High RIN1 expression is associated with poor prognosis in patients with gastric adenocarcinoma
Hai-Feng Yu • Gang Zhao • Zhi-Jun Ge • Dao-Rong Wang • Jie Chen • Yun Zhang • Tian-Zhou Zha • Kai Zhang • Miao Zhang • Yong-Fei Tan • Su-Jun Zhou • Chao Jiang
Received: 14 March 2012 / Accepted: 24 April 2012 / Published online: 6 May 2012
Ⓒ International Society of Oncology and BioMarkers (ISOBM) 2012
Abstract The aim of this study was to investigate the expres- sion and prognostic significance of RIN1 in gastric adenocar- cinoma. RIN1 expression was analyzed using quantitative real-time PCR (qRT-PCR), Western blotting, and immunohis- tochemical staining on tissue samples from a consecutive series of 315 gastric adenocarcinoma patients who underwent tumor resections between 2003 and 2006. The relationship between RIN1 expression, clinicopathological factors, and
Hai-Feng Yu, Gang Zhao, and Zhi-Jun Ge contributed equally to this paper.
H.-F. Yu : J. Chen : Y. Zhang : T.-Z. Zha : K. Zhang : M. Zhang : S.-J. Zhou (*) : C. Jiang (*)
Department of General Surgery, Yixing People’s Hospital, No. 75, Tongzhen Guan Rd,
Yixing, China
e-mail: [email protected] e-mail: [email protected]
G. Zhao
Department of Gastroenterology, Second Affiliated Hospital, Medical School of Xi’an Jiaotong University,
Xi’an 710004, China
Z.-J. Ge
Department of Anesthesiology, Yixing People’s Hospital, No. 75, Tongzhen Guan Rd,
Yixing, China
H.-F. Yu : D.-R. Wang (*) : J. Chen
Department of General Surgery, Northern Jiangsu People’s Hospital, No. 95, Nantong Western Rd,
Yangzhou 22500, China
e-mail: [email protected]
Y.-F. Tan
Department of Cardiothoracic Surgery, Yixing People’s Hospital, No. 75, Tongzhen Guan Rd,
Yixing, China
patient survival was investigated. qRT-PCR results showed that the RIN1 mRNA expression was higher in tumor tissue samples than in the adjacent normal tissues, and a corresponding increase in protein expression was confirmed by Western blotting. Immunohistochemical staining indicated that RIN1 is highly expressed in 54.3 % of gastric adenocar- cinomas. RIN1 expression levels were closely associated with tumor size, histological differentiation, tumor stage, and lymph node involvement. Kaplan–Meier survival analysis showed that high RIN1 expression exhibited a significant correlation with poor prognosis for gastric adenocarcinoma patients. Multivariate analysis revealed that RIN1 expression is an independent prognostic parameter for the overall survival rate of gastric adenocarcinoma patients. Our data suggest that RIN1 plays an important role in gastric adenocarcinoma pro- gression and that a high RIN1 expression predicts an unfavor- able prognosis in gastric adenocarcinoma patients.
Keywords RIN1 . Gastric adenocarcinoma . Prognosis . Survival . Diagnosis
Introduction
Currently, gastric adenocarcinoma is one of the most com- mon cancers worldwide and is one of the leading causes of cancer-related death in China [1]. Gastric adenocarcinoma encompasses many subtypes with distinct genetic and bio- logical features. Therefore, identification of new biological markers to determine the risk of poor prognosis is important for designing treatment strategies [2, 3].
Ras and Rab interactor 1 (RIN1) is a Ras effector protein that positively regulates endocytosis and cytoskeletal remod- eling through interacting with downstream Rab5 GTPases and the Abl tyrosine kinase [4–7]. RIN1 is strongly expressed in
mature forebrain neurons and moderately expressed in epithe- lial and hematopoietic cells [7, 8]. Consistent with this tissue distribution, a previous study using RIN1 knockout mice dem- onstrated a major physiological role for RIN1 in mature neu- rons [9]. Recent studies have indicated a potential role for altered RIN1 expression and function in tumor development and progression. Aberrant RIN1 expression has been found in a variety of human cancers, such as gastric, breast, and colo- rectal cancer; Wilms’ tumor; acute myeloid leukemia; and in several lung adenocarcinoma cell lines [10–15]. RIN1 over- expression is associated with poor prognosis and venous inva- sion in colorectal carcinoma [13]. Moreover, previous in vitro studies suggest that RIN1 positively regulates cell proliferation in lung adenocarcinoma cell lines through mediating the epi- dermal growth factor signaling pathway [14]. In addition, RIN1 mRNA was found to be highly expressed in gastric cancer cell lines [16]. Although a high level of RIN1 protein expression in gastric cancer cell lines has also been reported, the importance of RIN1 expression as a clinicopathological parameter and prognostic marker in gastric cancer remains unclear.
