Somatostatin (SS) and dopamine (DA) receptors have been highlighted as two critical regulators in the negative control of hormonal secretion in a wide group of human endocrine tumors. Both families of receptors belong to the superfamily of G protein-coupled receptors and share a number of structural and functional characteristics. Because of the generally reported high expression of somatostatin receptors (SSTRs) in neuroendocrine tumors (NET), somatostatin analogs (SSA) have a pronounced role in the medical therapy for this class of tumors, especially pituitary adenomas and well-differentiated gastroenteropancreatic NET (GEP NET). Moreover, NET express not only SSTR but also frequently dopamine receptors (DRs), and DA agonists targeting the D2 receptor (D2) have been demonstrated to be effective in controlling hormone secretion and cell proliferation in in vivo and in vitro studies. The treatment with SSAs combined with DA agonists has already been demonstrated efficacious in a subgroup of patients with GH-secreting pituitary adenomas and few reported cases of carcinoids. The recent availability of new selective and universal SSA and DA agonists, as well as the chimeric SS/DA compounds, may shed new light on the potential role of SSTR and D2 as combined targets for biotherapy in NET. This review provides an overview of the latest studies evaluating the expression of SSTR and DR in NET, focusing on their co-expression and the possible clinical implications of such co-expression. Moreover, the most recent insights in SSTR and D2 pathophysiology and the future perspectives for treatment with SSA, DA agonists, and SS/DA chimeric compounds are discussed.
Federico Gatto and Leo J Hofland
Thomas Cuny, Wouter de Herder, Anne Barlier and Leo J Hofland
Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) represent a group of heterogeneous tumors whose incidence increased over the past few years. Around half of patients already present with metastatic disease at the initial diagnosis. Despite extensive efforts, cytotoxic and targeted therapies have provided only limited efficacy for patients with metastatic GEP-NETs, mainly due to the development of a certain state of resistance. One factor contributing to both the failure of systemic therapies and the emergence of an aggressive tumor phenotype may be the tumor microenvironment (TME), comprising dynamic and adaptative assortment of extracellular matrix components and non-neoplastic cells, which surround the tumor niche. Accumulating evidence shows that the TME can simultaneously support both tumor growth and metastasis and contribute to a certain state of resistance to treatment. In this review, we summarize the current knowledge of the TME of GEP-NETs and discuss the current therapeutic agents that target GEP-NETs and those that could be of interest in the (near) future.
Anela Blažević, Johannes Hofland, Leo J Hofland, Richard A Feelders and Wouter W de Herder
Small intestinal neuroendocrine tumours (SI-NETs) are neoplasms characterized by their ability to secrete biogenic amines and peptides. These cause distinct clinical pathology including carcinoid syndrome, marked by diarrhoea and flushing, as well as fibrosis, notably mesenteric fibrosis. Mesenteric fibrosis often results in significant morbidity by causing intestinal obstruction, oedema and ischaemia. Although advancements have been made to alleviate symptoms of carcinoid syndrome and prolong the survival of patients with SI-NETs, therapeutic options for patients with mesenteric fibrosis are still limited. As improved insight in the complex pathogenesis of mesenteric fibrosis is key to the development of new therapies, we evaluated the literature for known and putative mediators of fibrosis in SI-NETs. In this review, we discuss the tumour microenvironment, growth factors and signalling pathways involved in the complex process of fibrosis development and tumour progression in SI-NETs, in order to elucidate potential new avenues for scientific research and therapies to improve the management of patients suffering from the complications of mesenteric fibrosis.
