Abstract
Background/Aim: Sulphite oxidase (SUOX) is a metalloenzyme that catalyses ATP synthesis via oxidative phosphorylation in the mitochondria. Although SUOX has been reported to affect the invasiveness and differentiation of cancer cells, its clinicopathological significance in pancreatic adenocarcinoma (PDAC) remains unclear. In this study, we investigated the utility of SUOX expression as a prognostic factor in PDAC. Patients and Methods: This study included 56 patients with PDAC who underwent pancreatic resection at the Kurume University Hospital between 2014 and 2018. SUOX immunohistochemistry was evaluated using tissue microarray specimens from patients. Patients were classified into a high SUOX expression group (≥10% of cells stained) or a low SUOX expression group (<10% of cells stained), and the associations of SUOX with clinicopathological characteristics and survival were analysed. Statistical analysis was performed using Cox regression analysis, the Kaplan–Meier method, and log-rank test. Results: SUOX was expressed in the cytoplasm of normal pancreatic ductal epithelium, pancreatic acinar cells, and islets of Langerhans. Although we did not find any significant correlation between SUOX expression and clinicopathological factors, SUOX was identified as an independent prognostic factor based on univariate and multivariate analyses. Pathological stage was also an independent prognostic factor. The high SUOX expression group showed a significantly poorer prognosis than the low SUOX expression group (p=0.018). Conclusion: SUOX-mediated mitochondrial metabolism in PDAC may be a factor influencing prognosis and SUOX may be a potential novel prognostic biomarker.
Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis among gastrointestinal malignancies and the percentage of patients diagnosed with PDAC has been gradually increasing in recent years (1-3). In PDAC cases, surgical resection is often difficult because metastasis or dissemination has occurred in many cases at initial diagnosis (2). However, since surgical resection is the first-line treatment for PDAC, the prognosis does not improve in many cases. In recent years, adjuvant multi-agent chemotherapy has been recognized as a standard treatment for advanced PDAC and has improved prognosis (2, 4, 5). However, the results are not yet satisfactory. In fact, the 5-year survival rate is extremely low (2, 6). Many studies have reported that metastasis and carbohydrate antigen 19-9 (CA19-9) levels are predictors of clinical prognosis (7-9). However, it is also essential to search for novel biomarkers for prognostic prediction.
PDAC is a neoplastic lesion arising from the pancreatic ductal epithelium and is mainly caused by mutations in KRAS, TP53, P16, and SMAD4/DPC4 (10); various signalling pathways have also been implicated in tumorigenesis (11). Moreover, a comprehensive analysis of RNA expression in PDAC revealed that the expression of many genes affects the prognosis of PDAC (12). Among them, sulphite oxidase (SUOX), an enzyme involved in mitochondrial function related to mitochondrial function, was shown to be a poor prognostic factor in PDAC (12).
The mitochondrial enzyme SUOX oxidizes sulphite that oxidizes sulphite to sulfuric acid, which is then used to drive ATP synthesis via oxidative phosphorylation. SUOX is a molybdenum cofactor (Moco)-dependent oxidoreductase located within the intermembrane space of mitochondria where it catalyses the oxidation of toxic sulphite to sulphate (13). Functional SUOX requires Moco and a cytochrome b5-type haem cofactor (14). An inability to oxidize sulphite results in isolated sulphite oxidase deficiency (15). However, there are few papers on the role of SUOX in cancer cells (16-19). In prostate cancer, a high SUOX expression group had a significantly earlier biochemical recurrence compared to a low SUOX expression group and showed a correlation with Ki-67 levels in immunohistochemistry (IHC) analysis and cell proliferation, indicating that SUOX affects cell proliferation (16). In contrast, low expression of SUOX in hepatocellular carcinoma (19) and tongue squamous cell cancer (17) has been related to tumour differentiation. In gastric adenocarcinoma, relatively low expression of SUOX was associated with a poor prognosis (18). As described above, SUOX IHC staining in various carcinomas showed that high and low SUOX expression play different roles in different developmental organs, and that SUOX activation requires correct homodimerization and mitochondrial maturation (20-22), suggesting that the activity of SUOX may be different in different organs.
