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ORIGINAL ARTICLE
Year : 2021  |  Volume : 17  |  Issue : 3  |  Page : 771-776

Overexpression of replication protein A3 is associated with unfavorable outcome in bladder urothelial carcinoma


1 Department of Urology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, China
2 Department of Urology, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China
3 Department of Radiotherapy, Shandong Second Provincial General Hospital (Shandong Provincial ENT Hospital), Jinan, Shandong, China

Date of Submission18-Feb-2021
Date of Acceptance18-Apr-2021
Date of Web Publication9-Jul-2021

Correspondence Address:
Jinhua Wang
Department of Radiotherapy, Shandong Second Provincial General Hospital (Shandong Provincial ENT Hospital), Jinan, Shandong 250021
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.jcrt_275_21

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 > Abstract 


Purpose: The replication protein A3 (RPA3) is a subunit of the RPA protein complex, which plays an essential role in multiple processes of DNA metabolism. However, the involvement of RPA3 bladder urothelial carcinoma (UC) prognosis has not yet been elucidated. The aim of our study is to investigate the prognostic role of RPA3 expression in patients with bladder UC.
Materials and Methods: Bladder UC tissue specimens from 155 consecutively treated patients who underwent surgery between 2013 and 2018 were evaluated. The RPA3 expression was determined by immunohistochemistry, Western blot, and correlated with clinicopathological parameters. The prognostic significance of RPA3 expression was explored using the univariate and multivariate survival analysis of 155 patients who were followed.
Results: A total of 155 tissue specimens “of patients” who were regularly followed with the mean 39.6 months (from 4 to 71 months). The expression of RPA3 was significantly associated with tumor grade (P = 0.031) and stage (P = 0.021), as well as tumor size (P = 0.034). In univariate analysis, RPA3 overexpression showed an unfavorable influence on recurrence-free survival with statistical significance (P < 0.01). TNM stage and grade also showed strong statistical relation with adverse recurrence-free survival (P < 0.01, P = 0.030). Multivariate analysis revealed that grade, stage, and RPA3 reactivity (P = 0.025, P < 0.01, P = 0.016) were identified as independent prognostic factors for recurrence-free survival in patients with bladder UC.
Conclusions: These results of this study proved that elevated expression of RPA3 was associated with worse clinical outcome in bladder UC patients. This finding suggested that RPA3 served as a potential prognostic biomarker, which could be useful to predict cancer evolution and may represent a novel therapeutic target for the intervention of bladder UC patients.

Keywords: Bladder urothelial carcinoma, prognosis, recurrence, replication protein A 3


How to cite this article:
Sun D, Zhang K, Fu Q, Zhang H, Liu S, Wang H, Xu Z, Wang J. Overexpression of replication protein A3 is associated with unfavorable outcome in bladder urothelial carcinoma. J Can Res Ther 2021;17:771-6

How to cite this URL:
Sun D, Zhang K, Fu Q, Zhang H, Liu S, Wang H, Xu Z, Wang J. Overexpression of replication protein A3 is associated with unfavorable outcome in bladder urothelial carcinoma. J Can Res Ther [serial online] 2021 [cited 2021 Jul 29];17:771-6. Available from: https://www.cancerjournal.net/text.asp?2021/17/3/771/321028




 > Introduction Top


Bladder urothelial carcinoma (UC) is the fifth most common malignancy worldwide with frequent recurrence and high mortality.[1] Prognostication and risk assessment are essential for treatment decision-making, patient counseling, and inclusion in clinical trials.[2] Currently, besides the clinical factors (grade and stage), a number of markers were expected to predict the prognosis of bladder UC, but few of them have been recognized as reliable methods.[3],[4],[5],[6] Thus, it is urgent to investigate effective markers that can accurately predict the prognosis of bladder UC with a high risk of recurrence and contribute to the development of anticancer target.

Replication protein A (RPA) is a single-stranded DNA-binding protein composed of three subunits: p70 (RPA1), p34 (RPA2), and p14 (RPA3). It is known to play an essential role in DNA replication, DNA repair, cell cycle checkpoint, and DNA recombination.[7] As critical members involved in maintaining genome integrity, RPAs are believed to be closely related to the development of human malignancy.[8],[9] Several studies have reported that down-regulation of RPA proteins correlate with a poor outcome and can be used as a prognostic biomarker in several cancer types.[10],[11] Moreover, the expression of RPA1 and RPA2 proteins appears to be useful prognostic indicators in colon cancer and bladder UC.[12],[13] Recently, RPA3 has been reported to be dysregulated in colorectal cancer.[14] Previous studies have shown that elevated expression of RPA3 is involved in tumorigenesis and associated with poor patient survival in gastric cancer and hepatocellular carcinoma.[15],[16] However, to the best of our knowledge, there is no data on the correlation between RPA3 expression and the prognosis of bladder UC.

