Cancer Cell, Volume 26 Supplemental Information Acetylation of Snail Modulates the Cytokinome of Cancer Cells to Enhance the Recruitment of Macrophages Dennis Shin-Shian Hsu, Hsiao-Jung Wang, Shyh-Kuan Tai, Chun-Hung Chou, Chia-Hsin Hsieh, Po-Hsien Chiu, Nien-Jung Chen, and Muh-Hwa Yang
27
Embed
Document S1. Article plus Supplemental Experimental Procedures ...
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Cancer Cell, Volume 26
Supplemental Information
Acetylation of Snail Modulates
the Cytokinome of Cancer Cells
to Enhance the Recruitment of Macrophages
Dennis Shin-Shian Hsu, Hsiao-Jung Wang, Shyh-Kuan Tai, Chun-Hung Chou, Chia-Hsin Hsieh, Po-Hsien Chiu, Nien-Jung Chen, and Muh-Hwa Yang
1
Supplemental Data
Figure S1, related to Figure 1. CBP interacts with Snail to remodel the chromatin on target genes.
2
(A) Quantitative chromatin immunoprecipitation (qChIP) for analyzing the enrichments of H3K4Ac (upper) and H3K14Ac (lower) on the promoter of ERCC1 and IL8 in FaDu cells transfected with Snail (FaDu-Snail) or a control vector (FaDu-CDH). Data represents means ± S.E.M. **p < 0.01. (B) qChIP for analyzing the enrichments of H3K18Ac (upper), H4R3me2 (middle), and H3K27me3 (lower) on the promoter of ERCC1, IL8, and CDH1 in FaDu cells treated with TGFβ 5 ng/ml for 7 days for induction of EMT or a vehicle control. Data represents means ± S.E.M. *p < 0.05. (C) Mapping the CBP-interacting domain on Snail. Left: schematic representation of the constructs. The interacting fragments are indicated as +. TRD, transcription regulatory domain. Z1-Z4, zinc finger domain. Right: immunoprecipitation-western blot in 293T cells co-transfected with pHA-CBP and different GST-Snail constructs to show the interaction between CBP and different Snail fragments. (D) Mapping the Snail-interacting domain on CBP. Left: schematic representation of the constructs. The interacting fragments are indicated as +. Right: immunoprecipitation-western blot in 293T cells co-transfected with GST-Snail and different FLAG-tagged CBP fragments to show the interaction between Snail and different CBP fragments.
3
Figure S2, related to Figure 2. Acetylation of Snail by CBP. (A)-(B) Proximity ligation assay. (A) The 293T cells were transfected with p53 expression vector (pRC-CMV-p53) with or without co-transfection of CBP expression vector (pHA-CBP), then were incubated with the anti-p53 and anti-acetyl lysine antibodies and detected by DuoLink probe. (B) The 293T cells were transfected
4
with different vectors expressing WT Snail or mutant Snail (K146R, K187R, or 2R) with or without co-transfection of CBP, and then were incubated with the anti-Snail and anti-acetyl lysine antibodies and detected by DuoLink probe. Red, Duolink fluorescence; blue, nuclei stained by DAPI. The negative control was the cells co-transfected with p53 and CBP (A), or WT Snail and CBP (B) and incubated with an anti-acetyl lysine antibody alone. The acetylated p53 detected by proximity ligation assay is a positive control for the study. Red, Duolink fluorescence; blue, nuclei stained by DAPI. Scale bar=10μm. (C) Validation of the anti-acetylated Snail lysine 146 and the anti-acetylated Snail lysine 187 antibodies. Dot blot analysis was used for showing the specificity of the anti-acetylated Snail lysine 146 antibody (upper) and anti-acetylated Snail lysine 187 antibody (lower). The biotin-tagged peptides around the sequence of Snail lysine 146/187 with or without acetylated modification on lysine 146/187 were used for experiments, and the peptide sequence was shown. The antibody specific for non-acetylated Snail lysine 146/187 was a negative control for experiment. (D) Western blot for detecting lysine 146- or lysine 187-acetylated Snail in FaDu-Snail transfectants receiving shRNA against different acetyltransferases or a control vector (pLKO). β-actin was a loading control.
