Clinical significance of SOX9 in human non-small cell lung cancer progression and overall patient survival
- Chun-Hui Zhou†2,
- Li-Ping Ye†1,
- Shi-Xing Ye1,
- Yan Li1,
- Xin-Yin Zhang1,
- Xin-Yu Xu1 and
- Li-Yun Gong1Email author
© Zhou et al; licensee BioMed Central Ltd. 2012
Received: 9 January 2012
Accepted: 3 March 2012
Published: 3 March 2012
Sex determining region Y (SRY)-related high mobility groupbox 9 (SOX9) is an important transcription factor required for development, which regulates the expression of target genes in the associated pathway. The aim of this study was to describe the expression of SOX9 in human non-small cell lung cancer (NSCLC) and to investigate the association between SOX9 expression and progression of NSCLC.
SOX9 protein and mRNA expression in normal human pneumonocytes, lung cancer cell lines, and eight pairs of matched lung cancer tissues and their adjacent normal lung tissues were detected by Western blotting and real-time reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemistry was used to determine SOX9 protein expression in 142 cases of histologically characterized NSCLC. Statistical analyses were applied to test for prognostic and diagnostic associations.
SOX9 in lung cancer cell lines was upregulated at both mRNA and protein levels, and SOX9 mRNA and protein were also elevated in NSCLC tissues compared with levels in corresponding adjacent non-cancerous lung tissues. Immunohistochemical analysis demonstrated a high expression of SOX9 in 74/142 (52.1%) paraffin-embedded archival lung cancer biopsies. Statistical analysis indicated that upregulation of SOX9 was significantly correlated with the histological stage of NSCLC (P = 0.017) and that patients with a high SOX9 level exhibited a shorter survival time (P < 0.001). Multivariate analysis illustrated that SOX9 upregulation might be an independent prognostic indicator for the survival of patients with NSCLC.
This work shows that SOX9 may serve as a novel and prognostic marker for NSCLC, and play a role during the development and progression of the disease.
Globally, lung cancer was the most commonly diagnosed cancer and the leading cause of cancer death in males, comprising 13% (1.6 million) of the total cases of cancer and 18% (1.4 million) of total cancer deaths in 2008 . The main clinical types of lung cancer are small cell lung cancer(SCLC) and non-small cell lung cancer (NSCLC). NSCLC represents almost 80% of lung cancer, which is the leading cause of cancer-related death in the world. The most common types of NSCLC are squamous cell lung carcinoma, adenocarcinoma, and large cell lung cancer. Surgical resection with adjuvant chemotherapy is the preferred approach for early stage NSCLC, while patients with advanced NSCLC are usually treated with chemotherapy or radiation therapy. Despite advances in treatment, the prognosis is generally poor. Following complete surgical resection of stage IA disease, 5-year survival of patients is 67%, but the 5-year survival rate of individuals with stage IV NSCLC is below 1% . One reason for such a low survival rate is that patients do not receive treatment early enough in disease progression for it to be effective, which is associated with the high metastasis character of NSCLC. Progression from low- to high -stage lung cancer is related to various molecular alterations. However, the cytogenetic and molecular data on various forms of NSCLC are still being investigated for better understanding the disease. The molecular mechanism underlying the progression of NSCLC requires further research, with a view to basing therapy on molecular signatures within tumors. There is significant clinical value in early detection and provision of effective interventions to treat NSCLC.
Sex determining region Y (SRY)-related high mobility group (HMG)-box 9 (SOX9) shares 70% amino acid homology to SRY through its HMG box, the domains of which are involved in the regulation of DNA-dependent processes, such as transcription and replication . SOX9 function was first identified as a key regulator of cartilage and male gonad development, with mutations in SOX9 causing campomelic dysplasia and autosomal sex reversal [4, 5]. Subsequently, it emerged that SOX9 has been found to be upregulated in several tumor types, such as lung adenocarcinoma, breast carcinoma, colorectal cancer, and prostate cancer [6–9]. However, the clinical and functional significance of SOX9 expression has not been characterized previously in all stages of NSCLC despite the recently reported correlation between upregulation of SOX9 and lung adenocarcinoma, and its association with cancer cell growth . In the present study, SOX9 expression was characterized in all stages of NSCLC from early to advanced. This study found that the expression level of SOX9 was correlated strongly with the histological stage and the survival time of NSCLC patients. In addition, the usefulness of SOX9 as a prognostic factor was evaluated by multivariate analysis. The data revealed that SOX9 could be a lung cancer-associated molecule with a prognostic value.
