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, 51 (2), 211-25

TRIB2 Acts Downstream of Wnt/TCF in Liver Cancer Cells to Regulate YAP and C/EBPα Function

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TRIB2 Acts Downstream of Wnt/TCF in Liver Cancer Cells to Regulate YAP and C/EBPα Function

Jiayi Wang et al. Mol Cell.

Abstract

Dysregulation of Wnt signaling is closely associated with human liver tumorigenesis. However, liver cancer-specific Wnt transcriptional programs and downstream effectors remain poorly understood. Here, we identify tribbles homolog 2 (TRIB2) as a direct target of Wnt/TCF in liver cancer and demonstrate that transcription of Wnt target genes, including TRIB2, is coordinated by the TCF and FoxA transcription factors in liver cancer cells. We show that Wnt-TRIB2 activation is critical for cancer cell survival and transformation. Mechanistically, TRIB2 promotes protein stabilization of the YAP transcription coactivator through interaction with the βTrCP ubiquitin ligase. Furthermore, we find that TRIB2 relieves the liver tumor suppressor protein C/EBPα-mediated inhibition of YAP/TEAD transcriptional activation in liver cancer cells. Altogether, our study uncovers a regulatory mechanism underlying liver cancer-specific Wnt transcriptional output, and suggests that TRIB2 functions as a signaling nexus to integrate the Wnt/β-catenin, Hippo/YAP, and C/EBPα pathways in cancer cells.

