The authors have declared that no competing interests exist.
Conceived and designed the experiments: LS SO BD BAM AM. Performed the experiments: LS SO BD RA JAB. Analyzed the data: LS SO BD BAM AM. Wrote the paper: LS AM.
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 (APCmin). While A20FL/FL villin-Cre mice exhibit uninflamed intestines without polyps, A20FL/FL villin-Cre APCmin/+ mice contain far greater numbers and larger colonic polyps than control APCmin 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.
Colon cancer is the second leading cause of cancer deaths in the USA; hence, understanding its pathogenesis remains a critical goal. Both germ line and somatic mutations in a number of genes have been described to contribute to colon cancer. These mutations contribute to colon carcinogenesis by perturbing cell cycling, cell survival, DNA repair and other critical cellular homeostasis functions
A20 is a potent anti-inflammatory enzyme, as global loss of A20 leads to multi-organ inflammation and perinatal lethality
While A20’s ability to inhibit NFκB signaling suggests that it may prevent inflammation association malignancies, A20’s capacity to inhibit cell death suggests that it might also promote tumorigenesis by protecting cancer cells from apoptosis. The discovery that bi-allelic somatic mutations of the A20 gene occur in up to 30% of Hodgkin’s lymphoma, MALT, marginal zone, and diffuse large B-cell lymphomas indicated that A20 functions as a tumor suppressor in B cells
Antibodies directed against A20 (Cell Signaling, 5630; Santa Cruz, sc-376564; Santa Cruz, sc-69980), Active β-catenin (Mllipore, 05–665), Axin (Cell Signaling, 2087), Total β-catenin (Santa Cruz, sc-1496), FLAG (Sigma, F3165), GAPDH (Millipore, MAB374), HA (Santa Cruz, sc-805), MYC (Santa Cruz, sc-789), and pan-ubiquitin (SC-8017) were used for immunoprecipitation and western blot as described below. RKO cells were obtained from the ATCC and maintained in DMEM (Cellgro) supplemented with 10% FCS (Atlanta Biologicals), 1% penicillin-streptomycin-glutamine (Cellgro), and 25 mM HEPES (Cellgro). Recombinant human wnt3a was purchased from R&D systems (5036-WN-010).
A20FL mice were generated in our laboratory and described previously
Freshly isolated intestinal epithelial cells or RKO cells were lysed in ice-cold 0.5% Triton X-100 (Sigma, BP151) containing 50 mM Tris HCl pH 7.4, 150 mM NaCl, and 10% glycerol and a protease inhibitor cocktail (Roche Complete EDTA-free). For studies concerning ubiquitin modifications, the lysis buffer was also supplemented with 10 mM orthophenanthroline (Sigma, P9375), 10 mM iodoacetamide (Sigma, I6125), and the proteasome inhibitor, MG132 (Enzo, BML-PI102) at a final concentration of 10 uM. The insoluble fraction was removed by centrifugation at 14,000 rpm for 25 minutes in a tabletop centrifuge. Protein concentrations were determined by BCA Assay (Thermo Scientific). For immunoprecipitation, equal amounts of protein were incubated overnight with 3 ug of antibody. Lysates were then immunoprecipitated using protein G Dynabeads (Invitrogen) according to the manufacturers instructions. Samples were resolved on NuPage precast 4–12% Bis-Tris gels (Invitrogen) and transferred to PVDF for western blot analysis.
siRNA to human A20 (5′ GAA GCU CAG AAU CAG AGA UUU 3′) or a control scrambled siRNA (5′ AAC GUA CGC GGA AUA CUU CGA 3′) was purchased from Dharmacon. The MegaTOPFlash luciferase reporter was a kind gift from Dr. Roel Nusse (Stanford University, CA). The pRL-Renilla vector and dual luciferase reporter assay system were obtained from Promega. RKO cells were seeded in 24-well tissue culture plates (Costar) in antibiotic-free media. Twenty-four hours prior to stimulation with recombinant human wnt3a, siRNA (30 pmoles), MegaTOPFlash (500 ng), and Renilla (0.1 ng) was transiently transfected using Lipofectamine (Invitrogen) according to the manufacturers instructions. Twelve hours prior to stimulation with recombinant human wnt3a, cells were cultured in media containing 2% fetal calf serum. Fresh media containing 2% FCS and 150 ng/mL wnt3a was added to the cells for eight hours prior to harvest and determination of luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity.
