Abstract
Background/Aim: The incidence of hepatocellular carcinoma (HCC) associated with metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatitis C virus sustained virologic response (HCV-SVR) are increasing. However, the mechanisms driving HCC development in these patients remain unclear. This study aimed to evaluate the role of cellular senescence and RNA editing in HCC by examining cyclin-dependent kinase inhibitor 2A (p16) and adenosine deaminase acting on RNA (ADAR1) expression. Patients and Methods: HCC specimens from patients with MASLD or HCV-SVR were analyzed by immunohistochemistry to assess p16 and ADAR1 expression. Statistical analyses were conducted using the Chi-squared test, Fisher’s exact test, and Mann–Whitney U-test. Survival analyses were performed using the Kaplan–Meier method, the log-rank test, and Cox regression analysis. Results: Among 122 patients, 59 (48.4%) had MASLD and 63 (51.6%) had HCV-SVR. p16 expression was observed in 69 cases (57.0%) in the noncancerous areas and 53 cases (44.5%) in the cancerous areas. ADAR1 expression was positive in 28 cases (23.5%) in the cancerous areas and significantly associated with p16 expression in the cancerous areas (p=0.039). Patients with p16 expression in the noncancerous areas were older (p=0.045) and had elevated serum ALT levels (p=0.024). p16 expression in the cancerous areas were correlated with a shorter recurrence-free survival (HR=1.65, 95%CI=1.00-2.73, p=0.046). Conclusion: Cellular senescence and RNA editing may play a key role in MASLD- and HCV-SVR-related HCC. p16 expression in the cancerous areas may serve as a prognostic biomarker for surgical outcomes.
- Hepatocellular carcinoma
- cellular senescence
- RNA dysregulation
- metabolic dysfunction-associated steatotic liver disease
- hepatitis C virus-sustained virologic response
Since the early 2020s, substantial epidemiological and clinical shifts have been observed in the underlying liver conditions associated with hepatocellular carcinoma (HCC). Historically, chronic viral hepatitis, particularly hepatitis B virus (HBV) and hepatitis C virus (HCV), was widely recognized as the predominant predisposing cause of HCC. However, the prevalence of viral liver diseases has significantly decreased, largely due to the widespread adoption of HBV vaccination and effective treatment of HCV with direct-acting antiviral agents (DAAs) (1, 2). In contrast, non-viral factors, such as metabolic dysfunction-associated steatotic liver disease (MASLD), predisposing to HCC have risen markedly. HCC associated with MASLD, driven by metabolic disorders, such as obesity and diabetes, has been increasingly frequently reported from both Western and Asian countries (2-4).
It has been presumed that the inflammatory reaction is mild in patients with MASLD or HCV-SVR, and yet some patients still progress to develop HCC. It has been reported that advanced liver fibrosis is one of the strongest promotors of liver carcinogenesis in patients with MASLD and in HCV patients showing sustained virologic response following treatment (HCV-SVR). The annual risk of development of HCC in patients with advanced liver fibrosis is approximately 2%-5% (5-7). However, HCC has also been reported in patients with only mild liver fibrosis, emphasizing the need for identifying additional predictive markers to better assess the risk in individual patients.
Several studies have investigated the mechanisms associated with HCC development and chronic liver inflammation. The NF-kappaB signaling pathway, STAT3 activation, a lymphotoxin-beta-driven pathway, and an immunosuppressive tumor microenvironment have been recognized as key contributors (8-10). However, these mechanisms are primarily linked to active viral hepatitis. The specific pathways involved in HCC development in patients with MASLD and HCV-SVR remain unclear, warranting further investigation. In our study, we focused on cellular senescence and RNA editing, which are considered as novel liver carcinogenic factors.
Patients and Methods
Between April 2013 and September 2021, a total of 459 liver resections for HCC were performed at our department. Among these, we retrospectively included cases of HCC associated with MASLD and HCV-SVR in this study. The study was conducted with the approval of the Ethical Review Board of Dokkyo Medical University Hospital (Approval ID: R-47-7), in accordance with the Ethical Guidelines for Clinical Research established by the Ministry of Health, Labour and Welfare, Japan. Informed consent was obtained from all the subjects prior to their participation in the study.
