The human embryonic kidney 293T (HEK293T) cells, and colon cancer SW480 and HCT116 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were maintained in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified incubator at 37°C with 5% CO₂. Cells in the logarithmic phase of growth were used for the subsequent experiments.

SR3029, D4476, MG149, NU9056, MG132 and cycloheximide (CHX) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). The following primary antibodies were used: anti-CK1δ, anti-β-catenin, anti-p300, anti-acetyl-lysine, IgG (Santa Cruz Biotechnology, Heidelberg, Germany), anti-CBP, anti-acetyl-β-catenin (K49), anti-phospho-β-catenin (S45), anti-Flag, anti-V5, anti-CK1ϵ, anti-GFP, anti-mouse IgG (Cell Signaling Technology, Danvers, MA), anti-Tip60 (Abcam, Cambridge, MA, USA), and anti-GAPDH antibody (Proteintech, Chicago, IL, USA). The Tip60-Flag, PCAF-Flag, p300-Flag and pEGFP-N1 plasmids were purchased from Vigenebio (Weizhen, Shandong, China). The SuperTopFlash reporter plasmid was provided by Karl Willert (University of California at San Diego, La Jolla, CA, USA). The expression plasmids encoding β-catenin, pCMXβgal (β galactosidase, β-gal), CK1α-V5, CK1δ-V5, CK1ϵ-V5, CK1α-Flag, CK1δ-Flag, CK1ϵ-Flag, CK1γ-Flag have been described previously (35, 36). For the construction of GFP-tagged CK1δ, the cDNAs encoding human CK1δ was amplified by PCR and subcloned into the BamHI/EcoRI site of pEGFP-N1 vector using ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China). The primer sequences used are as follows: CK1δ-GFP-sense, 5’-CTCGAGCTCAAGCTTCATGGAGCTGAGAGTCGGGAAC-3’; CK1δ-GFP-antisense, 5’-CTCACCATAAGGTGGCGACCGGTGTAGGTGCGTCGTG.

The CK1 shRNA oligos were cloned into pLKO.1-GFP vector, and the Tip60 shRNA oligos were inserted into pLKO.1-TRC vector. The resulting constructs were validated by sequencing. The shRNA oligos used have been previously described (37, 38). For lentiviral production and infection, HEK293T cells were transfected with a shRNA-expressing plasmid (10 µg), an envelope plasmid (pMD2.G, 2.5 µg) and a packaging plasmid (psPAX2, 7.5 µg) using Lipofectamine 2000 regent according to manufacturer’s instruction. At 48 h after transfection, viral supernatants were collected and filtered through a 0.45 μm filter. Virus was immediately added to SW480 and HCT116 cells with 8 µg/mL polybrene. After a week of infection, the GFP-positive cells were sorted by FACS AriaIII, and the knockdown of CK1δ or CK1ϵ was verified by Western blotting. For Tip60 deficient cells, cells were selected for stable expression in the presence of 3 µg/mL puromycin (Thermo Fisher Scientific, Waltham, MA, USA) after 72 h of infection. The puromycin-resistant stable clones were pooled, and Tip60 deficiency was confirmed by Western blotting.

HEK293T cells were transfected with the SuperTopFlash reporter, control vector, and the indicated expression plasmids in 24-well plates using Lipofectamine 2000 (Thermo Fisher, San Jose, California, USA). After 48 h, the cells were harvested and lysates were used to examine the expression of luciferase by a luciferase assay kit (Promega, Shanghai, China), according to the manufacturer’s instructions. The luciferase values were normalized using the β-gal internal control to determine the variation in transfection efficiency. The results are presented as means ± SD of at least three independent experiments. The values for luciferase activity were presented as fold induction over control.

The cells were lysed in RIPA buffer containing 50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF, protease inhibitors (Bimake, Beijing, China), and phosphatase inhibitors (Topscience, Shanghai, China). After quantifying the concentration of protein using the BCA protein assay kit (Vazyme, Nanjing, China), 30 µg of protein was loaded and separated by SDS-PAGE, followed by transferring to poly-vinylidenefluoride (PVDF) membranes (Millipore, Massachusetts, USA). Immunoblotting was performed with the indicated primary antibodies at 4°C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (ImmunoWay, Plano, USA) at room temperature for 2 h. After incubating the membranes with ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, Shanghai, China), immunolabeled proteins were detected using X-ray film or Chemiluminescent Imaging System (Tanon 5200, Shanghai, China).

