Int J Clin Exp Pathol 2015;8(4):3710-3718 www.ijcep.com /ISSN:1936-2625/IJCEP0006048

Original Article Decreased SATB2 expression is associated with metastasis and poor prognosis in human clear cell renal cell carcinoma Changcheng Guo*, Dabo Xiong*, Xudong Yao, Wenyu Gu, Haimin Zhang, Bin Yang, Bo Peng, Min Liu, Junhua Zheng Department of Urology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, People’s Republic of China. *Equal contributors. Received January 17, 2015; Accepted March 19, 2015; Epub April 1, 2015; Published April 15, 2015 Abstract: In this study, we investigate the expression and role of special AT-rich sequence-binding protein-2 (SATB2) in clear cell renal cell carcinoma (ccRCC) tissue, and to evaluate the clinical and prognostic significance of SATB2 protein in patients with ccRCC. The expression of SATB2 and SATB1 was examined in ccRCC tissue by Western blotting, real-time PCR and immunohistochemical staining. The association between clinicopathological features and SATB2 level was investigated. The correlation of SATB2 expression with overall survival was also analyzed. The expression of SATB2 protein in tumor tissues was much lower than that in paired normal tissues. The overall survival of the patients with high SATB2 expression was significantly higher than that of the low SATB2 expression group. Low or negative SATB2 expression was significantly correlated with AJCC staging and Furman grade in ccRCC. In contrast, the expression of SATB1 was significantly higher in adjacent tumor tissue than that in normal and tumor tissues. This study provides the first evidence of the expression and clinical significance of SATB2 in ccRCC. Our data suggest that SATB2 functions as a tumor suppressor in the development and progression of ccRCC, and is thereby implicated as a valuable prognostic marker for ccRCC patients. Keywords: SATB2, RCC, prognosis, SATB1

Introduction Renal cell carcinoma (RCC) is the most prevalent malignancy of the adult kidney, accounting for approximately 90-95% of all kidney neoplasm [1]. In the United States, the incidence of RCC has increased in the past few decades (between 2% and 4% annually) [2]. RCC accounted for an estimated 65,150 new cases and 13,680 deaths in 2013 [3]. Up to 30% of RCC patients present with metastatic disease and approximately 40% of patients present with recurrence after treatment for a localized tumor [4, 5]. RCC is heterogeneous and comprises several histological types, which have different genetic and clinicopathological features that determine clinical course and outcome. Clear cell RCC (ccRCC), also called conventional RCC, is the most common histological type of RCC, representing approximately80% of cases [6]. ccRCC is highly resistant to chemo-

therapy and radiotherapy, and approximately 10% of patients suffering from metastatic disease survive 5 years after diagnosis [7]. As a result, a better understanding the molecular mechanisms involved in the initiation and progression of ccRCC is required. Further identification of novel biomarkers associated with ccRCC progression and metastasis and their combined application with traditional diagnostic and prognostic parameters would contribute to the development of effective strategies for the prevention, early diagnosis and treatment of ccRCC [8]. Special AT-rich binding-sequence protein 2 (SATB2), a DNA binding protein, specifically binds nuclear matrix attachment regions involved in transcriptional regulation and chromatin remodeling [9]. SATB2 participates in a variety of important biological processes included cell differentiation [9], histone recruitment [10],

SATB2 and prognosis in renal cell carcinoma Table 1. Clinical characteristics of the patients with ccRCC Variables Total Sex Male Female Age < 45 45-65 > 65

Patients 73 45 28 19 32 22

Furman grade I II III IV 30 22 15 6 18 13 10 4 12 9 5 2 8 5 5 1 15 9 5 3 7 8 5 2

AJCC staging I II III IV 27 23 17 6 17 15 9 4 10 8 8 2 7 6 5 1 12 9 8 3 8 8 4 2

AJCC, American Joint Committee on Cancer (2002 version).

