RBPJ Inhibitor-1

Overexpression of RIN1 associates with tumor grade and progression in patients of bladder urothelial carcinoma

Guang-yi Shan • Zhe Zhang • Qi-guang Chen • Xiu-yue Yu • Guo-bin Liu • Chui-ze Kong

Abstract

Ras and Rab interactor 1 (RIN1) is an effector of H-Ras, which plays an important role in the development and progression of carcinomas, but it has not been reported in bladder cancer. Hence, the association of RIN1 expres- sion with prognosis of bladder urothelial carcinoma (UC) was examined. RIN1 mRNA and protein expression in 20 paired UCs and the adjacent normal tissues was detected by quantitative reverse transcription polymerase chain reaction and Western blot. The expression of RIN1 protein in 96 specimens of UCs and 22 specimens of adjacent normal bladder tissues were analyzed by immunohistochemistry. The overall survival (OS) was assessed by univariate and multivariate analysis. Moreover, the progression-free sur- vival (PFS) and recurrence-free survival (RFS), classified by the clinicopathologic features with RIN1 expression, were assessed by multivariate analysis. RIN1 mRNA and protein level was higher in UCs than in the adjacent normal tissues (P < 0.01). Enhanced RIN1 immunoexpression was associated with high histologic grades (P 00.046), cancer progression (P00.047) as well as Ki-67 expression (P00.023). Furthermore, the 5-year survival rate was 29% in the subgroup with high level of RIN1 expression, while it was 43% in the subgroup with normal level of RIN1 expres- sion (P<0.05). Importantly, RIN1 level was revealed as the significant independent prognostic factor for death (P00.023) and progression (P 00.003), but a weak contribution for recurrence (P00.063). Collectively, RIN1 expression could be a potential prognostic predictor for UC patients. Keywords RIN1 . Urothelial carcinoma . Prognosis . Recurrence . Progression Introduction Bladder cancer is one of the most deadly urological malig- nant tumors worldwide [1, 2]. It is the fourth most frequent neoplasia in men, clinically characterized by high recurrent rate and poor prognosis once tumors invade the muscular uroepithelial layer [1]. Urothelial carcinoma of bladder (UC) is the most common histopathologic type of bladder cancer. Although the treatment of bladder cancer has improved greatly in recent years, the mortality of this disease is still increasing [3]. Up to now, classical histopathological param- eters such as tumor stage or grade have been used as prog- nostic predictors and multiple biomarkers have been studied for their value as prognostic indicators. However, none of these factors have provided sufficient sensitivity and speci- ficity to imply the diverse behavior of bladder cancer. The RIN1 gene, located on chromosome 11q13.2, is a molecule consisting of a coding region of 2352 bp [4, 5]. The RIN1 protein was first isolated as a RAS effector [6, 7]. Subsequent research demonstrated that RIN1 is an ABL tyrosine kinase activator and a regulator of epithelial-cell adhesion and migration [8–10]. RIN1 was also reported to involve in regulating insulin receptor signal transduction pathways and IL3 receptor signal transduction pathway [11, 12]. Besides, RIN1 gene plays an important role in degradation [13–15]. Until now, there were only several reports about expression and biofunction of RIN1in human tumors. RIN1 expression was increased in primary colorec- tal cancers compared with the adjacent normal colorectal mucosa [16]. In breast cancer, RIN1 expression was reduced or silenced in tumor tissues and cell lines compared with adjacent normal breast tissues and normal breast glandular cells [17]. Hence, the results of RIN1 in different tumors are still controversial. Furthermore, there is no published report on expression of RIN1 in UC. So, in this study we aimed to explore the level of RIN1expression and its clinical signif- icance in human UCs. Materials and methods Patients and tissue specimens In this study, for qRT–PCR analysis, we collected 20 paired bladder urothelial carcinoma and corresponding normal tis- sues from the patients who underwent radical cystectomy between July 2010 and November 2010 in the First Affili- ated Hospital, China Medical University. The primary UC tissue and its corresponding normal bladder tissue were stored at −70°C immediately after resection for RNA extraction. In addition, 96 paraffin-embedded samples of bladder urothelial carcinoma and 22 specimens of adjacent normal bladder tissues were collected between 2004 and 2008. The 2004 World Health Organization (WHO) Consensus classi- fication and Staging System for bladder neoplasms were used to classify specimens [18]. All tumors were staged as non-invasive (pTa) tumors, tumors with invasion of the lamina propria (pT1), and tumors with