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Overexpressed Rce1 is positively correlated with tumor progression and predicts poor prognosis in prostate cancer
Overexpressed Rce1 is positively correlated with tumor progression and predicts poor prognosis in prostate cancer Liangliang Huang MD, Meicai Li MD, Delin Wang MD, PhD, Jiang He MD, Wenqiang Wu BS, Qiangfeng Zeng MD, Jianjun Li MD, Maolin Xiao MD, Jie Hu MD, Yunfeng He MD, PhD, Ying Li PhD, Li Mai PhD, Wujiang Liu MD, PhD PII: DOI: Reference:
S0046-8177(15)00354-8 doi: 10.1016/j.humpath.2015.08.021 YHUPA 3694
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
1 May 2015 18 August 2015 20 August 2015
Please cite this article as: Huang Liangliang, Li Meicai, Wang Delin, He Jiang, Wu Wenqiang, Zeng Qiangfeng, Li Jianjun, Xiao Maolin, Hu Jie, He Yunfeng, Li Ying, Mai Li, Liu Wujiang, Overexpressed Rce1 is positively correlated with tumor progression and predicts poor prognosis in prostate cancer, Human Pathology (2015), doi: 10.1016/j.humpath.2015.08.021
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ACCEPTED MANUSCRIPT Overexpressed Rce1 is positively correlated with tumor progression and predicts poor prognosis in prostate cancer
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Liangliang Huang MDa, Meicai Li MDa, Delin Wang MD, PhDa, Jiang He MDb, Wenqiang Wu
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BSc, Qiangfeng Zeng MDa, Jianjun Li MDa, Maolin Xiao MDa, Jie Hu MDa, Yunfeng He MD,
Department of Urology, The First Affiliated Hospital of Chongqing Medical University,
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a
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PhDa, Ying Li PhDd, Li Mai PhDe, and Wujiang Liu MD, PhDf
Chongqing, Chongqing 400016, People’s Republic of China Gastroenterology and Neurology Center, University-Town Hospital of Chongqing Medical
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b
University, Chongqing 401331
Department of Urology, Qijiang People’s Hospital of First Affiliated Hospital, Chongqing
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c
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e
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Medical University, Chongqing 400016
College of Life Science, Chongqing Medical University, Chongqing 400016
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Department of Biochemistry & Molecular Biology, Molecular Medicine & Cancer Research
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Center, Chongqing Medical University, Chongqing, 400016 Institute of Urology, The First Affiliated Hospital of Peking University, Beijing, 100000,
People’s Republic of China
Address for correspondence: Delin Wang, MD, PhD Department of Urology The First Affiliated Hospital of Chongqing Medical University Chongqing 400016, People’s Republic of China. E-mail: [email protected]
ACCEPTED MANUSCRIPT
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Running title: Rce1 as a Prostate Prognostic Biomarker
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Competing interests: The authors declare that they have no conflicts of interest.
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Funding: This project was supported by the National Natural Science Foundation of China (No.
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30972999), the Natural Science Foundation of Chongqing (No. CSTC2012JJA10162), the Health Bureau of Chongqing (No. 20132082), and the Health and Family Planning Commission
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Foundation of Chongqing (No. 20143061).
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Keywords:
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Biomarkers;
Rce1
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Prostate cancer;
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Prostate;
ACCEPTED MANUSCRIPT Summary Ras and a-factor-converting enzyme 1 (Rce1) have been reported to play a key role in the
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proteolysis processing of Ras proteins. The present study investigated the prognostic significance
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of Rce1 in patients with prostate cancer (PCa). The expressions of the mRNA and protein of Rce1 were analyzed in 12 pairs of PCa and benign prostatic hyperplasia (BPH) by quantitative
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real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively.
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Immunohistochemistry (IHC) was used to examine expression of Rce1 protein in 74 PCa tissues and 30 BPH tissues. The association between Rce1 expression and the specific clinicopathologic
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features was evaluated by χ2 tests. Kaplan-Meier and Cox proportional hazards regression models were used to analyze the data. We found that expression of Rce1 mRNA and protein was
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markedly higher in PCa tissues than in paired BPH tissues. Expression of Rce1 in PCa was
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strongly associated with clinicopathologic features. It was detected in 69/74 (93.24%) of PCa tissues by IHC, and it was found to be associated with Gleason score (P = .013), T class (P
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= .015), and distant metastasis (P = .044). Patients with PCa having higher Rce1 expression had
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substantially shorter survival times than patients with lower Rce1 expression. Univariate and multivariate analysis revealed that Rce1 was an independent prognostic factor. In conclusion, our study suggests that expression of Rce1 can serve as an independent biomarker for the prognosis of PCa patients.
ACCEPTED MANUSCRIPT 1. Introduction Prostate cancer (PCa) is the most common cancer in men worldwide and the second most
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common cause of cancer deaths in the USA [1]. Currently, the most important prognostic
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indicators are prostate-specific antigen (PSA), Gleason score, and TNM stage [2-4]; but the prognosis of PCa patients with the same indicator results can be completely different, and this
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adapt to different PCa patients is an urgent need.
