SOX2 overexpression correlates with poor prognosis in laryngeal squamous cell carcinoma

Auris Nasus Larynx 40 (2013) 481–486

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SOX2 overexpression correlates with poor prognosis in laryngeal squamous cell carcinoma Xia-bing Tang a,1, Xiao-hui Shen b,1, Lei Li c, Yi-fen Zhang d, Guo-qian Chen c,* a

Department of Otolaryngology, Head and Neck Surgery, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi 214023, Jiangsu, PR China Department of Otolaryngology, Head and Neck Surgery, Drum Tower Hospital Affiliated to Nanjing Medical University, Nanjing 210008, Jiangsu, PR China c Department of Medical Laboratory, Wuxi People’s Hospital Affiliated to Nanjing Medical University, Wuxi, Wuxi 214023, Jiangsu, PR China d Depatment of Pathology, Drum Tower Hospital Affiliated to Nanjing Medical University, Nanjing 210008, Jiangsu, PR China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 September 2012 Accepted 24 January 2013 Available online 23 February 2013

Objective: The aim of present study was to investigate the expression of SOX2, a key transcription factor, in LSCC and to assess its prognostic significance. Methods: SOX2 expression of 161 LSCC tissues was detected by immunohistochemistry using a tissue microarray and statistically analyzed for its correlation with clinicopathological charateristics and patient outcome. In addition, SOX2 expression was also observed in 20 self-paired fresh LSCC tissues by western blot. Results: SOX2 was overexpressed in LSCC tissues as compared to the corresponding adjacent normal tissues. SOX2 expression was significantly associated with tumour T classification (p < 0.001), clinical stage (p < 0.001), lymph node metastasis (p = 0.007) and recurrence (p = 0.001). Univariate analysis revealed that patients with high SOX2 expression were significantly related to overall survival (p < 0.001). Multivariate survival analysis further demonstrated that SOX2 expression was an independent prognostic factor for LSCC patients. Conclusion: SOX2 may contribute to the malignant progression of laryngeal squamous cell carcinoma (LSCC), and present as a useful prognostic marker and a potential therapeutic target for LSCC patients. ß 2013 Elsevier Ireland Ltd. All rights reserved.

Keywords: SOX2 transcription factor Laryngeal cancer Squamous cell carcinoma Prognosis

1. Introduction Head and neck cancer accounts for about 6% of all human cancers. There are approximately 47,560 new cases in the USA and at least 500,000 cases worldwide each year [1]. Laryngeal carcinoma, 90% to 95% of which is squamous cell carcinoma, is the eleventh-most-common cancer among men around the globe [2]. Laryngeal squamous cell carcinoma (LSCC), is one of the most frequent malignant neoplasm of the head and neck, and accounts for 1.5% of all cancers. Despite diagnostic and therapeutic advances including surgical management, radiotherapy, and chemotherapy, the 5-year survival rate still remains low owing to tumour recurrence and metastasis. The traditional prognostic factors (such as clinical stage, lymph node status) can not fully estimate patients’ survival. Therefore, it is essential to find reliable biological markers or marker combinations associated with tumour progression and

* Corresponding author. Tel.: +86 510 85350328; fax: +86 510 82700775. E-mail address: [email protected] (G.-q. Chen). 1 Both authors contributed equally to the article. 0385-8146/$ – see front matter . ß 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anl.2013.01.003

prognosis for developing novel strategies in the prevention and treatment of patients with LSCC. SOX2, a member of the SOX (SRY-related high mobility group box) family, is a key transcription factor involved in maintaining the pluripotency of embryonic stem cell in self-renewal and differentiation, and plays a critical role in determining the fate of stem cells [3,4]. It was suggested that tumours contain a cellular population that retains key stem cell properties [5], which have gene expression signatures closely related to embryonic stem cells [6]. Recent studies have demonstrated that SOX2, as a stem cell factor, has a potential function in tumorigenesis [7]. SOX2 is overexpressed in several human tumours including lung cancer, esophageal carcinoma, pancreatic carcinoma, breast cancer, ovarian carcinoma, glioma [8–14]. It has also been reported that SOX2 promotes the proliferation, clonogenicity, and tumorigenicity in cervical cancer cells and breast cancer cells both in vitro cell line and in vivo xenograft models [15,16]. However, the role of SOX2 in LSCC has not been investigated so far. In the present study, we detected SOX2 protein expression by immunohistochemistry and western blot, and analyzed its correlation with clinicopathological factors and prognosis, and

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further clarified independent prognostic factors affecting the overall survival of patients with LSCC.

