Cell cycle-dependent degradation of the methyltransferase SETD3 attenuates cell proliferation and liver tumorigenesis

Abstract

Histone modifications, including lysine methylation, are epigenetic marks that influence many biological pathways. Accordingly, many methyltransferases have critical roles in various biological processes, and their dysregulation is often associated with cancer. However, the biological functions and regulation of many methyltransferases are unclear. Here, we report that a human homolog of the methyltransferase SET (SU(var), enhancer of zeste, and trithorax) domain containing 3 (SETD3) is cell cycle-regulated; SETD3 protein levels peaked in S phase and were lowest in M phase. We found that the β-isoform of the tumor suppressor F-box and WD repeat domain containing 7 (FBXW7β) specifically mediates SETD3 degradation. Aligning the SETD3 sequence with those of well known FBXW7 substrates, we identified six potential non-canonical Cdc4 phosphodegrons (CPDs), and one of them, CPD1, is primarily phosphorylated by the kinase glycogen synthase kinase 3 (GSK3β), which is required for FBXW7β-mediated recognition and degradation. Moreover, depletion or inhibition of GSK3β or FBXW7β resulted in elevated SETD3 levels. Mutations of the phosphorylated residues in CPD1 of SETD3 abolished the interaction between FBXW7β and SETD3 and prevented SETD3 degradation. Our data further indicated that SETD3 levels positively correlated with cell proliferation of liver cancer cells and liver tumorigenesis in a xenograft mouse model, and that overexpression of FBXW7β counteracts the SETD3's tumorigenic role. We also show that SETD3 levels correlate with cancer malignancy, indicated by SETD3 levels that the 54 liver tumors are 2-fold higher than those in the relevant adjacent tissues. Collectively, these data elucidated that a GSK3β-FBXW7β-dependent mechanism controls SETD3 protein levels during the cell cycle and attenuates its oncogenic role in liver tumorigenesis.

Keywords: cell cycle; cell proliferation; liver cancer; methyltransferase; protein degradation; protein phosphorylation; protein stability.

Figure 1.

Protein levels of SETD3 fluctuate throughout the cell cycle. A, asynchronized (Asyn) or synchronized HeLa S3 cells at the G₁/S boundary, late prometaphase (M), or S phase (S) were subjected to Western blotting using the indicated antibodies. B and C, HeLa S3 cells were synchronized at the G₁/S boundary by a double thymidine arrest, released into fresh media, and harvested at the indicated times (T/T release). Protein levels were analyzed by Western blotting (B), and the cell cycle profile was assayed by FACS with propidium iodine (PI) staining (C). D and E, HeLa S3 cells were synchronized in late prometaphase by a thymidine-nocodazole arrest, released into fresh media, and harvested at the indicated times (T/N release). Protein levels were analyzed by Western blotting (D), and the cell cycle profile was assayed by FACS analysis with PI staining (E). F and G, HeLa S3 cells were synchronized in S phase by HU arrest, released into fresh media, and harvested at the indicated times (HU release). Protein levels were analyzed by Western blotting (F), and the cell cycle profile was assayed by FACS analysis with PI staining (G). The cyclin E1, cyclin B1, H3 phospho-S10, and Plk1 phospho-T210 antibodies were used for the cell cycle index. GAPDH and β-actin served as a loading control.

Figure 2.

