Efficacy of a Novel Second-Generation Somatostatin-Dopamine Chimera (TBR-065) in Human Medullary Thyroid Cancer: A Preclinical Study.

INTRODUCTION: Somatostatin and dopamine (DA) receptors have a pivotal role in controlling hormone secretion and cell proliferation in different neuroendocrine neoplasms, including medullary thyroid cancer (MTC). In the present preclinical study, we evaluated the anti-tumor activity of TBR-065 (formerly BIM-23B065), a second-generation somatostatin-DA chimera, in 2 human MTC cell lines. METHODS: The effects of lanreotide (LAN) and TBR-065 on cell growth and proliferation, calcitonin (CT) secretion, cell cycle, apoptosis, cell migration, and tumor-induced angiogenesis have been evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA flow cytometry with propidium iodide (PI), Annexin V-FITC/PI staining, electrochemiluminescence immuno assay, wound-healing assay, and zebrafish platform, respectively. RESULTS: TBR-065 exerted a more prominent anti-tumor activity than LAN in both MTC cell lines, as shown by inhibition of cell proliferation (maximal inhibition in TT: -50.3 and -37.6%, respectively; in MZ-CRC-1: -58.8 and -27%, respectively) and migration (in TT: -42.7 and -22.9%, respectively; in MZ-CRC-1: -75.5 and -58.2%, respectively). Only the new chimera decreased significantly the fraction of cells in S phase (TT: -33.8%; MZ-CRC-1: -18.8%) and increased cells in G2/M phase (TT: +13%; MZ-CRC-1: +30.5%). In addition, TBR-065 exerted a more prominent pro-apoptotic effect than LAN in TT cells. A concomitant decrease in CT secretion was observed after 2 days of incubation with both drugs, with a more relevant effect of TBR-065. However, neither LAN nor TBR-065 showed any effect on tumor-induced angiogenesis, as evaluated using a zebrafish/tumor xenograft model. DISCUSSION/CONCLUSION: In MTC cell lines, a second-generation somatostatin-DA analog, TBR-065, exerts a more relevant anti-tumor activity than LAN, through modulation of cell cycle, induction of apoptosis, and reduction in migration. Further studies are required to establish whether TBR-065 has comparable potent inhibitory effects on tumor growth in vivo.

Vandetanib versus Cabozantinib in Medullary Thyroid Carcinoma: A Focus on Anti-Angiogenic Effects in Zebrafish Model.

Medullary thyroid carcinoma (MTC) is a tumor deriving from the thyroid C cells. Vandetanib (VAN) and cabozantinib (CAB) are two tyrosine kinase inhibitors targeting REarranged during Transfection (RET) and other kinase receptors and are approved for the treatment of advanced MTC. We aim to compare the in vitro and in vivo anti-tumor activity of VAN and CAB in MTC. The effects of VAN and CAB on viability, cell cycle, and apoptosis of TT and MZ-CRC-1 cells are evaluated in vitro using an MTT assay, DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, respectively. In vivo, the anti-angiogenic potential of VAN and CAB is evaluated in Tg(fli1a:EGFP)y1 transgenic fluorescent zebrafish embryos by analyzing the effects on the physiological development of the sub-intestinal vein plexus and the tumor-induced angiogenesis after TT and MZ-CRC-1 xenotransplantation. VAN and CAB exert comparable effects on TT and MZ-CRC-1 viability inhibition and cell cycle perturbation, and stimulated apoptosis with a prominent effect by VAN in MZ-CRC-1 and CAB in TT cells. Regarding zebrafish, both drugs inhibit angiogenesis in a dose-dependent manner, in particular CAB shows a more potent anti-angiogenic activity than VAN. To conclude, although VAN and CAB show comparable antiproliferative effects in MTC, the anti-angiogenic activity of CAB appears to be more relevant.

Interferon-beta enhances sensitivity to gemcitabine in pancreatic cancer.

