Upregulated SOX9 expression indicates worse prognosis in solid tumors: a systematic review and meta-analysis

It was recently reported that increased SOX9 expression drives tumor growth and promotes cancer invasion during human tumorigenicity and metastasis. However, the prognostic value of SOX9 for the survival of patients with solid tumors remains controversial. The present meta-analysis was thus performed to highlight the link between dysregulated SOX9 expression and prognosis in cancer patients. A systematic literature search was conducted using the electronic databases PubMed, Web of Science and Embase to identify eligible studies. A random-effects meta-analytical model was employed to correlate SOX9 expression with overall survival (OS), disease-free survival (DFS) and clinicopathological features. In total, 17 studies with 3307 patients were eligible for the final analysis. Combined hazard ratios (HRs) and 95% confidence intervals (CIs) suggested that high SOX9 expression has an unfavourable impact on OS (HR = 1.66, 95% CI 1.36–2.02, P < 0.001) and DFS (HR = 3.54, 95% CI 2.29–5.47, P = 0.008) in multivariate analysis. Additionally, the pooled odds ratios (ORs) indicated that SOX9 over-expression is associated with large tumor size, lymph node metastasis, distant metastasis and a higher clinical stage. Overall, these results indicated that SOX9 over-expression in patients with solid tumors might be related to poor prognosis and could serve as a potential predictive marker of poor clinicopathological prognosis factor.


INTRODUCTION
SOX9 is a member of SOX [SRY (sex determining region Y)-related high mobility group (HMG) box] family and serves as a transcription factor that plays a central role in the development and differentiation of multiple cell lineages [1]. Discovery of SOX9 began with its function underlying campomelic dysplasia (CD), a rare genetic disorder characterized by bowing of the long bones [1]. In the past decade, the knowledge of SOX9 has developed rapidly. SOX9 plays a versatile role in chondrogenesis and skeletal development, in male gonad genesis, in differentiation of multiple organs, in ectoderm development, and in various solid tumors [2][3][4][5][6][7].
Increased SOX9 expression drives prostate cancer (PCa) tumor growth and angiogenesis and promotes prostate cancer invasion by reactivating the WNT/βcatenin signaling that mediates ductal morphogenesis in fetal prostate [8]. SOX9 overexpression significantly induces the proliferation and tumorigenicity of human esophageal squamous cell cancer (ESCC) cells by increasing the expression of phosphorylated Akt and its downstream targets such as phosphorylated forkhead box O (FOXO) 1 and phosphorylated FOXO3, two members of FOXO family of transcription factors [9]. Aberrant SOX9 expression contributes to the development of gastric cancer by inactivation of GKN1 as an early event [10]. Conversely, knockdown of SOX9 suppresses chondrosarcoma growth
In this study, we herein issued a comprehensive meta-analysis to appraise the prognostic significance of SOX9 overexpression in solid human tumors, and illustrate the clinical value of SOX9 as a prognostic indicator and potential therapeutic target for malignant tumor patients.

Study search information
The initial search identified 721 publications, of which, 30 studies were of acceptable relevance. However, eight of these studies were excluded because the absence of survival data, and five were excluded because of the absence of information about distinct data. Ultimately, 17 studies met the eligibility criteria and were included in the current meta-analysis ( Figure 1).

Correlations between SOX9 expression and OS
The pooled hazard ratio (HR) revealed that overexpressed SOX9 was significantly associated with poor overall survival (OS) for cancer victims in multivariate analysis (HR: 1.66, 95% CI: 1.36-2.02; Figure 2). However, a significant heterogeneity (I 2 = 62.5%, P = 0.001) was observed when using a random-effects model to analyze the pooled HR of the OSs.
To minimize heterogeneity, the subgroup analyses were performed according to the ethnics (Asian or not), case number (≥ 100 or not), NOS score (≥ 7 or not), follow-up time (≥ 120 m or not), antibody (various company), cut-off value (various scoring criteria). The pooled HRs and heterogeneities according to all these factors were presented in Table 2. Unfortunately, all these subgroup analyses demonstrated that there were no significant lower I 2 value when the P < 0.05. Therefore, subgroup analysis were failed to find the origin of high heterogeneity.