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mechanism is still confusing [5]. Hence, identification of a reliable prognostic biomarker that can
Despite the considerable evidence that has suggested that Ras proteins play a significant
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role in PCa progression [6-8], it is disappointing that attempts to disturb Ras competitively have often been ineffective. Recently, mediators of Ras signaling such as Rce1 (Ras and a-factor-
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converting enzyme 1) have been considered to promote a “Ras renaissance” [9]. Rce1, a type II
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cysteine aliphatic residue active on any amino acid with an appropriate sequence (CAAX: C, cysteine; A, aliphatic residue; X, any amino acid) prenyl endopeptidase, was first identified in
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Saccharomyces cerevisiae. It mediates the carboxyl terminal proteolysis step of Ras protein
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maturation [10,11]. A recent study proved the identity of Rce1 as a founding member of a novel family of glutamate-dependent intramembrane proteases [12]. Thus, it is possible to develop an inhibitor of Ras proteins if the structure and mechanism of Rce1 have been understood. In addition, much of the research has demonstrated the significance of Rce1 in some malignant diseases [13-16]. However, the expression and prognostic implications of Rce1 in PCa remain a question. In this study, quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry (IHC) were utilized to examine the expression of Rce1 in PCa and benign prostatic hyperplasia (BPH) specimens. We then investigated the correlation
ACCEPTED MANUSCRIPT between Rce1 expression and clinicopathologic features such as age, PSA concentration, Gleason score, T class, and distant metastasis. Meanwhile, we analyzed the association between
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Rce1 expression and overall survival of the PCa patients.
2. Materials and methods
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2.1. Patients and materials
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A total of 74 archived paraffin-embedded tissues from PCa patients who had undergone radical prostatectomy and 30 control cases (BPH) from transurethral resection of the prostate
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were obtained from the Department of Pathology and Urology, the First Affiliated Hospital of Chongqing Medical University. These patients had been treated between June 2009 and March
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2014. The cancer patients had not received chemotherapy, radiation therapy, or androgen
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deprivation before surgery. Samples were collected from areas of invasive adenocarcinoma, and the diagnosis was confirmed by two pathologists who were blinded to the clinical information.
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Tumor and clinical stages were defined according to the criteria of the 2002 TNM Classification
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and the Gleason system [17,18]. Overall survival duration was calculated as the surgery date to death or last follow-up. The clinicopathologic features of the patients are listed in Table 1. In addition, for qRT-PCR and Western blot analyses, 12 paired fresh PCa and BPH specimens were collected randomly and stored in liquid nitrogen immediately after resection. Written informed consent for the use of the tissues and clinical records had been obtained from all patients.
2.2. qRT-PCR Total RNA from 12 pairs of fresh surgically obtained PCa and BPH tissues was extracted using the RNAiso Plus reagent (Takara, Dalian, China) according to the manufacturer’s
ACCEPTED MANUSCRIPT instructions. The cDNA templates for qRT-PCR were synthesized from RNA samples. The extent of expression of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used
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as an endogenous control. The primer 5ʹ-CTC TGG AGG GAA CTC ACA GG-3ʹ and the
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reverse primer 5ʹ -GAA TCA TGG TCA GCA ACA GG-3ʹ were used to amplify the transcripts of RCE1. Gene expression was determined using SYBR Green PCR mix (Toyoko, Japan) and 10
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µL of template. The qRT-PCR was performed on a MyiQ.2 Two-Color Real-Time PCR
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Detection System (Bio-Rad [AU: location?]) using the following amplification conditions: 3 min at 95°C followed by 40 cycles of 30 s at 95°C and 30 s at 60°C. The relative amounts of Rce1
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expression were detected according to the manufacturer’s instructions, and the experiments were repeated in triplicate in the same reaction. The relative expression ratio was defined as the extent
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of expression of Rce1 mRNA relative to those of the internal reference gene, GAPDH mRNA.
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The fold changes between PCa and normal tissue pairs were analyzed by calculating the 2–△△Ct
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values.
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2.3. Western blot analysis
Total proteins from 12 pairs of fresh PCa and BPH tissues were homogenized with lysis buffer containing protease inhibitors (Sigma, St. Louis, MO USA). The protein concentration was determined by the Bradford assay (Bio-Rad) using a BCA Protein Assay Kit (Beyotime, Haimen, China). The protein samples were loaded under reducing conditions at the rate of 50 µg of total proteins and separated by 8% SDS-PAGE. The proteins on the gel were then transferred to PVDF membranes, and the membranes were blocked by 5% nonfat milk. Purified rabbit polyclonal anti-Rce1 antibody (1:150, sc-133951; Santa Cruz Biotechnology, Dallas, TX USA) and horseradish peroxidase–conjugated goat anti-rabbit IgG (1:1000; Zhongshan Golden Bridge
ACCEPTED MANUSCRIPT Bio-technology, Beijing, China) were used to detect the Rce1 protein. β-Actin was used as the equal protein loading control. A Western Bright ECL kit (Advansta, Menlo Park, CA USA) was
2.4. Evaluation of histopathological features and IHC
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used for the detection of horseradish peroxidase (HRP) in the antigen–antibody complex.