invasion of the muscularis propria or beyond (pT2–pT4). With regard to histologic type, the study included patients with papillary urothelial neoplasms of low malignant potential (PUNLMP), low-grade papillary urothelial carcinomas (LGPUC), and high-grade (papillary and non-papillary) urothelial carcino- mas (HGUC). The 96 patients included 69 males and 27 females from 38 to 71 years (mean, 55 years). Of these patients, 28 patients underwent radical cystectomy, 16 patients underwent partial cystectomy, and 52 patients underwent TURBT (transurethral resection of bladder tumor). For the use of these clinical materials, prior patient consent and the approval of the Institutional Review Board of China Medical University were obtained. The median follow-up time for overall survival was 49 months for patients still alive at the time of analysis, ranging from 5 to 76 months. Tumor recurrence was defined as a histologically confirmed tumor relapse during patient follow-up if this new tumor showed the same histologic grade and stage as the primary lesion, although it was not located at the same anatomical location in the bladder wall. Moreover, tumor progression was defined as an increase in histologic grade or tumor stage during follow-up of recur- rent disease or the formation of lymph node metastasis or systemic organ metastasis. RNA preparations and real-time PCR Total cellular RNA was extracted from tissue specimens using the RNeasy Plus Mini Kit from (Qiagen). Quantitative real-time polymerase chain reaction (qPCR) was done using SYBR Green PCR Master Mix (Applied Biosystems) in a total volume of 20 μl on a 7900 Real-Time PCR System (Applied Biosystems): 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. β-actin was used as the reference gene. The sequences for RIN1 and β-actin mRNA were as follows: RIN1, sense primer, 5′-GGCAG CAGAGGAGTAGCTTGA-3′, and antisense primer, 5′- GCTTGCTGGCGCTAAAAGG-3′ (NM. 004292, 91 bp); β-actin sense primer, 5′-TTAGTTGCGTTACACCCTTTC- 3′, and antisense primer, 5′-ACCTTCACCGTTCCAGTTT- 3′ (NM. 001101, 150 bp). The relative levels of gene expres- sion were represented as ΔCt=Ct gene-Ct reference, and the fold change of gene expression was computed by the 2−ΔΔCt method [19]. All tests were repeated in triplicate. Representa- tive results of mRNA contents of RIN1 expression in UCs and paired normal appearing tissues (NATs) were shown in Fig. 1a. Specifically, five normal specimens derived from these patients following radical resection were obtained, which was termed as normal bladder tissues out of the 5 cm location away from tumor tissues. Quantitative real-time PCR analysis of the mRNA level for RIN1 was performed by using SYBR Premix Ex assays (Transgen, Beijing, China). β-actin was used for normalization. Five normal specimens were mixed to- gether for mRNA extraction and normalized for detect- ing the normal average level of RIN1 expression; the fold changes were from the results of RIN1 expression in each UC or NAT compared with that in normal tissue (normalization to normal tissues). Individual outlines were also shown. Data were shown in triplicate with means (n020). Western blot Total protein from cells was extracted in lysis buffer (150 mM NaCl, 1% v/v NP-40, 0.1% SDS, 2 μg/ml apro- tinin, 1 mM PMSF) and quantified using the Bradford method. Fifty micrograms of total protein was separated by SDS–PAGE and then transferred to PVDF membrane (Millipore, Billerica, MA, USA). After blocking with 5% BSA, primary antibodies including rabbit polyclonal anti- RIN1 (1:300, BD Biosciences) and anti-β-actin (1:500, Santa Cruz Biotechnology) were incubated on the mem- branes overnight at 4°C. The membranes were then incu- bated for 2 h at 37°C with second antibodies (ZhongShan, China). Immunoreactive straps were identified using the ECL system (Pierce, Rockford, USA) as directed by the manufacturer. The DNR Imaging System was used to catch up the specific bands, and the optical density of each band protein level of RIN1expression was frequently overexpressed. c RIN 1 protein was detected by Western blot in 20 paired tissues. Protein expression of RIN1 in matched tumorous (T) and surrounding non- tumorous (N) tissues. Compared with non-tumorous tissues (N1–N3), the protein expression of RIN1 was increased in matched tumorous tissues (T1–T3). d The relative protein expression of RIN1, β-actin as an internal control (n020) (P 00.005). All P <0.05. Column—mean; bars—SD. *P <0.01. Statistical analysis was performed by paired t test and Student’s t test was measured using the Image J software. The ratio between the optical density of interest proteins and β-actin of the same sample was calculated as the relative content of pro- tein detected. The protein level of aberrant RIN1 expression in UCs and