Elvira Bakker

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

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.

Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron

The different segments of the nephron and glomerulus in the kidney balance the processes of water homeostasis, solute recovery, blood filtration, and metabolite excretion. When segment function is disrupted, a range of pathological features are presented. Little is known about nephron patterning during embryogenesis. In this study, we demonstrate that the early nephron is patterned by a gradient in β-catenin activity along the axis of the nephron tubule. By modifying β-catenin activity, we force cells within nephrons to differentiate according to the imposed β-catenin activity level, thereby causing spatial shifts in nephron segments. The β-catenin signalling gradient interacts with the BMP pathway which, through PTEN/PI3K/AKT signalling, antagonises β-catenin activity and promotes segment identities associated with low β-catenin activity. β-catenin activity and PI3K signalling also integrate with Notch signalling to control segmentation: modulating β-catenin activity or PI3K rescues segment identities normally lost by inhibition of Notch. Our data therefore identifies a molecular network for nephron patterning. DOI: http://dx.doi.org/10.7554/eLife.04000.001

Colorectal cancers choosing sides

In contrast to the majority of sporadic colorectal cancer which predominantly occur in the distal colon, most mismatch repair deficient tumours arise at the proximal side. At present, these regional preferences have not been explained properly. Recently, we have screened colorectal tumours for mutations in Wnt-related genes focusing specifically on colorectal location. Combining this analysis with published data, we propose a mechanism underlying the side-related preferences of colorectal cancers, based on the specific acquired genetic defects in β-catenin signalling.

Wnt5a promotes human colon cancer cell migration and invasion but does not augment intestinal tumorigenesis in Apc1638N mice

Whereas aberrant activation of canonical Wnt/β-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.

SOX2 redirects the developmental fate of the intestinal epithelium toward a premature gastric phenotype

Various factors play an essential role in patterning the digestive tract. During development, Sox2 and Cdx2 are exclusively expressed in the anterior and the posterior parts of the primitive gut, respectively. However, it is unclear whether these transcription factors influence each other in determining specification of the naïve gut endoderm. We therefore investigated whether Sox2 redirects the fate of the prospective intestinal part of the primitive gut. Ectopic expression of Sox2 in the posterior region of the primitive gut caused anteriorization of the gut toward a gastric-like phenotype. Sox2 activated the foregut transcriptional program, in spite of sustained co-expression of endogenous Cdx2. However, binding of Cdx2 to its genomic targets and thus its transcriptional activity was strongly reduced. Recent findings indicate that endodermal Cdx2 is required to initiate the intestinal program and to suppress anterior cell fate. Our findings suggest that reduced Cdx2 expression by itself is not sufficient to cause anteriorization, but that Sox2 expression is also required. Moreover, it indicates that the balance between Sox2 and Cdx2 function is essential for proper specification of the primitive gut and that Sox2 may overrule the initial patterning of the primitive gut, emphasizing the plasticity of the primitive gut.

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

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.

β-Catenin signaling dosage dictates tissue-specific tumor predisposition in Apc-driven cancer

Apc-driven tumor formation in patients and Apc-mutant mouse models is generally attributed to increased levels of β-catenin signaling. We and others have proposed that a specific level of β-catenin signaling is required to successfully initiate tumor formation, and that each tissue prefers different dosages of signaling. This is illustrated by APC genotype−tumor phenotype correlations in cancer patients, and by the different tumor phenotypes displayed by different Apc-mutant mouse models. Apc1638N mice, associated with intermediate β-catenin signaling, characteristically develop intestinal tumors (<10) and extra-intestinal tumors, including cysts and desmoids. Apc1572T mice associated with lower levels of β-catenin signaling are free of intestinal tumors, but instead develop mammary tumors. Although the concept of β-catenin signaling dosage and its impact on tumor growth among tissues is gaining acceptance, it has not been formally proven. Additionally, alternative explanations for Apc-driven tumor formation have been proposed. To obtain direct evidence for the dominant role of β-catenin dosage in tumor formation and tissue-specific tumor predisposition, we crossed Apc1638N mice with heterozygous β-catenin knockout mice, thereby reducing β-catenin levels. Whereas all the Apc1638N;Ctnnb1+/+ mice developed gastrointestinal tumors, none were present in the Apc1638N;Ctnnb1−/+ mice. Incidence of other Apc1638N-associated lesions, including desmoids and cysts, was strongly reduced as well. Interestingly, Apc1638N;Ctnnb1−/+ females showed an increased incidence of mammary tumors, which are normally rarely observed in Apc1638N mice, and the histological composition of the tumors resembled that of Apc1572T-related tumors. Hereby, we provide in vivo genetic evidence confirming the dominant role of β-catenin dosage in tumor formation and in dictating tumor predisposition among tissues in Apc-driven cancer.