transcription and reprogramming of expression profiles [11]. However, the role of SATB2 in most tumors is still unclear. Wang et al [12] reported that downregulated expression of SATB2 is associated with metastasis and poor prognosis in colorectal cancer. Recently, increased expression of SATB1 has been found in human renal cell carcinoma [8]. However, SATB2 expression and its clinical significance in RCC remain unclear. In the present study, we evaluatedSATB2 expression in clinical RCC tissue specimens by Western blot, immunohistochemistry, and realtime PCR. Furthermore, the correlation between SATB2 expression and clinicopathological characteristics was investigated. SATB1 expression was also detected in clinical RCC tissue specimens and its correlation with SATB2 expression was determined. Materials and methods Ethics statement This study was performed under a protocol approved by the Ethics Committee of Shanghai Tenth People’s Hospital, Tongji University School of Medicine (China). Written informed consent for participation in the study was obtained from each patient. Patients and samples Between January 2008 and August 2011, 75 patients were enrolled in the study in the Department of Shanghai Tenth People’s Hospital. None of the patients had received preoperative treatment. Laboratory and preoperative clinical data, including complete blood count, liver biochemistry, renal function, and tumor 3711

size, stage (American Joint Committee on Cancer (AJCC), 2002 version) and grade (Furhman grade I to IV), were assembled in a computerized database for each participant. Three different types of tissues from each RCC patient were processed immediately after surgical resection: tumor-free tissue > 5 cm far from the tumor edge (TF), adjacent nonmalignant tissue within 2 cm (AT), and tissue from the tumor (TT). Areas of tissue necrosis and hemorrhage were excluded. The specimens from each patient were divided into two parts. One part was snap-frozen immediately after resection and stored in liquid nitrogen until they were used for experiments. The other part was preserved in 10% formaldehyde solution and paraffin-embedded. Real-time PCR analysis Total RNA was extracted from the tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions, and was then used for the synthesis of first strand complementary DNA (cDNA) using the PrimeScript® 1st Strand cDNA Synthesis kit (TaKaRa Bio, Shiga, Japan). A total of 1 μl of the reverse transcription (RT) product was then used as the template to amplify specific SATB1 and SATB2fragments.Primers for human SATB1, SATB2 and β-actin genes were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA) and synthesized by Sangon (Shanghai, China). The basic information on primers, including gene name, forward primer, reverse primer and product size (bp) is presented in Table 1. Real-time PCRs were performed in triplicate for each sample in a 20 μl reaction mixture, containing 2 μl template DNA, 1 μl primers, 10 μl SYBR premix and 7 μl ddH2O, using an ExScript Real-time PCR kit (TaKaRa Bio). PCR was performed in a 7900HT Fast Real-Time PCR machine (Applied Biosystems) under the following conditions: 95°C for 30 s, then 40 cycles of 95°C for 5 s and 60°C for 30 s. Gene expression was presented using a modification of the 2-ΔΔCt method. Western blotting Total protein was extracted using lysis buffer. The protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Equal quantities of protein were separated by SDSInt J Clin Exp Pathol 2015;8(4):3710-3718