Primary antibodies and immunohistochemistry. Specimens from cancerous and noncancerous areas of the liver were collected from the resected surgical specimens. The specimens were fixed in 10% v/v formalin, cut into blocks, and embedded in paraffin. The paraffin blocks were sectioned into 4-μm-thick slices and subjected to immunohistochemical staining (IHC). IHC was performed using the BOND-MAX automated immunostaining machine and the Bond Polymer Refine Detection kit (Leica Biosystems, Newcastle, UK). The antibody used for p16 was a mouse monoclonal antibody (MA5-17142, Invitrogen, Waltham, MA, USA), while the antibody used for ADAR1 was a rabbit polyclonal antibody (HPA003890, Atlas Antibodies, Stockholm, Sweden). After dewaxing the sliced blocks, heat-induced epitope retrieval was performed using Bond Epitope Retrieval Solution 2 (pH 9.0 for 20 min) for p16 and Bond Epitope Retrieval Solution 1 (pH 6.0 for 10 min) for ADAR1. Primary antibody incubation was performed as follows: with a 1:400 dilution of the p16 antibody for 45 min, and a 1:500 dilution of the ADAR1 antibody for 45 min.
Immunohistochemical analysis. IHC findings were evaluated using 400× magnification. p16 expression was defined as positive if nuclear staining was present in at least 50% of the cells, as shown in Figure 1A-D. As a negative control, we stained a resected specimen of intrahepatic cholangiocarcinoma from an HCV Ab-negative patient without MASLD, representing normal liver tissue without any signs of hepatitis. Similarly, ADAR1 expression was defined as positive if staining was observed in both the cytoplasm and nucleus in at least 50% of the cells, as illustrated in Figure 2A and B.
p16 expression in hepatocellular carcinoma specimens by immunohistochemistry is shown as follows: (A) negative p16 expression in cancerous area, (B) positive p16 expression in cancerous area, (C) negative p16 expression in noncancerous area, (B) positive p16 expression in noncancerous area. Magnification: 400×.
ADAR1 expression in the cancerous area is shown by immunohistochemistry as follows: (A) negative p16 expression, (B) positive p16 expression in cancerous area. Magnification: 400×.
Postoperative surveillance. Routine postoperative surveillance was conducted every three months in the patients who underwent surgery. To detect HCC recurrence, serum levels of tumor markers, such as alpha-fetoprotein (AFP) and protein induced by vitamin K antagonist II (PIVKA-II) were measured at 3-month intervals. Helical dynamic computed tomography (HDCT) was also performed every three months or when any of the tumor marker levels exceeded the normal range. After three years postoperatively, the HDCT interval was extended to every 6 to 12 months.
Statistical analysis. The associations between the findings of IHC and clinicopathological characteristics were analyzed using the Chi-square test, Fisher’s exact test, or Mann–Whitney’s U-test, as appropriate. The clinical and pathological characteristics examined included the age (≤60, 61-70, ≥71 years), sex (male/female), liver disease background (MASLD/HCV-SVR), Child-Pugh class (A/B), and preoperative values of liver function test parameters and tumor markers, such as aspartate aminotransferase (AST, IU/l), alanine aminotransferase (ALT, IU/l), albumin (g/dl), prothrombin time (PT, %), indocyanine green retention rate at 15 min (ICGR15, %), alfa-fetoprotein (AFP, ng/ml), and protein induced by vitamin K absence or antagonist (PIVKA-II mAU/ml). Tumor characteristics, such as the size of the largest nodule (cm), tumor number (≥2/1), Tumor, Node, Metastasis (TNM) stage based on the Union for International Cancer Control (UICC) 8th edition classification, and tumor differentiation grade (well/moderate/others) were also analyzed.