SETD3 is degraded in a SCF^FBXW7β- and proteasome-dependent manner. A, proteasome-dependent degradation of SETD3. Cells were synchronized in M phase and then treated with DMSO or 20 μm MG132 for 5 h before harvesting. Protein levels were analyzed by Western blotting. Asynchronous cells served as a control. B, in vivo ubiquitination assays were performed using 293T cells co-transfected with SETD3-GFP and an empty vector or HA-ubiquitin with or without MG132 treatment. SETD3 was immunoprecipitated with a GFP antibody, and the ubiquitinated levels were detected by immunoblots with α-HA. Cell lysates (Input) served as a control. C, several E3 ligases were co-transfected with FLAG-SETD3 and GFP in 293T cells, and protein levels of SETD3 were examined. GFP levels served as a control. The expressions of the indicated E3 ligases were detected by immunoblotting. Asterisks represent the corresponding proteins of expressed E3 ligases. D, three isoforms of FBXW7 were individually co-transfected with SETD3-GFP and the GFP vector. Relative protein levels of SETD3 were examined. E, knockdown of FBXW7 increased endogenous SETD3 protein levels. HeLa cells were transfected with control siRNA or three validated siRNAs targeting FBXW7β. Endogenous SETD3 levels were analyzed by Western blotting with GAPDH as a loading control. F, cells transfected with siControl or siFBXW7β RNA were treated with CHX. SETD3 stability was monitored at the indicated time points. The levels of cyclin E1 were used as a control (left panel). Two independent experiments were quantified by densitometry of Western blot analysis using ImageJ and presented as mean ± S.D. (right panel). G, SETD3 interacts with subunits of the SCF^FBXW7β complex examined by reciprocal co-IP experiments. Co-IP assays were performed using SETD3 or FBXW7 antibody, and the subunits of the SCF^FBXW7β complex, including Cul1, Rbx1, FBXW7, and Skp1 (left panel), or SETD3 (right panel) were analyzed by Western blotting. Ten percent of the total cell lysates (Input) and nonspecific IgG IP served as a control. Asterisk represents the light chain of IgG. H, co-IP assay was performed using M2 FLAG resin in 293T cells expressing SETD3-GFP and FLAG-FBXW7β. The interaction between SETD3 and FBXW7β was analyzed by Western blotting. Five percent of total cell lysates (5% input) were loaded as a control. I, F-box domain of FBXW7β is required for SETD3 degradation. Empty vector, wild-type, or F-box deletion mutant of FBXW7β was co-transfected with SETD3-GFP and the GFP vector, and the protein levels of SETD3 were examined. J, pathological mutants of FBXW7β construct derived from somatic mutations showed reduced ability to destruct SETD3. Cells expressing SETD3-GFP were co-transfected with empty vector, WT, or FBXW7β-GST mutants. Exogenous SETD3 levels in the indicated cells were analyzed by Western blotting probed with α-GFP and α-SETD3 antibodies. K, 293T cells were transfected with an empty vector, SETD3-GFP, HA-ubiquitin, or FLAG-FBXW7β as indicated, followed by IP with α-GFP. The ubiquitinated SETD3 was detected by Western blotting with α-HA.

Figure 3.

SETD3 is degraded in a GSK3β-dependent manner. A, GFP vector and SETD3-GFP construct were co-transfected with an empty vector or FLAG-GSK3β into 293T cells. Relative levels of SETD3-GFP were analyzed by Western blotting. B, knockdown of GSK3β increased endogenous SETD3 levels. The 293T cells were transfected with control siRNA or two validated siRNAs targeting GSK3β. Protein levels of SETD3 and cyclin E1 were examined. C and D, inhibition of GSK3β increased SETD3 levels. HeLa cells were treated with different doses of the GSK3β inhibitor for 48 h in C or with 40 mm LiCl for 48 h in D. The endogenous SETD3 levels were examined by Western blotting. The cyclin E1 levels served as a control. The levels of phosphorylated GSK3β at Ser-9 were used to indicate the inhibitory efficiency of GSK3β. E, cells were treated with DMSO or a GSK3β inhibitor PL-02-061 in the presence of CHX. SETD3 stability was monitored at the indicated time points. F, in vitro ubiquitination assays were performed using individual recombinant proteins incubated with recombinant GST-SETD3, and the polyubiquitinated SETD3 bands were immunoblotted with an α-ubiquitin antibody.

Figure 4.

