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Notch as a Driver of Gastric Epithelial Cell Proliferation
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59 60 61 62 63 1 1,2 64 Elise S. Demitrack and Linda C. Samuelson 65 1 2 66 Department of Molecular and Integrative Physiology, Department of Internal Medicine, University of Michigan, 67 Ann Arbor, Michigan 68 GC remains a serious burden, with the National Cancer 69 SUMMARY Institute estimating 26,000 new cases and 11,000 deaths in 70 The Notch signaling pathway has emerged as a critical 2016. There are few effective treatment options for GC and 71 regulator of gastric stem cell homeostasis in both mouse and only 30% of patients will survive for 5 years or more. The 72 human stomachs. Notch promotes gastric stem/progenitor recent genetic profiling of almost 300 human gastric ade- 73 cell proliferation via activation of the NOTCH1 and NOTCH2 nocarcinomas in The Cancer Genome Atlas identified 4 74 receptors. Accordingly, constitutive Notch activation actimolecular GC subgroups: Epstein–Barr virus positive, mi- 75 vates gastric stem cells, resulting in gland fission, tissue crosatellite instability, genome stable, and chromosomal 76 expansion, and tumor formation. instability.2 Each subtype was associated with a distinct 77 molecular signature, providing valuable molecular clues that 78 can help to uncover drivers of tumorigenesis and identify 79 80 The gastric epithelium is sustained by a population of stem potential therapeutic targets. cells that replenish the various mature epithelial lineages A complementary approach to molecular profiling of 81 throughout adulthood. Regulation of stem and progenitor cell human gastric cancers is to decipher the key signaling 82 proliferation occurs via basic developmental signaling path- pathways that promote gastric stem/progenitor cell prolif- 83 ways, including the Notch pathway, which recently was eration during homeostasis. Basic developmental signaling 84 described to promote gastric stem cell proliferation in both pathways that regulate stem cell homeostasis can be key 85 the mouse and human beings. Current cancer theory pro- drivers of tumorigenesis in gastrointestinal (GI) tissues. For 86 poses that adult stem cells that maintain gastrointestinal example, Wnt pathway signaling is required to maintain Q1187 tissues accumulate mutations that promote cancerous intestinal stem cells and Wnt-activating mutations are 88 growth, and that basic signaling pathways, such as Notch, observed in more than 80% of colorectal cancers, primarily 89 which stimulate stem cell proliferation, can promote tumorvia mutation of the Wnt-negative regulator APC.3 Current 90 igenesis. Accordingly, constitutive Notch activation leads to theory is that Wnt-activating mutations accumulate in in- 91 unchecked cellular proliferation and gastric tumors in getestinal stem cells to initiate and maintain tumors.4 A similar 92 netic mouse models. Furthermore, there is emerging evi93 dence suggesting that the Notch pathway may be activated in unifying tumor-driving pathway required to maintain 94 some human gastric cancers, supporting a potential role for gastric stem cells has not been identified in stomach, 95 perhaps because of the variability between GC subtypes as Notch in gastric tumorigenesis. In this review, we first sum96 well as a comparatively rudimentary understanding of marize the current understanding of gastric stem cells 97 gastric stem cells. The identification of gastric stem cells in defined by genetic mouse studies, followed by discussion of the literature regarding Notch pathway regulation of gastric mouse models has set the stage to define the niche factors 98 stem cell function in the mouse and human beings. Notch regulating these cells and thereby potential pathways 99 100 action to maintain gastric epithelial cell homeostasis and the driving gastric cancer cell proliferation. In this review we summarize studies that have shown a 101 cellular consequences of dysregulated signaling to promote tumorigenesis are discussed, including studies associating key role for the Notch signaling pathway to maintain gastric 102 Notch activation with human gastric cancer. Finally, we stem cells. Notch function in both the corpus and antral 103 compare and contrast Notch function in the stomach with regions of the stomach are presented from analysis of 104 other gastrointestinal tissues, including the intestine, to mouse models, and mouse and human gastric organoids. 105 highlight the sensitivity of the stomach to Notch-induced The studies have shown that Notch functions to promote 106 tumors. (Cell Mol Gastroenterol Hepatol 2017;-:-–-; http:// stem/progenitor cell proliferation in the stomach. Accord- 107 dx.doi.org/10.1016/j.jcmgh.2017.01.012) ingly, constitutive Notch activation in gastric stem cells was 108 109 110 Keywords: Stomach; Homeostasis; Gastric Stem Cells; Gastric Abbreviations used in this paper: ADAM10, a disintegrin and 111 Cancer. metalloproteinase 10; GC, gastric cancer; GI, gastrointestinal; GSI, 112 g-secretase inhibitor; NICD, Notch receptor intracellular domain; TX, 113 tamoxifen. 114 © 2017 The Authors. Published by Elsevier Inc. on behalf of the AGA astric cancer (GC) is a significant human health Institute. This is an open access article under the CC BY-NC-ND 115 problem. Worldwide, it is the fifth most common license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 116 2352-345X cancer and the third leading cause of cancer-related deaths.
Notch as a Driver of Gastric Epithelial Cell Proliferation
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Despite a recent decrease in incidence in the United States,
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shown to induce gastric tumors in mouse.5,6 Moreover, there is emerging evidence of Notch pathway activation in some human gastric cancers.7–12 Hence, the Notch pathway may promote both normal and cancer cell proliferation in the stomach and thus it might provide potential therapeutic targets for treatment of GC.
The Notch Signaling Pathway The Notch pathway regulates key cellular processes such as proliferation and differentiation through communication between adjacent cells.13 Notch components are expressed broadly to regulate many homeostatic processes in adult tissues, including the GI tract.14 The Notch signaling pathway, present in all metazoans, is highly conserved. In mammals, there are 4 Notch receptors (NOTCH1–4) and 5 Notch ligands (Delta-like 1, 3, 4, and Jagged 1 and 2), all of which are single-pass transmembrane proteins.15 The signal-sending cells express Notch ligand and the signalreceiving cells express Notch receptor. Engagement of ligand and receptor initiates proteolytic cleavage events at the cell membrane to release an intracellular signaling fragment of the Notch receptor: Notch receptor intracellular domain (NICD). In the intestine, the initial Notch-receptor cleavage is performed by the membrane-localized sheddase a disintegrin and metalloproteinase 10 (ADAM10) to cleave the receptor ectodomain.16 ADAM10 function in the stomach has not yet been tested. After this receptor cleavage event by ADAM10, the NICD signaling fragment is released via intramembrane cleavage by the g-secretase complex. Subsequently, NICD translocates to the nucleus and assembles a transcription activation complex with the DNA binding protein RBP-Jk (also known as CSL) and coactivators, including MAML, to induce the transcription of target genes such as those in the hairy and enhancer of split (Hes)-related gene family. Direct gene targets vary with tissue context, with data suggesting that Hes1 and Olfm4 may be direct Notch target genes in stomach and intestine.5,17 As a consequence of the requirement for interactions between juxtaposed cells, Notch signaling communicates short-range signals. Furthermore, the signal is short-lived, with receptor destruction an integral aspect of the signaling process, and rapid degradation of NICD resulting from its PEST domain.15 Thus, Notch signaling is well suited as a niche pathway to regulate stem cell behavior in GI tissues.
Mouse Gastric Stem Cells The adult glandular stomach contains 2 regions: the corpus, whose primary function is the luminal secretion of acid and digestive enzymes, and the more distal antrum, which secretes the hormone gastrin. Distinct pools of actively cycling stem cells in each region fuel epithelial cell turnover throughout life. These active stem cells generate proliferating progenitors that differentiate into the various mature epithelial cell lineages of the stomach.14 In the
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corpus, adult stem cells thought to be located in the mid- 176 region of each gland generate progeny that migrate bidi- 177 rectionally to form the differentiated cell types, including 178 short-lived surface mucous cells, and longer-lived acid- 179 secreting parietal cells, endocrine cells, and zymogenic 180 lineage cells. In contrast, antral stem cells are located at the 181 gland base and generate surface mucous cells, endocrine 182 cells, including gastrin-producing G cells, and deep mucous 183 cells. In general, cellular turnover is more rapid in the 184 antrum than in the corpus, with a time frame of several days 185 vs several weeks or months (reviewed by Mills and 186 Shivdasani18). 187 Long-term lineage tracing in genetic mouse models has 188 been the gold standard approach for identifying stem cells 189 in the GI tract. With this approach, genetic markers have 190 been shown to define gastric stem cells that generate all of 191 the differentiated epithelial cell lineages, although discovery 192 in the stomach has lagged well behind parallel studies in the 193 intestine. After the discovery of LGR5 as a marker for in- 194 testinal stem cells,19 stem cells in the gastric antrum also 195 were shown to express LGR5 by observation of lineage 196 traces in Lgr5-EGFP-ires-CreERT2 mice more than 20 Q14197 months after Cre activation with tamoxifen (TX)20 (Table 1). 198 In addition, single Lgr5-GFPþ antral cells isolated from this 199 mouse strain were capable of initiating organoids with the 200 potential to differentiate into mature gastric epithelial cell 201 types, further supporting the conclusion that LGR5 marks an 202 active antral stem cell. Interestingly, Lgr5-GFP cells isolated 203 from stomach or intestine are both capable of forming long- 204 lived organoid lines, although they each retain regional 205 memory to form gastric or intestinal cell types despite 206 growth under similar culture conditions.20,21 This finding 207 suggests that GI tract stem cells are epigenetically marked to 208 follow prescribed region-specific differentiation programs 209 to generate mature epithelial cells. 210 Unfortunately, a specific marker for the active stem cell 211 in the adult corpus region of the stomach has not yet been 212 described. In contrast to the antrum, Lgr5-EGFP-ires- 213 CreERT2 does not mark active corpus stem cells; however, it 214 does mark progenitors in the immature neonatal stomach 215 that form adult corpus stem cells.20 Thus, the corpus stands 216 apart from more distal regions of the GI tract in regard to 217 expression of Lgr5. Recently, it was shown that the Wnt 218 signaling pathway drives gastric corpus patterning during 219 mouse stomach development, and that Wnt activation in 220 cultured human pluripotent stem cells induces differentia- 221 tion into corpus epithelial cell types,22 an important finding 222 that will further our understanding of pathway regulation of 223 adult gastric corpus stem cells. 224 In addition to specific stem cell markers, mouse genetic 225 alleles have been described that are expressed in differen- 226 tiated gastric cell types as well as in active stem cells. 227 Although not specific to stem cells, these Cre drivers still can 228 be useful tools to manipulate stem cell function because 229 they have been observed to generate long-term lineage 230 stripes after Cre activation (Table 1). These strains include 231 Sox2-CreER,23 Lrig1-CreERT2,24 CCK2R-CreERT,25 and eR1- 232 CreERT226 (Table 1). The Sox2 allele is particularly useful 233 because it is expressed in both corpus and antral stem cells, 234
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NOTE. Mouse Cre drivers expressed in gastric stem cells are categorized based on their contribution to normal homeostasis (active stem cell) or in response to injury (induced stem cell). Some Cre drivers also are expressed in gastric differentiated cell types. a Occasional slowly expanding lineage stripes appear to originate from differentiated chief cells, with accelerated expansion after loss of active stem/progenitor cells (Stange et al29). b Lineage stripes appear to originate from the isthmus region from slowly cycling cells, with accelerated expansion after loss of active stem/progenitor cells (Hayakawa et al30).