BACKGROUND: Adjuvant gemcitabine for pancreatic cancer has limited efficacy in the clinical setting. Impaired drug metabolism is associated with treatment resistance. We aimed to evaluate the chemosensitising effect of interferon-beta (IFN-ß). METHODS: BxPC-3, CFPAC-1, and Panc-1 cells were pre-treated with IFN-ß followed by gemcitabine monotherapy. The effect on cell growth, colony formation, and cell cycle was determined. RT-qPCR was used to measure gene expression. BxPC-3 cells were used in a heterotopic subcutaneous mouse model. RESULTS: IFN-ß increased sensitivity to gemcitabine (4-, 7.7-, and 1.7-fold EC50 decrease in BxPC-3, CFPAC-1, and Panc-1, respectively; all P < 0.001). Findings were confirmed when assessing colony formation. The percentage of cells in the S-phase was significantly increased after IFN-ß treatment only in BxPC-3 and CFPAC-1 by 12 and 7%, respectively (p < 0.001 and p < 0.05, respectively). Thereby, IFN-ß upregulated expression of the drug transporters SLC28A1 in BxPC-3 (252%) and SLC28A3 in BxPC-3 (127%) and CFPAC-1 (223%) (all p < 0.001). In vivo, combination therapy reduced tumor volume with 45% (P = 0.01). Both ex vivo and in vivo data demonstrate a significant reduction in the number of proliferating cells, whereas apoptosis was increased. CONCLUSIONS: For the first time, we validated the chemosensitising effects of IFN-ß when combined with gemcitabine in vitro, ex vivo, and in vivo. This was driven by cell cycle modulation and associated with an upregulation of genes involving intracellular uptake of gemcitabine. The use of IFN-ß in combination with gemcitabine seems promising in patients with pancreatic cancer and needs to be further explored.

Effect of the Tryptophan Hydroxylase Inhibitor Telotristat on Growth and Serotonin Secretion in 2D and 3D Cultured Pancreatic Neuroendocrine Tumor Cells.

Serotonin, a biologically active amine, is related to carcinoid syndrome in functioning neuroendocrine tumors (NETs). Telotristat ethyl is a novel inhibitor of the tryptophan hydroxylase (TPH), a key enzyme in the production of serotonin. While its use in patients with carcinoid syndrome and uncontrolled diarrhea under somatostatin analogs (SSAs) has been recently approved, in vitro data evaluating its effectiveness are lacking. For this reason, we aimed to evaluate the effect of telotristat as monotherapy, and in combination with SSAs, on proliferation and secretion in a NET cell line model. The human pancreatic NET cell lines BON-1/QGP-1 were used as 2D and 3D cultured models; somatostatin receptor and TPH mRNA expression, as well as the potential autocrine effect of serotonin on tumor cell proliferation using a 3D culture system were evaluated. Telotristat decreased serotonin production in a dose-dependent manner at a clinically feasible concentration, without affecting cell proliferation. Its combination with pasireotide, but not with octreotide, had an additive inhibitory effect on serotonin secretion. The effect of telotristat was slightly less potent, when BON-1 cells were co-treated with octreotide. Octreotide and pasireotide had no effect on the expression of TPH. Telotristat did not have an effect on mRNA expression of somatostatin receptor subtypes. Finally, we showed that serotonin did not have an autocrine effect on NET cell proliferation on the 3D cell model. These results suggest that telotristat is an effective drug for serotonin inhibition, but the effectiveness of its combination with SST2 (somatostatin receptor subtype 2)-preferring SSA should be evaluated in more detail.

Peptide receptor radionuclide therapy in patients with medullary thyroid carcinoma: predictors and pitfalls.

BACKGROUND: For progressive metastatic medullary thyroid carcinoma (MTC), the available treatment options with tyrosine kinase inhibitors result in grade 3-4 adverse events in a large number of patients. Peptide Receptor Radionuclide Therapy (PRRT), which has also been suggested to be a useful treatment for MTC, is usually well tolerated, but evidence on its effectivity is very limited. METHODS: Retrospective evaluation of treatment effects of PRRT in a highly selected group of MTC patients, with progressive disease or refractory symptoms. In addition, a retrospective evaluation of uptake on historical 111In-DTPA-octreotide scans was performed in patients with detectable tumor size > 1 cm. RESULTS: Over the last 17 years, 10 MTC patients were treated with PRRT. Four out of 10 patients showed stable disease at first follow-up (8 months after start of therapy) whereas the other 6 were progressive. Patients with stable disease were characterized by a combination of both a high uptake on 111In-DTPA-octreotide scan (uptake grade ≥ 3) and a positive somatostatin receptor type 2a (SSTR2a) expression of the tumor by immunohistochemistry. Retrospective evaluation of historical 111In-DTPA-octreotide scans of 35 non-treated MTC patients revealed low uptake (uptake grade 1) in the vast majority of patients 31/35 (89%) with intermediate uptake (uptake grade 2) in the remaining 4/35 (11%). CONCLUSIONS: PRRT using 177Lu-octreotate could be considered as a treatment in those patients with high uptake on 111In-DTPA-octreotide scan (uptake grade 3) and positive SSTR2a expression in tumor histology. Since this high uptake was present in a very limited number of patients, this treatment is only suitable in a selected group of MTC patients.