Anela Blažević, Wouter T Zandee, Gaston J H Franssen, Johannes Hofland, Marie-Louise F van Velthuysen, Leo J Hofland, Richard A Feelders and Wouter W de Herder
Mesenteric fibrosis (MF) surrounding a mesenteric mass is a hallmark feature of small intestinal neuroendocrine tumours (SI-NETs). Since this can induce intestinal obstruction, oedema and ischaemia, prophylactic resection of the primary tumour and mesenteric mass is often recommended. This study assessed the predictors for mesenteric metastasis and fibrosis and the effect of MF and palliative surgery on survival. A retrospective analysis of 559 patients with pathologically proven SI-NET and available CT-imaging data was performed. Clinical characteristics, presence of mesenteric mass and fibrosis on CT imaging and the effect of palliative abdominal surgery on overall survival were assessed. We found that MF was present in 41.4%. Older age, 5-HIAA excretion ≥67 μmol/24 h, serum CgA ≥121.5 μg/L and a mesenteric mass ≥27.5 mm were independent predictors of MF. In patients ≤52 years, mesenteric mass was less often found in women than in men (39% vs 64%, P = 0.002). Corrected for age, tumour grade, CgA and liver metastasis, MF was not a prognostic factor for overall survival. In patients undergoing palliative surgery, metastasectomy of mesenteric mass or prophylactic surgery did not result in survival benefit. In conclusion, we confirmed known predictors of MF and mesenteric mass and suggest a role for sex hormones as women ≤52 years have less often a mesenteric mass. Furthermore, the presence of MF has no effect on survival in a multivariate analysis, and we found no benefit of metastasectomy of mesenteric mass or prophylactic surgery on overall survival.
Peter M van Koetsveld, Giovanni Vitale, Richard A Feelders, Marlijn Waaijers, Diana M Sprij-Mooij, Ronald R de Krijger, Ernst-Jan M Speel, Johannes Hofland, Steven W J Lamberts, Wouter W de Herder and Leo J Hofland
Adrenocortical carcinoma (ACC) is an aggressive tumor with very poor prognosis. Novel medical treatment opportunities are required. We investigated the effects of interferon-β (IFN-β), alone or in combination with mitotane, on cell growth and cortisol secretion in primary cultures of 13 human ACCs, three adrenal hyperplasias, three adrenal adenomas, and in two ACC cell lines. Moreover, the interrelationship between the effects of IGF2 and IFN-β was evaluated. Mitotane inhibited cell total DNA content/well (representing cell number) in 7/11 (IC50: 38±9.2 μM) and cortisol secretion in 5/5 ACC cultures (IC50: 4.5±0.1 μM). IFN-β reduced cell number in 10/11 (IC50: 83±18 IU/ml) and cortisol secretion in 5/5 ACC cultures (IC50: 7.3±1.5 IU/ml). The effect of IFN-β on cell number included the induction of apoptosis. IFN-β strongly inhibited mRNA expression of STAR, CYP11A1, CYP17A1, and CYP11B1. Mitotane and IFN-β induced an additive inhibitory effect on cell number and cortisol secretion. IGF2 (10 nM) inhibited apoptosis and increased cell number and cortisol secretion. These effects were counteracted by IFN-β treatment. Finally, IFN-β inhibited IGF2 secretion and mRNA expression. In conclusion, IFN-β is a potent inhibitor of ACC cell growth in human primary ACC cultures, partially mediated by an inhibition of the effects of IGF2, as well as its production. The increased sensitivity of ACC cells to mitotane induced by treatment with IFN-β may open the opportunity for combined treatment regimens with lower mitotane doses. The inhibition of the expression of steroidogenic enzymes by IFN-β is a novel mechanism that may explain its inhibitory effect on cortisol production.
Aura D Herrera-Martínez, Leo J Hofland, María A Gálvez Moreno, Justo P Castaño, Wouter W de Herder and Richard A Feelders
Some biomarkers for functioning and non-functioning neuroendocrine neoplasms (NENs) are currently available. Despite their application in clinical practice, results should be interpreted cautiously. Considering the variable sensitivity and specificity of these parameters, there is an unmet need for novel biomarkers to improve diagnosis and predict patient outcome. Nowadays, several new biomarkers are being evaluated and may become future tools for the management of NENs. These biomarkers include (1) peptides and growth factors; (2) DNA and RNA markers based on genomics analysis, for example, the so-called NET test, which has been developed for analyzing gene transcripts in circulating blood; (3) circulating tumor/endothelial/progenitor cells or cell-free tumor DNA, which represent minimally invasive methods that would provide additional information for monitoring treatment response and (4) improved imaging techniques with novel radiolabeled somatostatin analogs or peptides. Below we summarize some future directions in the development of novel diagnostic and predictive/prognostic biomarkers in NENs. This review is focused on circulating and selected tissue markers.