The purpose of the present study was to examine the expression status of RIN1 mRNA and protein in gastric ade- nocarcinoma tissues and to evaluate whether the level of RIN1 expression correlates with the clinicopathological parameters and the prognosis of gastric adenocarcinoma patients.
Materials and methods
Patients and tissue specimens
A total of 315 gastric adenocarcinoma specimens and 40 corresponding non-tumor gastric tissues were collected from patients undergoing surgery between January 2003 and De- cember 2006 at the Northern Jiangsu People’s Hospital. None of the patients had received radiotherapy or chemotherapy prior to surgery. The histomorphology of all specimens was assessed by the Department of Pathology at the Yixing People’s Hospital. Gastric adenocarcinomas were graded based on the TNM stage classification (stages I–IV). Samples were taken from surgically removed tumors and matched non- cancerous tissue, snap-frozen in liquid nitrogen, and stored at
−80°C for RNA and protein analyses. Samples from the same tissues were embedded in paraffin after fixation in 10 % formalin for histological diagnosis. Clinical information, in- cluding sex, age, tumor size, TNM grade, WHO type, and lymph node involvement, was also collected. Follow-up in- formation for all participants was obtained every 3 months by telephone, during a visit to the clinic, or via a postal question- naire. During the follow-up period, overall survival was mea- sured from diagnosis to death or to the last follow-up (at 5 years). Death of a patient was ascertained by reporting from the family and verified by a review of public records.
Immunohistochemical analysis
Tissue specimens were subjected to immunohistochemical analysis using the avidin–biotin–peroxidase method. Sections were deparaffinized in xylene and dehydrated using a graded alcohol series before endogenous peroxidase activity was blocked with 0.5 % hydrogen peroxide in methanol for 10 min. Nonspecific binding was blocked by incubating sec- tions with 10 % normal goat serum in phosphate-buffered saline (PBS) for 1 h at room temperature. Without washing, sections were incubated with a polyclonal antibody against human RIN1 (1:300; SC-1971, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS at 4°C overnight. Following this, biotinylated goat anti-mouse immunoglobulin G (IgG; 1:400, Sigma, St. Louis, MO, USA) was incubated with the sections for 1 h at room temperature and detected using streptavidin–peroxidase. The brown color indicative of per- oxidase activity was developed by incubating sections with
⦁ % 3,3-diaminobenzidine (Sigma) in PBS with 0.05 % hydrogen peroxide for 5 min at room temperature. All tissue specimens were assessed separately by two pathologists under double-blind conditions, in which they had no prior knowl- edge of either the clinical or clinicopathological status of the specimens. RIN1 expression in gastric adenocarcinoma speci- mens was evaluated by scanning the entire tissue specimen under low magnification (×40) and was confirmed under high magnification (×200 and ×400). An immunoreactivity score system was applied as described elsewhere [17]. The percent- age of positive cells was scored as: 0, <5 % (negative); 1, 5– 25 % (sporadic); 2, 25–50 % (focal); 3, >50 % (diffuse). The staining intensity was scored as: 0—no staining; 1—weak staining; 2—moderate staining; and 3—strong staining. The RIN1 immunostaining score was calculated by multiplying the positive cell score by the staining intensity score and thus ranged from 0 to 9. High RIN1 expression was defined as a total score of ≥4 and low RIN1 expression level as a total score of
<4. Ki-67 immunoreactivity was evaluated as follows: 0, <20 % of tumor cells showing positive immunoreactivity; 1, 20–50 % of tumor cells showing positive immunoreactivity; and 2,
>50 % of tumor cells showing positive immunoreactivity. RNA isolation and qRT-PCR
Total RNA from 40 tumor and matched non-cancerous tissue samples stored at −80°C was isolated using the TRIzol reagent (Life Technologies, Rockville, MD, USA) according to the manufacturer’s instructions. Reverse transcription was per- formed using 1 μg of total RNA from each sample. qRT- PCR was performed with SYBR Green (Takara, Dalian, Chi- na) using a Real-Time Quantitative Thermal Block (Biometra, Göttingen, Germany). PCR primer sequences were based on the GenBank human RIN1 and β-actin gene sequences: RIN1 sense, 5-GGCAGCAGAGGAGTAGCTTGA-3; RIN1
antisense, 5-GCTTGCTGGCGCTAAAAGG-3; β-actin sense, 5-GTGGGGCGCCCCAGGCACCA-3; and β-actin antisense, 5-CTCCTTAATGTCACGCACGATTTC-3. The
RIN1 amplicon was 114 bp. Cycling conditions were: 95°C for 30 s to activate the polymerase; 40 cycles of 95°C for 20 s, 55°C for 15 s, and 72°C for 20 s; and a final extension at 72°C for 10 min. PCR specificity was confirmed by examining the dissociation curves. β-actin served as the internal control, and reactions were done in triplicate.
