Novellasdemunt L, Kucharska A, Jamieson C, Prange-Barczynska M, Baulies A, Antas P, van der Vaart J, Gehart H, Maurice MM, Li VS
PMID: 31867777 | DOI: 10.15252/embj.2019102771
The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis, regeneration and cancer, little is known about the post-translational regulation of LGR5. Here, we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation, increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically, we find that both NEDD4 and NEDD4L negatively regulate Wnt/?-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers, which subsequently enhances tumour predisposition and progression.
Jacob, JM;Di Carlo, SE;Stzepourginski, I;Lepelletier, A;Ndiaye, PD;Varet, H;Legendre, R;Kornobis, E;Benabid, A;Nigro, G;Peduto, L;
PMID: 35523143 | DOI: 10.1016/j.stem.2022.04.005
After birth, the intestine undergoes major changes to shift from an immature proliferative state to a functional intestinal barrier. By combining inducible lineage tracing and transcriptomics in mouse models, we identify a prodifferentiation PDGFRαHigh intestinal stromal lineage originating from postnatal LTβR+ perivascular stromal progenitors. The genetic blockage of this lineage increased the intestinal stem cell pool while decreasing epithelial and immune maturation at weaning age, leading to reduced postnatal growth and dysregulated repair responses. Ablating PDGFRα in the LTBR stromal lineage demonstrates that PDGFRα has a major impact on the lineage fate and function, inducing a transcriptomic switch from prostemness genes, such as Rspo3 and Grem1, to prodifferentiation factors, including BMPs, retinoic acid, and laminins, and on spatial organization within the crypt-villus and repair responses. Our results show that the PDGFRα-induced transcriptomic switch in intestinal stromal cells is required in the first weeks after birth to coordinate postnatal intestinal maturation and function.
Klingler, S;Hsu, KS;Hua, G;Martin, ML;Adileh, M;Baslan, T;Zhang, Z;Paty, PB;Fuks, Z;Brown, AM;Kolesnick, R;
PMID: 35260534 | DOI: 10.1172/jci.insight.153793
Recent data establish a logarithmic expansion of leucine rich repeat containing G protein coupled receptor 5-positive (Lgr5+) colonic epithelial stem cells (CESCs) in human colorectal cancer (CRC). Complementary studies using the murine 2-stage azoxymethane-dextran sulfate sodium (AOM-DSS) colitis-associated tumor model indicate early acquisition of Wnt pathway mutations drives CESC expansion during adenoma progression. Here, subdivision of the AOM-DSS model into in vivo and in vitro stages revealed DSS induced physical separation of CESCs from stem cell niche cells and basal lamina, a source of Wnt signals, within hours, disabling the stem cell program. While AOM delivery in vivo under non-adenoma-forming conditions yielded phenotypically normal mucosa and organoids derived thereof, niche injury ex vivo by progressive DSS dose escalation facilitated outgrowth of Wnt-independent dysplastic organoids. These organoids contained 10-fold increased Lgr5+ CESCs with gain-of-function Wnt mutations orthologous to human CRC driver mutations. We posit CRC originates by niche injury-induced outgrowth of normally suppressed mutated stem cells, consistent with models of adaptive oncogenesis.
Fatehullah, A;Terakado, Y;Sagiraju, S;Tan, TL;Sheng, T;Tan, SH;Murakami, K;Swathi, Y;Ang, N;Rajarethinam, R;Ming, T;Tan, P;Lee, B;Barker, N;
PMID: 34857912 | DOI: 10.1038/s41556-021-00793-9
Gastric cancer is among the most prevalent and deadliest of cancers globally. To derive mechanistic insight into the pathways governing this disease, we generated a Claudin18-IRES-CreERT2 allele to selectively drive conditional dysregulation of the Wnt, Receptor Tyrosine Kinase and Trp53 pathways within the gastric epithelium. This resulted in highly reproducible metastatic, chromosomal-instable-type gastric cancer. In parallel, we developed orthotopic cancer organoid transplantation models to evaluate tumour-resident Lgr5+ populations as functional cancer stem cells via in vivo ablation. We show that Cldn18 tumours accurately recapitulate advanced human gastric cancer in terms of disease morphology, aberrant gene expression, molecular markers and sites of distant metastases. Importantly, we establish that tumour-resident Lgr5+ stem-like cells are critical to the initiation and maintenance of tumour burden and are obligatory for the establishment of metastases. These models will be invaluable for deriving clinically relevant mechanistic insights into cancer progression and as preclinical models for evaluating therapeutic targets.
