Novellasdemunt, L;Kucharska, A;Baulies, A;Hutton, C;Vlachogiannis, G;Repana, D;Rowan, A;Suárez-Bonnet, A;Ciccarelli, F;Valeri, N;Li, VSW;
PMID: 36669491 | DOI: 10.1016/j.stemcr.2022.12.013
Adenomatous polyposis coli (APC) mutation is the hallmark of colorectal cancer (CRC), resulting in constitutive WNT activation. Despite decades of research, targeting WNT signaling in cancer remains challenging due to its on-target toxicity. We have previously shown that the deubiquitinating enzyme USP7 is a tumor-specific WNT activator in APC-truncated cells by deubiquitinating and stabilizing β-catenin, but its role in gut tumorigenesis is unknown. Here, we show in vivo that deletion of Usp7 in Apc-truncated mice inhibits crypt hyperproliferation and intestinal tumor development. Loss of Usp7 prolongs the survival of the sporadic intestinal tumor model. Genetic deletion, but not pharmacological inhibition, of Usp7 in Apc+/- intestine induces colitis and enteritis. USP7 inhibitor treatment suppresses growth of patient-derived cancer organoids carrying APC truncations in vitro and in xenografts. Our findings provide direct evidence that USP7 inhibition may offer a safe and efficacious tumor-specific therapy for both sporadic and germline APC-mutated CRC.
Cui, Y;Wu, H;Liu, Z;Ma, T;Liang, W;Zeng, Q;Chen, D;Qin, Q;Huang, B;Wang, MH;Huang, X;He, Y;Kuang, Y;Sugimoto, S;Sato, T;Wang, L;
PMID: 36373877 | DOI: 10.1002/path.6031
Radiation enteritis (RE) is a prevalent complication of radiotherapy for pelvic malignant tumors, characterized by severe intestinal epithelial destruction and progressive submucosal fibrosis. However, little is known about the pathogenesis of this disease and so far, there is no specific targeted therapy. Here, we report that CXCL16 is up-regulated in the injured intestinal tissues of RE patients and in a mouse model. Genetic deletion of Cxcl16 mitigates fibrosis and promotes intestinal stem cell-mediated epithelial regeneration after radiation injury in mice. Mechanistically, CXCL16 functions on myofibroblasts through its receptor CXCR6 and activates JAK3/STAT3 signaling to promote fibrosis, and meanwhile to transcriptionally modulate the levels of BMP4 and HGF in myofibroblasts. Moreover, we find that CXCL16 and CXCR6 auto- and cross-regulate themselves in positive feedback loops. Treatment with CXCL16 neutralizing monoclonal antibody attenuates fibrosis and improves the epithelial repair in RE mouse model. Our findings emphasize the important role of CXCL16 in the progression of RE, and suggest that CXCL16 signaling could be a potential therapeutic target for RE. This article is protected by
Molecular nutrition & food research
May, S;Greenow, KR;Higgins, AT;Derrick, AV;Taylor, E;Pan, P;Konstantinou, M;Nixon, C;Wooley, TE;Sansom, OJ;Wang, LS;Parry, L;
PMID: 36045438 | DOI: 10.1002/mnfr.202200234
Black raspberries (BRBs) have colorectal cancer (CRC) chemo-preventative effects. As CRC originates from an intestinal stem cell (ISC) this study has investigated the impact of BRBs on normal and mutant ISCs.Mice with an inducible Apcfl mutation in either the ISC (Lgr5CreERT2 ) or intestinal crypt (AhCre/VillinCreERT2 ) are fed a control or 10% BRB-supplemented diet. This study uses immunohistochemistry, gene expression analysis, and organoid culture to evaluate the effect of BRBs on intestinal homeostasis. RNAscope is performed for ISC markers on CRC adjacent normal colonic tissue pre and post BRB intervention from patients. 10% BRB diet has no overt effect on murine intestinal homeostasis, despite a reduced stem cell number. Following Apc ISC deletion, BRB diet extends lifespan and reduces tumor area. In the AhCre model, BRB diet attenuates the "crypt-progenitor" phenotype and reduces ISC marker gene expression. In ex vivo culture BRBs reduce the self-renewal capacity of murine and human Apc deficient organoids. Finally, the study observes a reduction in ISC marker gene expression in adjacent normal crypts following introduction of BRBs to the human bowel.BRBs play a role in CRC chemoprevention by protectively regulating the ISC compartment and further supports the use of BRBs in CRC prevention.