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Immunohistochemistry testing for Rce1 was performed by an assay kit (Zhongshan
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Golden Bridge Bio-technology, Beijing, China) according to the manufacturer’s instructions. Paraffin-embedded slides were deparaffinized in xylene and rehydrated with different
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concentrations of alcohol. The endogenous peroxidase activity of the sections was blocked with 3% hydrogen peroxide. Then sections were incubated sequentially with the Rce1 polyclonal
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antibody (1:200, sc-133951; Santa Cruz Biotechnology), Polymer Helper, and poly-HRP anti-
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rabbit IgG at room temperature. The primary antibodies were replaced by phosphate-buffered saline (PBS) for the negative controls. After staining with diaminobenzidine (DAB), the images
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were captured by the Olympus BX51 microscopic/DP71 Digital Camera System (Ina-shi,
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Nagano, Japan). The sections were evaluated blindly by two senior pathologists, and the following scoring system was used to evaluate Rce1 expression according to the intensity and percentage of positive cells [19]. The percentage was scored as 0 (no stained cells), 1 (1%–25% cells stained), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The intensity of all immunohistochemical stains was scored with a four-tier system (0 = negative, 1 = weak, 2 = moderate, and 3 = strong). A final immunoreactivity scores was obtained for each case by multiplying the percentage and the intensity score, the score being defined as follows: 0, negative; 1–3, weak; 4–6, moderate; and 8–12, strong. For further analysis, two immunoreactive groups were created: low, negative and weakly staining (score 0–3), and high, moderately and strongly
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2.5. Statistical analysis
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All statistical analyses were performed by the SPSS 19.0 statistical analysis software. P < .05 was considered statistically significant. The association of Rce1 expression with a PCa
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patient’s clinicopathologic features was assessed using the χ2 test. Survival curves were plotted
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using the Kaplan-Meier method and compared using the log-rank test. Survival data were
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evaluated using univariate and multivariate Cox regression analyses.
3. Results
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3.1. Elevated Rce1 mRNA by qRT-PCR and protein by Western blot in PCa tissues
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Expression of Rce1 mRNA was highly elevated in the 12 PCa tissues compared with the BPH tissues (Fig. 1A). Western blotting analysis also demonstrated overexpression of the Rce1
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protein in PCa tissues compared with the benign tissue counterparts (Fig. 1B).
3.2. Expression of Rce1 in PCa as determined by IHC Representative pictures of immunostaining of Rce1 protein are showed in Fig. 2. The staining showed a significant increase in Rce1 protein in PCa compared with BPH tissues. Among the 74 cases of PCa, we found 8 cases of high expression of Rce1, while 22 cases had moderate positive staining, 39 cases had weakly positive staining, and 5 cases had no staining. These findings suggest that Rce1 is commonly overexpressed in PCa, which correlates with the gene expression data.
ACCEPTED MANUSCRIPT 3.3. Relation between Rce1 expression and clinicopathologic parameters The association of Rce1 expression with the clinicopathologic features of PCa patients is
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summarized in Table 1. Statistical analysis was performed to evaluate the clinical significance of
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correlations between Rce1 expression and the clinicopathological findings of PCa, and Rce1 expression was found to be significantly associated with Gleason score (P = .013), T stage (P
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= .015), and distant metastasis (P = .044). There was no statistically significant correlation
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between Rce1 expression and age or PSA concentration (P = .351 and P = .272, respectively).
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3.4. Rce1 expression is correlated with cancer-specific deaths of PCa patients To investigate whether Rce1 expression in PCa was associated with patient survival,
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various statistical analyses were undertaken. The Kaplan–Meier analysis showed that patients
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with moderate or strong Rce1 expression had shorter survival times than patients with negative or weak Rce1 expression (P = .001 by log-rank test; Fig. 3). Moreover, as shown in Table 2, we
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found that Rce1 expression (HR = 3.193; P = .003), clinical T stage (HR= 2.490; P = .028) and
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Gleason score (HR = 2.773; P = .014) demonstrated significant effects on overall survival by univariate analysis. In multivariate analysis, the independent value of Rce1 in predicting the time to cancer-specific death was significant (HR = 3.163; P = .029), which was superior to such standard methods as Gleason score (HR = 2.193; P = .063) and clinical T stage (HR = 0.676; P = .510) at diagnosis. Our results strongly suggest that Rce1 expression is an independent prognostic predictor for PCa.
4. Discussion In the present study, we intended to investigate effects of Rce1 expression on the survival
ACCEPTED MANUSCRIPT of PCa patients. Our results demonstrated that the expression of Rce1 was elevated in PCa. Further analysis demonstrated that expression of Rce1 was associated with Gleason score, T
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class, and distant metastases. Importantly, high expression of Rce1 also was associated with a
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poor prognosis of PCa patients. These data suggest that Rce1 may play an important role in the progression of PCa and may serve as an independent prognostic biomarker for these patients.
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The processing of the “CAAX” motif has been observed in many proteins, including the
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Ras proteins [20]. In post-translational processing of Ras proteins, Rce1 removes the last three amino acids, which is very important for the association of Ras proteins with membranes [21,22].
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Mutationally activated forms of Ras are functionally associated with human cancers [23,24]. Because the affinity of GTP for Ras is extremely strong, it will be difficult to inhibit Ras
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competitively [25]. Thus, some mediators such as Ras regulatory proteins are particularly
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important. Among these mediators, Rce1 has engendered tremendous fascination, because it has a much wider specificity and can process all farnesylated and geranylgeranylated CAAX proteins.