NOTUM from Apc-mutant cells biases clonal competition to initiate cancer
Flanagan, DJ;Pentinmikko, N;Luopajärvi, K;Willis, NJ;Gilroy, K;Raven, AP;Mcgarry, L;Englund, JI;Webb, AT;Scharaw, S;Nasreddin, N;Hodder, MC;Ridgway, RA;Minnee, E;Sphyris, N;Gilchrist, E;Najumudeen, AK;Romagnolo, B;Perret, C;Williams, AC;Clevers, H;Nummela, P;Lähde, M;Alitalo, K;Hietakangas, V;Hedley, A;Clark, W;Nixon, C;Kirschner, K;Jones, EY;Ristimäki, A;Leedham, SJ;Fish, PV;Vincent, JP;Katajisto, P;Sansom, OJ;
PMID: 34079124 | DOI: 10.1038/s41586-021-03525-z
The tumour suppressor APC is the most commonly mutated gene in colorectal cancer. Loss of Apc in intestinal stem cells drives the formation of adenomas in mice via increased WNT signalling1, but reduced secretion of WNT ligands increases the ability of Apc-mutant intestinal stem cells to colonize a crypt (known as fixation)2. Here we investigated how Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We found that Apc-mutant cells are enriched for transcripts that encode several secreted WNT antagonists, with Notum being the most highly expressed. Conditioned medium from Apc-mutant cells suppressed the growth of wild-type organoids in a NOTUM-dependent manner. Furthermore, NOTUM-secreting Apc-mutant clones actively inhibited the proliferation of surrounding wild-type crypt cells and drove their differentiation, thereby outcompeting crypt cells from the niche. Genetic or pharmacological inhibition of NOTUM abrogated the ability of Apc-mutant cells to expand and form intestinal adenomas. We identify NOTUM as a key mediator during the early stages of mutation fixation that can be targeted to restore wild-type cell competitiveness and provide preventative strategies for people at a high risk of developing colorectal cancer.
Immunohistochemical Study of a Correlation between Pemphigus Vulgaris Activity Score and Stem Cell Control
The Egyptian Journal of Hospital Medicine
Bazid, H;Seleit, I;Abo Hegazy, S;Samaka, R;
| DOI: 10.21608/ejhm.2021.165168
BACKGROUND: Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disease. PV autoantibodies disrupt desmosomal adhesion and cause acantholysis. Previous researches have shown that stem cells are indirectly involved as a result of desmoglein deficiency. Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) as a follicular stem cell marker was evaluated in aim to correlate its intensity of expression with disease severity. OBJECTIVE: To correlate LGR5 intensity of expression with disease severity. PATIENT AND METHODS: This prospective cross sectional study was carried out on 20 PV patients. Patients were subjected to complete history taking, general, dermatological examination and assessment of disease severity by the Pemphigus Vulgaris Activity Score (PVAS), histopathological and immunohistochemical expression of LGR5 were done. RESULTS: All studied cases showed positive cytoplasmic basal LGR5 expression in patchy manner. 75% of cases had mild intensity of expression, 15% had moderate intensity and their Histo (H) score ranged from 50-130 with Mean ±SD 110±18.92. There were no significant correlation between PVAS scores ''skin, mucosa and total involvement'' and H score of LGR5 expression. CONCLUSION: The current study could shed a new light on the disease and its correlation with stem cells, LGR5 as a stem cell marker could be related to the healing process in PV. However, it didn't correlate PVAS scores either in skin, mucosa or total involvement.