SATB2 and prognosis in renal cell carcinoma and mouse anti-human SATB2 monoclonal antibody (Abcam) at a 1:200 dilution. Expression was detected using an Envision™ Detection kit, (peroxidase/DAB, rabbit/mouse; Gene Tech, Shanghai, China) in accordance with the manufacturer’s instructions. The slides were then stained with DAB, washed, counterstained with hematoxylin, dehydrated, treated with xylene and mounted. The followed method of interpreting protein staining was employed to evaluate SATB1 and SATB2 protein expression. Two independent observers scored the percent of positively staining cells per high-power field in the tissue Figure 1. Expression of SATB1 and SATB2 protein in 38 clear cell renal cell sections. The number of posicarcinoma (ccRCC) and paired normal tissue samples. Western blot analysis of (A) SATB1and (B) SATB2 in ccRCC. The protein expression levels of (C) tive cells per high-power field SATB1 and (D) SATB2 were quantified by comparing the grayscale levels of was assessed as -/+ = 0-10% each band in (A) and (B), respectively using Quantity One Software. (N: norstaining positively of cells, + = mal tissue, T: tumor tissue). 10-25% staining positively of cells, ++ = 25%-50% of cells, +++ = 50%-75% of cells, and ++++ = > 75% of polyacrylamide gel electrophoresis with triscells with positive staining. glycine running buffer, prior to being transferred to nitrocellulose membranes. Blots were blockStatistical analysis ed with 5% skimmed milk and 0.1% Tween in Tris-buffered saline, and incubated with antiStatistical analyses were performed using bodies against SATB1 (monoclonal antibody, SPSS 13.0 statistical software (SPSS, Chicago, Abcam, Cambridge, UK, 1:200) or SATB2 IL, USA). Data are presented as the mean ± (monoclonal antibody, Abcam, Cambridge, UK, standard deviation (SD) and analyzed using 1:200) independent t-tests. P < 0.05 was considered to indicate a statistically significant difference. Immunohistochemical staining of tissue sections Results Immunostaining was performed on paraffinembedded 4 μm sections of formalin-fixed tumor tissues, placed on chrome alum gelatincoated glass slides and dried for 30 min at 70°C. Following rehydration, the tissue sections were incubated in 3% hydrogen peroxide (Bio Basic, Amherst, NY, USA) to inhibit endogenous peroxidase activity. Following citrate buffer antigen retrieval, the sections were blocked by incubation in 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) (Bio Basic). Expression of SATB1 and SATB2 was assessed using mouse anti-human SATB1 (Abcam, Cambridge, UK) monoclonal antibody

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Clinicopathological characteristic of the selected patients A total of 73 patients with ccRCC, who were recruited from the Department of Urology, Shanghai Tenth People’s Hospital between July 2009 and June 2011, were included in this study. The clinical characteristic of these 73 patients are summarized in Table 1. SATB2 expression in ccRCC patients To determine the expression of SATB2 protein in ccRCC patients, Western blotting was per-

Int J Clin Exp Pathol 2015;8(4):3710-3718

SATB2 and prognosis in renal cell carcinoma

Figure 2. Quantitative PCR analysis of the expression of SATB1 and SATB2 in clear cell renal cell carcinoma (ccRCC) and adjacent normal tissue. Relationship between clinicopathological characteristics and SATB2 expression in patients with ccRCC (A, B); Relationship between AJCC staging and SATB2 expression in patients with ccRCC (C); Relationship between Furman grade and SATB2 expression in patients with ccRCC (D).

formed in38 ccRCC tissues with paired normal tissue. Compared with the paired normal tissues, significantly decreased SATB2 levels were detected in 36 of 38 ccRCC tissues (Figure 1B and 1D), while increased expression was detected in 2 of 38 ccRCC tissues (Figure 1B, Case 2). We then measured the expression of SATB2 transcripts in ccRCC patients by realtime PCR. Compared with TF tissue, SATB2 expression was significantly lower in AT and TT tissue, with decreased expression in 84% (61/73) of samples. The mean relative expression of SATB2 in AT and TT tissues were 0.73 ± 0.38 and 0.65 ± 0.29 (P < 0.0001, Figure 2B). To verify this observation, we further examined the expression of SATB2 protein in 73 paraffinembedded ccRCC samples with paired normal tissues by immunohistochemical analysis (Figure 3). There was a significant decrease in SATB2expression in the tumor tissue compared 3713

with that in the normal tissue (Table 2). Among the 73 paraffin-embedded tumor tissue samples, 54 showed negative nuclear or cytoplasmic staining of SATB2protein, while 11 showed mainly moderate staining and eight showed strong staining. Relationship between clinicopathological characteristics and SATB2 expression in patients with ccRCC The association betweenSATB2 expression and the clinicopathological outcomes are shown in Table 2. SATB2 expression exhibited a significantly negative correlation with Furman grade and AJCC staging (Figure 2C, 2D). Association between SATB2 expression and SATB1 expression To investigate the mechanism of SATB2 mediated regulation on prognosis of ccRCC, addiInt J Clin Exp Pathol 2015;8(4):3710-3718

