KDM3 epigenetically controls tumorigenic potentials of human colorectal cancer stem cells through Wnt/β-catenin signalling

Abstract

Human colorectal cancer stem cells (CSCs) are tumour initiating cells that can self-renew and are highly tumorigenic and chemoresistant. While genetic mutations associated with human colorectal cancer development are well-known, little is known about how and whether epigenetic factors specifically contribute to the functional properties of human colorectal CSCs. Here we report that the KDM3 family of histone demethylases plays an important role in tumorigenic potential and survival of human colorectal CSCs by epigenetically activating Wnt target gene transcription. The depletion of KDM3 inhibits tumorigenic growth and chemoresistance of human colorectal CSCs. Mechanistically, KDM3 not only directly erases repressive H3K9me2 marks, but also helps to recruit histone methyltransferase MLL1 to promote H3K4 methylation, thereby promoting Wnt target gene transcription. Our results suggest that KDM3 is a critical epigenetic factor in Wnt signalling that orchestrates chromatin changes and transcription in human colorectal CSCs, identifying potential therapeutic targets for effective elimination of CSCs.

Figure 1. siRNA screening of histone demethylases required for Wnt/β-catenin-mediated transcription.

(a) Analysis of luciferase activity of 293 T/Top cells treated with the indicated siRNA relative to those transfected with scramble siRNA. The relative luciferase activity was normalized against the protein concentration of each cell lysate sample. Values mean±s.d. from three independent experiments. (b) Real-time RT-PCR analysis of the relative expression of histone demethylases in human of 293 T/Top cells transfected with targeted siRNA relative to their expression in cells with scramble siRNA. Values are mean±s.d. of three independent experiments. (c) KDM3A, KDM3B and JMJD1C were knocked down by siRNA in 293 T cells. (d,e) Knockdown of endogenous KDM3A, KDM3B and JMJD1C inhibited AXIN2 and DKK1 expression induced by LiCl (d) or Wnt3a (e) in 293 T cells. Values are mean±s.d. of triplicate samples from a representative experiment. AB, siRNA for KDM3A/B; A+B+C, siRNA for KDM3A, KDM3B and JMJD1C. (f) KDM3A and KDM3B enhanced Topflash reporter activities induced by β-catenin in 293 T cells. Values are mean±s.d. for triplicate samples from a representative experiment. β-Cat; the mutant form of β-catenin. (g) Expression of KDM3A rescued Wnt3a-induced Topflash activity in 293 T cells depleted of KDM3A/B. Values are mean±s.d. of triplicate samples from a representative experiment. (h) Expression of KDM3A rescued AXIN2 and DKK1 expression in 293 T cells depleted of KDM3A/B. Values are mean±s.d. of triplicate samples from a representative experiment. (i,j) Wild-type KDM3A, but not KDM3Amut (KDM3A-H1120A), enhanced Topflash reporter activities induced by β-catenin (i) or Wnt3a in 293 T cells. Values are mean and±s.d. for triplicate samples from a representative experiment. *P<0.05; **P<0.01, unpaired two-tailed Student' s t-Test. (k) Western blot showed the knockdown of KDM3A/B by shRNA in HCT116 cells. (l) The knockdown of KDM3A/B inhibited the expression of Wnt target genes by Real-time RT-PCR. (m) Quantitative comparison of genes down-regulated by knockdown of KDM3A/B and Wnt/β-catenin target gene signatures in adenoma (left panel) and carcinoma (right panel) using GSEA. P<0.001, false discovery rate q<0.001.

Figure 2. KDM3A/B interact with β-catenin.

(a,b) KDM3A and KDM3B interacted with β-catenin when overexpressed in 293 T cells. (c) Endogenous β-catenin interacted with KDM3A and KDM3B in 293 T cells on Wnt3a stimulation. (d) Endogenous β-catenin interacted with KDM3A and KDM3B in HCT116 cells. (e) Glutathione S-transferase fusion β-catenin directly interacted with KDM3A and KDM3B in vitro.