Primary normal lung epithelial cells (NLEC) were established according to a previously report . In brief, surgical specimens from normal lung were promptly removed and transported aseptically in Hanks' solution (Invitrogen, Carlsbad, CA) with 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA) and 5 μg/ml gentamicin (Invitrogen, Carlsbad, CA). The tissue specimens were incubated with 1.5 units/ml dispase (Roche Molecular Biochemicals) at 4°C overnight, and the epithelium was dissected away and incubated with trypsin (Invitrogen, Carlsbad, CA). The reaction was stopped with soybean trypsin inhibitor (Sigma, Saint Louis, MI) and centrifuged. The pellet was resuspended in keratinocyte-SFM medium (KSFM) (Invitrogen, Carlsbad, CA) supplemented with 40 μg/ml bovine pituitary extract (Invitrogen, Carlsbad, CA), 1.0 ng/ml EGF (Invitrogen, Carlsbad, CA), 100 units/ml penicillin, 100 μg/ml streptomycin (Invitrogen, Carlsbad, CA), 5 μg/ml gentamycin, and 100 units/ml nyastatin (Invitrogen, Carlsbad, CA). NEEC cells were grown at 37°C and 5% CO2 with KSFM, with 40 μg/ml bovine pituitary extract, 1.0 ng/ml EGF, 100 units/ml penicillin, and 100 μg/ml streptomycin. Lung cancer cell lines, including SK-MES-1, NCI-H460, NCI-H358, NCI-H1650, NCI-H1975, NCI-H596 and PAa, were provided by American Type Culture Collection (ATCC) and grown in the Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, USA) with 10% fetal bovine serum (Invitrogen) at 37°C in a 5% CO2 atmosphere.
Patients and tissue specimens
Clinicopathologic characteristics of studied patient and expression of SOX9 in NSCLC
Squamous cell carcinoma
NSCLC histology (AJCC grade)
Survival (n = 89)
Survial time of low expression
Survival time of high expression
Expression of SOX9
RNA extraction and real-time reverse transcription-polymerase chain reaction
Total RNA from cultured cells was extracted using the TRIzol reagent (Invitrogen) and purified using the purelink RNA Mini Kit (Invitrogen) according to the manufacturer's instructions. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed to quantify the change of SOX9 mRNA level in lung cancer cell lines compared with that in normal human pneumonocytes. Real-time RT-PCR primers and probes for SOX9 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed with the assistance of the Primer Express version 2.0 software (Applied Biosystems).
SOX9 forward primer: 5'-CGAAATCAACGAGAAACTGGAC-3', SOX9 reverse primer: 5'-ATTTAGCACACTGATCACACG-3', SOX9 probe 5'-(FAM) CCATCATCCTCCACGCTTGCTCTG (TAMRA)-3', GAPDH forward primer: 5'-GACTCATGACCACAGTCCATGC-3', GAPDH reverse primer: 5'-AGAGGCAGGGATGATGTTCTG-3', GAPDH probe 5'-(FAM) CATCACTGCCACCCAGAAGACTGTG (TAMRA)-3'.
Expression data were normalized to the housekeeping gene GAPDH and calculated as 2-[(Ct of gene) - (Ct of GAPDH)], where Ct represents the threshold cycle for each transcript.
Cells were harvested in sampling buffer and boiled for 10 min. The procedure was perfomed similarly to previously described methods . Protein concentration was determined with the bicinchoninic acid (BCA) assay (Pierce, Rockford, USA) according to the manufacturer's instructions. Equal amounts of protein were separated electrophoretically on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, USA). The membrane was probed with an anti-SOX9 rabbit antibody (1:2,000 dilution; Millipore) and incubated with goat anti-rabbit immunoglobulin G (1:50,000 dilution; Pierce). Expression of SOX9 was determined with SuperSignal West Pico Chemiluminescent Substrate (Thermo, USA) according to the manufacturer's suggested protocol. The membranes were stripped and reprobed with an anti-actin mouse monoclonal antibody (1:2,000 dilution; Millipore) as a loading control.