Somatostatin Receptor Expression in GH-Secreting Pituitary Adenomas Treated with Long-Acting Somatostatin Analogues in Combination with Pegvisomant.

BACKGROUND: Growth hormone-secreting pituitary adenomas (somatotroph adenoma) predominantly express somatostatin receptors (SSTRs) subtypes 2 and 5. Higher SSTR2 expression on somatotroph adenomas results in a better response to somatostatin analogues (SSAs), which preferentially bind, but also downregulate, SSTR2. The effect of the combined treatment with SSAs and the GH receptor antagonist pegvisomant (PEGV) on SSTR expression in somatotroph adenomas is currently unknown. AIM OF THE STUDY: To assess SSTR2 and SSTR5 expression in three groups of somatotroph adenomas: drug-naive, treated with long-acting (LA) SSA monotherapy, or LA-SSA/PEGV combination therapy before surgery. Additionally, we evaluated the required PEGV dose to achieve insulin-like growth factor I (IGF-I) normalization in relation to the SSTR expression. MATERIALS AND METHODS: At our Pituitary Center Rotterdam, we selected acromegalic patients who underwent transsphenoidal neurosurgery. All patients were eventually treated with LA-SSA/PEGV combination therapy during their medical history. SSTR2 and SSTR5 expression in somatotroph adenoma tissues was determined using immunohistochemistry. RESULTS: Out of 39 somatotroph adenoma tissue samples, 23 were drug-naive, 9 received pretreatment with LA-SSA and 7 LA-SSA/PEGV combined treatment. SSTR2 expression was significantly higher in treatment-naive compared to combined treatment somatotroph adenomas (p = 0.048), while SSTR5 expression did not differ. Noteworthy, SSTR2 expression in naive somatotroph adenoma tissues was inversely correlated with the required PEGV dose to achieve IGF-I normalization during postsurgical medical treatment (ρ = -0.538, p = 0.024). CONCLUSION: In our specific cohort, the SSTR2 expression was lower in patients pretreated with LA-SSA/PEGV compared to the drug-naive acromegalic patients. Additionally, the SSTR2 expression in treatment-naive somatotroph adenoma tissues was inversely correlated with the required PEGV dose to achieve IGF-I normalization.

Long-term acquired everolimus resistance in pancreatic neuroendocrine tumours can be overcome with novel PI3K-AKT-mTOR inhibitors.

BACKGROUND: The mTOR-inhibitor everolimus improves progression-free survival in advanced pancreatic neuroendocrine tumours (PNETs). However, adaptive resistance to mTOR inhibition is described. METHODS: QGP-1 and BON-1, two human PNET cell lines, were cultured with increasing concentrations of everolimus up to 22 weeks to reach a dose of 1 µM everolimus, respectively, 1000-fold and 250-fold initial IC50. Using total DNA content as a measure of cell number, growth inhibitory dose-response curves of everolimus were determined at the end of resistance induction and over time after everolimus withdrawal. Response to ATP-competitive mTOR inhibitors OSI-027 and AZD2014, and PI3K-mTOR inhibitor NVP-BEZ235 was studied. Gene expression of 10 PI3K-Akt-mTOR pathway-related genes was evaluated using quantitative real-time PCR (RT-qPCR). RESULTS: Long-term everolimus-treated BON-1/R and QGP-1/R showed a significant reduction in everolimus sensitivity. During a drug holiday, gradual return of everolimus sensitivity in BON-1/R and QGP-1/R led to complete reversal of resistance after 10-12 weeks. Treatment with AZD2014, OSI-027 and NVP-BEZ235 had an inhibitory effect on cell proliferation in both sensitive and resistant cell lines. Gene expression in BON-1/R revealed downregulation of MTOR, RICTOR, RAPTOR, AKT and HIF1A, whereas 4EBP1 was upregulated. In QGP-1/R, a downregulation of HIF1A and an upregulation of ERK2 were observed. CONCLUSIONS: Long-term everolimus resistance was induced in two human PNET cell lines. Novel PI3K-AKT-mTOR pathway-targeting drugs can overcome everolimus resistance. Differential gene expression profiles suggest different mechanisms of everolimus resistance in BON-1 and QGP-1.

Effects of Somatostatin Analogs and Dopamine Agonists on Insulin-Like Growth Factor 2-Induced Insulin Receptor Isoform A Activation by Gastroenteropancreatic Neuroendocrine Tumor Cells.

