Michaela Krausova

Wnt signaling in adult intestinal stem cells and cancer.

Signaling initiated by secreted glycoproteins of the Wnt family regulates many aspects of embryonic development and it is involved in homeostasis of adult tissues. In the gastrointestinal (GI) tract the Wnt pathway maintains the self-renewal capacity of epithelial stem cells. The stem cell attributes are conferred by mutual interactions of the stem cell with its local microenvironment, the stem cell niche. The niche ensures that the threshold of Wnt signaling in the stem cell is kept in physiological range. In addition, the Wnt pathway involves various feedback loops that balance the opposing processes of cell proliferation and differentiation. Today, we have compelling evidence that mutations causing aberrant activation of the Wnt pathway promote expansion of undifferentiated progenitors and lead to cancer. The review summarizes recent advances in characterization of adult epithelial stem cells in the gut. We mainly focus on discoveries related to molecular mechanisms regulating the output of the Wnt pathway. Moreover, we present novel experimental approaches utilized to investigate the epithelial cell signaling circuitry in vivo and in vitro. Pivotal aspects of tissue homeostasis are often deduced from studies of tumor cells; therefore, we also discuss some latest results gleaned from the deep genome sequencing studies of human carcinomas of the colon and rectum.

Troy, a Tumor Necrosis Factor Receptor Family Member, Interacts With Lgr5 to Inhibit Wnt Signaling in Intestinal Stem Cells

BACKGROUND & AIMS The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice. METHODS We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc(+/Min) and Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization. RESULTS We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer-derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist. CONCLUSIONS TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.

Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling.

The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.

Monensin Inhibits Canonical Wnt Signaling in Human Colorectal Cancer Cells and Suppresses Tumor Growth in Multiple Intestinal Neoplasia Mice

The Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/β-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with β-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or β-catenin–induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of β-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling. Mol Cancer Ther; 13(4); 812–22. ©2014 AACR.

Efficient gene targeting of the Rosa26 locus in mouse zygotes using TALE nucleases

Gene targeting in mice mainly employs homologous recombination (HR) in embryonic stem (ES) cells. Although it is a standard way for production of genetically modified mice, the procedure is laborious and time‐consuming. This study describes targeting of the mouse Rosa26 locus by transcription activator‐like effector nucleases (TALENs). We employed TALEN‐assisted HR in zygotes to introduce constructs encoding TurboRFP and TagBFP fluorescent proteins into the first intron of the Rosa26 gene, and in this way generated two transgenic mice. We also demonstrated that these Rosa26‐specific TALENs exhibit high targeting efficiency superior to that of zinc‐finger nucleases (ZFNs) specific for the same targeting sequence. Moreover, we devised a reporter assay to assess TALENs activity and specificity to improve the quality of TALEN‐assisted targeting.

Generation of Two Modified Mouse Alleles of the Hic1 Tumor Suppressor Gene

HIC1 (hypermethylated in cancer 1) is a tumor suppressor gene located on chromosome 17p13.3, a region frequently hypermethylated or deleted in human neoplasias. In mouse, Hic1 is essential for embryonic development and exerts an antitumor role in adult animals. Since Hic1‐deficient mice die perinatally, we generated a conditional Hic1 null allele by flanking the Hic1‐coding region by loxP sites. When crossed to animals expressing Cre recombinase in a cell‐specific manner, the Hic1 conditional mice will provide new insights into the function of Hic1 in developing and mature tissues. Additionally, we used gene targeting to replace sequence‐encoding amino acids 186–893 of Hic1 by citrine fluorescent protein cDNA. We demonstrate that the distribution of Hic1‐citrine fusion polypeptide corresponds to the expression pattern of wild‐type Hic1. Consequently, Hic1‐citrine “reporter” mice can be used to monitor the activity of the Hic1 locus using citrine fluorescence. genesis 49:142‐151, 2011.

NKD1 marks intestinal and liver tumors linked to aberrant Wnt signaling.