For immunoprecipitation assay, cell lysates were harvested in 500 µL RIPA buffer supplemented with protease inhibitor, phosphatase inhibitor cocktails and 1 mM PMSF, followed by centrifugation at 15000 rpm for 15 min at 4°C. Protein concentration in the supernatant was quantified by the BCA Protein Assay Kit. The supernatant was incubated with specific primary antibody or control IgG at 4°C overnight, then further incubated with protein A/G magnetic beads (Biomake, Beijing, China) for 6 h. The immunoprecipitated pellet was washed with RIPA buffer at least three times, and proteins were eluted by boiling in SDS loading buffer. The eluted proteins (30 µg) were analyzed by SDS-PAGE and immunoblotting.

Total RNA was extracted using RNAiso Plus (TaKaRa, Kusatsu, Shiga, Japan) and then reverse-transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa, Kusatsu, Shiga, Japan) according to the manufacturer’s instructions. Prepared cDNA was then subjected to quantitative PCR analysis using 2×SYBR Green qPCR Master Mix (Promega, Shanghai, China). Real-time PCR assays were performed to quantify mRNA levels of human Survivin, Cyclin D1 and Prominin 1 (PROM1) genes. The comparative Ct method was used to analyze relative expression of genes. The data was presented as the fold change. The fold change was calculated as 2^-ΔΔCt, where ΔΔCt = ΔCt_treated -ΔCt_control. Ct is the cycle number at which fluorescence first exceeds the threshold. The ΔCt values from each target gene were obtained by subtracting the values for GAPDH Ct from the sample Ct. The primer pairs used for quantitative PCR amplification were as follows: Survivin sense, 5’-AGGACCACCGCATCTCTACAT-3’ and antisense, 5’-AAGTCTGGCTCGTTCTCAGTG-3’; Cyclin D1 sense, 5’-AATGACCCCGCACGATTTC-3’ and antisense, 5’-TCAGGTTCAGGCCTTGCAC-3’; PROM1 sense, 5’-AGGCTACTTTGAACATTATCTGC-3’ and antisense, 5’-GGCTTGTCATAACAGGATTGT-3’; GAPDH sense, 5’-CCAGAACATCATCCCTGCCTCTACT-3’ and antisense, 5’-GGTTTTTCTAGACGGCAGGTCAGGT.

SW480 cells were treated with 240 nM SR3029 for 24 h, and ChIP assay was performed with ChIP-IT Express Enzymatic Chromatin Immunoprecipitation Kit (Sigma-Aldrich, Merck, USA) according to the manufacturer’s protocols. The eluted DNA was amplified by quantitative PCR analysis using an ABI Prism 7500 Real-Time PCR System (Applied Biosystems, Foster city, CA, USA). Primer sequences were shown as follows: Survivin sense, 5’-GCGTTCTTTGAAAGCAGT-3’ and antisense, 5’-ATCTGGCGGTTAATGGCG-3’; Axin2 sense, 5’-TCTGGTAGCATTATGGCCATCGCA-3’ and antisense, 5’-AAAGTCCTCCAAGCCCAAATTCCC-3’; The antibodies used were anti-β-catenin and anti-mouse IgG.

SW480 and HCT116 cells were seeded in 96-well plates at a density of 5000 cells per well and cultured for 24 h. The cells were treated with SR3029 and MG149, alone or combined, at indicated concentrations for 48 h. Subsequently, 10 μL of MTT solution (5 mg/mL) was added into each well. The plate was further incubated for 4 h at 37°C and 100 μL of dimethyl sulfoxide (DMSO) was added to dissolve the insoluble formazan. The absorbance of the formazan solution was measured at 570 nm. Each treatment was performed in three replicates.

SW480 and HCT116 cells were plated on 96-well plates at a density of 5000 cells per well and cultured for 24 h. The cells were treated with SR3029 and MG149, alone or combined, at indicated concentrations for 48 h. The BrdU incorporation assay was performed using the Cell Proliferation ELISA BrdU Colorimetric Kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. BrdU incorporation was detected by measuring the absorbance at 450 nm. Each treatment was performed in three replicates.

Statistical analyses were conducted using GraphPad Prism 7 software (La Jolla, CA, USA). The experiments were repeated three times, and data were exhibited as mean ± standard deviation (SD). The Student’s t-test was applied to determine the significance of difference between the two groups. One-way analysis of variance (ANOVA) with Dunn’s multiple comparisons test were utilized to compare the means of several groups. P<0.05 was defined as statistically significant.