Dichotomal effect of space flight-associated microgravity on stress-activated protein kinases in innate immunity

Space flight strongly moderates human immunity but is in general well tolerated. Elucidation of the mechanisms by which zero gravity interacts with human immunity may provide clues for developing rational avenues to deal with exaggerated immune responses, e.g. as in autoimmune disease. Using two sounding rockets and one manned Soyuz launch, the influence of space flight on immunological signal transduction provoked by lipopolysaccharide (LPS) stimulation was investigated in freshly isolated peripheral blood monocytes and was compared to samples obtained from on-board centrifuge-loaded 1 g controls. The effect of microgravity on immunological signal transduction is highly specific, since LPS dependent Jun-N-terminal kinase activation is impaired in the 0 g condition, while the corresponding LPS dependent activation of p38 MAP kinase remains unaffected. Thus our results identify Jun-N-terminal kinase as a relevant target in immunity for microgravity and support using Jun-N-terminal kinase specific inhibitors for combating autoimmune disease.

Generation of an Inducible WNT5A Transgenic Mouse Model to Study the Contribution of Increased WNT5A Expression to Intestinal Tumor Growth

ence for detection of Ki67, BrdU, MUC2 and chromagranin A antigens, in combination with lgr5 mRNA fluorescence in situ hybridisation. Fluorescence labelling was visualised in 3D by confocal microscopy. RESULTS: In the native epithelium, the combination of OLFM4 immunocytochemistry and lgr5 mRNA in situ hybridisation revealed the presence of 10-20 comma-shaped cells that were positive for both lgr5 and OLFM4. This constituted up to 20% of the crypt cell population in the lower third of the crypt-axis. These slender, OLFM4+/ lgr5+cells were negative for MUC2 and chromagranin A and were ‘wrapped-around’ cells exhibiting a goblet cell-like morphology. 50% of the OLFM4+/lgr5+ presumptive stem cells were Ki67-positive. For cultured crypts, the presence and proliferative activity of OLFM4+/ lgr5+ cells was preserved under Wnt-stimulated conditions; more than 50% of OLFM4+ cells were positive for both BrdU and Ki67. Dkk1 (800ng/ml) markedly suppressed lgr5 and OLFM4 expression and abolished proliferation of detectable OLFM4+ cells, as did the absence of RSPO1/Wnt3A from the culture medium. DBZ-treatment (1 uM) eradicated OLFM4 expression and increased the number of MUC2+ goblet cells, as has been previously shown for In Vivo mouse studies. CONCLUSIONS: Canonical Wnt signals maintain the expression of intestinal stem cell markers and promote intestinal stem cell proliferation. Future studies will use this near-native culturemodel to investigate the status andmechanisms of stem cell-driven tissue renewal in the human colonic epithelium.

Abstract 4156: Generation of an inducible Wnt5a transgenic mouse model to study the contribution of increased Wnt5a expression to intestinal tumor growth

Introduction: The importance of the interaction between tumor cells and stromal tissue for tumor invasion and metastasis is becoming well-recognized lately. In this respect, tumor-associated macrophages are of special interest as they appear to promote angiogenesis and invasiveness. One of the factors expressed at increased levels by macrophages in intestinal tumors is noncanonical Wnt5a. Interestingly, despite the well known importance of Wnt signaling in colorectal cancer, Wnt5a is one out of only two Wnt ligands described to be upregulated in gastrointestinal tumors. In vitro data suggest that Wnt5a is involved in tumor cell growth and migration, angiogenesis and epithelial to mesenchymal transition, all key steps in cancer progression. At present, it is however unclear to which extent and through which mechanisms, Wnt5a contributes to the malignant behavior of colorectal tumors in vivo. Therefore, we have generated a mouse model in which expression of Wnt5a can be controlled in a tissue-specific and temporal fashion. Methods: We generated a transgenic mouse line carrying the Wnt5a gene driven by a doxycycline (dox)-responsive promoter, which was crossed with hnRNP-rtTA animals expressing the rtTA2 transcription factor in all their tissues. In this way a model was generated in which Wnt5a expression can be induced by dox administration in double transgenic animals. Subsequently, double transgenics were crossed with Apc1638N mice that develop gastrointestinal tumors spontaneously. These mice were provided with dox in their drinking water from the age of 3-8 months, where after they were analyzed for the development of intestinal tumors. Results: We identified transgenic lines showing a tightly controllable dox-induced expression of Wnt5a in vitro and in vivo. Whereas induced Wnt5a expression appears well-tolerated in adult mice, the induction during embryonic development results in a severe embryonic phenotype. Currently, the contribution of transgenic Wnt5a expression during the development of intestinal tumors in Apc mutant mice is being analyzed. Successful induction of Wnt5a expression in the developing tumors has been confirmed. Preliminary results suggest that induced Wnt5a expression underlies a shift in distribution pattern of the tumors along the gastrointestinal tract. Conclusions: We have successfully generated a transgenic model in which robust and tightly controllable Wnt5a expression can be induced upon dox administration. Although induced Wnt5a expression appears to be well-tolerated in adult animals, embryonic development is severely affected. Additionally we have successfully induced Wnt5a expression within the intestinal tumors developing in an Apc mutant model, of which we are currently investigating the possible contribution to tumor progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4156.