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High Gpx1 expression predicts poor survival in laryngeal squamous cell carcinoma
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High Gpx1 expression predicts poor survival in laryngeal squamous cell carcinoma Qicheng Zhang a,b, Hongli Xu c, Yiwen You b, Jie Zhang b, Renjie Chen a,d,* a Department of Otorhinolaryngology Head and Neck Surgery, The Second Clinical Medical College of Nanjing Medical University, Nanjing, Jiangsu Province, China b Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China c Department of Neurology, The Third People's Hospital of Nantong, Nantong, Jiangsu Province, China d Department of Otorhinolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 March 2017 Accepted 29 May 2017 Available online xxx
Objective: Several studies have demonstrated that abnormal glutathione peroxidases 1 (Gpx1) expression can influence the biological behavior of malignant cells. However, the roles of Gpx1 in laryngeal squamous cell carcinoma (LSCC) remain unknown. The purpose of this study is to analyze the Gpx1 expression and prognostic significance in LSCC patients. Methods: Gpx1 mRNA levels in laryngeal tissues were determined by qRT-PCR. Meanwhile, We examined the expression levels of Gpx1 protein in 140 primary tumor tissues and 28 cases of normal tissues by immunohistochemistry (IHC) analysis on tissue microarrays (TMA). Results: Our results revealed that the frequency of high Gpx1 was significantly higher in cancer tissue compared to normal surgical margins; Gpx1 expression correlated with clinical features and overall survival (OS). Gpx1 overexpression was significantly associated with lymph node metastasis (P = 0.023) and TNM stage (P = 0.008); Kaplan–Meier survival curves revealed that
Keywords: Gpx1 Laryngeal squamous cell carcinoma Prognosis Tissue microarrays (TMA)
1. Introduction Laryngeal cancer ranks as the second most common respiratory tract malignancy, and squamous cell carcinoma is its most common histopathological type [1,2]. In 2012, an estimated 3650 deaths worldwide were associated with laryngeal squamous cell carcinoma (LSCC) and the incidence of LSCC is increasing each year [3,4]. Depending on patient's conditions, several therapeutic strategies could be applied to these patients. However, the survival of these patients, particularly in advanced stage, has not significantly improved in the past several decades. In current clinical practice, some indexes such as tumor sub-sites, cervical lymph node metastases and histopathological grades are not sufficient to
* Corresponding author at: Department of Otorhinolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China. E-mail address: [email protected] (R. Chen).
accurately and completely evaluate the prognosis of these patients [5]. Therefore, the discovery of new biomarkers to accurately predict and further contribute to improving the prognosis of LSCC patients is important [6]. Glutathione peroxidases (GPXs), as selenoprotein-containing proteases, are a family of the body's antioxidant enzyme system [7]. They can maintain the metabolic balance of the reactive oxygen species (ROS) in the body to protect cells from oxidative DNA damage. At least eight GPX isoenzymes have been isolated from mammals [8]. Among these proteins, glutathione peroxidase 1 (Gpx1), also known as intracellular GPX, is one of the most abundant selenoproteins in the human body [9]. It can catalyze the conversion of glutathione (GSH) to oxidative glutathione (GSSG), reducing the toxic peroxides to non-toxic hydroxyl compounds and blocking the lipid peroxidation cascade, thereby protecting the cell structures and functions [9,10]. Previous studies have shown that aberrant Gpx1 expression could promote cell proliferation, migration, invasion and
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Please cite this article in press as: Zhang Q, et al. High Gpx1 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2017.05.012
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cisplatin resistance in oral, esophagus, lung, breast, bladder cancer [11,14]. However, Gpx1 expression and its prognostic role in LSCC remain unexplored to date. In the present study, Gpx1 mRNA level in laryngeal tissues was examined by qRTPCR; the expression of Gpx1 on tissue microarray was detected by immunohistochemistry (IHC). Furthermore, the relationship between Gpx1 expression and clinical pathological parameters were analyzed as well as overall survival (OS) in patients with LSCC. 2. Materials and methods 2.1. Tissue sample collection and clinical data collection Diagnosis of LSCC was made according to the World Health Organization (WHO) criteria [15] and TNM classification [UICC, 2009]. Totally, 140 hospitalized patients with primary LSCC were recruited from the Department of Otolaryngology/ Head and Neck Surgery, Affiliated Hospital of Nantong University, China, from year 2000 to 2009. Informed consents were obtained from these patients or their guardians. Clinical data of them were documented in detail. None of these patients received any types of preoperative adjuvant therapy (chemotherapy, radiation therapy or immunotherapy). Tissue samples resected from LSCC were prepared for TMAs. 19 cases were treated with carbon dioxide (CO2) laser surgery, 22 underwent a total laryngectomy and 99 had a partial laryngectomy. 67 cases underwent unilateral neck dissection and 28 had bilateral neck dissection (functional or radical neck dissection based on clinical and operative findings). The date of operation was used as the initial time of follow-up. Complete follow-up data was obtained up to May 2010. Twenty-five pairs of fresh LSCC and corresponding non-malignant tissues used for qRT-PCR were collected immediately after tumor excision during surgery from the Affiliated Hospital of Nantong University, China. The study protocol was approved by the Affiliated Hospital of Nantong University Ethics Committee.
