Impact of Nrf2 overexpression on cholangiocarcinoma treatment and clinical prognosis
Article information
Abstract
Purpose
Nrf2 regulates antioxidant protein expression and protects against drug toxicity and oxidative stress, whereas Keap1 controls Nrf2 activity. The Keap1-Nrf2 pathway affects the prognosis of various cancers, however, its effect on cholangiocarcinoma chemoresistance and prognosis remains unclear. This study aimed to determine whether the Keap1-Nrf2 pathway affects chemoresistance and prognosis of distal cholangiocarcinoma.
Methods
We investigated the correlation between Nrf2 and Keap1 expression and clinical characteristics and prognosis in 91 patients with distal cholangiocarcinoma who underwent curative surgery. Immunohistochemical staining was performed on paraffin blocks using primary antibodies against Nrf2 and Keap1. The relationship between Keap1 and Nrf2 protein expression levels, and clinical characteristics and prognosis was examined.
Results
Nrf2 expression was not associated with overall survival in patients who did not receive adjuvant chemotherapy (P=0.994). Among patients receiving adjuvant chemotherapy, the Nrf2 low expression group had a significantly longer median overall survival than the Nrf2 high expression group in Kaplan-Meier survival analysis (P=0.019). In multivariate analysis, high expression of Nrf2 was confirmed as an independent poor prognostic factor in the group receiving adjuvant chemotherapy (P=0.041).
Conclusion
This study suggests that Nrf2 overexpression reduces the efficacy of adjuvant chemotherapy in distal cholangiocarcinoma.
INTRODUCTION
Adjuvant chemotherapy is a crucial treatment option for decreasing the risk of cancer recurrence after surgical resection for malignancies such as cholangiocarcinoma and pancreatic cancer. Despite the availability of several chemotherapeutic agents that target different steps of cancer cell growth and proliferation, the efficacy of adjuvant chemotherapy varies depending on the type of cancer. Biliary tract cancer, including cholangiocarcinoma, are less responsive to adjuvant chemotherapy than other malignancies. Randomized controlled trials and systematic reviews of previous studies have suggested that adjuvant chemotherapy has a limited effect on extrahepatic bile duct cancer [1–3]. However, despite its limited efficacy, adjuvant chemotherapy has been demonstrated to enhance survival in some patients with biliary tract cancer [4–6].
One potential factor that may affect chemotherapy resistance and prognosis in cholangiocarcinoma is the Keap1 (Kelch-like ECH-associated protein)-Nrf2 (NF-E2 p45-related factor 2) pathway. The Keap1-Nrf2 pathway acts as a double-edged sword in cancer cells. Nrf2 activity protects cells and makes them resistant to oxidative and electrophilic stresses, whereas elevated Nrf2 activity helps in cancer cell survival and proliferation [7]. Unregulated NRF2 confers high-level resistance to anticancer drugs and reactive oxygen species and directs cancer cells toward metabolic reprogramming [8]. Therefore, Nrf2 has been studied as a potential therapeutic target molecule in cancer. This pathway plays a role in the production of antioxidant proteins that protect cells from oxidative stress and drug toxicity. However, cancer cells can hijack Nrf2 activity to support their malignant growth, making Nrf2 a potential therapeutic target [9–13]. Previous studies have demonstrated that the Keap1-Nrf2 pathway affects the prognosis of various cancers, including gallbladder cancer and pancreatic cancer [14–16]. However, the effect of the Keap1-Nrf2 pathway on chemoresistance and prognosis of cholangiocarcinoma remains unclear. Therefore, this study aimed to investigate whether the Keap1-Nrf2 pathway affects chemoresistance and prognosis in distal cholangiocarcinoma.
METHODS
This study adhered to the principles outlined in the Declaration of Helsinki. The study was approved by the Institutional Ethics Review Board of Ewha Womans University Mokdong Hospital (IRB No. EUMC 2015-07-023) and the informed consent was waived.
Patients
Of the 111 patients who underwent curative surgery for distal cholangiocarcinoma at a single center, 91 were enrolled in the study, while 20 were excluded because of distant lymph node metastasis, double primary cancer, or palliative surgery. All the included patients were histologically confirmed to have cholangiocarcinoma. Given that the objective of this study was to ascertain the correlation between Nrf2 and Keap1 expression and clinical characteristics and prognosis, the clinical data of each patient were further scrutinized. The inquiry encompassed an array of variables, such as sex, age, comorbidities (diabetes and hypertension), American Society of Anesthesiologists score, height, weight, body mass index, surgery date, discharge date, type of surgery, status of minimally invasive surgery, status of resection margin, degree of cell differentiation, lesion size, TNM stage (American Joint Committee on Cancer 7th edition). Additionally, the number of affected lymph nodes, tumor-positive lymph nodes, lymphovascular invasion, perineural invasion, preoperative chemotherapy, postoperative chemotherapy, postoperative radiotherapy, recurrence, recurrence site, death, and survival time were examined.
