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SOX4 expression levels in urothelial bladder carcinoma
Pathology – Research and Practice 207 (2011) 423–427
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.de/prp
Original article
SOX4 expression levels in urothelial bladder carcinoma夽 Sezgin Gunes a,∗ , Zeynep Yegin a , Yurdanur Sullu b , Recep Buyukalpelli c , Hasan Bagci a a
Department of Medical Biology and Genetics, Ondokuz Mayis University, Faculty of Medicine, 55139, Samsun, Turkey Department of Pathology, Ondokuz Mayis University, Faculty of Medicine, 55139, Samsun, Turkey c Department of Urology, Ondokuz Mayis University, Faculty of Medicine, 55139, Samsun, Turkey b
a r t i c l e
i n f o
Article history: Received 6 January 2011 Received in revised form 11 April 2011 Accepted 9 May 2011 Keywords: Bladder carcinoma SOX4 gene Gene expression
a b s t r a c t High levels of SOX4 gene expression have been reported in a variety of human cancers. The protein may function in the apoptosis pathway, leading to cell death as well as to tumorigenesis. The aim of this study was to investigate the levels of SOX4 expression in bladder cancer. Urinary bladder tumor samples were obtained from 57 bladder cancer and 13 normal bladder biopsies. The levels of SOX4 expression in bladder cancer were determined by immunohistochemistry and real-time PCR. SOX4 gene expression was increased 2.2 times in bladder tumors as compared with normal tissue. The presence of protein was confirmed by immunostaining. There were significant differences between immunostaining of bladder tumors and normal bladder tissue (P = 0.001). The present data suggest that SOX4 gene may have a role in bladder cancer tumorigenesis. © 2011 Elsevier GmbH. All rights reserved.
Introduction Bladder carcinoma (BC) is the second most common malignancy affecting the urinary system [1]. Numerous genetic and epigenetic alterations which are responsible for the transformed cell phenotype have been identified. According to the theory of cancer clonality, a long process of natural selection and clonal evolution finally represents the tumor phenotype arising from a single cell. Much effort has focused on identifying new genes that have roles in bladder carcinoma, and one of these genes that has drawn attention to in recent years is sex-determining region Y (SRY) related high mobility group (HMG) box (SOX4) gene (MIM ID *184430). It has been reported that the expression of SOX4 gene is increased in most bladder cancer cases [2]. SOX4 is a member of the SRY box (SOX) transcription factor family, which consists of 20 highly conserved transcription factors in humans. SOX4 gene is located on 6p22.3 and encodes a 47 kDa protein (474 amino acids) [3,4]. The protein coded by SOX4 has three major domains: a HMG box (aa 57–135), a glycine-rich region (aa 152–227), and a serine-rich region (SRR, aa 333–397). While the HMG box acts as DNA-binding region, SRR domain acts as transactivation domain. The glycine-rich region, which is located between the HMG box and SRR, is a part of the central domain, and this region has a function in promoting apoptotic cell death [5].
夽 Support: This study was supported by Ondokuz Mayis University Research Foundation (Project No. PYO.TIP.1901.08.557). ∗ Corresponding author. Tel.: +90 362 312 19 19x3164; fax: +90 362 457 60 41. E-mail address: [email protected] (S. Gunes). 0344-0338/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2011.05.005
SOX4 plays important roles in many developmental processes, such as embryonic cardiac development, thymocyte development, T cell differentiation pathway, and nervous system development [6–8]. The role of SOX4 in tumorigenesis is controversial. SOX4 upregulation has been associated with several types of cancers, including hepatic, breast, brain, lung, pancreatic, salivary gland, bladder, and ovarian cancers [3]. Aaboe et al. [2] were the first to delineate the upregulation (∼5-fold) of human transcription factor SOX4 in bladder carcinomas. To the best of our knowledge, since then there has been no report regarding the interaction between SOX4 expression and bladder carcinoma. This prompted us to examine the SOX4 expression by real-time PCR and immunohistochemistry in bladder tumor samples and compare our results with those of the previous findings. In the present study, qPCR and immunohistochemistry were employed in order to detect and verify the expression levels of SOX4 in patients with urinary bladder cancer. Materials and methods Subjects Fifty-seven patients with bladder carcinoma and 13 nonbladder cancer subjects attending the Urology Clinics of the Medical Faculty, Ondokuz Mayis University (OMU), between January 2007 and September 2010, were enrolled in this study. The patients signed an informed consent form after receiving information about the study. The study was approved by the ethical committee of the OMU. Table 1 presents the clinicopathological parameters of the
424
S. Gunes et al. / Pathology – Research and Practice 207 (2011) 423–427
Table 1 Clinicopathological characteristics of the subjects. Immunohistochemical analysis
qPCR analysis
Patients (n = 57) Controls (n = 13) Patients (n = 25) Controls (n = 3) Gender 52 Male Female 5 Age Mean 65.65 ± 1.69 Range 26–81 Tumor stage 7 Ta 33 T1 T2 16 T4 1 Tumor grade Low 31 26 High
9 4
24 1
3 –
63.66 ± 4.38 32–79
64.08 ± 2.46 40–86
68.33 ± 2.46 64–73
5 4 16 – 16 9