In this study, we evaluated the expression pattern of SUOX in PDAC and normal pancreatic tissues and investigated the role of SUOX as a prognostic factor in PDAC.
Patients and Methods
This was a retrospective cohort study using clinicopathological data of patients with PDAC from the Kurume University Hospital between 2014 and 2018. This study was approved by the Ethics Committee of the Kurume University (approval #18068). The pathological diagnoses of patients were re-examined, and all patients were pathologically diagnosed with PDAC. The exclusion criteria were as follows: 1) chemotherapy and/or radiotherapy before surgery, 2) PDAC with a special subtype, and 3) specimens could not be created for tissue microarray (TMA). Paraffin-embedded PDAC samples from 56 patients were used to construct a TMA containing 22 cores (two primary tissue cores per patient). All slides were consecutively sectioned to a 4 μm thickness and stained with haematoxylin and eosin. Histopathological evaluations were performed by two pathologists (Y.N. and N.K.). Histological diagnosis was performed according to the 2019 World Health Organization classification. Microvessel invasion (MVI) and neural invasion (NI) in the main section were assessed as mild grade (defined as £2) and severe grade (defined as ³3).
IHC staining. Paraffin-embedded tissue samples were sectioned at a 4 μm thickness, placed on a coated slide glass, and labelled with SUOX antibodies (×600, Abcam, Cambridge, MA, USA) using the BenchMark ULTRA (Ventana Automated Systems, Inc., Tucson, AZ, USA). Briefly, the slide was heat-treated using the Ventana’s ULTRA cell conditioning 1 retrieval solution (Ventana Automated Systems, Inc.) for 36 min at 95°C and then incubated with the SUOX antibodies for 32 min at 37°C. The automated system used the Ventana UltraVIEW DAB detection kit with incubation with the horseradish peroxidase-multimer secondary antibody for 30 min at 37°C followed by the addition of 3,3’-diaminobenzidine as the chromogen. We defined only cytoplasmic expression of SUOX as positive, and the percentage of positively stained neoplastic cells was also calculated and graded as follows: low expression, <10% of the neoplastic cells were stained; and high expression, ≥10% of the neoplastic cells were stained. These evaluations were conducted by two pathologists (Y.Y. and Y.K.). Normal pancreatic acinar cells and islets of Langerhans were used as positive controls. SUOX expression was also observed in pancreatic ductal epithelial cells (Figure 1).
Sulphite oxidase (SUOX) expression in pancreatic tissues. SUOX immunohistochemistry showing the expression of SUOX in normal pancreatic ductal epithelium (arrow), acinar cells (star), and islets of Langerhans (arrow head).
Statistical analysis. Data are presented as medians (interquartile ranges [IQRs]) or numbers (percentages). Clinicopathological variables were compared using the Wilcoxon rank-sum or Fisher’s exact tests. Overall survival (OS) was defined as the time from surgery to death from any cause. The survival function for OS was estimated using the Kaplan–Meier method. The log-rank test was used to compare differences in survival rates according to clinicopathological variables. A Cox regression model was used to evaluate the effect of SUOX expression while adjusting for potential confounding factors. All statistical analyses were performed using R version 4.1.2, and statistical significance was set at p<0.05.
Results
Patient characteristics and IHC staining. The clinicopathological characteristics of patients are summarized in Table I. PDAC with well or moderate differentiation was observed in 48 of 56 patients (85.7%). Severe MVI was observed in 22 patients (39.3%) and severe NI in 22 patients (39.3%). The pathological stage (pStage) was IA, IB, or IIB in 48 (85.7%) patients. The median follow-up time was 29.50 months (IQR=16.00, 38.75). Of the 56 patients, 13 patients (33.3%) had high SUOX expression, and 43 patients (66.7%) had low SUOX expression (Figure 2). Correlations between SUOX expression and clinicopathological characteristics are summarized in Table II. No correlation was observed between SUOX expression and any clinicopathological characteristic.