Surgery
In this study, two surgical methods, bile duct segmental resection and pancreatoduodenectomy, were used to treat distal cholangiocarcinoma [17]. Bile duct segmental resection involves the removal of the affected segment of the bile duct, along with regional lymph node dissection. This procedure is typically used when the tumor is localized to a small area of the bile duct and has not spread to nearby organs. On the other hand, pancreatoduodenectomy, also known as the Whipple procedure, is a more extensive surgery that involves the removal of the head of the pancreas, the duodenum, the gallbladder, and the bile duct, along with regional lymph node removal. This procedure is typically used when the tumor has spread to intrapancreatic bile duct or nearby organs. The choice of surgery depends on various factors, including the location and extent of the tumor and the overall health of the patient. We administered adjuvant chemotherapy and radiation therapy to the selected patients after surgery. The primary indication for these treatments was lymph node metastasis, followed by consideration of the patient’s age and overall health status, including underlying comorbidities. If a patient refused cancer treatment or radiation therapy, it was not administered.
Immunohistochemistry stain
Surgically resected specimens were used to create paraffin blocks containing multiple cores from both tumor and normal tissue. Immunohistochemical staining was performed on these blocks using DAKO Autostainer Plus (Agilent Technologies) and primary antibodies against Nrf2 and Keap1. Paraffin sections were deparaffinized using xylene and rehydrated with graded alcohol. Antigen retrieval was achieved by boiling the sections in 0.1 M citric acid buffer (pH 6.0) for 10 minutes using a decloaking chamber (Biocare Medical). Peroxidase was blocked for 10 minutes using peroxidase-blocking solution. The REAL EnVision Detection System, Peroxidase/DAB+ (K5007; DAKO) was used to visualize the staining. The primary antibodies used were anti-Nrf2 (sc-722; Santa Cruz Biotech, 1:100 dilution) and anti-Keap1 (10503-2-AP; ProteinTech, 1:200). All the antibodies were incubated for 30 minutes at room temperature [18].
Statistics
The degree of expression for each protein was scored by two experts who read the pathological slides for immunohistochemistry staining, and any discrepancies were resolved through consensus discussion. A 4-value intensity score (ranging from 0–3 positive) and the percentage of reactivity extent were used to determine the expression level of Keap1 and Nrf2. The respective values were multiplied together, which resulted in a possible score range from 0 to 300. This range was then categorized into two distinct levels of expression using a threshold score of 100. Scores below this threshold (<100) were classified as low or absent expression, while scores equal to or above this threshold (≥100) were classified as high expression. An analysis was then carried out to explore the relationship between the expression levels of Keap1 and Nrf2 and various clinical data. Categorical variables are expressed as numbers (percentages), while continuous variables are expressed as medians (ranges). The chi-square test and Mann-Whitney U test were used to compare categorical and continuous variables, respectively. Survival was calculated using the Kaplan-Meier method and Cox proportional hazard model from the date of surgical treatment, and differences in survival were assessed using the log-rank test. Statistical significance was set at P<0.05.
RESULTS
Demographics
A total of 91 patients with distal cholangiocarcinoma who underwent curative surgery between January 2000 and December 2012 were included in this study. The median age of the patients was 69 years (range, 47–88 years), and the median follow-up period was 23.9 months (range, 3.6–176.2 months).
We have categorized the overall patient demographics based on the administration of adjuvant chemotherapy. As a result, there was a statistically significant difference in the presence of lymph node metastasis between the group that received adjuvant chemotherapy and the group that did not. Additionally, the group that did not receive adjuvant chemotherapy was found to have a higher average age. This aligns with our institution’s approach, as mentioned in the methodology, of administering adjuvant chemotherapy to relatively younger patients who have lymph node metastasis or are in a good overall condition. There were no significant differences observed between the two groups in other variables (Table 1). And we summarized the differences among patient groups based on the expression levels of Nrf2 and Keap1 in Tables 2 and 3, respectively. The patients’ demographic characteristics showed that 31 patients belonged to the Keap1 low expression group, 60 patients belonged to the Keap1 high expression group, 51 patients belonged to the Nrf2 low expression group, and 40 patients belonged to the Nrf2 high expression group. The association between Keap1 and Nrf2 expression was not statistically significant (P=0.868). None of the patients had received neoadjuvant chemotherapy. Postoperative radiotherapy was not administered to any patients in the Keap1 low expression group, while nine (13%) patients in the Keap1 high expression group received radiotherapy (P=0.025). However, no significant differences were observed in sex, age, tumor stage, cell differentiation, adjuvant chemotherapy, resection margin status, operation time, or hospital stay (Table 2). Based on the hypothesis of our study, we assumed that the degree of Nrf2 expression could potentially impact the effectiveness of chemotherapy. Therefore, we categorized patients according to whether they had undergone adjuvant chemotherapy, and then compared the demographics while differentiating the differences in Nrf2 expression. As a result, no statistically significant differences were found in any of the categories (Table 3).
