Controlled gene expression in primary Lgr5 organoid cultures

Koo, Bon–Kyoung; Stange, Daniel E.; Sato, Toshiro; Karthaus, Wouter R.; Farin, Henner F.; Huch, Meritxell; Es, Johan H. van; Clevers, Hans

doi:10.1038/nmeth.1802

Cited by 285 publications

(243 citation statements)

References 13 publications

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“…1E). Because the ECD of ZNRF3 directly interacts with FZD receptors and ZNRF3 (27,28), these data imply that ZNRF3-ECDTM inhibits RSPO activity, most likely by hindering the interaction of RSPO-LGR4 with the Wnt signalosome. We tested this theory by determining the effect of overexpressing ZNRF3-ECDTM on the interaction between LGR4 and Wnt receptors and found that it blocked the formation of the supercomplex between LGR4 and LRP6 (Fig.…”

Section: Rspo-mentioning

confidence: 88%

“…Instead, it was shown that the RSPOs function as an extracellular bridge to bring LGR4 and E3 ligases RNF43/ZNRF3 together to form a ternary complex (LGR4-RSPO-RNF43/ZNRF3), which induces clearance of the E3 ligases (27). Because RNF43/ZRNF3 ubiquitinates Wnt receptors for degradation, their removal leads to elevated Wnt receptor levels and increased Wnt signaling (27,28). The importance of this ternary complex in potentiating Wnt signaling has now been revealed by multiple structural and biochemical studies (29)(30)(31), particularly by the finding that higher binding affinity of RSPO2/3 for RNF43/ZNRF3 accounts for their stronger potency in functional assays (32).…”

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confidence: 99%

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RSPO–LGR4 functions via IQGAP1 to potentiate Wnt signaling

Carmon

Gong

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

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R-spondins (RSPOs) and their receptor leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) play pleiotropic roles in normal and cancer development as well as the survival of adult stem cells through potentiation of Wnt signaling. Current evidence indicates that RSPO-LGR4 functions to elevate levels of Wnt receptors through direct inhibition of two membrane-bound E3 ligases (RNF43 and ZNRF3), which otherwise ubiquitinate Wnt receptors for degradation. Whether RSPO-LGR4 is coupled to intracellular signaling proteins to regulate Wnt pathways remains unknown. We identified the intracellular scaffold protein IQ motif containing GTPase-activating protein 1 (IQGAP1) as an LGR4-interacting protein that mediates RSPO-LGR4's interaction with the Wnt signalosome. IQGAP1 binds to and modulates the activities of a plethora of signaling molecules, including MAP kinases, Rho GTPases, and components of the Wnt signaling pathways. Interaction of LGR4 with IQGAP1 brings RSPO-LGR4 to the Wnt signaling complex through enhanced IQGAP1-DVL interaction following RSPO stimulation. In this configuration, RSPO-LGR4-IQGAP1 potentiates β-catenin-dependent signaling by promoting MEK1/2-medidated phosphorylation of LRP5/6 as well as β-catenin-independent signaling through regulation of actin dynamics. Overall, these findings reveal that RSPO-LGR4 not only induces the clearance of RNF43/ZNRF3 to increase Wnt receptor levels but also recruits IQGAP1 into the Wnt signaling complex, leading to potent and robust potentiation of both the canonical and noncanonical pathways of Wnt signaling.cell signaling | receptor activation | adhesion | migration T he four R-spondins (RSPO1-4) and three related leucinerich repeat-containing G-protein coupled receptors, LGR4, LGR5, and LGR6 (LGR4-6), constitute a ligand-receptor system that plays critical roles in development, stem cell survival, and oncogenesis (1-6). Mutations of RSPO1 and RSPO4 affect sex development and nail formation (7-9), respectively, and haplotype insufficiency of LGR4 in humans is associated with several diseases and other traits (10). In the mouse, knockouts of LGR4 or RSPOs are presented with severe developmental abnormalities, including neonatal/embryonic lethality accompanied by hypoplasia and defects in tubule elongation and branching in the kidney, lung, mammary gland, and testis (11-16).LGR5 and LGR6 are markers of adult stem cells in the intestine and other select solid tissues (2).LGR4 is frequently coexpressed with LGR5 or LGR6 and is required for the survival of crypt stem cells in the gut (4, 17). Recurrent, gain-of-expression gene fusions of RSPO2 (to EIF3E) and RSPO3 (to PTPRK) occur in a subset of colorectal cancers (18). In mouse mammary tumor virus-induced mouse models of breast and colon cancer, RSPO2 and RSPO3 were identified as two of the most commonly activated alleles (19-21). Ectopic expression of RSPO2/3 in mouse mammary epithelial cells led to an increase in invasiveness in vitro and in tumor formation and metastasis in vivo (20,22). LGR4 is up-reg...

