Purpose: Therapy resistance and associated liver disease make hepatocellular carcinomas (HCC) difficult to treat with traditional cytotoxic therapies, whereas newer targeted approaches offer only modest survival benefit. We focused on DNA-dependent protein kinase, DNA-PKcs, encoded by PRKDC and central to DNA damage repair by nonhomologous end joining. Our aim was to explore its roles in hepatocarcinogenesis and as a novel therapeutic candidate.

Experimental Design:PRKDC was characterized in liver tissues from of 132 patients [normal liver (n = 10), cirrhotic liver (n = 13), dysplastic nodules (n = 18), HCC (n = 91)] using Affymetrix U133 Plus 2.0 and 500 K Human Mapping SNP arrays (cohort 1). In addition, we studied a case series of 45 patients with HCC undergoing diagnostic biopsy (cohort 2). Histological grading, response to treatment, and survival were correlated with DNA-PKcs quantified immunohistochemically. Parallel in vitro studies determined the impact of DNA-PK on DNA repair and response to cytotoxic therapy.

Results: Increased PRKDC expression in HCC was associated with amplification of its genetic locus in cohort 1. In cohort 2, elevated DNA-PKcs identified patients with treatment-resistant HCC, progressing at a median of 4.5 months compared with 16.9 months, whereas elevation of activated pDNA-PK independently predicted poorer survival. DNA-PKcs was high in HCC cell lines, where its inhibition with NU7441 potentiated irradiation and doxorubicin-induced cytotoxicity, whereas the combination suppressed HCC growth in vitro and in vivo.

Conclusions: These data identify PRKDC/DNA-PKcs as a candidate driver of hepatocarcinogenesis, whose biopsy characterization at diagnosis may impact stratification of current therapies, and whose specific future targeting may overcome resistance. Clin Cancer Res; 21(4); 925–33. ©2014 AACR.

Translational Relevance

Hepatocellular carcinoma (HCC) has few treatment options and is the second most common cause of cancer death globally. This study demonstrates that an increase in DNA-PKcs, a key enzyme in DNA double-strand break repair, drives this deadly cancer. Increased DNA-PKcs copy number and expression were associated with the malignant process and predicted resistance to hepatic transarterial chemoembolization (TACE) therapy. We found that increased DNA-PK activity was an independent indicator of poor survival, and in preclinical studies, inhibition of DNA-PKcs profoundly radiosensitized and chemosensitized HCC.

Our study identifies DNA-PKcs as a candidate biomarker that potentially could be used to stratify patients for TACE therapy and identifies a novel candidate for future therapeutic inhibition to reverse radio- and chemoresistance in HCC. Targeting a “driving” process in hepatocarcinogenesis may ultimately offer significantly improved survival benefit.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer death (1). It arises on a background of chronic liver diseases, such as viral hepatitis, alcoholic liver disease (ALD), and, increasingly, non–alcoholic liver disease (NAFLD; ref. 2). Cirrhosis and advanced HCC stage at presentation in the majority of patients severely restricts both surgical and nonsurgical therapeutic options. Curative treatments are limited to patients with early cancers who are fit enough for resection, liver transplantation, or radiofrequency ablation (3). HCC resistance to conventional palliative cytotoxic agents is compounded by increased toxicity, attributed to hepatic metabolism and their reduced clearance in patients with impaired liver function. Sorafenib represents a major advance in the medical management of these patients (4), but the survival benefit is modest (4). The need to identify key drivers of hepatocarcinogenesis and chemoradioresistance, as well as biomarkers to target therapy effectively, is of paramount importance.

The most widely used palliative treatment for fit patients with intermediate stage HCC [Barcelona Clinic for Liver Cancer (BCLC) stage “B”] is transarterial chemoembolization (TACE). Its overall efficacy, however, is questionable and patient selection is key (3). Tumor-directed radiotherapy is promising (5, 6) but as yet has no proven benefit. Both radiotherapy and the cytotoxic drugs used in TACE cause DNA damage, to which the cell mounts a DNA damage response (DDR). DNA double-strand breaks (DSB) are the most cytotoxic and are repaired by two major pathways, with nonhomologous end joining (NHEJ) being the most active in both replicating and nonreplicating cells alike. Crucial to NHEJ is DNA-dependent protein kinase (DNA-PK), a heterodimeric enzyme consisting of Ku70, Ku80, and the catalytic subunit DNA-PKcs, encoded by protein kinase DNA-activated catalytic polypeptide (PRKDC). DNA-PK phosphorylates a variety of cellular proteins, including autophosphorylation of DNA-PKcs at serine2056 (7). Upregulated DNA repair activity is often evident in established cancers (8), potentially contributing to therapeutic resistance. DNA-PK inhibitors in preclinical development have been shown to sensitize human cancer cells and tumor xenografts to ionizing radiation (IR) and topoisomerase II poisons (9). NU7441 is a potent and selective DNA-PK inhibitor (in vitro IC50 = 14 nmol/L), demonstrating excellent sensitization in breast and colon cancer cell lines (10). The association between PRKDC expression and DNA-PK levels or activity in HCC is scant, but some evidence for an increase is documented (11, 12). The aim of the current study was to determine the prognostic significance of DNA-PK expression and activity in human HCC and explore the therapeutic potential of DNA-PK inhibition in vitro and in vivo.