In the present study, we analyzed the expression of RPA3 in bladder UC specimens by immunohistochemistry (IHC), Western blot analysis and investigated the correlation between RPA3 expression and clinicopathological characteristics, aiming to identify the evidence for its usefulness as a prognostic marker in patients with bladder UC.


 > Materials and Methods Top


Patients and tissue specimens

Bladder UC tissue samples and matched adjacent normal tissues were obtained from 155 patients between 2013 and 2018 in Shandong Provincial Hospital affiliated to Shandong First Medical University. None of the patients received local or systemic treatment before surgery. Written informed consent was obtained from all individual participants included in the study. All cases were reviewed by two experienced pathologists and assigned a histological grade and T-category according to the WHO 2004 classification.[17],[18] Superficial tumors were treated with maximal transurethral resection (TUR) followed by intravesical instillations. In contrast, muscle-invasive tumors were treated with maximal TUR followed by intravesical instillations and/or cystectomy. The duration of follow-up was calculated from the date of surgery to death or last follow-up, and patients were excluded if they had incomplete medical records or inadequate follow-up. Tissues were obtained from surgical specimens of bladder UC patients, and specimens were fixed in 4% buffered formalin and embedded in paraffin for analysis. Our study was approved by the Ethical Committees of the hospital.

Immunohistochemistry

IHC was carried out to elucidate the RPA3 expression in all 155 bladder UC samples. Sections of formalin-fixed, paraffin-embedded bladder UC tissues were cut to 4um thick slides in the Department of Pathology, Shandong Provincial Hospital. These slides were deparaffinized 2–3 times in xylene for 10 min each and hydrated through an ethanol gradient for 2 min each, and then were blocked in hydrogen peroxide in methanol for 10 min. After 10 min incubation in a steam pressure cooker containing a 0.01 M sodium citrate buffer, the samples completed antigen recovery. Then, the samples were blocked for 5 min and incubated with primary antibodies against RPA3 (Abcam, UK; 1:150) at 4°C overnight. In the following day, washed with phosphate-buffered saline (0.01M, PH 7), slides were cultured in biotinylated second antibody (goat anti-rabbit, Histostain-Plus Kits, ZSGB-BIO) and horseradish peroxidase for 30 min respectively, then stained with 5% diaminobenzidine for 1 min. Slides were counterstained with hematoxylin, differentiated in 1% acid alcohol, dehydrated, and cleared in xylene. Slides were routinely stained with hematoxylin and eosin to observe nucleus and cytoplasma.

Scoring of immunohistochemical staining

In evaluating immunoreactivity of RPA3 staining, the images were initially analyzed independently by two pathologists, who were blinded to the clinicopathological data and patients' outcomes. The positive fractions of RPA3 staining reactivity were assessed by positive cell proportion analysis, and at least 50 tumor cells were counted in four random regions of each section and the mean percentage of positive staining cells were evaluated. Both the extent and intensity of immunostaining were taken into consideration. The immunoreactivity was classified as follows: no staining reactivity (<10% positive tumor cells); low reactivity (10%–50% positive cells); and high reactivity (>50% positive cells).

Western blot analysis

Total protein from frozen tissues was isolated using RIPA lysis buffer (Beyotime, Nantong, China) and quantified using a BCA assay kit (Pierce, Rockford, IL, USA). We separated 30–50 μg of protein lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 10% to 15% Tris-HCl minigel and transferred them onto a polyvinylidene fluoride membrane following standard methods. After blocking, membranes were probed with appropriate dilutions of RPA3 antibodies overnight, followed by incubation with horseradish peroxidase-con-jugated secondary antibodies for 1 h. Proteins were visualized by the chemiluminescent detection system. Protein expression levels were normalized by β-actin. The intensity of protein expression was quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

Statistical data was analyzed using Statistical Package for Social Sciences (SPSS, version 17) software SPSS Inc., Chicago, IL, USA. The Pearson Chi-squared test was employed to explore the associations between RPA3 expression and clinicopathological parameters. Survival curves for recurrence-free survival were plotted with the Kaplan-Meier method, and the log-rank test was used to assess the significance of observed differences. Recurrence was defined as the time from the end of TUR or cystectomy until the first evidence of relapse. The patients who died during the follow-up period were treated as censored. For multivariate analysis, Cox's proportional hazards regression model was performed to assess the effect of various variables on outcome. P < 0.05 was considered to indicate statistical significance.