SATB2 and prognosis in renal cell carcinoma

Figure 3. Immunohistochemical analysis of the expression of SATB2 in ccRCC tumor tissues. SATB2 expression in normal tissues (A: magnification, × 100). SATB2 expression in normal tissues (B: magnification, × 200). SATB2 expression in adjacent tumor tissues (C: magnification, × 100). SATB2 expression in adjacent tumor tissues (D: magnification, × 200). SATB2 expression in tumor tissues (E: magnification, × 100). SATB2 expression in adjacent tumor tissues (F: magnification, × 200).

tional immunohistochemical staining were performed to detect the levels of SATB1, a homologue of SATB2, and to analyze the potential correlation between SATB1 and SATB2 expression in ccRCC tissues. We found that SATB1 levels were significantly down-regulated in ccRCC tissues compared with those in the paired normal tissues, which were in accordance with the expression of SATB2 (Figures 1A, 1C, 2A, 4). Correlation of SATB2 expression with overall survival We defined < 25% and > 25% positive immunohistochemical staining of SATB2 as low and 3714

high expression, respectively. Among the pa tients included in this study, the overall survival of the patients with high SATB2 expression (3-year overall survival rate, 85%) was significantly higher than that of the low SATB2 expression group (3-yearoverall survival rate, 60%) (P = 0.04, Figure 5). Discussion SATB2 is a novel AT-rich DNA binding protein, which regulates gene expression by altering chromatin structure.SATB2 interacts directly with the activity of transcription factors that regulating craniofacial development, cortical neuron differentiation and osteoblast differenInt J Clin Exp Pathol 2015;8(4):3710-3718

SATB2 and prognosis in renal cell carcinoma Table 2. Results from the immunohistochemistry analysis No. of patients 0 0 1 1 5 3 0 0 1 5 2 4 0 1 1 9 11 10 0 0 1 2 4 4 0 0 1 1 1 2 0 0 0 2 0 1

SATB2 TT N +/I I + IV IV ++ III (2)/II (2)/I III/II (3)/I +++ III (2)/II III/I (2) ++++ +/III IV + +/IV/III (2)/II (2) III (3)/II (2) ++ II/I IV/I +++ III/II (2)/I II (3)/I ++++ IV IV +/I I + + II (5)/I (4) II (5)/I (4) ++ IV/III (3)/II (3)/I (4) IV/III (3)/I (7) +++ IV/III (3)/II (2)/I(4) III (5)/II (4)/I ++++ +/I IV + ++ II (2) I (2) ++ IV/III/II/I III/II (2)/I +++ I (4) III (2)/II (2) ++++ +/II III + +++ I I ++ I I +++ I (2) II/I ++++ +/+ ++++ I (2) II/I ++ +++ I I ++++ Furman grade

AJCC staging

(-/+), 0-10%; (+), 10-25%; (++), 25–50%; (+++), 50-75%; and (++++), > 75% positive. AJCC, American Joint Committee on Cancer staging system.

tiation [13, 14]. In humans, SATB2 has been identified as a cleft palate geneon chromosome pair 2, which undergoes translocation and plays an important role in palate formation [15]. Recent studies conducted to evaluate the role of SATB2 in colorectal, breast, laryngeal 3715