subject population. Tumor samples were obtained directly from surgical material after the removal of the necessary amount of tissue by transurethral resection of the bladder for routine pathological examination. Tumor and normal tissues were collected in 0.9% filter sterilized NaCl, immediately snap frozen, and stored at −80 ◦ C until further RNA extraction. Immunohistochemistry In situ detection of SOX4 was performed for tissue samples from 57 patients with urothelial carcinoma and 13 non-bladder cancer subjects in the Department of Pathology. Hematoxylin–eosin sections of cases included in the study were re-evaluated and classified according to the WHO/ISUP (2004) and staged according to TNM (2002) [9]. According to the depth of invasion, 57 cases were classified: Ta is a non-invasive papillary tumor, T1 is tumor that invades subepithelial connective tissue, T2 is tumor that invades muscle, and T4 is tumor that invades any of the following: prostate, uterus, vagina, pelvic wall and abdominal wall. All tissues were fixed in a 10% buffered formaldehyde solution for 24 h and blocked in paraffin. Among the sections investigated, one block representing the tumor containing no hemorrhages or necrosis was chosen. The blocks were sectioned at 4 m in thickness and studied immunohistochemically using the streptavidin biotin peroxidase method using primary antibody against SOX4 (rabbit, polyclonal, S7318, Sigma–Aldrich). As positive controls, we used a thymus section. The best dilution rates were recorded by various attempts. SOX4 was diluted at 1/100. The endogenous peroxidase activity of deparaffinized sections was eliminated by incubation for 10 min in 3% hydrogen peroxide solution. Next, the sections were boiled for 35 min in citrate buffer and left to cool for 20 min. Blocking antibody was applied to the sections for 5 min and SOX4 primary antibody applied for 1 h. The sections were then incubated with biotinylated anti-immunoglobulin and streptavidin–peroxidase conjugate for 10 min each. A kit containing 3,3 -diamino benzidin (DAB) (DAKO, Carpinteria, USA) was used as the coloring agent. Finally, the sections were dyed with Mayer’s Hematoxylin for 60 min. Until DAB application, phosphate buffers (pH 7.6) were used in all stages, while distilled water was used following DAB application. The procedures were performed at room temperature. The results were evaluated using a Leica HMLB45 (Germany, 2000) light microscope considering cytoplasmic and nuclear dyeing. Assessment of the extensiveness and intensity of dyeing was performed semi-quantitatively in tumor areas selected in each block. The extensiveness of dyeing for SOX4 was graded as follows: dyeing of 0–5% of tumor cells as 0, dyeing of 6–25% of cells as 1, dyeing of 26–50% of the cells as 2, and dyeing of over 50% of cells as
3. Assessment of the intensity of dyeing was graded as follows: light yellow as 1, dark yellow as 2, and brown as 3. The values obtained from the sum of these two degrees were scored as: between 0 and 2 as negative (0), weak (1) for 3, moderate (2) for 4, and strong (3) for 5 and 6. RNA isolation Total RNA was extracted from bladder tissues using a commercially available kit (SV Total RNA Isolation System, Promega, Madison, WI), according to the manufacturer’s instructions. The final RNA preparations were diluted in 1/100 ratio in diethylpyrocarbonate-treated water and quantified by analyzing absorbance at 260 nm. The integrity of 28S and 18S rRNA was checked by electrophoresis of 1 g of total RNA in 1% agarose gel. RNA isolation was performed for all tumor samples. However, RNA of 32 samples could not be used because either the quantity of surgical tissue was limited or RNA was degradated. Only samples with clearly defined ribosomal peaks were used in the study. Reverse transcription Approximately 1 g of total RNA was used in 20 l of reverse transcription (RT) reaction mixture for cDNA synthesis with a RevertAidTM First Strand cDNA Synthesis Kit (MBI Fermentas, UK) and oligo-dT primers. RT reactions were performed for 1 h at 42 ◦ C as described in the commercial kit’s protocol. Determination of gene expression Real-time PCR was performed on SmartCyclerII (Cepheid, USA) using MaximaTM SYBR Green qPCR Master Mix (Fermentas, Glen Burnie, MD). Primer sequences were as follows: SOX4 (forward primer 5 -GTGAGCGAGATGATCTCGGG-3 ; reverse primer 5 -CAGGTTGGAGATGCTGGACTC), GAPDH (forward primer 5 -ACCACAGTCCATGCCATCAC-3 ; reverse primer 5 -TCCACCACCCTGTTGCTGTA-3 ) [2]. Master mixes were prepared and used in the PCR amplifications. A typical reaction mixture of 30 l consisted of 15 l SYBR Green master mix, 0.3 M of each of forward and reverse primers and 12.2 l of nuclease-free water. PCR amplification was done at 95 ◦ C for 10 min followed by 40 cycles of 95 ◦ C for 15 s, 60 ◦ C for 30 s, 72 ◦ C for 30 s and final melting curve from 60 ◦ C to 95 ◦ C with increments of 0.2 ◦ C/s. Primer specificity was confirmed by melting curve analysis and electrophoresis. PCR products were electrophoresed in 2% ethidium bromide-stained agarose gels, and the presence of the DNA bands in expected sizes was confirmed. Within each experiment, we loaded RNase-free water as our negative control, and samples were run as duplicates to control for variability in pipetting and cycling conditions, and the calculations were made on the average numbers of the duplicates. When the difference between duplicated samples exceeded 20%, these samples were quantified again. Computational analysis One microliter of cDNA product (equivalent to 50 ng RNA starting material) was used for assessing gene expression across groups by analyzing Ct values. The Cycle-Threshold (Ct ) is the numeric value for the cycle at which the product crossed the threshold. Reactions with lower Ct values contain more of the gene of interest since they took less time to amplify. On the other hand, samples with a higher Ct contain less of the gene of interest. The threshold cycle values for each target transcript was normalized to that of the gene encoding GAPDH by calculating the Ct according to the formula
S. Gunes et al. / Pathology – Research and Practice 207 (2011) 423–427
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Fig. 1. Cytoplasmic and nuclear immunohistochemical staining of SOX4 in T2, high-grade tumor, ×200 (A); in T1, low-grade tumor, ×200 (B); cytoplasmic staining in T1, high-grade tumor, ×200 (C) and cytoplasmic and nuclear immunostaining in only superficial cells in a control case, ×400 (D). target
Ct = Ct − CtGAPDH . The analysis of relative gene expression was performed by the comparative 2−Ct method [10].
