SETDB1 Accelerates Tumourigenesis by Regulating the WNT Signalling Pathway
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SETDB1 Accelerates Tumourigenesis by Regulating the WNT Signalling Pathway
We investigated the oncogenic role of SETDB1, focusing on non-small cell lung cancer (NSCLC), which has high expression of this protein. A total of 387 lung cancer cases were examined by immunohistochemistry; 72% of NSCLC samples were positive for SETDB1 staining, compared to 46% samples of normal bronchial epithelium (106 cases) (p <0.0001). The percentage of positive cells and the intensity of staining increased significantly with increased grade of disease. Forced expression of SETDB1 in NSCLC cell lines enhanced their clonogenic growth in vitro and markedly increased tumour size in a murine xenograft model, while silencing (shRNA) SETDB1 in NSCLC cells slowed their proliferation. SETDB1 positively stimulated activity of the WNT-β-catenin pathway and diminished P53 expression, resulting in enhanced NSCLC growth in vitro and in vivo. Our finding suggests that therapeutic targeting of SETDB1 may benefit patients whose tumours express high levels of SETDB1.
SETDB1; WNT; lung; tumourigenesis.
Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Conflict of interest statement
No conflict of interests was declared.
Fig. 1. Elevated expression of SETDB1 in NSCLC patients
A. Copy number alterations of SETDB1 across a variety of cancer types (TCGA and Tumorscape database). SETDB1 is located at the peak region of the amplicon in lung cancer in both lung cancer sample collection. Significance was defined as Q value (calculation was performed on the database servers). B. Heatmap schematic illustration of copy number amplification of SETDB1 in tumorscape lung cancer samples which harbor 1q21 amplification. Position of SETDB1 is indicated. C. Gain of SETDB1 DNA copy number correlates with an increased mRNA expression in the NSCLC samples. D. Elevated expression of SETDB1 was noted in 8 different lung cancer patient cohorts. Data were retrieved from GEO and EBI Gene Expression Atlas database (*, P≤0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). E. Kaplan-Meier plots of overall survival: comparison of cases with highest (20 patients) versus lowest (20 patients) expression of SETDB1 in NSCLC patients (GSE14814). P value was calculated by log-rank test. F. mRNA levels of SETDB1 (examined by Realtime PCR) in a set of 60 paired samples of NSCLC. Inner picture, elevated expression of SETDB1 correlates with the increasement of tumor grade.
Fig. 2. SETDB1 protein expression is elevated in NSCLC samples
A. Subtypes of lung cancer samples examined by immunohistochemistry (IHC). B. Representative examples of SETDB1 staining in NSCLC tumor sections. Upper panel: strong SETDB1 staining; lower panel: weak SETDB1 staining. C. Total of 387 NSCLC cases was analyzed by IHC. SETDB1 positive staining cells (upper row) and integrated intensities (lower row) in NSCLC samples compared to normal bronchial epithelium. D. SETDB1 positive staining cells (%) (upper row) and intensity of staining (lower row) correlates with the tumor grade. E. SETDB1 expression in different subtypes of lung cancer. Data are presented as percent positivity (upper row) and intensity of staining (lower row) is higher in each different subtype of cancer compared to normal.
Fig. 3. Aberrant expression of SETDB1 in NSCLC cells affects their proliferation in liquid culture, clonogenic growth in semi-soft cultures and tumor size in nude mice
A. Effect of stably silencing SETDB1 on NSCLC cell growth in liquid culture. MTT assay was performed in 96 wells plate. Mean ± SD of 6 wells. B. Effect of silencing of SETDB1 on colony formation of NSCLC cells. Cells were seeded into soft agar in triplicate, and colonies were counted after 21~28 days of culturing. Mean ± SD (3 wells) are expressed as percent variation relative to scramble shRNA infected cells (control). C. Effect of forced expression of SETDB1 on colony formation of PC14 and H1299 NSCLC cells. Cells were seeded into soft agar in triplicate dishes; and colonies were counted on day 28 of culture. Values are expressed as fold variation relative to GFP overexpressing cells (control). Results are mean ± SD of 3 experiments. Right panel, representative pictures of colonies of H1299 overexpressing SETDB1. D. Histone H3K9 methylation status of NSCLC cells with stable overexpression of SETDB1 (H1299 and A549) was examined by western blot using antibodies specific for H3K9me1, H3K9me2, and H3K9me3 (mono-, di- and tri- methylation of H3K9, respectively). Expression levels of histone H3 protein were used as an internal control. E. SETDB1 stimulates NSCLC tumor growth in mice. Photograph of tumors dissected from nude mice which were injected with H1299 cells overexpressing either GFP (upper row) or SETDB1 (lower row). Tumor cells (3 × 10
6) were suspended in a 1:1 mixture of fetal bovine serum and Matrigel (BD Labware) and injected into both flanks of five week-old nude mice. Tumors were removed on day 21 from initiation of the experiment. F. Weights of tumors are shown in Fig. 3 E. The bars show differences in average weight of tumors in two groups (mean ± S.D., n = 8). Difference of mean weights between control (GFP) and SETDB1 overexpressing tumors was statistically significant (p-value of 0.0003). G. Expression level of SETDB1 in the tumors with forced expression of either GFP or SETDB1. Proteins and mRNA were isolated from xenografts. Tumor mRNA values were measured by RT-PCR and normalized with β-actin. Values are expressed as fold variation of SETDB1 overexpressing tumors relative to GFP control tumors. SETDB1 protein was measured by western blot (inner picture).
