Eva Šloncová

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.

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.

Rapamycin-resistant phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in v-SRC-transformed hamster fibroblasts.

Increased phosphorylation of the translational repressor protein 4E‐BP1 was found in the cell line derived from the tumor induced in Syrian hamster by Rous sarcoma virus (RSV). This was accompanied by its dissociation from the complex with initiation factor eIF4E. The ribosomal S6 protein kinase p70S6k is supposed to be regulated by the same or a closely related rapamycin‐sensitive signalling pathway to that which modulates 4E‐BP1. Phosphorylation and activity of p70S6k were found to be also increased in RSV‐transformed H19 cells that express significantly higher amounts of the Src protein (p60src) relative to the non‐transformed hamster fibroblasts NIL‐2. The increased activity and phosphorylation of p70S6k were blocked by rapamycin, indicating that the rapamycin‐sensitive pathway is involved in its regulation in v‐src‐transformed hamster fibroblasts. In agreement with this, rapamycin reduced the expression of elongation factor eEF1α (whose translation is regulated by a rapamycin‐sensitive mechanism thought to involve p70S6k) and did not affect the production of a housekeeping protein, α‐tubulin, in these cells. Synthesis of Src protein was also inhibited in cells treated with rapamycin. However, treatment of cells with a concentration of rapamycin sufficient to completely inhibit the activity and phosphorylation of p70S6k resulted in only partial de‐phosphorylation of 4E‐BP1 and its re‐association with eIF4E in the transformed cells, indicating that additional rapamycin‐insensitive mechanisms/pathways are implicated in the control of 4E‐BP1 phosphorylation in RSV‐transformed hamster fibroblasts. Over‐expression of eIF4E favours cell proliferation and can lead to a transformed phenotype, while over‐expression of 4E‐BP1 has the opposite effect. The altered signalling to the phosphorylation of 4E‐BP1 in RSV‐transformed cells, which leads to its dissociation from eIF4E and thus relief of inhibition of eIF4E function, may therefore represent an important regulatory mechanism in malignant cell growth. Int. J. Cancer 81:963–969, 1999.

HIC1 Tumor Suppressor Loss Potentiates TLR2/NF-κB Signaling and Promotes Tissue Damage-Associated Tumorigenesis.

Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses “danger” signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells. Implications: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells. Mol Cancer Res; 13(7); 1139–48. ©2015 AACR.

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.

Changes of E-cadherin and beta-catenin in human and mouse intestinal tumours.

An immunohistochemical analysis of E-cadherin and β-catenin was performed in human colorectal cancer as well as in surrounding normal intestinal tissue. We also analysed the expression of these two cell adhesion proteins in transgenic Apc1638N mice as a model of human familial adenometous polyposis syndrome. In the normal intestinal mucosa of both species, E-cadherin and β-catenin were localized along the lateral plasma membrances of epithelial cells. In intestinal tumour cells, however, they were also present in the cytoplasm. The expression of both proteins was reduced in human and mouse tumours. The pattern of their distribution was frequently heterogenous with strongly positive cells in a mosaic of negative ones. Further, E-cadherin and β-catenin expression did not correlate to the Dukes staging of tumours and therefore neither can be used as prognostic criteria.

Initiation factor eIF2B not p70 S6 kinase is involved in the activation of the PI-3K signalling pathway induced by the v-src oncogene.

Our data show that in hamster fibroblasts transformed by Rous sarcoma virus (RSV), the phosphoinositide 3′‐kinase (PI‐3K)/Akt/glycogen synthase kinase 3 antiapoptotic pathway is upregulated and involved in increased protein synthesis through activation of initiation factor eIF2B. Upon inhibition of PI‐3K by wortmannin, phosphorylation of 70‐kDa ribosomal protein S6 kinase (p70 S6k) and its physiological substrate, ribosomal protein S6, decreased in the non‐transformed cells but not in RSV‐transformed cells. Thus PI‐3K, which is thought to be involved in regulation of p70 S6k, signals to p70 S6k in normal fibroblasts, but it does not appear to be an upstream effector of p70 S6k in fibroblasts transformed by v‐src oncogene, suggesting that changes in the PI‐3K signalling pathway upstream of p70 S6k are induced by RSV transformation.

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.

Wnt Effector TCF4 Is Dispensable for Wnt Signaling in Human Cancer Cells

T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

HIC1 Expression Distinguishes Intestinal Carcinomas Sensitive to Chemotherapy

Neoplastic growth is frequently associated with genomic DNA methylation that causes transcriptional silencing of tumor suppressor genes. We used a collection of colorectal polyps and carcinomas in combination with bioinformatics analysis of large datasets to study the expression and methylation of Hypermethylated in cancer 1 (HIC1), a tumor suppressor gene inactivated in many neoplasms. In premalignant stages, HIC1 expression was decreased, and the decrease was linked to methylation of a specific region in the HIC1 locus. However, in carcinomas, the HIC1 expression was variable and, in some specimens, comparable to healthy tissue. Importantly, high HIC1 production distinguished a specific type of chemotherapy-responsive tumors.