Figures

Fig. 1
Fig. 1. TRIB2 acts downstream of Wnt-βCatenin signaling in HCC
(A) Venn diagram showing overlapping of Wnt-TCF associated genes carrying unique TCF4 binding sites identified in HepG2 cells and human liver cancer samples. (B) qPCR analysis of selected Wnt target genes in HepG2 and LS174T cells with and without DN-TCF4 expression. Error bars indicate mean ± s.d. (C) Representative IHC images of βCatenin and TRIB2 staining showing HCC samples with membrane, cytoplasmic or nuclear βCatenin expression and the correlated levels of TRIB2 expression. Arrows point to tumor cells with nuclear βCatenin staining. Statistical analysis of the TMA data is shown in the bottom panel. (D) TRIB2 expression in HEK293T cells, Huh7, Hep3B and HepG2 cells. (E) Immunoblot analysis of TRIB2 expression in control and DN-TCF4-expressing HepG2 cells. (F-H) Wnt/βCatenin activation induces TRIB2 expression. Immunoblot analysis of TRIB2 in HL7702 (F) and Huh7 (G) cells with or without expression of DA-βCatenin. *: endogenous βCatenin; **: DA-βCatenin. Immunofluorecence staining of βCatenin and TRIB2 in Huh7 cells with or without Wnt1 over-expression (H). See also Figure S1, S2, S3 and Table S1, S2, S3.
Fig. 2
Fig. 2. TCF4 and FoxA control Wnt transcriptional output in liver cancer cells
(A) De novo motif analysis of TCF4 binding sites revealed the presence of the enriched TCF, FoxA and hepatocyte nuclear factor 1 (HNF1) motifs. (B) TCF4 and FoxA1/2 ChIP-seq analyses in the HepG2 genome revealed co-occupancy of TCF4 and FoxA1/2 on cis-regulatory regions. (C-F) FoxA expression in HCC and CRC. Representative IHC images of FoxA1 staining in HCC and CRC (C). The percentile of high FoxA1 expression in HCC (n=59) and CRC (n=48) samples (D). Immunoblot analysis of FoxA1, FoxA2, TRIB2 and Axin2 expression in LS174T and HepG2 cells (E). Relative mRNA levels of FoxA1, FoxA2 and FoxA3 in LS174T and HepG2 cells (F). *P<0.01, error bars indicates mean ± s.d. (G) Venn diagram showing distinct TCF downstream transcriptional programs in LS174T and HepG2 cells. (H) The percentile of DN-TCF4 and FoxA1/2-knockdown co-regulated genes in HepG2 and LS174T cells. (I) Immunoblot analysis of FoxA1, FoxA2 and TRIB2 in HepG2 cells with or without FoxA1/2 knockdown. See also Figure S4 and Table S4, S5, S6.
Fig. 3
Fig. 3. FoxA pioneering factors regulate TCF4 binding at distal enhancer
(A, B) Intersection of DNase-seq, FoxA1/2 and TCF4 ChIP-seq. 41315 FoxA1 peaks (42.7%) and 31285 FoxA2 peaks (43.6%) overlapped with DNase-seq peaks (A). 5.5% of TCF4 peaks and over than 80% of TCF4-FoxA1/2 co-occupied peaks overlapped with DNase-seq peaks (B). (C) Diagram showing a TCF4-occupied enhancer in the TRIB2 locus located 61.5kb downstream of the transcriptional starting site (TSS, +1). ChIP-PCR in HepG2 cells was performed with control IgG, TCF4 or FoxA1 antibody (ab) as indicated. (D) Sequential ChIP with antibody against TCF4 followed by antibody against FoxA1 (Re-ChIP) and vice versa. (E) The activity of the TRIB2-enhancer luciferase reporter (TRIB2-BS-Luc) and TOP-Flash reporter in HepG2 cells transfected with DN-TCF4. (F) The activity of the luciferase reporters driven by the wild-type (WT) or FoxA site-deleted (Del-FoxA) enhancer in stably-transfected HepG2 cells. (G, H) FoxA1/2 are required for TCF4 binding to the TRIB2 enhancer. TCF4 (G) and FoxA1 (H) ChIP-qPCR in HepG2 cells with FoxA1/2 knockdown or DN-TCF4 expression. Enrichment is calculated based upon qPCR relative to IgG control. (I, J) ChIP from control or FoxA1-overexpressing LS174T cells was performed with TCF4 or FoxA1 antibody as indicated. The presence of the enhancer was detected by PCR (I) and enrichment was measured by qPCR (J). (K) Immunoblot analysis of FoxA1 and TRIB2 expression in LS174T cells with or without FoxA1 overexpression. *P<0.05, **P<0.01, error bars indicate mean ± s.d. See also Figure S5.
Fig. 4
Fig. 4. TRIB2 is required for live cancer cell survival and transformation
