Correlation of clinicopathological features and LGR5 expression in colon adenocarcinoma.

Annals of Diagnostic Pathology

2019 May 03

Sato K, Uehara T, Iwaya M, Nakajima T, Miyagawa Y, Suga T, Ota H, Tanaka E.
PMID: - | DOI: 10.1016/j.anndiagpath.2019.05.002

Colon cancer stem cells (CSCs) are closely related to tumorigenesis and treatment response, and LGR5 is currently the most robust and reliable CSC marker in colorectal cancer (CRC). However, LGR5 expression in CRC tumor budding (TB) is not well understood. We examined the clinicopathological and prognostic significance of LGR5 in CRC TB. LGR5 expression was evaluated by RNAscope, a newly developed RNA in situ hybridization technique, using a tissue microarray consisting of 55 patient samples of TB in colon adenocarcinoma (CA) selected from the medical archives at our hospital. Patients were stratified into negative and positive LGR5 expression groups. Inflammatory cell infiltration was weaker and histological grade was lower in the LGR5-positive group compared with the LGR5-negative group (P = 0.0407 and P = 0.0436, respectively). There was a significant difference in OS between the LGR5-positive group and LGR5-negative group (log-rank test, P = 0.0088). Cox proportional hazards models revealed that the LGR5-positive group (Overall survival (OS) = 0.37, 95% CI: 0.17–0.79, P = 0.0101) had better OS. LGR5 expression may be affected by inflammatory cell infiltration in the budding area of CA and is an important potential marker of prognosis.

Rhythmic cilia changes support SCN neuron coherence in circadian clock

Science (New York, N.Y.)

2023 Jun 02

Tu, HQ;Li, S;Xu, YL;Zhang, YC;Li, PY;Liang, LY;Song, GP;Jian, XX;Wu, M;Song, ZQ;Li, TT;Hu, HB;Yuan, JF;Shen, XL;Li, JN;Han, QY;Wang, K;Zhang, T;Zhou, T;Li, AL;Zhang, XM;Li, HY;
PMID: 37262147 | DOI: 10.1126/science.abm1962

The suprachiasmatic nucleus (SCN) drives circadian clock coherence through intercellular coupling, which is resistant to environmental perturbations. We report that primary cilia are required for intercellular coupling among SCN neurons to maintain the robustness of the internal clock in mice. Cilia in neuromedin S-producing (NMS) neurons exhibit pronounced circadian rhythmicity in abundance and length. Genetic ablation of ciliogenesis in NMS neurons enabled a rapid phase shift of the internal clock under jet-lag conditions. The circadian rhythms of individual neurons in cilia-deficient SCN slices lost their coherence after external perturbations. Rhythmic cilia changes drive oscillations of Sonic Hedgehog (Shh) signaling and clock gene expression. Inactivation of Shh signaling in NMS neurons phenocopied the effects of cilia ablation. Thus, cilia-Shh signaling in the SCN aids intercellular coupling.

Translation initiation factor eIF2Bε promotes Wnt-mediated clonogenicity and global translation in intestinal epithelial cells

Stem cell research

2021 Aug 11

Smit, WL;de Boer, RJ;Meijer, BJ;Spaan, CN;van Roest, M;Koelink, PJ;Koster, J;Dekker, E;Abbink, TEM;van der Knaap, MS;van den Brink, GR;Muncan, V;Heijmans, J;
PMID: 34399164 | DOI: 10.1016/j.scr.2021.102499

Modulation of global mRNA translation, which is essential for intestinal stem cell function, is controlled by Wnt signaling. Loss of tumor supressor APC in stem cells drives adenoma formation through hyperactivion of Wnt signaling and dysregulated translational control. It is unclear whether factors that coordinate global translation in the intestinal epithelium are needed for APC-driven malignant transformation. Here we identified nucleotide exchange factor eIF2Bε as a translation initiation factor involved in Wnt-mediated intestinal epithelial stemness. Using eIF2BεArg191His mice with a homozygous point mutation that leads to dysfunction in the enzymatic activity, we demonstrate that eIF2Bε is involved in small intestinal crypt formation, stemness marker expression, and secreted Paneth cell-derived granule formation. Wnt hyperactivation in ex vivo eIF2BεArg191His organoids, using a GSK3β inhibitor to mimic Apc driven transformation, shows that eIF2Bε is essential for Wnt-mediated clonogenicity and associated increase of the global translational capacity. Finally, we observe high eIF2Bε expression in human colonic adenoma tissues, exposing eIF2Bε as a potential target of CRC stem cells with aberrant Wnt signaling.