Single-cell RNA sequencing of human nail unit defines RSPO4 onychofibroblasts and SPINK6 nail epithelium
Kim, HJ;Shim, JH;Park, JH;Shin, HT;Shim, JS;Jang, KT;Park, WY;Lee, KH;Kwon, EJ;Jang, HS;Yang, H;Lee, JH;Yang, JM;Lee, D;
PMID: 34099859 | DOI: 10.1038/s42003-021-02223-w
Research on human nail tissue has been limited by the restricted access to fresh specimen. Here, we studied transcriptome profiles of human nail units using polydactyly specimens. Single-cell RNAseq with 11,541 cells from 4 extra digits revealed nail-specific mesenchymal and epithelial cell populations, characterized by RSPO4 (major gene in congenital anonychia) and SPINK6, respectively. In situ RNA hybridization demonstrated the localization of RSPO4, MSX1 and WIF1 in onychofibroblasts suggesting the activation of WNT signaling. BMP-5 was also expressed in onychofibroblasts implicating the contribution of BMP signaling. SPINK6 expression distinguished the nail-specific keratinocytes from epidermal keratinocytes. RSPO4+ onychofibroblasts were distributed at close proximity with LGR6+ nail matrix, leading to WNT/β-catenin activation. In addition, we demonstrated RSPO4 was overexpressed in the fibroblasts of onychomatricoma and LGR6 was highly expressed at the basal layer of the overlying epithelial component, suggesting that onychofibroblasts may play an important role in the pathogenesis of onychomatricoma.
Human Type II Taste Cells Express ACE2 and are Infected by SARS-CoV-2
The American journal of pathology
Doyle, ME;Appleton, A;Liu, QR;Yao, Q;Mazucanti, CH;Egan, JM;
PMID: 34102107 | DOI: 10.1016/j.ajpath.2021.05.010
Chemosensory changes are well-reported symptoms of SARS-CoV-2 infection. The virus targets cells for entry by binding of its spike protein to cell-surface angiotensin-converting enzyme- 2 (ACE2). It was not known whether ACE2 is expressed on taste receptor cells (TRCs) nor if TRCs are infected directly. Using an in-situ hybridization (ISH) probe and an antibody specific to ACE2, ACE2 is present on a subpopulation of TRCs, namely, Type II cells in taste buds in taste papillae. Fungiform papillae (FP) of a SARS-CoV-2+ patient exhibiting symptoms of COVID-19, including taste changes, were biopsied. Based on ISH, replicating SARS-CoV-2 was present in Type II cells. Therefore, taste Type II cells provide a potential portal for viral entry that predicts vulnerabilities to SARS-CoV-2 in the oral cavity. The continuity and cell turnover of the patient's FP taste stem cell layer were disrupted during infection and had not completely recovered 6 weeks post symptom onset. Another patient suffering post-COVID-19 taste disturbances also had disrupted stem cells. These results demonstrate the possibility that novel and sudden taste changes frequently reported in COVID-19 may be the result of direct infection of taste papillae by SARS-CoV-2. This may result in impaired taste receptor stem cell activity and suggest more work is needed to understand the acute and post-acute dynamics of viral kinetics in the human taste bud.
Ziskin JL, Dunlap D, Yaylaoglu M, Fodor IK, Forrest WF, Patel R, Ge N, Hutchins GG, Pine JK, Quirke P, Koeppen H, Jubb AM (2013).
PMID: 22637696 | DOI: 10.1136/gutjnl-2011-301195.
OBJECTIVE:
Wnt/Tcf, Lgr5, Ascl2 and/or Bmi1 signalling is believed to define the mouse intestinal stem cell niche(s) from which adenomas arise. The aim of this study was to determine the relevance of these putative intestinal stem cell markers to human colorectal cancer.