5
6
Figure S3, related to Figure 3. The impact of Snail acetylation on target genes transactivation, stability, DNA binding ability, subcellular localization of Snail, and EMT. (A) Luciferase reporter assay. The 293T cells were co-transfected with the reporter plasmid containing ERCC1 (pXP2-ERCC1) promoter, pFLAG-Snail and pHA-CBP with or without the treatment of a HAT inhibitor C646 (6 μM for 8 hr). Data represents means ± S.E.M. *p < 0.05. (B) Quantitative RT-PCR for examining the expression of FN1 in FaDu-CDH, FaDu-Snail, and FaDu-Snail2R. Data represents means ± S.E.M. *p < 0.05. (C) Immunoprecipitation-western blot of OECM-1 cells. The lysates were immunoprecipitated with an anti-p65 antibody and immunoblotted with the indicated antibodies. (D) Immunoprecipitation-western blot of 293T cells co-transfected with pMT2T-p65, pHA-CBP, and pFLAG-Snail/pFLAG-Snail2R. The protein lysates were immunoprecipitated by an anti-FLAG antibody and immunoblotted with anti-FLAG and anti-p65 antibodies. (E) Immunoprecipitation-western blot of 23T cells co-transfected with pHA-CBP, pFLAG-Snail, and pMT2T-p65. The lysates were immunoprecipitated with an anti-HA antibody or an anti-p65 antibody and immunoblotted with the indicated antibodies. (F) ChIP and sequential ChIP assay in FaDu cells. The protein-DNA was cross-linked and immunoprecipitated by one or two antibodies as indicated. The DNA fragment containing the NF-κB binding site on the promoter of FN1 was amplified by PCR and analyzed by electrophoresis. (G) Luciferase reporter assay. The 293T cells were co-transfected with the reporter plasmid containing FN1
7
promoter (pGL4-FN1) and different plasmids as indicated. The red box is the p65 binding site, whereas the blue boxes indicate the Snail binding sites. Data represents means ± S.E.M. *p < 0.05. (H) Western blot of FLAG in 293T cells transfected with pFLAG-Snail (upper panel) or pFLAG-Snail2R (lower panel) after cyclohexamide (CHX) treatment for different periods. (I) Quantification of the western blot results in (H). (J) Immunoprecipitation-western blot for analyzing the phosphorylated or acetylated Snail in 293T cells transfected with WT Snail (pFLAG-Snail) or non-acetylatable Snail mutant (pFLAG-Snail2R) with or without co-transfection of pHA-CBP. Phospho-Ser/Thr, phosphorylated serine/threonine. (K) Analysis of polyubiquitinated FLAG-Snail in 293T cells co-transfected with pFLAG-Snail/pFLAG-Snail2R and pHA-ubiquitin. The FLAG-Snail was immunoprecipitated and analyzed for its polyubiquitination. Immunoglobulin G (IgG) was a control for immunoprecipitation. (L) Analysis of polyubiquitinated FLAG-Snail in 293T cells co-transfected with WT Snail or non-phosphorylatable Snail mutant (pFLAG-Snail6SA), pHA-CBP, and pHA-ubiquitin, and with or without the treatment of a HAT inhibitor C646 (6 μM for 8 hr). The FLAG-Snail was immunoprecipitated and analyzed for its polyubiquitination. Immunoglobulin G (IgG) was a control for immunoprecipitation. (M) EMSA. Left panel: western blot for indicating the expression of Snail or Snail2R in 293T cells for EMSA. Right panel: EMSA. Nuclear extracts from 293T cells under different transfection conditions were incubated with biotin-labeled probe containing the Snail binding site. Adding of the 30x non-labelled probe abrogated the shifted bands. The positions of shifted band are indicated. (N) Immunofluorescent staining of Snail in FaDu-CDH, FaDu-Snail, and FaDu-Snail2R cells. Green, Snail; blue, nuclei. Scale bar=100 μm. (O) Western blot of the EMT markers (E-cadherin, γ-catenin, and fibronectin) in FaDu-CDH, FaDu-Snail, and FaDu-Snail2R cells. β-actin was a loading control. (P) Immunofluorescent staining of E-cadherin and fibronectin in FaDu-CDH, FaDu-Snail, and FaDu-Snail2R cells. Red, E-cadherin; green, fibronectin; blue, nuclei. Scale bar=20 μm.
8
9
Figure S4, related to Figure 5. The mesenchymal genes and TNFA, CCL2, and CCL5 are target genes activated by acetylated Snail. (A) Quantitative RT-PCR for analyzing the expression of the mesenchymal genes ZEB1 and THBS1 in FaDu-CDH, FaDu-Snail, and FaDu-Snail2R. Data represents means ± S.E.M. *p < 0.05. (B) IPA analysis of the Snail-upregulated cytokine genes.
