Andrea Haegebarth

Identification of stem cells in small intestine and colon by marker gene Lgr5

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. It is currently believed that four to six crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined. Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5, also known as Gpr49) was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Here, using two knock-in alleles, we reveal exclusive expression of Lgr5 in cycling columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues. Using an inducible Cre knock-in allele and the Rosa26-lacZ reporter strain, lineage-tracing experiments were performed in adult mice. The Lgr5-positive crypt base columnar cell generated all epithelial lineages over a 60-day period, suggesting that it represents the stem cell of the small intestine and colon. The expression pattern of Lgr5 suggests that it marks stem cells in multiple adult tissues and cancers.

Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.

Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin.

Hair Today, Skin Tomorrow The epidermis of mammals contains hair follicles, sebaceous glands, and interfollicular epidermis, but it has not been clear how the development and repair of these structures is regulated. Snippert et al. (p. 1385) show that a stem-cell cluster in the hair follicle, characterized by the expression of Lgr6, a close homolog of the Lgr5 marker for stem cells in the small intestine and colon, resides directly above the hair bulge and gives rise to all cell lineages of the skin. Skin wounds in adult mice are repaired by Lgr6 stem cells in the hair follicles that flank the damage. After hair morphogenesis, Lgr6 stem cells give rise to epidermal and sebaceous gland lineages to generate fully differentiated new skin. Skin wounds can be repaired by primitive stem cells into fully differentiated tissue, complete with hairs and sebaceous glands. Mammalian epidermis consists of three self-renewing compartments: the hair follicle, the sebaceous gland, and the interfollicular epidermis. We generated knock-in alleles of murine Lgr6, a close relative of the Lgr5 stem cell gene. Lgr6 was expressed in the earliest embryonic hair placodes. In adult hair follicles, Lgr6+ cells resided in a previously uncharacterized region directly above the follicle bulge. They expressed none of the known bulge stem cell markers. Prenatal Lgr6+ cells established the hair follicle, sebaceous gland, and interfollicular epidermis. Postnatally, Lgr6+ cells generated sebaceous gland and interfollicular epidermis, whereas contribution to hair lineages gradually diminished with age. Adult Lgr6+ cells executed long-term wound repair, including the formation of new hair follicles. We conclude that Lgr6 marks the most primitive epidermal stem cell.

Transcription Factor Achaete Scute-Like 2 Controls Intestinal Stem Cell Fate

The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.

OLFM4 Is a Robust Marker for Stem Cells in Human Intestine and Marks a Subset of Colorectal Cancer Cells