BACKGROUND: Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) express insulin-like growth factor (IGF)-related factors [IGF1, IGF2; insulin receptor (IR)-A, IR-B; IGF-binding protein (IGFBP) 1-3] as well as somatostatin (SSTRs) and dopamine D2 receptors (D2Rs). OBJECTIVES: To (1) compare mRNA expression of IGF-related factors in human pancreatic NET (panNET) cell lines with that in human GEP-NETs to evaluate the usefulness of these cells as a model for studying the IGF system in GEP-NETs, (2) determine whether panNET cells produce growth factors that activate IR-A, and (3) investigate whether somatostatin analogs (SSAs) and/or dopamine agonists (DAs) influence the production of these growth factors. METHODS: In panNET cells (BON-1 and QGP-1) and GEP-NETs, mRNA expression of IGF-related factors was measured by quantitative real-time PCR. Effects of the SSAs octreotide and pasireotide (PAS), the DA cabergoline (CAB), and the dopastatin BIM-23A760 (all 100 nM) were evaluated at the IGF2 mRNA and protein level (by ELISA) and regarding IR-A bioactivity (by kinase receptor activation assay) in panNET cells. RESULTS: panNET cells and GEP-NETs had comparable expression profiles of IGF-related factors. Especially in BON-1 cells, IGF2 and IR-A were most highly expressed. PAS + CAB inhibited IGF2 (-29.5 ± 4.9%, p < 0.01) and IGFBP3 (-20.0 ± 4.0%, p < 0.01) mRNA expression in BON-1 cells. In BON-1 cells, IGF2 protein secretion was significantly inhibited with BIM-23A760 (-23.7 ± 3.8%). BON-1- but not QGP-1- conditioned medium stimulated IR-A bioactivity. In BON-1 cells, IR-A bioactivity was inhibited by BIM-23A760 and PAS + CAB (-37.8 ± 2.1% and -30.9 ± 4.1%, respectively, p < 0.0001). CONCLUSIONS: (1) The BON-1 cell line is a representative model for studying the IGF system in GEP-NETs, (2) BON-1 cells produce growth factors (IGF2) activating IR-A, and (3) combined SSTR and D2R targeting with PAS + CAB and BIM-23A760 suppresses IGF2-induced IR-A activation.

Is There an Additional Value of Using Somatostatin Receptor Subtype 2a Immunohistochemistry Compared to Somatostatin Receptor Scintigraphy Uptake in Predicting Gastroenteropancreatic Neuroendocrine Tumor Response?

BACKGROUND AND AIMS: It is unknown whether tumoral somatostatin receptor subtype 2a (sst2a) immunohistochemistry (IHC) has additional value compared to somatostatin receptor scintigraphy (SRS) uptake using OctreoScan® in predicting response to peptide receptor radiotherapy using 177Lu-octreotate (PRRT) in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs). The aims of this study were: (1) to establish the percentage of sst2a immunopositivity in GEP-NET samples of PRRT-treated patients, (2) to determine the relationship between best GEP-NET response using RECIST 1.0 criteria 1 year after PRRT and tumoral sst2a IHC, and (3) to compare characteristics of patients with sst2a IHC-negative and -positive tumors. METHODS: All 73 consecutive patients were selected for PRRT based on a positive SRS. Radiological response was scored according to RECIST 1.0 criteria. sst2a status was detected on tumor samples by IHC. RESULTS: In total, 93% of GEP-NET samples showed sst2a IHC positivity. No statistically significant relationship was observed between in vitro sst2a expression and in vivo best GEP-NET response 1 year after PRRT (p = 0.47). Sex, primary tumor site, disease stage, ENETS TNM classification, Ki-67 index, highest serum chromogranin-A level, and highest neuron-specific enolase level were not significantly different between patients with negative and positive sst2a tumoral IHC with the exception of age at diagnosis (p = 0.007). CONCLUSIONS: sst2a IHC of tumor samples has no additional value compared to SRS uptake using OctreoScan® in predicting tumor response after PRRT.

Influence of type-I Interferon receptor expression level on the response to type-I Interferons in human pancreatic cancer cells.

Pancreatic cancer is a highly aggressive malignancy with limited treatment options. Type-I interferons (e.g. IFN-α/-ß) have several anti-tumour activities. Over the past few years, clinical studies evaluating the effect of adjuvant IFN-α therapy in pancreatic cancer yielded equivocal results. Although IFN-α and -ß act via the type-I IFN receptor, the role of the number of receptors present on tumour cells is still unknown. Therefore, this study associated, for the first time, in a large panel of pancreatic cancer cell lines the effects of IFN-α/-ß with the expression of type-I IFN receptors. The anti-tumour effects of IFN-α or IFN-ß on cell proliferation and apoptosis were evaluated in 11 human pancreatic cell lines. Type-I IFN receptor expression was determined on both the mRNA and protein level. After 7 days of incubation, IFN-α significantly reduced cell growth in eight cell lines by 5-67%. IFN-ß inhibited cell growth statistically significant in all cell lines by 43-100%. After 3 days of treatment, IFN-ß induced significantly more apoptosis than IFN-α. The cell lines variably expressed the type-I IFN receptor. The maximal inhibitory effect of IFN-α was positively correlated with the IFNAR-1 mRNA (P < 0.05, r = 0.63), IFNAR-2c mRNA (P < 0.05, r = 0.69) and protein expression (P < 0.05, r = 0.65). Human pancreatic cancer cell lines variably respond to IFN-α and -ß. The expression level of the type-I IFN receptor is of predictive value for the direct anti-tumour effects of IFN-α treatment. More importantly, IFN-ß induces anti-tumour effects already at much lower concentrations, is less dependent on interferon receptor expression and seems, therefore, more promising than IFN-α.