The activity of the Wnt pathway undergoes complex regulation to ensure proper functioning of this principal signaling mechanism during development of adult tissues. The regulation may occur at several levels and includes both positive and negative feedback loops. In the present study we employed one of such negative feedback regulators, naked cuticle homolog 1 (Nkd1), to follow the Wnt pathway activity in the intestine and liver and in neoplasia originated in these organs. Using lineage tracing in transgenic mice we localized Nkd1 mRNA to the bottom parts of the small intestinal crypts and hepatocytes surrounding the central vein of the hepatic lobule. Furthermore, in two mouse models of intestinal tumorigenesis, Nkd1 expression levels were elevated in tumors when compared to healthy tissue. We utilized a collection of human intestinal polyps and carcinomas to confirm that NKD1 represents a robust marker of neoplastic growth. In addition, expression analysis of NKD1 in liver cancer showed that high expression levels of the gene distinguish a subclass of hepatocellular carcinomas related to aberrant Wnt signaling. Finally, our results were confirmed by bioinformatic analysis of large publicly available datasets that included gene expression profiling and high-throughput sequencing data of human colon and liver cancer specimens.

TALE-directed local modulation of H3K9 methylation shapes exon recognition.

In search for the function of local chromatin environment on pre-mRNA processing we established a new tool, which allows for the modification of chromatin using a targeted approach. Using Transcription Activator-Like Effector domains fused to histone modifying enzymes (TALE-HME), we show locally restricted alteration of histone methylation modulates the splicing of target exons. We provide evidence that a local increase in H3K9 di- and trimethylation promotes inclusion of the target alternative exon, while demethylation by JMJD2D leads to exon skipping. We further demonstrate that H3K9me3 is localized on internal exons genome-wide suggesting a general role in splicing. Consistently, targeting of the H3K9 demethylase to a weak constitutive exon reduced co-transcriptional splicing. Together our data show H3K9 methylation within the gene body is a factor influencing recognition of both constitutive and alternative exons.

snRNP proteins in health and disease

Split gene architecture of most human genes requires removal of intervening sequences by mRNA splicing that occurs on large multiprotein complexes called spliceosomes. Mutations compromising several spliceosomal components have been recorded in degenerative syndromes and haematological neoplasia, thereby highlighting the importance of accurate splicing execution in homeostasis of assorted adult tissues. Moreover, insufficient splicing underlies defective development of craniofacial skeleton and upper extremities. This review summarizes recent advances in the understanding of splicing factor function deduced from cryo-EM structures. We combine these data with the characterization of splicing factors implicated in hereditary or somatic disorders, with a focus on potential functional consequences the mutations may elicit in spliceosome assembly and/or performance. Given aberrant splicing or perturbations in splicing efficiency substantially underpin disease pathogenesis, profound understanding of the mis-splicing principles may open new therapeutic vistas. In three major sections dedicated to retinal dystrophies, hereditary acrofacial syndromes, and haematological malignancies, we delineate the noticeable variety of conditions associated with dysfunctional splicing and accentuate recurrent patterns in splicing defects.

Wnt Signaling Inhibition Deprives Small Intestinal Stem Cells of Clonogenic Capacity

The Wnt pathway plays a crucial role in self‐renewal and differentiation of cells in the adult gut. In the present study, we revealed the functional consequences of inhibition of canonical Wnt signaling in the intestinal epithelium. The study was based on generation of a novel transgenic mouse strain enabling inducible expression of an N‐terminally truncated variant of nuclear Wnt effector T cell factor 4 (TCF4). The TCF4 variant acting as a dominant negative (dn) version of wild‐type (wt) TCF4 protein decreased transcription of β‐catenin‐TCF4‐responsive genes. Interestingly, suppression of Wnt/β‐catenin signaling affected asymmetric division of intestinal stem cells (ISCs) rather than proliferation. ISCs expressing the transgene underwent several rounds of division but lost their clonogenic potential and migrated out of the crypt. Expression profiling of crypt cells revealed that besides ISC‐specific markers, the dnTCF4 production downregulated expression levels of epithelial genes produced in other crypt cells including markers of Paneth cells. Additionally, in Apc conditional knockout mice, dnTCF activation efficiently suppressed growth of Apc‐deficient tumors. In summary, the generated mouse strain represents a convenient tool to study cell‐autonomous inhibition of β‐catenin‐Tcf‐mediated transcription. genesis 54:101–114, 2016.