To examine the effect of CK1 family members on β-catenin-mediated Wnt signaling, the Wnt-responsive reporter SuperTopFlash was transfected into HEK293T cells with expression vector encoding β-catenin, together with CK1α, CK1δ, CK1ϵ and CK1γ expression plasmids, respectively. As shown in Figure 1A , CK1δ or CK1ϵ dramatically enhanced β-catenin-mediated transcription in a dose-dependent manner, while CK1δ or CK1ϵ alone had a moderate effect on the SuperTopFlash reporter activity. Comparatively, CK1α or CK1γ exerted little effect on β-catenin-mediated activity ( Figure 1A ). Since β-catenin can be acetylated at lysine 49 (K49), which is required for the activation of Wnt/β-catenin signaling (3, 5, 39, 40), we tested whether CK1 family members have any effect on the acetylation of β-catenin using an antibody against acetyl-K49 β-catenin. Our results showed that CK1δ or CK1ϵ significantly increased the K49 acetylation of exogenous β-catenin ( Figures 1B, C ). In contrast, CK1α or CK1γ had little effect on the acetyl-K49 β-catenin level ( Figures 1D, E ). Furthermore, we detected enhanced level of the K49 acetylation of endogenous β-catenin in HEK293T cells transfected with CK1δ or CK1ϵ, but not CK1α or CK1γ ( Figure 1F ). These results revealed that CK1δ and CK1ϵ may increase β-catenin-mediated transcriptional activity through regulating β-catenin acetylation.

Multiple histone acetyltransferases, including p300, CBP and PCAF, have been shown to acetylate β-catenin at different lysine residues, resulting in enhancing its transcriptional activity and up-regulating the expression of Wnt target genes (5–8). We then assessed whether CK1δ/ϵ could promote the interaction between β-catenin and some histone acetyltransferases. A coimmunoprecipitation assay was performed using an anti-β-catenin antibody in HEK293T cells that were transiently transfected with GFP-tagged CK1δ, Flag-tagged CK1ϵ or CK1α. The results showed that the expression of CK1δ or CK1ϵ did not affect the interaction between β-catenin and p300 or CBP or PCAF. Surprisingly, CK1δ or CK1ϵ could dramatically enhance the binding of β-catenin to Tip60 ( Figures 2A, B ), while CK1α had little effect on the interaction between β-catenin and Tip60 ( Figure 2C ). To further confirm the effect of CK1δ/ϵ and Tip60 on β-catenin acetylation, HEK293T cells were transfected with Tip60-Flag expression vector along with expression plasmids for GFP-CK1δ or Flag-CK1ϵ. Cell lysates were extracted from the transfected cells and immunoprecipitated with an anti-β-catenin antibody. Western blotting analysis showed that simultaneous expression of Tip60 and CK1δ or CK1ϵ markedly increased the acetylation of β-catenin at K49 compared to cells expressing Tip60 or CK1δ or CK1ϵ alone ( Figures 2D, E ). To test whether CK1δ or CK1ϵ could phosphorylate Tip60, HEK293T cells were transfected with Tip60-Flag expression plasmid along with expression vectors for CK1δ or CK1ϵ or CK1α, respectively. Total cell extracts were used for affinity purification by anti-Flag M2 agarose. We observed that anti-Flag M2 agarose could pull down Tip60-Flag, and the presence of CK1δ or CK1ϵ elevated the level of phosphorylated Tip60, detected by a pan phospho-serine antibody, while the expression of CK1α did not affect Tip60 phosphorylation ( Figures 2F–H ). These results suggest that the binding of CK1δ/ϵ to Tip60 induced Tip60 phosphorylation and facilitated the recruitment of Tip60 to β-catenin complex, resulting in the acetylation of β-catenin.

We next evaluated the effect of CK1δ/ϵ, β-catenin and Tip60, alone or combined, on the Wnt signaling pathway. The SuperTopFlash reporter was transfected into HEK293T cells together with CK1δ/ϵ, β-catenin and Tip60 expression plasmids, alone or combined, as indicated in Figure 3 . Overexpression of Tip60 significantly enhanced the SuperTopFlash reporter activity induced by CK1δ or CK1ϵ or β-catenin or CK1δ/β-catenin or CK1ϵ/β-catenin, while Tip60 alone had no any effect on the transcription of SuperTopFlash reporter ( Figures 3A, B ). Notably, p300 or PCAF had little effect on CK1δ/ϵ-mediated transcriptional activity ( Figures 3A, B ). These results indicated that Tip60 could positively modulate Wnt signaling in the presence of CK1δ/ϵ or/and β-catenin.

Considering that CK1δ and CK1ϵ are highly expressed in colon cancer, which is involved in advanced progression and poor prognosis (34, 41, 42), two colon cancer cell lines SW480 and HCT116 were employed to examine the interaction among CK1δ/ϵ, β-catenin and Tip60. These two cell lines have different types of mutations, SW480 cells harboring truncated mutation of APC and HCT116 cells with S45 mutation of β-catenin. The APC and β-catenin mutations are the most frequently occurred mutation types in components of the Wnt signaling pathway in colon cancer (43–45). The result of immunoprecipitation assay showed that the endogenous CK1δ, CK1ϵ, β-catenin and Tip60 proteins were immunoprecipitated with an anti-β-catenin antibody in SW480 and HCT116 cells ( Figures 4A, B ), suggesting that the CK1δ/CK1ϵ/β-catenin/Tip60 complex existed in colon cancer cells. To explore the effect of CK1δ/ϵ on the interaction between β-catenin and Tip60, lentivirus-mediated shRNAs were used to knockdown the expression of CK1δ and CK1ϵ, either alone or in combination, in colon cancer cells. Simultaneous knockdown of CK1δ and CK1ϵ dramatically attenuated the association of β-catenin with Tip60 in SW480 and HCT116 cells ( Figures 4C, D ). These results suggest that β-catenin may interact with Tip60 in a CK1δ/CK1ϵ-dependent manner.

