Given its fundamental role in development and cancer, the Wnt-β-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally ...found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively active mutants of β-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathway.
Oxygen sensing in mammalian cells is a conserved signaling pathway regulated by hypoxia inducible factor type 1 (HIF-1). Inadequate oxygen supply (hypoxia) is common to many pathological disorders ...where autophagy plays an import role. The aim of this study was the identification and characterization of novel HIF-1 target genes that promote autophagy during hypoxia.
Whole genome Chromatin Immune Precipitation from hypoxic HeLa cells was used to identify novel HIF-1 target genes. Hypoxia induced expression and transcription regulation was studied in wild type and HIF-deficient cells. siRNA silencing of candidate genes was used to establish their role during autophagy. Recombinant protein was used for screening immobilized glycosylated lipids to identify potential ligands.
We identified the Nucleotide Oligomerization Domain 2 (NOD2/CARD15) as a novel HIF-1 target and 3-O-sulfo-galactoceramide (sulfatide) and Mycobacterium sp. specific sulfolipid-1 as the first NOD2 ligands that both compete for binding to NOD2. Loss of NOD2 function impaired autophagy upstream of the autophagy inhibitor chloroquine by reducing the number of acidic vesicles. Inhibition of sulfatide synthesis elicited defects in autophagy similar to the NOD2 loss of function but did not influence NOD2-mediated NF-kB signaling.
Our findings suggest that the interaction of NOD2 with sulfatide may mediate the balance between autophagy and inflammation in hypoxic cells.
These findings may lead to a better understanding of complex inflammatory pathologies like Crohn's disease and tuberculosis where both NOD2 and hypoxia are implicated.
•NOD2 is a novel hypoxia induced HIF-1 target gene.•Sulfatide is the first endogenous NOD2 ligand.•Mycobacterium sulfolipid competes with sulfatide for NOD2 binding.•The sulfatide–NOD2 interaction uncouples autophagic and NF-kB related functions of NOD2.
Abstract
Introduction
Targeted drug treatment requires reliable companion diagnostics for therapy selection. Genomic and transcriptomic data can provide input for this, provided tools exist to ...convert this complex data into meaningful clinical information. We develop computational models of oncogenic pathways, to assess which one drives tumor growth in an individual patient and what is the causing (epi)genetic defect.
Computational pathway models
Based on a selection of experimentally validated direct target genes, we built initial models of the Wnt, ER, AR and Hedgehog pathways, covering their transcriptional program. We have modeled each pathway by a Bayesian network, which interprets the target genes’ mRNA levels (Affymetrix U133Plus2.0), and infers a probability that the respective pathway is active in a certain sample. Model parameters are based on literature insights and experimental data.
Results
A first Wnt model, calibrated on cell line data, validated perfectly on 32 normal colon samples and 32 colon adenomas from patients (GSE8671). A second Wnt model, calibrated on these 64 patient samples, correctly predicted no Wnt activity in all 44 normal colon samples, and Wnt activity in 97 of 101 colon cancer samples from GSE20916.
Next, we tested the second Wnt model on other cancer types. On 25 breast cancer cell lines from GSE12777 with known Wnt status, the model correctly identified the two samples with an active pathway. On two patient data sets (GSE12276, n=204; GSE21653, n=266) Wnt activity was predicted in a higher number of basal samples compared to other subtypes (p=0.021 and p=2.7e-5, respectively), in line with increasing evidence for Wnt activity in this subtype. Finally, tests on liver (GSE9843, GSE6764) and medulloblastoma sets (GSE10327) confirm the power of these models to predict Wnt pathway activity.
A first ER model was calibrated on estrogen-deprived and -stimulated MCF7 cell lines (GSE8697). Applied on breast cancer cell line data from GSE21618, increased incidence of ER pathway activity was found in tamoxifen-sensitive cell lines compared to resistant ones. On breast cancer patient data (GSE12276, GSE9195, GSE6532) the model showed no pathway activity in ER- samples, and an active ER pathway in 26-38% of the ER+ samples. Within the latter group, model-predicted ER activity correlated with improved survival. Clinical utility studies to correlate ER activity to hormone therapy response are in progress.
Finally, the AR model showed promising results on prostate cancer cell lines (GSE34211, GSE36133), as did the Hedgehog model on medulloblastoma samples (GSE10327).
Conclusion
Our computational pathway models predict functional activity of oncogenic pathways for an individual patient based on mRNA data, complementary to existing molecular and histopathology staining tests. Clinical utility for therapy response prediction is currently being validated with clinical partners.