Correlations between SOX9 expression and DFS
A significant correlation between over-expressed SOX9 and disease-free survival (DFS) was also observed in the patients with solid tumors in multivariate analysis (HR: 3.54, 95% CI: 2.29-5.47; Figure 3) in the randomeffects model with a significant heterogeneity (I 2 = 68.1%, P = 0.008).

Correlations between SOX9 expression and clinicopathological parameters
The clinical and pathological parameters collected from the eligible studies were presented in Supplementary  Table 1. Meanwhile, pooled results of the correlations were identified between over-expressed SOX9 and clinicopathological features of patients with solid tumors. No significant correlations between over-expressed SOX9 with gender and tumor differentiation were observed. However, the expression of SOX9 was positively associated with tumor size (OR: 1.58, 95% CI: 1.31-1.91), lymph node metastasis (OR: 1.61, 95% CI: 1.30-1.99), distant metastasis (OR: 1.53, 95% CI: 1.25-1.87) and a higher clinical stage (OR: 1.68, 95% CI: 1.33-2.12) in the random-effects model with significant heterogeneities (see Table 3 and Supplementary Figure 1).

Assessment of heterogeneity and sensitivity
There was significant heterogeneities (I 2 > 50%) between studies in OS and DFS analyses. So the randomeffect model was therefore adopted in these studies. A meta-regression analysis with published country, case number (≥ 100 or not), antibody (used for different companies) and cut-off value (scores or not) as covariates was conducted. All covariates were fit into the meta-regression model one at a time to identify potential sources of heterogeneity. In terms of OS and DFS, none of these covariates were verified as a significant source of heterogeneity (Table 4). Also, by successively omitting each study from the aggregated survival meta-analyses, sensitivity analysis was performed to evaluate the influence of each individual study on the pooled HR of OS and DFS ( Figure 4). The results revealed that the pooled estimates of the effect of over-expressed SOX9 on the OS and DFS of patients with solid tumors did not vary substantially with the exclusion of any individual study, which implies that the results of this meta-analysis are stable.

Publication bias
We constructed funnel plots and performed Begg's test to assess publication bias. As a result, the shape of the funnel plot for the OS, DFS and clinicopathological parameters seemed symmetrical in the multivariate