140 laryngeal cancer samples. As previously described, three TMAs were constructed in the Department of Pathology, the Affiliated Hospital of Nantong University [16]. Rabbit polyclonal anti-human Gpx1 antibody (dilution 1:100) (Abcam, ab22604) was used for IHC staining [16,17]. The immunohistochemistry results were examined and scored by two independent pathologists without knowing the patient’s clinical characteristics. Gpx1 expression was scored using the semiquantitative H-score method, taking both staining intensity and percentage of positive tumor cells into account [18]. The staining intensity of Gpx1 was graded as follows: 0 (absent), 1 (weak staining), 2 (moderate staining), and 3 (intense staining). The intensity and extent (percentage of the stained positive tumor cells) were multiplied to yield an intensity percentage score. Finally, the staining scale ranged from a minimum value of 0 (no staining) to a maximum value of 300 (100% cells with a staining intensity score of 3) [16,19]. 2.4. Statistical analysis Before statistical analysis, the continuous scores of Gpx1 protein expression were first converted into a two-level grading system (high or low) according to a specific cutoff value, which was selected to be significant in terms of OS using the X-tile software (The Rimm Lab at Yale University; http://www. tissuearray.org/rimmlab) [16,20]. Then, all statistical analyses were performed using SPSS 20.0 version software package. Comparison of Gpx1 mRNA expression between LSCC and non-cancerous tissues was analyzed with t-test. The relationship between Gpx1 expression and clinical parameters was calculated by Pearson Chi-square (x2) test. Kaplan–Meier method was used to establish the survival curves, and the logrank test was used for comparison. Univariate and multivariate analyses were calculated to examine the relationship between overall survival time and variables using a Cox’s proportional hazards regression model. A P-value of less than 0.05 was considered statistically significant.
2.2. RNA extraction and quantitative real-time PCR analysis
3. Results
Total RNA was extracted from tumor tissues obtained at surgery which were immediately frozen and stored at 80 C until use. cDNA was produced using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany, 04 896 866 001). Then 480 SYBR Green I Master (Roche, Germany, 04 707 516 001) was used for two-step quantitative real-time PCR. The primers were purchased from TaKaRa (Otsu, Japan), and GAPDH was used as an endogenous control. The sequences are shown as follows: Gpx1, (sense, 50 -GCGGGGCAAGGTACTACTTA30 ) and (anti-sense, 50 -CTCTTCGTTCTTGGCGTTCT-30 ). GAPDH, (sense, 50 -GCACCGTCAAGGCTGAGAAC-30 ) and (anti-sense, 50 -TGGTGAAGACGCCAGTGGA-30 ).
3.1. Gpx1 mRNA level in laryngeal tissues by quantitative realtime PCR
2.3. TMA construction and IHC analysis
In the initial phase, “70” was determined as the cutoff point via the X-Tile for TMAs data analysis [16]. Thus, scores of 70– 300 were identified as high expression, while scores of 0–70 were considered low expression. For all subsequent analyses,
A total of 168 formalin-fixed, paraffin-embedded tissues surgically resected from LSCC patients were prepared for this study. These included 28 matched normal surgical margins and
Gpx1 mRNA level was determined in 50 fresh frozen tissue samples by qRT-PCR test, including 25 cancerous tissue samples and 25 matched adjacent normal tissues. Relative Gpx1 mRNA expression level was normalized to the expression of housekeeping gene GAPDH. Gpx1 mRNA expression level was significantly higher in cancerous tissues than in adjacent normal tissues (P < 0.05) (Fig. 1). 3.2. IHC results of Gpx1 expression in laryngeal tissues
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Fig 1. Gpx1 mRNA level in 25 paired laryngeal tissues by qPCR. T: laryngeal squamous cell carcinoma tissues. N: adjacent normal tissues. (*P < 0.05).
Gpx1 expression were merely considered as either “high” or “low” according to this cutoff value [16]. Gpx1 protein showed only cytoplasmic staining (Fig. 2). Gpx1 overexpression was detected in 84 of 140 LSCC samples (60.00%) and in 11/28 normal surgical margins (39.29%). The frequency of high Gpx1 (Gpx1 + ) was significantly higher in cancer tissue compared to normal surgical margins (P = 0.044; Table 1).
3.3. Relationship between Gpx1 expression and clinical pathological parameters in LSCC Then, the correlation between Gpx1 protein expression and clinical parameters in LSCC patients was examined by x2 test. As shown in Table 2, Gpx1 expression was significantly associated with TNM stage (P = 0.008) and lymph node metastasis (P = 0.023). In contrast, Gpx1 exhibited no significant association with other clinicopathological parameters, including alcohol and tobacco consumption, age, gender,
Fig. 2. Representation of Gpx1 expression in LSCC and normal tissues (matched normal surgical margins) on TMA slides. a. LSCC with high Gpx1 (Gpx1+) expression; b. LSCC with no Gpx1 (Gpx1) expression; c. Matched normal surgical margins with no Gpx1 (Gpx1) expression; row 1 are Gpx1 staining with 40 (bar 500 mm) magnification and row 2 are Gpx1 staining with 400 (bar 50 mm) magnification, respectively.
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Table 1 IHC results of Gpx1 expression in laryngeal tissues. Groups
Total
Gpx1+ expression (%)
Gpx1 expression (%)
Pearson X2
P value
Corresponding non-malignant tissues Laryngeal Carcinoma
28 140
11 (39.29) 84 (60.00)
17 (60.71) 56 (40.00)
4.0746
0.044*
Gpx1+ represents high Gpx1 expression, and Gpx1 represents low Gpx1 expression. * p < 0.05.
tumor locations, histopathological grade and T classification (All P > 0.05; Table 2). 3.4. High Gpx1 expression predicts poor survival in LSCC by univariate and multivariate survival analysis Univariate analysis revealed that high Gpx1 expression (hazard ratio [HR] 3.057, 95% confidence interval [CI] 1.504– 6.215; P = 0.002) was associated with poor overall survival in LSCC patients. In addition, T Grade (HR 2.571, 95%CI 1.403– 4.712; P = 0.002), TNM stage (HR 3.238, 95%CI 1.701–6.163; P < 0.001), cervical lymph node metastasis (HR 3.982, 95%CI
2.082–7.616; P < 0.001), tumor location (HR1.813, 95%CI 1.127–2.918; P = 0.014), and histological grade (HR 2.253, 95%CI 1.154–4.399; P = 0.017) were also involved in impacting prognosis in LSCC patients. In multivariate analysis, the data revealed that high Gpx1 expression level (Gpx1+, HR 2.101, 95%CI 1.011–4.367; P = 0.047) was an independent prognostic factor of survival in LSCC patients (Table 3). Kaplan–Meier survival curves revealed that patients with high Gpx1 (Gpx1+) expression had worse prognoses than patients with low Gpx1 (Gpx1) expression (P = 0.001; Fig. 3).