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Section: Rspo-mentioning

confidence: 88%

mentioning

confidence: 99%

RSPO–LGR4 functions via IQGAP1 to potentiate Wnt signaling

Carmon

Gong

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

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“…Intestinal crypts from Apc flox/flox ; villin-Cre ERT and Apc flox/flox ; Lgr5-EGFP-IRES-Cre ERT mice (21) were isolated and cultured as described (9,10,47). Briefly, the crypts were embedded in Matrigel (BD Biosciences) and cultured in medium containing 100 ng/mL noggin, 50 ng/mL EGF (PeproTech), and 500 ng/mL mouse R-Spondin1 (R&D Systems).…”

Section: Methodsmentioning

confidence: 99%

Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-β

Wiener

Band

Kallio

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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In the majority of microsatellite-stable colorectal cancers (CRCs), an initiating mutation occurs in the adenomatous polyposis coli (APC) or β-catenin gene, activating the β-catenin/TCF pathway. The progression of resulting adenomas is associated with oncogenic activation of KRas and inactivation of the p53 and TGF-β/Smad functions. Most established CRC cell lines contain mutations in the TGF-β/Smad pathway, but little is known about the function of TGF-β in the early phases of intestinal tumorigenesis. We used mouse and human ex vivo 3D intestinal organoid cultures and in vivo mouse models to study the effect of TGF-β on the Lgr5 + intestinal stem cells and their progeny in intestinal adenomas. We found that the TGF-β-induced apoptosis in Apc-mutant organoids, including the Lgr5 + stem cells, was mediated by up-regulation of the BH3-only proapoptotic protein Bcl-2-like protein 11 (Bim). BH3-mimetic compounds recapitulated the effect of Bim not only in the adenomas but also in human CRC organoids that had lost responsiveness to TGF-β-induced apoptosis. However, wild-type intestinal crypts were markedly less sensitive to TGF-β than Apcmutant adenomas, whereas the KRas oncogene increased resistance to TGF-β via the activation of the Erk1/2 kinase pathway, leading to Bim down-regulation. Our studies identify Bim as a critical mediator of TGF-β-induced apoptosis in intestinal adenomas and show that the common progression mutations modify Bim levels and sensitivity to TGF-β during intestinal adenoma development.A ctivation of the β-catenin/TCF transcription factor (Wnt) pathway is the initiating event in the majority of human colorectal cancers (CRCs) and one of the key regulators of CRC pathogenesis (1, 2). The β-catenin level in cells is controlled through a multiprotein complex that contains the adenomatous polyposis coli (APC) protein. In 70-80% of individuals suffering from the sporadic version of CRC, both APC alleles are mutated and thus inactivated, resulting in elevated nuclear β-catenin levels (1). Recently, the intestinal stem cells have been identified as the cells of origin of CRC (3, 4).According to the model presented by Fearon and Vogelstein (5), the initiating APC or CTNNB1 mutation is followed by oncogenic activation of the KRAS gene and inactivation of the TGF-β signal transduction pathway and the TP53 tumor suppressor. Recent genome-wide analysis by the Cancer Genome Atlas Network has demonstrated the high frequency of mutations in the β-catenin/TCF, TGF-β, phosphoinositide 3-kinase (PI3K) and p53 pathways in CRC (6). Full understanding of the roles of the driver mutations in colorectal tumors has been hampered by lack of appropriate ex vivo model systems for studies of CRC progression, and thus it has been difficult to study the effects of TGF-β in intestinal adenoma cells at the early stages of tumor development. Although TGF-β receptors have been deleted in Apc-mutant mouse models of CRC (7,8), these studies do not reveal the mechanism of TGF-β action in intestinal adenomas or give further i...

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“…Organoids were removed from the Matrigel and mechanically dissociated. Dissociated organoids were infected with 100 mL of lentiviral suspension by spinoculation (23). The infected organoids were transferred to fresh Matrigel with a 1:4 split ratio.…”

Section: Organoid Growth Assaymentioning

confidence: 99%

miR-137 Regulates the Tumorigenicity of Colon Cancer Stem Cells through the Inhibition of DCLK1

Sakaguchi

Hisamori

Oshima

et al. 2016

Molecular Cancer Research

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miRNAs have important roles in regulating cancer stem cell (CSC) properties and are considered to be potential therapeutic targets. However, few studies have focused on miRNAs which are specifically related to colon CSCs. Here, a PCR-based miRNA profiling analysis of normal colon stem cells (NCSC) and colonÀ ) identified miRNAs which regulate colon CSC properties. Interestingly, miRNA-137 (miR-137) expression was downregulated in the colon CSCs compared with NCSCs, while doublecortin-like kinase 1 (DCLK1) mRNA was highly expressed in the colon CSCs but low in the NCSCs. In fact, DCLK1-positive cancer cells were widely distributed in clinically resected colon cancer specimens, while DCLK1-positve epithelial cells were rarely detected in normal colon tissues including the crypt bottoms. Luciferase assay and immunoblot analysis revealed that miR-137 regulated DCLK1 gene expression. Transduction of exogenous miR-137 suppressed the development of colon cancer organoids in vitro and the tumorigenicity of colon cancer cells in vivo without affecting the growth of normal intestinal organoids. Furthermore, the suppression of miR-137 enhanced the organoid development of normal colon cells. These data demonstrate that miR-137 has the capacity to suppress the tumorigenicity of colon CSCs and that maintained expression of miR-137 in NCSCs contributes to suppressing uncontrolled cell proliferation through the inhibition of DCLK1 expression.Implications: The miR-137/DCLK1 axis as an important regulator in NCSCs and colon CSCs; further understanding of this axis may foster the development of potential gene therapeutic strategies targeting colon CSCs.

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Controlled gene expression in primary Lgr5 organoid cultures

Cited by 285 publications

References 13 publications

RSPO–LGR4 functions via IQGAP1 to potentiate Wnt signaling

RSPO–LGR4 functions via IQGAP1 to potentiate Wnt signaling

Oncogenic mutations in intestinal adenomas regulate Bim-mediated apoptosis induced by TGF-β

miR-137 Regulates the Tumorigenicity of Colon Cancer Stem Cells through the Inhibition of DCLK1

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