Clinicopathological findings of 56 patients with pancreatic ductal adenocarcinoma (PDAC).
Sulphite oxidase (SUOX) expression in pancreatic ductal adenocarcinoma (PDAC). SUOX was expressed in the cytoplasm of tumour cells. High SUOX expression (A, B) was defined as expression in ≥10% of the population of tumour cells, and low SUOX expression (C, D) was defined as expression in <10% of the tumour cell population. (A, C): H&E staining; (B, D): SUOX staining.
Correlation between sulphite oxidase (SUOX) expression and clinicopathological characteristics in 56 patients with pancreatic ductal adenocarcinoma (PDAC).
SUOX is a biomarker for prognosis of PDAC. Kaplan–Meier curves demonstrated that OS was significantly shorter in patients with high SUOX expression than in those with low SUOX expression (p=0.018) (Figure 3A). In particular, less than half of the patients in the high SUOX expression group survived for more than 2 years. In addition, pStage was found to be a prognostic factor of OS. Patients with pStage III had worse OS than those with lower stages (p=0.036; Figure 3B). The results of the univariate and multivariate analyses of OS are shown in Table III. Based on the univariate analysis, SUOX expression [high vs. low: hazard ratio (HR)=2.604, 95% confidence interval (CI)=1.144-5.924, p=0.023] and pStage (I/IIB vs. III: HR=2.815, 95% CI=1.025-7.731, p=0.045) were significant predictors of OS. Based on multivariate analysis, SUOX expression (high vs. low: HR=2.623, 95% CI=1.146-6.006, p=0.023) and pStage (I/IIB vs. III: HR=2.848, 95%CI=1.024-7.920, p=0.045) were independent poor prognostic factors of OS in PDAC.
Kaplan–Meier curves of sulphite oxidase (SUOX) and pathological stage (pStage) in pancreatic ductal adenocarcinoma (PDAC). Kaplan–Meier curves showing the overall survival in patients based on SUOX expression (A) and pStage (B).
Univariate and multivariate analyses of overall survival (OS) of 56 patients with pancreatic ductal adenocarcinoma (PDAC).
Discussion
In this study, we performed a clinicopathological investigation of SUOX expression in PDAC. SUOX was expressed in the pancreatic ductal epithelium, but many PDAC tissues exhibited low expression. The high SUOX expression group had a significantly poorer prognosis than the low SUOX expression group, and SUOX was determined to be a prognostic factor based on both the univariate and multivariate analyses. These results are consistent with those of a previous comprehensive study using mRNA, which showed that high SUOX expression correlated with a poor prognosis in patients with PDAC (12). Overall, these results suggest that the mitochondrial function of tumour cells in PDAC is an important factor affecting patient prognosis.
SUOX is a mitochondrial enzyme involved in energy production (13). Mitochondria are responsible for supplying energy and building materials for new cells and also influence redox homeostasis, cancer signalling, innate immunity, and apoptosis regulation (23). Mitochondrial biogenesis and quality control are often enhanced in cancer cells. However, cancer cells preferentially carry out glycolysis, even in conditions of sufficient oxygen, resulting in lower mitochondrial activity. It is now believed that cancer cells do not have impaired mitochondrial function, although there are changes in biochemical metabolism, such as enhanced aerobic glycolysis (20). The mitochondria may also play important roles in PDAC. Mitochondrial iron-dependent immunometabolism influences carcinogenesis of PDAC, and mitochondria are also thought to play important roles in chemoresistance in PDAC by affecting apoptosis, metabolism, mtDNA metabolism, and mitochondrial dynamics (24). Interestingly, SUOX expression is low in hepatocellular carcinoma and tongue carcinoma in poorly differentiated tumours (17, 19). In the present study, although SUOX expression was observed in the normal pancreatic ductal epithelium, it was low in many PDAC tissues. Low SUOX expression has also been observed in many cases of prostate cancer, including PDAC (16). Therefore, low expression of SUOX may indicate that the homeostasis of mitochondrial function is lost due to carcinogenesis.