Hayakawa et al30 and Stange et al29
Stange et al29 Yes
Yes No, yesb
No
Yes
a,b
Yes No No
a
Qiao et al28
Matsuo et al26
Troy-eGFP-ires-CreERT2
No
a
No
-
Yes No No 12.4KVil-Cre
Yes Yes eR1-CreERT2
Yes Lrig1-CreERT2
Yes
Yes Sox2-CreER
Yes
-
-
Yes CCK2R-CreERT
No Yes Lgr5-EGFP-ires-CreERT2
No
-
a
No
Yes
Yes
Arnold et al23 Yes
Hayakawa et al25
Barker et al No
Yes (corpus)
20
Differentiated cells? Corpus Antrum Antrum
Corpus
Powell et al24 and Samuelson, unpublished data
Notch Drives Gastric Cell Proliferation
Strain name
Induced stem cell? Active stem cell?
Table 1.Genetic Mouse Strains Expressed in Adult Gastric Stem Cells
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but it is not expressed in the intestine. This is hugely ad- 294 vantageous for studying pathway regulation of gastric stem 295 cells because it will allow genetic manipulation of the 296 stomach without affecting the intestine, where changes to 297 stem cell function can limit animal viability. One consider- 298 ation is that most of the genetic mouse strains that have 299 been identified to mark gastric stem cells have not been 300 studied extensively, so the general utility of an individual 301 strain is uncertain. 302 Cellular plasticity is a hallmark of GI tissues, where 303 cellular reprogramming has been suggested to allow 304 committed progenitors or even mature cells to serve as 305 reserve stem cells when active stem cells are compro- 306 mised.27 The existence of dedicated quiescent, or reserve, 307 stem cell populations in the stomach has not been estab- 308 lished definitively. However, certain populations of non- 309 proliferating gastric epithelial cells have been shown to 310 expand during tissue injury and show active stem cell 311 lineage tracing activity. These include rare quiescent antral 312 stem cells that express a Villin Cre driver,28 and a small 313 population of differentiated corpus chief cells expressing 314 Troy-29 and Mist1-Cre drivers29,30 (Table 1). One of these 315 studies reported that Mist1-CreERT2 also is expressed in 316 rare quiescent stem cells in the isthmus of corpus glands 317 that can serve as the cell-of-origin for gastric cancer in 318 mouse models.30 Furthermore, recent studies have reported 319 that CCK2R-CreERT–expressing antral stem cells expand in 320 response to gastrin,25 and that LGR5þ antral stem cells can 321 expand in response to Helicobacter infection.31 322 One major challenge for stem cell studies in the stomach 323 is the extensive gastric cell toxicity observed in genetic 324 models using TX-regulated Cre drivers. TX targets the pa- 325 rietal cells, inducing rapid cell death, with increased corpus 326 progenitor cell proliferation and metaplasia following cell 327 death. These cellular changes are transient and normal Q15328 cellular homeostasis returns within 3 weeks.32 Parietal cells 329 are well known to be lynchpins in the stomach, required for 330 corpus epithelial cell homeostasis and maintenance of the 331 zymogenic cells in the gland base.27 Almost all of the genetic 332 alleles that have been described to manipulate gastric stem 333 cells in the mouse are TX-regulated Cre drivers (Table 1). 334 The TX-induced parietal cell damage is dose-dependent,32 335 and in many instances the observed lineage traces re- 336 ported for many of the gastric Cre drivers used doses that 337 would be expected to induce parietal cell damage. Thus, 338 there is likely to be an injury component to the response 339 that needs to be considered. To avoid parietal cell injury, 340 new genetic tools are needed that do not rely on acute TX 341 treatment to manipulate genes in gastric stem cells. 342 343 344 Notch Regulation of Gastric Epithelial Cell 345 Proliferation 346 Notch recently was established as a crucial pathway 347 regulating gastric stem cell proliferation and differentiation. 348 In the fetal mouse stomach, Notch pathway components 349 Notch1 and Jag2, and the Notch target gene Hes1, have been 350 mapped to the nascent gastric epithelium.6 Analysis of Hes1 351 mutant mice showed that the Notch pathway regulates 352
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cellular differentiation in the developing stomach, with excessive endocrine cell differentiation observed in perinatal mice.33 In the adult stomach, the consequences of Notch pathway inhibition and constitutive pathway activation also have been established from analysis of mouse models. Notch inhibition by g-secretase inhibitor (GSI) treatment in adult mice showed reduced progenitor cell proliferation in both corpus6,34 and antrum.5,6,35 Importantly, GSI treatment also reduced the number of proliferating Lgr5-GFP antral stem cells.5 Furthermore, single fluorescence-activated cell sorter–isolated Lgr5-GFP stem cells derived from GSI-treated mice initiated fewer organoids than stem cells derived from control mice, suggesting that Notch is required to support antral stem cell function.5 In contrast, constitutive Notch activation in LGR5þ antral stem cells using genetic overexpression of NICD induced stem and progenitor cell proliferation.5 Similarly, NICD expression in immature parietal cells in fetal mice and in adult corpus stem cells induced gastric epithelial cell proliferation.6,34 Furthermore, gastric organoids derived from Notch-activated adult corpus and antral stem cells grow faster, consistent with Notch pathway induction of stem and progenitor cell proliferation.5,34 Overall, these studies suggest that Notch promotes epithelial cell proliferation and is required to maintain stem cells in both corpus and antrum. Studies in both the mouse and human beings showed that Notch regulation of gastric antral and corpus progenitor cell proliferation occurs via NOTCH1 and NOTCH2 receptor signaling. Gene expression analysis has shown that NOTCH1 and NOTCH2 are the predominant Notch receptors expressed in the mouse and human stomach.34,35 Moreover, a Notch1-CreERT mouse strain induced full antral lineage stripes, indicating that active NOTCH1-receptor signaling occurs in antral stem cells.5,35 Inhibitory antibodies that target NOTCH1 or NOTCH2 significantly reduced stem and progenitor cell proliferation in mouse stomach and decreased growth of corpus and antral organoids, suggesting that both receptors promote gastric epithelial cell proliferation in vivo and in vitro.34,35 Furthermore, inhibition of NOTCH1 and NOTCH2 in organoids derived from human gastric glands from both corpus and antrum also led to reduced organoid growth, suggesting that the Notch pathway is crucial to support human gastric stem cell function.34,35 Importantly, the human and mouse studies concur, validating the mouse as a reliable model to probe pathway regulation of human gastric stem cells.