Full-length human A20 and Axin cDNA were obtained from OpenBiosystems. Murine full length A20 was cloned from mouse embryonic fibroblasts. N- and C-terminal truncation mutants were generated by FspI (NEB) digestion. The N-terminal fragment consists of amino acids 1–384 and terminates just prior to zinc finger 1. The C-terminal mutant consists of amino acids 369–775 and begins just prior to zinc finger 1. Full-length human and mouse, truncated murine A20 constructs and Axin were cloned into pCMV-3Tag (Agilent) vectors containing a N-terminal 3FLAG or 3MYC epitope tag respectively. A20 deficient RKO cells were generated by TALEN technology
Murine intestinal epithelial cells were isolated using the protocol of G.S. Evans with some modifications
Total RNA was isolated by Trizol (Invitrogen) according to the manufacturers instructions. One microgram of total RNA was used for reverse transcription using the Quantitech Reverse Transcription kit (Qiagen). Quantitative PCR was performed using the following Taqman primers (Applied Biosystems, Carlsbad CA): TNFAIP3 (Mm00437122_m1), Cyclin D1 (Mm00432359_m1), MYC (Mm00487803_m1), HPRT1 (Mm00446968_m1). Quantitative PCR was performed using the Gene Expression Master Mix (Applied Biosystems, 4369016) on an ABI 7300 using the following protocol: 50° 2 min, 95° 10 min, 95° 15 sec, 60° 1 min for 40 cycles. Ct values were normalized to HPRT1 values.
Resected intestinal tissue was fixed in 10% neutral-buffered formalin (Sigma) overnight. Tissue specimens were then embedded in paraffin and stained according to standard protocols.
Statistical analysis was performed with Graphpad Prism 4 (Graphpad Software, San Diego, CA). Comparisons between two groups were performed by two-tailed unpaired Student’s t-test. Multigroup comparisons were performed by one-way analysis of variance. P<0.05 was used as the threshold for statistical significance.
We first addressed potential epigenetic changes in A20 expression in colon carcinogenesis by searching NCBI’s Genome Expression Omnibus
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.
(A) immunoblot analysis of isolated IECs indicating efficient deletion of A20 from the small bowel (SB) and colon (C) of villin-Cre A20FL/FL APCmin/+ mice (fl/fl) compared to control villin-Cre A20+/+ APCmin/+ 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 APCmin mutation. (C) Tumor numbers in small intestines of A20 (fl/fl) and wild-type (+/+) mice harboring APCmin mutation. (D) Colon and small intestine lengths from (fl/fl) and wild-type (+/+) mice harboring APCmin 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 A20FL/FL APCmin/+ mice (fl/fl) and control villin-Cre A20+/+ APCmin/+ mice. 40X magnification shown.
As A20 restricts NFκB signaling, A20 might restrict colon tumorigenesis by preventing spontaneous intestinal inflammation in A20FL/FL villin-Cre APCmin/+ mice. However, similar to A20FL/FL villin-Cre and A20+/+ villin-Cre mice, no signs of spontaneous inflammation were present in A20FL/FL villin-Cre APCmin/+ and A20+/+ villin-Cre APCmin/+ mice, and there were no differences in intestinal length or mouse weights (
The APC gene is mutated in the majority of sporadic colorectal adenocarcinomas
Given A20’s pleiotropic functions in regulating ubiquitin dependent signals, we hypothesized that A20 might regulate wnt signaling. To investigate this possibility, we first used an siRNA approach to reduce A20 expression in RKO cells, a cell line with intact wnt induced β-catenin signaling
(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.
To better understand how A20 restricts β-catenin signaling, we investigated whether A20 binds to destruction complex proteins that regulate β-catenin ubiquitination. A20 co-precipitated with both heterologously expressed and endogenous Axin, the scaffolding protein that binds many destruction complex proteins (
As β-catenin protein levels are tightly regulated by the SCF/β-TRCP E3 ligase complex, we tested the role of A20 in regulating β-catenin ubiquitination and stability. Stimulation of both A20 siRNA and control siRNA knockdown RKO cells with wnt3a led to increased levels of total β-catenin, however, A20 deficient RKO cells accumulated significantly greater levels of the active dephosphorylated form of β-catenin when compared to control RKO cells (
Finally, to confirm that A20 expression in IECs regulates wnt/β-catenin signaling in vivo, we interbred A20FL/FL mice with mice expressing a tamoxifen-sensitive Cre recombinase under the villin gene promoter elements (villin-ER/Cre)
Villin-ER/Cre A20FL/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.
In summary, we have discovered that the ubiquitin-modifying enzyme A20 is a tumor suppressor for colon carcinogenesis. This tumor suppressor function appears to be due to a novel function for A20 in restricting wnt induced β-catenin signaling. The relevance of our findings to human disease is high, as reduced A20 expression is a common epigenetic finding in human adenomas, and as the increased tumorigenesis of A20FL/FL APCmin/+ mice appears to be selective for colonic tissue, where most human intestinal cancers occur. Our findings extend A20’s tumor suppressor function from B cell lymphomas to colon cancers. Finally, these studies demonstrate that A20, a potent regulator of multiple human autoimmune and inflammatory diseases, is also broadly important for preventing malignant transformation.
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The authors thank S. Robine for providing villin-ER/Cre mice, B. Wang and R. Nusse for helpful discussions and β-catenin reagents.