DESIGN:
19 putative intestinal stem cell markers, including Ascl2 and Lgr5, were identified from published data and an evaluation of a human colorectal gene expression database. Associations between these genes were assessed by isotopic in situ hybridisation (ISH) in 57 colorectal adenocarcinomas. Multiplex fluorescent ISH and chromogenic non-isotopic ISH were performed to confirm expression patterns. The prognostic significance of Lgr5 was assessed in 891 colorectal adenocarcinomas.
RESULTS:
Ascl2 and Lgr5 were expressed in 85% and 74% of cancers respectively, and expression was positively correlated (p=0.003). Expression of Bmi1 was observed in 47% of cancers but was very weak in 98% of cases with expression. Both Ascl2 and/or Lgr5 were positively correlated with the majority of genes in the signature but neither was correlated with Cdk6, Gpx2, Olfm4 or Tnfrsf19. Lgr5 did not have prognostic significance.
CONCLUSION:
These data suggest that 74-85% of colorectal cancers express a Lgr5/Ascl2 associated signature and support the hypothesis that they derive from Lgr5(+)/Ascl2(+) crypt stem cells, not Bmi1(+) stem cells. However, Olfm4 was not found to be a useful marker of Lgr5(+) cells in normal colon or tumours. In this large series, Lgr5 expression is not associated with increased tumour aggressiveness, as might be expected from a cancer stem cell marker.
Chen G, Gao C, Gao X, Zhang DH, Kuan SF, Burns TF, Hu J.
PMID: 29167314 | DOI: 10.1158/1535-7163.MCT-17-0561
One of the most encouraging developments in oncology has been the success of BRAF inhibitors in BRAF-mutant melanoma. However, in contrast to its striking efficacy in BRAF-mutant melanomas, BRAF inhibitor monotherapy is ineffective in BRAF-mutant colorectal cancer (CRC). While many studies on BRAF inhibitor resistance in CRC have focused on mechanisms underlying the reactivation of the EGFR/RAS/RAF/MEK/ERK pathway, the current study focuses on identifying novel adaptive signaling mechanisms, a fresh angle on CRC resistance to BRAF inhibition. We found that treatment with BRAF inhibitors (both current and next generation BRAF inhibitors) upregulated the Wnt/β-catenin pathway in BRAFV600E-mutant CRC cell lines through activating the cytoplasmic tyrosine kinase FAK (focal adhesion kinase). The results showed that FAK activation upon BRAF inhibitor treatment did not require EGFR (Epidermal Growth Factor Receptor) or ERK1/2 (extracellular-signal-regulated kinases1/2) activation, implying that BRAF inhibitor treatment-induced hyperactivation of Wnt signaling is "pathway reactivation"-independent. BRAF inhibition-induced Wnt pathway activation was further validated in preclinical models of BRAFV600E-mutant CRC including cell line xenograft model and a PDX (patient-derived xenograft) model. Combined inhibition of BRAF/Wnt pathways or BRAF/FAK pathways exerted strong synergistic antitumor effects in cell culture model and mouse xenograft model. Overall, the current study has identified activation of the Wnt/β-catenin pathway as a novel fundamental cause of colon cancer resistance to BRAF inhibition. Our results suggest that while complete vertical pathway blockade is pivotal for effective and durable control of BRAF-mutant CRC, co-targeting parallel adaptive signaling-the Wnt/β-catenin pathway-is also essential.
