PLoS One. 2015 May 21;10(5):e0127300.
Jang BG, Lee BL, Kim WH.
PMID: 26015511 | DOI: clincanres.3357.2014.
Gastric intestinal metaplasia (IM) is a highly prevalent preneoplastic lesion; however, the molecular mechanisms regulating its development remain unclear. We have previously shown that a population of cells expressing the intestinal stem cell (ISC) marker LGR5 increases remarkably in IM. In this study, we further investigated the molecular characteristics of these LGR5+ cells in IM by examining the expression profile of several ISC markers. Notably, we found that ISC markers-including OLFM4 and EPHB2-are positively associated with the CDX2 expression in non-tumorous gastric tissues. This finding was confirmed in stomach lesions with or without metaplasia, which demonstrated that OLFM4 and EPHB2 expression gradually increased with metaplastic progression. Moreover, RNA in situ hybridization revealed that LGR5+ cells coexpress several ISC markers and remained confined to the base of metaplastic glands, reminiscent to that of normal intestinal crypts, whereas those in normal antral glands expressed none of these markers. Furthermore, a large number of ISC marker-expressing cells were diffusely distributed in gastric adenomas, suggesting that these markers may facilitate gastric tumorigenesis. In addition, Barrett's esophagus (BE)-which is histologically similar to intestinal metaplasia-exhibited a similar distribution of ISC markers, indicating the presence of a stem cell population with intestinal differentiation potential. In conclusion, we identified that LGR5+ cells in gastric IM and BE coexpress ISC markers, and exhibit the same expression profile as those found in normal intestinal crypts. Taken together, these results implicate an intestinal-like stem cell population in the pathogenesis of IM, and provide an important basis for understanding the development and maintenance of this disease.
Jang BG, Lee BL, Kim WH. (2013).
PMID: 24340024 | DOI: 10.1371/journal.pone.0082390.
Lgr5 was identified as a promising gastrointestinal tract stem cell marker in mice. Lineage tracing indicates that Lgr5(+) cells may not only be the cells responsible for the origin of tumors; they may also be the so-called cancer stem cells. In the present study, we investigated the presence of Lgr5(+) cells and their biological significance in normal human gastric mucosa and gastric tumors. RNAscope, a newly developed RNA in situ hybridization technique, specifically labeled Lgr5(+) cells at the basal glands of the gastric antrum. Notably, the number of Lgr5(+) cells was remarkably increased in intestinal metaplasia. In total, 76% of gastric adenomas and 43% of early gastric carcinomas were positive for LGR5. Lgr5(+) cells were found more frequently in low-grade tumors with active Wnt signaling and an intestinal gland type, suggesting that LGR5 is likely involved in the very early stages of Wnt-driven tumorigenesis in the stomach. Interestingly, similar to stem cells in normal tissues, Lgr5(+) cells were often restricted to the base of the tumor glands, and such Lgr5(+) restriction was associated with high levels of intestinal stem cell markers such as EPHB2, OLFM4, and ASCL2. Thus, our findings show that Lgr5(+) cells are present at the base of the antral glands in the human stomach and that this cell population significantly expands in intestinal metaplasias. Furthermore, Lgr5(+) cells are seen in a large number of gastric tumors ; their frequent basal arrangements and coexpression of ISC markers support the idea that Lgr5(+) cells act as stem cells during the early stage of intestinal-type gastric tumorigenesis.
Senger S, Sapone A, Fiorentino MR, Mazzarella G, Lauwers GY, Fasano A.
PMID: 26649570 | DOI: 10.1371/journal.pone.0144634
Abstract
BACKGROUND:
In celiac disease (CD), intestinal epithelium damage occurs secondary to an immune insult and is characterized by blunting of the villi and crypt hyperplasia. Similarities between Hedgehog (Hh)/BMP4 downregulation, as reported in a mouse model, and CD histopathology, suggest mechanistic involvement of Hh/BMP4/WNT pathways in proliferation and differentiation of immature epithelial cells in the context of human intestinal homeostasis and regeneration after damage. Herein we examined the nature of intestinal crypt hyperplasia and involvement of Hh/BMP4 in CD histopathology.