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The molecular mechanisms that account for the association between Rce1 and PCa
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prognosis are unclear. Previous research demonstrated that Rce1 excision could lead to partial mislocalization of Ras away from the plasma membrane [26]. In mice, rce1 excision is incompatible with survival [13]. It was reported that Rce1 excision could accelerate the development of K-Ras–induced myeloproliferative disease but reduce the capacity of Rastransfected immortalized fibroblasts to form colonies [15,16]. In addition, mice with rce1 excision have a lethal cardiomyopathy [14]. These studies focus on the function of Rce1, but we find that the effect of Rce1 in different diseases is various and often significant, which may partly explain the association of Rce1 with the prognosis in PCa patients. Considering that previous investigators thought that the role of Rce1 was mainly modulation of Ras proteins and
ACCEPTED MANUSCRIPT that Ras-dependent signaling contributes to the development and progression of PCa, we argue that the molecular mechanisms of Rce1 in PCa may work primarily through Ras-dependent
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signaling. However, we cannot completely exclude Rce1-dependent signaling without the effect
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of Ras. A recent study found that the activity of Rce1 can be regulated by ubiquitin-specific protease 17 (USP17), which was mediated via the deubiquitination and relocalization of a novel
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isoform of Rce1 [27]. Thus, it is reasonable that there may be other regulators or effectors of
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Rce1, and further studies are needed to explore the molecular mechanisms of Rce1 action in PCa. There are some limitations to our study. First, because it is difficult to distinguish
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between PCa and adjacent normal prostate tissues after surgery, we assessed the expression of Rce1 in PCa and BPH tissues only after learning how in some previous studies. It is possible that
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this may not exactly demonstrate the differential expression of Rce1 between PCa and normal
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prostate. Therefore, we need a further investigation with PCa and adjacent normal prostate tissues. Second, we strictly selected the participants to control the potential heterogeneity, which
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led to a small number of cases (74 patients). A larger sample would be worthwhile for further
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analysis. Lastly, research on the cellular functions of Rce1 was absent in this study, but further studies in our laboratory are aimed at verifying the role of Rce1 in the development and progression of PCa.
In conclusion, we provide evidence, for the first time, that moderate or high expression of Rce1 is an independent prognostic factor in PCa patients. Our results suggest that Rce1 can be considered a novel biomarker for shortened survival time, and it may help clinical oncologists to render more rational and efficient treatment for PCa patients.
ACCEPTED MANUSCRIPT Ethical declaration The study protocol was carried out with the approval of the Ethics Committee of The First
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Affiliated Hospital of Chongqing Medical University.
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Fig. 1 qRT-PCR and Western blot analysis of Rce1 expression in paired PCa and BPH tissues. A,
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Fold changes (2-△△Ct values) by qRT-PCR showed upregulated expression of Rce1 mRNA in
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PCa cases compared with BPH tissues. B, Western blotting indicated upregulation of Rce1
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protein in 12 PCa tissues (T) in comparison with BPH tissues (N).
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Fig. 2 Representative photomicrographs of Rce1 immunoreactivity in PCa and BPH tissues. Representative negative expression of Rce1 (A) was observed in BPH tissues and lower-grade
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PCa. Rce1 weakly positive staining (B) and moderate staining (C) were present in most of the cancers. Diffuse cytoplasmic staining (D) was detected in some poorly differentiated cancers. E-
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H, Hematoxylin and eosin staining of corresponding cancer and hyperplastic tissues. Original
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magnification ×200.
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Fig. 3 Kaplan–Meier analysis of overall survival based on extent of Rce1 expression. Analyses
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for overall survival showed that patients with low Rce1 staining (negative and weak) had a significantly higher overall survival than did those with high expression (moderate and strong).
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Table 1 Association of Rce1 expression with clinicopathologic features of 74 prostate cancer patients Clinicopathological n (%) Rce1 protein expression (%) P variable Negative/weakly Moderate/strongly positive positive Age (y) .351 <65 25 (33.8) 13 (52.0) 12 (48.0) ≥65 49 (66.2) 31 (63.3) 18 (36.7) Preoperative PSA concentration .272a <10 6 (8.1) 5 (83.3) 1 (16.7) 10–20 26 (35.1) 17 (65.4) 9 (34.6) >20 42 (56.8) 22 (52.4) 20 (47.6) Gleason score .013 ≤6 21 (28.4) 15 (71.4) 6 (28.6) 7 28 (37.8) 20 (71.4) 8 (28.6) ≥8 25 (33.8) 9 (36.0) 16 (64.0) T stage .015 ≤T2 39 (52.7) 28 (71.8) 11 (28.2) T3 23 (31.1) 13 (56.5) 10 (43.5) T4 12 (16.2) 3 (25.0) 9 (75.0) Metastasis .044 M0 62 (83.8) 40 (64.5) 22 (35.5) M1 12 (16.2) 4 (33.3) 8 (66.7) Abbreviation: PSA, prostate specific antigen. a Calculated by Fisher’s χ2 test.
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Table 2 Univariate and multivariate analysis of overall patient survival Hazard ratio (95% CI) Univariate analysis Gleason score (≤7 vs. ≥8 ) 2.773 (1.227, 6.267) T stage (≤T2 vs. ≥T3) 2.490 (1.102, 5.626) PSA level (≤20 vs. >20 ng/100 mL) 0.361 (0.163, 0.799) Rce1 expression (low vs. high ) 3.193 (1.503, 6.784) Multivariate analysis Gleason score (≤7 vs. ≥8) 2.193 (0.959, 5.013) T stage (≤T2 vs. ≥T3 ) 0.676 (0.211, 2.164) PSA level (≤20 vs. >20) 0.359 (0.154, 0.835) Rce1 expression (low vs. high ) 3.165 (1.126, 8.901) Abbreviations: CI, confidence interval; PSA, prostate specific antigen.