Maria Cristina De Martino, Peter M van Koetsveld, Richard A Feelders, Diana Sprij-Mooij, Marlijn Waaijers, Steven W J Lamberts, Wouter W de Herder, Annamaria Colao, Rosario Pivonello and Leo J Hofland
Patients with adrenocortical carcinoma (ACC) need new treatment options. The aim of this study was to evaluate the effects of the mTOR inhibitors sirolimus and temsirolimus on human ACC cell growth and cortisol production. In H295, HAC15, and SW13 cells, we have evaluated mTOR, IGF2, and IGF1 receptor expressions; the effects of sirolimus and temsirolimus on cell growth; and the effects of sirolimus on apoptosis, cell cycle, and cortisol production. Moreover, the effects of sirolimus on basal and IGF2-stimulated H295 cell colony growth and on basal and IGF1-stimulated phospho-AKT, phospho-S6K1, and phospho-ERK in H295 and SW13 were studied. Finally, we have evaluated the effects of combination treatment of sirolimus with an IGF2-neutralizing antibody. We have found that H295 and HAC15 expressed IGF2 at a >1800-fold higher level than SW13. mTOR inhibitors suppressed cell growth in a dose-/time-dependent manner in all cell lines. SW13 were the most sensitive to these effects. Sirolimus inhibited H295 colony surviving fraction and size. These effects were not antagonized by IGF2, suggesting the involvement of other autocrine regulators of mTOR pathways. In H295, sirolimus activated escape pathways. The blocking of endogenously produced IGF2 increased the antiproliferative effects of sirolimus on H295. Cortisol production by H295 and HAC15 was inhibited by sirolimus. The current study demonstrates that mTOR inhibitors inhibit the proliferation and cortisol production in ACC cells. Different ACC cells have different sensitivity to the mTOR inhibitors. mTOR could be a target for the treatment of human ACCs, but variable responses might be expected. In selected cases of ACC, the combined targeting of mTOR and IGF2 could have greater effects than mTOR inhibitors alone.
Mieke E R Henfling, Aurel A Perren, Anja M Schmitt, Christiane M Saddig, Achim A Starke, Robert G Riedl, Yvonne M H Versleijen-Jonkers, Diana M Sprij-Mooij, Frans C S Ramaekers, Leo J Hofland and Ernst-Jan M Speel
Clinical and molecular studies have implicated epidermal growth factor receptor (EGFR), insulin-like growth factor (IGF) and target of rapamycin (mTOR) signaling pathways in the regulation of pancreatic neuroendocrine tumor (PanNET) growth. Interpretation and comparison of these studies is complex due to clinical and molecular tumor heterogeneity. We therefore focused in this study on insulinomas, which we examined for mRNA and protein expression of EGFR, IGF and mTOR signaling pathway components by quantitative real-time PCR (n = 48) and immunohistochemistry (n = 86). Findings were compared with normal pancreatic islets and correlated with histopathological data and clinical outcome. Insulinomas showed low EGFR and high IGF2 expression. IGFBP2, IGFBP3 and IGFBP6 mRNA levels were 2- to 4-folds higher than those in islets. High protein expression of IGF2, IGF1R and INSR (in 51–92% of the tumors) and low-to-moderate expression of mTORC1 pathway proteins p-S6k and p-4EBP1 (7–28% of the tumors) were observed. Correlations were found between (1) ERK1 mRNA expression and that of numerous IGF pathway genes, (2) p-ERK and IGF1R protein expression and (3) decrease of IGF pathway components and both metastatic disease and shorter 10-year disease-free survival. In conclusion, our observations suggest that high expression of IGF signaling pathway components is a hallmark of insulinomas, but does not necessarily lead to increased mTOR signaling. Reduced expression of IGF pathway components may be an adverse prognostic factor in insulinomas.
