Abstract
Background/Aim: Platinum-based drugs are the standard treatment for ovarian cancer, and platinum resistance is a major problem. A previous study has reported that the UBE2L6 expression is elevated in cisplatin-resistant cells, which in turn leads to cisplatin resistance by modulating the transcriptional expression of ABCB6. The present study aimed to investigate the expression of UBE2L6 and ABCB6 in ovarian carcinoma and to evaluate the association between these markers and platinum resistance. Patients and Methods: Ninety-two patients diagnosed with serous ovarian carcinoma (SOC) were enrolled in this study. Tissue samples were collected from these patients and analysed using immunohistochemistry to assess the expression of UBE2L6 and ABCB6. Results: UBE2L6 and ABCB6 staining was positive in 41 (44.6%) and 46 (50.0%) cases, respectively. UBE2L6 expression was statistically significantly associated with International Federation of Gynecology and Obstetrics (FIGO) stage (p=0.008). Both UBE2L6 and ABCB6 were significantly associated with platinum sensitivity (p<0.001 and p<0.001). A positive correlation was observed between the expression levels of UBE2L6 and ABCB6 (r=0.673, p<0.001). Progression-free survival (PFS) was significantly longer in the UBE2L6 negative group than that in the positive group (median PFS, 31.4 vs. 11.1 months, p<0.01) and in the ABCB6 negative group than that in the positive group (median PFS, 29.6 vs. 12.2 months, p<0.01). Conclusion: UBE2L6 and ABCB6 expression is associated with the prognosis of SOC. UBE2L6 and ABCB6 may be potential biomarkers of platinum-resistant ovarian cancer.
Platinum-based drugs, including cisplatin, carboplatin, and oxaliplatin, are used to treat various solid tumours, such as ovarian, testicular, head and neck, bladder, and lung (1, 2). Cytoreductive surgery and platinum-based chemotherapy, particularly carboplatin in combination with paclitaxel, are the current standard treatments for ovarian cancer (3). Although most ovarian cancers initially respond to these therapies, approximately 80% of cases develop platinum resistance (4). The sensitivity of ovarian cancer to platinum agents typically varies based on the histologic type, with platinum-based chemotherapy being more effective in treating serous carcinoma compared to clear cell or mucinous carcinoma (5). Due to the absence of noticeable symptoms in the early stages, ovarian cancer is usually diagnosed in advanced stages (FIGO stages III-IV) when symptoms like ascites effusion or other complications arise, which result in a poor prognosis (6). Anticancer drug therapy plays an important role in advanced ovarian cancer because it is difficult to completely remove all tumours surgically. In this regard, the acquisition of platinum drug resistance in ovarian cancer is an important issue.
Several studies have reported platinum resistance in ovarian cancer. Multiple mechanisms of resistance acquisition have been identified, such as inhibition of intracellular translocation of drugs, promotion of extracellular excretion, inactivation of DNA-platinum adducts, and promotion of nucleic acid repair systems (7, 8). Our previous study demonstrated that the ubiquitin-binding enzyme UBE2L6 and ATP-binding cassette (ABC) transporter ABCB6 are associated with cisplatin resistance at the cellular level (9). UBE2L6 expression was up-regulated in cisplatin-resistant cells, indicating that ABCB6 contributes to cisplatin resistance by regulating ABCB6 expression at the transcriptional level. However, the clinical relevance of these findings has not been established yet.
In this study, we aimed to examine the expression of UBE2L6 and ABCB6 in ovarian cancer samples and investigate the correlation between these markers and platinum resistance.
Patients and Methods
Case selection. We selected a total of 105 patients diagnosed with serous ovarian carcinoma (SOC) from the database of the Department of Obstetrics and Gynecology at the University of Occupational and Environmental Health Hospital in Kitakyusyu, Japan, between 1995 and 2015. Patients who received non-platinum anticancer agents or those who did not receive platinum agents were excluded, leaving us with a total of 92 patients. In some cases where neoadjuvant chemotherapy (NAC) was administered, surgical biopsy specimens were analysed before the start of NAC.
Clinicopathological parameters. The following clinicopathological parameters were evaluated: age at diagnosis, 2014 FIGO stage, histological grade, lymph node metastasis, and platinum sensitivity. Platinum resistance was defined as tumour progression during or within six months after the completion of the initial platinum therapy.