Figure 3. KDM3A and KDM3B control the tumorigenic potential of CSC-like cells through Wnt/β-catenin.

(a) The knockdown of KDM3A/B by siRNA in ALDH⁺HCT116 cells. (b) The knockdown of KDM3A/B inhibited the expression level of AXIN2, DKK1, CCND1 and MYC in ALDH⁺HCT116 cells, *P<0.05, **P<0.01, unpaired two-tailed student's t-test (n=3). (c,d) The Knockdown of KDM3A/B significantly inhibited tumorsphere formation of ALDH⁺HCT116 cells. The number of spheres were counted from five different fields and averaged. The results represent mean±s.d. from three independent experiments. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test (n=3). (e,f) The knockdown of KDM3A/B significantly inhibited tumorigenic potentials of ALDH⁺HCT116 cells in vivo. Values are mean±s.d. from a total of eight mice. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test. (g) Comparisons of tumour weights at the end of experiments. *P<0.05, **P<0.01, unpaired two-tailed student's t-test (n=16).

Figure 4. KDM3A/B are required to maintain tumorigenic potentials of human colorectal CSCs.

(a) Isolation of ALDH⁺EpCAM⁺ cells from human CRC tissues of patient case #1 (PS1) by FACS. (b,c) The knockdown of KDM3A/B inhibited tumorsphere formation of ALDH⁺EpCAM⁺ cells from PS1. **P<0.01, unpaired two-tailed student's t-test (n=3). (d) The knockdown of KDM3A/B by siRNA in ALDH⁺EpCAM⁺ cells from PS1. (e) The Topflash activity was significantly higher in ALDH⁺EpCAM⁺ cells than in ALDH⁻EpCAM⁺ cells from PS1. (f) The expression of Wnt target genes was higher in ALDH⁺EpCAM⁺ cells than in ALDH⁻EpCAM⁺. (g) The knockdown of KDM3A/B inhibited the expression of AXIN2, DKK1, CCND1 and MYC in ALDH⁺EpCAM⁺ cells from PS1 (h–j) The knockdown of KDM3A/B significantly inhibited tumour growth of ALDH⁺EpCAM⁺ cells in Nude mice. Tumour growth (i) was monitored for 3 weeks and the weight of the tumors from each group (j) were compared at the end of experiments. **P<0.01, unpaired two-tailed student's t-test (n=16). (k) The Topflash activity was significantly higher in ALDH⁺HCP1 cells than in ALDH⁻HCP1 cells. (l) The knockdown of KDM3A/B in ALDH⁺HCP1 cells inhibited the expression of AXIN2, DKK1, CCND1 and MYC. (m) The knockdown of KDM3A/B inhibited tumorsphere formation of ALDH⁺HCP1 in vitro. (n) The knockdown of KDM3A/B inhibited the tumorigenic potential of ALDH⁺HCP1 cells in vivo. **P<0.01, unpaired 2-tailed Student's t-test (n=16).

Figure 5. KDM3A/B promote chemoresistance in human CSCs.

(a) The knockdown of KDM3A/B enhanced cell death induced by cisplatin in ALDH⁺HCT116 cells. Values are mean±s.d. of triplicate samples from a representative experiment. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test (n=3). (b) The knockdown of KDM3A/B enhanced cell death induced by irinotecan in ALDH⁺HCT116 cells. Values are mean±s.d. of triplicate samples from a representative experiment. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test (n=3). (c) The knockdown of KDM3A/B enhanced the capase-3 activation and the cleavage of PARP-1 in ALDH⁺HCT116 induced by cisplatin. (d) The knockdown of KDM3A/B enhanced the capase-3 activation and the cleavage of PARP-1 in ALDH⁺HCT116 induced by irinotecan. (e) The knockdown of KDM3A/B enhanced cell death induced by cisplatin in ALDH⁺ cells from PS1. **P<0.01, unpaired two-tailed Student's t-test (n=3). (f) The knockdown of KDM3A/B enhanced cell death induced by irinotecan in ALDH⁺ cells from PS1. **P<0.01, unpaired 2-tailed Student's t-Test (n=3). (g) The knockdown of KDM3A/B enhanced the capase-3 activation and the cleavage of PARP-1 in ALDH⁺ cells from PS1 induced by cisplatin. (h) The knockdown of KDM3A/B enhanced the capase-3 activation and the cleavage of PARP-1 in ALDH⁺ cells from PS1 induced by irinotecan.