Citation Format: Wim Verhaegh, Henk van Ooijen, Marcia Alves de Inda, Kalyan Dulla, Ralf Hoffmann, Dianne van Strijp, Pantelis Hatzis, Hans Clevers, Anja van de Stolpe. Identifying tumor driving signaling pathways for companion diagnostics using computational pathway models. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 59. doi:10.1158/1538-7445.AM2013-59
A central point of regulation in the Wnt/β-catenin signalling pathway is the formation of the β-catenin destruction complex. Axin1, an essential negative regulator of Wnt signalling, serves as a ...scaffold within this complex and is critical for rapid turnover of β-catenin. To examine the mechanism by which Wnt signalling disables the destruction complex, we used an immunoprecipitation-coupled proteomics approach to identify novel endogenous binding partners of Axin1. We found mitogen-activated protein kinase kinase kinase 1 (MAP3K1) as an Axin1 interactor in Ls174T colorectal cancer (CRC) cells. Importantly, confirmation of this interaction in HEK293T cells indicated that the Axin1-MAP3K1 interaction is induced and modulated by Wnt stimulation. siRNA depletion of MAP3K1 specifically abrogated TCF/LEF-driven transcription and Wnt3A-driven endogenous gene expression in both HEK293T as well as DLD-1 CRC. Expression of ubiquitin ligase mutants of MAP3K1 abrogated TCF/LEF transcription, whereas kinase mutants had no effect in TCF-driven activity, highlighting the essential role of the MAP3K1 E3 ubiquitin ligase activity in regulation of the Wnt/β-catenin pathway. These results suggest that MAP3K1, previously reported as an Axin1 inter-actor in c-Jun NH2-terminal kinase pathway, is also involved in the canonical Wnt signalling pathway and positively regulates expression of Wnt target genes.
Hepatocyte nuclear factor 4 (HNF-4) is a key member of the transcription factor network regulating hepatocyte differentiation and function. Activation of the HNF-4 gene involves physical interaction ...between a distant enhancer and the proximal promoter region, bound by distinct sets of transcription factors. Here we report that, upon mitogen-activated protein (MAP) kinase activation, HNF-4 expression is downregulated in human hepatoma cells. This effect is mediated by the loss of CEBPalpha expression. During MAP kinase signaling, the recruitment of HNF-3beta and HNF-1alpha to the HNF-4 enhancer and RNA polymerase II to the proximal HNF-4 promoter was compromised. CBP, Brg1, and TFIIB were also dissociated from the HNF-4 regulatory regions, and the enhancer-promoter complex was disrupted. Interestingly, the extent of nucleosome acetylation did not decrease at either regulatory region, and HNF-6 and HNF-1alpha, as well as components of the TFIID, remained associated with the proximal promoter during the repressed state. The results point to an absolute requirement of enhancer-promoter communication for maintaining the active state of the HNF-4 gene and provide evidence for a molecular bookmarking mechanism, which may contribute to the prevention of permanent silencing of the locus during the repressed state.
Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/β-catenin complexes. In colorectal cancer, activating mutations in ...Wnt pathway components cause inappropriate activation of TCF4/β-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and β-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/β-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a β-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published β-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Calcineurin/NFAT signaling is involved in multiple aspects of skeletal
muscle development and disease. The myogenic basic helix-loop-helix
transcription factors, MyoD, myogenin, Myf5, and MRF4 ...specify the myogenic
lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T
cells) signaling is required for primary myogenesis by transcriptional
cooperation with the basic helix-loop-helix transcription factor MyoD.
Calcineurin/NFAT signaling is involved in myogenin expression in
differentiating myoblasts, where the myogenic regulatory factor MyoD
synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel
shift and chromatin immunoprecipitation assays, we identified two conserved
NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon
skeletal muscle differentiation. The transcriptional integration between
NFATc3 and MyoD is crucial for primary myogenesis
in vivo
, as
myogenin expression is weak in
myod
:
nfatc3
double null
embryos, whereas myogenin expression is unaffected in embryos with null
mutations for either factor alone. Thus, the combined findings provide a novel
transcriptional paradigm for the first steps of myogenesis, where a
calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with
MyoD during myogenesis.
Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/β-catenin complexes. In colorectal cancer, activating mutations in ...Wnt pathway components cause inappropriate activation of TCF4/β-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and β-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/β-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a β-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators to a large extent dedicated to Wnt target gene regulation. In contrast, previously published β-catenin coactivators p300 and BRG1 displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10, and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. We conclude that Mllt10/Af10-Dot1l are essential, largely dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase DOT1L may present an attractive candidate for drug targeting in colorectal cancer.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