In this study, high SUOX expression was an independent prognostic factor for PDAC. In particular, less than half of the patients in the SUOX high expression group survived for more than 2 years. This result is consistent with the poor prognosis of individuals in the high SUOX expression group, and the results of our previous comprehensive RNA microarray analysis (12). However, SUOX expression did not correlate with the degree of tumour differentiation. Thus, the metabolic function of mitochondria in PDAC cells can be considered as only a single factor affecting prognosis. However, there is also the possibility of compensatory changes in mitochondrial dysfunction as a result of cell carcinogenesis. This consideration requires a molecular pathology study of mitochondrial function. It is also interesting to note that the role of SUOX expression is different in different organs. High SUOX expression was determined to be associated with a poor prognosis in prostate cancer in an earlier study (16) and in PDAC in our study, whereas low expression was associated with a poor prognosis in gastric cancer (18). The reasons for these differences could be the differences in the surrounding environment of the cancer cells, or to the unique functions of each cancer cell. Some tumours, such as oncocytomas, are associated with defective mitochondria with pathogenic mtDNA mutations. Although the effects of mitochondrial hyperactivity are often unclear, there are two classes of mitochondria-rich oncocytic tumours: benign tumours with mitochondrial defects and malignant tumours with intact mitochondrial function (23). This is consistent with the fact that mitochondria play a role in promoting tumours. For example, the pattern of SUOX expression in many carcinomas, including PDAC, suggests that there may be tumours with normal mitochondrial function and tumours with loss of mitochondrial function. In fact, SUOX expression is reportedly low in poorly differentiated hepatocellular carcinoma and tongue carcinoma (17, 19). Therefore, the molecular pathological role of SUOX in carcinomas requires further investigation.
This study has certain limitations. First, although SUOX is a prognostic factor, it is not related to other clinicopathological factors. There are many studies on independent prognostic factors in PDAC, including clinicopathological factors such as surgical margins (25), lymph node metastasis (26), NI (27), MVI (28), and CA19-9 levels (29). However, SUOX expression is not correlated with these factors. Since SUOX is related to mitochondrial function, it is expected to have little relationship with clinicopathological factors. Therefore, it is necessary to study the relationship between SUOX and other factors related to mitochondrial function in the future. Second, the number of high-expression cases was small. Although the clinicopathological characteristics may not be fully understood owing to the small number of high SUOX-expression cases, there was a tendency for low SUOX-expression cases of prostate cancer as well (16). This suggests that there are characteristics of SUOX expression specific to the type of carcinoma, and the associated mechanism needs to be clarified.
In this study, we found that SUOX was expressed in pancreatic ductal epithelium, pancreatic acinar cells, and islets of Langerhans, although most of the PDAC tissues showed low expression. In contrast, there was a small number of patients with high SUOX expression, indicating that SUOX expression is an independent poor prognostic factor. The mitochondrial function of SUOX, as an enzyme involved in driving ATP synthesis by oxidative phosphorylation in PDAC, has an important influence on the prognosis had an important influence on the patient prognosis in this study, although mitochondrial function in PDAC cells remains unclear. This study focused on the association between clinicopathological factors and SUOX. Future studies are needed to investigate the molecular and biological role of SUOX in mitochondrial function in PDAC.
Acknowledgements
We would like to thank Editage (www.editage.com) for English language editing.
Footnotes
Authors’ Contributions
YN, JA, and YK designed the experiments; YN, YK, TO, MN, and MT performed the experiments; ES analysed the data; YO, HS, TH, and YA helped with the discussion; YN and JA. wrote the manuscript; and HY supervised the project. All Authors have reviewed the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received May 20, 2022.
- Revision received June 6, 2022.
- Accepted June 30, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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