Neuroendocrine tumours (NET) of the gastrointestinal tract and the lung are a rare and heterogeneous group of tumours. The molecular characterization and the clinical classification of these tumours have been evolving slowly and show differences according to organs of origin. Novel technologies such as next-generation sequencing revealed new molecular aspects of NET over the last years. Notably, whole-exome/genome sequencing (WES/WGS) approaches underlined the very low mutation rate of well-differentiated NET of all organs compared to other malignancies, while the engagement of epigenetic changes in driving NET evolution is emerging. Indeed, mutations in genes encoding for proteins directly involved in chromatin remodelling, such asDAXXandATRXare a frequent event in NET. Epigenetic changes are reversible and targetable; therefore, an attractive target for treatment. The discovery of the mechanisms underlying the epigenetic changes and the implication on gene and miRNA expression in the different subgroups of NET may represent a crucial change in the diagnosis of this disease, reveal new therapy targets and identify predictive markers. Molecular profiles derived from omics data including DNA mutation, methylation, gene and miRNA expression have already shown promising results in distinguishing clinically and molecularly different subtypes of NET. In this review, we recapitulate the major genetic and epigenetic characteristics of pancreatic, lung and small intestinal NET and the affected pathways. We also discuss potential epigenetic mechanisms leading to NET development.

Recent data suggest that Pancreatic Neuroendocrine Tumours (PanNETs) originate from α- or β-cells of the islets of Langerhans. The majority of PanNETs are non-functional and do not express cell-type specific hormones. In the current study we examine whether tumour DNA methylation (DNAme) profiling combined with genomic data is able to identify cell of origin and to reveal pathways involved in PanNET progression. We analyse genome-wide DNAme data of 125 PanNETs and sorted α- and β-cells. To confirm cell identity, we investigate ARX and PDX1 expression. Based on epigenetic similarities, PanNETs cluster in α-like, β-like and intermediate tumours. The epigenetic similarity to α-cells progressively decreases in the intermediate tumours, which present unclear differentiation. Specific transcription factor methylation and expression vary in the respective α/β-tumour groups. Depending on DNAme similarity to α/β-cells, PanNETs have different mutational spectra, stage of the disease and prognosis, indicating potential means of PanNET progression.

Increasing the efficacy of approved systemic treatments in metastasized pancreatic neuroendocrine tumors (PanNET) is an unmet medical need. The antiangiogenic tyrosine kinase inhibitor sunitinib is approved for PanNET treatment. In addition, sunitinib is a lysosomotropic drug and such drugs can induce lysosomal membrane permeabilization as well as autophagy. We investigated sunitinib-induced autophagy as a possible mechanism of PanNET therapy resistance. Sunitinib accumulated in lysosomes and induced autophagy in PanNET cell lines. Adding the autophagy inhibitor chloroquine reduced cell viability in cell lines and in primary cells isolated from PanNET patients. The same treatment combination reduced tumor burden in the Rip1Tag2 transgenic PanNET mouse model. The combination of sunitinib and chloroquine reduced recovery and induced apoptosis in vitro, whereas single treatments did not. Knockdown of key autophagy proteins in combination with sunitinib showed similar effect as chloroquine. Sunitinib also induced lysosomal membrane permeabilization, which further increased in the presence of chloroquine or knockdown of lysosome-associated membrane protein (LAMP2). Both combinations led to cell death. Our data indicate that chloroquine increases sunitinib efficacy in PanNET treatment via autophagy inhibition and lysosomal membrane permeabilization. We suggest that adding chloroquine to sunitinib treatment will increase efficacy of PanNET treatment and that such patients should be included in respective ongoing clinical trials. Mol Cancer Ther; 16(11); 2502–15. ©2017 AACR.

Pancreatic neuroendocrine tumors (PanNET) comprise two molecular subtypes, relatively benign islet tumors (IT) and invasive, metastasis-like primary (MLP) tumors. Until now, the origin of aggressive MLP tumors has been obscure. Herein, using multi-omics approaches, we revealed that MLP tumors arise from IT via dedifferentiation following a reverse trajectory along the developmental pathway of islet β cells, which results in the acquisition of a progenitor-like molecular phenotype. Functionally, the miR-181cd cluster induces the IT-to-MLP transition by suppressing expression of the Meis2 transcription factor, leading to upregulation of a developmental transcription factor, Hmgb3. Notably, the IT-to-MLP transition constitutes a distinct step of tumorigenesis and is separable from the classic proliferation-associated hallmark, temporally preceding accelerated proliferation of cancer cells. Furthermore, patients with PanNET with elevated HMGB3 expression and an MLP transcriptional signature are associated with higher-grade tumors and worse survival. Overall, our results unveil a new mechanism that modulates cancer cell plasticity to enable malignant progression. Significance: Dedifferentiation has long been observed as a histopathologic characteristic of many cancers, albeit inseparable from concurrent increases in cell proliferation. Herein, we demonstrate that dedifferentiation is a mechanistically and temporally separable step in the multistage tumorigenesis of pancreatic islet cells, retracing the developmental lineage of islet β cells. This article is highlighted in the In This Issue feature, p. 2355

Molecular mechanisms underlying the development and progression of pancreatic neuroendocrine tumors (PanNETs) are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this. In this study, we describe an in vitro 3-dimensional (3-D) human primary PanNET culture system as a novel preclinical model for more personalized therapy selection. We present a screening platform allowing multicenter sample collection and drug screening in 3-D cultures of human primary PanNET cells. We demonstrate that primary cells isolated from PanNET patients and cultured in vitro form islet-like tumoroids. Islet-like tumoroids retain a neuroendocrine phenotype and are viable for at least 2 weeks in culture with a high success rate (86%). Viability can be monitored continuously allowing for a per-well normalization. In a proof-of-concept study, islet-like tumoroids were screened with three clinically approved therapies for PanNET: sunitinib, everolimus and temozolomide. Islet-like tumoroids display varying in vitro response profiles to distinct therapeutic regimes. Treatment response of islet-like tumoroids differs also between patient samples. We believe that the presented human PanNET screening platform is suitable for personalized drug testing in a larger patient cohort, and a broader application will help in identifying novel markers predicting treatment response and in refining PanNET therapy.

Background: Pancreatic neuroendocrine neoplasms (PanNENs) fall into two subclasses: the well-differentiated, low- to high-grade pancreatic neuroendocrine tumors (PanNETs), and the poorly-differentiated, high-grade pancreatic neuroendocrine carcinomas (PanNECs). While recent studies suggest an endocrine descent of PanNETs, the origin of PanNECs remains unknown.Methods: We performed DNA methylation analysis for 57 PanNEN samples and found that distinct methylation profiles separated PanNENs into two major groups, clearly distinguishing high-grade PanNECs from other PanNETs including high-grade NETG3. DNA alterations and immunohistochemistry of cell-type markers PDX1, ARX, and SOX9 were utilized to further characterize PanNECs and their cell of origin in the pancreas.Results: Phylo-epigenetic and cell-type signature features derived from alpha, beta, acinar, and ductal adult cells suggest an exocrine cell of origin for PanNECs, thus separating them in cell lineage from other PanNENs of endocrine origin.Conclusions: Our study provides a robust and clinically applicable method to clearly distinguish PanNECs from G3 PanNETs, improving patient stratification.