Epigenetic Mechanisms Modulated by Glucocorticoids With a Focus on Cushing Syndrome.

In Cushing syndrome (CS), prolonged exposure to high cortisol levels results in a wide range of devastating effects causing multisystem morbidity. Despite the efficacy of treatment leading to disease remission and clinical improvement, hypercortisolism-induced complications may persist. Since glucocorticoids use the epigenetic machinery as a mechanism of action to modulate gene expression, the persistence of some comorbidities may be mediated by hypercortisolism-induced long-lasting epigenetic changes. Additionally, glucocorticoids influence microRNA expression, which is an important epigenetic regulator as it modulates gene expression without changing the DNA sequence. Evidence suggests that chronically elevated glucocorticoid levels may induce aberrant microRNA expression which may impact several cellular processes resulting in cardiometabolic disorders. The present article reviews the evidence on epigenetic changes induced by (long-term) glucocorticoid exposure. Key aspects of some glucocorticoid-target genes and their implications in the context of CS are described. Lastly, the effects of epigenetic drugs influencing glucocorticoid effects are discussed for their ability to be potentially used as adjunctive therapy in CS.

Somatostatin receptors and the associated intracellular machinery: the two sides of the coin.

Somatostatin receptors (SSTs) are widely expressed in pituitary tumors and neuroendocrine neoplasms (NENs) of different origins, i.e. the gastrointestinal tract and the thorax (lungs and thymus), thus representing a well-established target for medical treatment with SST ligands (SRLs). However, the response to SRLs is highly heterogeneous between tumors. Two main factors can contribute to this variability: (i) the differential SST expression among tumor types and (ii) the differential expression/modulation of the SST-related intracellular machinery. In this literature review, we provide an overview of available data on the variable expression of SSTs in pituitary tumors and NENs, together with the resulting clinical implications. Moreover, we aim to describe the complex intracellular machinery involved in SST signaling and trafficking. Particularly, we will focus on ß-arrestins and describe their role in receptor internalization and recycling, as well as the various functions of these scaffold molecules in tumor pathogenesis and progression. This review highlights the interplay between membrane receptors and intracellular machinery, together with its role in determining the clinical behavior of the tumor and the response to treatment in patients with pituitary tumors or NENs.

Protein kinase C-induced activin A switches adrenocortical steroidogenesis to aldosterone by suppressing CYP17A1 expression.

Functional zonation of the adrenal cortex is a consequence of the zone-specific expression of P450c17 (CYP17A1) and its cofactors. Activin and inhibin peptides are differentially produced within the zones of the adrenal cortex and have been implicated in steroidogenic control. In this study, we investigated whether activin and inhibin can function as intermediates in functional zonation of the human adrenal cortex. Activin A suppressed CYP17A1 expression and P450c17 function in adrenocortical cell lines as well as in primary adrenal cell cultures. Inhibin ßA-subunit mRNA and activin A protein levels were found to be increased up to 1,900-fold and 49-fold, respectively, after protein kinase C (PKC) stimulation through PMA or angiotensin II in H295R adrenocortical carcinoma cells. This was confirmed in HAC15 cells and for PMA in primary adrenal cell cultures. Both PMA and Ang II decreased CYP17A1 expression in the adrenocortical cell lines, whereas PMA concurrently suppressed CYP17A1 levels in the primary cultures. Inhibition of activin signaling during PKC stimulation through silencing of the inhibin ßA-subunit or blocking of the activin type I receptor opposed the PMA-induced downregulation of CYP17A1 expression and P450c17 function. In contrast, PKA stimulation through adrenocorticotrophin or forskolin increased expression of the inhibin α-subunit and betaglycan, both of which are antagonists of activin action. These data indicate that activin A acts as a PKC-induced paracrine factor involved in the suppression of CYP17A1 in the zona glomerulosa and can thereby contribute to functional adrenocortical zonation.