Neurotensin regulates proliferation and stem cell function in the small intestine in a nutrient-dependent manner
Cellular and molecular gastroenterology and hepatology
Rock, SA;Jiang, K;Wu, Y;Liu, Y;Li, J;Weiss, HL;Wang, C;Jia, J;Gao, T;Evers, BM;
PMID: 34560309 | DOI: 10.1016/j.jcmgh.2021.09.006
Intestinal stem cells (ISCs) are sensitive to dietary alterations and nutrient availability. Neurotensin (NT), a gut peptide localized predominantly to the small bowel and released by fat ingestion, stimulates the growth of intestinal mucosa under basal conditions and during periods of nutrient deprivation, suggesting a possible role for NT on ISC function.Lgr5-EGFP, NT wild type (Nt+/+) and Lgr5-EGFP, NT knockout (Nt-/-) mice were fed ad libitum (AL) or fasted for 48 h. Small intestine tissue and crypts were examined by gene expression analyses, fluorescence-activated cell sorting, western blot, immunohistochemistry, and crypt-derived organoid culture. Drosophila expressing NT in midgut enteroendocrine cells were fed a standard diet or low-energy diet and esg-GFP+ ISCs quantified via immunofluorescence.Loss of NT impaired crypt cell proliferation and ISC function in a manner dependent on nutrient status. Under nutrient-rich conditions, NT stimulated ERK1/2 signaling and the expression of genes that promote cell cycle progression, leading to crypt cell proliferation. Under conditions of nutrient depletion, NT stimulated WNT/ -catenin signaling and promoted an ISC gene signature, leading to enhanced ISC function. NT was required for the induction of WNT/ -catenin signaling and ISC-specific gene expression during nutrient depletion, and loss of NT reduced crypt cell proliferation and impaired ISC function and Lgr5 expression in the intestine during fasting. Conversely, the expression of NT in midgut enteroendocrine cells of Drosophila prevented loss of ISCs during nutrient depletion.Collectively, our findings establish an evolutionarily conserved role for NT in ISC maintenance during nutritional stress.
Induction of gastric cancer by successive oncogenic activation in the corpus
Douchi, D;Yamamura, A;Matsuo, J;Melissa Lim, YH;Nuttonmanit, N;Shimura, M;Suda, K;Chen, S;ShuChin, P;Kohu, K;Abe, T;Shioi, G;Kim, G;Shabbir, A;Srivastava, S;Unno, M;Bok-Yan So, J;Teh, M;Yeoh, KG;Huey Chuang, LS;Ito, Y;
PMID: 34391772 | DOI: 10.1053/j.gastro.2021.08.013
Metaplasia and dysplasia in the corpus are reportedly derived from dedifferentiation of chief cells. However, the cellular origin of metaplasia and cancer remained uncertain. Therefore, we investigated whether pepsinogen C-transcript expressing cells (PGC-transcript expressing cells) represent the cellular origin of metaplasia and cancer using a novel Pgc-specific CreERT2 recombinase mouse model.We generated a Pgc-mCherry-IRES-CreERT2 (Pgc-CreERT2) knock-in mouse model. Pgc-CreERT2/+ and Rosa-EYFP mice were crossed to generate Pgc-CreERT2/Rosa-EYFP (Pgc-CreERT2/YFP) mice. Gastric tissues were collected, followed by lineage-tracing experiments, histological and immunofluorescence staining. We further established Pgc-CreERT2;KrasG12D/+ mice and investigated whether PGC-transcript expressing cells are responsible for the precancerous state in gastric glands. To investigate cancer development from PGC-transcript expressing cells with activated Kras, inactivated Apc and Trp53 signaling pathways, we crossed Pgc-CreERT2/+ mice with conditional KrasG12D, Apcflox, Trp53flox mice.Expectedly, mCherry mainly labeled chief cells in the Pgc-CreERT2 mice. However, mCherry was also detected throughout the neck cell and isthmal stem/progenitor regions, albeit at lower levels. In the Pgc-CreERT2;KrasG12D/+ mice, PGC-transcript expressing cells with KrasG12D/+ mutation presented pseudopyloric metaplasia. The early induction of proliferation at the isthmus may reflect the ability of isthmal progenitors to react rapidly to Pgc-driven KrasG12D/+ oncogenic mutation. Furthermore, Pgc-CreERT2;KrasG12D/+;Apcflox/flox mice presented intramucosal dysplasia/carcinoma, while Pgc-CreERT2;KrasG12D/+;Apcflox/flox;Trp53flox/flox mice presented invasive and metastatic gastric carcinoma.The Pgc-CreERT2 knock-in mouse is an invaluable tool to study the effects of successive oncogenic activation in the mouse corpus. Time-course observations can be made regarding the responses of isthmal and chief cells to oncogenic insults. We can observe stomach-specific tumorigenesis from the beginning to metastatic development.