Patient cohorts

Cohort 1 included tissues from consented consecutive patients undergoing resection or liver transplantation at three university hospitals in the United States (Mount Sinai Hospital, New York, NY) and Europe (Hospital Clínic, Barcelona, Spain, and National Cancer Institute, Milan, Italy) as described previously (13, 14). Patients with extrahepatic spread were excluded. Specific analyses are as described in Fig. 1. Cohort 2 was a case series of 45 patients (Table 1) undergoing pretreatment diagnostic biopsy, either because there was no history/evidence of associated cirrhosis or because of radiological diagnostic doubt, from a total of 632 patients managed in Newcastle between 2000 and 2010 (2). Those who did not consent to the use of their surplus tissues after diagnostic purposes were excluded. Patients were followed until June 30, 2013.

Figure 1.

Increased PRKDC in HCC and amplification at the DNA locus. PRKDC (A) and ATM (B) mRNA expression levels were analyzed in 132 human liver tissues using Affymetrix U133 Plus 2.0 arrays and expressed as fold change relative to normal liver. Tissues included normal liver (n = 10), cirrhotic liver (n = 13), low-grade dysplastic nodules (LGDN; n = 10), high-grade dysplastic nodules (HGDN; n = 8), and HCV-related HCC (n = 91). PRKDC was significantly elevated in HCC; P = 0.0007. Tumor PRKDC locus copy number was determined using the Affymetrix 500 K Human Mapping Array (C). The maximum value of paired cirrhotic samples was used as a cutoff (mean DNA copy number in 0.5 Mb around PRKDC gene locus, cutoff 2.25). D, relationship between PRKDC locus copy number and mRNA levels (Spearman rank correlation ρ = 0.6; P = 10−7).

Figure 1.

Increased PRKDC in HCC and amplification at the DNA locus. PRKDC (A) and ATM (B) mRNA expression levels were analyzed in 132 human liver tissues using Affymetrix U133 Plus 2.0 arrays and expressed as fold change relative to normal liver. Tissues included normal liver (n = 10), cirrhotic liver (n = 13), low-grade dysplastic nodules (LGDN; n = 10), high-grade dysplastic nodules (HGDN; n = 8), and HCV-related HCC (n = 91). PRKDC was significantly elevated in HCC; P = 0.0007. Tumor PRKDC locus copy number was determined using the Affymetrix 500 K Human Mapping Array (C). The maximum value of paired cirrhotic samples was used as a cutoff (mean DNA copy number in 0.5 Mb around PRKDC gene locus, cutoff 2.25). D, relationship between PRKDC locus copy number and mRNA levels (Spearman rank correlation ρ = 0.6; P = 10−7).

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Table 1.

Clinical features of 45 patients undergoing diagnostic biopsy—cohort two

Variable
Age (mean ± SE) 67.07 ± 1.7 
Sex (male/female) 39/6 
BMI (mean ± SE) 28.8 ± 1.2 
Type 2 diabetes, yes/no 24/21 
Cirrhosis, yes/no 25/20 
Etiology  
 None 18 
 ALD 
 NAFLD 
 HCV 
 AIH 
 Hemochromatosis 
 Cryptogenic 
Edmondson grade, 1/2/3 13/23/9 
Size (cm; mean ± SE) 6.3 ± 0.70 
Number (mean ± SE) 2.47 ± 0.47 
PVT, yes/no 6/32 
Extra-hepatic disease, yes/no 6/39 
INR (mean ± SE) 1.02 ± 0.02 
Albumin (g/L; mean ± SE) 37.7 ± 0.89 
Bilirubin (μmol/L) 25.7 ± 10.50 
Ascites, yes/no 5/40 
Encephalopathy, yes/no 0/44 
Child–Pugh, A/B/C 39/5/1 
BCLC, A/B/C/D 14/9/20/2 
Median survival (months) 24.60 
Variable
Age (mean ± SE) 67.07 ± 1.7 
Sex (male/female) 39/6 
BMI (mean ± SE) 28.8 ± 1.2 
Type 2 diabetes, yes/no 24/21 
Cirrhosis, yes/no 25/20 
Etiology  
 None 18 
 ALD 
 NAFLD 
 HCV 
 AIH 
 Hemochromatosis 
 Cryptogenic 
Edmondson grade, 1/2/3 13/23/9 
Size (cm; mean ± SE) 6.3 ± 0.70 
Number (mean ± SE) 2.47 ± 0.47 
PVT, yes/no 6/32 
Extra-hepatic disease, yes/no 6/39 
INR (mean ± SE) 1.02 ± 0.02 
Albumin (g/L; mean ± SE) 37.7 ± 0.89 
Bilirubin (μmol/L) 25.7 ± 10.50 
Ascites, yes/no 5/40 
Encephalopathy, yes/no 0/44 
Child–Pugh, A/B/C 39/5/1 
BCLC, A/B/C/D 14/9/20/2 
Median survival (months) 24.60 

NOTE: Continuous data are presented as mean ± SE.

