An endogenous DNA adduct as a prognostic biomarker for hepatocarcinogenesis and its prevention by Theaphenon E in mice

CHEMICALS AND ENZYMES

All reagents purchased were of analytical or high-performance liquid chromatography grade.

ANIMALS

The details of the study design are listed in the Supporting Information.

QUANTIFICATION OF γ-OHPdG

The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been described.28

PATIENTS AND DATABASE

Paraffin-embedded liver biopsies or HCC specimens from patients who had liver biopsies or curative resection of HCC as part of standard medical care were obtained from Georgetown University Medical Center. Informed consent was obtained from all patients under an approved institutional review board protocol (1992-048). A secure database with proper safeguards was constructed for the management of patient data. An institutional review board approved the ethical, legal, and social implications of the project.

WHOLE-EXOME SEQUENCING

Genomic DNA extraction, library preparation, and whole-exome sequencing were performed by Otogenetics (Norcross, GA). Genomic mouse DNA samples were constructed into libraries and hybridized to Agilent SureSelect Mouse All Exon 51 Mb probes (Santa Clara, CA). Paired end sequencing was executed on the Illumina HiSeq 2500 (San Diego, CA), and raw data files were pushed through the DNAnexus standard exome analysis pipeline to complete alignment, quality control, coverage analysis, and variant calling.

IMMUNOHISTOCHEMICAL STAINING

Immunohistochemical detection of γ-OHPdG in healthy and cancerous tissues was performed using tissue microarrays (HLiv-HCC060CD-01 and HLiv-HCC180Sur-02; US Biomax, Rockville, MD), paraformaldehyde-fixed, and paraffin-embedded blocks of tumor tissue from cases operated at Georgetown University. All patients gave informed consent, and the study was authorized by the respective hospital ethics committees. Immunoscores were calculated by adding intensity and region of staining: 0, 1, 2, 3 correspond to no, weak, moderate, and high intensity staining, respectively, and 0, 1, 2, 3 correspond to no, focal, regional, and diffuse region staining, respectively.

STATISTICAL ANALYSIS

Differences in adduct levels were compared using the Student t test. Fisher's exact test was used to analyze categorical data. The Kaplan-Meier method was used to analyze the survival data, and the log-rank test was used to compare the survival between different groups. A z test was employed to calculate the P values of tumor incidences. P < 0.05 was considered statistically significant. The SAS software (SAS Inc., Cary, NC; version 9.3) was used for statistical analysis.

INCREASED LEVELS OF γ-OHPdG ARE ASSOCIATED WITH HEPATOCARCINOGENESIS IN Xpa^−/− MICE

The mutagenicity of γ-OHPdG has been demonstrated using in vitro assays,29 but the evidence of its role in carcinogenesis in vivo is elusive. We first examined the association between γ-OHPdG and liver tumorigenesis in Xpa^−/− mice deficient in nucleotide excision repair (NER).30 γ-OHPdG is the only endogenous DNA adduct known to be repaired by NER.31 Xpa^−/− C57/B6 mice are a model for skin carcinogenesis by ultraviolet-B irradiation. Intriguingly, these mice also develop spontaneous liver tumors.32 We proposed that the cumulated γ-OHPdG levels, due to the lack of its repair, contribute to hepatocarcinogenesis. To further enhance the spontaneous HCC, we backcrossed Xpa^−/− C57/B6 mice with C3H/HeNCrl mice, which have a high rate of spontaneous liver cancer (Supporting Fig. S1). Compared to wild-type (WT) controls, Xpa^−/− mice not only developed a higher incidence of liver tumors but also showed significantly larger tumor sizes and increased multiplicity (Fig. 1A,B). γ-OHPdG levels in liver DNA were quantified by LC-MS/MS. The data showed that it increased age-dependently in Xpa^−/− mice (P < 0.05; Fig. 1C). Mouse gender had no influence on γ-OHPdG levels (Supporting Fig. S3). We were able to see a statistically significant difference in the hepatic γ-OHPdG levels and liver cancer development between Xpa^−/− and WT mice (Fig. 1). Unlike Xpa^−/− mice, LEC rats are inflicted with increased LPO because of abnormal copper accumulation, mimicking that of human Wilson's disease.33 As a result, LEC rats develop acute hepatitis, followed by chronic hepatitis and eventually HCC. We found that γ-OHPdG levels in the livers of LEC rats were significantly higher than those of the WT Long-Evans rats (Supporting Fig. S4). These results provide evidence that the elevated liver γ-OHPdG levels are associated with an increased risk of HCC.