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there was increased expression of lineage-directing transcription factors FoxQ1 (surface mucous),36 Spdef (deep mucous),37 and Neurog3 (endocrine)38 in the Notchinhibited antral stomach.5,35 Similarly, expression of differentiated markers was increased in antral organoids treated with GSI or NOTCH1- and NOTCH2-receptor antibodies. In addition, Notch inhibition led to epithelial cell remodeling, with increased expression of secretory proteins normally associated with other regions of the GI tract, including intestine.35 In contrast to the general increase in antral epithelial cell differentiation observed after Notch inhibition, constitutive Notch activation via NICD expression in LGR5þ antral stem cells reduced differentiation, including decreased expression of transcription factors and markers for all epithelial cell lineages.5 Furthermore, antral organoids expressing NICD showed decreased expression of transcription factors and markers for endocrine and deep mucous cells. Together, the inhibition and activation data suggest that Notch inhibits overall differentiation in the antral stomach.5,35 This is in contrast to the intestine, where Notch functions to regulate cell fate choice, directing progenitor cells to adopt an absorptive rather than secretory cell fate via regulation of the transcription factor ATOH1.39–41 Although Notch signaling appears to suppress differentiation in the antrum, the role of Notch to regulate differentiation in the gastric corpus is less clear. Cellular turnover in the stomach varies regionally, with renewal of all antral epithelial cell lineages occurring within several weeks, and cellular renewal in the corpus occurring between several days (surface mucous) to several months (parietal, chief) (reviewed by Mills and Shivdasani18). Because Notch inhibition with GSI or NOTCH1- and NOTCH2-receptor antibodies results in rapid animal mortality,34 study of Notch regulation of complete corpus epithelial cellular renewal has not been feasible. However, it has been shown that although differentiation of surface mucous cells in the antrum is Notch-dependent,5,35 this same lineage in the gastric corpus does not appear to be affected by Notch inhibition,34 suggesting that corpus surface mucous cells are Notch-independent. Therefore, key molecular differences in corpus vs antral stem cell differentiation regulated by Notch signaling will be an important future area of research.
Notch and Gastric Tumorigenesis Notch Regulation of Gastric Epithelial Cell Differentiation In addition to regulation of proliferation, the Notch pathway regulates epithelial cell differentiation in the antral stomach. Notch signaling appears to act in a global manner, generally promoting progenitor cell proliferation and inhibiting differentiation. Analysis of mice after Notch inhibition via GSI treatment or antibody-mediated blockade of both NOTCH1 and NOTCH2 increased expression of all antral epithelial cell lineage markers, including surface mucous, deep mucous, and endocrine cells.5,35 Accordingly,
Notch regulation of gastric stem cell function in the adult mouse antrum has been more attainable than in the mouse corpus owing to the identification of LGR5 as an antral stem cell marker and the ability to track and manipulate these cells using the Lgr5-GFP-CreERT2 mouse model.20 Thus, in the mouse antrum, Notch is thought to regulate gastric stem cell number, as suggested by regulation of organoid establishment activity, and by observation of tissue expansion via increased proliferation, gland fission, and enhanced conversion of adult antral glands to monoclonality after Notch activation.5 Gland fission is a tissue growth phenomenon that normally occurs in the postnatal stomach to increase
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530 531 532 533 534 535 536 537 538 539 Notch and Gastric Cancer 540 Studies in mice have shown that NICD expression in 541 parietal progenitor cells,6 adult SOX2þ corpus stem cells,34 542 and LGR5þ antral stem cells5 lead to hyperplastic polyp 543 development in corpus or antrum. In human beings, Notch 544 pathway dysregulation, particularly via up-regulation of 545 ligands and/or receptors, has been observed in human 546 gastric cancer. Importantly, correlations between high 547 expression of NOTCH1, NOTCH2, and Jagged 1 have been 548 associated with gastric cancer morbidity.7,8,12 NOTCH1 549 activation in human gastric cancer has been associated with 550 Delta-like ligand 1 promoter methylation,11 suggesting that 551 epigenetic dysregulation of Notch signaling also may 552 contribute to gastric disease. Functional studies in human 553 gastric cancer cell lines show that pharmacologic Notch in- 554 hibition can reduce growth and invasion in lines that show 555 increased NOTCH1 expression.10 Notch manipulation also 556 can inhibit gastric tumor growth via regulation of PTEN Q16557 expression, where Notch inhibition with small interfering 558 RNA against NOTCH1 or GSI treatment leads to activation of 559 Human Gastric Cancer Stem Cells nuclear PTEN expression and growth inhibition and 560 Little is known about human gastric stem cells. Putative apoptosis of gastric cancer cells.9 Furthermore, Notch acti- 561 markers of gastric stem cells have been proposed based on vation via expression of NICD in the SC-M1 human gastric 562 their expression in cancer-initiating cells (also called cancer adenocarcinoma cell line promoted colony formation and 563 stem cells). The proliferative capacity and self-renewal tumor growth in mouse xenograft models through activa- 564 property of stem cells makes them susceptible to tumor- tion of cyclooxygenase-2 expression,12 suggesting that 565 initiating mutations, and therefore the transformation of Notch may initiate gastric tumorigenesis via cross-talk with 566 normal stem cells into cancer stem cells remains a likely other signaling pathways. Finally, the Notch pathway has 567 theory for tumorigenesis (reviewed by Vries et al4). Markers been shown to increase expression of CD4458 and CD133,59 568 of such cancer stem cells in the human stomach include putative markers of gastric cancer stem cells. Taken 569 CXCR4,46 and the cell surface markers CD133, also known as together, current data suggest significant Notch pathway 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 þ Figure 1. Notch activation in antral stem cells induces tumorigenesis. In the mouse, NICD activation in an LGR5 stem cell 585 (green) at the gland base results in increased stem cell proliferation and number followed by antral gland fission. This leads to 586 overall tissue expansion and eventual tumorigenesis. Such tumors are hyperproliferative and undifferentiated, expressing stem 587 and progenitor cell markers. 588 organ size with the growth of the organism.42 Fission is thought to occur in response to increased numbers of stem cells to spatially accommodate the increased cellular production,43 and results in a single glandular unit bifurcating, or splitting, into 2 individual glands. Gland fission during homeostatic conditions in the adult stomach is rare. The extensive gland fission that occurs as a result of constitutive NICD expression leads to tissue expansion and polyp formation within 6 months, showing that Notch activation in antral stem cells is tumorigenic (Figure 1). The NICDinduced tumors are highly proliferative, largely composed of undifferentiated cells that express progenitor cell markers, although interestingly not LGR5.5 This also has been shown in the mouse corpus, where constitutive NICD expression in parietal progenitor cells and in adult SOX2þ stem/progenitor cells induces hyperproliferative glands.6,34 The human metaplastic stomach contains clonal patches of epithelial cells, a process termed field cancerization.44,45 Whether this process is regulated by Notch signaling in the human stomach has not been shown, but our evidence from the mouse stomach suggests that unchecked signaling in pathways that regulate proliferation may give progenitor cells a selective advantage, leading to clonal expansion.5 In addition, in the human stomach it has been shown that a clonal mutational field can acquire additional new mutations, suggesting that gastric cancer may arise from a genetically advantageous proliferative subclone within the founder clonal field.44
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Prominin-147–49 and CD44,50 in particular CD44v651 and CD44v9.52 In addition, expression of SOX2 is altered in gastric cancer, although it is unclear whether SOX2 is oncogenic53,54 or has tumor-suppressing capabilities.55–57 Whether these markers also are expressed in normal human gastric stem cells is an important future question that will advance our understanding of human gastric epithelial cell biology.
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involvement in the regulation of gastric cancer cell growth, proliferation, and metastasis, highlighting Notch as a key pathway to explore further for the development of targeted therapies for gastric cancer treatment.
Notch Regulation of Gastric Versus Intestinal Stem Cells Gastric and intestinal stem cells are both regulated by Notch and Wnt, commonly using the same pathway components, such as Lgr5,19,20 and NOTCH1 and NOTCH2.35,60–64 However, these stem cell populations intrinsically are primed to perform functions specific to each tissue program. Thus, pathway regulation likely depends on specific molecular cues from the gastric or intestinal stem cell niche as it relates to overall tissue function. Interestingly, stem cells in stomach and intestine are differentially sensitive to Notch pathway activation in the context of tumorigenesis. Although constitutive expression of NICD in LGR5þ stem cells induces antral tumors with 100% penetrance, intestinal or colonic tumors are not observed.5 These findings suggest that Notch tone in the stomach and intestine differ, with gastric stem cells more sensitive to Notch-activating mutations than intestinal stem cells. Surprisingly, NICD-expressing intestinal stem cells appear to be lost, suggesting that they are at a competitive disadvantage to normal intestinal stem cells. These findings in mice would predict that Notch might play a larger role in the development of human gastric cancers than in human colon cancer. Both gastric and intestinal stem cells are affected similarly by Wnt pathway manipulation. Wnt activation via genetic deletion of Apc in mouse gastric and intestinal stem cells leads to epithelial cell hyperproliferation and tumorigenesis in both intestine65,66 and stomach.20,56 Patients with the Wnt activating genetic disorder familial adenomatous polyposis develop hundreds to thousands of intestinal adenomas that progress to colorectal cancer via accumulation of additional tumorigenic mutations (reviewed by Fearon67). Interestingly, almost all familial adenomatous polyposis patients also develop gastric polyps, typically fundic gland polyps, although they largely are benign and the risk for gastric adenocarcinoma development is low compared with the risk for colorectal cancer.68 This finding suggests that, in contrast to enhanced gastric stem cell sensitivity to Notch activation, the intestine may be more sensitive to Wnt activation than the stomach. Indeed, although adenomas are observed in both tissues after Apc deletion in LGR5 cells in mouse genetic models, the intestinal tumor burden is greater, limiting the duration of study owing to animal morbidity.20 Future studies will be necessary to determine how Notch and Wnt intersect to support the gastric and intestinal stem cell niche during normal tissue homeostasis and with diseases such as cancer.