Data were analyzed using the comparative threshold cycle method [18]. Relative RIN1 mRNA expression was calculated by the 2ΔCt method (ΔCt0 Ct of RIN1 − Ct of β-actin). The fold change in RIN1 expression in each tissue was defined as the ratio of relative RIN1 mRNA expression in tumor tissue to that in the corresponding normal tissues.
Western blot analysis
Total proteins were extracted from 40 surgically removed tumors and matched non-cancerous tissue samples stored at
−80°C using radioimmunoprecipitation assay buffer (Beyo-
time Institute of Biotechnology, Haimen, China), and protein concentrations were determined using a bovine serum albu- min standard curve. Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred to polyvinylidene fluoride membranes. Membranes were blocked with 5 % skimmed milk at room temperature for 2 h and then incubated overnight at 4°C with an anti-RIN1 (1:500, SC-1971, Santa Cruz Biotechnology) or anti-GAPDH antibody (1:1,000, Sigma-Aldrich), followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using an ECL plus chemiluminescence kit (Millipore, Bedford, MA, USA).
Statistical analysis
All statistical analyses were performed using SPSS software, version 17.0 (SPSS Inc., Chicago, IL, USA). Associations between RIN1 expression and clinicopathological variables were analyzed using the Mann–Whitney and Kruskal–Wallis tests. Survival curves were plotted using the Kaplan–Meier product limit method, and differences between the survival curves were tested using the log-rank test. Cox’s proportional hazards model was used to identify factors that had a significant influence on survival. Statistical significance was set at P<0.05.
Results
Analysis of RIN1 mRNA expression
The levels of RIN1 transcripts in 40 pairs of resected speci- mens (tumor tissue samples and matched adjacent non-
tumor tissue samples) from patients with gastric adenocar- cinomas were determined using qRT-PCR. We found that 24 of the 40 patients (60 %) had a higher RIN1 mRNA expres- sion in gastric adenocarcinoma tissue than in adjacent non- cancerous tissue (at least a 2.5-fold increase). In addition, the relative expression of RIN1 mRNA in gastric adenocar- cinoma specimens was significantly higher than in the corresponding normal tissues (0.00745 ± 0.002421 vs. 0.00343 ±0.001342, P<0.001; Fig. 1)
Analysis of RIN1 protein expression
RIN1 protein levels in resected gastric adenocarcinoma samples were measured by Western blotting. Increased RIN1 expression was detected in 26 of the 40 tumor tissue samples (65 %) compared with matched adjacent non-tumor tissue samples (P00.002; Fig. 2). These findings were con- sistent with the qRT-PCR data.
The degree of RIN1 immunohistochemical staining correlates with clinicopathological characteristics
To gain further insight into the downstream effects and prognostic value of increased RIN1 expression in gastric adenocarcinoma patients, paraffin-embedded sections (n 0315) of gastric adenocarcinomas confirmed by histopa- thology were examined by immunohistochemistry. RIN1 immunoreactivity varied between tumor tissue samples and matched adjacent non-tumor tissue samples. RIN1 localized to the cytoplasm of tumor cells. Overall, RIN1 expression was positive in 246 (78.1 %) gastric adenocarcinoma sam- ples and negative in 69 (21.9 %) gastric adenocarcinoma samples. RIN1 was highly expressed in 171 (54.3 %) and
Fig. 1 RIN mRNA expression in gastric adenocarcinoma and paired adjacent normal tissues examined by quantitative real-time RT-PCR and normalized to β-actin. Bars represent the means of RIN1 relative expression in cancer tissues and normal tissues, respectively. T tumor samples, NT non-tumor samples
Fig. 2 Western blot analysis of RIN1 expression in gastric adenocar- cinoma patients. Relative RIN1 expression in gastric adenocarcinoma tumor (T) tissues compared to adjacent non-tumor (N) tissues (n 040), assessed by Western blotting (P00.002). The results are shown as expression relative to GAPDH and are means (±SD) of three separate experiments
was expressed at lower levels in 144 (45.7 %) gastric ade- nocarcinoma patients (Fig. 3). The level of RIN1 expression in gastric adenocarcinoma correlated with tumor size, histo- logical differentiation, tumor stage, and lymph node involvement, but not with age or gender (Table 1).