Table 2 Relationship between Gpx1 expression and clinical pathological parameters in LSCC. x2
p value
23 (46.00) 33 (36.67)
1.1667
0.280
81 (59.12) 3 (100.00)
56 (40.88) 0 (0)
2.0438
74 32 34
44 (59.46) 22 (68.75) 18 (52.94)
30 (40.54) 10 (31.25) 16 (47.06)
0.8207
0.365
Alcohol consumption Yes No Unknown
52 54 34
32 (61.54) 34 (62.96) 18 (52.94)
20 (38.46) 20 (37.04) 16 (47.06)
0.0229
0.880
T classification T1 + T2 T3 + T4
88 52
49 (55.68) 35 (67.31)
39 (44.32) 17 (32.69)
1.8408
0.175
24 52 64
12 (50.00) 26 (50.00) 46 (71.88)
12 (50.00) 26 (50.00) 18 (28.13)
6.9271
0.008*
Lymph node metastasis Yes No
22 118
18 (81.82) 66 (55.93)
4 (18.18) 52 (44.07)
5.1772
0.023*
Histopathological grade High Moderate Low
54 72 14
29 (53.70) 44 (61.11) 11 (78.57)
25 (46.30) 28 (38.89) 3 (21.43)
1.4521
0.228
Tumor location Supraglottic Glottic Subglottic
51 75 14
27 (52.94) 45 (60.00) 12 (85.71)
24 (47.06) 30 (40.00) 2 (14.29)
4.9160
0.086
Groups
No.
High expression n (%)
Low expression n (%)
Total Age (years) <60 years 60 years
140
84 (60.00)
56 (40.00)
50 90
27 (54.00) 57 (63.33)
Gender Male Female
137 3
Tobacco consumption Yes No Unknown
TNM stage Stage I Stage II Stage III, IV
*
Gpx1
0.153
p < 0.05.
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Table 3 Univariate and multivariate analysis of prognostic markers for overall survival in LSCC patients. Variable
Gpx1 expression High vs Low Age (years) 60 years vs >60 years Tobacco consumption Yes vs No Alcohol consumption Yes vs No TNM stage Stage I, II vs Stage III, IV Lymph node metastasis Yes vs No Histopathological grade High vs Moderate vs Low Tumor location Glottic vs Supraglottic vs Subglottic T Grade T1 + 2 vs T3 + 4 *
Univariate analysis
Multivariate analysis
HR
p value
95% CI
HR
p value
95% CI
3.057
0.002*
1.504–6.215
2.101
0.047*
1.011–4.367
1.479
0.239
0.771–2.839
0.669
0.249
0.337–1.326
0.841
0.590
0.448–1.579
3.238
<0.001*
1.701–6.163
3.982
<0.001*
2.082–7.616
2.626
0.005*
1.335–5.167
2.253
0.017*
1.154–4.399
1.627
0.357
0.578–4.585
1.813
0.014*
1.127–2.918
1.062
0.880
0.486–2.320
2.571
0.002*
1.403–4.712
1.975
0.030*
1.066–3.657
p < 0.05.
4. Discussion As the second most common cancer in the head and neck region, the prognosis of LSCC patients, especially in advanced stage remains unsatisfactory [21]. Several clinical parameters play a guiding role in diagnosis and treatment of tumor, but they
can not accurately predict prognosis of laryngeal cancer [5]. Therefore, it is urgent to find promising biomolecules for precisely predicting LSCC prognosis [6]. Current studies have shown that oxidative stress involves in the genesis and development of many diseases, including cardiovascular disease [22], neuronal degeneration [23,24] and
Fig. 3. Survival curves of LSCC by the Kaplan–Meier method. The high Gpx1 expression group had significantly poorer prognosis than the low expression group (log-rank test, P < 0.05).