Survival analyses were performed using the Kaplan–Meier method, log-rank test, and univariate Cox regression analysis to investigate the association between the findings of IHC and the survival outcomes. All the statistical analyses were conducted using IBM SPSS Statistics version 29.0 software (IBM Co., New York, NY, USA). p-Values of <0.05 were considered as indicating statistical significance.
Results
Patient characteristics. We included a total of 122 patients in this study, including 97 men and 25 women. The age distribution was as follows: ≤60 years old: 11 patients (9.0%); 61-70 years, 49 patients (40.2%); >70 years, 62 patients (50.8%). Based on the liver functional reserve, the majority of patients were classified as Child-Pugh class A (113, 92.6%). Of all the patients, 24 (19.7%) had multiple HCCs.
Histopathologically, 44 tumors (36.1%) were classified as advanced-stage (>III), and 36 (29.5%) showed microvascular invasion. The degree of liver fibrosis was distributed as follows: No fibrosis (NL), 39 patients (32.0%); chronic hepatitis (CH), 56 patients (45.9%); liver cirrhosis (LC), 26 patients (21.3%) (Table I).
Preoperative background factors and pathological findings of overall cases.
Assessment of the findings of IHC analysis of the HCC specimens. p16 expression was detected in 69 cases (57.0%), while ADAR1 expression was detected in only five cases (4.1%) in the noncancerous areas. No p16 expression was detected in the normal liver tissue obtained from the intrahepatic cholangiocarcinoma specimens used as the negative control. In the cancerous areas, p16 expression was observed in 53 cases (44.5%) and ADAR1 expression in 28 cases (23.5%). Patients with ADAR1-positive tumors tended to exhibit p16 expression in the noncancerous areas as compared to those with ADAR1-negative tumors, although the difference was not statistically significant (71.4% vs. 50.5%, p=0.142, Table II). ADAR1 expression in the cancerous areas was significantly associated with p16 expression in the cancerous area (p=0.016, Table II).
Correlation between p16 and ADAR1 expression.
In one patient, no assessment of the noncancerous areas could be performed, as there were none in the specimen. Similarly, in three patients, no assessment of the cancerous areas could be performed due to tumor necrosis.
Association between IHC findings and clinicopathological characteristics. We investigated the association between the findings of our IHC analysis and the clinicopathological characteristics of the patients. Patients showing p16 expression in the noncancerous areas were more likely to be older (p=0.045) and to have elevated serum ALT (IU/l) levels (p<0.001) (Table III). However, neither p16 expression nor ADAR1 over-expression in the cancerous areas was associated with any of the clinicopathological characteristics of the patients (Table IV and Table V).
Correlation between expression of p16 in non-cancerous areas and preoperative background factors and pathological findings.
Correlation between expression of p16 in the cancerous areas and preoperative background factors and pathological findings.
Correlation between expression of ADAR1 in the cancerous areas and preoperative background factors and pathological findings.
Association between IHC findings and survival outcomes. The median follow-up period of the patients was 1,105 days (range=12-3,169 days). During the observation period, 21 patients died. The causes of death included cancer recurrence (17 patients), liver failure (2 patients), pneumonia (1 patient), and gastrointestinal perforation (1 patient). Overall, 83 patients developed tumor recurrence.
Neither p16 expression in noncancerous areas nor ADAR1 over-expression in the cancerous areas was associated with overall survival (OS) (Figure 3A and Figure 4A) or recurrence-free survival (RFS) (Figure 3B and Figure 4B). There was also no significant association between p16 expression in the cancerous areas and the OS [hazard ratio (HR)=1.97; 95% confidence interval (CI)=0.80-4.82; p=0.138] (Figure 5A). However, p16 expression in the cancerous areas was significantly associated with RFS (HR=1.65; 95%CI=1.00-2.73; p=0.046) (Figure 5B).
Association between p16 expression in noncancerous areas and post-surgical outcomes in hepatocellular carcinoma patients. Kaplan–Meier curves for overall survival (A) and recurrence-free survival (B) in patients with or without p16 expression in noncancerous areas.