RIN1 protein expression correlates with Ki-67 expression in gastric adenocarcinoma
As shown in Fig. 3, immunoreactive Ki-67 localized to the nuclei of tumor cells. We found that 47 % in gastric adeno- carcinomas were positive for Ki-67 protein expression and that there was a strong association between RIN1 overex- pression and a high Ki-67 labeling index (rs 00.247, P00.0015).
Correlation between RIN1 expression levels and patient survival
The prognostic effect of RIN1 on the overall survival rate of gastric adenocarcinoma patients was investigated by com- paring the survival rate of patients with high or low levels of RIN1 protein expression in tumors using Kaplan–Meier survival curves and the log-rank test. These tests showed that a high expression of RIN1 protein was a significant prognostic factor for poor overall survival of gastric adeno- carcinoma patients. The 5-year survival rates of gastric adenocarcinoma patients with a high or a low RIN1 protein
expression level were 31.6 and 64.6 %, respectively. This difference was statistically significant (P<0.001, log-rank test; Fig. 4).
Univariate and multivariate analyses
Univariate Cox regression analysis also showed that clinical variables, including tumor size, histological differentiation, tumor stage, lymph node involvement, and RIN1 expres- sion, significantly associated with overall survival (Table 2). Furthermore, multivariate Cox regression analyses were performed to evaluate the potential of RIN1 expression as an independent predictor for the overall survival of patients with gastric adenocarcinoma. Although other parameters failed to demonstrate independence, tumor size, histological differentiation, lymph node involvement, tumor stage, and RIN1 expression may play a role in predicting overall sur- vival in patients with gastric adenocarcinoma (Table 2).
Discussion
Despite many advances in diagnostic tumor imaging, com- bination chemotherapy, and radiation therapy, little im- provement has been achieved within the last decade in terms of the prognosis and quality of life for patients with gastric adenocarcinoma. Given the frequent failure of con- ventional treatment strategies, many cancer-related mole- cules have been characterized with the goal of developing novel anticancer therapies, including targeted drugs or anti- bodies and cancer vaccines [19, 20]. A number of studies have documented that RIN1 expression is upregulated in several human cancers, such as squamous cell carcinoma, colorectal cancer, and cervical cancer, through duplication or rearrangements of the RIN1 gene locus, suggesting that overexpression of RIN1 is involved in tumorigenesis for many types of cancer [11, 13, 15, 21, 22]. In contrast, however, Milstein et al. [12] demonstrated that RIN1 is silenced in breast tumor cell lines compared to cultured human mammary epithelial cells and that silencing likely
Fig. 3 Immunohistochemical staining of gastric adenocarcinoma tissues. a Representative sample showing high RIN1 expression in gastric adenocarcinoma tissue. b A representative sample
showing low RIN1 expression in gastric adenocarcinoma tissue. c Nuclear expression of Ki-67 in gastric adenocarcinoma tissue (×200 magnification)
Table 1 Relationship between RIN1 expression and clinicopath- ologic features of patients with
Clinicopathological variable Number Low RIN1
(total score, <4)
High RIN1 (total score, ≥4)
P value
gastric adenocarcinoma
n0144 n0171
Age (years)
<60 177 89 88 0.0653
≥60 138 55 83
Gender
Male 165 77 88 0.7219
Female 150 67 83
Tumor size (cm)
≤5 75 48 27 0.0003
>5 240 96 144
Histological grade
Well differentiated (G1) 23 15 8 0.0396
Moderately differentiated (G2) 136 67 69
Poorly differentiated (G3) 156 62 94
WHO type
Tubular 256 123 133 0.1089
Mucinous 13 7 6
Papillary 15 6 9
Signet ring cell 31 8 23
Lymph node involvement
0 60 42 18 0.0001
1 65 33 32
>1 190 69 121
TNM stage
I 34 20 14 0.0372
II 45 26 19
III 172 76 96
IV 64 22 42
Ki67
0 167 92 75 0.0015
1 72 35 37
2 76 23 53
Fig. 4 Overall survival rate of gastric adenocarcinoma patients relative to RIN1 expression levels in gastric adenocarcinoma tissue samples (Kaplan–Meier method) determined by immunohistochemical staining (P<0.001)
occurs via DNA methylation within the RIN1 promoter and/ or overexpression of the Snail transcription repressor [14]. In addition, RIN1 overexpression inhibits the initiation and progression of breast tumorigenesis both in vitro and in vivo, and RIN1 knockdown significantly enhances the invasive properties of breast tumor cells [14, 23]. Therefore, RIN1 has been characterized as a breast tumor suppressor protein that acts as a negative regulator of tumor cell inva- sive growth. It is therefore probable that RIN1 functions in a cell type-specific manner. Although a high-level expression of RIN1 in gastric cancer cell lines has also been reported, RIN1 expression in gastric adenocarcinoma tissues and its correlation with clinicopathological features have not yet been determined.