2. Materials and methods

WI, USA). Representative areas from formalin-fixed, paraffinembedded blocks were carefully selected on H&E-stained sections and tissue cores with a diameter of 0.6 mm were taken from each block. The tissue cores were arrayed on a recipient paraffin block. 5 mm sections of this tissue array block were cut and mounted onto charged Super-Frost Plus glass slides.

2.1. Patients and tissue specimens 2.3. Immunohistochemistry A total of 161 patients with LSCC, who underwent a surgical resection and were histologically confirmed at the Department of Otolaryngology, Drum Tower Hospital affiliated to Nanjing Medical University between 2002 and 2006, were enrolled in this retrospective study. None of the patients had received preoperative radiotherapy, immunotherapy or chemotherapy. The histological classification of tumours was evaluated based on the classification system of the World Health Organization, and the postoperative pathological staging was made according to the 2002 TNM classification of malignant tumours by the International Union Against Cancer. All the formalin-fixed paraffin-embedded tissue samples were retrieved from the Pathology Department. Twenty pairs of LSCC tissues and corresponding adjacent normal tissues were frozen in liquid nitrogen and stored at 80 8C below zero for western blot. The study was approved by the Research Ethics Committee of Drum Tower Hospital affiliated to Nanjing Medical University. Informed consent was obtained from all patients. The main clinical and pathological variables of all patients were described in Table 1. 2.2. Tissue microarray construction The tissue microarray (TMA) was constructed as previously described [12]. TMA composed of 161 LSCC tissues was created using a manual tissue arrayer (Beecher Instruments, Sun Prairie, Table 1 Correlation between SOX2 protein expression and clinicopathological characteristics. Characteristics

n

Age (years) <60 63 60 98 Sex Male 152 Female 9 Smoking Yes 107 No 54 Alcohol intake Yes 91 No 70 Tumour site Supraglottic 57 Glottic 94 Subglottic 10 Tumour differentiation Well 51 Moderately 102 Poorly 8 T classification T1+T2 80 T3+T4 81 Clinical stage I–II 65 III–IV 96 Lymph node metastasis Yes 41 No 120 Recurrence Yes 93 No 68

SOX2 expression

p value

0–3

4–5

6–7

30 43

12 10

21 45

0.150

70 3

21 1

61 5

0.661

46 27

16 6

45 21

0.649

40 33

14 8

37 29

0.761

21 50 2

8 12 2

28 33 5

0.194

26 46 1

6 12 4

19 44 3

0.080

50 23

13 9

17 49

<0.001

43 30

10 12

11 55

<0.001

10 63

7 15

24 42

0.007

32 41

11 11

50 16

0.001

Immunohistochemical stains were performed on paraffinembedded sections of the TMA. The tissue sections were deparaffinized in xylene, and rehydrated through graded ethanol. Antigen retrieval was carried out using a microwave oven in 10 mmol/L citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubating sections in methanol containing 3% hydrogen peroxide. The sections were washed and incubated with anti-human SOX2 polyclonal antibody (1:100 dilution; Epitomics, Inc., USA) at 4 degrees Celsius overnight, followed by incubation with horseradish peroxidase(HRP) conjugated goat anti-rabbit IgG and 3,30 -diaminobenzidine (Dako), and then counterstained with haematoxylin, dehydration and mounted with glycerol gelatin. Negative controls were prepared by omitting the primary antibody under the same experimental conditions. 2.4. Evaluation of staining TMA sections were evaluated and scored by two doubleblinded independent and experienced pathologists, who were unaware of clinical data and outcome. Nuclear expression of SOX2 was scored semiquantitatively by the combination of intensity (scored as 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and proportion of positively stained tumour cells in 5 high power field (scored as (0) 0%, (1) 1–25%, (2) 26–50%, (3) 51–75% and (4) 76–100%). The sum of staining intensity score and percentage of positive tumour cells score used as the final staining score (ranges: 0–7) for SOX2. Tumours were categorized into three groups (scored 0–3, 4–5 and 6–7) based on the final staining score and tumours with a final staining score of >3 were considered to be positive. The expression of SOX2 was thus divided into low expression (scored 0–5) and high expression (scored 6–7). There was no discrepancy in overall interpretation of immunohistochemistry results between the two pathologists. 2.5. Western blot analysis Twenty pairs of LSCC tissues and corresponding adjacent normal tissues were subjected to western blot analysis. The total protein was extracted from fresh frozen tissues samples lysed in RIPA buffer (1  PBS, 1% NP40, 0.1% SDS, 5 mmol/L EDTA, 0.5% sodium deoxycholate, and 1 mmol/L sodium orthovanadate) with protease inhibitors. Protein concentration was quantified by bicinchoninic acid protein assay kit (Beyotime, China). Equal amounts of proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking in 5% fat-free milk in Tris-buffered saline Tween-20, the blotting membranes were incubated with rabbit anti-human SOX2 polyclonal antibody (1:10,000 dilution; Epitomics, Inc., USA) at 4 8C overnight. After washing, they were incubated with HRP conjugated goat anti-rabbit IgG (Biotech, China) for 1 h at room temperature. Bands were finally visualized by employing the enhanced chemiluminescence (ECL, Millipore, Bedford, MA, USA). An mouse anti-b tubulin monoclonal antibody (1:2000 dilution, Abmart, USA) was performed as an internal loading control. The relative level of SOX2 was conducted by comparison with the corresponding internal loading control. All experiments were carried out in triplicate.