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tumors, etc. [25,26]. Under physiological conditions, there are two antioxidant defense systems in the body, namely the enzymes and non-enzyme defense system. GPXs, a family of selenoprotein-containing antioxidant enzymes, can efficiently eliminate hydrogen peroxide and other ROS in the body and protect cells from the oxidative DNA damage. Nevertheless, the GPXs expressions appear obviously aberrant in the pathological process of cancers [10]. Several studies have demonstrated that abnormal GPXs expression can influence the biological behavior of malignant cells [11–14]. As a member of GPXs family, Gpx1 is the most abundant selenoproteins in the human body due to its presence in a wide range of tissues. The gene encoding Gpx1 is located in the chromosome 3q21 region and consists of two exons and a 1.42 kb region. The binding site of the nuclear factor kappa B (NF-kB) and the transcription factor AP1 is present on the promoter of the Gpx1-encoding gene. By combining with the Gpx1 promoter region, NF-kB and AP1 may regulate Gpx1 transcription and expression [11,13,27]. Previous studies have suggested that Gpx1 overexpression promotes growth, migration, invasion and cisplatin resistance in esophageal squamous cell carcinoma (ESCC) in vitro [13]. Downregulation of Gpx1 expression by Gpx1-siRNA suppressed three cell lines biological functions in salivary adenoid cystic carcinoma (SACC) [11]. Similarly, Gpx1 exhibited the effects of oncogene in breast and lung cancer [12,28,29]. Increased expression of Gpx1 in tumors may be explained by upregulation of oxidative stress. However, other studies have showed a contradictory result. The high level of GPXs as a tumor suppressor was correlated with better diseasespecific survival, especially for advanced stage patients with buccal mucosal squamous cell carcinoma [14]. Additionally, some studies have described the relationship between the polymorphism of Gpx1 and laryngeal cancer [30,31]. But Coskun et al. made a conclusion that variants of GPx1 should not be considered as a risk factor of LSCC [30]. Aynali et al. also demonstrated that there was no significant difference between laryngeal cancer and control groups regarding GPx1 Pro198Leu polymorphisms [31]. To date, no data are available about the roles of Gpx1 in patients with LSCC. To investigate the roles of Gpx1 in LSCC patients, we examined Gpx1 mRNA levels by qRT-PCR. Then we designed to investigate the Gpx1 expression by IHC analysis on TMAs slides. In addition, we correlated Gpx1 expression with clinical indexes and overall survival. Our results revealed that Gpx1 mRNA expression level was significantly higher in cancerous tissues than in adjacent normal tissues. And the frequency of Gpx1-overexpression was much higher in cancer tissues than in normal surgical margins. Elevated Gpx1 expression level was significantly associated with cervical lymph node metastasis and tumor TNM stage, indicating that it was correlated with aggressive phenotypes. Both univariate and multivariate analysis showed an association between Gpx1 expression level and survival in LSCC patients. To explain why Gpx1 act as an oncogene, we speculate that [1]: as we all know, tumor cells stay at a higher level of oxidative stress. They produce more ROS or its intermediate products than normal cells, resulting in tumor mutation, invasion and metastasis.
Then, more ROS require the body to produce more antioxidant enzymes, such as GPX1, to reduce the toxic peroxides. Consequently, Gpx1 expression was up- regulated [2]. ROS also have a cytotoxic effect on tumor cells. Overexpressing GPx1 scavenge ROS, reducing the cytotoxic effect on tumor cells. This is the first study that Gpx1 might be used to precisely predict the prognosis of postoperative patients with LSCC. However, the underlying mechanism of Gpx1 roles remains unexplored. Thus, prospective studies in vitro and in vivo are needed to elucidate the molecular mechanisms regulating Gpx1 expression. 5. Conclusion In summary, our study reveals that elevated Gpx1 expression level correlates with poor OS in patients with LSCC. Data from the present study support the idea that Gpx1 expression may be applied to precisely predict the prognosis in patients with LSCC. Authors’ contributions Qicheng Zhang and Hongli Xu designed the research and wrote the paper; Yiwen You, Hongli Xu and Jie Zhang participated in the Clinical data collection; Qicheng Zhang and Renjie Chen contributed to the final data analysis and prepared figures. All authors read and approved the final manuscript. References [1] Iizuka Y, Yoshimura M, Inokuchi H, Matsuo Y, Nakamura A, Mizowaki T, et al. Recurrence patterns after postoperative radiotherapy for squamous cell carcinoma of the pharynx and larynx. Acta Otolaryngol 2015;135(January (1)):96–102. [2] Ye K, Xu JH, Sun YF, Lin JA, Zheng ZG. Characteristics and clinical significance of lymph node metastases near the recurrent laryngeal nerve from thoracic esophageal carcinoma. Genet Mol Res 2014;13(August (3)):6411–9. [3] Chu EA, Kim YJ. Laryngeal cancer: diagnosis and preoperative workup. Otolaryngol Clin North Am 2008;41(August (4)):673–95. [4] Braakhuis BJ, Leemans CR, Visser O. Incidence and survival trends of head and neck squamous cell carcinoma in the Netherlands between 1989 and 2011. Oral Oncol 2014;50(January (7)):670–5. [5] Papadas TA, Alexopoulos EC, Mallis A, Jelastopulu E, Mastronikolis NS, Goumas P. Survival after laryngectomy: a review of 133 patients with laryngeal carcinoma. Eur Arch Otorhinolaryngol 2010;267 (7):1095–101. [6] Xu Y, Lin YP, Yang D, Zhang G, Zhou HF. Clinical significance of miR149 in the survival of patients with laryngeal squamous cell carcinoma. Biomed Res Int 2016;2016:8561251. [7] Malandrakis EE, Exadactylos A, Dadali O, Golomazou E, Klaoudatos S, Panagiotaki P. Molecular cloning of four glutathione peroxidase (GPx) homologs and expression analysis during stress exposure of the marine teleost Sparus aurata. Comp Biochem Physiol B Biochem Mol Biol 2014;168(Febuary):Error: FPage (53) is higher than LPage (–61)!. [8] Brigelius-Flohe R, Maiorino M. Glutathione peroxidases. Biochim Biophys Acta 2013;1830(May (5)):3289–303. [9] Liu GD, Sheng Z, Wang YF, Han YL, Zhou Y, Zhu JQ. Glutathione peroxidase 1 expression, malondialdehyde levels and histological alterations in the liver of Acrossocheilus fasciatus exposed to cadmium chloride. Gene 2016;578(March (2)):210–8. [10] Cao M, Mu X, Jiang C, Yang G, Chen H, Xue W. Single-nucleotide polymorphisms of Gpx1 and MnSOD and susceptibility to bladder
