SH3BP4 Regulates Intestinal Stem Cells and Tumorigenesis by Modulating β-Catenin Nuclear Localization.

Cell Rep.

2019 Feb 26

Antas P, Novellasdemunt L, Kucharska A, Massie I, Carvalho J, Oukrif D, Nye E, Novelli M, Li VSW.
PMID: 30811977 | DOI: 10.1016/j.celrep.2019.01.110

Wnt signals at the base of mammalian crypts play a pivotal role in intestinal stem cell (ISC) homeostasis, whereas aberrant Wnt activation causes colon cancer. Precise control of Wnt signal strength is governed by a number of negative inhibitory mechanisms acting at distinctlevels of the cascade. Here, we identify the Wnt negative regulatory role of Sh3bp4 in the intestinal crypt. We show that the loss of Sh3bp4 increases ISC and Paneth cell numbers in murine intestine and accelerates adenoma development in Apcmin mice. Mechanistically, human SH3BP4 inhibits Wnt signaling downstream of β-catenin phosphorylation and ubiquitination. This Wnt inhibitory role is dependent on the ZU5 domain of SH3BP4. We further demonstrate that SH3BP4 is expressed at the perinuclear region to restrict nuclear localization of β-catenin. Our data uncover the tumor-suppressive role of SH3BP4 that functions as a negative feedback regulator of Wnt signaling through modulating β-catenin's subcellular localization.

Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation.

Nat Commun.

2019 Mar 06

Zhao B, Chen Y, Jiang N, Yang L, Sun S, Zhang Y, Wen Z, Ray L, Liu H, Hou G, Lin X.
PMID: 30842416 | DOI: 10.1038/s41467-019-09060-w

Lgr5+ stem cells are crucial to gut epithelium homeostasis; however, how these cells are maintained is not fully understood. Zinc finger HIT-type containing 1 (Znhit1) is an evolutionarily conserved subunit of the SRCAP chromosome remodeling complex. Currently, the function of Znhit1 in vivo and its working mechanism in the SRCAP complex are unknown. Here we show that deletion of Znhit1 in intestinal epithelium depletes Lgr5+ stem cells thus disrupts intestinal homeostasis postnatal establishment and maintenance. Mechanistically, Znhit1 incorporates histone variant H2A.Z into TSS region of genes involved in Lgr5+ stem cell fate determination, including Lgr5, Tgfb1 and Tgfbr2, for subsequent transcriptional regulation. Importantly, Znhit1 promotes the interaction between H2A.Z and YL1 (H2A.Z chaperone) by controlling YL1 phosphorylation. These results demonstrate that Znhit1/H2A.Z is essential for Lgr5+ stem cell maintenance and intestinal homeostasis. Our findings identified a dominant role of Znhit1/H2A.Z in controlling mammalian organ development and tissue homeostasis in vivo.

Single-Cell Analysis of the Liver Epithelium Reveals Dynamic Heterogeneity and an Essential Role for YAP in Homeostasis and Regeneration.

Cell Stem Cell.

2019 May 09

Pepe-Mooney BJ, Dill MT, Alemany A, Ordovas-Montanes J, Matsushita Y, Rao A, Sen A, Miyazaki M, Anakk S, Dawson PA, Ono N, Shalek AK, van Oudenaarden A, Camargo FD.
PMID: 31080134 | DOI: 10.1016/j.stem.2019.04.004

The liver can substantially regenerate after injury, with both main epithelial cell types, hepatocytes and biliary epithelial cells (BECs), playing important roles in parenchymal regeneration. Beyond metabolic functions, BECs exhibit substantial plasticity and in some contexts can drive hepatic repopulation. Here, we performed single-cell RNA sequencing to examine BEC and hepatocyte heterogeneity during homeostasisand after injury. Instead of evidence for a transcriptionally defined progenitor-like BEC cell, we found significant homeostatic BEC heterogeneity that reflects fluctuating activation of a YAP-dependent program. This transcriptional signature defines a dynamic cellular state during homeostasis and is highly responsive to injury. YAP signaling is induced by physiological bile acids (BAs), required for BEC survival in response to BA exposure, and is necessary for hepatocyte reprogramming into biliary progenitors upon injury. Together, these findings uncover molecular heterogeneity within the ductal epithelium and reveal YAP as a protective rheostat and regenerative regulator in the mammalian liver.