 > Results Top


Relative expression of RPA3 and its relationship with clinicopathological features

The nuclear staining was observed in bladder UC cells [Figure 1]a and [Figure 1]b. Positive staining signals of RPA3 were detected in 72 (46.5%) patients with low immunoreactivity and in 83 (53.5%) patients with high immunoreactivity, respectively. As shown in [Figure 1], RPA3 was significantly up-regulated in cancerous tissues compared with adjacent normal urothelium tissues. Besides, Western blotting was used to detect the expression of RPA3 in bladder UC tissues and adjacent normal tissues. Compared with adjacent normal tissues, UC tissues showed significantly increased RPA3 expression, and high-grade bladder UC had a stronger RPA3 expression level than low grade [Figure 2]. The association between clinicopathological characteristics and tumor cell RPA3 expression (low vs. high) is summarized in [Table 1]. The relation between RPA3 expression and tumor size, grade and stage was statistically significant (P = 0.034, P = 0.031, P = 0.021, respectively). There was no significant correlation of the RPA3 expression with patients' age, gender, and multifocality (P = 0.435, P = 0.401, P = 0.342, respectively). We have found that patients with high RPA3 expression (>50% positive cells) had poorer survival rates, therefore a worse outcome, than patients with low RPA3 immunostaining (10%–50% positive cells).
Figure 1: Immunohistochemistry of replication protein A3 in bladder urothelial carcinoma samples. Representative examples of urothelial carcinoma with low replication protein A3 staining reactivity (a: ×400) and high replication protein A3 staining reactivity (b: ×400), as evaluated by immunohistochemistry. Representative examples of adjacent normal tissues with no replication protein A3 staining were shown (c: ×400). Cells with yellow labeled nuclei were considered as replication protein A3 positive cells. Scale bar 50 μm

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Figure 2: The expression of replication protein A3 in patients with bladder urothelial carcinoma was significantly higher than that in adjacent normal tissues at protein level, and high grade urothelial carcinoma had stronger replication protein A3 expression level than low grade bars represent standard deviation *P < 0.05 versus normal; ** P < 0.05, high grade versus low grade

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Table 1: Correlations of replication protein A3 reactivity with clinical and pathological characteristics

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Survival analysis

At the end of follow-up, the following period of 155 patients ranged from 4 to 71 months (median time, 34.5 months). In the univariate analysis, the Kaplan–Meier survival curves showed that the recurrence-free survival was significantly worse with high RPA3 expression than those with low expression [P < 0.01, [Figure 3]]. Strong associations between recurrence-free survival and grade, TNM stage and RPA3 reactivity were found statistically significant [P = 0.030, P < 0.01, P < 0.01 [Table 2]]. Furthermore, multivariate analysis using a Cox proportional hazard model demonstrated that grade, TNM stage and RPA3 reactivity were identified as independent prognostic predictors with adverse significance in patients with UC [P = 0.025, P < 0.01, P = 0.016 [Table 3]], while age, gender, tumor size and multifocality were excluded as independent predictors with no statistical significance [P = 0.117, P = 0.293, P = 0.082, P = 0.074 [Table 3]].
Figure 3: Kaplan-Meier survival curves show recurrence-free survival of 155 patients with bladder urothelial carcinoma, according to replication protein A3 staining reactivity (low, 10%.50%; high, >50%), log rank test P < 0.01. Blue and green curves indicate low and high reactivity of replication protein A3 separately

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Table 2: Univariate analyses (Kaplan-Meier method; log-rank test) of the recurrence-free survival in the 155 bladder urothelial carcinoma cases

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Table 3: Multivariate analysis for predictors of survival (Cox's proportional hazards regression model)

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 > Discussion Top


Bladder UC is the second-most common genitourinary tumor and is a significant cause of morbidity and mortality.[1] Nonmuscle invasive bladder UC is likely to have a favorable prognosis, but frequent recurrence and progression are the typical adverse outcomes, even after curative resection followed by chemotherapeutic intravesical instillations.[19] The standard treatment for muscle-invasive bladder UC is radical cystectomy and bilateral pelvic lymph node dissection. Although surgery may be curative, about 50% of patients with muscle-invasive bladder UC develop metastases within 2 years and subsequently die of the disease.[20],[21] Although considerable progress in diagnosis, therapy, and understanding the biological behaviors of bladder UC, some aspects of the prognosis still remain unclear, especially concerning the prognostic markers of the tumor recurrence and patient survival. Consequently, significant attention has been directed to the elucidation of novel prognostic markers, which can be related to tumor recurrence and progression to predict the prognosis of bladder UC patients.

