Genome analysis identifies differences in the transcriptional targets of duodenal versus pancreatic neuroendocrine tumours

Abstract

ObjectiveGastroenteropancreatic neuroendocrine tumours (GEP-NETs) encompass a diverse group of neoplasms that vary in their secretory products and in their location within the gastrointestinal tract. Their prevalence in the USA is increasing among all adult age groups.AimTo identify the possible derivation of GEP-NETs using genome-wide analyses to distinguish small intestinal neuroendocrine tumours, specifically duodenal gastrinomas (DGASTs), from pancreatic neuroendocrine tumours.DesignWhole exome sequencing and RNA-sequencing were performed on surgically resected GEP-NETs (discovery cohort). RNA transcript profiles available in the Gene Expression Omnibus were analysed using R integrated software (validation cohort). Digital spatial profiling (DSP) was used to analyse paraffin-embedded GEP-NETs. Human duodenal organoids were treated with 5 or 10 ng/mL of tumor necrosis factor alpha (TNFα) prior to qPCR and western blot analysis of neuroendocrine cell specification genes.ResultsBoth the discovery and validation cohorts of small intestinal neuroendocrine tumours induced expression of mesenchymal and calcium signalling pathways coincident with a decrease in intestine-specific genes. In particular, calcium-related, smooth muscle and cytoskeletal genes increased in DGASTs, but did not correlate with MEN1 mutation status. Interleukin 17 (IL-17) and tumor necrosis factor alpha (TNFα) signalling pathways were elevated in the DGAST RNA-sequencing. However, DSP analysis confirmed a paucity of immune cells in DGASTs compared with the adjacent tumour-associated Brunner’s glands. Immunofluorescent analysis showed production of these proinflammatory cytokines and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) by the tumours and stroma. Human duodenal organoids treated with TNFα induced neuroendocrine tumour genes, SYP, CHGA and NKX6.3.ConclusionsStromal–epithelial interactions induce proinflammatory cytokines that promote Brunner’s gland reprogramming.

GFAP-directed Inactivation of Men1 Exploits Glial Cell Plasticity in Favor of Neuroendocrine Reprogramming

“…Previous work by our group demonstrated that non-cell autonomous loss of menin protein in enteric glial cells induces gastrin hormone expression [13,14] . Furthermore, human duodenal NETs are known to express glial cell markers, with some cells within the tumor exhibiting expression of both the neuroendocrine marker Syp and the glial-specific protein S100b [14,60] . We recently used digital spatial profiling to characterize neuroglial features in a small subset of human duodenal NETs [60] .…”

“…In identifying molecular features unique to the tumor-naïve hyperplastic antrum, we focused on a subset of genes enriched in our study and previously reported to be upregulated in human duodenal gastrinomas (DGASTs), including Hap1, Mn1 , and Uchl1 [60] . Among these, the neuronal protein Huntingtin-associated protein (Hap1) is also expressed by enteroendocrine cells (EECs) within the GI tract and was recently reported to be expressed in G cells but not in other EEC lineages of the pyloric antrum [61] .…”

“…Furthermore, human duodenal NETs are known to express glial cell markers, with some cells within the tumor exhibiting expression of both the neuroendocrine marker Syp and the glial-specific protein S100b [14,60] . We recently used digital spatial profiling to characterize neuroglial features in a small subset of human duodenal NETs [60] . Tumors exhibited reduced expression of mature neuronal and glial cell markers compared to the adjacent Brunner’s glands from which 60% of these tumors are reported to originate [9] .…”

Gastrin: From Physiology to Gastrointestinal Malignancies

“…Immunofluorescent analysis indicated strong immunoreactivity of tumor cells, Brunner's glands, and the tumor stroma for both cytokines and downstream pSTAT3 activation. 149 Both IL-17 and TNF⍺ are known to activate downstream targets through NF-κB and pSTAT3 signaling pathways. Furthermore, previous studies have shown that STAT3 binds the SYP promoter, suggesting a direct mechanism for cytokine-induced neuroendocrine reprogramming.…”

“… 150–152 In support of this, treatment of normal human duodenal organoids with TNF⍺ stimulated NF-κB and pSTAT3 activation and these events coincided with increased expression of neuroendocrine transcripts SYP, CHGA , and the gastrin-specification factor NKX6.3 . 149 , 153 , 154 Cytokine-mediated regulation of NKX6.3 is underscored by in silico analysis identifying an NF-κB binding site in the 5’ UTR of the NKX6.3 promoter. Taken together, these observations suggest a role for inflammatory cytokines in potential reprogramming of the Brunner's glands in favor of neuroendocrine differentiation and tumorigenesis ( Figure 2 ).…”