LIG4 mediates Wnt signalling-induced radioresistance

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.


Supplementary Figure 1. Isolation of GFP High cells.
(A) Schematic diagram of cell isolation based on Wnt signaling activity. Colorectal cancer (CRC) cell lines were stably transduced with lentivirus encoding ionatenin reporter (7TGP). After selection, cells were processed for fluorescenceactivated cell sorting (FACS), based on GFP expression. Parental HCT116 cells (left panel) served as negative controls for cell sorting. Scale bars = 100 μm.
(B and C) Isolation of GFP High cells from CRC cells. After FACS, HCT116-7TGP cells were further cultured. GFP High (B) and GFP Low cells (C). Scale bars = 50 μm. (D) Increased β-catenin expression in GFP High cells. Immunofluorescent (IF) staining of SW620-7TGP cells. Scale bars = 20 μm. Arrows: cells highly expressing β-catenin. (E and F) Upregulation of βcatenintarget genes in GFP High cells. IF staining of SW620-7TGP cells for βcatenin (D), CD44 (E), and CD133 (F). Scale bars = 20 μm. Arrows: cells highly expressing CD44 or CD133. (G and H) Increased sphere formation of GFP High cells. GFP High and GFP Low HCT116-7TGP cells were grown in low attachment condition for 14 days. Images (G) and quantification of the number and size of spheres (H). Scale bars = 200 μm; Student's ttest; N = 3. Error bars = SEM.

Supplementary Figure 2. No difference in cell proliferation between GFP High and GFP Low cells
HCT116-7TGP cells were sorted based on GFP expression using FACS. 5,000 cells were plated and counted at day 1, 3, and 5, using Biorad cell counter (TC10). Student's t-test; N = 3. Error bars = SEM.

Supplementary Figure 5. Validation of TCE cassette
Prior to generating TERTTCE knock-in mESCs and mouse model, we characterized tdTomato-CreERT2 (TCE) cassette. (A) Illustration of TCE fusion protein. pA: poly (A) signal. (B and C) Nuclear translocation of TCE by 4-hydroxy-tamoxigen (4OHT) treatment. HeLa cells were transiently transfected with TCE-pcDNA3.1 plasmid. 24 hours after transfection, cells were treated with 100 μM 4OHT (in ethanol) and analyzed for IF staining of TCE (tdTomato red fluorescent signal). 24 hours after 4OHT treatment, TCE was completely localized in the nucleus. At 48 hours after 4OHT treatment, the cytosolic TCE was also observed (B). However, tdTomato (a negative control) displayed subcellular localization both in the nucleus and in the cytosol, regardless of 4OHT treatment (C). Figure 6. Establishment of TERT TCE genetically engineered mouse model (A) Gene targeting (knock-in) strategy. We constructed targeting vector containing 5' homologous arm, tdTomato-CreERT2-pA, Neo cassette (for positive selection) flanked by FRT sites, 3' homologous arm, and diphtheria toxin A (DTA) (for negative selection) cassette. Of note, TCE cassette was inserted into TERT allele in-frame. Linearized targeting plasmids were electroporated into G4 mESCs to generate TERT TCE-Neo mESCs. mESCs were injected into the blastocysts to generate chimeric mice. Mice confirmed for germ line transmission were further bred with FLPeR deletor strain to remove Neo selection cassette (TERT TCE strain). (B) Genotyping of TERT TCE strain. For genotyping, insertion of 5' and 3' homologous arms were confirmed by PCR of genomic DNA of TERT TCE-Neo mESCs. Clones #1, 8, and 10 were selected for blastocyst injection.

Supplementary Figure 7. Change in cell adhesion components by radiation
(A) Localization change of p120-catenin by radiation. Mice were treated with WBI (10 Gy). 24 hours later, mouse small intestine samples were collected for immunostaining for p120-catenin (Cell Signaling). Of note, in normal intestine, p120-catenin is specifically associated with E-cadherin. However, WBI-treated small intestine displays the cytosolic and the cell adhesion-associated p120-catenin.
(B and C) The decrease of E-cadherin by WBI. Untreated and WBI (10 Gy)-treated mouse small intestine samples were subjected to immunostaining of E-cadherin (B). Using two different antibodies for E-cadherin detection similarly exhibited the significant decrease of E-cadherin by WBI. Consistently, immunoblotting of intestinal epithelial cells isolated from mouse small intestine (control and WBI) also showed the decreased level of E-cadherin (C). Representative images were shown. All images were captured under the same exposure time for comparison. Scale bars = 20 μm.