between SOX4 expression and clinical variables such as recurrence, progression, and tumor-specific survival (Table 3).
Statistical analysis
Expression profile of SOX4 in bladder carcinoma
Data were analyzed using SPSS for Windows (version 15). To calculate relative fold-changes between patient and control samples, we utilized the delta C(T) method, calculated as follows: {[C(T)(SOX4)] − [C(T)(GAPDH)]} [10]. The Mann–Whitney test was carried out to compare SOX4 gene expression levels between highgrade and low-grade tumors. P < 0.05 was considered statistically significant.
We did a gene expression analysis using quantitative real-time PCR and determined the expression of SOX4 in comparison with that of the housekeeping gene GAPDH. Gene expression profiles were generated for 25 bladder tumor samples and 3 control bladder samples. The expression change was calculated based on the 2−Ct method as proposed by Livak and Schmittgen [10]. We found a moderate 2.2-fold upregulation of SOX4 in bladder tumors, as compared with normal bladder tissues, at all stages of bladder cancer (Fig. 2). It was also evident that in some tumor samples, SOX4 expression was downregulated as compared with those of normal bladder.
Results SOX4 expression by immunohistochemistry Of 57 tumors examined, 33 were classified as T1, 16 as T2, 7 as Ta, and 1 as T4 according to the WHO/ISUP (2004) and staged according to TNM (2002) (Table 1). SOX4 was expressed in all tumor samples and in 6 (46%) control specimens (P = 0.001). SOX4 was strongly expressed in 49 (86%), moderately expressed in 4 (7%), and weakly expressed in 1 (2%), and not expressed in 3 (5%) tumor samples. Expression of SOX4 protein was observed in both the cytoplasmic and nuclear cell compartments. SOX4 was strongly expressed in 1 (8%), moderately expressed in 3 (23%), and weakly expressed in 2 (15%) control samples (Fig. 1). Seven (54%) control samples were SOX4-negative. In control cases, expression of SOX4 was observed only in superficial cells. We did not find any correlation between SOX4 expression and tumor stage (P = 0.636) and grade (P = 0.887) (Table 2). There was no significant correlation
No correlation between tumor grades and SOX4 expression We also aimed to investigate whether there was an expression difference between high-grade and low-grade tumors. Previously, it was shown by Aaboe et al. [2] that higher SOX4 expression results in better survival in bladder tumor patients. We thought that we could make an assumption about prognosis on the basis of expression differences between high-grade and low-grade tumors. Using the Mann–Whitney test, we compared the expression differences between high-grade and low-grade tumors. Low-grade tumors exhibited a slight increase in SOX4 expression when compared with the high-grade ones (mean ranks were 14.06 and 11.11, respectively). However, the differences were not statistically significant.
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Table 2 Immunohistochemical analysis of SOX4 in patients with bladder carcinoma and controls. Pathological classification
Stage Ta (n = 7) T1 (n = 33) T2 (n = 16) T4 (n = 1) Grade Low (n = 32) High (n = 25) All tumors (n = 57) Controls (n = 13) a
SOX4 Negative (%)
Weak (%)
Moderate (%)
Strong (%)
Pa
– 3 (9) – –
– 1 (3) – –
– 2 (6) 2 (12) –
7 (100) 27 (82) 14 (88) 1 (100)
0.636
3 (9) – 3 (5)
– 1 (4) 1 (2)
2 (6) 2 (8) 4 (7)
27 (85) 22 (88) 49 (86)
7 (54)
2 (15)
3 (23)
1 (8)
0.887
Fisher’s exact test.
