XLF-mediated NHEJ activity in hepatocellular carcinoma therapy resistance

Abstract

Background: DNA repair pathways are used by cancer cells to overcome many standard anticancer treatments, causing therapy resistance. Here, we investigated the role of XRCC4-like factor (XLF), a core member of the non-homologous end joining (NHEJ) repair pathway, in chemoresistance in hepatocellular carcinoma (HCC).

Methods: qRT-PCR analysis and western blotting were performed to detect expression levels of genes and proteins related to NHEJ. NHEJ repair capacity was assessed in vitro (cell-free) and in vivo by monitoring the activity of the NHEJ pathway. Cell viability and IC50 assays were used to measure sensitivity to drug therapy. A xenograft HCC model was used to develop methods of targeting XLF-induced chemosensitization. Clinicopathological analysis was conducted on patients with HCC treated with transarterial chemoembolization (TACE).

Results: Many conventional cancer chemotherapeutics induce DNA double-strand breaks (DSBs). HCC cells respond to these breaks by increasing their NHEJ activity, resulting in resistance. XLF-knockdown cells show an inhibition of NHEJ activity in both cell-free and live-cell assays as well as a high level of unrepaired cellular DSBs. These results indicate that XLF facilitates DNA end-joining and therefore promotes NHEJ activity in cancer cells. Consequently, knockdown of XLF significantly chemosensitized resistant cells both in vitro and in xenograft tumors. A low rate of XLF genomic alteration was found in patients with primary HCC, but XLF expression was induced after drug treatment. Clinically, a high level of XLF expression is significantly associated with advanced HCC and shorter overall survival.

Conclusion: Chemotherapy-induced overexpression of XLF and XLF-mediated enhancements in NHEJ activity contribute to chemoresistance in HCC cells and patients with HCC. Targeting XLF to modulate DSB repair could enhance drug sensitivity and may be a therapeutically useful addition to conventional therapy.

Keywords: Chemoresistance; DNA repair; HCC; NHEJ activity; XLF.

Fig. 1

Chemosensitivity in HCC cells is associated with drug-induced increases in NHEJ activity. a Following conventional therapy regimens with the drugs cisplatin (cis), oxaliplatin (oxa) doxorubicin (dox), as well as treatment with 5-FU, DSB foci in PLC and 97 L cells were localized using an anti-γH2AX antibody. b Quantification of the percentage of γH2AX foci-positive cells. Data are reported as the mean ± SD from 2 independent experiments. c Statistical comparison of the percentage of γH2AX-positive cells between PLC and 97 L groups at 9 h and 48 h after drug treatment. A total of 1000 cells were randomly selected for counting. The mean ± SD was from 3 independent experiments. p values ≤0.05 and ≤0.01 were denoted as * and **, respectively. d In vitro NHEJ activity was assayed after oxaliplatin and doxorubicin treatment and quantified by plasmid-based quantitative PCR (see methods). NHEJ activity from 3 independent experiments was statistically analyzed using a paired Student’s t test. e HCC cells were transfected with the plasmid pEGFP-PEM1-Ad2, followed by drug treatment. In vivo NHEJ activity was calculated based on the frequency of GFP-positive cells normalized to the transfection efficiency. Drug-induced NHEJ activity was counted as a ratio of drug-induced activity versus control activity. Representative data are reported as the mean ± SD, n = 3. f PLC and 97 L cells were treated with cisplatin (1 μg/ml), oxaliplatin (1 μg/ml), or doxorubicin (0.2 μg/ml). Cell viability (%) was determined using a Cell Counting Kit 8 and standardized against cells without drug treatment. Representative data are reported as the mean ± SD, n = 3. g The therapy drug (oxaliplatin) response curve and the IC50 concentration for the PLC and 97 L cells were analyzed from 2 independent experiments using GraphPad 6.0

Fig. 2

Liver CD133+ CSCs possess high intrinsic NHEJ capacity. a Statistical comparison of drug sensitivity in CD133+ and CD133− liver cancer cells measured by cell viability assays. b Expression levels of the NHEJ genes Ku, DNA-PK, and XLF in CD133+ and CD133− HCC cells determined by qRT-PCR. Expression levels were normalized to the reference gene 18S rRNA. The data are reported as the mean ± SD obtained from 2 experiments with duplicates. c Huh7 cells were transfected with pEGFP-PEM1-Ad2 plasmid, followed by cisplatin, oxaliplatin, or doxorubicin treatment. GPF+ gating was based on CD133-PE-positive (upper part) and CD133-PE-negative (lower part) populations. Live-cell NHEJ activity is presented as the percentage of GFP+ cells within CD133+ and CD133− cell populations and was normalized to the transfection efficiency. d Statistical comparison of drug-induced NHEJ activity in vivo of c between CD133+ and CD133− cells from 2 independent experiments

Fig. 3

Knockdown of XLF chemosensitizes resistant 97 L cells by causing inhibition of NHEJ activity. a In vitro NHEJ activity was assayed and quantified in shRNA-Con and shRNA-XLF lentivirus-infected 97 L cells from 3 independent experiments. b In vivo NHEJ activity was calculated as the percentage of GFP-positive cells and normalized to transfection efficiency for the shCon and shXLF group, which were transfected with pEGFP-PEM1-Ad2 plasmid and treated with or without oxaliplatin. The data are reported as the mean ± SD, n = 4. c Quantification of the percentage of cells with <5 and >5 γΗ2ΑX foci per nucleus at 26 h after drug treatment for the siRNA-Con and siRNA-XLF groups (left panel). The data are reported as the mean ± SD, n = 2. Image of γH2AX speckles with <5 and >5 foci per nucleus (right panel). d Statistical comparison of drug sensitivity based on colony formation assay results from shCon and shXLF cells. Number of colonies is reported as the mean ± SD from 2 independent experiments. e Gene expression levels (left panel) and protein levels (right panel) of LIG4 and ERCC1 were determined by qRT-PCR and WB, respectively, in shCon and shXLF HCC cells. f Cisplatin response curves and IC50 concentrations for siCon, siXLF, siERCC1, and siXLF + siERCC1 cells, respectively. Statistical comparison was between siXLF, siERCC1, and siXLF + siERCC1 versus siCon, respectively, with treatment by cisplatin 1 or 5 μg/ml. * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 4

XLF knockdown restores drug sensitivity in HCC xenograft model. a Experimental setup used to create the xenograft model. shCon and shXLF 97 L cells (1 × 10⁶) were subcutaneously injected into nude mice. Oxaliplatin (4 mg/kg/week or 10 mg/kg/week) was administered to the mice by intra-peritoneal injection. b Tumor volume was reported in mm³ as the mean ± SD and statistically compared between shCon and shXLF tumors. c Representative images of xenograft tumor. d In vitro NHEJ activity in nuclear proteins extracted from xenograft tumor tissue. The statistical comparison was based on the mean ± SD from 3 independent experiments

Fig. 5

Clinical significance of XLF expression. a Genomic alteration frequency for XLF was calculated using cBioPortal [http://www.cbioportal.org] from the TCGA (193 patients) and AMC (231 patients) databases. Tumor types are indicated at the bottom and ordered by the frequency of samples harboring mutation, deletion, amplification and multiple alterations. The XLF mutation rate in HCC is highlighted. b Statistical comparison of XLF gene expression in tumor tissue between patients with HCC who underwent TACE (n = 31) and those who did not (n = 22). c Kaplan-Meier analysis of overall survival between patients with HCC with high and low expression of XLF, who all underwent TACE treatment. d Kaplan-Meier analysis of disease-free survival for patients with HCC as in c