Epigenetic reprogramming has been associated with the functional plasticity of cancer-initiating cells (CICs); however, the regulatory pathway has yet to be elucidated. A siRNA screen targeting known epigenetic genes revealed that G9a profoundly impairs the chemo-resistance, self-renewal and metastasis of CICs obtained from patients with colorectal cancer (CRC). Patients with elevated G9a were shown to face a high risk of relapse and poor survival rates. From a mechanistic perspective, G9a binds with and stabilizes RelB, thereby recruiting DNA methyltransferase 3 on the Let-7b promoter and repressing its expression. This leads to the activation of the K-RAS/β-catenin pathway and regulates self-renewal and function of CICs. These findings indicate that the G9a/RelB/Let-7b axis acts as a critical regulator in the maintenance of CIC phenotypes and is strongly associated with negative clinical outcomes. Thus, these findings may have diagnostic as well as therapeutic implications for the treatment of chemotherapy-resistant or metastatic CRC.
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We thank C.-S. Chen (Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan) for the gift of K-RASG12D and K-RASG12V plasmid. We also thank the Technology Commons in the College of Life Science and Center for Systems Biology, National Taiwan University, for instrument support for cell sorting. This work was supported by grants from the National Science Council, Taiwan (NSC 099-2811-C-099-003; NSC 100-2811-C-099-003; NSC 102-2321-B-002-034), the Ministry of Science and Technology, Taiwan (MOST 104-231-B-002-006; MOST 104-2320-B-002-070-MY3; MOST 104-2911-I-002-302); and National Taiwan University (NTU 104-R7559-6; NTU 104R8952-1; NTU 104R7602).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Validation of the stem-like and differentiation properties in LGR5-RFP+ and CK20-GFP+ cells.
(a) RT-qPCR for the stemness gene (left panel) and differentiation gene (right panel) in LGR5-RFP+ and CK20-GFP+cells. Data represent mean ± S. D. n = 3 independent experiment (each experiment contains 3 technical replicates). (b) Sphroid formation (left panel) and TOP/FOP reporter activity (right panel) in LGR5-RFP+ and CK20-GFP+cells. Data represent mean ± S. D. n = 3 independent experiment (each experiment contains 3 technical replicates). (c) RT-qPCR to illustrated the mRNA levels of knockdown efficacy of 19 epigenetic regulators. Data represent mean ± S. D. n = 3 independent experiment (each experiment contains 3 technical replicates).
Supplementary Figure 2 G9a activate AKT signalling and β-catenin phosphorylation in K-RAS mutant CCICs.
(a) Western blotting analysis of indicated antibody in CCICs versus non-CCICs in APC wt and mutated CRC cells. (b) Let-7b expression in (a). n = 3 for each group; P < 0.01 by Student’s t-test. (c) Upper: Western blot analysis of p-AKT, AKT, p- β-catenin and β-catenin in K-RAS wildtype and mutated CRC cells treatment with different dosage of specific PI3K inhibitor, XL147. Lower: TOP/FOP activity assay. Data represent mean ± S. D. n = 3 independent experiment (each experiment contains 3 technical replicates). (d) Western blot analysis of p-AKT, AKT, p- β-catenin and β-catenin in K-RAS wildtype and mutated CRC cells transient transfection with shAKT plasmid. Lower: TOP/FOP activity assay. Data represent mean ± S. D. n = 3 independent experiment (each experiment contains 3 technical replicates). (e) RT-qPCR of K-RAS expreesion in CD133− and CD133+ cells. Uncropped images of blots of a, c, d, are shown in Supplementary Fig. 6.
(a) Chip-Seq peaks over chromosomes. (b) Overlap between the top 20,000 genomic location (1-kb windown) bounds by G9a, H3K9me2 and shG9a-reexpressed DNG9a as determined using Illumina. GEO accession number: GSE82131.
Supplementary Figure 4 GLP, the obligatory G9a partner, plays as a bridge to stabilize G9a protein in functional roles in CCICs.
(a) Upper: Western blot analysis of G9a, GLP, H3K9me2 and RelB in CCICs transient transfection with shG9a, shGLP, G9a or DNG9a. Lower: sphere formation analysis. n = 3 independent experiments (each experiment contains 5 technical replicates). (b) Upper: Western blot analysis of G9a, GLP, H3K9me2 and RelB in CCICs transient transfection with shGLP, G9a and DNG9a. Lower: sphere formation analysis. n = 3 independent experiments (each experiment contains 5 technical replicates). (c) Western blot analysis of G9a, GLP, H3K9me2, RelB, K-RAS, p-β-catenin and β-catenin in CCICs transfection with shG9a GLP expression plasmid. (d) RT-qPCR for Let-7b expression in (c). n = 3 independent experiment (each experiment contains 3 technical replicates). (e) TOP/FOP activity assay in (c). Data represent mean ± s.d.n = 3 independent experiment (each experiment contains 3 technical replicates). (f) Sphere formation analysis in (c). n = 3 (each experiment contains 5 technical replicates). (g) Uncropped images of blots of a–c are shown in Supplementary Fig. 6.
(a) Western blot analysis of G9a, GLP, H3K9me2, RelB, KRAS, p-β-catenin and β-catenin in CCICs transfection with two different sequence of shG9a and shGLP plasmid in CCICs. (b) RT-qPCR for Let-7b expression in (a). n = 3 independent experiment (each experiment contains 3 technical replicates). (c) TOP/FOP activity assay in (a). Data represent mean ± S. D. n = 3 independent experiment (each experiment contains 3 technical replicates). (d) Sphere formation in (a) n = 3 (each experiment contains 5 technical replicates). (e) Representative image of immunohistochemical staining for G9a, RelB and K-RAS. Scale bars, 200 μm. (g) G9a-RelB-Let-7b-K-RAS-β-catenin signalling in CCICs.
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Cha, ST., Tan, CT., Chang, CC. et al. G9a/RelB regulates self-renewal and function of colon-cancer-initiating cells by silencing Let-7b and activating the K-RAS/β-catenin pathway. Nat Cell Biol 18, 993–1005 (2016). https://doi.org/10.1038/ncb3395
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Retraction Note: G9a/RelB regulates self-renewal and function of colon-cancer-initiating cells by silencing Let-7b and activating the K-RAS/β-catenin pathway
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