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2.6. Follow-up All the patients with LSCC were discharged from hospital without suffering major perioperative complications and evaluated at the outpatient clinic at a 3–6 month interval. Recurrence and metastasis were diagnosed by physical examination, imaging evaluation and pathological studies. The follow-up time ranged from 8 to 60 months, with a median time of 46.3 months. Overall survival (OS) was measured from the onset of treatment to the date of death or the survival status at the last date of follow-up. 2.7. Statistical analysis All statistical analyses were carried out using the SPSS 18.0 software package (SPSS Inc., Chicago, IL, USA). The result of relative level of SOX2 in western blot analysis was evaluated by using the Student’s t-test. Correlations of SOX2 expression with clinicopathological characteristics were assessed by Pearson’s x2 test. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. Cox regression multivariate analysis was performed to identify independent prognostic factors. p < 0.05 was considered statistically significant. 3. Results 3.1. SOX2 protein expression in LSCC tissues To investigate SOX2 protein expression in LSCC, immunohistochemistry was initially performed in 161 paraffin-embedded TMA sections. Positive SOX2 immunostaining was predominantly observed in the nuclei of carcinoma cells (Fig. 1). Positive expression of SOX2 protein was detected in 88 (54.7%) primary tumour samples. Among tumour samples, SOX2 expression was scored 0–3 in 73 (45.3%), 4–5 in 22 (13.7%) and 6–7 in 66(41.0%) specimens. Low expression of SOX2 was found in 95(59.0%; Fig. 1A–D). High expression of SOX2 was found in 66 (41.0%; Fig. 1E and F). To confirm the expression levels of SOX2 seen by immunostaining in the specimens from our TMA, we examined the expression of SOX2 protein by western blot analysis in 20 randomly selected pairs of LSCC tissues and their corresponding adjacent normal tissues. In 11 of 20 (55.0%) LSCC patients, SOX2 protein was upregulated in tumour tissues compared with their adjacent normal tissues (Fig. 2). 3.2. Correlation between SOX2 protein expression and clinicopathological characteristics

Fig. 1. Immunohistochemical analysis of SOX2 expression in LSCC tissues. (A and B) Negative expression of SOX2 in some LSCC tissues (100 and 400, respectively). (C and D) Low expression of SOX2 in some LSCC tissues (100 and 400, respectively).(E and F) High expression of SOX2 in some LSCC tissues (100 and 400, respectively).

expression of SOX2 (p < 0.001; Fig. 3 a), indicating that the high expression of SOX2 was correlated with a shorter survival time. Cox regression multivariate analysis including age, sex, smoking, alcohol intake, tumour site, tumour differentiation, T classification, clinical stage, lymph node metastasis and SOX2 expression was performed. Our results demonstrated that SOX2 expression had a significant correlation with LSCC prognosis, and it was found to be an independent prognostic factor of outcomes in patients with LSCC after tumour resection (hazard ratio = 1.911, 95% confidence interval (CI) = 1.037–3.522, p = 0.038) (Table 2).

