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. 2008 Sep 25;455(7212):552-6.
doi: 10.1038/nature07310. Epub 2008 Sep 14.

E2F1 represses beta-catenin transcription and is antagonized by both pRB and CDK8

Erick J Morris  1 , Jun-Yuan Ji, Fajun Yang, Luisa Di Stefano, Anabel Herr, Nam-Sung Moon, Eun-Jeong Kwon, Kevin M Haigis, Anders M Näär, Nicholas J Dyson
Affiliations

E2F1 represses beta-catenin transcription and is antagonized by both pRB and CDK8

Erick J Morris et al. Nature. .

Abstract

The E2F1 transcription factor can promote proliferation or apoptosis when activated, and is a key downstream target of the retinoblastoma tumour suppressor protein (pRB). Here we show that E2F1 is a potent and specific inhibitor of beta-catenin/T-cell factor (TCF)-dependent transcription, and that this function contributes to E2F1-induced apoptosis. E2F1 deregulation suppresses beta-catenin activity in an adenomatous polyposis coli (APC)/glycogen synthase kinase-3 (GSK3)-independent manner, reducing the expression of key beta-catenin targets including c-MYC. This interaction explains why colorectal tumours, which depend on beta-catenin transcription for their abnormal proliferation, keep RB1 intact. Remarkably, E2F1 activity is also repressed by cyclin-dependent kinase-8 (CDK8), a colorectal oncoprotein. Elevated levels of CDK8 protect beta-catenin/TCF-dependent transcription from inhibition by E2F1. Thus, by retaining RB1 and amplifying CDK8, colorectal tumour cells select conditions that collectively suppress E2F1 and enhance the activity of beta-catenin.