Association between ADAR1 expression in cancerous areas on post-surgical outcomes in hepatocellular carcinoma patients. Kaplan–Meier survival analysis for overall survival (A) and recurrence-free survival (B) based on ADAR1 expression in cancerous areas.
Association between p16 expression in cancerous areas and post-surgical outcomes in hepatocellular carcinoma patients. Kaplan–Meier curves for (A) overall survival (A) and recurrence-free survival (B) in patients with p16 expression in cancerous areas.
Discussion
In HCC specimens obtained from patients with MASLD and HCV-SVR, p16 expression in the cancerous areas was significantly associated with ADAR1 expression in the same areas (Table II). In addition, patients with p16 expression in noncancerous areas of the specimens were more likely to be older and to have elevated serum ALT levels (Table III). These findings suggest that aging-related cellular senescence and chronic liver impairment may contribute to liver carcinogenesis through RNA dysfunction in these patients.
Finally, p16 expression in the cancerous areas was associated with a poor RFS, indicating that p16 could serve as a novel biomarker for predicting survival outcomes in patients with MASLD- and HCV-SVR-related HCC (Figure 3).
Although not statistically significant, patients with ADAR1-positive tumors were more likely to exhibit p16 expression in the noncancerous areas of the HCC specimens as compared to those with ADAR1-negative tumors (71.4% vs. 50.5%, Table II). This finding suggests a potential link between RNA editing and cellular senescence in liver carcinogenesis in patients with MASLD and HCV-SVR.
ADAR1 is an RNA-editing enzyme that converts adenosine to inosine, potentially altering protein function. RNA editing of AZIN1, mediated by ADAR1, is observed in approximately 30% of HCC cases and could contribute to liver carcinogenesis (11). Cellular senescence has been reported to impair RNA processing, inhibit RNA splicing, and reduce RNA degradation, leading to RNA instability (12-14). These disruptions may up-regulate ADAR1 expression, ultimately promoting HCC development.
Cellular senescence, mediated by p16 expression, typically suppresses carcinogenesis by inducing cell cycle arrest in response to DNA damage (15). However, our findings suggest that cellular senescence and RNA editing may serve as novel hallmarks of HCC development, particularly in patients with MASLD and HCV-SVR.
Expression of p16 in the noncancerous areas in the specimens in our study was associated not only with advanced age, but also with chronic liver function impairment (Table III). Previous studies have reported that chronic inflammation promotes cellular senescence, a phenomenon that was particularly pronounced in the liver as compared with other organs (16, 17). However, evidence exploring the association between cellular senescence and liver pathology using human specimens remains limited. Notably, in this study, liver cellular senescence was observed in 50% of the cases of HCV-SVR. Hepatitis associated with MASLD and HCV-SVR is generally considered as being mild, but the split of cases between positive and negative p16 expression may be due to differences in the disease duration.
Our findings indicate that advanced age and elevated serum ALT levels could act as surrogate markers of liver cell senescence in patients with MASLD and HCV-SVR. Interestingly, p16 expression in the noncancerous areas of the specimens in our study was not associated with pathological liver fibrosis (Table III), suggesting that cellular senescence could be useful for predicting HCC development even in patients with mild liver fibrosis.
The Japanese HCV guidelines identify advanced age, elevated serum ALT levels, and liver fibrosis as risk factors for the development of HCC following HCV-SVR. In contrast, the Japanese MASLD guidelines primarily use presence/absence of liver fibrosis alone to stratify the risk of development of HCC in MASLD patients (18, 19). Our results suggest that incorporating age and serum ALT levels into the risk stratification system may enhance the predictive accuracy for HCC development in patients with MASLD.