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High Gpx1 expression predicts poor survival in laryngeal squamous cell carcinoma Qicheng Zhang a,b, Hongli Xu c, Yiwen You b, Jie Zhang b, Renjie Chen a,d,* a Department of Otorhinolaryngology Head and Neck Surgery, The Second Clinical Medical College of Nanjing Medical University, Nanjing, Jiangsu Province, China b Department of Otorhinolaryngology Head and Neck Surgery, Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China c Department of Neurology, The Third People's Hospital of Nantong, Nantong, Jiangsu Province, China d Department of Otorhinolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 March 2017 Accepted 29 May 2017 Available online xxx
Objective: Several studies have demonstrated that abnormal glutathione peroxidases 1 (Gpx1) expression can influence the biological behavior of malignant cells. However, the roles of Gpx1 in laryngeal squamous cell carcinoma (LSCC) remain unknown. The purpose of this study is to analyze the Gpx1 expression and prognostic significance in LSCC patients. Methods: Gpx1 mRNA levels in laryngeal tissues were determined by qRT-PCR. Meanwhile, We examined the expression levels of Gpx1 protein in 140 primary tumor tissues and 28 cases of normal tissues by immunohistochemistry (IHC) analysis on tissue microarrays (TMA). Results: Our results revealed that the frequency of high Gpx1 was significantly higher in cancer tissue compared to normal surgical margins; Gpx1 expression correlated with clinical features and overall survival (OS). Gpx1 overexpression was significantly associated with lymph node metastasis (P = 0.023) and TNM stage (P = 0.008); Kaplan–Meier survival curves revealed that
Keywords: Gpx1 Laryngeal squamous cell carcinoma Prognosis Tissue microarrays (TMA)
1. Introduction Laryngeal cancer ranks as the second most common respiratory tract malignancy, and squamous cell carcinoma is its most common histopathological type [1,2]. In 2012, an estimated 3650 deaths worldwide were associated with laryngeal squamous cell carcinoma (LSCC) and the incidence of LSCC is increasing each year [3,4]. Depending on patient's conditions, several therapeutic strategies could be applied to these patients. However, the survival of these patients, particularly in advanced stage, has not significantly improved in the past several decades. In current clinical practice, some indexes such as tumor sub-sites, cervical lymph node metastases and histopathological grades are not sufficient to
* Corresponding author at: Department of Otorhinolaryngology Head and Neck Surgery, The Second Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China. E-mail address: [email protected] (R. Chen).
accurately and completely evaluate the prognosis of these patients [5]. Therefore, the discovery of new biomarkers to accurately predict and further contribute to improving the prognosis of LSCC patients is important [6]. Glutathione peroxidases (GPXs), as selenoprotein-containing proteases, are a family of the body's antioxidant enzyme system [7]. They can maintain the metabolic balance of the reactive oxygen species (ROS) in the body to protect cells from oxidative DNA damage. At least eight GPX isoenzymes have been isolated from mammals [8]. Among these proteins, glutathione peroxidase 1 (Gpx1), also known as intracellular GPX, is one of the most abundant selenoproteins in the human body [9]. It can catalyze the conversion of glutathione (GSH) to oxidative glutathione (GSSG), reducing the toxic peroxides to non-toxic hydroxyl compounds and blocking the lipid peroxidation cascade, thereby protecting the cell structures and functions [9,10]. Previous studies have shown that aberrant Gpx1 expression could promote cell proliferation, migration, invasion and
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Please cite this article in press as: Zhang Q, et al. High Gpx1 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2017.05.012
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cisplatin resistance in oral, esophagus, lung, breast, bladder cancer [11,14]. However, Gpx1 expression and its prognostic role in LSCC remain unexplored to date. In the present study, Gpx1 mRNA level in laryngeal tissues was examined by qRTPCR; the expression of Gpx1 on tissue microarray was detected by immunohistochemistry (IHC). Furthermore, the relationship between Gpx1 expression and clinical pathological parameters were analyzed as well as overall survival (OS) in patients with LSCC. 2. Materials and methods 2.1. Tissue sample collection and clinical data collection Diagnosis of LSCC was made according to the World Health Organization (WHO) criteria [15] and TNM classification [UICC, 2009]. Totally, 140 hospitalized patients with primary LSCC were recruited from the Department of Otolaryngology/ Head and Neck Surgery, Affiliated Hospital of Nantong University, China, from year 2000 to 2009. Informed consents were obtained from these patients or their guardians. Clinical data of them were documented in detail. None of these patients received any types of preoperative adjuvant therapy (chemotherapy, radiation therapy or immunotherapy). Tissue samples resected from LSCC were prepared for TMAs. 19 cases were treated with carbon dioxide (CO2) laser surgery, 22 underwent a total laryngectomy and 99 had a partial laryngectomy. 67 cases underwent unilateral neck dissection and 28 had bilateral neck dissection (functional or radical neck dissection based on clinical and operative findings). The date of operation was used as the initial time of follow-up. Complete follow-up data was obtained up to May 2010. Twenty-five pairs of fresh LSCC and corresponding non-malignant tissues used for qRT-PCR were collected immediately after tumor excision during surgery from the Affiliated Hospital of Nantong University, China. The study protocol was approved by the Affiliated Hospital of Nantong University Ethics Committee.