METHODS AND FINDINGS:
Immunohistochemistry, qPCR and in situ hybridization were used to study a cohort of 24 healthy controls (HC) and 24 patients with diagnosed acute celiac disease (A-CD) intestinal biopsies. In A-CD we observed an increase in cells positive for Leucin-rich repeat-containing G protein-coupled receptor 5 (LGR5), an epithelial stem cell specific marker and expansion of WNT responding compartment. Further, we observed alteration in number and distribution of mesenchymal cells, predicted to be part of the intestinal stem cells niche. At the molecular level we found downregulation of indian hedgehog (IHH) and other components of the Hh pathway, but we did not observe a concurrent downregulation of BMP4. However, we observed upregulation of BMPs antagonists, gremlin 1 and gremlin 2.
CONCLUSIONS:
Our data suggest that acute CD histopathology partially recapitulates the phenotype reported in Hh knockdown models. Specifically, Hh/BMP4 paradigm appears to be decoupled in CD, as the expansion of the immature cell population does not occur consequent to downregulation of BMP4. Instead, we provide evidence that upregulation of BMP antagonists play a key role in intestinal crypt hyperplasia. This study sheds light on the molecular mechanisms underlying CD histopathology and the limitations in the use of mouse models for celiac disease.
De Cian MC, Gregoire EP, Le Rolle M, Lachambre S, Mondin M, Bell S, Guigon CJ, Chassot AA, Chaboissier MC
PMID: 32341451 | DOI: 10.1038/s41418-020-0547-7
R-spondin2 (RSPO2) is a member of the R-spondin family, which are secreted activators of the WNT/?-catenin (CTNNB1) signaling pathway. In the mouse postnatal ovary, WNT/CTNNB1 signaling is active in the oocyte and in the neighboring supporting cells, the granulosa cells. Although the role of Rspo2 has been previously studied using in vitro experiments, the results are conflicting and the in vivo ovarian function of Rspo2 remains unclear. In the present study, we found that RSPO2/Rspo2 expression is restricted to the oocyte of developing follicles in both human and mouse ovaries from the beginning of the follicular growth. In mice, genetic deletion of Rspo2 does not impair oocyte growth, but instead prevents cell cycle progression of neighboring granulosa cells, thus resulting in an arrest of follicular growth. We further show this cell cycle arrest to be independent of growth promoting GDF9 signaling, but rather associated with a downregulation of WNT/CTNNB1 signaling in granulosa cells. To confirm the contribution of WNT/CTNNB1 signaling in granulosa cell proliferation, we induced cell type specific deletion of Ctnnb1 postnatally. Strikingly, follicles lacking Ctnnb1 failed to develop beyond the primary stage. These results show that RSPO2 acts in a paracrine manner to sustain granulosa cell proliferation in early developing follicles. Taken together, our data demonstrate that the activation of WNT/CTNNB1 signaling by RSPO2 is essential for oocyte-granulosa cell interactions that drive maturation of the ovarian follicles and eventually female fertility
Sci Rep. 2015 Mar 2;5:8654.
Baker AM, Graham TA, Elia G, Wright NA, Rodriguez-Justo M.
PMID: 25728748 | DOI: 10.1038/srep08654
LGR5 is known to be a stem cell marker in the murine small intestine and colon, however the localization of LGR5 in human adenoma samples has not been examined in detail, and previous studies have been limited by the lack of specific antibodies. Here we used in situ hybridization to specifically examine LGR5 mRNA expression in a panel of human adenoma and carcinoma samples (n = 66). We found that a small number of cells express LGR5 at the base of normal colonic crypts. We then showed that conventional adenomas widely express high levels of LGR5, and there is no evidence of stereotypic cellular hierarchy. In contrast, serrated lesions display basal localization of LGR5, and the cellular hierarchy resembles that of a normal crypt. Moreover, ectopic crypts found in traditional serrated adenomas show basal LGR5 mRNA, indicating that they replicate the stem cell organization of normal crypts with the development of a cellular hierarchy. These data imply differences in the stem cell dynamics between the serrated and conventional pathways of colorectal carcinogenesis. Furthermore we noted high LGR5 expression in invading cells, with later development of a stem cell niche in adenocarcinomas of all stages.
Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, Watanabe T, Kanai T, Sato T.
PMID: 25706875 | DOI: 10.1038/nm.3802.
Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.
Nature. 2014 Dec 4;516(7529):121-5.
Ranade SS, Woo SH, Dubin AE, Moshourab RA, Wetzel C, Petrus M, Mathur J, Bégay V, Coste B, Mainquist J, Wilson AJ, Francisco AG, Reddy K, Qiu Z, Wood JN, Lewin GR, Patapoutian A.
PMID: 25471886 | DOI: 10.1038/nature13980.
The sense of touch provides critical information about our physical environment by transforming mechanical energy into electrical signals1. It is postulated that mechanically activated cation channels initiate touch sensation, but the identity of these molecules in mammals has been elusive2. Piezo2 is a rapidly adapting, mechanically activated ion channel expressed in a subset of sensory neurons of the dorsal root ganglion and in cutaneous mechanoreceptors known as Merkel-cell–neurite complexes3, 4. It has been demonstrated that Merkel cells have a role in vertebrate mechanosensation using Piezo2, particularly in shaping the type of current sent by the innervating sensory neuron4, 5, 6; however, major aspects of touch sensation remain intact without Merkel cell activity4, 7. Here we show that mice lacking Piezo2 in both adult sensory neurons and Merkel cells exhibit a profound loss of touch sensation. We precisely localize Piezo2 to the peripheral endings of a broad range of low-threshold mechanoreceptors that innervate both hairy and glabrous skin. Most rapidly adapting, mechanically activated currents in dorsal root ganglion neuronal cultures are absent in Piezo2 conditional knockout mice, and ex vivo skin nerve preparation studies show that the mechanosensitivity of low-threshold mechanoreceptors strongly depends on Piezo2. This cellular phenotype correlates with an unprecedented behavioural phenotype: an almost complete deficit in light-touch sensation in multiple behavioural assays, without affecting other somatosensory functions. Our results highlight that a single ion channel that displays rapidly adapting, mechanically activated currents in vitro is responsible for the mechanosensitivity of most low-threshold mechanoreceptor subtypes involved in innocuous touch sensation. Notably, we find that touch and pain sensation are separable, suggesting that as-yet-unknown mechanically activated ion channel(s) must account for noxious (painful) mechanosensation.
Hilkens J, Timmer NC, Boer M, Ikink GJ, Schewe M, Sacchetti A, Koppens MA, Song JY, Bakker ER.
PMID: 27511199 | DOI: 10.1136/gutjnl-2016-311606
Abstract
OBJECTIVE:
The gross majority of colorectal cancer cases results from aberrant Wnt/β-catenin signalling through adenomatous polyposis coli (APC) or CTNNB1 mutations. However, a subset of human colon tumours harbour, mutually exclusive with APC and CTNNB1 mutations, gene fusions in RSPO2 or RSPO3, leading to enhanced expression of these R-spondin genes. This suggested that RSPO activation can substitute for the most common mutations as an alternative driver for intestinal cancer. Involvement of RSPO3 in tumour growth was recently shown in RSPO3-fusion-positive xenograft models. The current study determines the extent into which solely a gain in RSPO3 actually functions as a driver of intestinal cancer in a direct, causal fashion, and addresses the in vivo activities of RSPO3 in parallel.
DESIGN:
We generated a conditional Rspo3 transgenic mouse model in which the Rspo3 transgene is expressed upon Cre activity. Cre is provided by cross-breeding with Lgr5-GFP-CreERT2 mice.
RESULTS:
Upon in vivo Rspo3 expression, mice rapidly developed extensive hyperplastic, adenomatous and adenocarcinomatous lesions throughout the intestine. RSPO3 induced the expansion of Lgr5+ stem cells, Paneth cells, non-Paneth cell label-retaining cells and Lgr4+ cells, thus promoting both intestinal stem cell and niche compartments. Wnt/β-catenin signalling was modestly increased upon Rspo3 expression and mutant Kras synergised with Rspo3 in hyperplastic growth.
