Franken, P.F.

Alterations in Wnt-β-catenin and Pten signalling play distinct roles in endometrial cancer initiation and progression (Article)

2013-03-18T00:00:00Z

Endometrioid endometrial cancer arises through a gradual series of histological changes, each accompanied by specific alterations in gene expression and activity. Activation of the Wnt-β-catenin pathway and loss of PTEN activity are frequently observed in endometrial cancers. However, the specific roles played by alterations in these pathways in the initiation and progression of endometrial cancer are currently unclear. Here, we investigated the effects of loss of Pten and Apc gene function in the mouse endometrium by employing tissue-specific and inducible mutant alleles, followed by immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of their corresponding cancerous lesions. Loss of the Apc function in the endometrium leads to cytoplasmic and nuclear β-catenin accumulation in association with uterine hyperplasia and squamous cell metaplasia, but without malignant transformation. Loss of Pten function also resulted in squamous metaplasia but, in contrast to loss of Apc function, it initiates endometrial cancer. On the other hand, loss of Apc function in the endometrium accelerates Pten-driven endometrial tumourigenesis. Analysis of compound heterozygous mice confirmed that somatic loss of the wild-type Pten allele represents the rate-limiting initiation step in endometrial cancer. Simultaneous loss of Pten and Apc resulted in endometrial cancer characterized by earlier onset and a more aggressive malignant behaviour. These observations are indicative of the synergistic action between the Wnt-β-catenin and Pten signalling pathways in endometrial cancer onset and progression.

Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice (Article)

2012-09-01T00:00:00Z

Wnt5a is essential during embryonic development, as indicated by mouse . Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of . Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible . Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5. dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in . Wnt5a knockout mice. However, Wnt5a induction from 13.5. dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5. dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages.

Early morbidity encountered in the dietary-related mouse model of Barrett's esophagus: A question of zinc? (Article)

2011-07-01T00:00:00Z

Recently, a mouse model for Barrett's esophagus based on a zinc-deficient diet supplemented with deoxycholic bile acids has been published. The aim of this study was to attempt to reproduce these data and extend them by employing genetically modified mice and intraperitoneal iron supplementation. The study design encompassed six experimental groups (wild type, Apc-mutant and Smad4-mutant mice, with or without iron injections), with all animals fed with the zinc-deficient diet supplemented with deoxycholic bile acids. All treatments were started at 3-5 weeks of age (the majority [78%] at 5 weeks). Animals were scheduled for euthanasia at two distinct time points, namely at 3 and 6 months of age. All mice showed signs of considerable distress already 4 weeks after the start of the modified diets, and had to be euthanized before the first evaluation time point (mean age 9.3 weeks, range 5-15 weeks). No differences were observed between wild type and genetically modified mice, or between animals with or without iron supplementation. On histological examination, we could not detect any lesions (Barrett's esophagus-like or tumors) other than esophagitis. In the currently presented experimental settings, we were not able to reproduce the mouse model according to which Barrett's-like lesions could be detected in animals fed with the zinc-deficient diet supplemented with deoxycholic bile acids. © 2010 Copyright the Authors. Journal compilation

β-catenin tyrosine 654 phosphorylation increases Wnt signalling and intestinal tumorigenesis (Article)

2011-02-09T00:00:00Z

Objective: Deregulation of the Wnt signalling pathway by mutations in the Apc or β-catenin genes underlies colorectal carcinogenesis. As a result, β-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear β-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate β-catenin at tyrosine residues, which is thought to increase β-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of β-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design: A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous β-catenin gene was introduced. Results: This study provided in vivo evidence that β-cateninE654 is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the β-cateninE654 targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of βcatenin. Surprisingly, the expression of β-cateninE654 did not affect histological grade or induce tumour invasiveness. Conclusions: A thus far unknown mechanism was uncovered in which Y654 phosphorylation of β-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that β-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.

Reproducibility, accuracy, and predictors of accuracy for the detection of coronary atherosclerotic plaque composition by computed tomography: An ex vivo comparison to intravascular ultrasound (Article)

2010-11-01T00:00:00Z

The MUTYH gene encodes a DNA glycosylase involved in base excision repair (BER). Biallelic pathogenic MUTYH variants have been associated with colorectal polyposis and cancer. The pathogenicity of a few variants is beyond doubt, including c.536A>G/p.Tyr179Cys and c.1187G>A/p.Gly396Asp (previously c.494A>G/p.Tyr165Cys and c.1145G>A/p.Gly382Asp). However, for a substantial fraction of the detected variants, the clinical significance remains uncertain, compromising molecular diagnostics and thereby genetic counseling. We have established an interactive MUTYH gene sequence variant database () with the aim of collecting and sharing MUTYH genotype and phenotype data worldwide. To support standard variant description, we chose NM_001128425.1 as the reference sequence. The database includes records with variants per individual, linked to available phenotype and geographic origin data as well as records with in vitro functional and in silico test data. As of April 2010, the database contains 1968 published and 423 unpublished submitted entries, and 230 and 61 unique variants, respectively. This open-access repository allows all involved to quickly share all variants encountered and communicate potential consequences, which will be especially useful to classify variants of uncertain significance.