140 laryngeal cancer samples. As previously described, three TMAs were constructed in the Department of Pathology, the Affiliated Hospital of Nantong University [16]. Rabbit polyclonal anti-human Gpx1 antibody (dilution 1:100) (Abcam, ab22604) was used for IHC staining [16,17]. The immunohistochemistry results were examined and scored by two independent pathologists without knowing the patient’s clinical characteristics. Gpx1 expression was scored using the semiquantitative H-score method, taking both staining intensity and percentage of positive tumor cells into account [18]. The staining intensity of Gpx1 was graded as follows: 0 (absent), 1 (weak staining), 2 (moderate staining), and 3 (intense staining). The intensity and extent (percentage of the stained positive tumor cells) were multiplied to yield an intensity percentage score. Finally, the staining scale ranged from a minimum value of 0 (no staining) to a maximum value of 300 (100% cells with a staining intensity score of 3) [16,19]. 2.4. Statistical analysis Before statistical analysis, the continuous scores of Gpx1 protein expression were first converted into a two-level grading system (high or low) according to a specific cutoff value, which was selected to be significant in terms of OS using the X-tile software (The Rimm Lab at Yale University; http://www. tissuearray.org/rimmlab) [16,20]. Then, all statistical analyses were performed using SPSS 20.0 version software package. Comparison of Gpx1 mRNA expression between LSCC and non-cancerous tissues was analyzed with t-test. The relationship between Gpx1 expression and clinical parameters was calculated by Pearson Chi-square (x2) test. Kaplan–Meier method was used to establish the survival curves, and the logrank test was used for comparison. Univariate and multivariate analyses were calculated to examine the relationship between overall survival time and variables using a Cox’s proportional hazards regression model. A P-value of less than 0.05 was considered statistically significant.
2.2. RNA extraction and quantitative real-time PCR analysis
3. Results
Total RNA was extracted from tumor tissues obtained at surgery which were immediately frozen and stored at 80 C until use. cDNA was produced using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany, 04 896 866 001). Then 480 SYBR Green I Master (Roche, Germany, 04 707 516 001) was used for two-step quantitative real-time PCR. The primers were purchased from TaKaRa (Otsu, Japan), and GAPDH was used as an endogenous control. The sequences are shown as follows: Gpx1, (sense, 50 -GCGGGGCAAGGTACTACTTA30 ) and (anti-sense, 50 -CTCTTCGTTCTTGGCGTTCT-30 ). GAPDH, (sense, 50 -GCACCGTCAAGGCTGAGAAC-30 ) and (anti-sense, 50 -TGGTGAAGACGCCAGTGGA-30 ).
3.1. Gpx1 mRNA level in laryngeal tissues by quantitative realtime PCR
2.3. TMA construction and IHC analysis
In the initial phase, “70” was determined as the cutoff point via the X-Tile for TMAs data analysis [16]. Thus, scores of 70– 300 were identified as high expression, while scores of 0–70 were considered low expression. For all subsequent analyses,
A total of 168 formalin-fixed, paraffin-embedded tissues surgically resected from LSCC patients were prepared for this study. These included 28 matched normal surgical margins and
Gpx1 mRNA level was determined in 50 fresh frozen tissue samples by qRT-PCR test, including 25 cancerous tissue samples and 25 matched adjacent normal tissues. Relative Gpx1 mRNA expression level was normalized to the expression of housekeeping gene GAPDH. Gpx1 mRNA expression level was significantly higher in cancerous tissues than in adjacent normal tissues (P < 0.05) (Fig. 1). 3.2. IHC results of Gpx1 expression in laryngeal tissues
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Fig 1. Gpx1 mRNA level in 25 paired laryngeal tissues by qPCR. T: laryngeal squamous cell carcinoma tissues. N: adjacent normal tissues. (*P < 0.05).
Gpx1 expression were merely considered as either “high” or “low” according to this cutoff value [16]. Gpx1 protein showed only cytoplasmic staining (Fig. 2). Gpx1 overexpression was detected in 84 of 140 LSCC samples (60.00%) and in 11/28 normal surgical margins (39.29%). The frequency of high Gpx1 (Gpx1 + ) was significantly higher in cancer tissue compared to normal surgical margins (P = 0.044; Table 1).
3.3. Relationship between Gpx1 expression and clinical pathological parameters in LSCC Then, the correlation between Gpx1 protein expression and clinical parameters in LSCC patients was examined by x2 test. As shown in Table 2, Gpx1 expression was significantly associated with TNM stage (P = 0.008) and lymph node metastasis (P = 0.023). In contrast, Gpx1 exhibited no significant association with other clinicopathological parameters, including alcohol and tobacco consumption, age, gender,
Fig. 2. Representation of Gpx1 expression in LSCC and normal tissues (matched normal surgical margins) on TMA slides. a. LSCC with high Gpx1 (Gpx1+) expression; b. LSCC with no Gpx1 (Gpx1) expression; c. Matched normal surgical margins with no Gpx1 (Gpx1) expression; row 1 are Gpx1 staining with 40 (bar 500 mm) magnification and row 2 are Gpx1 staining with 400 (bar 50 mm) magnification, respectively.
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Table 1 IHC results of Gpx1 expression in laryngeal tissues. Groups
Total
Gpx1+ expression (%)
Gpx1 expression (%)
Pearson X2
P value
Corresponding non-malignant tissues Laryngeal Carcinoma
28 140
11 (39.29) 84 (60.00)
17 (60.71) 56 (40.00)
4.0746
0.044*
Gpx1+ represents high Gpx1 expression, and Gpx1 represents low Gpx1 expression. * p < 0.05.
tumor locations, histopathological grade and T classification (All P > 0.05; Table 2). 3.4. High Gpx1 expression predicts poor survival in LSCC by univariate and multivariate survival analysis Univariate analysis revealed that high Gpx1 expression (hazard ratio [HR] 3.057, 95% confidence interval [CI] 1.504– 6.215; P = 0.002) was associated with poor overall survival in LSCC patients. In addition, T Grade (HR 2.571, 95%CI 1.403– 4.712; P = 0.002), TNM stage (HR 3.238, 95%CI 1.701–6.163; P < 0.001), cervical lymph node metastasis (HR 3.982, 95%CI
2.082–7.616; P < 0.001), tumor location (HR1.813, 95%CI 1.127–2.918; P = 0.014), and histological grade (HR 2.253, 95%CI 1.154–4.399; P = 0.017) were also involved in impacting prognosis in LSCC patients. In multivariate analysis, the data revealed that high Gpx1 expression level (Gpx1+, HR 2.101, 95%CI 1.011–4.367; P = 0.047) was an independent prognostic factor of survival in LSCC patients (Table 3). Kaplan–Meier survival curves revealed that patients with high Gpx1 (Gpx1+) expression had worse prognoses than patients with low Gpx1 (Gpx1) expression (P = 0.001; Fig. 3).
