A20 expression in IECs restricts colon tumorigenesis.

<p>(A) immunoblot analysis of isolated IECs indicating efficient deletion of A20 from the small bowel (SB) and colon (C) of villin-Cre A20^FL/FL APC^min/+ mice (fl/fl) compared to control villin-Cre A20^+/+ APC^min/+ mice (+/+) mice. GAPDH is shown as a loading control. (B) Tumor number (left panel) and aggregate tumor size (right panel) in colons of A20 (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. (C) Tumor numbers in small intestines of A20 (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. (D) Colon and small intestine lengths from (fl/fl) and wild-type (+/+) mice harboring APC^min mutation. Each point represents one mouse. Lines indicate mean values. (f) Hematoxylin and eosin staining (upper panels) and Ki-67 and cleaved caspase-3 immunostaining (lower panels) of colonic sections from villin-Cre A20^FL/FL APC^min/+ mice (fl/fl) and control villin-Cre A20^+/+ APC^min/+ mice. 40X magnification shown.</p

A20 supports β-catenin ubiquitination and degradation through an interaction with the destruction box.

<p>(A) Luciferase assay showing transcriptional activity of a β-catenin dependent TCF/LEF4 reporter in RKO cells. Cells were treated with A20 specific or control siRNA and recombinant human wnt3a (rhwnt3a) as indicated. Relative luciferase units (RLU) are shown. **indicates p<0.01. (B) Co-precipitation of A20 with Axin. RKO cells transfected with the indicated expression plasmids were lysed, immunoprecipiated (IP) for the indicated epitope tag, and immunoblotted (IB) for the indicated proteins. Cells were stimulated with rhwnt3a or control as indicated for four hours. Input levels of MYC, FLAG, and GAPDH are shown as controls below. (C) Co-precipitation of partial A20 proteins with Axin. Co-transfection experiments as in (B). Input levels of MYC, FLAG, and GAPDH shown below. (D) A20 suppresses wnt3a stimulated induction of β-catenin expression. Immunoblot analyses of active and total β-catenin expression in RKO cells treated with A20 specific or control siRNA. A20 and GAPDH levels shown below as loading control. (E) A20 supports wnt3a stimulated β-catenin ubiquitination. RKO cells were treated with A20 specific or control siRNAs and wnt3a for the indicated times. Lysates were immunoprecipitated for β-catenin followed by immunoblotting for ubiquitin. Input amounts of beta-catenin, A20, and GAPDH proteins shown below as controls. All data are representative of three or more independent experiments.</p

Pseudoclefts and other specificational copular sentences

<div><p>A20 is a ubiquitin-editing enzyme that is known to regulate inflammatory signaling and cell death. However, A20 mutations are also frequently found in multiple malignancies suggesting a potential role as a tumor suppressor as well. We recently described a novel role for A20 in regulating the wnt-beta-catenin signaling pathway and suppressing colonic tumor development in mice. The underlying mechanisms for this phenomenon are unclear. To study this, we first generated A20 knockout cell lines by genome-editing techniques. Using these cells, we show that loss of A20 causes dysregulation of wnt-dependent gene expression by RNAseq. Mechanistically, A20 interacts with a proximal signaling component of the wnt-signaling pathway, receptor interacting protein kinase 4 (RIPK4), and regulation of wnt-signaling by A20 occurs through RIPK4. Finally, similar to the mechanism by which A20 regulates other members of the receptor interacting protein kinase family, A20 modifies ubiquitin chains on RIPK4 suggesting a possible molecular mechanism for A20’s control over the wnt-signaling pathway.</p></div

A20 Restricts Wnt Signaling in Intestinal Epithelial Cells and Suppresses Colon Carcinogenesis

<div><p>Colon carcinogenesis consists of a multistep process during which a series of genetic and epigenetic adaptations occur that lead to malignant transformation. Here, we have studied the role of A20 (also known as TNFAIP3), a ubiquitin-editing enzyme that restricts NFκB and cell death signaling, in intestinal homeostasis and tumorigenesis. We have found that A20 expression is consistently reduced in human colonic adenomas than in normal colonic tissues. To further investigate A20’s potential roles in regulating colon carcinogenesis, we have generated mice lacking A20 specifically in intestinal epithelial cells and interbred these with mice harboring a mutation in the adenomatous polyposis coli gene (APC^min). While A20^FL/FL villin-Cre mice exhibit uninflamed intestines without polyps, A20^FL/FL villin-Cre APC^min/+ mice contain far greater numbers and larger colonic polyps than control APC^min mice. We find that A20 binds to the β-catenin destruction complex and restricts canonical wnt signaling by supporting ubiquitination and degradation of β-catenin in intestinal epithelial cells. Moreover, acute deletion of A20 from intestinal epithelial cells in vivo leads to enhanced expression of the β-catenin dependent genes cyclinD1 and c-myc, known promoters of colon cancer. Taken together, these findings demonstrate new roles for A20 in restricting β-catenin signaling and preventing colon tumorigenesis.</p> </div

Human colonic adenomas express less A20 than normal colonic mucosa.