Discussion In the present study, qPCR and immunohistochemistry were employed in order to detect and verify the expression levels of SOX4 in patients with urinary bladder cancer. Immunohistochemistry revealed that there were significant differences between immunostaining of bladder tumors and normal tissue. SOX4 protein was expressed in both nuclei and cytoplasm. On the other hand, we did not find any correlation between SOX4 expression and clinical variables, tumor stage, and grade. We found a moderate total increase (2.2-fold) in SOX4 gene expression in bladder tumors as compared with the normal bladder tissues. Immunohistochemistry results of a previous study have also shown that SOX4 was highly upregulated in bladder cancer, and SOX4 protein was expressed in a cancer cell-specific manner [2]. Aaboe et al. [2] also reported that their microarray analysis of SOX4 protein showed that T1 tumors were strongly positive when compared with Ta tumors, but no difference was found between Ta and T2–4 tumors. Controversially, in our study, there was no correlation between SOX4 protein expression and tumor stage and grade. Although we evaluated 57 bladder tumor samples by immunohistochemistry, our limitation was that a relatively low number of tumor samples were amenable for RNA studies since we had to exclude more than half of the samples collected, either because of
limited surgical tissue quantity or because of lack of RNA integrity. Though the fold-expression level was not as high as that of Aaboe et al. [2], we found a moderate total increase (2.2-fold) in SOX4 gene expression in bladder tumors as compared with that in normal bladder tissues. The SOX4 may play roles in the development of cancer cell. One possibility is that SOX4 may function as tumor suppressor gene. Pan et al. [11] stated that SOX4 is a new DNA damage sensor that is required for p53 activation. SOX4 binds transcription factor p53 and thereby prevents its degradation by blocking Mdm2-mediated ubiquitination and degradation. These observations seem to provide a clear explanation for the role played by SOX4 in promoting cell cycle arrest, apoptosis, and inhibiting tumor growth via interaction with p53. On the other hand, Liu et al. [4] reported SOX4 as an oncogene for human prostate cancer cells. Hur et al. [5] also indicated that overexpression of SOX4 protein was closely associated with hepatocarcinogenesis in human hepatocellular cancer (HCC), and suggested that the interaction of SOX4 with p53 modulates p53-mediated transcription at the Bax promoter, and via the suppression of Bax gene expression apoptosis is inhibited. Medina et al. [12] emphasize the oncogenic properties of SOX4 and show the interaction between gene amplification at 6p and SOX4 overexpression in lung cancer. Their results showed that most tumors with gene amplification also resulted in increase in gene expression. On the other hand, some tumor cells, especially small cell lung cancer (SCLC) type, showed an increase in gene expression without gene amplification, which may imply other mechanisms of SOX4 overexpression. Aaboe et al. [2] explained that they did not find any significant correlation between chromosomal amplification of the region 6p22 and SOX4 gene overexpression. Amplification is not the only mechanism to explain overexpression. There may be multiple mechanisms such as decrease in destruction of mRNA and/or protein or upregulated transcription [13–15]. In the study of de Bont et al. [16], SOX4 expression was associated with a favorable outcome in medulloblastoma, however, these findings do not correlate with the study of Neben et al. [17], who showed that SOX4
Table 3 Clinical parameters and SOX4 expression. Clinical parameters Recurrence Yes (n = 16) No (n = 35) Progression Yes (n = 11) No (n = 40) Tumor-specific survival Survival (n = 41) Death (n = 7) Fig. 2. The expression status of the SOX4 gene in patients compared with controls.
a
Fisher’s exact test.
Negative/weak (%)
Strong (%)
Pa
3 (5.9) 5 (9.8)
13 (25.5) 30 (58.8)
0.49
3 (5.9) 5 (9.8)
8 (15.7) 35 (68.6)
0.22
7 (14.6) 1 (2.1)
34 (70.8) 6 (12.5)
0.67
S. Gunes et al. / Pathology – Research and Practice 207 (2011) 423–427
was among the genes in which expression levels correlate with unfavorable survival. Therefore, the precise mechanism that SOX4 may act as a tumor suppressor or an oncogene seems to be related to the context and is dependent on external signals. It may be possible that the balance of proapoptotic and antiapoptotic SOX4 targets may shape a cellular fate. Recently, using luciferase assays, Dyrskjøt et al. [18] showed that the interaction between miR-129 and SOX4. SOX4 was downregulated at the 48-h time point upon pre-miR-129 transfection, and this reflects the potential of SOX4 as a direct target for miR-129. The interpretation of this newer study is important for revalueing both the study of Aaboe et al. [2] and studies performed thereafter. Dyrskjøt et al. [18] explained that high levels of miR-129 (corresponding low SOX4 levels) may be indicative of poor outcome. These findings may be quite important in directing the future studies and investigating the potential effects of micro-RNAs that may target SOX4 and shape the prognosis of bladder carcinoma. As we described in our results, in some tumors, SOX4 expression was downregulated in cancer tissues as compared with those of normal bladder. In the light of these new studies, the interactions of micro-RNAs with their targets, especially the miR-129–SOX4, can be helpful for future researchers to plan combined expression analysis studies. Thus, a deeper insight into the role of SOX4 expression and the potential regulators of SOX4 in bladder carcinoma might be gained. Besides SOX4 gene itself, epigenetic regulations can also lead to the changes in SOX4 expression. For instance, epigenetic repression caused by promoter methylation of miR-129-2 gene has been shown in both endometrial and gastric cancer. SOX4 expression was associated with metastasis, poor differentiation, and hypermethylation of miR-129-2 [3]. In conclusion, our data indicate that SOX4 transcription is moderately upregulated in bladder cancer, and SOX4 protein is expressed in bladder cancer in a specific manner. Although this study included only 25 patients samples for qPCR, it increases our knowledge of the relative expression pattern of SOX4. Further studies involving a large number of patients to determine the precise role of the SOX4 protein in tumorigenesis are desirable. Conflict of interest statement All authors state that they do not have any financial interest or connections, direct or indirect that might raise the question of bias in the present work. References [1] O.P. Vrooman, J.A. Witjes, Molecular markers for detection, surveillance and prognostication of bladder cancer, Int. J. Urol. 16 (2009) 234–243.
