Are Facultative Reserve ISCs the Cellular Origin of Familial Small Intestinal Neuroendocrine Tumors?

Abstract

See “Polyclonal crypt genesis and development of familial small intestinal neuroendocrine tumors,” by Sei Y, Feng J, Zhao X, et al, on page 140. See “Polyclonal crypt genesis and development of familial small intestinal neuroendocrine tumors,” by Sei Y, Feng J, Zhao X, et al, on page 140. Serotonin-producing neuroendocrine tumors of the small intestine (SI-NETs; or mid-gut carcinoids) are well-differentiated, slow-growing tumors. Although SI-NETs are relatively uncommon, they represent the most frequent tumor of the distal small intestine.1Lundqvist M. Wilander E. A study of the histopathogenesis of carcinoid tumors of the small intestine and appendix.Cancer. 1987; 60: 201-206Google Scholar, 2Moyana T.N. Satkunam N. A comparative immunohistochemical study of jejunoileal and appendiceal carcinoids. Implications for histogenesis and pathogenesis.Cancer. 1992; 70: 1081-1088Google Scholar, 3Cunningham J.L. Janson E.T. The biological hallmarks of ileal carcinoids.Eur J Clin Invest. 2011; 41: 1353-1360Google Scholar, 4Solcia E. Vanoli A. Histogenesis and natural history of gut neuroendocrine tumors: present status.Endocr Pathol. 2014; 25: 165-170Google Scholar, 5Chung D.C. The enigma of carcinoids.Gastroenterology. 2015; 149: 14-15Google Scholar Clinical diagnosis of SI-NET is difficult owing to their slow tumor growth and mild symptoms, and many patients with SI-NET are often diagnosed late with advanced disease and a poor prognosis.1Lundqvist M. Wilander E. A study of the histopathogenesis of carcinoid tumors of the small intestine and appendix.Cancer. 1987; 60: 201-206Google Scholar, 2Moyana T.N. Satkunam N. A comparative immunohistochemical study of jejunoileal and appendiceal carcinoids. Implications for histogenesis and pathogenesis.Cancer. 1992; 70: 1081-1088Google Scholar, 3Cunningham J.L. Janson E.T. The biological hallmarks of ileal carcinoids.Eur J Clin Invest. 2011; 41: 1353-1360Google Scholar, 4Solcia E. Vanoli A. Histogenesis and natural history of gut neuroendocrine tumors: present status.Endocr Pathol. 2014; 25: 165-170Google Scholar, 5Chung D.C. The enigma of carcinoids.Gastroenterology. 2015; 149: 14-15Google Scholar Earlier studies proposed that these tumors were derived from intraepithelial enteroendocrine cells (EECs).1Lundqvist M. Wilander E. A study of the histopathogenesis of carcinoid tumors of the small intestine and appendix.Cancer. 1987; 60: 201-206Google Scholar, 2Moyana T.N. Satkunam N. A comparative immunohistochemical study of jejunoileal and appendiceal carcinoids. Implications for histogenesis and pathogenesis.Cancer. 1992; 70: 1081-1088Google Scholar The primary candidate for the cellular origin of SI-NETs is the serotonin-producing enterochromaffin cells (EC); however, the exact histogenesis of SI-NETS has not been defined. In this issue of Gastroenterology, Sei et al6Sei Y. Feng J. Zhao X. et al.Polyclonal crypt genesis and development of familial small intestinal neuroendocrine tumors.Gastroenterology. 2016; 151: 140-151Google Scholar hypothesize that SI-NETs originate from EC cells expressing reserve intestinal stem cell (ISC) markers found primarily at +4 position in crypts and that tumors develop through a multifocal and polyclonal process. Most SI-NETs occur sporadically and, to date, no common molecular genetic basis for this disease has been found. Familial SI-NETs are even more rare and commonly present with multiple synchronous primary tumors. Recent genetic analysis of one familial SI-NET group demonstrated that SI-NET susceptibility was transmitted in an autosomal-dominant manner.7Sei Y. Zhao X. Forbes J. et al.A hereditary form of small intestinal carcinoid associated with a germline mutation in inositol polyphosphate multikinase.Gastroenterology. 2015; 149: 67-78Google Scholar All individuals with SI-NET in this family had a germ-line mutation in the inositol polyphosphate multikinase (IPMK) gene, which truncates the protein. Although mutant IPMK had reduced kinase activity and altered cellular localization that was linked to decreased p53-mediated apoptosis and increased cell survival, it has not been proven directly that this IPMK mutation is causal for SI-NET tumor development. Importantly, this IPMK mutation has not been detected in other SI-NET families.7Sei Y. Zhao X. Forbes J. et al.A hereditary form of small intestinal carcinoid associated with a germline mutation in inositol polyphosphate multikinase.Gastroenterology. 