10
(C) Quantitative RT-PCR for validating the expression of different cytokine genes in HCT15-Snail vs HCT15-control. Data represents means ± S.E.M. *p < 0.05. (D) Left: quantitative RT-PCR for analyzing the expression of IL6 in FaDu-CDH, FaDu-Snail, and FaDu-Snail2R. Right: ELSIA for analyzing the level of secreted IL6 in the corresponding cells. Data represents means ± S.E.M. *p < 0.05. (E) ELSIA for analyzing the level of secreted TNFα (left), CCL2 (middle), and CCL5 (right) in FaDu-CDH, FaDu-Snail, and FaDu-Snail2R. Data represents means ± S.E.M. *p < 0.05. (F) Western blot analysis of Snail, TNFα, CCL2, and CCL5 in OECM-1 cells receiving shRNA against SNAI1 (sh-Snail) or a scrambled sequence (sh-scr). β-actin was a loading control. The fold change of target proteins was shown. (G) Quantitative RT-PCR analysis of the mRNA level of SNAI1, TNFA, CCL2, and CCL5 in OECM-1 cells receiving sh-Snail vs. sh-scr. Data represents means ± S.E.M. *P < 0.05. (H) Western blot analysis of Snail, TNFα, CCL2, and CCL5 in HT-29 cells receiving shRNA against SNAI1 (sh-Snail) or a scrambled sequence (sh-scr). β-actin was a loading control. The fold change of target proteins was shown. (I) Quantitative RT-PCR analysis of the mRNA level of SNAI1, TNFA, CCL2, and CCL5 in HT-29 cells receiving sh-Snail vs. sh-scr. Data represents means ± S.E.M. *p < 0.05. (J) Quantitative ChIP for analyzing the occupancy of Snail on the promoter of TNFA, CCL2, and CCL5 in FaDu cells transfected with Snail (FaDu-Snail) or a control vector (FaDu-CDH). The organization of TNFA, CCL2, and CCL5 and the primer-amplified region was shown. TSS, transcription start site. E1, E2, E3, and E4 indicate E-boxes. IgG was a control for immunoprecipitation. Data represents means ± S.E.M. *p < 0.05. (K) Quantitative RT-PCR assay for analyzing the relative mRNA level of TNFA, CCL2, ERCC1, and IL8 in in FaDu cells transfected with WT Snail (FaDu-Snail), non-acetylatable Snail (FaDu-Snail2R), or an empty vector (FaDu-CDH) with or without Etanercept treatment (25 μg/ml for 8hr). Data represents means ± S.E.M. *p < 0.05. (L) Quantitative RT-PCR for analyzing the relative mRNA expression of CCND1 in FaDu-CDH vs. FaDu-Snail cells treated with the NFκB inhibitor parthenolide (PAR)(10 μM for 8hr). The data is presented as the percentage of target genes repression after PAR treatment compared with the cells without PAR treatment. Data represents means ± S.E.M. (M) Quantitative RT-PCR for analyzing the relative mRNA expression of CCL2, CCL5, and IL8 in FaDu-CDH vs. FaDu-Snail cells treated with the NFκB inhibitor parthenolide (PAR)(10 μM for 8 hr). The data is presented as the percentage of target genes repression after PAR treatment compared with the cells without PAR treatment. Data represents means ± S.E.M. *p < 0.05. (N) Quantitative RT-PCR for analyzing the relative mRNA expression of TNFA, CCL2, and CCL5 in OECM-1 cells receiving shRNA against Snail (OECM1-sh-Snail) or a scrambled sequence (OECM1-sh-scr). The data is presented as the folds of mRNA induction by TNFα compared with the cells without TNFα treatment. Data represents means ± S.E.M. *p < 0.05. Table S1, related to Figure 5. Provided as a separated Excel file. Upregulated and downregulated genes in cDNA microarray analysis of FaDu-Snail vs. FaDu-Snail2R.
Table S2, related to Figure 5. Provided as a separated Excel file. PCR array for analyzing the expression of 84 cytokine genes in FaDu-Snail vs. FaDu-CDH and HT29-scr vs. HT29-Snail.
11
Figure S5, related to Figure 6. Acetylated Snail promotes M2-like polarization and recruitment of tumor-associated macrophages. (A) Quantitative RT-PCR for analyzing the relative mRNA level of MRC1 in CD14+ human monocytes (upper) or a human monocytic cell line THP1 (lower) incubated with the conditioned medium from FaDu cells expression WT Snail (FaDu-Snail), acetylation sites-mutated Snail (FaDu-Snail2R), or an empty vector (FaDu-
12
CDH) for 3 days. Data represents means ± S.E.M. **p < 0.01. (B) Flow cytometry for analyzing the expression of mannose receptor (MR) in CD14+ human monocytes incubated with the conditioned medium from FaDu-Snail, acetylation FaDu-Snail2R, or FaDu-CDH for 3 days. The cells were incubated with an anti-mannose receptor antibody (lower panel) or an isotype IgG control (upper panel) and subject to flow cytometry analysis. The percentage of mannose receptor-positive cells is shown in each panel. (C) Western blot for detecting acetylated Snail lysine 187 in the 4T1-formed orthotopic tumors. Ctrl, 4T1-control cells-formed tumor; Snail, 4T1-Snail cells-formed tumors; Snail2R, 4T1-Snail2R cells-formed tumors. Each group contains 6 mice (Ctr1(1)-(6), Snail(1)-(6), and Snail2R(1)-(6)). β-actin was a loading control. (D) Immunohistochemistry of arginase 1 in 4T1-formed orthotopic tumors. Scale bar=100 μm.