Rintatolimod: a potential treatment in patients with pancreatic cancer expressing Toll-like receptor 3.

Pancreatic cancer has a dismal prognosis, and treatment options for patients with locally advanced or metastatic disease are limited. Early tumor progression after standard chemo- and or radiotherapy remains a major concern in managing these patients. Treating pancreatic cancer patients with the Toll-like receptor 3 (TLR-3) agonist rintatolimod (Ampligen®) was effective in boosting the immune response. Rintatolimod acts via the TLR-3 receptor on several immune cells. However, the TLR-3 expression pattern in pancreatic cancer cells and how rintatolimod affects pancreatic cancer cells have not yet been investigated. The TLR-3 protein and mRNA expression were evaluated in thirteen PDAC tissue samples as well as in the human PDAC (hPDAC) cell lines CFPAC-1, MIAPaCa-2, and PANC-1 using immunohistochemistry and multiplexed gene expression analysis, respectively. The direct anti-tumor effects of rintatolimod were investigated using a proliferation and migration assay after different incubation time points with increasing concentrations of rintatolimod (ranging from 0.05 to 0.4 mg/ml). The TLR-3 protein and mRNA expression were heterogeneous between the PDAC tissue samples and the three hPDAC cell lines. TLR-3 protein and mRNA expression were high in CFPAC-1, moderate in MIAPaCa-2, and undetectable in PANC-1. Rintatolimod three-day treatment resulted in significantly reduced proliferation of CFPAC-1 cells compared to vehicle-treated control cells. In addition, after 24 hours, rintatolimod-treated CFPAC-1 cells showed less cell migration compared to vehicle-treated control cells, although this difference was not statistically significant. Lastly, we identified fifteen genes, altered with a Log2 FOC > |1.0| in rintatolimod-treated CFPAC-1 cells, which were significantly related to three transcription factors (NFKB1, RELA, and SP1) regulating the TLR-3 signaling pathway. In conclusion, we propose that rintatolimod treatment might have a direct TLR-3-dependent anti-tumoral effect on pancreatic cancer cells expressing TLR-3.

The Role of the IGF2 Methylation Score in Diagnosing Adrenocortical Tumors with Unclear Malignant Potential-Feasibility of Formalin-Fixed Paraffin-Embedded Tissue.

The differentiation between benign and malignant adrenocortical tumors based on pathological assessment can be difficult. We present a series of 17 patients with unclear malignant tumors, of whom six had recurrent or metastatic disease. The assessment of the methylation pattern of insulin-like growth factor 2 (IGF2) regulatory regions in fresh frozen material has shown to be valuable in determining the malignancy of adrenocortical tumors, although this has not been elaborately tested in unclear malignant tumors. Since fresh frozen tissue was only available in six of the patients, we determined the feasibility of using formalin-fixed paraffin-embedded (FFPE) tissue for this method. We isolated DNA from FFPE tissue and matched the fresh frozen tissue of three patients with adrenocortical carcinoma. Methylation patterns of IGF2 regulatory regions were determined by pyrosequencing using different amounts of bisulfite-converted DNA (5 ng, 20 ng, 40 ng). Compared to fresh frozen tissue, FFPE tissue had a higher failure rate (fresh frozen 0%; FFPE 18.5%) and poor-to-moderate replicability (fresh frozen rho = 0.89-0.99, median variation 1.6%; FFPE rho = -0.09-0.85, median variation 7.7%). There was only a poor-to-moderate correlation between results from fresh frozen and FFPE tissue (rho = -0.28-0.70, median variation 13.2%). In conclusion, FFPE tissue is not suitable for determining the IGF2 methylation score in patients with an unclear malignant adrenocortical tumor using the currently used method. We, therefore, recommend fresh frozen storage of resection material for diagnostic and biobank purposes.

Peripheral glucocorticoid receptor antagonism by relacorilant with modest HPA axis disinhibition.