Abbreviations: AIH, autoimmune hepatitis; BMI, body mass index; INR, international normalized ratio; PVT, portal vein thrombosis.

Immunohistochemistry

Using formalin-fixed paraffin-embedded tissues, HCC grading was by two pathologists (15). A detailed immunohistochemistry (IHC) protocol is described in Supplementary Methods. Briefly, antigen retrieval was with an Antigen Access Unit (A. Menarini Diagnostics). Antibodies: anti–DNA-PKcs (rabbit polyclonal, H-163; 1:500; Santa Cruz Biotechnology), anti-phosphorylated Ser2056 DNA-PKcs (rabbit polyclonal, ab20407; 1:500; Abcam), anti-ATM (rabbit polyclonal, MAT3-4G10/8; 1:800; Sigma), anti-phosphorylated Ser1981 ATM (rabbit polyclonal, AF1655; 1:300; R&D Systems). Sections were analyzed using Aperio Image analysis. Hepatocyte nuclei were identified using a modified nuclear algorithm and staining quantified in pixels after background subtraction. The selection of normal versus tumor areas was by a pathologist, whereas the application of the quantification algorithm was by supervised researchers. Both pathologists and researchers were blinded until the study endpoint.

Cell lines and in vitro assays

HCC cell lines SNU-182, SNU-475, HepG2, Hep3B, Huh7 (ATCC), and PLC/PRF/5 (ECACC) were maintained as per suppliers guidelines. All cell lines were authenticated (LGC Standards) and free of Mycoplasma contamination (MycoAlert Assay; Cambrex Bio Science). Mean change in gene expression (±SEM), using Human DNA Repair PCR Profiler Arrays (SA Biosciences; Qiagen), was expressed as ΔΔCt relative to HPRT1. Western blotting was as described previously (16). Image acquisition/densitometry was performed using a G-box chemiluminescent image analyzer (Syngene). γH2AX and RAD51 foci detection was as previously described (17). Cell survival was assessed by colony formation and automated counting, normalized to untreated control (±SEM; ref. 16). ShRNA-mediated knockdown of DNA-PKcs and subsequent analysis of double-strand break repair (DSBR) activity using the traffic light reporter system (18, 19) are detailed in Supplementary Methods.

Xenograft model

Female nude mice (CD1 nu/nu; Charles River) were maintained as previously described (16). Huh7 cells (1 × 107 in 50 μL culture medium) were implanted subcutaneously. NU7441 (10 mg/kg i.p.) and/or doxorubicin (2 mg/kg i.p.) were administered to tumor-bearing mice daily for 5 days.

Statistical analysis

Data were analyzed using SPSS statistics (version 19.0). A two-way ANOVA was used for clonogenic and immunofluorescence assays, paired t tests were used to compare tumor versus normal tissue, and a log-rank test (Mantel–Cox) and Cox proportional hazards regression were used for survival analyses.

Chemicals

Chemicals were from Sigma unless stated otherwise. Antibodies: anti-RAD51 antibody (rabbit polyclonal, sc-8349; Santa Cruz Biotechnology); Ku70 (monoclonal, ab3114), Ku80 (monoclonal, ab3107), from Abcam; actin (mouse, monoclonal, Ab-1; Calbiochem, Merck Biosciences); anti-γH2AX (monoclonal, 05-636; Millipore).

Amplification of PRKDC in HCC in association with increased mRNA levels

Expression of genes involved in the DDR was evaluated in a cohort of 132 samples (13, 14) of normal, chronically diseased, and tumor liver tissues (Fig. 1A). PRKDC was upregulated 2.4-fold in HCC relative to noncancerous liver (P = 0.0007), whereas the mRNA level of ATM (Ataxia Telangiectasia Mutated kinase), central to the DDR involving both homologous recombination repair (HRR) and NHEJ, was unchanged (Fig. 1B). The PRKDC gene locus showed copy-number gains in 55% of HCCs [56/101 samples compared with 83 paired cirrhotic hepatitis C virus (HCV)–positive samples; Fig. 1C]. PRKDC copy number correlated significantly with gene expression (Spearman rho = 0.6, P = 1 × 10−7, Fig. 1D). There was no correlation between PRKDC mRNA levels and patient outcome. In a small number of supplementary cases from the Newcastle HPB Research Tissue bank, tumor-specific PRKDC locus amplification determined by Multiplex Ligation-dependent Probe Amplification (MPLA) was associated with DNA-PKcs protein overexpression shown in Supplementary Fig. S1.