(A) Immunoblot analysis of TRIB2 levels in HepG2 cells expressing shRNA constructs against GFP (control) or TRIB2 (shTRIB2 #1, shTRIB2 #2, or a combination of shTRIB2 #1+#2, referred as shTRIB2). (B) Phase-contrast images of HepG2 cells expressing shRNA against GFP or TRIB2. Cells were plated at a density of 5000 per well and imaged six days later. (C-E) TRIB2 knockdown induces apoptosis and blocks proliferation in HepG2 cells, but not in HL7702 cells, measured by cleaved caspase 3 staining (C), caspase 3/7 activity (D) and MTT-based proliferation assay (E). (F) Anchorage-independent soft-agar colony formation in HepG2 cells with TRIB2 knockdown or DN-TCF4 expression, in the absence or presence of a mouse TRIB2 expression vector that is resistant to shRNA targeting human TRIB2. (G-J) Silencing TRIB2 expression in HepG2 cells inhibits tumor growth in vivo. Images of the tumors formed in nude mice induced by HepG2 cells expressing shGFP (H), shTRIB2 (I) or DN-TCF4 (J) are shown. Tumor volumes were measured for 45 days after injection (G) (n=5 mice per group). (K) Caspase 3/7 activity in DN-TCF4-expressing HepG2 cells with and without TRIB2 expression. (L) Caspase 3/7 activity in Huh7 cells expressing DA-βCatenin or shRNA against TRIB2, with or without Doxorubicin (Doxo) treatment. *P<0.05, **P<0.01, error bars indicate mean ± s.d.
Fig. 5
Fig. 5. TRIB2 protects YAP from βTrCP-mediated degradation
(A-B) Analysis of YAP mRNA (A) and protein (B) levels upon DN-TCF4 expression in HepG2 cells. *P<0.01, error bars indicates mean ± s.d. Immunoblot analysis of YAP in HL7702 cells with or without DA-βCatenin expression (B). (C) qPCR analysis of YAP, CTGF and ANKRD1 expression in HepG2 cells. *P<0.01, error bars indicates mean ± s.d. (D) Immunoblot analysis of YAP and phosphorylated YAP (p-YAP) in HepG2 cells with or without TRIB2 knockdown. (E) Quantification of p-YAP/total YAP ratio. (F) YAP intracellular localization in HepG2 cells with or without TRIB2 knockdown. (G) Ubiquitination of YAP in HepG2 cells with TRIB2 knockdown. (H-J) TRIB2 stabilizes YAP. In HepG2 cells expressing TRIB2 (H) or shRNA against TRIB2 (I), protein synthesis was blocked by treatment of CHX for the indicated time. Relative YAP protein levels were quantified by YAP/GAPDH ratio (J). (K) Immunoblot analysis of endogenous YAP in HepG2 cells with TRIB2 or βTrCP knockdown. (L) TRIB2 blocks Cul1-induced YAP degradation in transfected HEK293T cells. (M) TRIB2 binds to endogenous βTrCP in HepG2 cells. (N) TRIB2 C-terminal domain is responsible for βTrCP interaction. Immunoblot analysis shows that full length TRIB2, but not C-terminal deletion mutant (TRIB2ΔC), binds to βTrCP. See also Figure S6, S7.
Fig. 6
Fig. 6. C/EBPα regulates YAP transcriptional activity
(A) Immunoblot analysis of C/EBPα in HepG2 cells with or without TRIB2 shRNAs. (B-D) Caspase-3/7 activity (B), cell proliferation (C), and colony formation (D) in HepG2 cells with C/EBPα over-expression or YAP knockdown (shYAP#1 and shYAP#2). (E) Immunoblot analysis of C/EBPα in HepG2 cells expressing shRNAs against TRIB2 or β-TrCP. (F) Immunoblot analysis of YAP and C/EBPα in HepG2 cells overexpressing C/EBPα or shRNA against C/EBPα. (G) C/EBPα inhibits YAP/TEAD-induced gene transcription, measured by a Gal4-TEAD4/UAS-Luciferase reporter in HepG2 cells. qPCR (H) and immunoblot (I) analyses of CTGF expression in HepG2 cells with or without C/EBPα expression. (J) C/EBPα blocks YAP binding to the CTGF promoter. ChIP using anti-YAP antibody was performed in HepG2 cells with or without C/EBPα overexpression. The βTubulin genomic region was used as a negative control. (K) Immunoblot analysis of C/EBPα in HL7702 cells with or without DA-βCatenin expression. (L) qPCR analysis of C/EBPα and CTGF mRNA in HL7702 cells with or without C/EBPα knock-down. *P<0.05, **P<0.01, error bars indicate mean ± s.d.
Fig. 7
Fig. 7. C/EBPα inhibits YAP/TEAD interaction
(A) Diagram showing the PPGY motif of C/EBPα, adjacent to the CDK interacting domain (CID). DBD: DNA binding domain. (B) C/EBPα binds to endogenous YAP. (S) shorter exposure; (L) longer exposure. (C-D) PPGY and WW domains mediate C/EBPα and YAP interaction. Co-IP of wild-type or mutated YAP and C/EBPα proteins in transfected HEK293T cells. (E) C/EBPα blocks YAP/TEAD4 interaction. In transfected HEK293 cells, TEAD4 was immunoprecipitated with an anti-Myc antibody, and immunoblot analysis of YAP was done by an anti-Flag antibody. (F) The inhibitory activity of C/EBPα and C/EBPα-ΔPPGY on YAP-mediated transcription, measured by a TEAD4-luciferase reporter in HepG2 cells. (G, H) Caspase-3/7 activity (G) and proliferation (H) of HepG2 cells expressing C/EBPα, C/EBPα-ΔPPGY and YAP. *P<0.05, **P<0.01, error bars indicate mean ± s.d. (I-K) Representative images of soft-agar colony formation in control (I), C/EBPα-expressing (J), or C/EBPα-ΔPPGY-expressing (K) HepG2 cells. (L) Model showing a role for TRIB2 in controlling HCC cell survival and transformation by interlinking the Wnt/TCF, Hippo/YAP and C/EBPα pathways.

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