i H m c O d i a c m i b r t he epithelium of the small intestine and colon is the most rapidly self-renewing tissue in mammals. Proiferative cells are confined to crypts, and differentiated ell types predominantly occupy the villi in the small ntestine and the surface epithelium in the colon. We ecently demonstrated the existence of a long-lived pool f cycling stem cells defined by Lgr5 expression. These ells are intermingled with postmitotic Paneth cells at rypt bottoms in the small intestine. In the colon, they eside at crypt bottoms and are intermingled with goblet ells. Whereas knockin alleles of Lgr5 have been instruental in defining these cells, Lgr5 mRNA or protein evels are too low to serve as faithful markers. Olfactomein-4 (Olfm4) has emerged from a gene signature for these gr5 stem cells as a robust marker for murine small ntestinal stem cells. Herein, we show that OLFM4 is ighly expressed in crypt base columnar cells in human mall intestine and colon. Moreover, OLFM4 is expressed n cells within adenocarcinomas of the colon. We propose hat OLFM4 can serve as a useful marker for Lgr5-type tem cells in human small intestine and colon. Intestinal crypts contain stem cells and their transitmplifying (TA) daughter cells. Cells exiting the prolifertive crypts onto the villi terminally differentiate into nterocytes, goblet cells, and enteroendocrine cells. Panth cells escape the crypt–villus flow by migrating to crypt ottoms where they live for several weeks. With the exeption of stem cells and Paneth cells, the murine small ntestinal epithelium is renewed approximately every 5 ays.1 Because of the intimate connection between Wnt ignaling, crypt biology,2– 4 and colon cancer,5,6 we have reviously attempted to unravel the TCF4 target gene rogram activated by this pathway in crypts and colorectal umors.7–9 Lgr5 emerged from these studies as a candiate stem cell marker. Using Cre-mediated genetic tracng, we demonstrated that Lgr5 marks long-lived, multiotent stem cells.10,11 Each crypt bottom harbors about 6 f these small, cycling cells intermingled with Paneth ells. Although Lgr5 stem cells occasionally occupy a osition directly above the Paneth cells, they seem to be istinct from another proposed stem cell population ocated at the so called 4 position12,13; Lgr5 stem cells re not particularly radiation-sensitive and do not retain NA labels.1,10 Recently, it was shown that Bmi1 expresion marks cells at position 4 relative to the crypt ottom. Lineage tracing has revealed that Bmi1 cells ark pluripotent stem cells that replenish the epithelium ith similar kinetics to Lgr5 stem cells.14 With real-time uantitative polymerase chain reaction analysis, we have ound that Bmi1RNA is enriched in Lgr5 stem cells,15 uggesting that Bmi1 and Lgr5 stem cells represent overapping populations. We previously determined a differential gene expresion profile for Lgr5 stem cells and their immediate aughters, by GFP-based sorting of epithelial cells from solated crypts of Lgr5-EGFP-ires-CreERT2 mice.15 When ndividual genes were tested by in situ hybridization nalysis, Olfm4 emerged as a highly specific and robust arker for Lgr5 stem cells. Olfm4 was not expressed in urine colon.15 Yet, human OLFM4 is enriched in human olon crypts as demonstrated by microarray analysis.16 he OLFM4 gene encodes a secreted molecule with unnown function originally cloned from human myelolasts.17 Recently, it was shown that Xenopus ONT1, an LFM4 family member, acts as a BMP antagonist.18 We obtained paraffin sections of normal human small ntestine and colon and performed in situ hybridizations. uman OLFM4 is expressed in comparable fashion to urine Olfm4 in the small intestine, that is, in crypt base olumnar cells (Figure 1A and B). Although murine lfM4 is not expressed in the colon, a specific signal was etected in human colonic epithelium (Figure 1C), remniscent of Lgr5 expression in the colon.10 OLFM4 was lso highly expressed in subsets of cells within colorectal arcinomas (Figure 1D). OLFM4 expression in these tuor cells was much higher than the expression observed n wild-type crypt base columnar cells at flanking crypt ottoms. -Catenin staining on serial sections clearly evealed the location of malignant tissue elements within he preparation (Figure 1E).

Wnt Signaling, Lgr5, and Stem Cells in the Intestine and Skin

Stem cells hold great promise for regenerative medicine, but have remained elusive in many tissues because of a lack of adequate definitive markers. Progress in mouse genetics has provided the tools for characterization and validation of stem cell markers by functional and/or lineage tracing assays. The Wnt target gene Lgr5 has been recently identified as a novel stem cell marker of the intestinal epithelium and the hair follicle. In the intestine, Lgr5 is exclusively expressed in cycling crypt base columnar cells. Genetic lineage-tracing experiments revealed that crypt base columnar cells are capable of self-renewal and multipotency, thus representing genuine intestinal stem cells. In the stem cell niche of the murine hair follicle, Lgr5 is expressed in actively cycling cells. Transplantation and lineage tracing experiments have demonstrated that these Lgr5(+ve) cells maintain all cell lineages of the hair follicle throughout long periods of time and can build entire new hair follicles. Expression of Lgr5 in multiple other organs indicates that it may represent a global marker of adult stem cells. This review attempts to provide a comprehensive overview of the stem cell compartments in the intestine and skin with a focus on the cycling, yet long-lived and multipotent, Lgr5(+ve) stem cell populations.