An Nfic-Hedgehog Signaling Cascade Regulates Tooth Root Development

Liu Y, Feng J, Li J, Zhao H, Ho TV, Chai Y.
PMID: 26293299

Coordination between the Hertwig’s Epithelial Root Sheath (HERS) and apical papilla (AP) is crucial for proper tooth root development. The Hedgehog (Hh) signaling pathway and Nfic are both involved in tooth root development, however their relationship has yet to be elucidated. Here we establish a time course of mouse molar root development by histological staining of sections and demonstrate that Hh signaling is active before and during root development in the AP and HERS using Gli1 reporter mice. The proper pattern of Hh signaling activity in the AP is critical for the proliferation of dental mesenchymal cells, because either inhibition with Hh inhibitors or constitutive activation of Hh signaling activity in transgenic mice leads to decreased proliferation in the AP and shorter roots. Moreover, Hh activity is elevated in Nfic-/- mice, a root defect model, whereas RNA sequencing and in situ hybridization show that Hh attenuator Hhip is down-regulated. ChIP and RNAscope analyses suggest that Nfic binds to the promoter region of Hhip. Treatment of Nfic-/- mice with Hh inhibitor partially restores cell proliferation, AP growth and root development. Taken together, our results demonstrate that an Nfic-Hhip-Hh signaling pathway is critical for apical papilla growth and proper root formation. This discovery provides insight into the molecular mechanisms regulating tooth root development.

Fasting Activates Fatty Acid Oxidation to Enhance Intestinal Stem Cell Function during Homeostasis and Aging

Cell Stem Cell

2018 May 03

Mihaylova MM, Cheng CW, Cao AQ, Tripathi S, Mana MD, Bauer-Rowe KE, Abu-Remaileh M, Clavain L, Erdemir A, Lewis CA, Freinkman E, Dickey AS, La Spada AR, Huang Y, Bell GW, Deshpande V, Carmeliet P, Katajisto P, Sabatini DM, Yilmaz ÖH.
PMID: - | DOI: 10.1016/j.stem.2018.04.001

Diet has a profound effect on tissue regeneration in diverse organisms, and low caloric states such as intermittent fasting have beneficial effects on organismal health and age-associated loss of tissue function. The role of adult stem and progenitor cells in responding to short-term fasting and whether such responses improve regeneration are not well studied. Here we show that a 24 hr fast augments intestinal stem cell (ISC) function in young and aged mice by inducing a fatty acid oxidation (FAO) program and that pharmacological activation of this program mimics many effects of fasting. Acute genetic disruption of Cpt1a, the rate-limiting enzyme in FAO, abrogates ISC-enhancing effects of fasting, but long-term Cpt1a deletion decreases ISC numbers and function, implicating a role for FAO in ISC maintenance. These findings highlight a role for FAO in mediating pro-regenerative effects of fasting in intestinal biology, and they may represent a viable strategy for enhancing intestinal regeneration.

The ESCRT-III Protein CHMP1A Mediates Secretion of Sonic Hedgehog on a Distinctive Subtype of Extracellular Vesicles.

Cell Rep.

2018 Jul 24

Coulter ME, Dorobantu CM, Lodewijk GA, Delalande F, Cianferani S, Ganesh VS, Smith RS, Lim ET, Xu CS, Pang S, Wong ET, Lidov HGW, Calicchio ML, Yang E, Gonzalez DM, Schlaeger TM, Mochida GH, Hess H, Lee WA, Lehtinen MK, Kirchhausen T Haussler D Jacobs FMJ
PMID: 30044992 | DOI: 10.1016/j.celrep.2018.06.100

Endosomal sorting complex required for transport (ESCRT) complex proteins regulate biogenesis and release of extracellular vesicles (EVs), which enable cell-to-cell communication in the nervous system essential for development and adult function. We recently showed human loss-of-function (LOF) mutations in ESCRT-III member CHMP1A cause autosomal recessive microcephaly with pontocerebellar hypoplasia, but its mechanism was unclear. Here, we show Chmp1a is required for progenitor proliferation in mouse cortex and cerebellum and progenitor maintenance in human cerebral organoids. In Chmp1a null mice, this defect is associated with impaired sonic hedgehog (Shh) secretion and intraluminal vesicle (ILV) formation in multivesicular bodies (MVBs). Furthermore, we show CHMP1A is important for release of an EV subtype that contains AXL, RAB18, and TMED10 (ART) and SHH. Our findings show CHMP1A loss impairs secretion of SHH on ART-EVs, providing molecular mechanistic insights into the role of ESCRT proteins and EVs in the brain.