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

Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches

Nat Commun

2020 Jan 17

Kim JE Fei L, Yin WC, Coquenlorge S, Rao-Bhatia A, Zhang X, Shi SSW, Lee JH, Hahn NA, Rizvi W, Kim KH, Sung HK, Hui CC, Guo G, Kim TH
PMID: 31953387 | DOI: 10.1038/s41467-019-14058-5

Stomach and intestinal stem cells are located in discrete niches called the isthmus and crypt, respectively. Recent studies have demonstrated a surprisingly conserved role for Wnt signaling in gastrointestinal development. Although intestinal stromal cells secrete Wnt ligands to promote stem cell renewal, the source of stomach Wnt ligands is still unclear. Here, by performing single cell analysis, we identify gastrointestinal stromal cell populations with transcriptome signatures that are conserved between the stomach and intestine. In close proximity to epithelial cells, these perictye-like cells highly express telocyte and pericyte markers as well as Wnt ligands, and they are enriched for Hh signaling. By analyzing mice activated for Hh signaling, we show a conserved mechanism of GLI2 activation of Wnt ligands. Moreover, genetic inhibition of Wnt secretion in perictye-like stromal cells or stromal cells more broadly demonstrates their essential roles in gastrointestinal regeneration and development, respectively, highlighting a redundancy in gastrointestinal stem cell niches.

L1CAM defines the regenerative origin of metastasis-initiating cells in colorectal cancer

Nat Cancer

2020 Jan 13

Karuna Ganesh, Harihar Basnet, Yasemin Kaygusuz, Ashley M. Laughney, Lan He, Roshan Sharma, Kevin P. O�Rourke, Vincent P. Reuter, Yun-Han Huang, Mesruh Turkekul, Ekrem Emrah Er, Ignas Masilionis, Katia Manova-Todorova, Martin R. Weiser, Leonard B. Saltz, Julio Garcia-Aguilar, Richard Koche, Scott W. Lowe, Dana Pe�er, Jinru Shia & Joan Massagu�
| DOI: 10.1038/s43018-019-0006-x

Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM+ cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAMhigh cells partially overlap with LGR5high stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin�REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAMlow to an L1CAMhigh state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells.

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.

Amino acid transporter SLC7A5 regulates Paneth cell function to affect the intestinal inflammatory response

bioRxiv : the preprint server for biology

2023 Jan 24

Bao, L;Fu, L;Su, Y;Chen, Z;Peng, Z;Sun, L;Gonzalez, FJ;Wu, C;Zhang, H;Shi, B;Shi, YB;
PMID: 36789439 | DOI: 10.1101/2023.01.24.524966

The intestine is critical for not only processing and resorbing nutrients but also protecting the organism from the environment. These functions are mainly carried out by the epithelium, which is constantly being self-renewed. Many genes and pathways can influence intestinal epithelial cell proliferation. Among them is mTORC1, whose activation increases cell proliferation. Here, we report the first intestinal epithelial cell-specific knockout ( ΔIEC ) of an amino acid transporter capable of activating mTORC1. We show that the transporter, SLC7A5, is highly expressed in mouse intestinal crypt and Slc7a5 ΔIEC reduces mTORC1 signaling. Surprisingly, Slc7a5 ΔIEC mice have increased cell proliferation but reduced secretory cells, particularly mature Paneth cells. scRNA-seq and electron microscopic analyses revealed dedifferentiation of Paneth cells in Slc7a5 ΔIEC mice, leading to markedly reduced secretory granules with little effect on Paneth cell number. We further show that Slc7a5 ΔIEC mice are prone to experimental colitis. Thus, SLC7A5 regulates secretory cell differentiation to affect stem cell niche and/or inflammatory response to regulate cell proliferation.

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.

Post-fast refeeding enhances intestinal stem cell-mediated regeneration and tumourigenesis through mTORC1-dependent polyamine synthesis

Research square

2023 Jan 10

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.

Intestinal Apc-inactivation induces HSP25 dependency

EMBO molecular medicine

2022 Nov 02

van Neerven, SM;Smit, WL;van Driel, MS;Kakkar, V;de Groot, NE;Nijman, LE;Elbers, CC;Léveillé, N;Heijmans, J;Vermeulen, L;
PMID: 36321561 | DOI: 10.15252/emmm.202216194

The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc-mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc-driven transformation, using both in vitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high-risk individuals.

Pages

	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

Search form

Probes for LGR5

Content for comparison

Gene

Product

Research area

Category

Pages

Advanced Cell Diagnostics

Bio-Techne

Advanced Cell Diagnostics China