Am J Physiol Gastrointest Liver Physiol.
Zaborin A, Krezalek M, Hyoju S, DeFazio JR, Setia N, Belogortseva N, Bindokas VP, Guo Q, Zaborina O, Alverdy JC.
PMID: 27979825 | DOI: 10.1152/ajpgi.00294.2016
Cecal crypts represent a unique niche that are normally occupied by the commensal microbiota. Due to their density and close proximity to stem cells, microbiota within cecal crypts may modulate epithelial regeneration. Here it is demonstrated that surgical stress, a process that invariably involves a short period of starvation, antibiotic exposure and tissue injury, results in cecal crypt evacuation of their microbiota. Crypts devoid of their microbiota display pathophysiological features characterized by abnormal stem cell activation as judged by Lgr5 staining, abnormal stem cell distribution with cells migrating toward the tips of the crypts, and an increase in TUNEL positive cells. In addition, crypts are devoid of their microbiota also display loss of their regenerative capacity as assessed by their ability to form organoids ex vivo. When a four (4) member human pathogen community isolated from the stool of a critically ill patient is introduced into the cecum of mice with empty crypts, crypts become occupied by the introduced pathogens and develop persistent and abnormal Lgr5 expression and severe crypt cell disruption. Fecal microbiota transplantation restores the cecal crypts' microbiota, normalizes the Lgr5 pattern, and reestablishes its regenerative capacity. Taken together, these findings define an emerging role for the microbiota within cecal crypts to maintain epithelial cell homeostasis in a manner that may enhance recovery in response to the physiological stress imposed by the process of surgery.
Qin, D;Liu, S;Lu, Y;Yan, Y;Zhang, J;Cao, S;Chen, M;Chen, N;Huang, W;Wang, L;Chen, X;Zhang, L;
PMID: 36168631 | DOI: 10.7150/thno.74194
Background: Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is a target gene of Wnt/β-Catenin which plays a vital role in hepatic development and regeneration. However, the regulation of Lgr5 gene and the fate of Lgr5 + cells in hepatic physiology and pathology are little known. This study aims to clarify the effect of metabolic nuclear receptors on Lgr5 + cell fate in liver. Methods: We performed cell experiments with primary hepatocytes, Hep 1-6, Hep G2, and Huh 7 cells, and animal studies with wild-type (WT), farnesoid X receptor (FXR) knockout mice, peroxisome proliferator-activated receptor α (PPARα) knockout mice and Lgr5-CreERT2; Rosa26-mTmG mice. GW4064 and CDCA were used to activate FXR. And GW7647 or Wy14643 was used for PPARα activation. Regulation of Lgr5 by FXR and PPARα was determined by QRT-PCR, western blot (WB) and RNAscope in situ hybridization (ISH) and immunofluorescence (IF), luciferase reporter assay, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Diethyl 1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate (DDC) diet was used to induce liver injury. Results: Pharmacologic activation of FXR induced Lgr5 expression, whereas activation of PPARα suppressed Lgr5 expression. Furthermore, FXR and PPARα competed for binding to shared site on Lgr5 promoter with opposite transcriptional outputs. DDC diet triggered the transition of Lgr5 + cells from resting state to proliferation. FXR activation enhanced Lgr5 + cell expansion mainly by symmetric cell division, but PPARα activation prevented Lgr5 + cell proliferation along with asymmetric cell division. Conclusion: Our findings unravel the opposite regulatory effects of FXR and PPARα on Lgr5 + cell fate in liver under physiological and pathological conditions, which will greatly assist novel therapeutic development targeting nuclear receptors.