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Figures

Figure 1
Figure 1. Functional antagonism between E2F1 and β-catenin/TCF-signaling
(a) Normal Drosophila wing of Act88F-Gal4/+ genotype. A dE2F1-induced apoptotic wing phenotype (b) is strongly suppressed by co-expression of arm (d) or pan/dTCF (e), phenocopied by expression of dominant-negative dTCF (dTCFΔN) (f), and strongly synergizes with sgg/GSK3 co-expression (h). Expression of arm (c), sgg (g), or pan (not shown) alone does not induce a wing phenotype. (i–l) GMR-mediated eye-specific expression of dE2F1/dDP strongly suppresses a rough eye phenotype induced by activated-arm (arm*; S44Y) expression. All phenotypes were compared in female progeny from F1 crosses conducted at 25°C versus control (w1118 background). (m) E2F1 expression activates a canonical pE2F4B-luciferase reporter while abrogating the S33Y-β-catenin-mediated activation of pTopFLASH. (n) Inhibition of pTopFLASH activity is specific to E2F1. (o) E2F1 dominantly inhibits pTopFLASH activation by p73. All data is expressed as mean ± s.d. (n = 3) of normalized relative light units (NRLU) of luciferase.
Figure 2
Figure 2. E2F1 abrogates Wnt signaling by modulating β-catenin target gene expression and inducing the GSK3-independent degradation of β-catenin
(a) In Saos2-TR-E2F1 cells, E2F1 represses c-myc levels without affecting the levels of TCF1 or TCF4. (b–d) E2F1 modulates the expression of endogenous Wnt target genes (qPCR analysis after Tet-induced E2F1 expression at 24 hours). (e) The kinetics of E2F1-induced AXIN2 and SIAH1 expression mirrors the E2F1 activation of CCNE1/Cyclin E and CCNA1/Cyclin A, and precedes E2F1-induced apoptosis. (f) E2F1 represses the expression of Wnt targets in DLD1 colorectal cancer cells. Levels of mRNA were normalized to GAPDH and the effect of E2F1 is depicted as the ratio between samples after pCMV-empty or pCMV-E2F1 (1 µg each) expression. (g, h) E2F1 induced β-catenin degradation (control expression from the same lysates shown in Supplementary Figure 4f) is both GSK3- and caspase-independent. Saos2 cells were treated with control (DMSO), GSK3 inhibitors (20 µM SB216763 and 5 mM LiCl), or the caspase inhibitor peptide BOC-aspartyl-FMK (BAF; 100 µM) with or without Tet-induction of E2F1 (Western blot). (i) Co-expression of Bcl-2 (25 ng), pRb (10–25 ng), stabilized tumor-derived β-catenin mutants (10–25 ng), or the GSK3β inhibitor SB216763 (15 uM), is sufficient to partially-rescue E2F1-induced apoptosis. Cell death was depicted as percent inhibition of EGFP loss at 48 hours after transfection with pCMV-E2F1 or pCMV-empty (100 ng each) along with EGFP expression construct. All data is expressed as mean ± s.d. (n = 3).
Figure 3
Figure 3. pRB inactivation abrogates β-catenin/TCF-dependent transcription
(a–b)High levels of pRB and β-catenin colocalize within the tumor epithelium of an ApcMin colonic tumor. (c) Endogenous pRB was depleted for 6 days in U2OS-shRb cells (containing DOX-inducible short-hairpin-Rb and GFP transgenes) (d) pRB depletion activates E2F and represses TCF activity. Basal and activated E2F and TCF activity was determined by transfection of their respective luciferase reporter plasmids (for 24 hours) in the absence (basal) or presence (activated) of E2F1 or S33Y-β-catenin expression constructs. (e) In SW480 colorectal cancer cells, E2F1 expression is sufficient to activate E2F activity and repress endogenous β-catenin activity in a dose-dependent manner. (f) Expression of short-hairpin pRB (400 ng) is sufficient to repress pTopFLASH activity in three different colorectal cancer cell lines (SW480, DLD1, and HCT116). (g) pRB inactivation reduces clonogenic survival of SW480 cells and is partially rescued by S33Y-β-catenin/TCF1E, but not Bcl-2 expression. SW480 cells were Amaxa nucleoporated with either control LLP-GFP or -Rb silencing constructs (1 µg each) with or without S33Y-β-catenin (250 ng), TCF1E (250 ng), or Bcl-2 (500 ng) expression constructs, and survival was determined at day 5 by MTT assay. All data is expressed as mean ± s.d. (n = 3; *p < 0.015 by T-test).
Figure 4
Figure 4. CDK8 antagonizes E2F1 activity
(a–c)The dCdk8c01804 mutant dominantly suppresses a rough eye phenotype caused by the eye-specific expression of a dE2F1RNAi transgene. (d) The expression of the dE2F1 target genes, PCNA and MCM5 are upregulated in dCdk8 (null or hypomorphic alleles) or dCycC null mutant Drosophila larvae, while the expression of the dE2F2 target gene vasa is unaffected. (e) dCDK8 physically interacts with dE2F1 by GST-pulldown assay. (f) Co-immunoprecipitation of human E2F1 and CDK8 from Saos2 whole-cell extracts. As control, E2F1 does not associate with the CRSP70 small-Mediator subunit. (g) CDK8 binds to and specifically phosphorylates E2F1. Kinase-assay was performed following CDK8 or CRSP70 (as control) immunoprecipitation from Saos2 cells. E2F1, E2F4, or DP1 were re-immunoprecipitated and resolved in 12% SDS-PAGE. (h) The expression of wild-type CDK8, but not kinase-dead, abrogates the inhibitory effects of E2F1 on β-catenin/TCF-dependent transcription. pTopFLASH assays were determined at 48 hours in co-transfection experiments with 200 ng CDK8 or CDK8KD in Saos2 cells. (i) Expression of short-hairpin pRB (400 ng) and CDK8KD (300 ng) co-operatively repress pTopFLASH activity in SW480 colorectal cancer cells. All data is expressed as mean ± s.d. (n = 3).
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Comment in

  • Cancer: Entangled pathways.
    Bernards R. Bernards R. Nature. 2008 Sep 25;455(7212):479-80. doi: 10.1038/455479a. Nature. 2008. PMID: 18818647 No abstract available.

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