Glucocorticoid stress hormones are produced in response to hypothalamic-pituitary-adrenal (HPA) axis activation. Glucocorticoids are essential for physiology and exert numerous actions via binding to the glucocorticoid receptor (GR). Relacorilant is a highly selective GR antagonist currently undergoing a phase 3 clinical evaluation for the treatment of endogenous Cushing's syndrome. It was found that increases in serum adrenocorticotropic hormone (ACTH) and cortisol concentrations after relacorilant treatment were substantially less than the increases typically observed with mifepristone, but it is unclear what underlies these differences. In this study, we set out to further preclinically characterize relacorilant in comparison to the classical but non-selective GR antagonist mifepristone. In human HEK-293 cells, relacorilant potently antagonized dexamethasone- and cortisol-induced GR signaling, and in human peripheral blood mononuclear cells, relacorilant largely prevented the anti-inflammatory effects of dexamethasone. In mice, relacorilant treatment prevented hyperinsulinemia and immunosuppression caused by increased corticosterone exposure. Relacorilant treatment reduced the expression of classical GR target genes in peripheral tissues but not in the brain. In mice, relacorilant induced a modest disinhibition of the HPA axis as compared to mifepristone. In line with this, in mouse pituitary cells, relacorilant was generally less potent than mifepristone in regulating Pomc mRNA and ACTH release. This contrast between relacorilant and mifepristone is possibly due to the distinct transcriptional coregulator recruitment by the GR. In conclusion, relacorilant is thus an efficacious peripheral GR antagonist in mice with only modest disinhibition of the HPA axis, and the distinct properties of relacorilant endorse the potential of selective GR antagonist treatment for endogenous Cushing's syndrome.

Dissecting the In Vitro Efficacy of Octreotide and Cabergoline in GH- and GH/PRL-Secreting Pituitary Tumors.

CONTEXT: Cabergoline (CAB) is an off-label medical therapy for acromegaly, overshadowed by first-generation somatostatin receptor ligands, eg, octreotide (OCT). OBJECTIVE: This was a head-to-head comparison between OCT and CAB in inhibiting growth hormone (GH) secretion in primary cultures of GH- and GH/prolactin (PRL)-secreting tumors; we also investigated the role of somatostatin (SST) and dopamine type 2 (D2R) receptor expression. METHODS: We evaluated the antisecretory effect of OCT and CAB, together with receptor mRNA expression, in 23 tumor cultures obtained from acromegaly patients referred to the Erasmus Medical Center (Rotterdam, The Netherlands). GH concentrations in cell culture media were determined after 72-hour OCT and CAB treatment (10 nM). RESULTS: OCT showed a slightly higher efficacy compared with CAB (GH decrease -39.5% vs -32.5%, P = 0.079). The effect of the 2 drugs was superimposable in GH/PRL co-secreting tumors (-42.1% vs -44.8%), where SST1 and D2R had a higher expression compared with the pure GH-secreting tumors (P = 0.020 and P = 0.026). OCT was more effective than CAB in 8/23 cultures, while CAB was more effective than OCT in 3/23 (CAB+ group). In CAB+ tumors, SST1 expression was higher compared with the other groups (P = 0.034). At receiver operating characteristic (ROC) curve analysis, SST1 and D2R discriminated between GH and GH/PRL co-secretion (AUC 0.856, P = 0.013; AUC 0.822, P = 0.024). SST1 was the best predictor of CAB response (≥50% GH reduction, AUC 0.913, P = 0.006; 80% sensitivity, 94% specificity). CONCLUSION: OCT is 5% to 10% more effective than CAB in vitro. SST1 mRNA expression can represent a reliable marker of GH/PRL co-secreting tumors showing a preferential response to CAB treatment.

Epigenetic regulation of SST2 expression in small intestinal neuroendocrine tumors.

Background: Somatostatin receptor type 2 (SST2) expression is critical for the diagnosis and treatment of neuroendocrine tumors and is associated with improved patient survival. Recent data suggest that epigenetic changes such as DNA methylation and histone modifications play an important role in regulating SST2 expression and tumorigenesis of NETs. However, there are limited data on the association between epigenetic marks and SST2 expression in small intestinal neuroendocrine tumors (SI-NETs). Methods: Tissue samples from 16 patients diagnosed with SI-NETs and undergoing surgical resection of the primary tumor at Erasmus MC Rotterdam were analysed for SST2 expression levels and epigenetic marks surrounding the SST2 promoter region, i.e. DNA methylation and histone modifications H3K27me3 and H3K9ac. As a control, 13 normal SI-tissue samples were included. Results: The SI-NET samples had high SST2 protein and mRNA expression levels; a median (IQR) of 80% (70-95) SST2-positive cells and 8.2 times elevated SST2 mRNA expression level compared to normal SI-tissue (p=0.0042). In comparison to normal SI-tissue, DNA methylation levels and H3K27me3 levels were significantly lower at five out of the eight targeted CpG positions and at two out of the three examined locations within the SST2 gene promoter region of the SI-NET samples, respectively. No differences in the level of activating histone mark H3K9ac were observed between matched samples. While no correlation was found between histone modification marks and SST2 expression, SST2 mRNA expression levels correlated negatively with DNA methylation within the SST2 promoter region in both normal SI-tissue and SI-NETs (p=0.006 and p=0.04, respectively). Conclusion: SI-NETs have lower SST2 promoter methylation levels and lower H3K27me3 methylation levels compared to normal SI-tissue. Moreover, in contrast to the absence of a correlation with SST2 protein expression levels, significant negative correlations were found between SST2 mRNA expression level and the mean level of DNA methylation within the SST2 promoter region in both normal SI-tissue and SI-NET tissue. These results indicate that DNA methylation might be involved in regulating SST2 expression. However, the role of histone modifications in SI-NETs remains elusive.