Summary and Future Directions/Questions Notch functions to balance cellular proliferation and differentiation in the stomach, with Notch inhibition leading to reduced stem and progenitor cell proliferation and
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increased differentiation, although Notch activation results in increased stem and progenitor cell proliferation and reduced differentiation. Furthermore, genetic overexpression of NICD induces tissue expansion via gland fission and gastric tumors. Thus, Notch appears to be a central signaling pathway that promotes gastric stem cell proliferation and expansion. Furthermore, Notch pathway activation also has been reported in human gastric cancer, suggesting that this pathway may prove to be a useful therapeutic target for treatment of some human gastric cancers. Several important questions remain unanswered that undoubtedly will further our understanding of Notch pathway function in mouse and human gastric stem cells. These questions include the following: How is the Notch niche defined? What niche cells present ligand to gastric stem cells? What are the key Notch ligands in the stomach? How does Notch regulate gastric stem cell self-renewal and proliferation? How does Notch cooperate with Wnt and other signaling pathways to both maintain homeostasis and promote tumorigenesis? What are the molecular markers for human gastric stem cells? Can Notch pathway inhibition be effective for gastric cancer treatment?
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Notch Drives Gastric Cell Proliferation
10. Li LC, Peng Y, Liu YM, et al. Gastric cancer cell growth and epithelial-mesenchymal transition are inhibited by gamma-secretase inhibitor DAPT. Oncol Lett 2014; 7:2160–2164. 11. Piazzi G, Fini L, Selgrad M, et al. Epigenetic regulation of delta-like1 controls Notch1 activation in gastric cancer. Oncotarget 2011;2:1291–1301. 12. Yeh TS, Wu CW, Hsu KW, et al. The activated Notch1 signal pathway is associated with gastric cancer progression through cyclooxygenase-2. Cancer Res 2009; 69:5039–5048. 13. Bray SJ. Notch signalling in context. Nat Rev Mol Cell Biol 2016;17:722–735. 14. Demitrack ES, Samuelson LC. Notch regulation of gastrointestinal stem cells. J Physiol 2016;594: 4791–4803. 15. Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 2009; 137:216–233. 16. Tsai YH, VanDussen KL, Sawey ET, et al. ADAM10 regulates Notch function in intestinal stem cells of mice. Gastroenterology 2014;147:822–834 e13. 17. VanDussen KL, Carulli AJ, Keeley TM, et al. Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells. Development 2012;139:488–497. 18. Mills JC, Shivdasani RA. Gastric epithelial stem cells. Gastroenterology 2011;140:412–424. 19. Barker N, van Es JH, Kuipers J, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007;449:1003–1007. 20. Barker N, Huch M, Kujala P, et al. Lgr5(þve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 2010;6:25–36. 21. Sato T, Vries RG, Snippert HJ, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009;459:262–265. 22. McCracken KW, Aihara E, Martin B, et al. Wnt/betacatenin promotes gastric fundus specification in mice and humans. Nature 2017;541:182–187. 23. Arnold K, Sarkar A, Yram MA, et al. Sox2(þ) adult stem and progenitor cells are important for tissue regeneration and survival of mice. Cell Stem Cell 2011;9:317–329. 24. Powell AE, Wang Y, Li Y, et al. The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor. Cell 2012;149:146–158. 25. Hayakawa Y, Jin G, Wang H, et al. CCK2R identifies and regulates gastric antral stem cell states and carcinogenesis. Gut 2015;64:544–553. 26. Matsuo J, Kimura S, Yamamura A, et al. Identification of stem cells in the epithelium of the stomach corpus and antrum of mice. Gastroenterology 2017; 152:218–231. 27. Mills JC, Sansom OJ. Reserve stem cells: differentiated cells reprogram to fuel repair, metaplasia, and neoplasia in the adult gastrointestinal tract. Sci Signal 2015;8:re8. 28. Qiao XT, Ziel JW, McKimpson W, et al. Prospective identification of a multilineage progenitor in murine stomach epithelium. Gastroenterology 2007;133: 1989–1998.
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29. Stange DE, Koo BK, Huch M, et al. Differentiated Troyþ chief cells act as reserve stem cells to generate all lineages of the stomach epithelium. Cell 2013; 155:357–368. 30. Hayakawa Y, Ariyama H, Stancikova J, et al. Mist1 expressing gastric stem cells maintain the normal and neoplastic gastric epithelium and are supported by a perivascular stem cell niche. Cancer Cell 2015; 28:800–814. 31. Sigal M, Rothenberg ME, Logan CY, et al. Helicobacter pylori activates and expands Lgr5(þ) stem cells through direct colonization of the gastric glands. Gastroenterology 2015;148:1392–1404 e21. 32. Huh WJ, Khurana SS, Geahlen JH, et al. Tamoxifen induces rapid, reversible atrophy, and metaplasia in mouse stomach. Gastroenterology 2012;142:21–24 e7. 33. Jensen J, Pedersen EE, Galante P, et al. Control of endodermal endocrine development by Hes-1. Nat Genet 2000;24:36–44. 34. Demitrack ES, Gifford GB, Keeley TM, et al. NOTCH1 and NOTCH2 regulate epithelial cell proliferation in mouse and human gastric corpus. Am J Physiol Gastrointest Liver Physiol 2017;312:G133–G144. 35. Gifford GB, Demitrack ES, Keeley TM, et al. Notch1 and Notch2 receptors regulate mouse and human gastric antral epithelial cell homoeostasis. Gut 2016. Epub ahead of print. 36. Verzi MP, Khan AH, Ito S, et al. Transcription factor foxq1 controls mucin gene expression and granule content in mouse stomach surface mucous cells. Gastroenterology 2008;135:591–600. 37. Horst D, Gu X, Bhasin M, et al. Requirement of the epithelium-specific Ets transcription factor Spdef for mucous gland cell function in the gastric antrum. J Biol Chem 2010;285:35047–35055. 38. Lee CS, Perreault N, Brestelli JE, et al. Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity. Genes Dev 2002; 16:1488–1497. 39. Shroyer NF, Helmrath MA, Wang VY, et al. Intestinespecific ablation of mouse atonal homolog 1 (Math1) reveals a role in cellular homeostasis. Gastroenterology 2007;132:2478–2488. 40. VanDussen KL, Samuelson LC. Mouse atonal homolog 1 directs intestinal progenitors to secretory cell rather than absorptive cell fate. Dev Biol 2010;346:215–223. 41. Yang Q, Bermingham NA, Finegold MJ, et al. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science 2001;294:2155–2158. 42. Nomura S, Kaminishi M, Sugiyama K, et al. Clonal analysis of isolated intestinal metaplastic glands of stomach using X linked polymorphism. Gut 1998; 42:663–668. 43. Loeffler M, Bratke T, Paulus U, et al. Clonality and life cycles of intestinal crypts explained by a state dependent stochastic model of epithelial stem cell organization. J Theor Biol 1997;186:41–54. 44. Gutierrez-Gonzalez L, Graham TA, Rodriguez-Justo M, et al. The clonal origins of dysplasia from intestinal
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metaplasia in the human stomach. Gastroenterology 2011;140:1251–1260 e1–6. McDonald SA, Greaves LC, Gutierrez-Gonzalez L, et al. Mechanisms of field cancerization in the human stomach: the expansion and spread of mutated gastric stem cells. Gastroenterology 2008;134:500–510. Fujita T, Chiwaki F, Takahashi RU, et al. Identification and characterization of CXCR4-positive gastric cancer stem cells. PLoS One 2015;10:e0130808. Hashimoto K, Aoyagi K, Isobe T, et al. Expression of CD133 in the cytoplasm is associated with cancer progression and poor prognosis in gastric cancer. Gastric Cancer 2014;17:97–106. Wen L, Chen XZ, Yang K, et al. Prognostic value of cancer stem cell marker CD133 expression in gastric cancer: a systematic review. PLoS One 2013; 8:e59154. Zhao P, Li Y, Lu Y. Aberrant expression of CD133 protein correlates with Ki-67 expression and is a prognostic marker in gastric adenocarcinoma. BMC Cancer 2010; 10:218. Ghaffarzadehgan K, Jafarzadeh M, Raziee HR, et al. Expression of cell adhesion molecule CD44 in gastric adenocarcinoma and its prognostic importance. World J Gastroenterol 2008;14:6376–6381. da Cunha CB, Oliveira C, Wen X, et al. De novo expression of CD44 variants in sporadic and hereditary gastric cancer. Lab Invest 2010;90:1604–1614. Hirata K, Suzuki H, Imaeda H, et al. CD44 variant 9 expression in primary early gastric cancer as a predictive marker for recurrence. Br J Cancer 2013;109:379–386. Hutz K, Mejias-Luque R, Farsakova K, et al. The stem cell factor SOX2 regulates the tumorigenic potential in human gastric cancer cells. Carcinogenesis 2014; 35:942–950. Matsuoka J, Yashiro M, Sakurai K, et al. Role of the stemness factors sox2, oct3/4, and nanog in gastric carcinoma. J Surg Res 2012;174:130–135. Otsubo T, Akiyama Y, Yanagihara K, et al. SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis. Br J Cancer 2008;98:824–831. Sarkar A, Huebner AJ, Sulahian R, et al. Sox2 suppresses gastric tumorigenesis in mice. Cell Rep 2016; 16:1929–1941. Wang S, Tie J, Wang R, et al. SOX2, a predictor of survival in gastric cancer, inhibits cell proliferation and metastasis by regulating PTEN. Cancer Lett 2015; 358:210–219. Li LC, Wang DL, Wu YZ, et al. Gastric tumor-initiating CD44þ cells and epithelial-mesenchymal transition are
