LIG4 mediates Wnt signalling-induced radioresistance

Abstract

Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with radioresistance in colorectal cancer (CRC) cells and intestinal stem cells (ISCs). We find that LIG4, a DNA ligase in DNA double-strand break repair, is a direct target of β-catenin. Wnt signalling enhances non-homologous end-joining repair in CRC, which is mediated by LIG4 transactivated by β-catenin. During radiation-induced intestinal regeneration, LIG4 mainly expressed in the crypts is conditionally upregulated in ISCs, accompanied by Wnt/β-catenin signalling activation. Importantly, among the DNA repair genes, LIG4 is highly upregulated in human CRC cells, in correlation with β-catenin hyperactivation. Furthermore, blocking LIG4 sensitizes CRC cells to radiation. Our results reveal the molecular mechanism of Wnt signalling-induced radioresistance in CRC and ISCs, and further unveils the unexpected convergence between Wnt signalling and DNA repair pathways in tumorigenesis and tissue regeneration.

Figure 1. Association of Wnt signalling and radioresistance in CRC cells.

(a) CRC cell sorting by Wnt signalling activity. HCT116-7TGP cells were sorted based on GFP expression (GFP^High and GFP^Low) by FACS. HCT116 parental cells served as a negative control for cell sorting. (b,c) Increased cell survival in GFP^High than GFP^Low cells. HCT116-7TGP (GFP^High and GFP^Low) cells were treated with IR (4 Gy). Two weeks later, cells were fixed for crystal violet staining (b). Colony number quantification of HCT116-7TGP and SW620-7TGP cells after IR (4 Gy) (c). *P<0.05. Student's t-test; N=3; error bars=±s.e.m. (d) Radioresistance in GFP^High CRC cells compared with GFP^Low cells. Clonogenic cell survival assay of SW620-7TGP and HCT116-7TGP cells. After IR (2, 4 and 8 Gy), sorted cells (GFP^High and GFP^Low) were seeded based on plating efficiency. Two weeks later, the number of colonies was quantified. Student's t-test; N=3; error bars=±s.e.m. (e) Radiosensitization of CRC cells by iCRT14. CRC cells (SW620, HCT116, HT15, COLO205, HCC2998 and KM12) were pretreated with iCRT14 (24 h) and IR (0, 2, 4 and 8 Gy). Two weeks later, colonies were counted for quantification. Student's t-test; N=3; error bars=±s.e.m.

Figure 2. LIG4 transactivation by β-catenin.

(a) Identification of β-catenin target genes. DNA repair genes that are upregulated by β-catenin (465 genes) and downregulated by iCRT14 (50 μM for 24 h; 57 genes) were identified. Notably, LIG4 is both upregulated by β-catenin and downregulated by iCRT14. Fold change > 2; P<0.005. Student's t-test; N=3. (b) Identification of NHEJ genes downregulated by β-catenin inhibition. HCT116 cells were treated with iCRT14 (50 μM for 24 h) and analyzed for expression of NHEJ repair genes using qRT-PCR. Student's t-test; N=3; error bars=±SEM. (c) Downregulation of LIG4 by iCRT14 in CRC cells. CRC cells (HCT116, SW620, HT15, KM12 and COLO205) were treated with iCRT14 (50 μM for 24h) and analyzed by qRT-PCR. AXIN2 served as a positive control for the β-catenin target gene. Student's t-test; N=3; *P<0.05; error bars=±s.e.m. (d) Upregulation of LIG4 by Wnt3A in IECs. Human IECs (FHC and CCD841CoN) and 293T cells were treated with Wnt3A (200 ng ml⁻¹ for 24 h) and analysed by qRT–PCR. Student's t-test; N=3; *P<0.05; error bars=±s.e.m. (e) LIG4 promoter analysis. Conserved noncoding sequences (CNS) found in both the mouse and human LIG4 promoter were analysed for potential TCF/LEF-binding elements (TBEs; balloons). UTR: untranslated region. (f) β-catenin transcriptional complex occupies LIG4 promoter. HCT116 cells were analysed by ChIP assays. ChIP amplicons (1–8) were detected by ChIP-PCR. (g,h) Upregulation of LIG4 in GFP^High cells. SW620-7TGP CRC cells were sorted into GFP^High and GFP^Low cells, and clonally selected for semi-quantitative RT–PCR of LIG4 expression (g). ImageJ analysis of LIG4 expression normalized by hypoxanthine phosphoribosyltransferase 1 (HPRT) (h). Student's t-test; N=3; *P<0.05; error bars=±s.e.m.