Cellular and molecular gastroenterology and hepatology
Chen, ME;Naeini, SM;Srikrishnaraj, A;Drucker, DJ;Fesler, Z;Brubaker, PL;
PMID: 35218981 | DOI: 10.1016/j.jcmgh.2022.02.011
Leucine-rich repeat-containing G-protein coupled receptor-5 (Lgr5)+/olfactomedin-4 (Olfm4)+ intestinal stem cells (ISCs) in the crypt-base are crucial for homeostatic maintenance of the epithelium. The gut hormone, glucagon-like peptide-21-33 (GLP-2), stimulates intestinal proliferation and growth; however, the actions of GLP-2 on the Lgr5+ ISCs remain unclear. The aim of this study was to determine whether and how GLP-2 regulates Lgr5+ ISC cell cycle dynamics and number.Lgr5-eGFP-IRES-creERT2 mice were acutely administered human Gly2-GLP-2, or the GLP-2 receptor antagonist, GLP-23-33. Intestinal epithelial-insulin-like growth factor-1 receptor knockout and control mice were treated chronically with hGly2-GLP-2. Cell cycle parameters were determined by EdU, BrdU, Ki67 and phosphohistone-3 labeling and cell cycle gene expression.Acute hGly2-GLP-2 treatment increased the proportion of eGFP+EdU+/OLFM4+EdU+ cells by 11-22% (p<0.05), without affecting other cell cycle markers. hGly2-GLP-2 treatment also increased the ratio of eGFP+ cells in early-to-late S-phase by 97% (p<0.001), and increased the proportion of eGFP+ cells entering S-phase by 218% (p<0.001). hGly2-GLP-2 treatment induced jejunal expression of genes involved in cell cycle regulation (p<0.05), and increased expression of Mcm3 in the Lgr5-expressing cells by 122% (p<0.05). Conversely. GLP-23-33 reduced the proportion of eGFP+EdU+ cells by 27% (p<0.05), as well as the expression of jejunal cell cycle genes (p<0.05). Finally, chronic hGly2-GLP-2 treatment increased the number of OLFM4+ cells/crypt (p<0.05), in an intestinal epithelial insulin-like growth factor-1 receptor-dependent manner.These findings expand the actions of GLP-2 to encompass acute stimulation of Lgr5+ ISC S-phase entry through the GLP-2R, and chronic induction of Lgr5+ ISC expansion through downstream intestinal insulin-like growth factor-1 signaling.
Qi, Y;Lee, NJ;Ip, CK;Enriquez, R;Tasan, R;Zhang, L;Herzog, H;
PMID: 35167990 | DOI: 10.1016/j.molmet.2022.101455
Aguti-related protein (AGRP) neurons in the arcuate nucleus of the hypothalamus (ARC), which co-express neuropeptide Y (NPY), are key regulators of feeding and energy homeostasis. However, the precise role NPY has within these neurons and the specific pathways that it control are still unclear. In this article, we aimed to determine what aspects of feeding behaviour and energy homeostasis are controlled by NPY originating from AGRP neurons and which Y-receptor pathways are utilised to fulfil this function.Novel conditional Agrpcre/+;Npylox/lox knockout mice were generated and comprehensively phenotyped, both under standard chow as well as high-fat-diet conditions. Designer receptor exclusively activated by designer drugs (DREADD) technology was used to assess the altered responses on feeding and energy homeostasis control in the absence of NPY in these neurons. Rescue experiments utilising Npy1r- and Npy2r-selective NPY ligands were performed to assess which component of the energy homeostasis control is dependent by which specific Y-receptor pathway.We show that the specific deletion of Npy only in AGRP neurons leads to a paradoxical mild obese phenotype associated with reduced locomotion and energy expenditure and increased feeding and Respiratory Quotient (RQ) that remain elevated under a positive energy balance. The activation of Npy-deficient AGRP neurons via DREADD's is still able to drive feeding, yet with a delayed onset. Additionally, Clozapine-N-oxide (CNO) treatment reduces locomotion without impacting on energy expenditure. Rescue experiments re-introducing Npy1r- and Npy2r-selective NPY ligands revealed that the increased feeding and RQ are mostly driven by Npy1r, whereas energy expenditure and locomotion are controlled by Npy2r signalling.Together, these results demonstrate that NPY originating from AGRP neurons is not only critical to initiate but also for continuously driving feeding, and we for the first time identify which Y-receptor controls which pathway.