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Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.de/prp
Original article
SOX4 expression levels in urothelial bladder carcinoma夽 Sezgin Gunes a,∗ , Zeynep Yegin a , Yurdanur Sullu b , Recep Buyukalpelli c , Hasan Bagci a a
Department of Medical Biology and Genetics, Ondokuz Mayis University, Faculty of Medicine, 55139, Samsun, Turkey Department of Pathology, Ondokuz Mayis University, Faculty of Medicine, 55139, Samsun, Turkey c Department of Urology, Ondokuz Mayis University, Faculty of Medicine, 55139, Samsun, Turkey b
a r t i c l e
i n f o
Article history: Received 6 January 2011 Received in revised form 11 April 2011 Accepted 9 May 2011 Keywords: Bladder carcinoma SOX4 gene Gene expression
a b s t r a c t High levels of SOX4 gene expression have been reported in a variety of human cancers. The protein may function in the apoptosis pathway, leading to cell death as well as to tumorigenesis. The aim of this study was to investigate the levels of SOX4 expression in bladder cancer. Urinary bladder tumor samples were obtained from 57 bladder cancer and 13 normal bladder biopsies. The levels of SOX4 expression in bladder cancer were determined by immunohistochemistry and real-time PCR. SOX4 gene expression was increased 2.2 times in bladder tumors as compared with normal tissue. The presence of protein was confirmed by immunostaining. There were significant differences between immunostaining of bladder tumors and normal bladder tissue (P = 0.001). The present data suggest that SOX4 gene may have a role in bladder cancer tumorigenesis. © 2011 Elsevier GmbH. All rights reserved.
Introduction Bladder carcinoma (BC) is the second most common malignancy affecting the urinary system [1]. Numerous genetic and epigenetic alterations which are responsible for the transformed cell phenotype have been identified. According to the theory of cancer clonality, a long process of natural selection and clonal evolution finally represents the tumor phenotype arising from a single cell. Much effort has focused on identifying new genes that have roles in bladder carcinoma, and one of these genes that has drawn attention to in recent years is sex-determining region Y (SRY) related high mobility group (HMG) box (SOX4) gene (MIM ID *184430). It has been reported that the expression of SOX4 gene is increased in most bladder cancer cases [2]. SOX4 is a member of the SRY box (SOX) transcription factor family, which consists of 20 highly conserved transcription factors in humans. SOX4 gene is located on 6p22.3 and encodes a 47 kDa protein (474 amino acids) [3,4]. The protein coded by SOX4 has three major domains: a HMG box (aa 57–135), a glycine-rich region (aa 152–227), and a serine-rich region (SRR, aa 333–397). While the HMG box acts as DNA-binding region, SRR domain acts as transactivation domain. The glycine-rich region, which is located between the HMG box and SRR, is a part of the central domain, and this region has a function in promoting apoptotic cell death [5].
夽 Support: This study was supported by Ondokuz Mayis University Research Foundation (Project No. PYO.TIP.1901.08.557). ∗ Corresponding author. Tel.: +90 362 312 19 19x3164; fax: +90 362 457 60 41. E-mail address: [email protected] (S. Gunes). 0344-0338/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2011.05.005
SOX4 plays important roles in many developmental processes, such as embryonic cardiac development, thymocyte development, T cell differentiation pathway, and nervous system development [6–8]. The role of SOX4 in tumorigenesis is controversial. SOX4 upregulation has been associated with several types of cancers, including hepatic, breast, brain, lung, pancreatic, salivary gland, bladder, and ovarian cancers [3]. Aaboe et al. [2] were the first to delineate the upregulation (∼5-fold) of human transcription factor SOX4 in bladder carcinomas. To the best of our knowledge, since then there has been no report regarding the interaction between SOX4 expression and bladder carcinoma. This prompted us to examine the SOX4 expression by real-time PCR and immunohistochemistry in bladder tumor samples and compare our results with those of the previous findings. In the present study, qPCR and immunohistochemistry were employed in order to detect and verify the expression levels of SOX4 in patients with urinary bladder cancer. Materials and methods Subjects Fifty-seven patients with bladder carcinoma and 13 nonbladder cancer subjects attending the Urology Clinics of the Medical Faculty, Ondokuz Mayis University (OMU), between January 2007 and September 2010, were enrolled in this study. The patients signed an informed consent form after receiving information about the study. The study was approved by the ethical committee of the OMU. Table 1 presents the clinicopathological parameters of the
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S. Gunes et al. / Pathology – Research and Practice 207 (2011) 423–427
Table 1 Clinicopathological characteristics of the subjects. Immunohistochemical analysis
qPCR analysis
Patients (n = 57) Controls (n = 13) Patients (n = 25) Controls (n = 3) Gender 52 Male Female 5 Age Mean 65.65 ± 1.69 Range 26–81 Tumor stage 7 Ta 33 T1 T2 16 T4 1 Tumor grade Low 31 26 High
9 4
24 1
3 –
63.66 ± 4.38 32–79
64.08 ± 2.46 40–86
68.33 ± 2.46 64–73
5 4 16 – 16 9