Progesterone inhibition of Wnt/β-catenin signaling in normal endometrium and endometrial cancer (Article)

2009-09-15T00:00:00Z

Purpose. Wnt signaling regulates the fine balance between stemness and differentiation. Here, the role of Wnt signaling to maintain the balance between estrogen-induced proliferation and progesterone-induced differentiation during the menstrual cycle, as well as during the induction of hyperplasia and carcinogenesis of the endometrium, was investigated. Experimental Design: Endometrial gene expression profiles from estradiol (E2) and E 2 + medroxyprogesterone acetate-treated postmenopausal patients were combined with profiles obtained during the menstrual cycle (PubMed; GEO DataSets). Ishikawa cells were transfected with progesterone receptors and Wnt inhibitors dickkopf homologue 1 (DKK1) and forkhead box O1 (FOXO1), measuring Wnt activation. Expression of DKK1 and FOXO1 was inhibited by use of sequence-specific short hairpins. Furthermore, patient samples (hormone-treated endometria, hyperplasia, and endometrial cancer) were stained for Wnt activation using nuclear β-catenin and CD44. Results: In vivo, targets and components of the Wnt signaling pathway (among them DKK1 and FOXO1) are regulated by E2 and progesterone. In Wnt-activated Ishikawa cells, progesterone inhibits Wnt signaling by induction of DKK1 and FOXO1. Furthermore, using siRNA-mediated knockdown of both DKK1 and FOXO1, progesterone inhibition of Wnt signaling was partly circumvented. Subsequently, immunohistochemical analysis of the Wnt target gene CD44 showed that progesterone acted as an inhibitor of Wnt signaling in hyperplasia and in well-differentiated endometrial cancer. Conclusion: Progesterone induction of DKK1 and FOXO1 results in inhibition of Wnt signaling in the human endometrium. This Wnt inhibitory effect of progesterone is likely to play a rate-limiting role in the maintenance of endometrial homeostasis and, on its loss, in tumor onset and progression toward malignancy.

Generation of a tightly regulated doxycycline-inducible model for studying mouse intestinal biology (Article)

2009-07-01T00:00:00Z

To develop a sensitive and inducible system to study intestinal biology, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the 12.4 kb murine Villin promoter. The newly generated Villin-rtTA2-M2 mice were then bred with the previously developed tetOHIST1H2BJ/GFP model to assess inducibility and tissues-pecificity. Expression of the histone H2B-GFP fusion protein was observed exclusively upon doxycycline induction and was uniformly distributed throughout the intestinal epithelium. The Villin-rtTA2-M2 was also found to drive transgene expression in the developing mouse intestine. Furthermore, we could detect transgene expression in the proximal tubules of the kidney and in a population of alleged gastric progenitor cells. By administering different concentrations of doxycycline, we show that the Villin-rtTA2-M2 system drives transgene expression in a dosage-dependent fashion. Thus, we have generated a novel doxycycline-inducible mouse model, providing a valuable tool to study the effect of different gene dosages on intestinal physiology and pathology.

Wnt pathway-related gene expression during malignant progression in ulcerative colitis (Article)

2007-08-01T00:00:00Z

Long-standing ulcerative colitis (UC) has been associated with a high risk of developing colonic adenocarcinoma. Importantly, both low- and high-grade dysplasia are strongly related to the presence or development of malignancy. The canonical Wnt/β-catenin signaling pathway is of crucial importance in cancer development and progression, but its role in UC-related carcinogenesis remains to be determined. We evaluated the immunolabeling patterns of β-catenin, as well as the products of Wnt-associated cancer genes E-cadherin, cyclin D1 and c-myc, along the dysplasia-carcinoma pathway in UC. For this purpose, immunohistochemistry (IHC) was performed on 18 adenocarcinomas and 17 dysplasias, derived from 21 patients. We found that intracellular β-catenin accumulation, the hallmark of Wnt signaling activation, is observed in dysplasia, together with enhanced labeling of nuclear protein cyclin D1 and reduction of membranous labeling of E-cadherin. c-myc displayed moderate immunolabeling in the (pre)malignant lesions. Thus, the Wnt pathway is activated in early stages of malignant progression in UC. Furthermore, upregulation of the oncogene cyclin D1 and downregulation of tumor suppressor E-cadherin also occurs in the (pre)neoplastic state. This may contribute to the high potential for malignant degeneration of dysplasia in UC-related colitis.

The CHEK2 1100delC mutation identifies families with a hereditary breast and colorectal cancer phenotype (Article)

2003-01-01T00:00:00Z

Because of genetic heterogeneity, the identification of breast cancer-susceptibility genes has proven to be exceedingly difficult. Here, we define a new subset of families with breast cancer characterized by the presence of colorectal cancer cases. The 1100delC variant of the cell cycle checkpoint kinase CHEK2 gene was present in 18% of 55 families with hereditary breast and colorectal cancer (HBCC) as compared with 4% of 380 families with non-HBCC (P<.001), thus providing genetic evidence for the HBCC phenotype. The CHEK2 1100delC mutation was, however, not the major predisposing factor for the HBCC phenotype but appeared to act in synergy with another, as-yet-unknown susceptibility gene(s). The unequivocal definition of the HBCC phenotype opens new avenues to search for this putative HBCC-susceptibility gene.