Molecular characterization and prospective isolation of human fetal cochlear hair cell progenitors

Nat Commun.

2018 Oct 02

Roccio M, Perny M, Ealy M, Widmer HR, Heller S, Senn P.
PMID: 30279445 | DOI: 10.1038/s41467-018-06334-7

Sensory hair cells located in the organ of Corti are essential for cochlear mechanosensation. Their loss is irreversible in humans resulting in permanent hearing loss. The development of therapeutic interventions for hearing loss requires fundamental knowledge about similarities and potential differences between animal models and human development as well as the establishment of human cell based-assays. Here we analyze gene and protein expression of the developing human inner ear in a temporal window spanning from week 8 to 12 post conception, when cochlear hair cells become specified. Utilizing surface markers for the cochlear prosensory domain, namely EPCAM and CD271, we purify postmitotic hair cell progenitors that, when placed in culture in three-dimensional organoids, regain proliferative potential and eventually differentiate to hair cell-like cells in vitro. These results provide a foundation for comparative studies with otic cells generated from human pluripotent stem cells and for establishing novel platforms for drug validation.

A Novel Organoid Model of Damage and Repair Identifies HNF4? as a Critical Regulator of Intestinal Epithelial Regeneration

Cell Mol Gastroenterol Hepatol.

2020 Mar 05

Montenegro-Miranda PS, van der Meer JHM, Jones C, Meisner S, Vermeulen JLM, Koster J, Wildenberg ME, Heijmans J, Boudreau F, Ribeiro A, van den Brink GR, Muncan V
PMID: 32145468 | DOI: 10.1016/j.jcmgh.2020.02.007

BACKGROUND & AIMS: Recent evidence has suggested that the intact intestinal epithelial barrier protects our body from a range of immune-mediated diseases. The epithelial layer has an impressive ability to reconstitute and repair upon damage and this process of repair increasingly is seen as a therapeutic target. In vitro models to study this process in primary intestinal cells are lacking. METHODS: We established and characterized an in vitro model of intestinal damage and repair by applying ?-radiation on small-intestinal organoids. We then used this model to identify novel regulators of intestinal regeneration. RESULTS: We identified hepatocyte nuclear factor 4? (HNF4?) as a pivotal upstream regulator of the intestinal regenerative response. Organoids lacking Hnf4a were not able to propagate in vitro. Importantly, intestinal Hnf4a knock-out mice showed impaired regeneration after whole-body irradiation, confirming intestinal organoids as a valuable alternative to in vivo studies. CONCLUSIONS: In conclusion, we established and validated an in vitro damage-repair model and identified HNF4? as a crucial regulator of intestinal regeneration

EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids

JCI insight

2023 Feb 23

Childs, CJ;Holloway, EM;Sweet, CW;Tsai, YH;Wu, A;Vallie, A;Eiken, MK;Capeling, MM;Zwick, RK;Palikuqi, B;Trentesaux, C;Wu, JH;Pellon-Cardenas, O;Zhang, CJ;Glass, IA;Loebel, C;Yu, Q;Camp, JG;Sexton, JZ;Klein, OD;Verzi, MP;Spence, JR;
PMID: 36821371 | DOI: 10.1172/jci.insight.165566

Epithelial organoids derived from intestinal tissue, called 'enteroids', recapitulate many aspects of the organ in vitro, and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identify an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells, feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown and EREG-grown enteroids show that EGF-enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine-like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

FAK loss reduces BRAFV600E-induced ERK phosphorylation to promote intestinal stemness and cecal tumor formation

Research square

2023 Feb 01

Gao, C;Ge, H;Kuan, SF;Cai, C;Lu, X;Esni, F;Schoen, R;Wang, J;Chu, E;Hu, J;
PMID: 36778401 | DOI: 10.21203/rs.3.rs-2531119/v1

BRAFV600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a "just-right" level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vill-Cre;BRAFV600E/+;Fakfl/fl mice, Fak deletion maximized BRAFV600E's oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation resulted in increased mRNA expression and stability of Lgr4, promoting intestinal stemness and cecal tumor formation. Together, our findings show that a "just-right" ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.

USP7 inactivation suppresses APC-mutant intestinal hyperproliferation and tumor development

Stem cell reports

2022 Dec 29

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.

CXCL16 inhibits epithelial regeneration and promotes fibrosis during the progression of radiation enteritis

The Journal of pathology

2022 Nov 14

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

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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