Conentionally, tumor grade and stage are the most accurate prognostic factors in the evaluation of bladder UC, but they cannot always predict the true tumor biological potential, as tumors of the same stage and grade can have completely different clinical courses.[4] Furthermore, bladder UC is a heterogeneous spectrum of diseases with different biological and clinical behaviors, determined by distinct molecular alterations.[22] Increasing studies have discussed molecular prognostic biomarkers for bladder cancer. Research showed that MicroRNA-451 was reported to act as a tumor suppressor in bladder cancer and might be a novel indicator for its prognosis.[23] Zhang et al. demonstrated that the expression of RNA-binding protein SAM68 was increased in muscle-invasive bladder cancer, which appeared to be a potential predictor for the disease.[24] However, the majority of molecular and genetic factors do not have notable impact significance in bladder UC and there are still controversies about the use of particular markers as prognostic factors.

RPA is a highly conserved multi-subunit protein involved in DNA replication, recombination, and repair in eukaryotic cells. Under normal conditions, human cells can respond to various genotoxic stresses by launching an array of biological processes, including DNA damage checkpoints, DNA damage repair, transcriptional regulation, and apoptosis.[25] Defects in efficiently and appropriately repairing DNA damage result in genomic instability and potentially tumorigenesis. Accumulating evidence shows that RPA protein levels are associated with patient survival in several cancer types. In esophageal carcinoma patients, lower expression of RPA1 and RPA2 was associated with poor outcome.[10] Downregulation of RPA2 was found to correlate with a poor outcome in nonmuscle-invasive bladder urothelial tumors.[13] Dai et al. found that elevated expression of RPA3 promoted tumor progression and was correlated with shorter survival in gastric cancer patients.[15] Another previous study reported that high RPA3 expression was associated with poorer patient survival and appeared to be a useful prognostic indicator in patients with nasopharyngeal carcinoma.[26] These results suggest that the functions of RPA proteins in cancer development are tumor type dependent, and there may be some subtle differences among RPA family members. Unfortunately, there were few reports about the relationship between RPA3 and clinical prognosis in patients with bladder UC.

In the present study, we have elucidated that RPA3 overexpression determined by IHC and Western blot, is an important prognostic factor in bladder UC. This was the first demonstration that RPA3 expression correlated with clinical outcome in bladder UC, and the observation provided the opportunity to consider potential clinical applications as a prognostic marker. The nuclear expression of RPA3 was detected by IHC staining, and RPA3 was found to be up-regulated in bladder UC tissues compared with normal urothelium tissues [Figure 1]. Furthermore, the increased expression of RPA3 was detected in tumor tissues by WB [Figure 2]. In addition, the results indicated that RPA3 overexpression has a negative correlation with clinical prognosis in bladder UC patients. The higher RPA3 expression cases tended to have a poorer outcome in terms of recurrence-free survival than the lower RPA3 expression cases by drawing survival curves. By Cox multivariate regression study, significant and independent prognostic factors in this analysis included tumor stage, grade, and RPA3 reactivity, while age, gender, multifocality, and tumor size were omitted as independent prognostic factors for bladder UC. These results suggested the clinical significance of RPA3 as a potential predictive biomarker for bladder UC patients' survival.


 > Conclusions Top


The main finding of this study suggested that higher expression levels of RPA3 were significantly associated with worse clinical outcome, and may serve as a marker for prognostic values, especially bladder UC recurrence. Future investigations including more patients with further analysis regarding the association between RPA3 and clinicopathological features of patients are needed to extend the value of the current study and to provide more insight into the potential role of RPA3 as a novel target for anticancer therapy in bladder UC.

Acknowledgment

This study was supported by the grants Key Research and Development Program of Shandong Province (No. 2018GSF118142), Medical Science and Technology Development Plan Project of Shandong Province (No. 2017WS289), and Natural Science Foundation of Shandong Province (No. ZR2017BH036).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

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