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884 885 59. 886 887 888 60. 889 890 891 61. 892 893 894 62. 895 896 897 898 899 63. 900 901 902 64. 903 904 905 65. 906 907 908 66. 909 910 911 912 913 67. 914 915 68. 916 917 918 919 920 921 Received November 23, 2016. Accepted January 28, 2017. 922 Correspondence 923 Q1 Address correspondence to: Linda C. Samuelson, MD, Department of Molecular and Integrative Physiology, University of Michigan, 109 Zina 924 Pitcher Place, 2041 BSRB, Ann Arbor, Michigan 48109. e-mail: Q2 925 [email protected]; fax: ---. 926 Conflicts of interest 927 Q3 The authors disclose no conflicts. 928 Funding 929 Supported by National Institutes of Health F32-DK093340, MICHR PTSP CTSA Q4 930 UL1TR000433, National Institutes of Health T32-HD007505, and an AACRDDF Career Development Award (E.S.D.); and by research support through Q5931 National Institutes of Health P01-DK062041, National Institutes of Health 932 5R01-DK096972, NCI P50-CA130810, and the American Gastroenterological 933 Association Research Foundation (L.C.S.). 934 935 936 937 938 939 940 941 942 inhibited by gamma-secretase inhibitor DAPT. Oncol Lett 2015;10:3293–3299. Konishi H, Asano N, Imatani A, et al. Notch1 directly induced CD133 expression in human diffuse type gastric cancers. Oncotarget 2016;7:56598–56607. Carulli AJ, Keeley TM, Demitrack ES, et al. Notch receptor regulation of intestinal stem cell homeostasis and crypt regeneration. Dev Biol 2015;402:98–108. Fre S, Hannezo E, Sale S, et al. Notch lineages and activity in intestinal stem cells determined by a new set of knock-in mice. PLoS One 2011;6:e25785. Riccio O, van Gijn ME, Bezdek AC, et al. Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2. EMBO Rep 2008; 9:377–383. Tran IT, Sandy AR, Carulli AJ, et al. Blockade of individual Notch ligands and receptors controls graft-versus-host disease. J Clin Invest 2013;123:1590–1604. Wu Y, Cain-Hom C, Choy L, et al. Therapeutic antibody targeting of individual Notch receptors. Nature 2010; 464:1052–1057. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 2009; 457:608–611. Powell AE, Vlacich G, Zhao ZY, et al. Inducible loss of one Apc allele in Lrig1-expressing progenitor cells results in multiple distal colonic tumors with features of familial adenomatous polyposis. Am J Physiol Gastrointest Liver Physiol 2014;307:G16–G23. Fearon ER. Molecular genetics of colorectal cancer. Annu Rev Pathol 2011;6:479–507. Arnason T, Liang WY, Alfaro E, et al. Morphology and natural history of familial adenomatous polyposis-associated dysplastic fundic gland polyps. Histopathology 2014; 65:353–362.
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59 60 61 62 63 1 1,2 64 Elise S. Demitrack and Linda C. Samuelson 65 1 2 66 Department of Molecular and Integrative Physiology, Department of Internal Medicine, University of Michigan, 67 Ann Arbor, Michigan 68 GC remains a serious burden, with the National Cancer 69 SUMMARY Institute estimating 26,000 new cases and 11,000 deaths in 70 The Notch signaling pathway has emerged as a critical 2016. There are few effective treatment options for GC and 71 regulator of gastric stem cell homeostasis in both mouse and only 30% of patients will survive for 5 years or more. The 72 human stomachs. Notch promotes gastric stem/progenitor recent genetic profiling of almost 300 human gastric ade- 73 cell proliferation via activation of the NOTCH1 and NOTCH2 nocarcinomas in The Cancer Genome Atlas identified 4 74 receptors. Accordingly, constitutive Notch activation actimolecular GC subgroups: Epstein–Barr virus positive, mi- 75 vates gastric stem cells, resulting in gland fission, tissue crosatellite instability, genome stable, and chromosomal 76 expansion, and tumor formation. instability.2 Each subtype was associated with a distinct 77 molecular signature, providing valuable molecular clues that 78 can help to uncover drivers of tumorigenesis and identify 79 80 The gastric epithelium is sustained by a population of stem potential therapeutic targets. cells that replenish the various mature epithelial lineages A complementary approach to molecular profiling of 81 throughout adulthood. Regulation of stem and progenitor cell human gastric cancers is to decipher the key signaling 82 proliferation occurs via basic developmental signaling path- pathways that promote gastric stem/progenitor cell prolif- 83 ways, including the Notch pathway, which recently was eration during homeostasis. Basic developmental signaling 84 described to promote gastric stem cell proliferation in both pathways that regulate stem cell homeostasis can be key 85 the mouse and human beings. Current cancer theory pro- drivers of tumorigenesis in gastrointestinal (GI) tissues. For 86 poses that adult stem cells that maintain gastrointestinal example, Wnt pathway signaling is required to maintain Q1187 tissues accumulate mutations that promote cancerous intestinal stem cells and Wnt-activating mutations are 88 growth, and that basic signaling pathways, such as Notch, observed in more than 80% of colorectal cancers, primarily 89 which stimulate stem cell proliferation, can promote tumorvia mutation of the Wnt-negative regulator APC.3 Current 90 igenesis. Accordingly, constitutive Notch activation leads to theory is that Wnt-activating mutations accumulate in in- 91 unchecked cellular proliferation and gastric tumors in getestinal stem cells to initiate and maintain tumors.4 A similar 92 netic mouse models. Furthermore, there is emerging evi93 dence suggesting that the Notch pathway may be activated in unifying tumor-driving pathway required to maintain 94 some human gastric cancers, supporting a potential role for gastric stem cells has not been identified in stomach, 95 perhaps because of the variability between GC subtypes as Notch in gastric tumorigenesis. In this review, we first sum96 well as a comparatively rudimentary understanding of marize the current understanding of gastric stem cells 97 gastric stem cells. The identification of gastric stem cells in defined by genetic mouse studies, followed by discussion of the literature regarding Notch pathway regulation of gastric mouse models has set the stage to define the niche factors 98 stem cell function in the mouse and human beings. Notch regulating these cells and thereby potential pathways 99 100 action to maintain gastric epithelial cell homeostasis and the driving gastric cancer cell proliferation. In this review we summarize studies that have shown a 101 cellular consequences of dysregulated signaling to promote tumorigenesis are discussed, including studies associating key role for the Notch signaling pathway to maintain gastric 102 Notch activation with human gastric cancer. Finally, we stem cells. Notch function in both the corpus and antral 103 compare and contrast Notch function in the stomach with regions of the stomach are presented from analysis of 104 other gastrointestinal tissues, including the intestine, to mouse models, and mouse and human gastric organoids. 105 highlight the sensitivity of the stomach to Notch-induced The studies have shown that Notch functions to promote 106 tumors. (Cell Mol Gastroenterol Hepatol 2017;-:-–-; http:// stem/progenitor cell proliferation in the stomach. Accord- 107 dx.doi.org/10.1016/j.jcmgh.2017.01.012) ingly, constitutive Notch activation in gastric stem cells was 108 109 110 Keywords: Stomach; Homeostasis; Gastric Stem Cells; Gastric Abbreviations used in this paper: ADAM10, a disintegrin and 111 Cancer. metalloproteinase 10; GC, gastric cancer; GI, gastrointestinal; GSI, 112 g-secretase inhibitor; NICD, Notch receptor intracellular domain; TX, 113 tamoxifen. 114 © 2017 The Authors. Published by Elsevier Inc. on behalf of the AGA astric cancer (GC) is a significant human health Institute. This is an open access article under the CC BY-NC-ND 115 problem. Worldwide, it is the fifth most common license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 116 2352-345X cancer and the third leading cause of cancer-related deaths.
Notch as a Driver of Gastric Epithelial Cell Proliferation
G
Despite a recent decrease in incidence in the United States,
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shown to induce gastric tumors in mouse.5,6 Moreover, there is emerging evidence of Notch pathway activation in some human gastric cancers.7–12 Hence, the Notch pathway may promote both normal and cancer cell proliferation in the stomach and thus it might provide potential therapeutic targets for treatment of GC.
The Notch Signaling Pathway The Notch pathway regulates key cellular processes such as proliferation and differentiation through communication between adjacent cells.13 Notch components are expressed broadly to regulate many homeostatic processes in adult tissues, including the GI tract.14 The Notch signaling pathway, present in all metazoans, is highly conserved. In mammals, there are 4 Notch receptors (NOTCH1–4) and 5 Notch ligands (Delta-like 1, 3, 4, and Jagged 1 and 2), all of which are single-pass transmembrane proteins.15 The signal-sending cells express Notch ligand and the signalreceiving cells express Notch receptor. Engagement of ligand and receptor initiates proteolytic cleavage events at the cell membrane to release an intracellular signaling fragment of the Notch receptor: Notch receptor intracellular domain (NICD). In the intestine, the initial Notch-receptor cleavage is performed by the membrane-localized sheddase a disintegrin and metalloproteinase 10 (ADAM10) to cleave the receptor ectodomain.16 ADAM10 function in the stomach has not yet been tested. After this receptor cleavage event by ADAM10, the NICD signaling fragment is released via intramembrane cleavage by the g-secretase complex. Subsequently, NICD translocates to the nucleus and assembles a transcription activation complex with the DNA binding protein RBP-Jk (also known as CSL) and coactivators, including MAML, to induce the transcription of target genes such as those in the hairy and enhancer of split (Hes)-related gene family. Direct gene targets vary with tissue context, with data suggesting that Hes1 and Olfm4 may be direct Notch target genes in stomach and intestine.5,17 As a consequence of the requirement for interactions between juxtaposed cells, Notch signaling communicates short-range signals. Furthermore, the signal is short-lived, with receptor destruction an integral aspect of the signaling process, and rapid degradation of NICD resulting from its PEST domain.15 Thus, Notch signaling is well suited as a niche pathway to regulate stem cell behavior in GI tissues.