NATs is shown in Fig. 1c and d. Box plots described the relative expression of RIN1 protein level. The ends of the boxes define the 25th and 75th percentiles. A line indicated the median, and bars defined the 5th and 95th percentiles. Immunohistochemistry The immunohistochemical study was carried out on 4-μm- thick formalin-fixed paraffin-embedded tissue sections. Immunostaining was performed using the avidin–biotin– peroxidase complex method (Ultrasensitive™, MaiXin, Fuzhou, China). The sections were deparaffinized in xy- lene, rehydrated with graded alcohol, and then boiled in 0.01 M citrate buffer (pH 6.0) for 90 s with an auto- clave. Hydrogen peroxide (0.3%) was applied to block endogenous peroxide activity, and the sections were in- cubated with normal goat serum to reduce non-specific binding. Tissue sections were incubated with RIN1 rabbit polyclonal antibody with an optimal dilution of 1:200 (H00009610-D01P, Novus Biologicals, Littleton, CO, USA) and Ki-67/MIB-1 monoclonal antibody (MaiXin, Fuzhou, China). In the negative control, primary antibody was replaced by the non-immune mouse IgG of the same isotype. The specimens were incubated with primary antibody overnight at 4°C. Biotinylated goat anti-rabbit serum IgG was used as a secondary antibody. After washing, the sections were incubated with streptavidin– biotin conjugated with horseradish peroxidase, and the perox- idase reaction was developed with 3,3′-diaminobenzidine tetrahydrochloride. The immunoreaction was subjectively evaluated by two experienced pathologists by using a combined score system. Five views were examined per slide, and 100 cells were observed per view at ×400 magnification. Nuclear and/or cytoplasmic immunostaining in tumor cells was considered positive staining. The positive reaction was scored into four levels for both the intensity of staining and the percentage of positive cells. Grades according to the intensity of the staining included 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown), while the percentages of positive cells were separated into 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The scores of each sample were multiplied to give a final score of 0–12. Since the scores in all 22 cases of normal bladder tissues were all ≤2, we regarded the scores less than 3 as “normal expression”, while scores of 3 or more were consid- ered as “overexpression”. For Ki-67, the status of Ki-67 nuclear expression was assessed as the percentage of Ki-67 primary UC and adjacent normal appearing specimens using qRT–PCR. A total of 16 of 20 (80%) UCs showed up- regulated RIN1 mRNA expression, when compared with their adjacent normal bladder tissues (Fig. 1). The mean expression value of RIN1 mRNA in cancer tissues (11.48 ± 5.71; mean ± SD, normalized by β-actin gene expression and normal expression level) was significantly higher than the value (4.56 ± 1.95) in the adjacent paired normal appearing tissues (P 00.004, Fig. 1b). Then, we examined the protein level of RIN1 expression in 20 positive cells stained in each tumor. The median value of this series (41% of positive cells) was used as the cut-off value to differentiate tumors with low (<41%) from tumors with high (≥41%) ratio of cell proliferation. Clinical outcome assessment Overall survival (OS) was defined as the time from diagno- sis to the date of death or when censored at the latest date if patients were still alive. Progression-free survival (PFS) was defined as the time from diagnosis to the date of local failure or distant metastasis. Recurrence-free survival (RFS) was defined as the time from diagnosis to the date of relapse or recurrence, respectively. Statistical analysis Statistical analysis was done with the SPSS software (SPSS Standard version 16.0; SPSS, Inc.). The association of RIN1 protein expression with UC patients’ clinicopathologic features was assessed by the chi-square test. The t test was used to compare data from the densitometry analysis of mRNA and protein expression. For survival analysis, we analyzed all UC patients by Kaplan–Meier analysis, and differences in the sur- vival of subgroups of patients were compared using Mantel’s log-rank test. The multivariate Cox proportional hazards model was used to estimate the hazard ratios and 95% CIs for patient OS, PFS, and RFS. Variables in the model included age, gender, grade, tumor size, multiplicity tumor stage, the status of recur- rence, progression, and lymphatic metastasis, and the protein level of Ki-67 and RIN1 expression. All P values quoted were two-sided, and P <0.05 was considered statistically significant. Results High expression of RIN1 at mRNA and protein level in UCs To investigate the status of RIN1 expression in UC, we studied RIN1 mRNA and protein expression in 20 pairs of paired