Immunohistochemistry. Formalin-fixed, paraffin-embedded tissue blocks from 92 SOC cases were cut into 4 μM-thick sections. The sections were then deparaffinised. Antigen retrieval was performed at 121°C for 15 min in 10 mM citrate buffer (pH 6.0). Primary antibody staining was carried out using a rabbit monoclonal antibody against UBE2L6 (Proteintech, Rosemont, IL, USA) diluted to 1:250 for 60 min at room temperature, and a rabbit polyclonal antibody against ABCB6 (Abcam, Cambridge, UK) diluted to 1:200 for 90 min at room temperature. Histofine Simple Stain MAX PO (R) (Nichirei, Tokyo, Japan) was used to visualise the secondary antibody binding. The slides were counterstained with Meyer’s haematoxylin. Immunostained areas were scored from 0 to 3 based on staining intensity. The scores were defined as follows: 0 (no staining), 1 (weak), 2 (moderate), 3 (strong). The staining scores were classified into negative (scores 0 and 1) and positive (scores 2 and 3) groups (Figure 1). Two gynaecologic oncologists, who were blinded to patient outcomes and clinical and pathologic parameters, independently analysed the scores.
Statistical analyses. Statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) (10). Clinicopathological data were evaluated using the Mann–Whitney U-test, and the strength of association between the two variables was investigated using Spearman’s rank correlation. The correlation coefficients were categorised into poor (<0.40), moderate (0.40 to 0.59), good (0.60 to 0.74), and excellent (0.75 to 1.0) based on their absolute values. Progression-free survival (PFS) was assessed using the Kaplan–Meier method and log-rank test. PFS was defined as the duration between the first treatment and objective disease progression or the last follow-up, with disease progression objectively determined through imaging studies instead of elevated tumour markers. p<0.05 was considered statistically significant.
Ethics approval. Ethical approval for this study was granted by the Review Board of the University Hospital of Occupational and Environmental Health on Ethical Issues (approval no. 030901). Informed consent was obtained from all the participants.
Results
Characteristics of patients. Ninety-two SOC patients were analysed, and the clinicopathological data are shown in Table I. The median age was 61 years (range=34-83 years), and 51.0% of the patients were over 60 years old. The majority of patients had advanced cancer (stages III and IV, 83.7%) or high-grade SOC (87.0%). Lymph node metastasis was detected in 38 (41.3%) patients. Twenty-six patients (28.2%) achieved platinum resistance after initial treatment.
Immunohistochemical staining. Of the total number of patients, 41 (44.6%) and 46 (50.0%) tested positive for UBE2L6 and ABCB6, respectively (Table II). UBE2L6 expression was found to be significantly associated with FIGO stage (p=0.008). Both UBE2L6 and ABCB6 expression were significantly associated with platinum sensitivity (p<0.001 and p<0.001), while no significant correlation was observed with histological grade (p=0.408 and p=0.542) or lymph node metastasis (p=0.926 and p=0.266). UBE2L6 and ABCB6 expression levels were moderately correlated with platinum resistance (r=0.466, p<0.001 and r=0.474, p<0.001). A positive correlation was observed between UBE2L6 and ABCB6 expression (r=0.673, p<0.001).
Prognosis. The median follow-up duration was 46 months (range=1-303 months). The median PFS was 31.4 months in the UBE2L6-negative group and 11.1 months in UBE2L6-positive group (Figure 2A). The UBE2L6-negative group had significantly longer PFS than the positive group (p<0.01). Additionally, the median PFS was 29.6 months in the ABCB6-negative group and 12.2 months in the ABCB6-positive group (Figure 2B). Similarly, PFS was significantly longer in the ABCB6-negative group than that in the positive group (p<0.01). We further assessed PFS in the advanced stage group and found that both UBE2L6 and ABCB6 was significantly associated with longer PFS in the negative group compared to the positive group (UBE2L6: median PFS 28.8 vs. 10.8 months, p<0.01; ABCB6: median PFS 26.4 vs. 11.1 months, p<0.01).
Discussion
This study is the first to establish the association between UBE2L6 and ABCB6, and their correlation with platinum resistance in ovarian cancer specimens. Our findings indicate a positive correlation between UBE2L6 and ABCB6 expression in SOC specimens. Notably, we observed that elevated levels of both UBE2L6 and ABCB6 were significantly linked with a shorter PFS in SOC patients. PFS was used as an endpoint in this study as it was unlikely to be influenced by various types of treatment apart from platinum-based drugs, unlike overall survival in ovarian cancer.