Aura D Herrera-Martínez, Rosanna van den Dungen, Fadime Dogan-Oruc, Peter M van Koetsveld, Michael D Culler, Wouter W de Herder, Raúl M Luque, Richard A Feelders and Leo J Hofland
Control of symptoms related to hormonal hypersecretion by functioning neuroendocrine tumors (NETs) is challenging. New therapeutic options are required. Since novel in vitro tumor models seem to better mimic the tumor in vivo conditions, we aimed to study the effect of somatostatin and dopamine receptor agonists (octreotide and cabergoline, respectively) and novel somatostatin-dopamine chimeric multi-receptor drugs (BIM-065, BIM-23A760) using 2D (monolayer) and 3D (spheroids) cultures. Dose–response studies in 2D and 3D human pancreatic NET cell cultures (BON-1 and QGP-1) were performed under serum-containing and serum-deprived conditions. Cell proliferation, somatostatin and dopamine receptor expression (SSTs and D2R), apoptosis, lactate dehydrogenase, as well as serotonin and chromogranin A (CgA) release were assessed. The following results were obtained. 3D cultures of BON-1/QGP-1 allowed better cell survival than 2D cultures in serum-deprived conditions. SSTs and D2R mRNA levels were higher in the 3D model vs 2D model. Octreotide/cabergoline/BIM-065/BIM-23A760 treatment did not affect cell growth or spheroid size. In BON-1 2D-cultures, only BIM-23A760 significantly inhibited CgA release –this effect being more pronounced in 3D cultures. In BON-1 2D cultures, cabergoline/BIM-065/BIM-23A760 treatment decreased serotonin release (maximal effect up to 40%), being this effect again more potent in 3D cultures (up to 67% inhibition; with BIM-23A760 having the most potent effects). In QGP-1, cabergoline/BIM-065 treatment decreased serotonin release only in the 3D model. In conclusion, cultures of NET 3D spheroids represent a promising method for evaluating cell proliferation and secretion in NET cell-line models. Compared to 2D models, 3D models grow relatively serum independent. In 3D model, SST-D2R multi-receptor targeting drugs inhibit CgA and serotonin secretion, but not NET cell growth.
Tullio Florio, Federica Barbieri, Renato Spaziante, Gianluigi Zona, Leo J Hofland, Peter M van Koetsveld, Richard A Feelders, Günter K Stalla, Marily Theodoropoulou, Michael D Culler, Jesse Dong, John E Taylor, Jacques-Pierre Moreau, Alexandru Saveanu, Ginette Gunz, Henry Dufour and Philippe Jaquet
Dopamine D2 and somatostatin receptors (sstrs) were reported to affect non-functioning pituitary adenoma (NFPA) proliferation in vitro. However, the reported results differ according to the experimental conditions used. We established an experimental protocol allowing reproducible evaluation of NFPA cell proliferation in vitro, to test and compare the antiproliferative effects of dopamine and somatostatin analogs (alone or in combination) with the activity of the dopamine–somatostatin chimeric molecule BIM-23A760. The protocol was utilized by four independent laboratories, studying 38 fibroblast-deprived NFPA cell cultures. Cells were characterized for GH, POMC, sstr1–sstr5, total dopamine D2 receptor (D2R) (in all cases), and D2 receptor long and short isoforms (in 15 out of 38 cases) mRNA expression and for α-subunit, LH, and FSH release. D2R, sstr3, and sstr2 mRNAs were consistently observed, with the dominant expression of D2R (2.9±2.6 copy/copy β-glucuronidase; mean±s.e.m.), when compared with sstr3 and sstr2 (0.6±1.0 and 0.3±0.6 respectively). BIM-23A760, a molecule with high affinity for D2R and sstr2, significantly inhibited [3H]thymidine incorporation in 23 out of 38 (60%) NFPA cultures (EC50=1.2 pM and E max=−33.6±3.7%). BIM-23A760 effects were similar to those induced by the selective D2R agonist cabergoline that showed a statistically significant inhibition in 18 out of 27 tumors (compared with a significant inhibition obtained in 17 out of 27 tumors using BIM-23A760, in the same subgroup of adenomas analyzed), while octreotide was effective in 13 out of 27 cases. In conclusion, superimposable data generated in four independent laboratories using a standardized protocol demonstrate that, in vitro, chimeric dopamine/sstr agonists are effective in inhibiting cell proliferation in two-thirds of NFPAs.