CPD motif is required for SETD3 degradation. A, alignment of six putative CPD sequences in SETD3. The numbers represent the first amino acid position, and the amino acids in red indicate the ones mutated to alanine. B, wild-type or six putative CPD mutants of SETD3-GFP (named m1–m6) were co-transfected with either the empty vector or FBXW7β and GSKβ accompanied by the GFP vector in 293T cells. SETD3 protein levels were analyzed by Western blotting. C, wild-type or four CPD mutants (m1–m4) of SETD3-GFP were co-transfected with FLAG-FBXW7β in 293T cells. FBXW7β was subjected to IP with α-FLAG, and associated SETD3-GFP proteins were analyzed by immunoblotting with α-GFP. D and E, double CPD mutant of SETD3 (m1 + m3) was examined by protein stability (D) or binding with FBXW7β (E), compared with wild-type or single mutants. F, in vitro kinase assays were performed using recombinant wild-type (WT) or a CPD1 mutant (m1) of GST-SETD3 incubated with different doses of GSK3β protein in the presence or absence of ³²P-labeled γ-ATP. The phosphorylation signals of SETD3 were examined by autoradiography. The amounts of SETD3 protein used in each reaction were monitored by Coomassie Brilliant Blue staining (CBB).

Figure 5.

SETD3 promotes cellular proliferation and tumorigenesis in liver cancer cells. A, relative SETD3 protein levels in various liver cell lines were examined by Western blotting with β-actin as a loading control. B, SETD3 protein stability in the indicated liver cell lines treated with CHX was examined by Western blotting (left panel), and relative SETD3 protein levels were plotted (right panel). C–F, cellular proliferation and colony formation assays were performed in the indicated HepG2 cell lines of either knockdown SETD3 by shRNA (C and D) or overexpression of SETD3 and FBXW7β (E and F). Cell growth curves were plotted over several consecutive days in C and E. Colony formation assays using the indicated cell lines were performed in D and F. Quantifications of colony numbers from the representative images (3 × 3 cm) from three independent plates are shown in D and F (right panel). G and H, SETD3 and FBXW7β regulate tumorigenicity of the liver cancer cells in nude mice. Images show tumor formation in nude mice (G) and the dissected tumors 27 days after injection (H). Tumor masses for each group are shown. Data are presented as mean ± S.E. *, p < 0.05; ***, p < 0.001, n.s., not significant.

Figure 6.

SETD3 protein levels are correlated with liver tumor. A and B, 54 pairs of human liver samples including adjacent tissues (P) and tumor tissues (T) were analyzed. 20 pairs of representative images using Western blot analysis examining SETD3 protein levels are shown in A. Arabic numerals represent individual patient case number. Average protein levels of SETD3 in the tumor tissues compared with the corresponding adjacent tissues were quantified in B. C and D, representative IHC staining images of SETD3 in human liver cancer and adjacent tissues obtained from hepatocellular carcinoma (HCC) tissue microarrays shown in C (53 pairs, 106 spots, each pair contains a cancer tissue and an adjacent tissue). We omitted seven pairs of spoiled dots in the microarray chips, and scores of the SETD3 staining in 46 pairs of human liver cancer and adjacent tissues were plotted in D. E and F, representative IHC staining images of SETD3 tissues with different clinicopathological stages (Grade I–III) obtained from the same tissue arrays shown in E. Scores of the staining of SETD3 in different stages of human liver cancer were plotted as the F. n, tested tissue numbers in individual clinical stages. Data are presented as the mean ± S.E. H&E (hematoxylin-eosin) staining results were provided by the Shanghai Outdo Biotech Co. Square frame, enlarged images. Scale bar, H&E staining, 100 μm; SETD3 staining, 50 μm.

Figure 7.

Model depicting how the GSK3β-CSF^FBXW7β-SETD3 axis regulates cell cycle progression and attenuates liver tumorigenesis (see details in the text).