Table 2 Relationship between Gpx1 expression and clinical pathological parameters in LSCC. x2
p value
23 (46.00) 33 (36.67)
1.1667
0.280
81 (59.12) 3 (100.00)
56 (40.88) 0 (0)
2.0438
74 32 34
44 (59.46) 22 (68.75) 18 (52.94)
30 (40.54) 10 (31.25) 16 (47.06)
0.8207
0.365
Alcohol consumption Yes No Unknown
52 54 34
32 (61.54) 34 (62.96) 18 (52.94)
20 (38.46) 20 (37.04) 16 (47.06)
0.0229
0.880
T classification T1 + T2 T3 + T4
88 52
49 (55.68) 35 (67.31)
39 (44.32) 17 (32.69)
1.8408
0.175
24 52 64
12 (50.00) 26 (50.00) 46 (71.88)
12 (50.00) 26 (50.00) 18 (28.13)
6.9271
0.008*
Lymph node metastasis Yes No
22 118
18 (81.82) 66 (55.93)
4 (18.18) 52 (44.07)
5.1772
0.023*
Histopathological grade High Moderate Low
54 72 14
29 (53.70) 44 (61.11) 11 (78.57)
25 (46.30) 28 (38.89) 3 (21.43)
1.4521
0.228
Tumor location Supraglottic Glottic Subglottic
51 75 14
27 (52.94) 45 (60.00) 12 (85.71)
24 (47.06) 30 (40.00) 2 (14.29)
4.9160
0.086
Groups
No.
High expression n (%)
Low expression n (%)
Total Age (years) <60 years 60 years
140
84 (60.00)
56 (40.00)
50 90
27 (54.00) 57 (63.33)
Gender Male Female
137 3
Tobacco consumption Yes No Unknown
TNM stage Stage I Stage II Stage III, IV
*
Gpx1
0.153
p < 0.05.
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Table 3 Univariate and multivariate analysis of prognostic markers for overall survival in LSCC patients. Variable
Gpx1 expression High vs Low Age (years) 60 years vs >60 years Tobacco consumption Yes vs No Alcohol consumption Yes vs No TNM stage Stage I, II vs Stage III, IV Lymph node metastasis Yes vs No Histopathological grade High vs Moderate vs Low Tumor location Glottic vs Supraglottic vs Subglottic T Grade T1 + 2 vs T3 + 4 *
Univariate analysis
Multivariate analysis
HR
p value
95% CI
HR
p value
95% CI
3.057
0.002*
1.504–6.215
2.101
0.047*
1.011–4.367
1.479
0.239
0.771–2.839
0.669
0.249
0.337–1.326
0.841
0.590
0.448–1.579
3.238
<0.001*
1.701–6.163
3.982
<0.001*
2.082–7.616
2.626
0.005*
1.335–5.167
2.253
0.017*
1.154–4.399
1.627
0.357
0.578–4.585
1.813
0.014*
1.127–2.918
1.062
0.880
0.486–2.320
2.571
0.002*
1.403–4.712
1.975
0.030*
1.066–3.657
p < 0.05.
4. Discussion As the second most common cancer in the head and neck region, the prognosis of LSCC patients, especially in advanced stage remains unsatisfactory [21]. Several clinical parameters play a guiding role in diagnosis and treatment of tumor, but they
can not accurately predict prognosis of laryngeal cancer [5]. Therefore, it is urgent to find promising biomolecules for precisely predicting LSCC prognosis [6]. Current studies have shown that oxidative stress involves in the genesis and development of many diseases, including cardiovascular disease [22], neuronal degeneration [23,24] and
Fig. 3. Survival curves of LSCC by the Kaplan–Meier method. The high Gpx1 expression group had significantly poorer prognosis than the low expression group (log-rank test, P < 0.05).