2015; 149: 67-78Google Scholar The lack of common genetic mutations in both familial and sporadic SI-NET suggest that tumor development is diverse in nature and highlights the need for further investigations into the mechanism of SI-NET tumor development. In the small intestine, LGR5+ stem cells located at the crypt base continuously divide and give rise to transit-amplifying progenitors cells within the crypt that undergo cell lineage specification into committed Notchhigh enterocytes (HES1+) and Notchlow secretory (ATOH1+) progenitors, respectively. These committed progenitors then give rise to all differentiated post-mitotic epithelial cell types that line the crypt–villus axis. Atoh1 is a basic helix–loop–helix transcription factor and ATOH1+ secretory progenitors are responsible for generating all differentiated secretory cell types including goblet, Paneth, and EECs.8Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration.Nat Rev Mol Cell Biol. 2014; 15: 19-33Google Scholar Neurogenin3 (NEUROG3) is another basic helix–loop–helix transcription factor that acts downstream of ATOH1 and all EECs in the intestine are derived from NEUROG3+ secretory progenitors. Although NEUROG3 is obligatory for early endocrine lineage specification, further EEC subtype specification and differentiation relies on a complex network of transcription factors including NEUROD1, PAX4, NKX2.2, and so on. NEUROD1 is of particular interest because it functions in later stages of EEC differentiation.9Li H.J. Ray S.K. Singh N.K. et al.Basic helix-loop-helix transcription factors and enteroendocrine cell differentiation.Diabetes Obes Metab. 2011; 13 Suppl 1: 5-12Google Scholar Traditionally, EECs have been classified according to the principal hormone they produce, but it is now clear that EEC populations are far more complex with considerable overlap. In general, different EEC populations show either intestine-specific expression or broader expression throughout the gut. Serotonin-producing EC are an example of the latter and are distributed widely in the epithelium of the stomach, small bowel, and colon. An important feature of EC cells is that they use the rate-limiting enzyme tryptophan hydroxylase-1 (TPH1) to synthesize serotonin. Serotonin is an important mediator of normal gut physiology associated with motility, enteric neurogenesis, mucosal growth/maintenance, and intestinal inflammation. Abnormal regulation of serotonin in the gut has been implicated with a diverse array of gastrointestinal disorders, such as inflammatory bowel disease and irritable bowel syndrome.10Gershon M.D. 5-Hydroxytryptamine (serotonin) in the gastrointestinal tract.Curr Opin Endocrinol Diabetes Obes. 2013; 20: 14-21Google Scholar Rapidly cycling LGR5+ stem cells are responsible for maintenance of day-to-day crypt homeostasis. However, upon injury or damage to LGR5+ cells, facultative reserve stem cell populations can be mobilized to replenish the LGR5+ stem cell pool.8Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration.Nat Rev Mol Cell Biol. 2014; 15: 19-33Google Scholar For example, a recent study showed that slow cycling label-retaining cells (LRCs) express reserve ISC markers, Tert, HopX, and Lrig1, but also Lgr5 and markers of Paneth cell and EEC lineages. Although these slow cycling LRCs do not contribute to normal crypt homeostasis, they can be induced to become rapidly cycling stem cells upon injury.11Buczacki S.J. Zecchini H.I. Nicholson A.M. et al.Intestinal label-retaining cells are secretory precursors expressing Lgr5.Nature. 2013; 495: 65-69Google Scholar In addition, other EEC secretory progenitors (NEUROG3+ CHGA+) found at the +4 position or lower in the crypt have been described to express many reserve ISC markers, suggesting that they may also have the potential to function as facultative reserve stem cells.12Roche K.C. Gracz A.D. Liu X.F. et al.SOX9 maintains reserve stem cells and preserves radioresistance in mouse small intestine.Gastroenterology. 2015; 149: 1553-1563 e10Google Scholar, 13Schonhoff S.E. Giel-Moloney M. Leiter A.B. Neurogenin 3-expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types.Dev Biol. 2004; 270: 443-454Google Scholar, 14Sei Y. Lu X. Liou A. et al.A stem cell marker-expressing subset of enteroendocrine cells resides at the crypt base in the small intestine.Am J Physiol Gastrointest Liver Physiol. 2011; 300: G345-G356Google Scholar In the mouse small intestine, both Lgr5- and Bmi1-expressing cells are susceptible to Wnt-dependent adenoma formation, but there is limited information on whether LRCs or other facultative reserve stem cells can be transformed. In the current manuscript, Sei et al from the National Institutes of Health have investigated whether ECs located at +4 position or lower in the crypt have reserve stem cell characteristics and, if so, whether they are involved in the development of familial SI-NET. To examine this possible relationship, the authors analyzed jejunal–ileal tissue specimens from 14 patients with familial SI-NETs. They initially characterized stem cell marker expression in normal EC cells. In normal ileum, dual-labeling in situ hybridization and immunochemistry revealed that a significant subset of THP1+ EC cells found at or below the +4 position expressed NEUROG3, NEUROD1, and CHGA and had overlapping expression of the rapidly cycling ISC gene LGR5 and reserve ISC genes BMI1 and HOPX. Although these results suggest secretory progenitors expressing reserve ISC markers are conserved in mice and humans, it awaits further investigation as to whether these EC cells truly represent facultative reserve stem cells with the ability to be mobilized and dedifferentiate into LGR5+ stem cells. Further studies are also needed to understand the relationship between EC cells expressing reserve ISC markers and their more differentiated counterparts. Strikingly, when Sei et al performed the same expression analysis in familial SI-NETs, they found all TPH1+ NEUROD1+ tumor cells to have abundant expression of reserve marker genes BMI1, HOPX, LRIG1, and DCLK1, but negligible expression of