The association between SOX2 protein expression and clinicopathological parameters of patients with LSCC was assessed by Pearson’s x2 test. As shown in Table 1, SOX2 protein expression was significantly associated with tumour T classification (p < 0.001), clinical stage (p < 0.001), lymph node metastasis (p = 0.007) and recurrence (p = 0.001), respectively. However, no significant relationship between SOX2 protein level and variables such as age (p = 0.150), sex (p = 0.661), smoking (p = 0.649), alcohol intake (p = 0.761), tumour site (p = 0.194) and tumour differentiation (p = 0.080). 3.3. Correlation between SOX2 protein expression and patients’ survival As determined by Kaplan–Meier method, the expression of SOX2 in LSCC tissues was significantly correlated with overall survival. The log-rank test showed that the survival time was significantly different between groups with high and low

Fig. 2. Western blot analysis of SOX2 expression in fresh LSCC tissues. (A) Western blot of SOX2 levels in LSCC tissues (T), corresponding adjacent normal tissues (NT). (B) The abundance of SOX2 protein was demonstrated relative to the level of btubulin protein.

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Fig. 3. Survival analysis of LSCC patients. Kaplan–Meier estimated of overall survival in all patients according to SOX2 expression (A), lymph node status (B) and SOX2 expression/lymph node status (C). The log-rank test was applied to calculate p-value.

It has been reported that lymph node metastasis was an important prognostic factor in patients with LSCC. Thus, we investigated the correlation between lymph node metastasis and patients’ prognosis in LSCC. Our results revealed that lymph node metastasis had a significant correlation with overall survival (p < 0.001; Fig. 3b). A subset analysis was carried out by combining SOX2 expression with lymph node status. We divided patients into 4 groups: high expression/lymph node (+), high expression/lymph node (), low expression/lymph node (+) and low expression/ lymph node (), and calculated the survival curves. The results showed that the survival time was shorter in group with high expression/lymph node (+) than high expression/lymph node () (p = 0.044), and it was significantly shorter in group with low expression/lymph node (+) than low expression/lymph node () (p < 0.001) (Fig. 3c). Our results revealed that lymph node metastasis was significantly associated with prognosis, and was an independent prognostic factor of outcomes (p = 0.004; Table 2). 4. Discussion Stem cells are primitive cells found in almost all organ systems. The key features of stem cells are their ability to renew themselves and to differentiate into a spectrum of specialized cell types. Stem cells can be classified as embryonic stem cells which are present in

embryonic development and adult stem cells which are present in adult tissues. In adult organs, stem cells may normally function as progenitor cells to repair injury or to replenish cells in tissues with high turn-over. However, they may also contribute to development of neoplasia if the body surveillance mechanism fails [17]. There is a strong belief that most cancers are derived from stem cells in the current. SOX2 is an essential transcription factor, which not only has role during neurogenesis and embryonic foregut development, but also allows reprogramming of adult cells to pluripotent stem cells [3,4]. More recently, studies have identified SOX2 as a lineagesurvival oncogene and is required for proliferation and anchorageindependent growth of cancer cell lines in lung and esophageal squamous cell carcinoma [11]. SOX2 promotes proliferation, clonogenicity, and tumorigenicity of cervical cancer cells, and similar results have been reported in breast cancer [15,16]. In addition, SOX2 has also been reported to be upregulated in several kinds of human solid tumours including lung cancer, esophageal carcinoma, pancreatic carcinoma, breast cancer, ovarian carcinoma, glioma. Taken together, SOX2 might serve as a novel candidate oncogene in carcinogenesis. To date, this study is the first report demonstrating that SOX2 is overexpressed in human LSCC tissues. We investigated the expression of SOX2 in paraffin-embedded TMA sections by immunochemistry and observed positive SOX2 stains were

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Table 2 Univariate and multivariate analyses of prognostic factors in LSCC patients. Variables

Age (years) <60 60 Sex Male Female Smoking Yes No Alcohol intake Yes No Tumour site Supraglottic Glottic Subglottic Tumour differentiation Well Moderately Poorly T classification T1+T2 T3+T4 Clinical stage I–II III–IV Lymph node metastasis Yes No SOX2 expression High Low

Univariate analysis

Multivariate analysis

Median survival(months)