tissues. Interestingly, RIN1 was also strongly expressed in the UCs. To assess RIN1 expression in a large scale of UCs, a TMA containing samples including 96 UCs and 22 NATs, with corresponding clinical data, including age, gender, tumor size, tumor grade, etc., was used (Table 1). This TMA underwent IHC staining for RIN1 was expected from previous studies. Moreover, 54.1% (52/96) of UCs had strong RIN1 expression com- pared with NATs (6/22, 27.2%) that expressed high RIN1, confirming that aberrant RIN1 was a common molecular event in UC development. Relationship between RIN1 protein expression and clinicopathological features The RIN1 protein appeared to be expressed in both cytoplasm and nucleus of cancer cells (Fig. 2b, c, d). While in non- cancerous tissues, RIN1 protein staining was present in few cells, and the protein staining was weak (≤2 score). According to our evaluation criteria, they were categorized as normal expression (Fig. 2a). More interestingly, 96 UC samples eligi- ble for analysis showed a statistically significant positive cor- relation between RIN1 expression and Ki-67 as well as high grade, aggressive stage and progression (P00.023, P00.023, P00.046, and P00.047, respectively, Table 2), but was not correlated with age, gender, size, multiplicity, recurrence, or lymphatic metastasis (P>0.05). The positive rate of RIN1 protein expression was 15.4%, 48.0%, and 65.5% in different bladder tumors with histopathologic grade PUNLMP, LGPUC, and HGUC, respectively (P<0.05, χ2 test), while in the stage of pTa, pT1, and pT2–pT4, the positive rate of RIN1 protein was 39.5%, 57.7%, and 68.7%, respectively (P<0.05). To determine whether or not RIN1 expression was associated with cell proliferation in UC, Ki-67, a widely used cellular proliferation marker, was examined by IHC staining in our study. Ki-67 was often expressed together with RIN1 and was associated with cellular malignancy (Spearman correlation R00.232, P00.023; Fig. 2). RIN1 predicted clinical outcome of UCs Data from univariate analysis showed that there was a positive association between RIN1 expression and overall survival (P00.046, log rank). Patients with high level of RIN1expression had a poorer outcome (median ± SD, 43.6± 4.2 months) compared with patients with normal level of RIN1 expression (median ± SD, 55.3 ± 4.4 months). Kaplan–Meier analysis and the log-rank test were used to calculate the effect of RIN1 expression on survival. The 5- year survival in the group of high RIN1 expression was 29%, but it was 43% in the group of normal RIN1 expres- sion. The normal RIN1 expression group had longer survival, whereas the high RIN1 expression group had shorter survival (Fig. 3a).The log-rank test showed that overall survival (OS) was different between these two groups (P00.046). Kaplan– Meier estimates also corroborated a correlation between the overexpression of RIN1 protein immunoreactivity and a shorter progression-free survival (PFS) (P00.036) (Fig. 3c). However, no statistical association could be demonstrated regarding recurrence-free survival (P 00.261) (Fig. 3b). According to multivariate Cox proportional hazards model (Table 3), this analysis also demonstrated that high RIN1 expression was associated with a significant hazard ratio of 3.22 (95% CI 1.17–8.88) in OS. Furthermore, we analyzed the relationship between RIN1 level and PFS and RFS with 5- year follow-up. As shown in Table 3, a statistically significant difference was observed for PFS between patients with high and normal RIN1 level (P00.003); the hazard ratio with high RIN1 was 20.18 (95% CI 2.79–146.11). Moreover, the hazard ratio for RFS was 3.97 (95% CI 0.93–16.95, P00.063). Discussion Many studies have reported that the malignant transforma- tion of bladder tissue involves various gene changes. Vari- ous factors determine cell functions such as proliferation, differentiation, and invasion, most of which are thought to be involved in the activation of intracellular signal transduc- tion molecules to exert their function. In the 1990s, the currently well-known main pathways of proliferation signal transduction from the cell membrane to cytoplasm, i.e., growth factor–tyrosine kinase receptor–low molecular weight G protein (Ras)–RAF1, Ral GDS, AF6, Nore1, and PI3–kinase pathway, were identified, and their im- portance was widely recognized [20–28]. These path- ways were well conserved throughout evolution from yeasts to mammals and are said to be involved in not only the proliferation but also the fate and function control of various cells. Also, the RIN1 protein has been characterized as a downstream effector of H-Ras [5, 7, 29]. This gene encodes RIN1 protein, which structurally contains an SH2 domain toward the N-terminal end, and binds to ABL protein, a non-receptor