P .014 .028 .012 .003 .063 .510 .017 .029
S0046-8177(15)00354-8 doi: 10.1016/j.humpath.2015.08.021 YHUPA 3694
To appear in:
Human Pathology
Received date: Revised date: Accepted date:
1 May 2015 18 August 2015 20 August 2015
Please cite this article as: Huang Liangliang, Li Meicai, Wang Delin, He Jiang, Wu Wenqiang, Zeng Qiangfeng, Li Jianjun, Xiao Maolin, Hu Jie, He Yunfeng, Li Ying, Mai Li, Liu Wujiang, Overexpressed Rce1 is positively correlated with tumor progression and predicts poor prognosis in prostate cancer, Human Pathology (2015), doi: 10.1016/j.humpath.2015.08.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Overexpressed Rce1 is positively correlated with tumor progression and predicts poor prognosis in prostate cancer
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Liangliang Huang MDa, Meicai Li MDa, Delin Wang MD, PhDa, Jiang He MDb, Wenqiang Wu
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BSc, Qiangfeng Zeng MDa, Jianjun Li MDa, Maolin Xiao MDa, Jie Hu MDa, Yunfeng He MD,
Department of Urology, The First Affiliated Hospital of Chongqing Medical University,
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a
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PhDa, Ying Li PhDd, Li Mai PhDe, and Wujiang Liu MD, PhDf
Chongqing, Chongqing 400016, People’s Republic of China Gastroenterology and Neurology Center, University-Town Hospital of Chongqing Medical
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b
University, Chongqing 401331
Department of Urology, Qijiang People’s Hospital of First Affiliated Hospital, Chongqing
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c
d
e
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Medical University, Chongqing 400016
College of Life Science, Chongqing Medical University, Chongqing 400016
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Department of Biochemistry & Molecular Biology, Molecular Medicine & Cancer Research
f
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Center, Chongqing Medical University, Chongqing, 400016 Institute of Urology, The First Affiliated Hospital of Peking University, Beijing, 100000,
People’s Republic of China
Address for correspondence: Delin Wang, MD, PhD Department of Urology The First Affiliated Hospital of Chongqing Medical University Chongqing 400016, People’s Republic of China. E-mail: [email protected]
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Running title: Rce1 as a Prostate Prognostic Biomarker
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Competing interests: The authors declare that they have no conflicts of interest.
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Funding: This project was supported by the National Natural Science Foundation of China (No.
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30972999), the Natural Science Foundation of Chongqing (No. CSTC2012JJA10162), the Health Bureau of Chongqing (No. 20132082), and the Health and Family Planning Commission
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Foundation of Chongqing (No. 20143061).
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Keywords:
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Biomarkers;
Rce1
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Prostate cancer;
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Prostate;
ACCEPTED MANUSCRIPT Summary Ras and a-factor-converting enzyme 1 (Rce1) have been reported to play a key role in the
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proteolysis processing of Ras proteins. The present study investigated the prognostic significance
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of Rce1 in patients with prostate cancer (PCa). The expressions of the mRNA and protein of Rce1 were analyzed in 12 pairs of PCa and benign prostatic hyperplasia (BPH) by quantitative
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real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively.
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Immunohistochemistry (IHC) was used to examine expression of Rce1 protein in 74 PCa tissues and 30 BPH tissues. The association between Rce1 expression and the specific clinicopathologic
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features was evaluated by χ2 tests. Kaplan-Meier and Cox proportional hazards regression models were used to analyze the data. We found that expression of Rce1 mRNA and protein was
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markedly higher in PCa tissues than in paired BPH tissues. Expression of Rce1 in PCa was
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strongly associated with clinicopathologic features. It was detected in 69/74 (93.24%) of PCa tissues by IHC, and it was found to be associated with Gleason score (P = .013), T class (P
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= .015), and distant metastasis (P = .044). Patients with PCa having higher Rce1 expression had
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substantially shorter survival times than patients with lower Rce1 expression. Univariate and multivariate analysis revealed that Rce1 was an independent prognostic factor. In conclusion, our study suggests that expression of Rce1 can serve as an independent biomarker for the prognosis of PCa patients.
ACCEPTED MANUSCRIPT 1. Introduction Prostate cancer (PCa) is the most common cancer in men worldwide and the second most
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common cause of cancer deaths in the USA [1]. Currently, the most important prognostic
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indicators are prostate-specific antigen (PSA), Gleason score, and TNM stage [2-4]; but the prognosis of PCa patients with the same indicator results can be completely different, and this
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adapt to different PCa patients is an urgent need.
