNET cells. Dopamine agonists (DAs) can also be used if SSA treatment is not effective. This study aimed to determine differences in transcriptome signatures induced by SSA/DA therapy in PitNET tissue. We selected tumor tissue from twelve patients with somatotropinomas, with half of the patients receiving SSA/DA treatment before surgery and the other half treatment naive. Transcriptome sequencing was then carried out to identify differentially expressed genes (DEGs) and their protein-protein interactions, using pathway analyses. We found 34 upregulated and six downregulated DEGs in patients with SSA/DA treatment. Three tumor development promoting factors <i>MUC16, MACC1</i>, and <i>GRHL2</i>, were significantly downregulated in therapy administered PitNET tissue; this finding was supported by functional studies in GH3 cells. Protein-protein interactions and pathway analyses revealed extracellular matrix involvement in the antiproliferative effects of this type of the drug treatment, with pronounced alterations in collagen regulation. Here, we have demonstrated that somatotropinomas can be distinguished based on their transcriptional profiles following SSA/DA therapy, and SSA/DA treatment does indeed cause changes in gene expression. Treatment with SSA/DA significantly downregulated several factors involved in tumorigenesis, including <i>MUC16, MACC1</i>, and <i>GRHL2.</i> Genes that were upregulated, however, did not have a direct influence on antiproliferative function in the PitNET cells. These findings suggested that SSA/DA treatment acted in a tumor suppressive manner and furthermore, collagen related interactions and pathways were enriched, implicating extracellular matrix involvement in this anti-tumor effect of drug treatment.