Loss of the Tumor Suppressor CYLD Enhances Wnt/β-Catenin Signaling through K63-Linked Ubiquitination of Dvl

The mechanism by which Wnt receptors transduce signals to activate downstream beta-catenin-mediated target gene transcription remains incompletely understood but involves Frizzled (Fz) receptor-mediated plasma membrane recruitment and activation of the cytoplasmic effector Dishevelled (Dvl). Here, we identify the deubiquitinating enzyme CYLD, the familial cylindromatosis tumor suppressor gene, as a negative regulator of proximal events in Wnt/beta-catenin signaling. Depletion of CYLD from cultured cells markedly enhances Wnt-induced accumulation of beta-catenin and target gene activation. Moreover, we demonstrate hyperactive Wnt signaling in human cylindroma skin tumors that arise from mutations in CYLD. At the molecular level, CYLD interacts with and regulates K63-linked ubiquitination of Dvl. Enhanced ubiquitination of the polymerization-prone DIX domain in CYLD-deficient cells positively links to the signaling activity of Dvl. Together, our results argue that loss of CYLD instigates tumor growth in human cylindromatosis through a mechanism in which hyperubiquitination of polymerized Dvl drives enhancement of Wnt responses.

Dynamics of Lgr6(+) Progenitor Cells in the Hair Follicle, Sebaceous Gland, and Interfollicular Epidermis.

Summary The dynamics and interactions between stem cell pools in the hair follicle (HF), sebaceous gland (SG), and interfollicular epidermis (IFE) of murine skin are still poorly understood. In this study, we used multicolor lineage tracing to mark Lgr6-expressing basal cells in the HF isthmus, SG, and IFE. We show that these Lgr6+ cells constitute long-term self-renewing populations within each compartment in adult skin. Quantitative analysis of clonal dynamics revealed that the Lgr6+ progenitor cells compete neutrally in the IFE, isthmus, and SG, indicating population asymmetry as the underlying mode of tissue renewal. Transcriptional profiling of Lgr6+ and Lgr6− cells did not reveal a distinct Lgr6-associated gene expression signature, raising the question of whether Lgr6 expression requires extrinsic niche signals. Our results elucidate the interrelation and behavior of Lgr6+ populations in the IFE, HF, and SG and suggest population asymmetry as a common mechanism for homeostasis in several epithelial skin compartments.

Novel Class of Potent and Cellularly Active Inhibitors Devalidates MTH1 as Broad-Spectrum Cancer Target

MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival.

SIAH ubiquitin ligases regulate breast cancer cell migration and invasion independent of the oxygen status

Seven-in-absentia homolog (SIAH) proteins are evolutionary conserved RING type E3 ubiquitin ligases responsible for the degradation of key molecules regulating DNA damage response, hypoxic adaptation, apoptosis, angiogenesis, and cell proliferation. Many studies suggest a tumorigenic role for SIAH2. In breast cancer patients SIAH2 expression levels correlate with cancer aggressiveness and overall patient survival. In addition, SIAH inhibition reduced metastasis in melanoma. The role of SIAH1 in breast cancer is still ambiguous; both tumorigenic and tumor suppressive functions have been reported. Other studies categorized SIAH ligases as either pro- or antimigratory, while the significance for metastasis is largely unknown. Here, we re-evaluated the effects of SIAH1 and SIAH2 depletion in breast cancer cell lines, focusing on migration and invasion. We successfully knocked down SIAH1 and SIAH2 in several breast cancer cell lines. In luminal type MCF7 cells, this led to stabilization of the SIAH substrate Prolyl Hydroxylase Domain protein 3 (PHD3) and reduced Hypoxia-Inducible Factor 1α (HIF1α) protein levels. Both the knockdown of SIAH1 or SIAH2 led to increased apoptosis and reduced proliferation, with comparable effects. These results point to a tumor promoting role for SIAH1 in breast cancer similar to SIAH2. In addition, depletion of SIAH1 or SIAH2 also led to decreased cell migration and invasion in breast cancer cells. SIAH knockdown also controlled microtubule dynamics by markedly decreasing the protein levels of stathmin, most likely via p27Kip1. Collectively, these results suggest that both SIAH ligases promote a migratory cancer cell phenotype and could contribute to metastasis in breast cancer.