Increased HCC nuclear DNA-PKcs and treatment resistance

Nuclear DNA-PKcs protein levels assessed by IHC in paired tumor and nontumor liver from an independent cohort of 45 patients (Table 1) were scored as negative or grades one to three based on the positive pixel count (Fig. 2A). Most hepatocyte and HCC nuclei were positive, but the percentage of grade 3 nuclei was higher in tumor tissues (normal hepatocytes 33 ± 5%, versus 50 ± 5% of HCC nuclei; P = 0.001) and increased stepwise with the histological grade (Fig. 2B). The HCC DNA-PKcs level, or percentage of grade 3 nuclei, was not associated with overall survival (data not shown). In a subset analysis of patients receiving palliative doxorubicin in the form of TACE as their first-line therapy (n = 26; Supplementary Table S1), the time to radiological progression (EASL guidelines 2001; ref. 20) after the first treatment was significantly shorter in those with high DNA-PKcs (>48% HCC nuclei DNA-PKcs grade 3) compared with those with lower DNA-PKcs (median, 4.5 months vs. 16.9 months, P = 0.011, Kaplan–Meier; Fig. 2C). The high DNA-PKcs association was independent of tumor size, which was the only other factor also predictive of time to radiological progression in this selected subset of patients by univariate analysis [P = 0.034; multivariate Cox regression: DNA-PKcs grade 3+ HR 2.5, confidence intervals (CI), 1.0–6.0; P = 0.041; tumor size HR, 1.12; CI, 0.97–1.23; P = 0.12]. The BCLC stage (20), combining HCC features, liver function, and patient performance, rather than any single factor, was predictive of survival in this treated group (Fig. 2D and E). There were no differences in ATM levels between HCC and paired nontumor liver tissues.

Figure 2.

Increased DNA-PKcs protein levels in HCC and a shorter time to progression in patients receiving palliative TACE. DNA-PKcs protein levels were determined immunohistochemically in 45 paired normal and HCC paraffin-embedded tissues from cases described in Table 1. Hepatocyte nuclei were detected using an Aperio Imagescope nuclear algorithm. Level of nuclear expression was determined by pixel intensity, scored as unelevated (0, blue), low (1, yellow), moderate (2, orange), or high (3, red). Application of the algorithm is shown in two paired normal and HCC tissues (A). DNA-PK was detected in the majority of hepatocyte nuclei, and the percentage of total positive nuclei (score 1–3) is shown as a percentage of nuclei scoring 3+ within each Edmondson tumor grade (normal, n = 45; grade 1, n = 13; grade 2, n = 23; grade 3, n = 9). Grade 3+ nuclei increased significantly with tumor grade (B). Time to radiological progression (TTP) in patients receiving first-line treatment with doxorubicin TACE (patients without extra-hepatic disease, no portal vein thrombosis, Child–Pugh grade A, unsuitable for first-line radiofrequency ablation) is shown in C, where “high DNA-PKcs” cases were those with >48% HCC nuclei grade 3+ (n = 14; range, 48%–99%), compared with “low DNA-PKcs” cases, with <48% grade 3+ (n = 12; range, 0%–43%). Median time to radiological progression was 4.5 versus 16.9 months (P = 0.011, Kaplan–Meier). Survival within this selected group was not significantly associated with any individual factor, including nuclear DNA-PK level (D), but was predicted by BCLC stages A–C, combining tumor, liver function, and performance status (P = 0.017, Kaplan–Meier; E).

Figure 2.

Increased DNA-PKcs protein levels in HCC and a shorter time to progression in patients receiving palliative TACE. DNA-PKcs protein levels were determined immunohistochemically in 45 paired normal and HCC paraffin-embedded tissues from cases described in Table 1. Hepatocyte nuclei were detected using an Aperio Imagescope nuclear algorithm. Level of nuclear expression was determined by pixel intensity, scored as unelevated (0, blue), low (1, yellow), moderate (2, orange), or high (3, red). Application of the algorithm is shown in two paired normal and HCC tissues (A). DNA-PK was detected in the majority of hepatocyte nuclei, and the percentage of total positive nuclei (score 1–3) is shown as a percentage of nuclei scoring 3+ within each Edmondson tumor grade (normal, n = 45; grade 1, n = 13; grade 2, n = 23; grade 3, n = 9). Grade 3+ nuclei increased significantly with tumor grade (B). Time to radiological progression (TTP) in patients receiving first-line treatment with doxorubicin TACE (patients without extra-hepatic disease, no portal vein thrombosis, Child–Pugh grade A, unsuitable for first-line radiofrequency ablation) is shown in C, where “high DNA-PKcs” cases were those with >48% HCC nuclei grade 3+ (n = 14; range, 48%–99%), compared with “low DNA-PKcs” cases, with <48% grade 3+ (n = 12; range, 0%–43%). Median time to radiological progression was 4.5 versus 16.9 months (P = 0.011, Kaplan–Meier). Survival within this selected group was not significantly associated with any individual factor, including nuclear DNA-PK level (D), but was predicted by BCLC stages A–C, combining tumor, liver function, and performance status (P = 0.017, Kaplan–Meier; E).