Robustelli Test, E;Sena, P;Locatelli, AG;Carugno, A;di Mercurio, M;Moggio, E;Gambini, DM;Arosio, MEG;Callegaro, A;Morotti, D;Gianatti, A;Vezzoli, P;
PMID: 34989043 | DOI: 10.1111/pde.14903
Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, an increasing number of chilblain-like lesions (ChLL) have been increasingly reported worldwide. To date, the causal link between ChLL and SARS-CoV-2 infection has not been unequivocally established.In this case series, we present demographic, clinical, laboratory, and histopathological information regarding 27 young patients with a clinical diagnosis of ChLL who referred to the Dermatology Unit of Papa Giovanni XXIII Hospital, Bergamo, Italy, from 1 April 2020 to 1 June 2020.The mean age was 14.2 years, and 21 patients (78%) experienced mild systemic symptoms a median of 28 days before the onset of cutaneous lesions. ChLL mostly involved the feet (20 patients - 74%). Among acral lesions, we identified three different clinical patterns: (i) chilblains in 20 patients (74%); (ii) fixed erythematous macules in 4 children (15%); (iii) erythrocyanosis in 3 female patients (11%). Blood examinations and viral serologies, including parvovirus B19, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and coxsackievirus were normal in all. Three patients (11%) underwent nasopharyngeal swab for RT-PCR for SARS-CoV-2 showing only 1 positive. Histopathological examinations of 7 skin biopsies confirmed the clinical diagnosis of chilblains; vessel thrombi were observed only in 1 case. Our findings failed to demonstrate the direct presence of SARS-CoV-2 RNA in skin biopsies, both with real-time polymerase chain reaction (RT-PCR) and RNAscope in situ hybridization (ISH).Limited number of cases, unavailability of laboratory confirmation of COVID-19 in all patients, potential methodological weakness, and latency of skin biopsies in comparison to cutaneous lesions onset.These observations may support the hypothesis of an inflammatory pathogenesis rather than the presence of peripheral viral particles. Although, we could not exclude an early phase of viral endothelial damage followed by an IFN-I or complement-mediated inflammatory phase. Further observations on a large number of patients are needed to confirm this hypothesis.
Helicobacter pylori Activate and Expand Lgr5+ Stem Cells Through Direct Colonization of the Gastric Glands (check out Movie S4 when it gets out)
Gastroenterology. 2015 Feb 25.
Sigal M, Rothenberg ME, Logan CY, Lee JY, Honaker RW, Cooper RL, Passarelli B, Camorlinga M, Bouley DM, Alvarez G, Nusse R, Torres J, Amieva MR
Background & Aims Helicobacter pylori infection is the main risk factor for gastric cancer. We characterized the interactions of H pylori with gastric epithelial progenitor and stem cells in humans and mice and investigated how these interactions contribute to H pylori-induced pathology. Methods We used quantitative confocal microscopy and 3-dimensional reconstruction of entire gastric glands to determine the localizations of H pylori in stomach tissues from humans and infected mice. Using lineage tracing to mark cells derived from Lgr5+ stem cells (Lgr5-eGFP-IRES-CreERT2/Rosa26-TdTomato mice) and in situ hybridization, we analyzed gastric stem cell responses to infection. Isogenic H pylori mutants were used to determine the role of specific virulence factors in stem cell activation and pathology. Results H pylori grow as distinct bacterial microcolonies deep in the stomach glands and interact directly with gastric progenitor and stem cells in tissues from mice and humans. These gland-associated bacteria activate stem cells, increasing the number of stem cells, accelerating Lgr5+ stem cell proliferation, and upregulating expression of stem cell-related genes. Mutant bacteria with defects in chemotaxis that are able to colonize the stomach surface but not the antral glands in mice do not activate stem cells. Moreover, bacteria that are unable to inject the contact-dependent virulence factor CagA into the epithelium colonized stomach glands in mice, but did not activate stem cells or produce hyperplasia to the same extent as wild-type H pylori. Conclusions H pylori colonize and manipulate the progenitor and stem cell compartments, which alters turnover kinetics and glandular hyperplasia. Bacterial ability to alter the stem cells has important implications for gastrointestinal stem cell biology and H pylori-induced gastric pathology.
Cell Mol Gastroenterol Hepatol.
Meijer BJ1, Giugliano FP, Baan B, van der Meer JHM, Meisner S, van Roest M, Koelink PJ, de Boer RJ, Jones N, Breitwieser W, van der Wel NN, Wildenberg ME, van den Brink GR, Heijmans J, Muncan V
PMID: 31958521 | DOI: 10.1016/j.jcmgh.2020.01.005
BACKGROUND & AIMS:
Activation factor-1 transcription factor family members activating transcription factors 2 and 7 (ATF2 and ATF7) have highly redundant functions owing to highly homologous DNA binding sites. Their role in intestinal epithelial homeostasis and repair is unknown. Here, we assessed the role of these proteins in these conditions in an intestine-specific mouse model.