Immunohistochemical analysis was performed to study altered protein expression in 142 human lung cancer tissues. The procedures were carried out in a similar manner to previously described methods . Paraffin-embedded specimens were cut into 4 μm sections and baked at 65°C for 30 minutes. The sections were deparaffinized with xylenes and rehydrated. Sections were submerged into ethylenediaminetetraacetic acid antigenic retrieval buffer and microwaved for antigenic retrieval. The sections were treated with 3% hydrogen peroxide in methanol to quench the endogenous peroxidase activity, followed by incubation in 1% bovine serum albumin to block non-specific binding. Rabbit anti-SOX9 (1:50 dilution; Millipore) was incubated with the sections at 4°C overnight. Primary antibody was replaced by normal goat serum in the negative controls. After washing, the tissue sections were treated with biotinylated anti-rabbit secondary antibody (Zymed, San Francisco, USA) followed by a further incubation with streptavidin-horseradish peroxidase complex (Zymed). The tissue sections were immersed in 3-amino-9-ethyl carbazole and counterstained using 10% Mayer's hematoxylin, dehydrated, and mounted in Crystal Mount (Sigma). The degree of immunostaining of formalin-fixed, paraffin-embedded sections was viewed and scored separately by two independent investigators, who were blinded to the histopathological features and patient details of the samples. Scores were determined by combining the proportion of positively stained tumor cells and the intensity of staining. The scores given by the two independent investigators were averaged for further comparative evaluation of SOX9 expression. The proportion of positively stained tumor cells was staged as follows: 0 (no positive tumor cells), 1 (<10% positive tumor cells), 2 (10-50% positive tumor cells), and 3 (>50% positive tumor cells). The cells at each intensity of staining were recorded on a scale of 0 (no staining), 1 (weak staining, light yellow), 2 (moderate staining, yellowish brown), and 3 (strong staining, brown). The staining index was calculated as follows: staining index = staining intensity × proportion of positively stained tumor cells. Using this method of assessment, the expression of SOX9 in lung cancers was evaluated using the staining index (scored as 0, 1, 2, 3, 4, 6, or 9). High and low expression of SOX9 were defined using cutoff values based on a measure of heterogeneity with the log-rank test statistics with respect to overall survival; an optimal cutoff value was identified. A staining index score of ≥ 6 was used to define tumors with high expression and a staining index ≤ 4 was used to define tumors with low expression of SOX9.
Immunohistochemical staining for protein expression in tumor and normal tissues was quantitatively analyzed with the Olympus BX51 image analysis system assisted with the CellSens Dimension 1.5 Imaging software. The stained sections were evaluated at × 200 magnification and 10 representative staining fields per section were analyzed to verify the mean absorbance, which represents the strength of staining signals as measured per positive pixels. The mean absorbance data were analyzed statistically using t test to compare the average mean absorbance difference between different groups of tissues; a P < 0.05 was considered significant.
All statistical analyses were carried out using the statistical software package, SPSS, version 17.0 (IBM SPSS, Chicago, USA). The χ2 test was used to analyze the relationship between SOX9 expression and the clinicopathological characteristics. Bivariate correlations between study variables were calculated by Spearman rank correlation coefficients. Survival curves were plotted with the Kaplan-Meier method and compared by the log-rank test. Survival data were evaluated using univariate and multivariate Cox regression analyses. In all cases, P < 0.05 was considered statistically significant.
Increased expression of SOX9 in NSCLC
Correlation between increased expression of SOX9 and malignancy of NSCLC
Expression of SOX9 protein and histological staging of NSCLC
Correlation between the clinicalpathologic features and expressions of SOX9
Low or none
Squamous cell carcinoma
NSCLC histology (AJCC grade)
I and II
III and IV
Survival (n = 89)
Spearman correlation analysis between SOX9 and clinical pathologic factors
Association between SOX9 expression and patient prognosis
Univariate and multivariate analysis of different prognostic parameters in patients with NSCLC by Cox-regression analysis
95% confidence interval
NSCLC histology (AJCC grade)
The major finding of our study is that the progression of human NSCLC is related to upregulation of SOX9 expression. Although, a previous report has described a correlation between the expression of SOX9 mRNA and protein levels with lung adenocarcinoma , this study represents the first demonstration that SOX9 mRNA and protein are upregulated in all stages of human NSCLC and that this degree of upregulation increases as NSCLC progresses to advanced stages.