Figure 3. LIG4 mediates Wnt signalling-induced radioresistance.

(a,b) Increase of unrepaired DNA damage by Wnt signalling inhibition. HCT116 cells were pretreated with dimethylsulfoxide (DMSO; vehicle control) or iCRT14 (50μM for 24h) and were treated with IR (4 Gy). At each time point, cells were collected for IF staining for phospho-γH2AX (A); quantitative analysis of DNA damage foci (b). Student's t-test; N=3; *P<0.05; error bars=±s.e.m.; scale bars=20μm. (c,d) Decreased DNA damage foci formation by Wnt signalling. Human IECs (FHC and CCD841CoN) were pretreated with Wnt3A (200 ng ml⁻¹ for 24 h) and were subjected to IR (4 Gy). After 24 h, cells were analysed for DNA damage foci formation. IF staining (c); quantitative analysis (d). Student's t-test; N=3; *P<0.05; error bars=±s.e.m.; scale bars=20μm. (e,f) Inhibition of LIG4 blocks Wnt3A-induced radioresistance. CCD841CoN IECs were pretreated with Wnt3A (200 ng ml⁻¹ for 24 h) and subjected to IR (4 Gy) in the absence or presence of SCR7 (10 μM); quantitative analysis (f). Student's t-test; N=3; *P<0.05; error bars=±s.e.m.; scale bars=20μm. (g–i) Reduced NHEJ activity after Wnt signalling inhibition. SW620-7TGP GFP^High cells were co-transfected with linearized tdTomato-expressing plasmids (g) CMV, cytomegalovirus. Twenty-four hours after transfection, cells were analysed by FACS (h) and were quantified (i). Student's t-test; N=3; *P<0.05; NS (not significant, P≥0.05); error bars=±s.e.m. (j–l) LIG4 expression rescues Wnt signalling inhibition-induced radiosensitization. iCRT14-treated HCT116 cells (control: empty vector; stably expressing wild-type (WT) LIG4 or R278H mutant LIG4) were exposed to IR (4 Gy). After 24 h, cells were analysed for γH2AX foci formation; IF staining (j), statistical analysis (k) and crystal violet staining (14 days) (l). Student's t-test; N=3; *P<0.05; NS (not significant, P≥0.05); error bars=±s.e.m.; scale bars=20μm. For b,d,f and k, the number of phospho-γH2AX foci were counted in more than 20 nuclei from the three biological replicas.

Figure 4. Expression of LIG4 in the intestine.