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mechanism is still confusing [5]. Hence, identification of a reliable prognostic biomarker that can
Despite the considerable evidence that has suggested that Ras proteins play a significant
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role in PCa progression [6-8], it is disappointing that attempts to disturb Ras competitively have often been ineffective. Recently, mediators of Ras signaling such as Rce1 (Ras and a-factor-
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converting enzyme 1) have been considered to promote a “Ras renaissance” [9]. Rce1, a type II
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cysteine aliphatic residue active on any amino acid with an appropriate sequence (CAAX: C, cysteine; A, aliphatic residue; X, any amino acid) prenyl endopeptidase, was first identified in
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Saccharomyces cerevisiae. It mediates the carboxyl terminal proteolysis step of Ras protein
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maturation [10,11]. A recent study proved the identity of Rce1 as a founding member of a novel family of glutamate-dependent intramembrane proteases [12]. Thus, it is possible to develop an inhibitor of Ras proteins if the structure and mechanism of Rce1 have been understood. In addition, much of the research has demonstrated the significance of Rce1 in some malignant diseases [13-16]. However, the expression and prognostic implications of Rce1 in PCa remain a question. In this study, quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry (IHC) were utilized to examine the expression of Rce1 in PCa and benign prostatic hyperplasia (BPH) specimens. We then investigated the correlation
ACCEPTED MANUSCRIPT between Rce1 expression and clinicopathologic features such as age, PSA concentration, Gleason score, T class, and distant metastasis. Meanwhile, we analyzed the association between
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Rce1 expression and overall survival of the PCa patients.
2. Materials and methods
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2.1. Patients and materials
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A total of 74 archived paraffin-embedded tissues from PCa patients who had undergone radical prostatectomy and 30 control cases (BPH) from transurethral resection of the prostate
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were obtained from the Department of Pathology and Urology, the First Affiliated Hospital of Chongqing Medical University. These patients had been treated between June 2009 and March
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2014. The cancer patients had not received chemotherapy, radiation therapy, or androgen
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deprivation before surgery. Samples were collected from areas of invasive adenocarcinoma, and the diagnosis was confirmed by two pathologists who were blinded to the clinical information.
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Tumor and clinical stages were defined according to the criteria of the 2002 TNM Classification
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and the Gleason system [17,18]. Overall survival duration was calculated as the surgery date to death or last follow-up. The clinicopathologic features of the patients are listed in Table 1. In addition, for qRT-PCR and Western blot analyses, 12 paired fresh PCa and BPH specimens were collected randomly and stored in liquid nitrogen immediately after resection. Written informed consent for the use of the tissues and clinical records had been obtained from all patients.
2.2. qRT-PCR Total RNA from 12 pairs of fresh surgically obtained PCa and BPH tissues was extracted using the RNAiso Plus reagent (Takara, Dalian, China) according to the manufacturer’s
ACCEPTED MANUSCRIPT instructions. The cDNA templates for qRT-PCR were synthesized from RNA samples. The extent of expression of human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used
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as an endogenous control. The primer 5ʹ-CTC TGG AGG GAA CTC ACA GG-3ʹ and the
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reverse primer 5ʹ -GAA TCA TGG TCA GCA ACA GG-3ʹ were used to amplify the transcripts of RCE1. Gene expression was determined using SYBR Green PCR mix (Toyoko, Japan) and 10
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µL of template. The qRT-PCR was performed on a MyiQ.2 Two-Color Real-Time PCR
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Detection System (Bio-Rad [AU: location?]) using the following amplification conditions: 3 min at 95°C followed by 40 cycles of 30 s at 95°C and 30 s at 60°C. The relative amounts of Rce1
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expression were detected according to the manufacturer’s instructions, and the experiments were repeated in triplicate in the same reaction. The relative expression ratio was defined as the extent
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of expression of Rce1 mRNA relative to those of the internal reference gene, GAPDH mRNA.
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The fold changes between PCa and normal tissue pairs were analyzed by calculating the 2–△△Ct
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values.
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2.3. Western blot analysis
Total proteins from 12 pairs of fresh PCa and BPH tissues were homogenized with lysis buffer containing protease inhibitors (Sigma, St. Louis, MO USA). The protein concentration was determined by the Bradford assay (Bio-Rad) using a BCA Protein Assay Kit (Beyotime, Haimen, China). The protein samples were loaded under reducing conditions at the rate of 50 µg of total proteins and separated by 8% SDS-PAGE. The proteins on the gel were then transferred to PVDF membranes, and the membranes were blocked by 5% nonfat milk. Purified rabbit polyclonal anti-Rce1 antibody (1:150, sc-133951; Santa Cruz Biotechnology, Dallas, TX USA) and horseradish peroxidase–conjugated goat anti-rabbit IgG (1:1000; Zhongshan Golden Bridge
ACCEPTED MANUSCRIPT Bio-technology, Beijing, China) were used to detect the Rce1 protein. β-Actin was used as the equal protein loading control. A Western Bright ECL kit (Advansta, Menlo Park, CA USA) was
2.4. Evaluation of histopathological features and IHC
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used for the detection of horseradish peroxidase (HRP) in the antigen–antibody complex.