the rapidly cycling ISC marker LGR5. The authors then utilized the multifocal nature of tumor formation within familial SI-NETs to perform a comprehensive analysis of ISC marker gene expression at different stages of tumor development. Early THP1+ tumor clusters at the crypt base coexpressed the ISC marker LGR5, whereas more advanced aberrant crypts containing endocrine clusters and intraepithelial tumor cells had lost LGR5 expression. By contrast, reserve ISC markers BMI1 and HOPX were expressed abundantly in all tumor stages, including extraepithelial tumor nests. Interestingly, SI-NETs were also positive for other endocrine markers CHGA and tachykinin1, but other non-EC prohormones glucagon and neurotensin were not found, providing supporting evidence that SI-NETs originate from serotonin-producing EC cells. Intriguingly, a single case of sporadic SI-NET showed a similar reserve ISC marker expression profile. Finally, the authors convincingly show using mitochondrial DNA-based clonality analysis that familial SI-NETs originate from the crypt bottom in a multifocal and polyclonal fashion. Based on these observations, Sei et al proposed that SI-NET tumors likely arise from EC cells expressing reserve ISCs makers as well as low LGR5 levels at the +4 position, but they rapidly transition and lose LGR5 expression during tumor progression. Numerous recent studies have reported plasticity and reprogramming of LRCs, facultative reserve stem cells and other more differentiated progenitor cells in the intestine. Upon loss of LGR5+ ISCs, many of these cells can dedifferentiate and replenish the LGR5+ ISC compartment.8Barker N. Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration.Nat Rev Mol Cell Biol. 2014; 15: 19-33Google Scholar However, whether all of these different facultative reserve stem cell populations can be transformed is not known.15Mills J.C. Sansom O.J. Reserve stem cells: Differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract.Sci Signal. 2015; 8: re8Google Scholar Wnt signaling is not required for EEC specification or terminal differentiation in mice; however, it is interesting to note that Wnt activation in early Neurog3-expressing endocrine progenitors can induce slow-growing adenomas expressing serotonin in the small intestine.16Wang Y. Giel-Moloney M. Rindi G. et al.Enteroendocrine precursors differentiate independently of Wnt and form serotonin expressing adenomas in response to active beta-catenin.Proc Natl Acad Sci U S A. 2007; 104: 11328-11333Google Scholar By contrast, Wnt activation in NeuroD1-expressing EEC cells, which represent a later stage of EEC differentiation, did not produce tumors.16Wang Y. Giel-Moloney M. Rindi G. et al.Enteroendocrine precursors differentiate independently of Wnt and form serotonin expressing adenomas in response to active beta-catenin.Proc Natl Acad Sci U S A. 2007; 104: 11328-11333Google Scholar In light of the fact that the familial SI-NET tumors express well-differentiated endocrine markers, NEUROD1, CHGA, THP-1, and tachykinin, it raises the question about the cellular origin of tumor initiation. Although familial SI-NET is transmitted in an autosomal-dominant manner, the long delay to tumor formation and its multifocal and polyclonal nature suggest that the germline mutation, which must be carried by all LGR5+ stem cells, is not sufficient to initiate tumor formation directly and that additional mutations or environmental effects are required. This is reminiscent of the 2-step mutation model of tumor formation recently reviewed by Mills and Sansom.15Mills J.C. Sansom O.J. Reserve stem cells: Differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract.Sci Signal. 2015; 8: re8Google Scholar In this model, quiescent or slow cycling EC progenitors expressing reserve ISC markers would be expected to accumulate additional mutations before transformation, but whether these cells then undergo direct transformation or require dedifferentiation to a more proliferative state permissive for transformation will need to be determined.15Mills J.C. Sansom O.J. Reserve stem cells: Differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract.Sci Signal. 2015; 8: re8Google Scholar The results from Sei et al clearly demonstrate the possible involvement of EC progenitors expressing reserve ISC markers in SI-NET tumor development. Further understanding of these events may provide therapeutic opportunities for the treatment of both familial and sporadic SI-NET. Polyclonal Crypt Genesis and Development of Familial Small Intestinal Neuroendocrine TumorsGastroenterologyVol. 151Issue 1PreviewSmall intestinal neuroendocrine tumors (SI-NETs) are serotonin-secreting well-differentiated neuroendocrine tumors believed to originate from enterochromaffin (EC) cells. Intestinal stem cell (ISC) are believed to contribute to the formation of SI-NETs, although little is known about tumor formation or development. We investigated the relationship between EC cells, ISCs, and SI-NETs. Full-Text PDF Covering the CoverGastroenterologyVol. 151Issue 1PreviewIn this phase 2 randomized clinical trial, a twice-daily subcutaneous synthetic ghrelin agonist, relamorelin, significantly reduced vomiting frequency and severity and accelerated gastric emptying in patients with moderate-to-severe diabetic gastroparesis. Full-Text PDF