Novel diagnostic biomarkers and therapeutic options for neuroendocrine tumors

textabstractNeuroendocrine tumors (NETs) represent a heterogeneous group of rare, slow-growing neoplasms, originating from enterochromaffin cells. Their annual incidence has progressively increased, although it is not known whether this is a true increase in NET incidence, the result of increased use of (improved) diagnostic procedures, or a combination of both. While metastatic disease is frequently observed at diagnosis, survival in NET patients has increased, which may be related to the advances in diagnostic and therapeutic procedures. NETs require appropriate standardized diagnostic procedures to assure early diagnosis, monitor disease progression and guide an optimal treatment. Additionally, medical treatment options in NETs have significantly increased and improved in the last years. This thesis evaluates a number of novel diagnostic and therapeutic options for patients with NET. Chapter 1 provides a general overview about NETs, emphasizes the applicability and limitations of the current diagnostic biomarkers and summarizes some putative novel biomarkers in NETs. At the same time, this chapter describes current and novel therapeutic options for functioning a non-functioning NETs. The chapter introduces the main content of this thesis: novel diagnostic and therapeutic options for NETs. Chapter 1 ends with an overview of the main aims of this thesis. Chapter 2 comprehensively evaluates the expression of several components of the somatostatin/cortistatin and ghrelin systems in a large series of well-characterized typical and atypical lung carcinoids. To the best of our knowledge, this study represents the first systematic characterization of the components of these regulatory systems in samples from lung carcinoids (LCs), in which a comparison is made with adjacent non-tumor regions and normal lung tissue. In this study, we observed that the expression of somatostatin, of some receptor subtypes (SSTs), and of the ghrelin receptor GHSR gradually increases from normal to non-tumoral adjacent and tumor tissue. Moreover, the presence of the truncated isoforms SST5TMD4, In1-ghrelin and the ghrelin receptor GHSR1b are first reported in LCs. Higher expression levels of ghrelin-O-acyltransferase (GOAT) were observed in tumors with necrosis, which were tumors with a larger size and higher capacity of peritumoral invasion and distant metastasis. These data suggest that a dysregulation of ghrelin system may be involved in the development/progression of these tumors. Chapter 3 evaluates the expression of the somatostatin/cortistatin system components in gastroenteropancreatic- (GEP-) NETs. The observed overexpression of SST5 in tumor tissue compared to adjacent non-tumor and normal tissue, as well as its clinical relation with vascular and nerve invasion suggests the putative role of SST5 as a target for treating aggressive GEP-NETs. Furthermore, it emphasizes that the evaluation of SSTs profile in GEP-NETs may provide additional information for clinical decision making, including the use of second-generation somatostatin analogs (SSAs). The most novel and relevant finding of chapter 4 is the marked overexpression of GOAT in GEP-NET samples. Whereas the expression of this enzyme is almost absent in the corresponding normal tissues, it is present in adjacent non-tumoral tissue and notably overexpressed tumor tissues. Additionally, its overexpression was demonstrated using immunohistochemistry. Importantly, this enzyme was associated to larger tumors, especially in gastrointestinal-NETs. These findings, in combination with previous publications, suggest a putative role of GOAT as a diagnostic biomarker in GEP-NETs. Chapter 5 evaluates the effect of ketoconazole on proliferation, cell cycle, apoptosis and secretion of adrenocorticotropic hormone (ACTH) or serotonin and chromogranin in an ACTH-producing bronchial NET and a non-ACTH producing pancreatic NET cell line, respectively. Ketoconazole exerted a predominant pro-apoptotic or cytotoxic effect, depending on the cell line. Additionally, ketoconazole induced changes in the cell cycle progression, e.g. it increases G0/G1 phase in both cell lines and an arrest in G2/M phase in the pancreatic NET cell line. Furthermore, a non-previously reported inhibitory effect of the SSA pasireotide on serotonin secretion by pancreatic NET cells was reported, which is probably related to the predominant SST5 expression in this cell line. In chapter 6, a comprehensive characterization of two pancreatic NET models was performed using two- (2D; monolayer) and three- dimensional (3D; spheroids) culture systems in different medium conditions. According to our results, NET 3D spheroid cultures represent a promising method for evaluating cell proliferation and secretion in NET cell lines, even in serum-deprived conditions. A non-previously reported dynamic expression of SSTs and dopamine receptor (D2R) was observed during growth of both 2D and 3D cultures. The effects of the somatostatin analog octreotide, the dopamine agonist cabergoline and of two novel SSTs-D2R multi-receptor targeting drugs (BIM-065, BIM-23A760) were evaluated using both culture systems. Cabergoline and SSTs-D2R multi-receptor targeting drugs, but not octreotide, inhibited CgA and serotonin secretion, but not NET cell growth. This suggests that the effect of the SSTs-D2R multi-receptor targeting drugs on secretion is mediated by D2R, and may indicate a putative role of dopamine agonists for treating D2R expressing functioning NETs, including carcinoid syndrome. Chapter 7 shows the in vitro effects of the novel tryptophan hydoxylase inhibitor telotristat on pancreatic NET cells. Telotristat potently inhibited serotonin release in a dose-dependent manner at a clinically feasible concentration. Its combination with pasireotide, but not with octreotide, had an additive effect on serotonin secretion. In 3D cultured pancreatic NET cells, serotonin secretion was completely blocked by telotristat, whereas this drug did not influence cell growth. These data suggest that serotonin has no autocrine/paracrine effect on cell growth in this 3D PNET model. In chapter 8 decreased expression of SSTs was observed in tumor tissue of GEP-NET patients with type 2 diabetes, compared to non-diabetic GEP-NET patients. This decreased expression was not observed in type 2 diabetic patients that were treated with metformin. The in vitro experiments showed an antiproliferative effect of biguanides and statins in NET cell lines, with consequent increased apoptosis and decreased cell migration. Since metformin and statins are low cost commercially available drugs, with a large experience in their clinical use, they may represent putative options for adjuvant therapy in NETs. In Chapter 9, the results and conclusions of the studies included in this thesis, are discussed in relation to current and possible novel future diagnostic modalities, as well as therapeutic options for NET patients.