Mouse Gastric Stem Cells The adult glandular stomach contains 2 regions: the corpus, whose primary function is the luminal secretion of acid and digestive enzymes, and the more distal antrum, which secretes the hormone gastrin. Distinct pools of actively cycling stem cells in each region fuel epithelial cell turnover throughout life. These active stem cells generate proliferating progenitors that differentiate into the various mature epithelial cell lineages of the stomach.14 In the
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corpus, adult stem cells thought to be located in the mid- 176 region of each gland generate progeny that migrate bidi- 177 rectionally to form the differentiated cell types, including 178 short-lived surface mucous cells, and longer-lived acid- 179 secreting parietal cells, endocrine cells, and zymogenic 180 lineage cells. In contrast, antral stem cells are located at the 181 gland base and generate surface mucous cells, endocrine 182 cells, including gastrin-producing G cells, and deep mucous 183 cells. In general, cellular turnover is more rapid in the 184 antrum than in the corpus, with a time frame of several days 185 vs several weeks or months (reviewed by Mills and 186 Shivdasani18). 187 Long-term lineage tracing in genetic mouse models has 188 been the gold standard approach for identifying stem cells 189 in the GI tract. With this approach, genetic markers have 190 been shown to define gastric stem cells that generate all of 191 the differentiated epithelial cell lineages, although discovery 192 in the stomach has lagged well behind parallel studies in the 193 intestine. After the discovery of LGR5 as a marker for in- 194 testinal stem cells,19 stem cells in the gastric antrum also 195 were shown to express LGR5 by observation of lineage 196 traces in Lgr5-EGFP-ires-CreERT2 mice more than 20 Q14197 months after Cre activation with tamoxifen (TX)20 (Table 1). 198 In addition, single Lgr5-GFPþ antral cells isolated from this 199 mouse strain were capable of initiating organoids with the 200 potential to differentiate into mature gastric epithelial cell 201 types, further supporting the conclusion that LGR5 marks an 202 active antral stem cell. Interestingly, Lgr5-GFP cells isolated 203 from stomach or intestine are both capable of forming long- 204 lived organoid lines, although they each retain regional 205 memory to form gastric or intestinal cell types despite 206 growth under similar culture conditions.20,21 This finding 207 suggests that GI tract stem cells are epigenetically marked to 208 follow prescribed region-specific differentiation programs 209 to generate mature epithelial cells. 210 Unfortunately, a specific marker for the active stem cell 211 in the adult corpus region of the stomach has not yet been 212 described. In contrast to the antrum, Lgr5-EGFP-ires- 213 CreERT2 does not mark active corpus stem cells; however, it 214 does mark progenitors in the immature neonatal stomach 215 that form adult corpus stem cells.20 Thus, the corpus stands 216 apart from more distal regions of the GI tract in regard to 217 expression of Lgr5. Recently, it was shown that the Wnt 218 signaling pathway drives gastric corpus patterning during 219 mouse stomach development, and that Wnt activation in 220 cultured human pluripotent stem cells induces differentia- 221 tion into corpus epithelial cell types,22 an important finding 222 that will further our understanding of pathway regulation of 223 adult gastric corpus stem cells. 224 In addition to specific stem cell markers, mouse genetic 225 alleles have been described that are expressed in differen- 226 tiated gastric cell types as well as in active stem cells. 227 Although not specific to stem cells, these Cre drivers still can 228 be useful tools to manipulate stem cell function because 229 they have been observed to generate long-term lineage 230 stripes after Cre activation (Table 1). These strains include 231 Sox2-CreER,23 Lrig1-CreERT2,24 CCK2R-CreERT,25 and eR1- 232 CreERT226 (Table 1). The Sox2 allele is particularly useful 233 because it is expressed in both corpus and antral stem cells, 234
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No Mist1-CreERT2
NOTE. Mouse Cre drivers expressed in gastric stem cells are categorized based on their contribution to normal homeostasis (active stem cell) or in response to injury (induced stem cell). Some Cre drivers also are expressed in gastric differentiated cell types. a Occasional slowly expanding lineage stripes appear to originate from differentiated chief cells, with accelerated expansion after loss of active stem/progenitor cells (Stange et al29). b Lineage stripes appear to originate from the isthmus region from slowly cycling cells, with accelerated expansion after loss of active stem/progenitor cells (Hayakawa et al30).
Hayakawa et al30 and Stange et al29
Stange et al29 Yes
Yes No, yesb
No
Yes
a,b
Yes No No
a
Qiao et al28
Matsuo et al26
Troy-eGFP-ires-CreERT2
No
a
No
-
Yes No No 12.4KVil-Cre
Yes Yes eR1-CreERT2
Yes Lrig1-CreERT2
Yes
Yes Sox2-CreER
Yes
-
-
Yes CCK2R-CreERT
No Yes Lgr5-EGFP-ires-CreERT2
No
-
a
No
Yes
Yes
Arnold et al23 Yes
Hayakawa et al25
Barker et al No
Yes (corpus)
20
Differentiated cells? Corpus Antrum Antrum
Corpus
Powell et al24 and Samuelson, unpublished data
Notch Drives Gastric Cell Proliferation
Strain name
Induced stem cell? Active stem cell?
Table 1.Genetic Mouse Strains Expressed in Adult Gastric Stem Cells
235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293
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but it is not expressed in the intestine. This is hugely ad- 294 vantageous for studying pathway regulation of gastric stem 295 cells because it will allow genetic manipulation of the 296 stomach without affecting the intestine, where changes to 297 stem cell function can limit animal viability. One consider- 298 ation is that most of the genetic mouse strains that have 299 been identified to mark gastric stem cells have not been 300 studied extensively, so the general utility of an individual 301 strain is uncertain. 302 Cellular plasticity is a hallmark of GI tissues, where 303 cellular reprogramming has been suggested to allow 304 committed progenitors or even mature cells to serve as 305 reserve stem cells when active stem cells are compro- 306 mised.27 The existence of dedicated quiescent, or reserve, 307 stem cell populations in the stomach has not been estab- 308 lished definitively. However, certain populations of non- 309 proliferating gastric epithelial cells have been shown to 310 expand during tissue injury and show active stem cell 311 lineage tracing activity. These include rare quiescent antral 312 stem cells that express a Villin Cre driver,28 and a small 313 population of differentiated corpus chief cells expressing 314 Troy-29 and Mist1-Cre drivers29,30 (Table 1). One of these 315 studies reported that Mist1-CreERT2 also is expressed in 316 rare quiescent stem cells in the isthmus of corpus glands 317 that can serve as the cell-of-origin for gastric cancer in 318 mouse models.30 Furthermore, recent studies have reported 319 that CCK2R-CreERT–expressing antral stem cells expand in 320 response to gastrin,25 and that LGR5þ antral stem cells can 321 expand in response to Helicobacter infection.31 322 One major challenge for stem cell studies in the stomach 323 is the extensive gastric cell toxicity observed in genetic 324 models using TX-regulated Cre drivers. TX targets the pa- 325 rietal cells, inducing rapid cell death, with increased corpus 326 progenitor cell proliferation and metaplasia following cell 327 death. These cellular changes are transient and normal Q15328 cellular homeostasis returns within 3 weeks.32 Parietal cells 329 are well known to be lynchpins in the stomach, required for 330 corpus epithelial cell homeostasis and maintenance of the 331 zymogenic cells in the gland base.27 Almost all of the genetic 332 alleles that have been described to manipulate gastric stem 333 cells in the mouse are TX-regulated Cre drivers (Table 1). 334 The TX-induced parietal cell damage is dose-dependent,32 335 and in many instances the observed lineage traces re- 336 ported for many of the gastric Cre drivers used doses that 337 would be expected to induce parietal cell damage. Thus, 338 there is likely to be an injury component to the response 339 that needs to be considered. To avoid parietal cell injury, 340 new genetic tools are needed that do not rely on acute TX 341 treatment to manipulate genes in gastric stem cells. 342 343 344 Notch Regulation of Gastric Epithelial Cell 345 Proliferation 346 Notch recently was established as a crucial pathway 347 regulating gastric stem cell proliferation and differentiation. 348 In the fetal mouse stomach, Notch pathway components 349 Notch1 and Jag2, and the Notch target gene Hes1, have been 350 mapped to the nascent gastric epithelium.6 Analysis of Hes1 351 mutant mice showed that the Notch pathway regulates 352
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cellular differentiation in the developing stomach, with excessive endocrine cell differentiation observed in perinatal mice.33 In the adult stomach, the consequences of Notch pathway inhibition and constitutive pathway activation also have been established from analysis of mouse models. Notch inhibition by g-secretase inhibitor (GSI) treatment in adult mice showed reduced progenitor cell proliferation in both corpus6,34 and antrum.5,6,35 Importantly, GSI treatment also reduced the number of proliferating Lgr5-GFP antral stem cells.5 Furthermore, single fluorescence-activated cell sorter–isolated Lgr5-GFP stem cells derived from GSI-treated mice initiated fewer organoids than stem cells derived from control mice, suggesting that Notch is required to support antral stem cell function.5 In contrast, constitutive Notch activation in LGR5þ antral stem cells using genetic overexpression of NICD induced stem and progenitor cell proliferation.5 Similarly, NICD expression in immature parietal cells in fetal mice and in adult corpus stem cells induced gastric epithelial cell proliferation.6,34 Furthermore, gastric organoids derived from Notch-activated adult corpus and antral stem cells grow faster, consistent with Notch pathway induction of stem and progenitor cell proliferation.5,34 Overall, these studies suggest that Notch promotes epithelial cell proliferation and is required to maintain stem cells in both corpus and antrum. Studies in both the mouse and human beings showed that Notch regulation of gastric antral and corpus progenitor cell proliferation occurs via NOTCH1 and NOTCH2 receptor signaling. Gene expression analysis has shown that NOTCH1 and NOTCH2 are the predominant Notch receptors expressed in the mouse and human stomach.34,35 Moreover, a Notch1-CreERT mouse strain induced full antral lineage stripes, indicating that active NOTCH1-receptor signaling occurs in antral stem cells.5,35 Inhibitory antibodies that target NOTCH1 or NOTCH2 significantly reduced stem and progenitor cell proliferation in mouse stomach and decreased growth of corpus and antral organoids, suggesting that both receptors promote gastric epithelial cell proliferation in vivo and in vitro.34,35 Furthermore, inhibition of NOTCH1 and NOTCH2 in organoids derived from human gastric glands from both corpus and antrum also led to reduced organoid growth, suggesting that the Notch pathway is crucial to support human gastric stem cell function.34,35 Importantly, the human and mouse studies concur, validating the mouse as a reliable model to probe pathway regulation of human gastric stem cells.