To address these issues, we first investigated RIN1 mRNA and protein expression in gastric adenocarcinoma
Table 2 Univariate analysis and multivariate analysis identifies
Variables Univariate analysis Multivariate analysis
factors influencing the overall
survival rate of gastric adeno- carcinoma patients HR 95%CI P value HR 95%CI P value
RIN1 1.987 1.023–3.674 0.015 1.876 1.082–3.543 0.014
Age 0.867 0.435–1.435 0.354
Gender 0.679 0.367–1.231 0.442
Tumor size 1.342 0.897–1.964 0.008 1.231 0.987–2.856 0.013
Histologic grade 1.213 0.768–2.324 0.014 1.356 0.823–1.978 0.012
Lymph node involvement 1.456 0.986–2.546 0.035 1.678 1.123–2.134 0.014
Tumor stage 1.465 0.967–1.956 0.013 1.213 0.684–1.563 0.005
Ki67 1.342 1.123–1.678 0.003 1.751 1.134–2.341 0.007
specimens by qRT-PCR and Western blotting, respectively. RIN1 transcript and protein levels were determined in 40 pairs of resected specimens (tumor tissue samples and matched adjacent non-tumor tissue samples) from gastric adenocarcinoma patients. We observed that RIN1 mRNA and protein levels were significantly increased in 24 (60 %) and 26 (65 %) tumor tissue samples, respectively, compared with adjacent non-tumor tissues. In addition, the RIN1 protein and mRNA showed similar expression patterns in the matched samples. These observations support the hypothesis that RIN1 may function as an oncogene in gastric adenocarcinoma and also suggest that RIN1 may play an important role in the tumorigenesis of gastric adenocarcinoma.
Furthermore, in a relatively large series of gastric adeno- carcinoma patients (n 0315), we observed that a high RIN1 expression associated with poor tumor differentiation, large tumor size, NM stage, and lymph node metastasis. This strengthens the hypothesis that RIN1 acts as an oncogene in gastric adenocarcinoma. The Ki-67 antigen is a cell proliferation marker; Ki-67 expression strictly correlates with cell cycle progression and can be observed in the G1, S, and G2 phases and mitotic cells. In this study, we observed that a high RIN1 overexpression associated with a high Ki-67 index.
In the Kaplan–Meier survival analysis, the overall survival period of patients with tumors with a high RIN1 expression was significantly shorter than that of patients with a low RIN1 expression. Univariate analyses showed that an increased RIN1 expression in gastric adenocarcinoma tissues is significantly associated with the overall survival rate. Moreover, multivariate analysis demonstrated that RIN1 expression, together with some traditional prognostic factors such as tumor size, lymph node status, and NM stage, is an independent risk factor in the prognosis of gastric adenocar- cinoma patients. These results suggest that the detection of increased RIN1 expression might help identify gastric adeno- carcinoma patients with a poor prognosis and could therefore be a novel prognostic marker for gastric adenocarcinoma patients.
In conclusion, our results indicate that a high RIN1 expression in gastric adenocarcinoma may be important for tumor progression and thus serves as an independent biomarker for poor survival. Therefore, a high RIN1 expres- sion identifies high-risk patients and is a potential novel therapeutic target for gastric adenocarcinoma.
Conflicts of interest None.
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