A Potential Role of Neuroendocrine Tumor Gene MEN1 in Reserve Intestinal Stem Cell (rISC) Proliferation

Abstract

IntroductionNeuroendocrine Tumors (NETs) are rare, slow‐growing neoplasias that feature an excess of hormone‐producing endocrine cells. The occurrence of NETs has long been linked to loss‐of‐function mutations of the tumor suppressor gene, Men1, which encodes the transcription‐factor associating protein menin. Evidence suggests that cancers originate from stem cell populations that acquire driver mutations, resulting in tissue hyperplasia. In the intestine, the cell of origin and underlying mutations giving rise to Gastrointestinal NETs (GINETs) is unknown. The intestinal epithelium is renewed by two populations of stem cells (ISCs), one that actively proliferates (aISCs) and one that is a quiescent ‘reserve’ population (rISC). rISCs express high levels of the transcription factor, SOX9, and also exhibit stem cell as well as endocrine‐like gene expression profiles.HypothesisWe hypothesize that the Men1 gene suppresses stem cell proliferation in the intestinal crypt compartment, and a loss‐of‐function mutation in ISCs catalyzes GI‐NET tumorigenesis.ResultsKi67 immunohistochemistry staining of a tamoxifen‐induced Men1 conditional knockout from a Men1fl/fl:Sox9‐IRES‐CreERT2:Sox9EGFP mouse model trends towards an expansion of the proliferative zone. Interestingly, Pyronin Y flow cytometric analysis of Sox9high cells derived from Men1 knockouts indicates elevated levels of RNA consistent with an active cell cycle, supporting Men1‐dependent cycle re‐entry of the Sox9high population. A FACS (fluorescence activated cell sorting) cell cycle assay using single Sox9high (rISCs) dissociated from a Sox9EGFP mouse demonstrates that Sox9high cells in S/G2/M cell‐cycle state preferentially form organoids compared to the more numerous G0/G1 population. Future studies will evaluate whether the loss of Men1 drives Sox9high cells to preferentially enter S/G2/M, indicating an increase in proliferative potential.ConclusionsEarly evidence of enhanced proliferation within the intestinal crypt compartment suggests that the conditional deletion of Men1 in Sox9 expressing stem cell populations may lead to hyperplasia. While Pyronin Y expression indicates a ‘ramping‐up’ of RNA production prior to cell‐cycle, future studies will evaluate the role of Men1 in driving Sox9high cells into S/G2/M phase. This result would indicate that these cells have undergone a transformation in proliferative potential consistent with our observed increase in organoid formation efficiency of the rISC population. Longitudinal in vivo studies are in progress to evaluate NET formation due to Men1 deletion alone and in combination with other candidate tumor suppressor genes.Support or Funding InformationKevin was supported through funding by the American Physiological Society's Undergraduate Summer Research Fellowship. Additional funding was provided by the University Cancer Research Fund Development Grant. Special thanks for the support provided from the following: Magness Lab, UNC Gastrointestinal Stem Cell Group, UNC Advanced Analytics Core, UNC Zeiss Confocal Microscopy Core, and NIH T35‐DK007386.