<p>Expression of A20 (top panel), cyclin D1 (middle panel) and c-myc (bottom panel) mRNAs in normal colonic mucosa and colonic adenomas, as quantitated by the Genome Expression Omnibus (GDS2947). Relative expression levels are shown. **indicates p<0.01.</p

Gene expression changes after wnt3a stimulation in the presence or absence of A20.

<p>A) Volcano plot showing the differentially downregulated (blue) and upregulated (red) genes in A20 knockdown versus control knockdown RKO after 24 hours of treatment with wnt3a. The top five upregulated and downregulated genes are labelled. B) RNA expression of A20 in control (WT) or A20 siRNA knockdown (KD) C) Pathway analysis showing dysregulation of genes related to EGFR signaling in RKO cells stimulated with wnt3a after A20 siRNA knockdown.</p

Regulation of wnt-signaling by A20 through RIPK4.

<p>A) Wild-type (WT RKO) or A20 knockout RKO (KO RKO) were transfected with a TCF4-luciferase reporter and either a control or RIPK4 siRNA. Cells were stimulated with wnt3a for 8 hours. Luciferase activity was measured and normalized to Renilla luciferase. B) Wild-type (WT RKO) or A20 knockout RKO (KO RKO) were transfected with a TCF4-luciferase reporter and either a control or a RIPK4-K51R plasmid at different concentrations. Cells were stimulated with wnt3a for 8 hours. Luciferase activity was measured and normalized to Renilla luciferase. C) Wild-type (WT RKO) or A20 knockout RKO (KO RKO) were transfected with MYC-tagged RIPK4 and an HA-tagged K48-only ubiquitin construct. Cells were stimulated with wnt3a for the indicated time points. Lysates were subjected to immunoprecipitation with an antibody against MYC and then blotted with an antibody against HA. Inputs are shown below. Tubulin is shown as a loading control. ** = p < 0.01. Each panel is representative of at least three independent experiments.</p

A20 interacts with RIPK4.

<p>A) Mammalian-2-hybrid assay showing interaction of A20 and RIPK4, but not A20 with GFP (as a negative control) or RIPK4 with GFP (as a negative control). Interaction of p53 and SV40 shown as a positive control. B) Wild-type RKO cells were stimulated with either TNF or wnt3a for 4 hours. Cells were lysed and subject to immunoprecipitation of A20 using two separate antibodies (B5 or 4H16) or an isotype control. Immunoprecipitates were subject to Western blot for RIPK4. Inputs for A20, RIPK4, and GAPDH as a loading control are shown below. C) Flag-tagged A20 (FLAG-A20), MYC-tagged full-length RIPK4 (FL), RIPK4 N-terminal domain (NT), RIPK4 C-terminal domain (CT), and control vector were expressed in RKO cells. Cell lysates were subject to immunoprecipitation with an antibody towards MYC and then blotted with an antibody against FLAG (upper) or immunoprecipitation with an antibody towards FLAG and then blotted with an antibody against MYC (lower). Inputs shown below. GAPDH shown as a loading control. D) MYC-tagged RIPK4, FLAG-tagged full-length A20 (FL), FLAG-tagged A20 N-terminal domain (NT), FLAG-tagged A20 C-terminal domain (CT), and a control vector were expressed in RKO cells. Cell lysates were subject to immunoprecipitation with an antibody towards MYC and then blotted with an antibody against FLAG. Inputs shown below. GAPDH shown as a loading control. ** = p < 0.01. Each panel is representative of at least three independent experiments.</p

Acute deletion of A20 from IECs leads to increased levels of Cyclin D1 and MYC mRNA in vivo.

<p>Villin-ER/Cre A20^FL/FL (fl/fl) and control Villin-ER/Cre A20^+/+ (+/+) were injected with 1 mg of tamoxifen daily for 5 days. IECs were then isolated and studied for expression of A20 (upper panel), Cyclin D1 (middle panel), and MYC (lower panel) mRNAs by qPCR. Each point represents one mouse. *indicates p<0.05; **indicates p<0.01.</p