subject population. Tumor samples were obtained directly from surgical material after the removal of the necessary amount of tissue by transurethral resection of the bladder for routine pathological examination. Tumor and normal tissues were collected in 0.9% filter sterilized NaCl, immediately snap frozen, and stored at −80 ◦ C until further RNA extraction. Immunohistochemistry In situ detection of SOX4 was performed for tissue samples from 57 patients with urothelial carcinoma and 13 non-bladder cancer subjects in the Department of Pathology. Hematoxylin–eosin sections of cases included in the study were re-evaluated and classified according to the WHO/ISUP (2004) and staged according to TNM (2002) [9]. According to the depth of invasion, 57 cases were classified: Ta is a non-invasive papillary tumor, T1 is tumor that invades subepithelial connective tissue, T2 is tumor that invades muscle, and T4 is tumor that invades any of the following: prostate, uterus, vagina, pelvic wall and abdominal wall. All tissues were fixed in a 10% buffered formaldehyde solution for 24 h and blocked in paraffin. Among the sections investigated, one block representing the tumor containing no hemorrhages or necrosis was chosen. The blocks were sectioned at 4 m in thickness and studied immunohistochemically using the streptavidin biotin peroxidase method using primary antibody against SOX4 (rabbit, polyclonal, S7318, Sigma–Aldrich). As positive controls, we used a thymus section. The best dilution rates were recorded by various attempts. SOX4 was diluted at 1/100. The endogenous peroxidase activity of deparaffinized sections was eliminated by incubation for 10 min in 3% hydrogen peroxide solution. Next, the sections were boiled for 35 min in citrate buffer and left to cool for 20 min. Blocking antibody was applied to the sections for 5 min and SOX4 primary antibody applied for 1 h. The sections were then incubated with biotinylated anti-immunoglobulin and streptavidin–peroxidase conjugate for 10 min each. A kit containing 3,3 -diamino benzidin (DAB) (DAKO, Carpinteria, USA) was used as the coloring agent. Finally, the sections were dyed with Mayer’s Hematoxylin for 60 min. Until DAB application, phosphate buffers (pH 7.6) were used in all stages, while distilled water was used following DAB application. The procedures were performed at room temperature. The results were evaluated using a Leica HMLB45 (Germany, 2000) light microscope considering cytoplasmic and nuclear dyeing. Assessment of the extensiveness and intensity of dyeing was performed semi-quantitatively in tumor areas selected in each block. The extensiveness of dyeing for SOX4 was graded as follows: dyeing of 0–5% of tumor cells as 0, dyeing of 6–25% of cells as 1, dyeing of 26–50% of the cells as 2, and dyeing of over 50% of cells as
3. Assessment of the intensity of dyeing was graded as follows: light yellow as 1, dark yellow as 2, and brown as 3. The values obtained from the sum of these two degrees were scored as: between 0 and 2 as negative (0), weak (1) for 3, moderate (2) for 4, and strong (3) for 5 and 6. RNA isolation Total RNA was extracted from bladder tissues using a commercially available kit (SV Total RNA Isolation System, Promega, Madison, WI), according to the manufacturer’s instructions. The final RNA preparations were diluted in 1/100 ratio in diethylpyrocarbonate-treated water and quantified by analyzing absorbance at 260 nm. The integrity of 28S and 18S rRNA was checked by electrophoresis of 1 g of total RNA in 1% agarose gel. RNA isolation was performed for all tumor samples. However, RNA of 32 samples could not be used because either the quantity of surgical tissue was limited or RNA was degradated. Only samples with clearly defined ribosomal peaks were used in the study. Reverse transcription Approximately 1 g of total RNA was used in 20 l of reverse transcription (RT) reaction mixture for cDNA synthesis with a RevertAidTM First Strand cDNA Synthesis Kit (MBI Fermentas, UK) and oligo-dT primers. RT reactions were performed for 1 h at 42 ◦ C as described in the commercial kit’s protocol. Determination of gene expression Real-time PCR was performed on SmartCyclerII (Cepheid, USA) using MaximaTM SYBR Green qPCR Master Mix (Fermentas, Glen Burnie, MD). Primer sequences were as follows: SOX4 (forward primer 5 -GTGAGCGAGATGATCTCGGG-3 ; reverse primer 5 -CAGGTTGGAGATGCTGGACTC), GAPDH (forward primer 5 -ACCACAGTCCATGCCATCAC-3 ; reverse primer 5 -TCCACCACCCTGTTGCTGTA-3 ) [2]. Master mixes were prepared and used in the PCR amplifications. A typical reaction mixture of 30 l consisted of 15 l SYBR Green master mix, 0.3 M of each of forward and reverse primers and 12.2 l of nuclease-free water. PCR amplification was done at 95 ◦ C for 10 min followed by 40 cycles of 95 ◦ C for 15 s, 60 ◦ C for 30 s, 72 ◦ C for 30 s and final melting curve from 60 ◦ C to 95 ◦ C with increments of 0.2 ◦ C/s. Primer specificity was confirmed by melting curve analysis and electrophoresis. PCR products were electrophoresed in 2% ethidium bromide-stained agarose gels, and the presence of the DNA bands in expected sizes was confirmed. Within each experiment, we loaded RNase-free water as our negative control, and samples were run as duplicates to control for variability in pipetting and cycling conditions, and the calculations were made on the average numbers of the duplicates. When the difference between duplicated samples exceeded 20%, these samples were quantified again. Computational analysis One microliter of cDNA product (equivalent to 50 ng RNA starting material) was used for assessing gene expression across groups by analyzing Ct values. The Cycle-Threshold (Ct ) is the numeric value for the cycle at which the product crossed the threshold. Reactions with lower Ct values contain more of the gene of interest since they took less time to amplify. On the other hand, samples with a higher Ct contain less of the gene of interest. The threshold cycle values for each target transcript was normalized to that of the gene encoding GAPDH by calculating the Ct according to the formula
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Fig. 1. Cytoplasmic and nuclear immunohistochemical staining of SOX4 in T2, high-grade tumor, ×200 (A); in T1, low-grade tumor, ×200 (B); cytoplasmic staining in T1, high-grade tumor, ×200 (C) and cytoplasmic and nuclear immunostaining in only superficial cells in a control case, ×400 (D). target
Ct = Ct − CtGAPDH . The analysis of relative gene expression was performed by the comparative 2−Ct method [10].