13
Figure S6, related to Figure 7. Immunohistochemistry of arginase 1 for indicating the TAMs in LLC1-formed tumors. Scale bar=100 μm.
14
Figure S7, related to Figure 8. Correlation between SNAI1, CCL2, and CCL5 in public array database, and the association between acetylated Snail and tumor-associated macrophages in lung cancer samples. (A) Left: a heatmap showing the relative level of CDH1, SNAI1, CCL2, and CCL5 in cancer cells lines from NCI-60 panel. Right: a table showing the correlation between the level of CDH1, SNAI1, CCL2, and CCL5 in cells lines from NCI-60 panel. (B) Summary of the expression of SNAI1, CCL2, and CCL5 in human head and neck cancer originated from different sites. LSCC, laryngeal squamous cell carcinoma; OSCC, oral cavity squamous cell carcinoma; OPSCC, oropharyngeal squamous cell carcinoma; TSCC, tongue squamous cell carcinoma; TsSCC, tonsillar squamous cell carcinoma. The data is obtained from Hensen dataset. (C) Immunohistochemistry using an anti-acetylated Snail lysine 187 antibody for recognizing acetylated Snail and an anti-CD163 antibody for detecting tumor-associated macrophages in non-small cell lung cancer samples. Case 1 is a representative case of double positive, whereas case 2 is a representative case of double negative. Scale bar for acetylated Snail staining=100 μm, for CD163=200 μm.
15
Table S3, related to Figure 8. Provided as a separated Excel file. Characteristics of 15 head and neck cancer patients analyzed by immunohistochemistry and proximity ligation assay. Table S4, related to Figure 8. Provided as a separated Excel file. Characteristics of 82 head and neck cancer patients for immunohistochemical analysis of acetylated Snail and TAMs.
16
Supplemental Experimental Procedures Cell lines, plasmids, and reagents. The human head and neck cancer cell line FaDu, the human embryonic
kidney cell line 293T, the human colon cancer cell line HCT15, the BALB/C mouse breast carcinoma cell line
4T1, and the C57BL/6 mouse lung carcinoma cell line LLC1 were originally from ATCC. The human head and
neck cancer cell line OECM-1 was provided by Dr. Kuo-Wei Chang (National Yang-Ming University of
Taiwan). The human lung adenocarcinoma cell lines CL1-1 and CL1-5 were obtained from Professor Pan-Chyr
Yang (National Taiwan University, Taipei, Taiwan). The pFLAG-CBP, pHA-CBP, pHA-EECBP, pHA-AACBP,
and SnailS6A expression vectors and the MEFs from Ikkα, Ikkβ, and Ikkγ-KO mice were gifts from Professor
Mien-Chie Hung (M.D. Anderson Cancer Center, Houston, TX, USA). The pRC-CMV-p53 plasmid was
provided by Professor Fung-Fang Wang (National Yang-Ming University, Taipei, Taiwan). The pMT2T-p65
plasmid was provided by Professor Kou-Juey Wu (National Yang-Ming University, Taipei, Taiwan). The
pcDNA3-Snail, pSUPER-sh-Snail, and pSUPER-scramble have been previously described (Hsu et al., 2010).
The GST-Snail, pFLAG-Snail, pCDH-Snail, and pHA-Ajuba plasmids were generated by inserting full-length
cDNA (SNAI1: NM_005985; AJUBA: NM_032876) into the pGEX4T1, pFLAG-CMV2, pCDH-CMV-MCS-
EF1-puro, and pHA-MEX vectors, respectively. The pLKO.1-shLuc, CBP shRNA (TRCN000006487), p300
• Hensen, E.F., De Herdt, M.J., Goeman, J.J., Oosting, J., Smit, V.T., Cornelisse, C.J., and Baatenburg de Jong, R.J. (2008). Gene-expression of metastasized versus non-metastasized primary head and neck squamous cell carcinomas: A pathway-based analysis. BMC Cancer 8, 168.
• Ross, D.T., Scherf, U., Eisen, M.B., Perou, C.M., Rees, C., Spellman, P., Iyer, V., Jeffrey, S.S., Van de Rijn, M., Waltham, M., et al. (2000). Systematic variation in gene expression patterns in human cancer cell lines. Nat. Genet. 24, 227-235.
• Yang, M.H., Hsu, D.S., Wang, H.W., Wang, H.J., Lan, H.Y., Yang, W.H., Huang, C.H., Kao, S.Y., Tzeng, C.H., Tai, S.K., et al. (2010). Bmi1 is essential in Twist1-induced epithelial-mesenchymal transition. Nat. Cell Biol. 12, 982-992.