CONCLUSIONS:
We provide in vivo evidence that RSPO3 stimulates the crypt stem cell and niche compartments and drives rapid intestinal tumorigenesis. This establishes RSPO3 as a potent driver of intestinal cancer and proposes RSPO3 as a candidate target for therapy in patients with colorectal cancer harbouring RSPO3 fusions.
Stem Cell Reports. 2015 Jun 3.
Finkbeiner SR, Hill DR, Altheim CH, Dedhia PH, Taylor MJ, Tsai YH, Chin AM, Mahe MM, Watson CL, Freeman JJ, Nattiv R, Thomson M, Klein OD, Shroyer NF, Helmrath MA, Teitelbaum DH, Dempsey PJ, Spence JR.
PMID: 26067134
Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation.
Nakagawa A, Adams CE, Huang Y, Hamarneh SR, Liu W, Von Alt KN, Mino-Kenudson M, Hodin RA, Lillemoe KD, Fernández-Del Castillo C, Warshaw AL, Liss AS.
PMID: 26856877 | DOI: 10.1038/srep20390
Absorptive and secretory cells of the small intestine are derived from a single population of Lgr5-expressing stem cells. While key genetic pathways required for differentiation into specific lineages have been defined, epigenetic programs contributing to this process remain poorly characterized. Members of the BET family of chromatin adaptors contain tandem bromodomains that mediate binding to acetylated lysines on target proteins to regulate gene expression. In this study, we demonstrate that mice treated with a small molecule inhibitor of BET bromodomains, CPI203, exhibit greater than 90% decrease in tuft and enteroendocrine cells in both crypts and villi of the small intestine, with no changes observed in goblet or Paneth cells. BET bromodomain inhibition did not alter the abundance of Lgr5-expressing stem cells in crypts, but rather exerted its effects on intermediate progenitors, in part through regulation of Ngn3 expression. When BET bromodomain inhibition was combined with the chemotherapeutic gemcitabine, pervasive apoptosis was observed in intestinal crypts, revealing an important role for BET bromodomain activity in intestinal homeostasis. Pharmacological targeting of BET bromodomains defines a novel pathway required for tuft and enteroendocrine differentiation and provides an important tool to further dissect the progression from stem cell to terminally differentiated secretory cell.
Nonomura K, Woo SH, Chang RB, Gillich A, Qiu Z, Francisco AG, Ranade SS, Liberles SD, Patapoutian A.
PMID: 28002412 | DOI: 10.1038/nature20793
Respiratory dysfunction is a notorious cause of perinatal mortality in infants and sleep apnoea in adults, but the mechanisms of respiratory control are not clearly understood. Mechanical signals transduced by airway-innervating sensory neurons control respiration; however, the physiological significance and molecular mechanisms of these signals remain obscured. Here we show that global and sensory neuron-specific ablation of the mechanically activated ion channel Piezo2 causes respiratory distress and death in newborn mice. Optogenetic activation of Piezo2+ vagal sensory neurons causes apnoea in adult mice. Moreover, induced ablation of Piezo2 in sensory neurons of adult mice causes decreased neuronal responses to lung inflation, an impaired Hering-Breuer mechanoreflex, and increased tidal volume under normal conditions. These phenotypes are reproduced in mice lacking Piezo2 in the nodose ganglion. Our data suggest that Piezo2 is an airway stretch sensor and that Piezo2-mediated mechanotransduction within various airway-innervating sensory neurons is critical for establishing efficient respiration at birth and maintaining normal breathing in adults.
Imada, S;Shin, H;Khawaled, S;Meckelmann, S;Whittaker, C;Correa, R;Pradhan, D;Calibasi, G;Nascentes, LN;Allies, G;Wittenhofer, P;Schmitz, O;Roper, J;Vinolo, M;Cheng, CW;Tasdogan, A;Yilmaz, ÃM;
PMID: 36711807 | DOI: 10.21203/rs.3.rs-2320717/v1
For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.