We next check the effect of CK1δ/ϵ on β-catenin acetylation at K49 in colon cancer cells. As shown in Figures 5A, B , the level of the acetylation of β-catenin at K49 was dramatically reduced in two colon cancer cell lines (SW480 and HCT116) with knockdown of CK1δ and CK1ϵ simultaneously ( Figures 5A, B ). Moreover, treatment with small molecular CK1 inhibitors (SR3029, D4476 and longdaysin) markedly decreased the K49 acetylation of β-catenin in SW480 and HCT116 cells, while depletion of CK1δ/ϵ or treatment with small molecular CK1 inhibitors had little effect on the expression of Tip60 and β-catenin ( Figures 5C–H and Figures S1A–H ). In contrast, proteasome inhibitor MG132 increased the expression of acetylated β-catenin and total β-catenin in SW480 and HCT116 cells ( Figures S2A, B ). Consistently, treatment with the protein synthesis inhibitor CHX did not alter the kinetics of β-catenin degradation in CK1δ/ϵ-knockdown SW480 cells ( Figure S2C ). However, we detected significantly decreased level of β-catenin acetylation at K49 in CK1δ/ϵ-knockdown cells ( Figure S2C ). These results indicated that CK1δ and CK1ϵ could enhance the acetylation of β-catenin, but had little effect on its stability in colon cancer cells.

We further assessed whether the inhibition of CK1δ/ϵ activity by SR3029 had any effect on β‐catenin binding to the Wnt target gene promoter. The ChIP assay was performed to detect β‐catenin binding to the promoters of two known Wnt target genes, Survivin and Axin2, in SW480 cells. Our results showed that SR3029 treatment significantly reduced β‐catenin binding to the promoters of Survivin and Axin2 ( Figures S3A, B ). We also examined the effect of CK1δ and CK1ϵ on colon cancer cell viability and proliferation. Knockdown of either CK1δ or CK1ϵ decreased the viability and proliferation in SW480 and HCT116 cells. Simultaneous knockdown of CK1δ and CK1ϵ exerted greater inhibition of viability and proliferation in both cells ( Figures S4A–D ). Taken together, these results illustrated that silencing of CK1δ/1ϵ or treatment with CK1 inhibitor could reduce the β-catenin acetylation and suppress β‐catenin binding to the promoters of Wnt target genes, resulting in the inhibition of colon cancer cell viability and proliferation.

To determine whether Tip60 is able to regulate the acetylation of β-catenin, we silenced the expression of Tip60 using lentivirus-mediated shRNAs in colon cancer cells. Depletion of Tip60 downregulated the level of β-catenin acetylation at K49 without influencing total β-catenin expression in SW480 and HCT116 cells ( Figures 6A, B ). Two small molecular Tip60 inhibitors MG149 and NU9056 were used to block the activity of Tip60 in SW480 and HCT116 cells. The K49 acetylation of β-catenin was significantly reduced upon treatment with either MG149 or NU9056 ( Figures 6C–F ). Collectively, our results illustrated that Tip60 could regulate β-catenin acetylation in colon cancer cells.

To assess the potential consequence of combined inhibition of CK1δ/ϵ and Tip60 activities, we tested the effect of combined treatment with CK1δ/ϵ inhibitor SR3029 and Tip60 inhibitor MG149 on β-catenin acetylation, the transcription of Wnt target genes, cell viability and proliferation in colon cancer cells. SW480 and HCT116 cells were treated with SR3029 (240 nM) and MG149 (5 and 10 μM), alone or combined. Our results showed that combined treatment markedly downregulated the level of β-catenin acetylation at K49 ( Figures 7A, B ) and the transcription of Wnt target genes Survivin, Cyclin D1 and PROM1 ( Figures S5A–F ) compared with either drug alone. Furthermore, combined treatment of SR3029 and MG149 was more effective than either drug alone in inhibiting viability and proliferation in SW480 and HCT116 cells ( Figures 7C–F ). These results revealed that combined inhibition of CK1δ/ϵ and Tip60 activities exert a more profound inhibitory effect on β-catenin acetylation, the transcription of Wnt target genes, the viability and proliferation in colon cancer cells.