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Elevated HCC DNA-PKcs activity was independently associated with poor survival

DNA-PK activity, assessed by IHC detection of pDNA-PKcss2056, was less prevalent and did not correlate significantly with levels of the native protein (data not shown). However, pDNA-PKcss2056 was elevated in tumor versus nontumor liver (71 ± 7% positive nuclei vs. 29 ± 12%, respectively, P = 0.003) and increased with histological tumor grade (P = 0.011; Fig. 3A). The median survival of patients with low tumor pDNA-PKcss2056 (<25% 3+ nuclei; n = 26) was 35 months compared with 9.9 months in those with >25% 3+ nuclei (n = 19; P = 0.007, Kaplan–Meier; Fig. 3B). HCC pDNA-PKcss2056 was independently associated with survival (P = 0.006; HR, 2.91; 95% CI, 1.37–6.17; Supplementary Table S2). ATM activity (autophosphorylation at serine1981; ref. 21) was unchanged in HCC compared with nontumor liver and was not associated with tumor grade or survival.

Figure 3.

Increased tumor pDNA-PK and poorer survival. IHC pDNA-PKcsserine2056 levels were determined by pixel intensity, scored as described in Fig. 2. Compared with total DNA-PK, fewer nuclei scored positive for pDNA-PKcsserine2056 overall. The % mean ± SEM of positive total nuclei (scores 1–3 combined) and nuclei scoring 3+ within each Edmondson tumor stage are shown in A. B, median survival of patients grouped as high (>25% 3+ nuclei, n = 19) was 9.9 months, compared with low (<25% 3+ nuclei, n = 26) tumor pDNA-PK (35 months; P = 0.007, Kaplan–Meier, P = 0.006 by multivariate Cox regression, including other tumor factors that were significant by univariate analysis: tumor size, tumor number, presence of extra-hepatic disease, presence of portal vein thrombosis).

Figure 3.

Increased tumor pDNA-PK and poorer survival. IHC pDNA-PKcsserine2056 levels were determined by pixel intensity, scored as described in Fig. 2. Compared with total DNA-PK, fewer nuclei scored positive for pDNA-PKcsserine2056 overall. The % mean ± SEM of positive total nuclei (scores 1–3 combined) and nuclei scoring 3+ within each Edmondson tumor stage are shown in A. B, median survival of patients grouped as high (>25% 3+ nuclei, n = 19) was 9.9 months, compared with low (<25% 3+ nuclei, n = 26) tumor pDNA-PK (35 months; P = 0.007, Kaplan–Meier, P = 0.006 by multivariate Cox regression, including other tumor factors that were significant by univariate analysis: tumor size, tumor number, presence of extra-hepatic disease, presence of portal vein thrombosis).

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DNA-PK expression and activity in a panel of HCC cell lines

Measurement of DDR gene mRNA levels in a panel of 6 HCC cell lines revealed high PRKDC, XRCC6 (Ku70), and XRCC5 (Ku80), with Hep3B and PLC/PRF/5 having the highest and lowest expression, respectively (Fig. 4A). Other genes involved in NHEJ (XRCC4, LIG4, and XRCC6BP1; Supplementary Fig. S2A) and ATM (Fig. 4A) were not as highly expressed. The expression of genes involved in HRR was modest, and there was no indication that nucleotide excision repair, base excision repair, or mis-match repair gene expression was altered across the panel (Supplementary Fig. S2B–S2D). Western blot analysis of DNA-PKcs, Ku70, Ku80 (Fig. 4B and C), XRCC4, Ligase 4, and ATM (Supplementary Fig. S3) revealed abundant DNA-PKcs in all cell lines. DNAP-PK function is critical to the DDR and cell survival and treatment with IR (10 Gy) induced variable activation of DNA-PK (pDNA-PKser2056) and ATM (pATMser1981; Fig. 4B and C). Irradiation did not affect the levels of Ku70, Ku80, XRCC4, or Ligase 4. To establish a relationship between DNA-PK expression and NHEJ activity, the level of DNA-PKcs was suppressed (shRNA), causing a corresponding reduction in NHEJ repair, assessed using the traffic light reporter system (18), in HuH7 cells (Supplementary Fig. S4 and Supplementary Methods).

Figure 4.

Expression of DNA-PK and related genes in HCC cell lines and inhibition of DNA-PK and ATM activity by specific inhibitors. DDR gene expression shown as ΔΔCt was determined by quantitative real-time PCR super-arrays relative to HPRT. High levels of DNA-PKcs and partners XRCC5 and XRCC6 were detected (A). Quantification of protein levels for DNA-PK (B) and ATM (C) was by Western blotting relative to β-actin, and activity was determined using phospho-specific antibodies. DNA-PK or ATM activation following 10 Gy irradiation was inhibited in each of the cell lines in the presence of 1 μmol/L DNA-PK inhibitor NU7441 (B) or 10 μmol/L ATM inhibitor KU55933 (C), respectively. Basal control activity (C) is also shown for comparison. NU7441 (gifted from Celine Cano, Newcastle University), KU55933 (from Marc Hummersone, KuDOS), and doxorubicin were dissolved in dimethyl sulfoxide.

