Multiomic analyses delineate human neuroendocrine tumor cell states in relation to normal enteroendocrine cell ontogeny
Pratik N.P. Singh, Elsa Hadj Bachir, James R. Howe, Andrew M. Bellizzi, Paloma Cejas, Shariq Madha-Krause, Charles B. Epstein, Jennifer A. Chan, Bradley Bernstein, Matthew H. Kulke, Qiao Zhou, Ramesh A. Shivdasani
Pratik N.P. Singh, Elsa Hadj Bachir, James R. Howe, Andrew M. Bellizzi, Paloma Cejas, Shariq Madha-Krause, Charles B. Epstein, Jennifer A. Chan, Bradley Bernstein, Matthew H. Kulke, Qiao Zhou, Ramesh A. Shivdasani
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Research Article Development Gastroenterology Oncology

Multiomic analyses delineate human neuroendocrine tumor cell states in relation to normal enteroendocrine cell ontogeny

Abstract

Cancers reflect aberrant growth and differentiation of normal cell populations. Biological understanding of small intestine neuroendocrine tumors (SI-NETs) is hampered because their closest normal counterparts, enteroendocrine cells (EECs), constitute tiny fractions of intestinal epithelium. Recent characterization of adult human EEC ontogeny from intestinal stem cells can help overcome that limitation. Transient expression of the transcription factor gene ASCL1 normally ensures proper timing and fidelity of well-differentiated EECs, which express NEUROD1. Here, we report that SI-NETs resembled mature enterochromaffin cells; however, individual tumor cells coexpressed stem/progenitor genes, harboring each differentiation state along the EEC trajectory except ASCL1+ precursors. We found that enhancers normally active, and others inactive, during EEC differentiation underlie aberrant SI-NET gene activity. SI-NETs uniformly expressed NEUROD1 but lacked ASCL1, owing to inaccessible chromatin and repressive H3K27me3 marking at the ASCL1 locus. Multiple cyclin-dependent kinase inhibitor (CDKi) genes were similarly silenced, other than CDKN1B, the only gene recurrently mutated in SI-NETs. Deletion of CDKN1B altered cell cycle kinetics during human EEC differentiation, and deletions of ASCL1 or CDKN1B activated certain genes that are expressed in SI-NETs but not in the normal EEC trajectory. We propose that a limited CDKi repertoire and absence of ASCL1-dependent constraints on EEC maturation together explain unique SI-NET characteristics.

Authors

Pratik N.P. Singh, Elsa Hadj Bachir, James R. Howe, Andrew M. Bellizzi, Paloma Cejas, Shariq Madha-Krause, Charles B. Epstein, Jennifer A. Chan, Bradley Bernstein, Matthew H. Kulke, Qiao Zhou, Ramesh A. Shivdasani

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Figure 6

Expression and regulation of CDKN1B and other cell cycle regulators in normal EEC differentiation and SI-NETs.

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Expression and regulation of CDKN1B and other cell cycle regulators in n...
See also Supplemental Figures 7 and 8. (A) CDKs and their inhibitors (CDKi) in cell cycle control and their mRNA expression in SI-NETs. RB phosphorylation by specific CDKs releases E2F to transcribe G1 and S phase genes. NEUROG3 activates CDKN1A, and approximately 10% of SI-NETs have inactivating CDKN1B mutations. Among CDKi genes, SI-NETs express low levels of CDKN2A and CDKN1C. CDKN1A and CDKN1B levels are at least an order of magnitude higher than other CDKN genes; only CDKN1B levels exceed those seen at any stage in normal EEC differentiation. The box-and-whisker plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. (B) H3K27me3 marking, inaccessible chromatin, and absence of H3K27ac signify epigenetic silencing of CDKN2A, CDKN2B, and CDKN1C loci in SI-NETs. Conversely, CDKN1B lacks H3K27me3, and multiple accessible cis-elements carry H3K27ac marks. CDKN1A lacks H3K27me3 or H3K27ac, but many sites accessible in normal EEC differentiation are inaccessible in SI-NETs. Dashed boxes outline promoters. (C) Cell cycle phases (mean ± SEM, n ≥ 3 independent experiments) in control (scrambled gRNA-edited), ASCL1-null, CDKN1B-null, and double mutant hISCNeurog3 cells. S phase, quantified by EdU flow cytometry in mCherry+ cells, dropped steeply after Tam exposure (Neurog3 activation) in all lines. G2/M phase was prolonged in CDKN1B-null cells. (D) RT-qPCR analysis of ASCL1-null, CDKN1B-null, and double-null hISCNeurog3 cells 6 days after Neurog3 activation, relative to control cells edited with scrambled gRNA. mRNA levels normalized to GAPDH are shown for proliferative and EEC markers and genes expressed in both SI-NETs and in vitro differentiated ASCL1-null cells. Data are shown as mean ± SEM, n ≥ 3 independent experiments; note different y axes (dotted lines mark 2-fold elevations in mRNA). *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test). (E) Proposed basis for SI-NET properties: absence of ASCL1 from epigenetic silencing accelerates terminal EEC differentiation, while epigenetic CDKi repression and CDKN1B mutation may combine to overcome replication arrest in differentiated cells.