Characteristics of patients according to adjuvant chemotherapy
Characteristics of patients according to the expression of Keap1 and Nrf2
Characteristics of patients according to expression of Nrf2 with or without chemotherapy
There was no predetermined regimen for the chemotherapy, and it was administered by an oncology specialist. The types of chemotherapy varied greatly, with four patients receiving oral chemotherapy such as Xeloda, five patients receiving 5-fluorouracil (5-FU) and cisplatin, three patients treated with 5-FU and leucovorin, six patients administered with gemcitabine, one patient with cisplatin, one patient with epirubicin, and three patients treated at other hospitals for which the regimen was unknown. Given the variety of chemotherapy regimens, it was not possible to conduct an analysis of the differences in Kea1 and Nrf2 expression for each regimen.
Immunohistochemistry
Immunohistochemistry staining with Keap1 and Nrf2 specific antibodies was performed successfully for all cases by an experienced pathologist. Staining intensity and reactivity extension values were evaluated by two experts, and a consensus was reached. Cases with multiplication result of intensity score and reactivity extension value less than 100 were classified as having low or absent Keap1 and Nrf2 expression, whereas cases with multiplication result score of 100 or more were categorized as having high Keap1 and Nrf2 expression (Fig. 1).
Immunohistochemistry results of Keap1 and Nrf2 proteins (×200), with high magnification images in small squares (×400). (A) Negative Keap1 expression. (B) Low Keap1 expression. (C) High Keap1 expression. (D) Negative Nrf2 expression. (E) Low Nrf2 expression. (F) High Nrf2 expression.
Survival
For patients who did not receive adjuvant chemotherapy, there was no significant difference in overall survival between the Keap1 low expression group and Keap1 high expression group or between the Nrf2 low expression group and Nrf2 high expression group. For patients who received adjuvant chemotherapy, the median overall survival was significantly longer for the Nrf2 low expression group (31.3 months) than for the Nrf2 high expression group (21.1 months) (P=0.019) (Fig. 2). However, there was no significant difference between the Keap1 low and Keap1 high expression groups. On multivariate analysis, Nrf2 high expression was found to be an independent prognostic factor in patients with distal cholangiocarcinoma who received adjuvant chemotherapy (Table 4).
The Kaplan-Meyer overall survival (OS) analysis based on Keap1 and Nrf2 expression levels in patients with distal cholangiocarcinoma. (A, B) OS according to the expression level of Keap1 without and with adjuvant chemotherapy, respectively. (C, D) OS according to the expression level of Nrf2 without and with adjuvant chemotherapy, respectively.
Univariate and multivariate analyses using Cox regression proportional hazard model of OS after adjuvant chemotherapy in distal bile duct cancer patients
DISCUSSION
The Keap1-Nrf2 pathway is a regulatory system that modulates the activity of Nrf2, a protein responsible for controlling antioxidant expression and offers protection against drug toxicity and oxidative stress. The Keap1 protein governs Nrf2 activity, and the Keap1-Nrf2 pathway has been demonstrated to influence the prognosis of various cancers [19]. And there are bright and dark sides of Keap1-Nrf2 pathway. While it plays a protective role in normal cells by producing antioxidative enzymes and preventing malignant transformation, cancer cells often exploit this pathway to protect themselves against the oxidative stress of chemotherapy and support their malignant growth (Fig. 3) [20]. In cholangiocarcinoma, the effect of this pathway on chemoresistance and prognosis remains unclear. Nonetheless, studies have indicated that unregulated Nrf2 provides high-level resistance to anticancer drugs and reactive oxygen species, steers cancer cells toward metabolic reprogramming [10]. Keap1 deletion hastens mutant K-ras/p53-driven cholangiocarcinoma, emphasizing the complex interplay between this pathway and carcinogenesis [21].
Keap1-Nrf2 pathway modulates the production of antioxidant enzymes. There are bright and dark sides of the Keap1-Nrf2 pathway. While it plays a protective role in normal cells by producing antioxidative enzymes and preventing malignant transformation, cancer cells often exploit the pathway to protect themselves against the oxidative stress of chemotherapy and support their malignant growth.