Figure 4.

Expression of DNA-PK and related genes in HCC cell lines and inhibition of DNA-PK and ATM activity by specific inhibitors. DDR gene expression shown as ΔΔCt was determined by quantitative real-time PCR super-arrays relative to HPRT. High levels of DNA-PKcs and partners XRCC5 and XRCC6 were detected (A). Quantification of protein levels for DNA-PK (B) and ATM (C) was by Western blotting relative to β-actin, and activity was determined using phospho-specific antibodies. DNA-PK or ATM activation following 10 Gy irradiation was inhibited in each of the cell lines in the presence of 1 μmol/L DNA-PK inhibitor NU7441 (B) or 10 μmol/L ATM inhibitor KU55933 (C), respectively. Basal control activity (C) is also shown for comparison. NU7441 (gifted from Celine Cano, Newcastle University), KU55933 (from Marc Hummersone, KuDOS), and doxorubicin were dissolved in dimethyl sulfoxide.

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Effect of DNA-PK and ATM inhibitors on the repair of DNA DSBs

The effect of DNA-PK and ATM inhibitors, NU7441 and KU55933, on DSB repair was investigated in the cell lines that showed a substantial IR induction of ATM and/or DNA-PK activity. DNA DSB induction and repair by HRR were visualized by γH2AX and RAD51 foci, respectively (Fig. 5). The IR-induced rapid and substantial (5- to 10-fold) increase, then gradual decline, in γH2AX foci was followed by an increase in RAD51 foci, peaking at 24 hours, thereafter gradually declining. NU7441 slightly delayed the time to reach peak γH2AX and hindered resolution of the foci. This was particularly notable in PLC/PRF/5 cells where the foci were still 5x baseline at 24 hours (Fig. 5A). Interestingly, inhibition of NHEJ by NU7441 caused a very substantial increase in RAD51 foci (Fig. 5A, C, and E), a marker of HRR, for which ATM is presumed to be key. Contrary to expectation, the ATM inhibitor KU55933 did not suppress RAD51 foci (Fig. 5B, D, and F).

Figure 5.

Irradiation induced DSB repair, retarded by DNA-PK or ATM inhibition. PLC/PR5, Hep3B, or HuH7 cells were exposed to 2 Gy IR treatment in the absence (solid lines) or presence (broken lines) of 1 μmol/L NU7441 (A, C, E) or 10 μmol/L KU55933 (B, D, F) for 1 hour before, during, and after IR. DSBs visualized by γH2AX foci (black symbols and lines), and RAD51 foci (gray symbols and lines) as a measure of HRR, were detected immunohistochemically over a 56-hour time course and quantified using ImageJ software. Data are mean ± SEM of a minimum of three independent experiments.

Figure 5.

Irradiation induced DSB repair, retarded by DNA-PK or ATM inhibition. PLC/PR5, Hep3B, or HuH7 cells were exposed to 2 Gy IR treatment in the absence (solid lines) or presence (broken lines) of 1 μmol/L NU7441 (A, C, E) or 10 μmol/L KU55933 (B, D, F) for 1 hour before, during, and after IR. DSBs visualized by γH2AX foci (black symbols and lines), and RAD51 foci (gray symbols and lines) as a measure of HRR, were detected immunohistochemically over a 56-hour time course and quantified using ImageJ software. Data are mean ± SEM of a minimum of three independent experiments.

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Doxorubicin sensitivity, radiosensitivity, and the effect of NU7441 and KU55933

Measurement of the survival of HCC cells exposed to IR and doxorubicin (Supplementary Fig. S5) revealed that HepG2 cells, with high levels of DNA-PK and ATM, were highly radio- and chemoresistant. SNU-182 cells were the most chemosensitive, consistent with their low DNA-PK and ATM activity. SNU-182 cells grew slowly, and the more modest induction of DNA-PK autophosphorylation after irradiation may reflect different temporal kinetics of its activation. Otherwise, ATM and DNA-PK expression and activity were not critical determinants of sensitivity to IR or doxorubicin. NU7441 significantly potentiated IR-induced cytotoxicity by 3- to 40-fold and doxorubicin cytotoxicity by 2- to 50-fold (Fig. 6A–D; Supplementary Fig. S6). Inhibition of ATM by KU55933 caused an up to 44-fold radiosensitization and 10-fold chemosensitization (Supplementary Fig. S7).

Figure 6.