Fig. 4. Inverse correlation between the protein levels of P53 and SETDB1
A. Silencing TP53 enhanced the colony formation of A549 cells (wild type P53) having forced expression of SETDB1. B. A549 NSCLC cells were either stably overexpressing SETDB1 (upper row) or stably overexpressing SETDB1 and silencing TP53 (lower row). These two cohorts of cells (5 × 10
6) were injected into the opposite flanks of nude mice. Tumors were photographed (Panel B) and weighed (Panel C) on day 35 after initiation of the study. D. SETDB1 expression levels in the HCT116 TP53 +/+ and TP53 −/− HCT116 cells. Left panel, SETDB1 mRNA expression; right panel, western blot of the same cells. E. Protein levels (western blot) of P53 and SETDB1 in NSCLC cells (PC14, HCC1975, H23) with either stable overexpression or silencing of SETDB1.
Fig. 5. Altered gene expression in NSCLC cells with forced expression of SETDB1
A. RNA array data was transformed into heat-map of gene expression in H1299 NSCLC cells stably expressing SETDB1. Green and red represents down-regulation and up-regulation of gene expression, respectively. Total 711 genes were down-regulated and 522 genes were upregulated after overexpression of SETDB1. B. Pathway enrichment analysis between SETDB1 and control GFP overexpressing H1299 cells using the KEGG database. Top 10 significantly enriched pathways are presented in the graph (*, P≤0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001). C. RT-PCR validation of expression of genes related to the WNT signaling pathway in SETDB1 overexpressing H1299 cells. Relative mRNA amounts were normalized to β-actin. D. Recruitment of SETDB1 to the promoter region of APOE and IGFBP4 was examined by ChIP. Nucleotide segments 2 ~ 2.3 kb upstream and 600 ~ 1000 bp downstream from the starting site (ATG) were divided into 14 (APOE) and 10 (IGFBP4) regions, enrichment of each fragment (250~300 bp) were examined by PCR. Enrichment occurred in the promoter regions of P
A 3, P A 7, P A 10 and P A 14 of the APOE promoter (left panel) and regions of P I 2, P I 5, P I 6 and P I 8 of the IGFBP4 promoter (right panel). TSS, transcriptional start site; ATG, protein start codon. Negative results of promoter regions P A 8 (APOE) and P I 7 (IGFBP4) were shown as controls for respective genes. E. mRNA levels of IGFBP4, APOE and β-catenin in SETDB1 silenced H1299 cells. Relative mRNA amounts were normalized with β-actin. Error bars indicate SD (n = 4).
Fig. 6. Protein levels of β-catenin in the NSCLC cells with either stably overexpressed or silenced SETDB1
A. Western analysis of β-catenin accumulation in NSCLC cell lines with either silenced or overexpressed SETDB1. Ctrl, sh-Scramble; sh1,2,3, three shRNA targeting to SETDB1. B. Relative TOP/FOP activities when overexpressing SETDB1 in H1299 cells. The stably forced expression of either SETDB1 or GFP containing cells were transfected with either pGL-TOP or pGL-FOP luciferase-reporter constructs. Luciferase activities were measured 72 hours after the transfection and normalized to the corresponding co-transfected Renilla luciferase activity. Data are shown as the ratio between TOP/FOP and Renilla. Error bars represent SD of three independent experiments. C. Silencing of IGFBP4, LRP8 or FZD1 reduced the WNT activity in H1299 NSCLC cells. Cells stably infected with either IGFBP4, LRP8 or FZD1 were transfected with either TOP or FOP, as well as Renilla control vector, and the levels of WNT pathway activity were determined by TOP/FOP assay. D. Cytoplasmic and nuclear fraction of H1299 cells stably overexpressing either GFP or SETDB1. Cells were grown on 10 cm dishes to 50% ~ 70% confluence, and the cytoplasmic and nuclear proteins were prepared as described in Materials and Methods. Cytoplasmic α-tubulin and nuclear histone H3 was used as controls of protein fractionation. E. Western analysis of alternations of downstream genes (c-MYC, Cyclin D1) of the WNT pathway in SETDB1 stably overexpressing H1299 NSCLC cells. F. Effect of stable silencing of β-catenin on clonogenic growth of H1299 cells overexpressing either GFP (control) or SETDB1. Values are expressed as % of GFP control cells, mean ± SD (three independent experiments). The silencing efficiency of β-catenin was determined by Western blot (right side). G. Clonogenic growth of either GFP or SETDB1 forced expressing H1299 cells after stable silencing of FZD1, LRP8 or IGFBP4. Values are expressed as % of GFP control cells. Experiments were performed in triplicate, and results are presented as mean ± SD. H. Schematic illustration of the proposed signaling interaction of SETDB1 and WNT pathway.
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Biomarkers, Tumor / genetics
Biomarkers, Tumor / metabolism
Carcinoma, Non-Small-Cell Lung / enzymology
Carcinoma, Non-Small-Cell Lung / genetics
Carcinoma, Non-Small-Cell Lung / pathology
Gene Expression Regulation, Neoplastic
Lung Neoplasms / enzymology
Lung Neoplasms / genetics
Lung Neoplasms / pathology
Protein Methyltransferases / genetics
Protein Methyltransferases / metabolism
Tumor Suppressor Protein p53 / genetics
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Wnt Signaling Pathway / genetics
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