and oral carcinomas [12, 13, 16, 17] have yielded highly controversial results. Therefore, in the present study, we focused on SATB2 expression patterns and their relationship with the clinicopathological features of ccRCC patients. Our study, which provides new insight into the role of SATB2 in ccRCC progression, reveals the first evidence that SATB2 expression is much lower in carcinoma tissues than that in normal tissue in RCC. Furthermore, our findings implicate SATB2 as a prognostic factor for RCC patients. As shown in the present study, SATB2 expression was significantly lower in tumor tissue compared to that in paired normal tissue at both the RNA and protein levels. Moreover, low levels of SATB2 expression correlated with high AJCC staging and high Furman grade. Our results also indicated that SATB2 protein expression levels were closely correlated with overall survival. This result indicates that low SATB2 expression levels in renal tissue are related to poor prognosis. However, due to the small number of patients in our study, further studies are required to verify these findings and to establish SATB2 as a reliable clinical predictor of outcome in RCC patients. SATB1, which is a close homolog of SATB2, overexpressed in a variety of tumor types including breast cancer [18], laryngeal squamous cell carcinoma [19], gastric cancer [20], and malignant cutaneous melanoma [21]. In this study, we found higher expression ofSATB1mRNA and protein in adjacent nonmalignant tissue than that in tumor tissue and normal tissue. However, Chao et al. reported that levels of SATB1 mRNA and protein were dramatically increased in human ccRCC tissues, and that upregulation of SATB1 was significantly associated with depth of invasion, lymph node status and TNM stage [8]. It can be speculated that this discrepancy arises from our selection of adjacent nonmalignant tissue located approximately 2 cm from tumor because of difficulties in distinguishing the adjacent nonmalignant tissue from the tumor and normal tissues. Moreover, although we aimed to exclude necrotic and hemorrhagic tissues from the sample, some necrosis of the tumor tissue was unavoidable. Unfortunately, further discussion of this discrepancy is not possible because the report by Chan et al. did not include any Int J Clin Exp Pathol 2015;8(4):3710-3718

SATB2 and prognosis in renal cell carcinoma

Figure 4. Expression of SATB1 in ccRCC tumor tissues. SATB1 expression in normal tissues (A: magnification, × 100). SATB1 expression in normal tissues (B: magnification, × 200). SATB1 expression in adjacent tumor tissues (C: magnification, × 100). SATB1 expression in adjacent tumor tissues (D: magnification, × 200). SATB1 expression in tumor tissues (E: magnification, × 100). SATB1 expression in adjacent tumor tissues (F: magnification, × 200).

detail regarding the selection of tissue samples. Nevertheless, our data clearly demonstrate low expression of SATB1 in normal tissue, and that SATB1 upregulation was significantly associated with poor prognosis. These observations indicate that SATB1 andSATB2 are associated with opposite effects in ccRCC, although the specific mechanisms require further studies clarification. Immunohistochemistry has several advantages compared to analysis of mRNA levels in that it allows for assessment of candidate protein bio-

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markers in a morphological and sub-cellular context [22]. Studies have demonstrated that even a small fraction of SATB1positive cells by detected by immunohistochemistry is sufficient to confer a poor prognosis in some tumor types [18, 23]. Moreover, results from studies on the prognostic value of SATB1 mRNA levels have shown discrepant results in relation to its protein expression [24, 25]. In the present study, we used immunohistochemical analysis to evaluate the expression of SATB2 and its correlation with clinicopathological characteristics and overall survival. Although a significant

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SATB2 and prognosis in renal cell carcinoma References [1]

Figure 5. Survival curves for patients with high versus low SATB2-expressing carcinoma. The 3-year overall survival rate was 85% in the high SATB2 protein expression group (green line) (n = 19), but only 60% in the low expression group (blue line) (n = 54), (P = 0.04).

association was found between SATB2 expression and poor prognosis, the small number of participants and short follow-up time prevented us forming precise conclusions regarding the potential prognostic value of SATB2 expression in ccRCC. Moreover, a correlation between Furman grade and AJCC staging and the expression of SATB2 was identified in this study. However, due to the small number of samples in each stage, no significant difference was found among the groups; therefore, the detailed mechanisms underlying the functions of SATB2 in ccRCC require further investigation. Conclusion This study provides the first description of the expression and clinical significance of SATB2 in ccRCC and provides evidence suggesting that SATB2 functions as a tumor suppressor in the development and progression of ccRCC. Thus, SATB2 is implicated as a valuable prognostic marker for ccRCC patients. Disclosure of conflict of interest None. Address correspondence to: Dr. Min Liu or Dr. Junhua Zheng, Department of Urology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, NO. 301, Yan-chang Road, Shanghai 200072, People’s Republic of China. Tel: +86 21 66307508; Fax: +86 21 66301655; E-mail: [email protected] (ML); Tel: +86 21 63307478; Fax: +86 21 66301655; E-mail: zhengjh0471@sina. com (JHZ)

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