between SOX4 expression and clinical variables such as recurrence, progression, and tumor-specific survival (Table 3).
Statistical analysis
Expression profile of SOX4 in bladder carcinoma
Data were analyzed using SPSS for Windows (version 15). To calculate relative fold-changes between patient and control samples, we utilized the delta C(T) method, calculated as follows: {[C(T)(SOX4)] − [C(T)(GAPDH)]} [10]. The Mann–Whitney test was carried out to compare SOX4 gene expression levels between highgrade and low-grade tumors. P < 0.05 was considered statistically significant.
We did a gene expression analysis using quantitative real-time PCR and determined the expression of SOX4 in comparison with that of the housekeeping gene GAPDH. Gene expression profiles were generated for 25 bladder tumor samples and 3 control bladder samples. The expression change was calculated based on the 2−Ct method as proposed by Livak and Schmittgen [10]. We found a moderate 2.2-fold upregulation of SOX4 in bladder tumors, as compared with normal bladder tissues, at all stages of bladder cancer (Fig. 2). It was also evident that in some tumor samples, SOX4 expression was downregulated as compared with those of normal bladder.
Results SOX4 expression by immunohistochemistry Of 57 tumors examined, 33 were classified as T1, 16 as T2, 7 as Ta, and 1 as T4 according to the WHO/ISUP (2004) and staged according to TNM (2002) (Table 1). SOX4 was expressed in all tumor samples and in 6 (46%) control specimens (P = 0.001). SOX4 was strongly expressed in 49 (86%), moderately expressed in 4 (7%), and weakly expressed in 1 (2%), and not expressed in 3 (5%) tumor samples. Expression of SOX4 protein was observed in both the cytoplasmic and nuclear cell compartments. SOX4 was strongly expressed in 1 (8%), moderately expressed in 3 (23%), and weakly expressed in 2 (15%) control samples (Fig. 1). Seven (54%) control samples were SOX4-negative. In control cases, expression of SOX4 was observed only in superficial cells. We did not find any correlation between SOX4 expression and tumor stage (P = 0.636) and grade (P = 0.887) (Table 2). There was no significant correlation
No correlation between tumor grades and SOX4 expression We also aimed to investigate whether there was an expression difference between high-grade and low-grade tumors. Previously, it was shown by Aaboe et al. [2] that higher SOX4 expression results in better survival in bladder tumor patients. We thought that we could make an assumption about prognosis on the basis of expression differences between high-grade and low-grade tumors. Using the Mann–Whitney test, we compared the expression differences between high-grade and low-grade tumors. Low-grade tumors exhibited a slight increase in SOX4 expression when compared with the high-grade ones (mean ranks were 14.06 and 11.11, respectively). However, the differences were not statistically significant.
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Table 2 Immunohistochemical analysis of SOX4 in patients with bladder carcinoma and controls. Pathological classification
Stage Ta (n = 7) T1 (n = 33) T2 (n = 16) T4 (n = 1) Grade Low (n = 32) High (n = 25) All tumors (n = 57) Controls (n = 13) a
SOX4 Negative (%)
Weak (%)
Moderate (%)
Strong (%)
Pa
– 3 (9) – –
– 1 (3) – –
– 2 (6) 2 (12) –
7 (100) 27 (82) 14 (88) 1 (100)
0.636
3 (9) – 3 (5)
– 1 (4) 1 (2)
2 (6) 2 (8) 4 (7)
27 (85) 22 (88) 49 (86)
7 (54)
2 (15)
3 (23)
1 (8)
0.887
Fisher’s exact test.