Figure 6. KDM3A/B bind to Wnt target gene promoters to erase H3K9me2 marks.

(a) The bar chat shows the distribution of distances between KDM3B or H3K9me2 peaks and TSS. (b) Average levels of KDM3B and H3K9me2 enrichment within ± 5 kb of TSS in HCT116/ABsh-1 cells were compared to HCT116/Scrsh cells. (c) Gene ontology analysis of enriched H3K9me2 target genes. The x-axis value is − log₁₀ of binomial raw P value. (d) Average levels of KDM3B and H3K9me2 around the Wnt target genes transcription start site in HCT116/Scrsh cells were compared to HCT116/ABsh-1 cells. (e–h) Representative view of ChIP-seq results in AXIN2, DKK1, CCND1 and MYC gene by Genome Browser.

Figure 7. KDM3A/B directly erase H3K9 methylation and promote H3K4 methylation by MLL1.

(a) ChIP assays confirmed that the knockdown of KDM3A/B abolished KDM3A/B enrichments on the AXIN2 promoter in ALDH⁺HCP1 cells. (b) ChIP assays confirmed that the knockdown of KDM3A/B increased the levels of H3K9me2 on the AXIN2 promoter in ALDH⁺HCP1 cells. (c) ChIP assays showed that the knockdown of KDM3A/B did not affect β-catenin binding on the AXIN2 promoter in ALDH⁺HCP1 cells. (d) ChIP assays confirmed that the knockdown of KDM3A/B abolished KDM3A/B enrichments on the DKK1 promoter in ALDH⁺HCP1 cells. (e) ChIP assays confirmed that the knockdown of KDM3A/B increased the levels of H3K9me2 on the DKK1 promoter in ALDH⁺HCP1 cells. *P<0.05, **P<0.01, unpaired two-tailed Student's t-test (n=3). (f) ChIP assays showed that the knockdown of KDM3A/B did not affect β-catenin binding on the DKK1 promoter in ALDH⁺HCP1 cells. *P<0.05, **P<0.01, unpaired 2-tailed Student's t-test (n=3). (g) ChIP assays showed that the knockdown of KDM3A/B reduced the levels of H3K4me3 on the AXIN2 promoter in CSCs isolated HCP1 cells. (h) ChIP assays showed that the knockdown of KDM3A/B reduced the recruitment of MLL1 to the AXIN2 promoter in ALDH⁺HCP1 cells. (i) ChIP assays showed that the knockdown of KDM3A/B reduced the levels of H3K4me3 on the DKK1 promoter in ALDH⁺HCP1 cells. (j) ChIP assays showed that the knockdown of KDM3A/B reduced the recruitment of MLL1 to the DKK1 promoter in ALDH⁺HCP1 cells. (k) ChIP assays showed that KDM3A/B knockdown reduced BCL9/BCL9L occupancies on the AXIN2 promoter in ALDH⁺HCP1 cells. (l) ChIP assays showed that KDM3A/B knockdown reduced PYGO2 occupancies on the AXIN2 promoter in ALDH⁺HCP1 cells. (m) ChIP assays showed that KDM3A/B knockdown reduced BCL9/BCL9L occupancies on the DKK1 promoter in ALDH⁺HCP1 cells. (n) ChIP assays showed that KDM3A/B knockdown reduced PYGO2 occupancies on the DKK1 promoter in ALDH⁺HCP1 cells.