NU7441 potentiates chemotherapy and radiation-induced cytotoxicity and delays xenograft tumor growth. HCC cells were exposed to IR 1 or 2 Gy (A, B) or doxorubicin 10 or 100 nmol/L (C, D) and incubated for 24 hours before reseeding in drug-free medium and incubation for 14 to 21 days for colony formation. Colony forming assays with (white bars) or without (black bars) 1 μmol/L NU7441 for 1 hour before IR exposure and for 24 hours after, or DMSO (black bars) versus 1 μmol/L NU7441 (black bars) for 1 hour before doxorubicin and for 24 hours after demonstrate NU7441 potentiation, with reduced cell survival. In the absence of IR or doxorubicin, the combination of NU7441 (1 μmol/L) and KU55933 (10 μmol/L) inhibits cell survival (E). Data are plotted as mean ± SEM for a minimum of 3 independent experiments, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Mice with measurable Huh7 xenografts were treated (i.p.) with saline (control, n = 9), 10 mg/kg NU7441 (n = 9),2 mg/kg doxorubicin (n = 9), or both 10 mg/kg NU7441 and 2 mg/kg doxorubicin (n = 9) daily for five days (F). Increasing tumor volume was delayed in treatment groups compared with control animals, with the trend in the combination treatment group in keeping with modest potentiation.

Figure 6.

NU7441 potentiates chemotherapy and radiation-induced cytotoxicity and delays xenograft tumor growth. HCC cells were exposed to IR 1 or 2 Gy (A, B) or doxorubicin 10 or 100 nmol/L (C, D) and incubated for 24 hours before reseeding in drug-free medium and incubation for 14 to 21 days for colony formation. Colony forming assays with (white bars) or without (black bars) 1 μmol/L NU7441 for 1 hour before IR exposure and for 24 hours after, or DMSO (black bars) versus 1 μmol/L NU7441 (black bars) for 1 hour before doxorubicin and for 24 hours after demonstrate NU7441 potentiation, with reduced cell survival. In the absence of IR or doxorubicin, the combination of NU7441 (1 μmol/L) and KU55933 (10 μmol/L) inhibits cell survival (E). Data are plotted as mean ± SEM for a minimum of 3 independent experiments, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Mice with measurable Huh7 xenografts were treated (i.p.) with saline (control, n = 9), 10 mg/kg NU7441 (n = 9),2 mg/kg doxorubicin (n = 9), or both 10 mg/kg NU7441 and 2 mg/kg doxorubicin (n = 9) daily for five days (F). Increasing tumor volume was delayed in treatment groups compared with control animals, with the trend in the combination treatment group in keeping with modest potentiation.

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Although NU7441 or KU55933 alone had only a marginal effect on survival, the two together profoundly inhibited survival, particularly in HepG2 and Huh7 cells (Fig. 6E; Supplementary Fig. S8). The combination of these inhibitors also contributed additional chemo- and radiopotentiation compared with either alone (Supplementary Fig. S9).

The rank order of sensitivity to doxorubicin compared with IR in the cell lines was markedly different, as was sensitization by NU7441 and KU55933. This was not due to effects on doxorubicin efflux as nuclear accumulation was similar in all cells and was not increased by either NU7441 or KU55933. In contrast, verapamil, an established MDR inhibitor, increased the nuclear accumulation of doxorubicin in all cell lines (Supplementary Fig. S10).

In vivo studies

Huh7 cells, which reliably formed subcutaneous xenografts and had high DNA-PK levels and activity, were used to explore chemosensitization by NU7441 in vivo. The poor solubility of KU55933 precluded in vivo evaluation of ATM inhibition. Neither doxorubicin nor NU7441, alone or in combination, caused any significant weight loss in the mice (Supplementary Fig. S11A). Scheduled killing based on tumor burden was 7 days after treatment allocation in the control group versus 12 in the NU7441 + doxorubicin group (Kaplan–Meier, P = 0.043; Supplementary Fig. S11B). In mice treated with NU7441 + doxorubicin, the increasing tumor volume trend was modestly reduced compared with control over the study period (Fig. 6F).

Here, we show increased PRKDC mRNA expression in HCC in association with amplification of the PRKDC locus, supporting copy-number gain as a potential mechanism. Having demonstrated tumor-specific overexpression of DNA-PKcs, the protein encoded by PRKDC, in association with MPLA-defined PRKDC locus amplification in resected HCC cases, we demonstrated increased HCC DNA-PKcs in a second cohort of patients undergoing pretreatment diagnostic biopsies. Importantly, increased DNA-PKcs expression was associated with a shorter time to progression in patients receiving cytotoxic TACE therapy. These data support a role for DNA-PK in resistance of HCC to cytotoxic therapy. In all patients in the second cohort, DNA-PK activation (pDNA-PKcss2056) in the diagnostic pretreatment biopsy was independently associated with poorer patient survival. This suggests that activation of DNA-PKcs following gene amplification contributes to tumor progression. Although higher levels of genomic stress associated with inflammation and reactive oxygen species are well recognized in patients with chronic liver disease, and may predispose to carcinogenesis, recent evidence confirms even higher levels of oxidative DNA damage (22, 23) in HCC tissues. Thus, endogenous activation of DNA-PK may reflect high levels of oxidative stress–induced tumor DNA damage in a subgroup of patients with a particularly poor prognosis. DNA-PK stabilization of c-Myc (11), and promotion of genomic instability through competition with high-fidelity HRR (24), are candidate contributory mechanisms. Taken together, these data identify DNA-PK amplification and elevated expression, in the presence of either endogenous or exogenous activation, as a candidate driver of hepatocarcinogenesis or therapy resistance, respectively.