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Immunohistochemistry testing for Rce1 was performed by an assay kit (Zhongshan
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Golden Bridge Bio-technology, Beijing, China) according to the manufacturer’s instructions. Paraffin-embedded slides were deparaffinized in xylene and rehydrated with different
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concentrations of alcohol. The endogenous peroxidase activity of the sections was blocked with 3% hydrogen peroxide. Then sections were incubated sequentially with the Rce1 polyclonal
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antibody (1:200, sc-133951; Santa Cruz Biotechnology), Polymer Helper, and poly-HRP anti-
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rabbit IgG at room temperature. The primary antibodies were replaced by phosphate-buffered saline (PBS) for the negative controls. After staining with diaminobenzidine (DAB), the images
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were captured by the Olympus BX51 microscopic/DP71 Digital Camera System (Ina-shi,
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Nagano, Japan). The sections were evaluated blindly by two senior pathologists, and the following scoring system was used to evaluate Rce1 expression according to the intensity and percentage of positive cells [19]. The percentage was scored as 0 (no stained cells), 1 (1%–25% cells stained), 2 (26%–50%), 3 (51%–75%), and 4 (76%–100%). The intensity of all immunohistochemical stains was scored with a four-tier system (0 = negative, 1 = weak, 2 = moderate, and 3 = strong). A final immunoreactivity scores was obtained for each case by multiplying the percentage and the intensity score, the score being defined as follows: 0, negative; 1–3, weak; 4–6, moderate; and 8–12, strong. For further analysis, two immunoreactive groups were created: low, negative and weakly staining (score 0–3), and high, moderately and strongly
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2.5. Statistical analysis
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All statistical analyses were performed by the SPSS 19.0 statistical analysis software. P < .05 was considered statistically significant. The association of Rce1 expression with a PCa
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patient’s clinicopathologic features was assessed using the χ2 test. Survival curves were plotted
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using the Kaplan-Meier method and compared using the log-rank test. Survival data were
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evaluated using univariate and multivariate Cox regression analyses.
3. Results
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3.1. Elevated Rce1 mRNA by qRT-PCR and protein by Western blot in PCa tissues
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Expression of Rce1 mRNA was highly elevated in the 12 PCa tissues compared with the BPH tissues (Fig. 1A). Western blotting analysis also demonstrated overexpression of the Rce1
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protein in PCa tissues compared with the benign tissue counterparts (Fig. 1B).
3.2. Expression of Rce1 in PCa as determined by IHC Representative pictures of immunostaining of Rce1 protein are showed in Fig. 2. The staining showed a significant increase in Rce1 protein in PCa compared with BPH tissues. Among the 74 cases of PCa, we found 8 cases of high expression of Rce1, while 22 cases had moderate positive staining, 39 cases had weakly positive staining, and 5 cases had no staining. These findings suggest that Rce1 is commonly overexpressed in PCa, which correlates with the gene expression data.
ACCEPTED MANUSCRIPT 3.3. Relation between Rce1 expression and clinicopathologic parameters The association of Rce1 expression with the clinicopathologic features of PCa patients is
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summarized in Table 1. Statistical analysis was performed to evaluate the clinical significance of
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correlations between Rce1 expression and the clinicopathological findings of PCa, and Rce1 expression was found to be significantly associated with Gleason score (P = .013), T stage (P
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= .015), and distant metastasis (P = .044). There was no statistically significant correlation
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between Rce1 expression and age or PSA concentration (P = .351 and P = .272, respectively).
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3.4. Rce1 expression is correlated with cancer-specific deaths of PCa patients To investigate whether Rce1 expression in PCa was associated with patient survival,
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various statistical analyses were undertaken. The Kaplan–Meier analysis showed that patients
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with moderate or strong Rce1 expression had shorter survival times than patients with negative or weak Rce1 expression (P = .001 by log-rank test; Fig. 3). Moreover, as shown in Table 2, we
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found that Rce1 expression (HR = 3.193; P = .003), clinical T stage (HR= 2.490; P = .028) and
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Gleason score (HR = 2.773; P = .014) demonstrated significant effects on overall survival by univariate analysis. In multivariate analysis, the independent value of Rce1 in predicting the time to cancer-specific death was significant (HR = 3.163; P = .029), which was superior to such standard methods as Gleason score (HR = 2.193; P = .063) and clinical T stage (HR = 0.676; P = .510) at diagnosis. Our results strongly suggest that Rce1 expression is an independent prognostic predictor for PCa.
4. Discussion In the present study, we intended to investigate effects of Rce1 expression on the survival
ACCEPTED MANUSCRIPT of PCa patients. Our results demonstrated that the expression of Rce1 was elevated in PCa. Further analysis demonstrated that expression of Rce1 was associated with Gleason score, T
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class, and distant metastases. Importantly, high expression of Rce1 also was associated with a
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poor prognosis of PCa patients. These data suggest that Rce1 may play an important role in the progression of PCa and may serve as an independent prognostic biomarker for these patients.
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The processing of the “CAAX” motif has been observed in many proteins, including the
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Ras proteins [20]. In post-translational processing of Ras proteins, Rce1 removes the last three amino acids, which is very important for the association of Ras proteins with membranes [21,22].
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Mutationally activated forms of Ras are functionally associated with human cancers [23,24]. Because the affinity of GTP for Ras is extremely strong, it will be difficult to inhibit Ras
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competitively [25]. Thus, some mediators such as Ras regulatory proteins are particularly
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important. Among these mediators, Rce1 has engendered tremendous fascination, because it has a much wider specificity and can process all farnesylated and geranylgeranylated CAAX proteins.