SOC accounts for more than 70% of epithelial ovarian cancers (11). Platinum sensitivity in ovarian cancer differs according to the histologic type, and SOC is more sensitive to platinum. Our study was limited to cases of SOC, for which the transition from platinum sensitivity to resistance was more easily observable. This situation makes it easier to clearly distinguish between platinum-sensitive and platinum-resistant cases.
In a previous study, UBE2L6 was found to be highly expressed in cisplatin-resistant cells by comparing gene expression profiles (9). UBE2L6-silenced cells exhibited cisplatin sensitivity, whereas the sensitivity of UBE2L6-over-expressing cells did not change. High expression of ABCB6, a gene associated with UBE2L6 expression, was detected in cisplatin-resistant cells. Additionally, ABCB6 expression was found to be down-regulated upon suppression of UBE2L6. In addition, the ABCB6 promoter activity was suppressed in UBE2L6-silenced cells. These results suggested that UBE2L6 regulates the transcriptional activity of ABCB6 and may be involved in platinum resistance. Based on the results of a previous study, we confirmed an association between UBE2L6 or ABCB6 expression and platinum drug resistance in SOC specimens.
UBE2L6 is a ubiquitin-conjugating enzyme that conjugates ubiquitin-like proteins and interferon-stimulated gene 15 (ISG15). ISG15 regulates the p53 signalling pathway and plays an important role in DNA replication in response to DNA damage (12). Several reports have been published on the association between ISG15 and cisplatin resistance. Chen et al. reported that ISG15 over-expression increases cisplatin resistance in nasopharyngeal carcinoma cells (13). However, some reports contradict this result. Huo et al. revealed that ISG15 silencing increases cisplatin resistance by activating p53-mediated cell DNA repair (14).
ABCB6 is an ABC transporter involved in resistance to various drugs by promoting its excretion out of cells (15, 16). While the exact mechanism has yet to be determined, it is hypothesised that ABCB6 may promote drug resistance by transporting molecules created by intracellular reactive oxygen species, which are induced by anticancer drugs, into the extracellular space (15). Wang et al. reported that ISG15 suppressed the translation of ABCC2, a multidrug resistance-associated protein 2, and enhanced the drug sensitivity of cisplatin-resistant ovarian cancer cells (17). Few studies have reported a relationship between ISG15 and ABC transporters in cisplatin-resistant cells. The precise mechanism by which ISGylation of p53 regulates ABCB6 expression in cisplatin-resistant cells remains unclear.
In this study, both UBE2L6 and ABCB6 expression correlated weakly with platinum resistance; however, UBE2L6 and ABCB6 were strongly correlated, which suggests that UBE2L6 expression increases ABCB6 expression in patients with SOC. Both UBE2L6 and ABCB6 were significantly associated with prolonged PFS in the negative group. This result suggests that the acquisition of platinum resistance in patients could have an impact on their prognosis.
Although our study found significant differences in PFS between UBE2L6/ABCB6 positive and negative tumours, the study is limited by the small sample size of patients evaluated. Nevertheless, our findings suggest that UBE2L6 and ABCB6 can be useful predictors of platinum resistance despite the small number of patients evaluated. Second, our study was limited to immunohistochemical analysis; therefore, it remains a semi-subjective and semi-quantitative assessment. Third, in the previous experiment, the study subjects were cervical cancer cells and cisplatin resistance was evaluated; however, the clinical specimens in this study were SOC, most of which were treated using carboplatin. Fourth, we restricted the assessment of platinum resistance in this study to patients who relapsed or progressed within 6 months after the initial platinum administration. However, it is possible to obtain a stronger correlation between platinum resistance and the expression of UBE2L6 or ABCB6 by evaluating platinum resistance after the second or subsequent platinum therapy. Therefore, our findings on the association between these markers and platinum resistance in ovarian cancer patients may be further improved with a more comprehensive evaluation of platinum resistance beyond the first therapy.
Our findings suggest that the expression of UBE2L6 and ABCB6 is associated with the prognosis of ovarian cancer and supports previous findings at the cellular level. This study suggests that UBE2L6 and ABCB6 are potential biomarkers of platinum-resistant ovarian cancer and that molecular target drugs for these markers may be effective in platinum-resistant patients.
Footnotes
Authors’ Contributions
MS and TK contributed to the conception and design of this study and collected the clinical data. MS, MM, and HH drafted the manuscript. CK, HI, and KY provided expert opinion and edited the manuscript. All the Authors have read and approved the final manuscript.
Conflicts of Interest
The Authors have no conflicts of interest to declare in relation to this study.
- Received May 13, 2023.
- Revision received May 31, 2023.
- Accepted June 1, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).