(a) Expression of LIG4 in mouse small intestine samples; IF staining. Scale bars, 50μm. (b) Illustration of ISCs in the small intestine. ISCs located at position 4 (+4) are indicated by arrows, and CBC ISCs are indicated by arrowheads hereafter. (c) Expression of LIG4 in the human small intestine. 3,3′-Diaminobenzidine substrate staining. (d,e) Expression of LIG4 in the crypt of the mouse small intestine. IF staining (d) and qRT–PCR (e) of Lgr5⁺ and Lgr5⁻ cells isolated from the crypts of Lgr5CreERT2 strain. AXIN2 served as a positive control for Wnt/β-catenin signalling activity. Student's t-test; N=3; error bars=±s.e.m.; scale bars=20μm. (f) Generation of the TERT^TCE strain. TCE was inserted into the TERT allele-coding sequence in frame. (g,h) Expression of TCE in mouse ESCs. Low (g) and high (h) magnification. Scale bars=100μm. (i,j) TCE expression in the crypt of mouse small intestine samples (TERT^TCE mouse). Sagittal (i) and transverse (j) sections. Scale bars=20μm. (k,l) Expression of LIG4 in the TCE-expressing cells in the crypt of mouse small intestine samples (TERT^TCE mouse). IF staining (k) and qRT–PCR (l) of TERT⁺ cells isolated from TERT^TCE. Student's t-test; N=3; error bars=±s.e.m.; scale bars=20μm. (m,n) WBI-induced DNA damage formation. Mice were treated with WBI (10 Gy; hereafter). Notably, CBC ISCs (arrowheads) disappeared 24 h after WBI. IF staining (m); quantification of phospho-γH2AX⁺ cells per crypts (the number of crypts counted ≥40) (n). Student's t-test; N=3; error bars=±s.e.m.; scale bars=20μm. (o) Apoptosis of CBC cells. Samples of mouse small intestine treated with WBI. Scale bars=20μm. (p) Mitosis of IECs after WBI. Mitotic cells (arrows); haematoxylin and eosin staining. Scale bars=20μm. (q) LIG4 upregulation in mitotic cells (arrows). CBC ISCs (arrowheads). Scale bars=20μm. (r) WBI-induced activation of β-catenin. Of note, WBI (10 Gy) rapidly induces β-catenin's localization change from the cell adhesion to the cytosol and the nucleus 1 and 24 h after WBI. Scale bars=20μm. (s,t) CD44, a β-catenin target gene, upregulation by WBI. One hour after WBI, CD44 expression is diffused. Twenty-four hours after WBI, CD44 expression is considerably upregulated in IECs at transit-amplifying zone but not in Paneth cells. IF staining (s); qRT–PCR of crypts (10 Gy, 24 h). Student's t-test; N=3; error bars=±s.e.m.; scale bars=20μm. For comparative analysis, images for each figure were captured under the same exposure time. DAPI, 4,6-diamidino-2-phenylindole.

Figure 5. Upregulation of LIG4 in CRC cells.

(a) cBioPortal analysis of the expression of NHEJ repair genes in CRC cells. Compared with the other NHEJ genes, LIG4 is highly upregulated in CRC (17%). The cancer genome atlas (TCGA) 2012; 195 cases. (b) Oncomine analysis of LIG4 expression in CRC cells. P<0.05; fold change >2. (c) Expression of LIG4 in CRC cell lines. Immunoblot analysis of LIG4 in CCD841CoN IECs and CRC cell lines. (d) Upregulation of LIG4 in CRC cells. Immunostaining of human CRC tissue microarray samples for LIG4; 3,3′-diaminobenzidine substrate staining. Human colorectal adenocarcinoma samples (#1–3). Scale bars=20μm. (e) Correlation between LIG4 and β-catenin upregulation. Immunostaining of human CRC tissue microarray for LIG4 and β-catenin (β-cat). Pearson correlation coefficient was calculated. R=0.7135; P<0.00001; scale bars=20μm.

Figure 6. Radiosensitization of CRC cells by blockade of LIG4.

(a) Depletion of endogenous LIG4 using shRNAs. HCT116 cells were stably transduced by lentiviruses encoding shRNAs against LIG4. Immunoblot assays. (b) Inhibition of cell survival by LIG4 depletion. HCT116 and SW620 (shGFP (control) and shLIG4) were treated with IR (4 Gy). Fourteen days after IR, cell survival was quantified by crystal violet staining. Student's t-test; N=3; *P<0.05; error bars=±s.e.m. (c,d) Decreased colony survival of CRC cells treated with SCR7. CRC cells were treated with SCR7 (10μM), were subjected to IR (4 Gy), and were grown for 14 days. Crystal violet staining of HCT116 cells (c) and quantification of colony survival of CRC cell lines based on optical density (d). Student's t-test; N=3; error bars=±s.e.m. (e) Radiosensitization by SCR7. CRC cells (HCT116, HT15 and SW620) pretreated with SCR7 (10μM, 24h) were treated with IR (0, 2, 4, and 8 Gy) and analyzed by clonogenic cell survival assays. Student's t-test; N=3; error bars=±s.e.m.

Figure 7. Model of Wnt/radiosensitization by SCR7.

In the normal intestine, LIG4 expression is upregulated in intestinal crypts, where Wnt signalling is active. After genotoxic stress (IR), highly proliferative CBC ISCs undergo apoptosis. By an unknown mechanism, IR activates β-catenin, which leads to the upregulation of LIG4 during intestinal regeneration.