Cholangiocarcinoma is linked to a worse prognosis compared to other cancers [22]. Prognostic factors for cholangiocarcinoma include location, margin status, vascular invasion, lymph node metastases, extension to the gallbladder, histology, gender, and serum albumin and bilirubin levels [4]. Among these factors, poor response to adjuvant chemotherapy is a primary contributor to the dismal prognosis observed in cholangiocarcinoma [2,23]. However, recent randomized controlled trials have shown that adjuvant chemotherapy can improve relapse-free survival in biliary tract cancer patients, including those with cholangiocarcinoma [24].
The findings of the present study indicated a correlation between Nrf2 overexpression and poor prognosis in patients with distal cholangiocarcinoma, with Nrf2 overexpression emerging as a significant predictor of chemoresistance. Furthermore, Nrf2 has been identified as a potential factor influencing the therapeutic efficacy of adjuvant chemotherapy following surgery. Notably, Nrf2 has been acknowledged as a potential cancer treatment target in other carcinomas, such as glioblastoma. However, the Keap1-Nrf2 pathway is crucial in normal cells, and overcoming the challenges associated with therapeutically targeting this pathway presents a considerable obstacle [25].
With advances in genetic analysis technology, precision medicine has emerged as a promising approach to the treatment of various carcinomas. However, cholangiocarcinoma presents unique challenges, owing to its heterogeneity and, diverse gross and histological features. While applying precision medicine to cholangiocarcinoma is considered difficult, recent efforts have been made to identify and target specific mutations in this cancer [26,27]. Nrf2 is a transcription factor that responds to environmental stimuli, primarily oxidative stress. Under oxidative stress, Keap1 dissociates from Nrf2, allowing Nrf2 to migrate to the nucleus and promote the production of cytoprotective enzymes. When a chemotherapeutic agent enters the cell, Keap1 dissociates from Nrf2, which fosters the translation of antioxidant and drug-transporting enzymes. This mechanism can inhibit various chemotherapeutic agents, including 5-fluorouracil and doxorubicin [12,25]. Previous studies have reported Nrf2 activity rates of 25%, 53%, and 61% in pancreatic and gallbladder cancers, respectively [14,15,28]. These rates are similar to those found in the present study.
Translational research, aimed at bridging the gap between fundamental scientific discoveries and clinical applications, serves as a crucial component in understanding and addressing chemoresistance in cholangiocarcinoma. This study suggests that the Keap1-Nrf2 pathway may play a clinically significant role in cholangiocarcinoma development. The results of this study demonstrated that Nrf2 overexpression is associated with poor prognosis in patients with distal cholangiocarcinoma. Consequently, targeting Nrf2 may enhance the chemotherapy efficacy in cholangiocarcinoma patients. These findings could contribute to the development of novel treatments targeting the Keap1-Nrf2 pathway in cholangiocarcinoma, potentially improving patient outcomes and survival rates.
Recent studies have proposed that inhibiting Nrf2 expression in vitro can augment chemotherapy effectiveness, underscoring the importance of this study as it suggests a novel approach to cholangiocarcinoma treatment [29,30]. By identifying patients who may benefit from effective chemotherapy or boost chemotherapeutic effects through Nrf2 suppression, patient survival rates can be improved. However, it is essential to note that the study’s sample size was limited, and various chemotherapy regimens were not investigated. In this study, only approximately one-fourth of the patients received anticancer treatment. However, it is worth noting that recent studies, such as the BILCAP trial, have reported the effectiveness of anticancer treatments, leading to an increasing emphasis on their use [6]. As the field of chemotherapy for cholangiocarcinoma continues to evolve and treatment options become more established, it is anticipated that future research can be conducted with more sophisticated study designs. Furthermore, there is a possibility of subjective evaluation of immunohistochemical staining results by researchers. Additionally, due to their diverse nature, antioxidative enzymes, which are products of the Keap1-Nrf2 pathway, were not analyzed in this study, complicating the assessment. Further studies considering these limitations are necessary to corroborate the findings of this study.
In conclusion, recent research on the Keap1-Nrf2 pathway has demonstrated the feasibility of selecting effective chemotherapy based on Nrf2 expression levels in patients with various cancers, indicating a new direction for carcinoma treatment by selecting patients for effective chemotherapy or by enhancing the chemotherapeutic effect through Nrf2 inhibition. This study revealed that Nrf2 expression in distal cholangiocarcinoma was similar to that in other cancers, confirming the possibility that Keap1 and Nrf2 expression is associated with chemotherapy effects.
Notes
No potential conflict of interest relevant to this article was reported.
FUNDING
None.