Disruption of the Men1 locus in epithelial and endocrine tissues fails to generate the full spectrum of gastroenteropancreatic neuroendocrine tumors (GEP-NETs), raising the possibility of a potential stromal source for these cancers. Neural crest-derived glial cells were previously implicated in neuroendocrine tumors arising in the pituitary and pancreas, yet these studies lacked a clear mechanism for these events. Here, we investigated the hypothesis that Men1-driven Hedgehog (HH) signaling redirects the glial cell fate to give rise to neuroendocrine tumors in the gastrointestinal tract.

Hyperactivation of the HH signaling pathway in human GEP-NETs was evaluated using immunofluorescent staining and clinicogenomic databases. Men1 was deleted in the glial lineage by expressing Cre recombinase downstream of the human GFAP and Sox10 promoters. Overexpression of HH signaling proteins in mouse GEP-NETs was confirmed by immunofluorescent staining and immunoblot analysis. We generated human and mouse GEP-NET tumoroids and exposed them to agonists and inhibitors of HH signaling. HH activation of Men1-deficient glial cells was blocked by deleting the gene encoding primary ciliary protein KIF3A required for transducing SHH signaling.

We demonstrated that human GEP-NETs overexpress HH signaling pathway components, including SHH and its cognate receptor PTCH1. We showed that patient-derived GEP-NET tumoroids proliferate in response to SHH pathway agonists. In contrast, pharmacologic inhibition of GLI1/2, but not inhibition of SMO alone, attenuated tumoroid growth. Genetic deletion of Men1 in GFAP⁺ and SOX10⁺ glial cells caused the development of pancreatic and intestinal NETs that overexpress HH proteins. Further use of tdTomato⁺ mice demonstrated the involvement of GFAP⁺ and SOX10⁺ glial cells in these tumors. Tumoroid cultures of mouse pancreatic, duodenal, and jejunal NETs recapitulated the drug response shown by patient-derived tumoroids. Lastly, Men1-deficient enteric glial cultures showed a glial-to-neuroendocrine transition that was alleviated upon HH inhibition, and these events were reproduced in genetic mice harboring GFAP⁺ cells with impaired primary cilia.

Our study implicates the HH signaling pathway in GEP-NET development and underscores a glial cell of origin for these tumors.