Recent cogent evidence has provided a link between SOX9 and cancer development and progression [14, 15], and the upregulation of SOX9 has been observed in several types of solid tumors, including lung adenocarcinoma, breast carcinoma, colorectal cancer, and prostate cancer [6–9]. In addition, there is marked inhibition of differentiation, coupled with an expanded domain of expression of SOX9 protein in Nmyc overexpressing lung . It has been reported that the induction of SOX9 expression could be induced through various mechanisms. Dysregulation of tissue development pathways can be conducive to cancer initiation and progression. As part of a developmental pathway, elevation of SOX9 in prostate neoplasia promotes tumor cell proliferation . Moreover, as a transcription factor, SOX9 is linked to the hedgehog pathways and may play a role in the development of malignant peripheral nerve sheath tumor in patients . Ling et al. reported that despite SOX9 levels being high during periods of prenatal urothelial development in mouse bladders, SOX9 was diminished and quiescent with maturation after birth, but was rapidly induced by a variety of injuries and urothelial cancer . All these findings suggest that SOX9 may play important roles in cancer development and progression, which prompted the authors to ask whether it is also clinically associated with the progression of NSCLC. To address this question, studies were performed to characterize the expression of SOX9 in NSCLC cell lines and clinical lung cancer tissues. The data show that upregulation of SOX9 mRNA and protein is a common and frequent event in both NSCLC cell lines and human lung cancer tissues. Comparative analyses of SOX9 mRNA and protein in lung cancer tissues and their paired adjacent normal tissue have provided strong support for the identified upregulation of SOX9 in NSCLC. Moderate to strong cytoplasmic staining of SOX9 was displayed in tumor cells from 135/142 (95.1%) paraffin-embedded archived NSCLC biopsy samples in comparison with the adjacent non-cancerous cells, which expressed little, if any, SOX9.
Further analysis of the relationship between SOX9 staining and the clinicopathological characteristics of patients showed a significant correlation between SOX9 expression and the histopathological staging of NSCLC. This revealed that SOX9 levels were higher in advanced stages of the disease, supporting the hypotheses that SOX9 may play a role in the progression of NSCLC and that it could represent a biomarker that identifies subsets of lung-cancer patients with more aggressive disease. It is of particular note that patients with high SOX9 expression had shorter survival time, suggesting the possibility of using SOX9 as a predictor for patient prognosis and survival.
In a more detailed survival study, univariate and multivariate analyses demonstrated that high expression of SOX9 is a predictor of poor prognosis for lung-cancer patients. It is of note that there is a significant correlation between shorter overall survival times of patients and high SOX9 expression in both the early histological stage subgroup (stages I and II) and the late histological stage subgroup (stages III and IV), suggesting that SOX9 may be a useful prognostic marker for all stages of NSCLC.
Although several lines of evidence have suggested that SOX9 might be involved in cancer development and progression, only a few studies have linked SOX9 to lung cancer. Knockdown of SOX9 has been found to decrease the proliferation rate of lung cancer cell lines and significantly attenuate the tumorigenicity of lung adenocarcinoma . Despite the above finding, the precise pathway that SOX9 uses to inhibit the differentiation of NSCLC and promote lung cancer development and progression remains unclear. Based on the findings from this and other studies, further investigation is warranted to validate whether SOX9 can be used as a novel therapeutic target for NSCLC.
This study was supported by grants from The Natural Science Foundation of China (No.81101501), The Science and Technology Bureau of ShenZhen City grants(No.JC200903120125A), The Health Bureau of Guang Zhou City grants(No. 2009-YB-163), The Natural Science Foundation of ShenZhen University (No. 200921), The Natural Science Foundation of Guangzhou medical University (No.2008C06), the Laboratory Opening Grants of Shenzhen University(2011 year).
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