95% CI

49.1 44.5

44.9–53.3 40.5–48.4

46.8 37.2

Hazard ratio

95% CI

p value

0.301

1.252

0.698–2.246

0.450

43.8–49.8 23.4–51.0

0.151

0.617

0.237–1.604

0.322

47.2 44.6

43.6–50.8 39.5–49.6

0.378

1.020

0.570–1.823

0.948

48.2 43.8

44.5–51.9 39.1–48.5

0.269

0.635

0.358–1.128

0.121

45.2 47.3 42.9

40.1–50.3 43.6–51.1 29.7–56.1

0.738

0.871

0.575–1.321

0.517

48.8 46.1 32.5

43.9–53.6 42.4–49.8 17.5–47.5

0.082

0.699

0.423–1.156

0.163

52.4 40.3

49.0–55.8 35.9–44.7

<0.001

0.492

0.196–1.237

0.131

55.0 40.4

51.9–58.1 36.3–44.5

<0.001

1.370

0.414–4.536

0.606

33.5 50.7

27.3–39.8 47.7–53.6

<0.001

2.582

1.354–4.925

0.004

37.5 52.7

32.6–42.5 49.7–55.7

<0.001

1.911

1.037–3.522

0.038

predominantly in the nuclei of carcinoma cells and was detected in 54.7% primary tumour samples. High expression of SOX2 was found in 41.0% cases of LSCC tissues. To confirm the expression levels of SOX2 seen by immunostaining, we examined the expression of SOX2 by Western blot analysis. In 55.0% LSCC patients, SOX2 expression was significantly higher in tumour tissues compared with their adjacent normal tissues. These results are consistent with previous studies in which SOX2 is overexpressed in lung squamous cell cancer, esophageal squamous cell carcinoma, colorectal cancer and basal cell-like breast carcinomas [11–13,18]. However, in gastric cancers and choriocarcinomas, SOX2 was downregulated by aberrant DNA methylation [19,20]. We also examined the correlation between SOX2 protein expression and clinicopathological characteristics, found that SOX2 expression was significantly associated with tumour T classification, clinical stage, lymph node metastasis, and recurrence in patients with LSCC, which indicated that SOX2 expression may be associated with progression in LSCC. A role for SOX2 in cancer progression has been reported by several groups. SOX2 was found to be involved in later events of carcinogenesis, such as invasion and metastasis of pancreatic intraepithelial neoplasias [8]. SOX2 expression was significantly correlated with lymph node metastasis and the stage of tumour invasion in gastric cancer [21]. Further study of the oncogenic function of SOX2 and underlying molecular mechanisms accounting for SOX2 being associated with progression still remain to be elucidated. We require engineered animal and organotypic tissue culture models. With regard to the prognostic value of SOX2 in patients with LSCC, it remains unknown. In the present, prognostic evaluation is mainly based on traditional prognostic factors (such as tumour site, clinical stage, lymph node status). Recent studies have suggested that other factors, such as molecular and cellular characteristics of the primary tumours, may improve our ability to prognosticate [22].

p value

Our investigation revealed that the expression of SOX2 in LSCC was correlated with poor clinical outcome. Multivariate survival analysis demonstrated that SOX2 was an independent prognostic factor of outcomes in patients with LSCC after tumour resection, although a study demonstrated that SOX2 protein overexpression is associated with better outcome in squamous cell lung cancer [13]. Consistently, most findings suggested that SOX2 expression could be a marker of poor prognosis in stage I lung adenocarcinomas, oral tongue squamous cell carcinoma, gastric carcinoma [12,23,24]. One of the reasons for this discrepancy might be the difference in sample sizes and methods used to assess the SOX2 status, including the use of different antibodies. Moreover, our findings indicated that lymph node metastasis had a significant correlation with overall survival, and was an important prognostic factor in patients with LSCC, which are in agreement with previous studies [25]. A subset analysis was also carried out by combining SOX2 expression with lymph node status. The results demonstrated that patients with the phenotype of high expression/lymph node (+) had poorer overall survival than high expression/lymph node (), and patients with the phenotype of low expression/lymph node (+) had poorer overall survival than low expression/lymph node (). However, overall survival had no difference between high expression/lymph node (+) and low expression/lymph node (+). Therefore, the evaluation of SOX2 alone or the lymph node status may further provide new information for patients’ prognosis. In summary, SOX2 overexpression is associated with progression and poor prognosis in LSCC. SOX2 appears to be a therapeutically promising target molecule and it might be a useful indicator for predicting the prognosis of LSCC. Conflict of interest We declare that we have no conflict of interest.

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