GC>TA IS THE DOMINANT SOMATIC MUTATION IN HCC OF Xpa^−/− MICE

Studies have shown that γ-OHPdG causes predominantly GC>TA and GC>AT mutations.21, 22 We reasoned that the increased γ-OHPdG in the livers of Xpa^−/− mice can lead to a somatic mutation pattern in which GC>TA and GC>AT mutations are the most frequent alterations. We obtained the mutation frequencies through comparing liver tumor nodules versus adjacent normal liver tissues from two Xpa^−/− mice using whole-exome next-generation sequencing. Whole-exome sequencing produced a mean yield of 23.8 million reads or 2.5 gigabases of data per sample with 94.1% >Q30. Samples were sequenced to a mean coverage of 29X, and 99.4% of the reads were mapped to the target regions. We found 60 and 100 variants in the two liver nodules (Fig. 2A), with GC>TA mutation as the dominant alteration, accounting for 92% and 86% mutations, respectively (Fig. 2B). While examining the Sorting Intolerant from Tolerant prediction scores, we also noted that >35% of the variants in both samples were predicted as deleterious mutations (Fig. 2C). The high GC>TA mutation frequency in the Xpa^−/− mouse liver tumors implies that γ-OHPdG may play a role in the mutagenesis of HCC development. Different from other solid tumors in which CG>TA transitions are the highest variations in the mutation spectrum, with the exception of lung cancer, variants in human HCC also showed an overrepresentation of GC>TA transversion.1 We identified a number of mutant genes within mouse liver nodules that were reported in human HCC, including ABCA1, CSMD1, LAMA2, TRRAP, and TRANK1,1 suggesting that this model is relevant to human liver carcinogenesis (Supporting Table S1).

ANTIOXIDANTS SUPPRESS LIVER γ-OHPdG LEVELS AND HCC DEVELOPMENT IN Xpa^−/− MICE

To further assess the role of γ-OHPdG in HCC development, we next studied if suppression of γ-OHPdG formation in the mouse liver would inhibit hepatocarcinogenesis. Xpa^−/− mice yield 100% liver cancer incidence at the end of the 72-week bioassay. Three antioxidants (Theaphenon E, α-lipoic acid, and α-tocopherol) were used.34 To determine whether these antioxidants suppress liver γ-OHPdG levels, Xpa^−/− mice were fed diets containing antioxidants: α-lipoic acid (2 g/kg), Theaphenon E (20 g/kg), and α-tocopherol (1.8 g/kg)28, 35-37 (details of the bioassay are in Supporting Information). We observed a significant decrease of γ-OHPdG in the livers of mice fed the antioxidant diets with different potencies: Theaphenon E > α-lipoic acid > α-tocopherol (Fig. 3). No significant changes of γ-OHPdG levels were found in the lungs, a nontarget organ. We also examined the ratio of reduced to oxidized glutathione, an indicator of oxidative stress. The increases in the ratio of reduced to oxidized glutathione in the liver tissues from the mice fed different antioxidants for 32 weeks are consistent with the decreases of γ-OHPdG (Fig. 3C). We have reported in LEC rats that Theaphenon E, but not α-tocopherol, suppresses γ-OHPdG formation.28

