Vladimir Korinek

XTcf-3 Transcription Factor Mediates β-Catenin-Induced Axis Formation in Xenopus Embryos

XTcf-3 is a maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1. The N-terminus of XTcf-3 binds to beta-catenin. Microinjection of XTcf-3 mRNA in embryos results in nuclear translocation of beta-catenin. The beta-catenin-XTcf-3 complex activates transcription in a transient reporter gene assay, while XTcf-3 by itself is silent. N-terminal deletion of XTcf-3 (delta N) abrogates the interaction with beta-catenin, as well as the consequent transcription activation. This dominant-negative delta N mutant suppresses the induction of axis duplication by microinjected beta-catenin. It also suppresses endogenous axis specification upon injection into the dorsal blastomeres of a 4-cell-stage embryo. We propose that signaling by beta-catenin involves complex formation with XTcf-3, followed by nuclear translocation and activation of specific XTcf-3 target genes.

A Novel Tankyrase Inhibitor Decreases Canonical Wnt Signaling in Colon Carcinoma Cells and Reduces Tumor Growth in Conditional APC Mutant Mice

Increased nuclear accumulation of β-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/β-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the β-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the β-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the β-catenin destruction complex, followed by increased degradation of β-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of β-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/β-catenin signaling through inhibiting the PARP domain of TNKS1/2.

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.

A Novel Tankyrase Small-Molecule Inhibitor Suppresses APC Mutation–Driven Colorectal Tumor Growth

Most colorectal cancers (CRC) are initiated by mutations of APC, leading to increased β-catenin-mediated signaling. However, continued requirement of Wnt/β-catenin signaling for tumor progression in the context of acquired KRAS and other mutations is less well-established. To attenuate Wnt/β-catenin signaling in tumors, we have developed potent and specific small-molecule tankyrase inhibitors, G007-LK and G244-LM, that reduce Wnt/β-catenin signaling by preventing poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting β-catenin destabilization. We show that novel tankyrase inhibitors completely block ligand-driven Wnt/β-catenin signaling in cell culture and display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines. It was previously unknown whether the level of AXIN protein stabilization by tankyrase inhibition is sufficient to impact tumor growth in the absence of normal APC activity. Compound G007-LK displays favorable pharmacokinetic properties and inhibits in vivo tumor growth in a subset of APC-mutant CRC xenograft models. In the xenograft model most sensitive to tankyrase inhibitor, COLO-320DM, G007-LK inhibits cell-cycle progression, reduces colony formation, and induces differentiation, suggesting that β-catenin-dependent maintenance of an undifferentiated state may be blocked by tankyrase inhibition. The full potential of the antitumor activity of G007-LK may be limited by intestinal toxicity associated with inhibition of Wnt/β-catenin signaling and cell proliferation in intestinal crypts. These results establish proof-of-concept antitumor efficacy for tankyrase inhibitors in APC-mutant CRC models and uncover potential diagnostic and safety concerns to be overcome as tankyrase inhibitors are advanced into the clinic.

Spatial and temporal regulation of Wnt/β-catenin signaling is essential for development of the retinal pigment epithelium

Wnt/beta-catenin signaling is highly active in the dorsal retinal pigment epithelium (RPE) during eye development. To study the role of Wnt/beta-catenin signaling in the RPE development we used a conditional Cre/loxP system in mice to inactivate or ectopically activate Wnt/beta-catenin signaling in the RPE. Inactivation of Wnt/beta-catenin signaling results in transdifferentiation of RPE to neural retina (NR) as documented by downregulation of RPE-specific markers Mitf and Otx2 and ectopic expression of NR-specific markers Chx10 and Rx, respectively. In contrast, ectopic activation of Wnt/beta-catenin signaling results in the disruption of the RPE patterning, indicating that precise spatial and temporal regulation of Wnt/beta-catenin signaling is required for normal RPE development. Using chromatin immunoprecipitation (ChIP) and reporter gene assays we provide evidence that Otx2 and RPE-specific isoform of Mitf, Mitf-H, are direct transcriptional targets of Wnt/beta-catenin signaling. Combined, our data suggest that Wnt/beta-catenin signaling plays an essential role in development of RPE by maintaining or inducing expression of Mitf and Otx2.

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.

HIC1 attenuates Wnt signaling by recruitment of TCF-4 and β-catenin to the nuclear bodies

The hypermethylated in cancer 1 (HIC1) gene is epigenetically inactivated in cancer, and in addition, the haploinsufficiency of HIC1 is linked to the development of human Miller–Dieker syndrome. HIC1 encodes a zinc‐finger transcription factor that acts as a transcriptional repressor. Additionally, the HIC1 protein oligomerizes via the N‐terminal BTB/POZ domain and forms discrete nuclear structures known as HIC1 bodies. Here, we provide evidence that HIC1 antagonizes the TCF/β‐catenin‐mediated transcription in Wnt‐stimulated cells. This appears to be due to the ability of HIC1 to associate with TCF‐4 and to recruit TCF‐4 and β‐catenin to the HIC1 bodies. As a result of the recruitment, both proteins are prevented from association with the TCF‐binding elements of the Wnt‐responsive genes. These data indicate that the intracellular amounts of HIC1 protein can modulate the level of the transcriptional stimulation of the genes regulated by canonical Wnt/β‐catenin signaling.

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.

Dazap2 modulates transcription driven by the Wnt effector TCF-4

A major outcome of the canonical Wnt/β-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with β-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/β-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.