Previous studies have reported that ADAR1 expression in HCC tumors is associated with a worse prognosis (20-22). However, our findings showed that ADAR1 expression in the cancerous areas was not significantly correlated with the survival outcomes. In the aforementioned previous studies, active viral hepatitis predominantly characterized the liver background. This difference in liver background may explain the discrepancy in survival outcomes between our study and these previous studies. Further research is needed to clarify whether ADAR1 expressed in HCCs functions as a carcinogenic trigger or promotes tumor progression.
Unexpectedly, p16 expression in cancerous areas was significantly associated with a poorer RFS. It remains unclear whether these results indicate that the tumor has evaded cell cycle arrest or that p16 exhibits a function(s) different from its conventional role. Additionally, our results demonstrated that the p16 expression in the tumors was also associated with ADAR1 expression. This finding suggests that RNA editing may play a role in regulating p16 function in the tumors. In breast cancer, p16 expression in the tumors has been reported to promote proliferation and cell migration through activation of the IL-6/JAK2/STAT3 signaling pathway (23). Animal studies using cultured melanoma and colorectal cancer cells have also shown that p16 expression causes exhaustion and apoptosis in tumor-infiltrating CD8+ T cells, thereby reducing tumor immunity (24). Given our findings and these previous reports, we speculate that p16 expressed in HCC tumors may exhibit a function different from its conventional role.
A potential limitation of the present study was its retrospective design, and its conduct on a small cohort of patients from a single institution. Although MASLD is fundamentally distinct from HCV-SVR in terms of the pathogenesis, we were unable to analyze them separately due to the small number of subjects. We observed positive p16 expression in nearly 50% of our study subjects with MASLD/HCV-SVR; however, the odds ratios for liver carcinogenesis in MASLD and HCV-SVR remain unclear. Because our study was retrospective, it was not possible to elucidate how cellular senescence leads to RNA editing in liver carcinogenesis. Additionally, the role and significance of p16 expression in HCC associated with MASLD and HCV-SVR remain poorly understood. The subtypes of ADAR present in the liver are ADAR1 and ADAR2 (20). Previous studies have reported that in HCC, over-expression of ADAR1 promotes tumor progression, whereas ADAR2 acts as a tumor suppressor (20). Similarly, in another malignant tumor, it has been suggested that ADAR2 expression may also contribute to tumor suppression (25). In this study, we did not investigate ADAR2, and therefore, we were unable to clarify the impact of cellular senescence on ADAR2 expression. Further studies are needed to elucidate the relationship between not only ADAR1 but also ADAR2 and cellular senescence. To address these limitations, further studies on larger cohorts and with a prospective design are required in the future.
Conclusion
p16 expression in noncancerous areas in the resected specimens was observed in approximately 50% of cases of MASLD- and HCV-SVR-related HCC. Patients with p16 expression in noncancerous areas were more likely to be older and have elevated serum ALT levels. Additionally, p16 expression in cancerous areas was associated with tumor RNA editing as well as a poorer RFS.
Acknowledgements
The Authors thank the Department of Diagnostic Pathology for preparing the specimens used in our study.
Footnotes
Authors’ Contributions
Conceptualization: Shotaro Miyashita, Takayuki Shimizu, Taku Aoki. Data curation: Maiko Niki, Shun Sato, Genki Tanaka, Takamune Yamaguchi, Kwang Hwa Park, Takatsugu Matsumoto, Takayuki Shiraki, Shozo Mori. Funding acquisition: Taku Aoki. Investigation: Shotaro Miyashita, Takayuki Shimizu. Staining procedures: Shotaro Miyashita, Takayuki Shimizu. Observation of specimens: Shotaro Miyashita, Takayuki Shimizu, Taku Aoki. Project administration: Taku Aoki. Resources: Taku Aoki. Supervision: Taku Aoki. Writing of the original draft: Shotaro Miyashita.
Conflicts of Interest
The Authors have no conflicts of interests to declare in relation to this study.
Funding
This study was supported in part by 21fk0z1009250101/JAPAN Agency for Medical Research and Development (AMED).
- Received December 7, 2024.
- Revision received December 16, 2024.
- Accepted December 18, 2024.
- Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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