Proteomic analysis of small intestinal neuroendocrine tumors and mesenteric fibrosis.

Mesenteric metastases in small intestinal neuroendocrine tumors (SI-NETs) are associated with mesenteric fibrosis (MF) in a proportion of patients. MF can induce severe abdominal complications, and an effective preventive treatment is lacking. To elucidate possible novel therapeutic targets, we performed a proteomics-based analysis of MF. The tumor cell and stromal compartment of primary tumors and paired mesenteric metastases of SI-NET patients with MF (n = 6) and without MF (n = 6) was analyzed by liquid chromatography-mass spectrometry-based proteomics. Analysis of differential protein abundance was performed. Collagen alpha-1(XII) (COL12A1) and complement component C9 (C9) expression was evaluated by immunohistochemistry (IHC) in mesenteric metastases. A total of 2988 proteins were identified. Unsupervised hierarchical clustering showed close clustering of paired primary and mesenteric tumor cell samples. Comparing MF to non-MF samples, we detected differentially protein abundance solely in the mesenteric metastasis stroma group. There was no differential abundance of proteins in tumor cell samples or primary tumor stroma samples. Analysis of the differentially abundant proteins (n = 36) revealed higher abundance in MF samples of C9, various collagens and proteoglycans associated with profibrotic extracellular matrix dysregulation and signaling pathways. Proteins involved in fatty acid oxidation showed a lower abundance. COL12A1 and C9 were confirmed by IHC to have significantly higher expression in MF mesenteric metastases compared to non-MF. In conclusion, proteome profiles of SI-NETs with and without MF differ primarily in the stromal compartment of mesenteric metastases. Analysis of differentially abundant proteins revealed possible new signaling pathways involved in MF development. In conclusion, proteome profiles of SI-NETs with and without MF differ primarily in the stromal compartment of mesenteric metastases. Analysis of differentially abundant proteins revealed possible new signaling pathways involved in MF development.

Applying HDACis to increase SSTR2 expression and radiolabeled DOTA-TATE uptake: from cells to mice.

AIMS: The aim of our study was to determine the effect of histone deacetylase (HDAC) inhibitors (HDACis) on somatostatin type-2 receptor (SSTR2) expression and [111In]In-/[177Lu]Lu-DOTA-TATE uptake in vitro and in vivo. MATERIALS AND METHODS: The human cell lines NCI-H69 (small-cell lung carcinoma) and BON-1 (pancreatic neuroendocrine tumor) were treated with HDACis (i.e. entinostat, mocetinostat (MOC), LMK-235, CI-994 or panobinostat (PAN)), and SSTR2 mRNA expression levels and [111In]In-DOTA-TATE uptake were measured. Furthermore, vehicle- and HDACi-treated NCI-H69 and BON-1 tumor-bearing mice were injected with radiolabeled DOTA-TATE followed by biodistribution studies. Additionally, SSTR2 and HDAC mRNA expression of xenografts, and of NCI-H69, BON-1, NCI-H727 (human pulmonary carcinoid) and GOT1 (human midgut neuroendocrine tumor) cells were determined. KEY FINDINGS: HDACi treatment resulted in the desired effects in vitro. However, no significant increase in tumoral DOTA-TATE uptake was observed after HDACi treatment in NCI-H69 tumor-bearing animals, whereas tumoral SSTR2 mRNA and/or protein expression levels were significantly upregulated after treatment with MOC, CI-994 and PAN, i.e. a maximum of 2.1- and 1.3-fold, respectively. Analysis of PAN-treated BON-1 xenografts solely demonstrated increased SSTR2 mRNA expression levels. Comparison of HDACs and SSTR2 expression in BON-1 and NCI-H69 xenografts showed a significantly higher expression of 6/11 HDACs in BON-1 xenografts. Of these HDACs, a significant inverse correlation was found between HDAC3 and SSTR2 expression (Pearson r = -0.92) in the studied cell lines. SIGNIFICANCE: To conclude, tumoral uptake levels of radiolabeled DOTA-TATE were not enhanced after HDACi treatment in vivo, but, depending on the applied inhibitor, increased SSTR2 expression levels were observed.