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tumors, etc. [25,26]. Under physiological conditions, there are two antioxidant defense systems in the body, namely the enzymes and non-enzyme defense system. GPXs, a family of selenoprotein-containing antioxidant enzymes, can efficiently eliminate hydrogen peroxide and other ROS in the body and protect cells from the oxidative DNA damage. Nevertheless, the GPXs expressions appear obviously aberrant in the pathological process of cancers [10]. Several studies have demonstrated that abnormal GPXs expression can influence the biological behavior of malignant cells [11–14]. As a member of GPXs family, Gpx1 is the most abundant selenoproteins in the human body due to its presence in a wide range of tissues. The gene encoding Gpx1 is located in the chromosome 3q21 region and consists of two exons and a 1.42 kb region. The binding site of the nuclear factor kappa B (NF-kB) and the transcription factor AP1 is present on the promoter of the Gpx1-encoding gene. By combining with the Gpx1 promoter region, NF-kB and AP1 may regulate Gpx1 transcription and expression [11,13,27]. Previous studies have suggested that Gpx1 overexpression promotes growth, migration, invasion and cisplatin resistance in esophageal squamous cell carcinoma (ESCC) in vitro [13]. Downregulation of Gpx1 expression by Gpx1-siRNA suppressed three cell lines biological functions in salivary adenoid cystic carcinoma (SACC) [11]. Similarly, Gpx1 exhibited the effects of oncogene in breast and lung cancer [12,28,29]. Increased expression of Gpx1 in tumors may be explained by upregulation of oxidative stress. However, other studies have showed a contradictory result. The high level of GPXs as a tumor suppressor was correlated with better diseasespecific survival, especially for advanced stage patients with buccal mucosal squamous cell carcinoma [14]. Additionally, some studies have described the relationship between the polymorphism of Gpx1 and laryngeal cancer [30,31]. But Coskun et al. made a conclusion that variants of GPx1 should not be considered as a risk factor of LSCC [30]. Aynali et al. also demonstrated that there was no significant difference between laryngeal cancer and control groups regarding GPx1 Pro198Leu polymorphisms [31]. To date, no data are available about the roles of Gpx1 in patients with LSCC. To investigate the roles of Gpx1 in LSCC patients, we examined Gpx1 mRNA levels by qRT-PCR. Then we designed to investigate the Gpx1 expression by IHC analysis on TMAs slides. In addition, we correlated Gpx1 expression with clinical indexes and overall survival. Our results revealed that Gpx1 mRNA expression level was significantly higher in cancerous tissues than in adjacent normal tissues. And the frequency of Gpx1-overexpression was much higher in cancer tissues than in normal surgical margins. Elevated Gpx1 expression level was significantly associated with cervical lymph node metastasis and tumor TNM stage, indicating that it was correlated with aggressive phenotypes. Both univariate and multivariate analysis showed an association between Gpx1 expression level and survival in LSCC patients. To explain why Gpx1 act as an oncogene, we speculate that [1]: as we all know, tumor cells stay at a higher level of oxidative stress. They produce more ROS or its intermediate products than normal cells, resulting in tumor mutation, invasion and metastasis.
Then, more ROS require the body to produce more antioxidant enzymes, such as GPX1, to reduce the toxic peroxides. Consequently, Gpx1 expression was up- regulated [2]. ROS also have a cytotoxic effect on tumor cells. Overexpressing GPx1 scavenge ROS, reducing the cytotoxic effect on tumor cells. This is the first study that Gpx1 might be used to precisely predict the prognosis of postoperative patients with LSCC. However, the underlying mechanism of Gpx1 roles remains unexplored. Thus, prospective studies in vitro and in vivo are needed to elucidate the molecular mechanisms regulating Gpx1 expression. 5. Conclusion In summary, our study reveals that elevated Gpx1 expression level correlates with poor OS in patients with LSCC. Data from the present study support the idea that Gpx1 expression may be applied to precisely predict the prognosis in patients with LSCC. Authors’ contributions Qicheng Zhang and Hongli Xu designed the research and wrote the paper; Yiwen You, Hongli Xu and Jie Zhang participated in the Clinical data collection; Qicheng Zhang and Renjie Chen contributed to the final data analysis and prepared figures. All authors read and approved the final manuscript. References [1] Iizuka Y, Yoshimura M, Inokuchi H, Matsuo Y, Nakamura A, Mizowaki T, et al. Recurrence patterns after postoperative radiotherapy for squamous cell carcinoma of the pharynx and larynx. Acta Otolaryngol 2015;135(January (1)):96–102. [2] Ye K, Xu JH, Sun YF, Lin JA, Zheng ZG. Characteristics and clinical significance of lymph node metastases near the recurrent laryngeal nerve from thoracic esophageal carcinoma. Genet Mol Res 2014;13(August (3)):6411–9. [3] Chu EA, Kim YJ. Laryngeal cancer: diagnosis and preoperative workup. Otolaryngol Clin North Am 2008;41(August (4)):673–95. [4] Braakhuis BJ, Leemans CR, Visser O. Incidence and survival trends of head and neck squamous cell carcinoma in the Netherlands between 1989 and 2011. Oral Oncol 2014;50(January (7)):670–5. [5] Papadas TA, Alexopoulos EC, Mallis A, Jelastopulu E, Mastronikolis NS, Goumas P. Survival after laryngectomy: a review of 133 patients with laryngeal carcinoma. Eur Arch Otorhinolaryngol 2010;267 (7):1095–101. [6] Xu Y, Lin YP, Yang D, Zhang G, Zhou HF. Clinical significance of miR149 in the survival of patients with laryngeal squamous cell carcinoma. Biomed Res Int 2016;2016:8561251. [7] Malandrakis EE, Exadactylos A, Dadali O, Golomazou E, Klaoudatos S, Panagiotaki P. Molecular cloning of four glutathione peroxidase (GPx) homologs and expression analysis during stress exposure of the marine teleost Sparus aurata. Comp Biochem Physiol B Biochem Mol Biol 2014;168(Febuary):Error: FPage (53) is higher than LPage (–61)!. [8] Brigelius-Flohe R, Maiorino M. Glutathione peroxidases. Biochim Biophys Acta 2013;1830(May (5)):3289–303. [9] Liu GD, Sheng Z, Wang YF, Han YL, Zhou Y, Zhu JQ. Glutathione peroxidase 1 expression, malondialdehyde levels and histological alterations in the liver of Acrossocheilus fasciatus exposed to cadmium chloride. Gene 2016;578(March (2)):210–8. [10] Cao M, Mu X, Jiang C, Yang G, Chen H, Xue W. Single-nucleotide polymorphisms of Gpx1 and MnSOD and susceptibility to bladder
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Please cite this article in press as: Zhang Q, et al. High Gpx1 expression predicts poor survival in laryngeal squamous cell carcinoma. Auris Nasus Larynx (2017), http://dx.doi.org/10.1016/j.anl.2017.05.012