Having demonstrated that antioxidants can suppress γ-OHPdG formation, we examined the relationships of γ-OHPdG with hepatocarcinogenesis in a 72-week lifetime tumor bioassay in Xpa^−/− mice. Mice randomized into four groups were fed diets containing antioxidants throughout the bioassay period.28, 35-37 Theaphenon E showed a strong inhibition with a remarkable reduction of tumor incidence to 14% from 100% in mice on the control diet and a significant decrease in tumor numbers and size (Fig. 4B-D). α-Lipoic acid also decreased HCC incidence but less effectively, whereas α-tocopherol had no significant effect. The potency of antioxidants to inhibit HCC showed a close correlation with their effects on γ-OHPdG levels. The protective effects of antioxidants against HCC development were similar in both male and female mice (Supporting Fig. S5), consistent with their suppressing effects on γ-OHPdG in both genders (Supporting Fig. S3). We compared γ-OHPdG adduct levels in nontumorous livers from the control and Theaphenon E groups at 72 weeks (Supporting Fig. S6). Consistent with observations at earlier time points, a significant reduction was observed in the Theaphenon E group. Although a significant decrease in body weight gain was observed in the Theaphenon E group (Supporting Fig. S6A), probably related to thermogenesis and fat oxidation caused by green tea extract,38 no food consumption difference was noted and mice in this group were leaner and healthy without any overt adverse effects (Supporting Fig. S6B).

γ-OHPdG LEVELS CORRELATE WITH DEN-INDUCED HEPATOCARCINOGENESIS IN MICE

The relationship of γ-OHPdG in HCC was further examined in C57/B6 mice involving a single injection of procarcinogen DEN. Mice develop poorly differentiated HCC nodules within 32 weeks after DEN exposure.39 A time-dependent development of steatosis was observed in DEN-injected mice fed the control diet. Malignant liver nodules were observed after 8 months (Fig. 5). To determine whether γ-OHPdG levels are associated with hepatocarcinogenesis, we measured γ-OHPdG in the livers obtained at four time intervals, shortly before and 8, 16, and 24 weeks after DEN injection, by LC-MS/MS. Similar to Xpa^−/− mice, an age-dependent increase of γ-OHPdG was seen in the livers of these mice but not in the lungs (a nontarget tissue) (Fig. 5B). These results are consistent with previous reports that DEN causes oxidative stress and induces LPO.40 Similar to the results in Xpa^−/− mice, Theaphenon E showed a remarkable suppression of HCC formation in these mice by decreasing tumor size from 10 mm to <1 mm, tumor multiplicity from 30 to 3 nodules per mouse, and incidence from 100% to 40% (Fig. 6).

γ-OHPdG AS A PROGNOSTIC BIOMARKER FOR HCC RECURRENCE IN PATIENTS

The results described above demonstrated that γ-OHPdG is closely associated with liver carcinogenesis in the animal models. To determine whether γ-OHPdG serves as a prognostic biomarker for the survival of HCC patients, we studied liver samples from a set of 90 HCC patients who underwent surgery without adjuvant therapy between August 2006 and November 2009. These patients were followed up for 4-7 years. We found that the high γ-OHPdG levels in the liver cancerous tissues were strongly correlated (P < 0.0001) with poorer survival in these patients (Fig. 7A,B). We then recruited 45 patients and examined the relationship of γ-OHPdG in the liver cancerous tissues from these patients after surgical resection with their recurrence-free survival. We did not observe a correlation between cancerous tissues and adjacent normal tissue in the levels of γ-OHPdG (Supporting Fig. S8). Patients with low γ-OHPdG tumors (immunohistochemistry score ≤3) experienced a significantly prolonged HCC recurrence-free survival compared to patients with high γ-OHPdG tumors (immunohistochemistry sore >3) (P = 0.007; Fig. 7C,D). After 2 years of follow-up from the date of curative surgical resection, the probability of no cancer recurrence in patients with low γ-OHPdG tumors was 75%, while the probability of no cancer recurrence in patients with high γ-OHPdG tumors was only 13% (P = 0.0002; Supporting Table S2). We also compared the levels of γ-OHPdG in the recurrent tumors to levels in primary HCC and found that 80% showed similar levels of γ-OHPdG compared to their primary HCC tumors (Fig. 7E). The data from the clinical studies strongly support γ-OHPdG as a biomarker for predicting the risk of human HCC recurrence.