Similarly, HCC cell lines also had high levels of PRKDC mRNA and DNA-PKcs, with lower expression of other NHEJ genes and ATM, supporting a specific role for DNA-PK, rather than a general one for DDR genes, in hepatocarcinogenesis. Variation in the protein levels between the cell lines unrelated to the mRNA expression was in keeping with translation and protein stability being dysregulated in cancer (25). Neither DNA-PK nor ATM expression/activity predicted the rate of DSB repair or chemo- or radiosensitivity in the HCC cell lines, reflecting the complexity and multifactorial nature of the DDR. Nevertheless, suppression of DNA-PKcs levels in HuH7 cells reduced repair of I-SceI–induced DSBs by NHEJ. Furthermore, inhibition of DNA-PK activity retarded the resolution of DSBs, sensitizing HCC cells to the effects of doxorubicin and IR.

Both DNA-PK suppression and inhibition conferred a shift toward HRR, consistent with the hypothesis that NHEJ and HRR compete for DNA breaks (26). In contrast with previous reports in other cells (27, 28), KU55933 did not suppress IR-induced RAD51 focus formation, suggesting that ATM kinase activity is not essential for HRR in HCC. Our subsequent demonstration that the combination of NU7441 and KU55933 had a substantial impact on HCC cell survival in the absence of a DNA-damaging agent is indicative of synthetic lethality, as neither alone was significantly cytotoxic. These observations support recent studies indicating that ATM deficiency confers sensitivity to DNA-PK inhibition (29). Furthermore, the ability of NU7441 and KU55933 to chemo- and/or radiosensitize in all the cell lines was encouraging for their potential as anticancer therapeutics. Although extending these studies to the in vivo setting was hampered by the rapid growth of the tumor xenografts, the combination of NU7441 with doxorubicin did suppress the rate of xenograft growth.

These data are novel and suggest that (i) the combination of inhibitors of DNA repair pathway inhibitors may induce HCC cell death and (ii) the potentiation of tumor damage by cytotoxic agents may facilitate their use at doses much less toxic to nontumor liver, warranting further exploration. Of more immediate clinical relevance is the evidence that HCC DNA-PKcs expression by IHC in pretreatment diagnostic biopsy material predicts responsiveness to TACE. TACE treatment has been controversial, although in fit (BCLC B) patients, there is overall survival benefit (3). Here, we report a biomarker that may identify patients with more aggressive tumors, potentially with a lesser response to TACE. Because confirmatory HCC biopsy is not routinely performed, our present observational study is hampered by a small cohort size of radiologically atypical patients, as well as heterogeneity in terms of HCC stage. However, in line with the management of other nonresectable cancers, where tissue-based molecular testing guides decision making (30), we suggest that the time is approaching where biopsy characterization of HCC will be needed for better patient stratification.

A. Villanueva is a consultant/advisory board member for Bayer Pharmaceuticals. D. Newell reports receiving a commercial research grant from and is a consultant/advisory board member for Astex Pharmaceuticals. No potential conflicts of interest were disclosed by the other authors.

Conception and design: L. Cornell, J.M. Munck, L. Ogle, H.D. Thomas, D. Newell, N.J. Curtin, H.L. Reeves

Development of methodology: L. Cornell, J.M. Munck, L. Ogle, D. Televantou, H.D. Thomas, J. Jackson, N.J. Curtin, H.L. Reeves

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L. Cornell, J.M. Munck, A. Villanueva, L. Ogle, C.E. Willoughby, H.D. Thomas, J. Rose, D.M. Manas, G.I. Shapiro, N.J. Curtin, H.L. Reeves

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L. Cornell, J.M. Munck, C. Alsinet, A. Villanueva, L. Ogle, C.E. Willoughby, D. Televantou, H.D. Thomas, A.D. Burt, G. Shapiro, N.J. Curtin, H.L. Reeves

Writing, review, and/or revision of the manuscript: L. Cornell, J.M. Munck, C. Alsinet, A. Villanueva, L. Ogle, H.D. Thomas, D. Newell, D.M. Manas, G.I. Shapiro, N.J. Curtin, H.L. Reeves

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L. Cornell, D. Televantou, H.D. Thomas, N.J. Curtin, H.L. Reeves

Study supervision: L. Cornell, D. Newell, N.J. Curtin, H.L. Reeves

The authors thank Professor John Lunec and Dr. Debbie Hicks for help with MLPA analyses.

L. Cornell was supported by the patient support group, LIVErNORTH. H.L. Reeves and the creation of the Newcastle University Gastroenterology Research Tissue Bank were supported by the European Community's Seventh Framework Programme (FP7/2001-2013) under grant agreement HEALTH-F2-2009-241762 for the project FLIP. J.M. Munck, D. Televantou, H.D. Thomas, J. Jackson, and D. Newell were supported by programme grants from Cancer Research UK (CR UK) and Newcastle Experimental Cancer Medicine Center.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data