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The molecular mechanisms that account for the association between Rce1 and PCa
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prognosis are unclear. Previous research demonstrated that Rce1 excision could lead to partial mislocalization of Ras away from the plasma membrane [26]. In mice, rce1 excision is incompatible with survival [13]. It was reported that Rce1 excision could accelerate the development of K-Ras–induced myeloproliferative disease but reduce the capacity of Rastransfected immortalized fibroblasts to form colonies [15,16]. In addition, mice with rce1 excision have a lethal cardiomyopathy [14]. These studies focus on the function of Rce1, but we find that the effect of Rce1 in different diseases is various and often significant, which may partly explain the association of Rce1 with the prognosis in PCa patients. Considering that previous investigators thought that the role of Rce1 was mainly modulation of Ras proteins and
ACCEPTED MANUSCRIPT that Ras-dependent signaling contributes to the development and progression of PCa, we argue that the molecular mechanisms of Rce1 in PCa may work primarily through Ras-dependent
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signaling. However, we cannot completely exclude Rce1-dependent signaling without the effect
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of Ras. A recent study found that the activity of Rce1 can be regulated by ubiquitin-specific protease 17 (USP17), which was mediated via the deubiquitination and relocalization of a novel
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isoform of Rce1 [27]. Thus, it is reasonable that there may be other regulators or effectors of
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Rce1, and further studies are needed to explore the molecular mechanisms of Rce1 action in PCa. There are some limitations to our study. First, because it is difficult to distinguish
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between PCa and adjacent normal prostate tissues after surgery, we assessed the expression of Rce1 in PCa and BPH tissues only after learning how in some previous studies. It is possible that
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this may not exactly demonstrate the differential expression of Rce1 between PCa and normal
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prostate. Therefore, we need a further investigation with PCa and adjacent normal prostate tissues. Second, we strictly selected the participants to control the potential heterogeneity, which
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led to a small number of cases (74 patients). A larger sample would be worthwhile for further
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analysis. Lastly, research on the cellular functions of Rce1 was absent in this study, but further studies in our laboratory are aimed at verifying the role of Rce1 in the development and progression of PCa.
In conclusion, we provide evidence, for the first time, that moderate or high expression of Rce1 is an independent prognostic factor in PCa patients. Our results suggest that Rce1 can be considered a novel biomarker for shortened survival time, and it may help clinical oncologists to render more rational and efficient treatment for PCa patients.
ACCEPTED MANUSCRIPT Ethical declaration The study protocol was carried out with the approval of the Ethics Committee of The First
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Affiliated Hospital of Chongqing Medical University.
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Fig. 1 qRT-PCR and Western blot analysis of Rce1 expression in paired PCa and BPH tissues. A,
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Fold changes (2-△△Ct values) by qRT-PCR showed upregulated expression of Rce1 mRNA in
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PCa cases compared with BPH tissues. B, Western blotting indicated upregulation of Rce1
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protein in 12 PCa tissues (T) in comparison with BPH tissues (N).
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Fig. 2 Representative photomicrographs of Rce1 immunoreactivity in PCa and BPH tissues. Representative negative expression of Rce1 (A) was observed in BPH tissues and lower-grade
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PCa. Rce1 weakly positive staining (B) and moderate staining (C) were present in most of the cancers. Diffuse cytoplasmic staining (D) was detected in some poorly differentiated cancers. E-
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H, Hematoxylin and eosin staining of corresponding cancer and hyperplastic tissues. Original
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magnification ×200.
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Fig. 3 Kaplan–Meier analysis of overall survival based on extent of Rce1 expression. Analyses
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for overall survival showed that patients with low Rce1 staining (negative and weak) had a significantly higher overall survival than did those with high expression (moderate and strong).
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Table 1 Association of Rce1 expression with clinicopathologic features of 74 prostate cancer patients Clinicopathological n (%) Rce1 protein expression (%) P variable Negative/weakly Moderate/strongly positive positive Age (y) .351 <65 25 (33.8) 13 (52.0) 12 (48.0) ≥65 49 (66.2) 31 (63.3) 18 (36.7) Preoperative PSA concentration .272a <10 6 (8.1) 5 (83.3) 1 (16.7) 10–20 26 (35.1) 17 (65.4) 9 (34.6) >20 42 (56.8) 22 (52.4) 20 (47.6) Gleason score .013 ≤6 21 (28.4) 15 (71.4) 6 (28.6) 7 28 (37.8) 20 (71.4) 8 (28.6) ≥8 25 (33.8) 9 (36.0) 16 (64.0) T stage .015 ≤T2 39 (52.7) 28 (71.8) 11 (28.2) T3 23 (31.1) 13 (56.5) 10 (43.5) T4 12 (16.2) 3 (25.0) 9 (75.0) Metastasis .044 M0 62 (83.8) 40 (64.5) 22 (35.5) M1 12 (16.2) 4 (33.3) 8 (66.7) Abbreviation: PSA, prostate specific antigen. a Calculated by Fisher’s χ2 test.
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Table 2 Univariate and multivariate analysis of overall patient survival Hazard ratio (95% CI) Univariate analysis Gleason score (≤7 vs. ≥8 ) 2.773 (1.227, 6.267) T stage (≤T2 vs. ≥T3) 2.490 (1.102, 5.626) PSA level (≤20 vs. >20 ng/100 mL) 0.361 (0.163, 0.799) Rce1 expression (low vs. high ) 3.193 (1.503, 6.784) Multivariate analysis Gleason score (≤7 vs. ≥8) 2.193 (0.959, 5.013) T stage (≤T2 vs. ≥T3 ) 0.676 (0.211, 2.164) PSA level (≤20 vs. >20) 0.359 (0.154, 0.835) Rce1 expression (low vs. high ) 3.165 (1.126, 8.901) Abbreviations: CI, confidence interval; PSA, prostate specific antigen.
P .014 .028 .012 .003 .063 .510 .017 .029