METHODS:
We performed in vivo and ex vivo experiments using Villin-CreERT2Atf2fl/flAtf7ko/ko mice. We investigated the effects of intestinal epithelium-specific deletion of the Atf2 DNA binding region in Atf7-/- mice on cellular proliferation, differentiation, apoptosis, and epithelial barrier function under homeostatic conditions. Subsequently, we exposed mice to 2% dextran sulfate sodium (DSS) for 7 days and 12 Gy whole-body irradiation and assessed the response to epithelial damage.
RESULTS:
Activating phosphorylation of ATF2 and ATF7 was detected mainly in the crypts of the small intestine and the lower crypt region of the colonic epithelium. Under homeostatic conditions, no major phenotypic changes were detectable in the intestine of ATF mutant mice. However, on DSS exposure or whole-body irradiation, the intestinal epithelium showed a clearly impaired regenerative response. Mutant mice developed severe ulceration and inflammation associated with increased epithelial apoptosis on DSS exposure and were less able to regenerate colonic crypts on irradiation. In vitro, organoids derived from double-mutant epithelium had a growth disadvantage compared with wild-type organoids, impaired wound healing capacity in scratch assay, and increased sensitivity to tumor necrosis factor-?-induced damage.
CONCLUSIONS:
ATF2 and ATF7 are dispensable for epithelial homeostasis, but are required to maintain epithelial regenerative capacity and protect against cell death during intestinal epithelial damage and repair.
Cellular and molecular gastroenterology and hepatology
Li, C;Zhou, Y;Wei, R;Napier, DL;Sengoku, T;Alstott, MC;Liu, J;Wang, C;Zaytseva, YY;Weiss, HL;Wang, Q;Evers, BM;
PMID: 36584817 | DOI: 10.1016/j.jcmgh.2022.12.012
The Intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation, how glycolysis contributes to intestinal epithelial homeostasis remains to be defined.Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 MAPK, the transcription factor atonal homolog 1 (ATOH1), and intestinal cell differentiation markers lysozyme, mucin 2, and chromogranin A were determined by western blot, qPCR or IF and IHC staining.HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout (KO) or inhibition of glycolysis resulted in increased numbers of Paneth, goblet, and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically, HK2 KO resulted in activation of p38 MAPK and increased expression of ATOH1; inhibition of p38 MAPK signaling attenuated the phenotypes induced by HK2 KO in intestinal organoids. HK2 KO significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 KO.Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 MAPK/ATOH1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function.
Cloft, S;Uni, Z;Wong, E;
| DOI: 10.1016/j.psj.2023.102495
Mature small intestines have crypts populated by stem cells which produce replacement cells to maintain the absorptive villus surface area. The embryonic crypt is rudimentary and cells along the villi are capable of proliferation. By 7 d post-hatch the crypts are developed and are the primary sites of proliferation. Research characterizing the proliferative expansion of the small intestine during the peri-hatch period is lacking. The objective of this study was to profile the changes of genes that are markers of stem cells and proliferation: Olfactomedin 4 (Olfm4), Leucine-rich repeat containing G protein-coupled receptor 5 (Lgr5), and marker of proliferation Ki67 from embryonic day 17 to 7 d post-hatch using quantitative PCR and in situ hybridization (ISH). The expression of the stem cell marker genes differed. Olfm4 mRNA increased while Lgr5 mRNA decreased post-hatch. Ki67 mRNA decreased post-hatch in the duodenum and was generally the greatest in the ileum. The ISH was consistent with the quantitative PCR results. Olfm4 mRNA was only seen in the crypts and increased with morphological development of the crypts. In contrast Lgr5 mRNA was expressed in the crypt and the villi in the embryonic periods but became restricted to the intestinal crypt during the post-hatch period. Ki67 mRNA was expressed throughout the intestine pre-hatch, but then expression became restricted to the crypt and the center of the villi. The ontogeny of Olfm4, Lgr5 and Ki67 expressing cells show that proliferation in the peri-hatch intestine changes from along the entire villi to being restricted within the crypts.