tyrosine kinase, and a domain which binds to H-Ras, and 14-3-3 protein is present at the C-terminal end. It is known that RIN1 protein interacts directly with H-Ras and competes with RAF1, that 14-3-3 protein acts as a negative regulator of membrane localization of RIN1 protein, that the critical serine of RIN1 is a substrate for protein kinase D, and that RIN1 protein enhances the transforming properties of ABL [30, 31]. Furthermore, the RIN family has been identified as several guanine nucleotide exchange factors (GEFs) for Rab5 and shown to possess unique biochem- ical properties, and Rab5 plays important roles in mem- brane budding and trafficking in the early endocytic pathways, and the activation of this GTPase is mediated by GEFs at each of the transport steps [32–35]. These observations have led to the speculation that RIN1 pro- tein is a biologically important factor. First, we analyzed the state of RIN1 gene expression in paired tissues of UCs and found that the RIN1 gene was highly expressed in UCs. Next, analysis of RIN1 expression in specimens of blad- der cancers resected in our department showed that the expression was increased in 54.2% of primary bladder can- cer lesions compared with the adjacent normal tissues. In- creased RIN1 gene expression was associated with a significantly lower survival rate, suggesting that overexpres- sion of RIN1 was contributed to the malignant potential of UCs, as well as the positive association with the grade of histology. Although detailed studies of the RIN1 gene are needed in the future, we first found in this study that the RIN1 gene was abnormally expressed in many bladder cancer specimens by IHC staining and that its abnormal expression was associated with a decreased survival rate. These findings will serve as a step forward to defining the degree of malignancy of bladder cancer. Here, we reported that RIN1 expression was overex- pressed in a large proportion of bladder cancer tissues. RIN1, isolated as a RAS effector, is expressed in several tumor tissues and cell lines. It has been demonstrated that RIN1 protein was down-regulated in breast cancer and cell lines as the potential suppressor gene. In addition to tran- scription repressor-based silencing, DNA methylation of the RIN1 promoter was observed in a breast tumor cell line [17], whereas its mRNA expression was up-regulated in lung cancer and colorectal cancer tissues compared with normal tissues [15–17]. In this study, we report that RIN1 is overexpressed in human UCs compared with adjacent normal tissues. The overexpression of RIN1 protein was correlated with tumor classification, stage, progression, and prognosis. Consistent with previous reports of colorec- tal cancer, overexpression of RIN1 protein indicated poor prognosis for patients with bladder cancer. The 5-year survival rate was significantly different between the two groups, and the results showed that the higher expression of RIN1 protein is accompanied with poorer outcome. In lung adenocarcinoma A549 cell lines, up-regulation of RIN1 may contribute to their proliferative nature [15]. In concert with the above conclusion, we found that cases showing high levels of RIN1 expression also had strong Ki-67 signals (P00.023). This result suggests a potential important role of RIN1 in the control of cell proliferation, an activity that might be responsible, at least in part, for tumor- igenesis and/or progression of UC. Meanwhile, our study displayed that enhanced RIN1 expression was related to bladder cancer stage, classification, and progression. As regards tumor stage, non-invasive urothelial carcinoma (pTa tumors) had the lowest expression level of RIN1 com- pared with invasive neoplasms (pT1–pT4) while pT2–pT4 tumors had higher level of RIN1 expression than pT1 tumors. Similarly, HGUC had the higher level of RIN1 than LGPUC and PUNLMP. All the above results implied that the RIN1 may function as a putative oncogene in bladder cancer tumorigenesis and development processes. However, this is contrast to the report in breast cancer that RIN1 is a breast tumor suppressor gene and RIN1 expression is si- lenced in several breast cancer cell lines [17]. In addition, a previous study in lung adenocarcinoma suggested that up- regulation of RIN1 could promote epidermal growth factor (EGF) signaling in a manner important for proliferation [15]. Obviously, RIN1 may have distinct expression levels and diverse impact on cancer cells in different cancer types. The above analyses and evidence presented here further support the model that RIN1 can participate in the potenti- ation or attenuation of cell signaling processes through its modulation of endocytic trafficking events in a context- specific manner [15]. 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