Discussion In the present study, qPCR and immunohistochemistry were employed in order to detect and verify the expression levels of SOX4 in patients with urinary bladder cancer. Immunohistochemistry revealed that there were significant differences between immunostaining of bladder tumors and normal tissue. SOX4 protein was expressed in both nuclei and cytoplasm. On the other hand, we did not find any correlation between SOX4 expression and clinical variables, tumor stage, and grade. We found a moderate total increase (2.2-fold) in SOX4 gene expression in bladder tumors as compared with the normal bladder tissues. Immunohistochemistry results of a previous study have also shown that SOX4 was highly upregulated in bladder cancer, and SOX4 protein was expressed in a cancer cell-specific manner [2]. Aaboe et al. [2] also reported that their microarray analysis of SOX4 protein showed that T1 tumors were strongly positive when compared with Ta tumors, but no difference was found between Ta and T2–4 tumors. Controversially, in our study, there was no correlation between SOX4 protein expression and tumor stage and grade. Although we evaluated 57 bladder tumor samples by immunohistochemistry, our limitation was that a relatively low number of tumor samples were amenable for RNA studies since we had to exclude more than half of the samples collected, either because of
limited surgical tissue quantity or because of lack of RNA integrity. Though the fold-expression level was not as high as that of Aaboe et al. [2], we found a moderate total increase (2.2-fold) in SOX4 gene expression in bladder tumors as compared with that in normal bladder tissues. The SOX4 may play roles in the development of cancer cell. One possibility is that SOX4 may function as tumor suppressor gene. Pan et al. [11] stated that SOX4 is a new DNA damage sensor that is required for p53 activation. SOX4 binds transcription factor p53 and thereby prevents its degradation by blocking Mdm2-mediated ubiquitination and degradation. These observations seem to provide a clear explanation for the role played by SOX4 in promoting cell cycle arrest, apoptosis, and inhibiting tumor growth via interaction with p53. On the other hand, Liu et al. [4] reported SOX4 as an oncogene for human prostate cancer cells. Hur et al. [5] also indicated that overexpression of SOX4 protein was closely associated with hepatocarcinogenesis in human hepatocellular cancer (HCC), and suggested that the interaction of SOX4 with p53 modulates p53-mediated transcription at the Bax promoter, and via the suppression of Bax gene expression apoptosis is inhibited. Medina et al. [12] emphasize the oncogenic properties of SOX4 and show the interaction between gene amplification at 6p and SOX4 overexpression in lung cancer. Their results showed that most tumors with gene amplification also resulted in increase in gene expression. On the other hand, some tumor cells, especially small cell lung cancer (SCLC) type, showed an increase in gene expression without gene amplification, which may imply other mechanisms of SOX4 overexpression. Aaboe et al. [2] explained that they did not find any significant correlation between chromosomal amplification of the region 6p22 and SOX4 gene overexpression. Amplification is not the only mechanism to explain overexpression. There may be multiple mechanisms such as decrease in destruction of mRNA and/or protein or upregulated transcription [13–15]. In the study of de Bont et al. [16], SOX4 expression was associated with a favorable outcome in medulloblastoma, however, these findings do not correlate with the study of Neben et al. [17], who showed that SOX4
Table 3 Clinical parameters and SOX4 expression. Clinical parameters Recurrence Yes (n = 16) No (n = 35) Progression Yes (n = 11) No (n = 40) Tumor-specific survival Survival (n = 41) Death (n = 7) Fig. 2. The expression status of the SOX4 gene in patients compared with controls.
a
Fisher’s exact test.
Negative/weak (%)
Strong (%)
Pa
3 (5.9) 5 (9.8)
13 (25.5) 30 (58.8)
0.49
3 (5.9) 5 (9.8)
8 (15.7) 35 (68.6)
0.22
7 (14.6) 1 (2.1)
34 (70.8) 6 (12.5)
0.67
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was among the genes in which expression levels correlate with unfavorable survival. Therefore, the precise mechanism that SOX4 may act as a tumor suppressor or an oncogene seems to be related to the context and is dependent on external signals. It may be possible that the balance of proapoptotic and antiapoptotic SOX4 targets may shape a cellular fate. Recently, using luciferase assays, Dyrskjøt et al. [18] showed that the interaction between miR-129 and SOX4. SOX4 was downregulated at the 48-h time point upon pre-miR-129 transfection, and this reflects the potential of SOX4 as a direct target for miR-129. The interpretation of this newer study is important for revalueing both the study of Aaboe et al. [2] and studies performed thereafter. Dyrskjøt et al. [18] explained that high levels of miR-129 (corresponding low SOX4 levels) may be indicative of poor outcome. These findings may be quite important in directing the future studies and investigating the potential effects of micro-RNAs that may target SOX4 and shape the prognosis of bladder carcinoma. As we described in our results, in some tumors, SOX4 expression was downregulated in cancer tissues as compared with those of normal bladder. In the light of these new studies, the interactions of micro-RNAs with their targets, especially the miR-129–SOX4, can be helpful for future researchers to plan combined expression analysis studies. Thus, a deeper insight into the role of SOX4 expression and the potential regulators of SOX4 in bladder carcinoma might be gained. Besides SOX4 gene itself, epigenetic regulations can also lead to the changes in SOX4 expression. For instance, epigenetic repression caused by promoter methylation of miR-129-2 gene has been shown in both endometrial and gastric cancer. SOX4 expression was associated with metastasis, poor differentiation, and hypermethylation of miR-129-2 [3]. In conclusion, our data indicate that SOX4 transcription is moderately upregulated in bladder cancer, and SOX4 protein is expressed in bladder cancer in a specific manner. Although this study included only 25 patients samples for qPCR, it increases our knowledge of the relative expression pattern of SOX4. Further studies involving a large number of patients to determine the precise role of the SOX4 protein in tumorigenesis are desirable. Conflict of interest statement All authors state that they do not have any financial interest or connections, direct or indirect that might raise the question of bias in the present work. References [1] O.P. Vrooman, J.A. Witjes, Molecular markers for detection, surveillance and prognostication of bladder cancer, Int. J. Urol. 16 (2009) 234–243.
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