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there was increased expression of lineage-directing transcription factors FoxQ1 (surface mucous),36 Spdef (deep mucous),37 and Neurog3 (endocrine)38 in the Notchinhibited antral stomach.5,35 Similarly, expression of differentiated markers was increased in antral organoids treated with GSI or NOTCH1- and NOTCH2-receptor antibodies. In addition, Notch inhibition led to epithelial cell remodeling, with increased expression of secretory proteins normally associated with other regions of the GI tract, including intestine.35 In contrast to the general increase in antral epithelial cell differentiation observed after Notch inhibition, constitutive Notch activation via NICD expression in LGR5þ antral stem cells reduced differentiation, including decreased expression of transcription factors and markers for all epithelial cell lineages.5 Furthermore, antral organoids expressing NICD showed decreased expression of transcription factors and markers for endocrine and deep mucous cells. Together, the inhibition and activation data suggest that Notch inhibits overall differentiation in the antral stomach.5,35 This is in contrast to the intestine, where Notch functions to regulate cell fate choice, directing progenitor cells to adopt an absorptive rather than secretory cell fate via regulation of the transcription factor ATOH1.39–41 Although Notch signaling appears to suppress differentiation in the antrum, the role of Notch to regulate differentiation in the gastric corpus is less clear. Cellular turnover in the stomach varies regionally, with renewal of all antral epithelial cell lineages occurring within several weeks, and cellular renewal in the corpus occurring between several days (surface mucous) to several months (parietal, chief) (reviewed by Mills and Shivdasani18). Because Notch inhibition with GSI or NOTCH1- and NOTCH2-receptor antibodies results in rapid animal mortality,34 study of Notch regulation of complete corpus epithelial cellular renewal has not been feasible. However, it has been shown that although differentiation of surface mucous cells in the antrum is Notch-dependent,5,35 this same lineage in the gastric corpus does not appear to be affected by Notch inhibition,34 suggesting that corpus surface mucous cells are Notch-independent. Therefore, key molecular differences in corpus vs antral stem cell differentiation regulated by Notch signaling will be an important future area of research.
Notch and Gastric Tumorigenesis Notch Regulation of Gastric Epithelial Cell Differentiation In addition to regulation of proliferation, the Notch pathway regulates epithelial cell differentiation in the antral stomach. Notch signaling appears to act in a global manner, generally promoting progenitor cell proliferation and inhibiting differentiation. Analysis of mice after Notch inhibition via GSI treatment or antibody-mediated blockade of both NOTCH1 and NOTCH2 increased expression of all antral epithelial cell lineage markers, including surface mucous, deep mucous, and endocrine cells.5,35 Accordingly,
Notch regulation of gastric stem cell function in the adult mouse antrum has been more attainable than in the mouse corpus owing to the identification of LGR5 as an antral stem cell marker and the ability to track and manipulate these cells using the Lgr5-GFP-CreERT2 mouse model.20 Thus, in the mouse antrum, Notch is thought to regulate gastric stem cell number, as suggested by regulation of organoid establishment activity, and by observation of tissue expansion via increased proliferation, gland fission, and enhanced conversion of adult antral glands to monoclonality after Notch activation.5 Gland fission is a tissue growth phenomenon that normally occurs in the postnatal stomach to increase
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530 531 532 533 534 535 536 537 538 539 Notch and Gastric Cancer 540 Studies in mice have shown that NICD expression in 541 parietal progenitor cells,6 adult SOX2þ corpus stem cells,34 542 and LGR5þ antral stem cells5 lead to hyperplastic polyp 543 development in corpus or antrum. In human beings, Notch 544 pathway dysregulation, particularly via up-regulation of 545 ligands and/or receptors, has been observed in human 546 gastric cancer. Importantly, correlations between high 547 expression of NOTCH1, NOTCH2, and Jagged 1 have been 548 associated with gastric cancer morbidity.7,8,12 NOTCH1 549 activation in human gastric cancer has been associated with 550 Delta-like ligand 1 promoter methylation,11 suggesting that 551 epigenetic dysregulation of Notch signaling also may 552 contribute to gastric disease. Functional studies in human 553 gastric cancer cell lines show that pharmacologic Notch in- 554 hibition can reduce growth and invasion in lines that show 555 increased NOTCH1 expression.10 Notch manipulation also 556 can inhibit gastric tumor growth via regulation of PTEN Q16557 expression, where Notch inhibition with small interfering 558 RNA against NOTCH1 or GSI treatment leads to activation of 559 Human Gastric Cancer Stem Cells nuclear PTEN expression and growth inhibition and 560 Little is known about human gastric stem cells. Putative apoptosis of gastric cancer cells.9 Furthermore, Notch acti- 561 markers of gastric stem cells have been proposed based on vation via expression of NICD in the SC-M1 human gastric 562 their expression in cancer-initiating cells (also called cancer adenocarcinoma cell line promoted colony formation and 563 stem cells). The proliferative capacity and self-renewal tumor growth in mouse xenograft models through activa- 564 property of stem cells makes them susceptible to tumor- tion of cyclooxygenase-2 expression,12 suggesting that 565 initiating mutations, and therefore the transformation of Notch may initiate gastric tumorigenesis via cross-talk with 566 normal stem cells into cancer stem cells remains a likely other signaling pathways. Finally, the Notch pathway has 567 theory for tumorigenesis (reviewed by Vries et al4). Markers been shown to increase expression of CD4458 and CD133,59 568 of such cancer stem cells in the human stomach include putative markers of gastric cancer stem cells. Taken 569 CXCR4,46 and the cell surface markers CD133, also known as together, current data suggest significant Notch pathway 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 þ Figure 1. Notch activation in antral stem cells induces tumorigenesis. In the mouse, NICD activation in an LGR5 stem cell 585 (green) at the gland base results in increased stem cell proliferation and number followed by antral gland fission. This leads to 586 overall tissue expansion and eventual tumorigenesis. Such tumors are hyperproliferative and undifferentiated, expressing stem 587 and progenitor cell markers. 588 organ size with the growth of the organism.42 Fission is thought to occur in response to increased numbers of stem cells to spatially accommodate the increased cellular production,43 and results in a single glandular unit bifurcating, or splitting, into 2 individual glands. Gland fission during homeostatic conditions in the adult stomach is rare. The extensive gland fission that occurs as a result of constitutive NICD expression leads to tissue expansion and polyp formation within 6 months, showing that Notch activation in antral stem cells is tumorigenic (Figure 1). The NICDinduced tumors are highly proliferative, largely composed of undifferentiated cells that express progenitor cell markers, although interestingly not LGR5.5 This also has been shown in the mouse corpus, where constitutive NICD expression in parietal progenitor cells and in adult SOX2þ stem/progenitor cells induces hyperproliferative glands.6,34 The human metaplastic stomach contains clonal patches of epithelial cells, a process termed field cancerization.44,45 Whether this process is regulated by Notch signaling in the human stomach has not been shown, but our evidence from the mouse stomach suggests that unchecked signaling in pathways that regulate proliferation may give progenitor cells a selective advantage, leading to clonal expansion.5 In addition, in the human stomach it has been shown that a clonal mutational field can acquire additional new mutations, suggesting that gastric cancer may arise from a genetically advantageous proliferative subclone within the founder clonal field.44
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Prominin-147–49 and CD44,50 in particular CD44v651 and CD44v9.52 In addition, expression of SOX2 is altered in gastric cancer, although it is unclear whether SOX2 is oncogenic53,54 or has tumor-suppressing capabilities.55–57 Whether these markers also are expressed in normal human gastric stem cells is an important future question that will advance our understanding of human gastric epithelial cell biology.
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involvement in the regulation of gastric cancer cell growth, proliferation, and metastasis, highlighting Notch as a key pathway to explore further for the development of targeted therapies for gastric cancer treatment.
Notch Regulation of Gastric Versus Intestinal Stem Cells Gastric and intestinal stem cells are both regulated by Notch and Wnt, commonly using the same pathway components, such as Lgr5,19,20 and NOTCH1 and NOTCH2.35,60–64 However, these stem cell populations intrinsically are primed to perform functions specific to each tissue program. Thus, pathway regulation likely depends on specific molecular cues from the gastric or intestinal stem cell niche as it relates to overall tissue function. Interestingly, stem cells in stomach and intestine are differentially sensitive to Notch pathway activation in the context of tumorigenesis. Although constitutive expression of NICD in LGR5þ stem cells induces antral tumors with 100% penetrance, intestinal or colonic tumors are not observed.5 These findings suggest that Notch tone in the stomach and intestine differ, with gastric stem cells more sensitive to Notch-activating mutations than intestinal stem cells. Surprisingly, NICD-expressing intestinal stem cells appear to be lost, suggesting that they are at a competitive disadvantage to normal intestinal stem cells. These findings in mice would predict that Notch might play a larger role in the development of human gastric cancers than in human colon cancer. Both gastric and intestinal stem cells are affected similarly by Wnt pathway manipulation. Wnt activation via genetic deletion of Apc in mouse gastric and intestinal stem cells leads to epithelial cell hyperproliferation and tumorigenesis in both intestine65,66 and stomach.20,56 Patients with the Wnt activating genetic disorder familial adenomatous polyposis develop hundreds to thousands of intestinal adenomas that progress to colorectal cancer via accumulation of additional tumorigenic mutations (reviewed by Fearon67). Interestingly, almost all familial adenomatous polyposis patients also develop gastric polyps, typically fundic gland polyps, although they largely are benign and the risk for gastric adenocarcinoma development is low compared with the risk for colorectal cancer.68 This finding suggests that, in contrast to enhanced gastric stem cell sensitivity to Notch activation, the intestine may be more sensitive to Wnt activation than the stomach. Indeed, although adenomas are observed in both tissues after Apc deletion in LGR5 cells in mouse genetic models, the intestinal tumor burden is greater, limiting the duration of study owing to animal morbidity.20 Future studies will be necessary to determine how Notch and Wnt intersect to support the gastric and intestinal stem cell niche during normal tissue homeostasis and with diseases such as cancer.
Summary and Future Directions/Questions Notch functions to balance cellular proliferation and differentiation in the stomach, with Notch inhibition leading to reduced stem and progenitor cell proliferation and
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increased differentiation, although Notch activation results in increased stem and progenitor cell proliferation and reduced differentiation. Furthermore, genetic overexpression of NICD induces tissue expansion via gland fission and gastric tumors. Thus, Notch appears to be a central signaling pathway that promotes gastric stem cell proliferation and expansion. Furthermore, Notch pathway activation also has been reported in human gastric cancer, suggesting that this pathway may prove to be a useful therapeutic target for treatment of some human gastric cancers. Several important questions remain unanswered that undoubtedly will further our understanding of Notch pathway function in mouse and human gastric stem cells. These questions include the following: How is the Notch niche defined? What niche cells present ligand to gastric stem cells? What are the key Notch ligands in the stomach? How does Notch regulate gastric stem cell self-renewal and proliferation? How does Notch cooperate with Wnt and other signaling pathways to both maintain homeostasis and promote tumorigenesis? What are the molecular markers for human gastric stem cells? Can Notch pathway inhibition be effective for gastric cancer treatment?
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metaplasia in the human stomach. Gastroenterology 2011;140:1251–1260 e1–6. McDonald SA, Greaves LC, Gutierrez-Gonzalez L, et al. Mechanisms of field cancerization in the human stomach: the expansion and spread of mutated gastric stem cells. Gastroenterology 2008;134:500–510. Fujita T, Chiwaki F, Takahashi RU, et al. Identification and characterization of CXCR4-positive gastric cancer stem cells. PLoS One 2015;10:e0130808. Hashimoto K, Aoyagi K, Isobe T, et al. Expression of CD133 in the cytoplasm is associated with cancer progression and poor prognosis in gastric cancer. Gastric Cancer 2014;17:97–106. Wen L, Chen XZ, Yang K, et al. Prognostic value of cancer stem cell marker CD133 expression in gastric cancer: a systematic review. PLoS One 2013; 8:e59154. Zhao P, Li Y, Lu Y. Aberrant expression of CD133 protein correlates with Ki-67 expression and is a prognostic marker in gastric adenocarcinoma. BMC Cancer 2010; 10:218. Ghaffarzadehgan K, Jafarzadeh M, Raziee HR, et al. Expression of cell adhesion molecule CD44 in gastric adenocarcinoma and its prognostic importance. World J Gastroenterol 2008;14:6376–6381. da Cunha CB, Oliveira C, Wen X, et al. De novo expression of CD44 variants in sporadic and hereditary gastric cancer. Lab Invest 2010;90:1604–1614. Hirata K, Suzuki H, Imaeda H, et al. CD44 variant 9 expression in primary early gastric cancer as a predictive marker for recurrence. Br J Cancer 2013;109:379–386. Hutz K, Mejias-Luque R, Farsakova K, et al. The stem cell factor SOX2 regulates the tumorigenic potential in human gastric cancer cells. Carcinogenesis 2014; 35:942–950. Matsuoka J, Yashiro M, Sakurai K, et al. Role of the stemness factors sox2, oct3/4, and nanog in gastric carcinoma. J Surg Res 2012;174:130–135. Otsubo T, Akiyama Y, Yanagihara K, et al. SOX2 is frequently downregulated in gastric cancers and inhibits cell growth through cell-cycle arrest and apoptosis. Br J Cancer 2008;98:824–831. Sarkar A, Huebner AJ, Sulahian R, et al. Sox2 suppresses gastric tumorigenesis in mice. Cell Rep 2016; 16:1929–1941. Wang S, Tie J, Wang R, et al. SOX2, a predictor of survival in gastric cancer, inhibits cell proliferation and metastasis by regulating PTEN. Cancer Lett 2015; 358:210–219. Li LC, Wang DL, Wu YZ, et al. Gastric tumor-initiating CD44þ cells and epithelial-mesenchymal transition are
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884 885 59. 886 887 888 60. 889 890 891 61. 892 893 894 62. 895 896 897 898 899 63. 900 901 902 64. 903 904 905 65. 906 907 908 66. 909 910 911 912 913 67. 914 915 68. 916 917 918 919 920 921 Received November 23, 2016. Accepted January 28, 2017. 922 Correspondence 923 Q1 Address correspondence to: Linda C. Samuelson, MD, Department of Molecular and Integrative Physiology, University of Michigan, 109 Zina 924 Pitcher Place, 2041 BSRB, Ann Arbor, Michigan 48109. e-mail: Q2 925 [email protected]; fax: ---. 926 Conflicts of interest 927 Q3 The authors disclose no conflicts. 928 Funding 929 Supported by National Institutes of Health F32-DK093340, MICHR PTSP CTSA Q4 930 UL1TR000433, National Institutes of Health T32-HD007505, and an AACRDDF Career Development Award (E.S.D.); and by research support through Q5931 National Institutes of Health P01-DK062041, National Institutes of Health 932 5R01-DK096972, NCI P50-CA130810, and the American Gastroenterological 933 Association Research Foundation (L.C.S.). 934 935 936 937 938 939 940 941 942 inhibited by gamma-secretase inhibitor DAPT. Oncol Lett 2015;10:3293–3299. Konishi H, Asano N, Imatani A, et al. Notch1 directly induced CD133 expression in human diffuse type gastric cancers. Oncotarget 2016;7:56598–56607. Carulli AJ, Keeley TM, Demitrack ES, et al. Notch receptor regulation of intestinal stem cell homeostasis and crypt regeneration. Dev Biol 2015;402:98–108. Fre S, Hannezo E, Sale S, et al. Notch lineages and activity in intestinal stem cells determined by a new set of knock-in mice. PLoS One 2011;6:e25785. Riccio O, van Gijn ME, Bezdek AC, et al. Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2. EMBO Rep 2008; 9:377–383. Tran IT, Sandy AR, Carulli AJ, et al. Blockade of individual Notch ligands and receptors controls graft-versus-host disease. J Clin Invest 2013;123:1590–1604. Wu Y, Cain-Hom C, Choy L, et al. Therapeutic antibody targeting of individual Notch receptors. Nature 2010; 464:1052–1057. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 2009; 457:608–611. Powell AE, Vlacich G, Zhao ZY, et al. Inducible loss of one Apc allele in Lrig1-expressing progenitor cells results in multiple distal colonic tumors with features of familial adenomatous polyposis. Am J Physiol Gastrointest Liver Physiol 2014;307:G16–G23. Fearon ER. Molecular genetics of colorectal cancer. Annu Rev Pathol 2011;6:479–507. Arnason T, Liang WY, Alfaro E, et al. Morphology and natural history of familial adenomatous polyposis-associated dysplastic fundic gland polyps. Histopathology 2014; 65:353–362.
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