DISCUSSION
The transcription factor SOX9 is a member of SOX family proteins which contain a highly conserved HMG domain that was first identified in Sry, an essential factor involved in mammalian male sex determination [43]. In general, proteins containing a domain with 50% or higher amino acid similarity to the HMG are referred to as SOX proteins. Around 20 SOX proteins have been confirmed in mice and humans, and are grouped A through H based on the structural homology outside of their HMG boxes. SOX9 belongs to SoxE proteins [44] and exerts its function in sex determination, cell differentiation during embryonic development, and cell maintenance and specification during adult life of mice and human [2].
Since the first record on the analysis of SOX9 expression in human cancer published in 1997 [45], more than hundreds studies have explored the role of SOX9 expression in tumors in larger patient groups. SOX9 is over-expressed in various human malignancies and growing evidence demonstrates its association with human solid tumor growth [9,10]. Conversely, knockdown of SOX9 provides inhibition of chondrosarcoma growth and migration, and induces apoptosis and cell cycle arrest [26]. The meta-analysis presented herein is the first comprehensive description of all reported survival data from 3307 solid tumor patients from 17 eligible studies, which met the inclusion criteria, investigating the impact of SOX9 expression in human tumors on prognosis. For all studies, SOX9 expression was detected by IHC. By meta-analysis of the 17 studies, we identified the pool HRs which indicated that SOX9 was a factor in poor prognosis in various cancers. Because there is no significant heterogeneity among our included studies, so we did not perform further subgroup analyses.
For the reasons of SOX9 overexpression correlated with poor prognosis in various solid tumors, we summarized as follows: i) Downregulated expression of E-cadherin and increased expression of βcatenin, which are key factors for epithelial-mesenchymal transition (EMT) in gastric cancers, by SOX9 overexpression. Aberrant SOX9 expression inactivates the activity of gastrokine 1 (GKN1) [46]. Inactivation of GKN1 downregulates expression of E-cadherin and increases expression of βcatenin in gastric cancers [46]. Besides that, SOX9 activates TGFβ/Smad signaling, activation of this signaling pathway upregulates snail expression, which in turn triggers EMT, resulting in down-regulation of E-cadherin and increased expression of βcatenin [47]. Overexpression of βcatenin leads to the induction of EMT in gastric cancers and partially restores the colonyforming potential in squamous cell cancer (SCC) development [48]. ii) SOX9 is important in maintaining the properties of cancer stem cell (CSC) in various tumors. The hedgehog (Hh) pathway is involved in CSC maintenance in various tumors [49]. Glioma-associated oncogene homolog 1 (Gli1) is a key mediator of the Hh pathway; involved in CSC maintenance [49]. Gli1 expression is correlated with    the expression of stemness genes, SOX9, and cell cycle regulators such as p21, cyclin D1, cyclin E1, and NF-κB, which are strongly linked to worse clinical outcome and independent poor prognostic factors in overall survival and disease-free survival in ESCC [19]. iii) Enhanced transcription of SOX9 responsive genes during tumorgenecity. SOX9 is showed to bind to 4293 genes in common between the mouse and bovine genomes [50]. Most of these genes are already known to be involved in sex determination. Moreover, transcriptomic (RNA-seq) analysis of foetal testes from SOX9 knockout mice showed that SOX9 not only regulates transcription of its target genes directly, but also influences their RNA splicing [50]. Thus, in great possibility, the overexpressed SOX9 might results in disordered gene expression in tumorgenecity. For example, SOX9 transcriptionally activated FOXK2, which belongs to the fork head DNA binding protein family, has been shown to play a critical role in tumorigenesis, high expression of FOXK2 is significantly correlated with poor survival of colorectal cancer [51]. iv) SOX9 promotes osteosarcoma (OS) cell growth by inhibiting the promoter activity of the CLDN8 gene and down-regulating CLDN8 expression, which functions as an oncogenic factor and was up-regulated in OS cells [14]; Overexpression of SOX9 in adult mouse prostate epithelia induces an early high-grade prostate intraepithelial neoplasia (PIN) lesion, indicating that SOX9 augments the loss of PTEN, which is a factor vital for tumor formation [52].
Additionally, no publication bias was observed. Our meta-analysis results involve several important implications. First, it shows that over-expressed SOX9 was positively related to poor OS and DFS in solid tumor patients. Second, pooled results of the correlations were identified between over-expressed SOX9 and clinicopathological features of patients with solid tumors, indicating that SOX9 may serve as a promising therapeutic target. Third, our results showed the expression of SOX9 was positively associated with lymph node metastasis, large tumor size, distant metastasis and a higher clinical stage. We can explain this result by SOX9's ability to enhance prostate cancer (PCa) tumor growth, promote tumor cell proliferation, invasion and metastasis [31]. Because of its involvement in these processes, SOX9 is likely to be causally involved in tumor progression and, consequently, increased levels of SOX9 would be expected to indicate a poor prognosis. Finally, it highlights the potential clinical application of SOX9 as a valuable prognostic biomarker.
This meta-analysis was properly performed, however, further analysis with several limitations would be considered in the future. Firstly, need more trials to analysis; second, some of the survival data were extracted from Kaplan-Meier curves and might be less reliable than a direct analysis of variance; third, we need to search more non-English publications. In addition, the possible existence of unpublished studies could also result in  potential publication bias. In general, concerning these limitations mentioned above, a larger cohort sample size, adjusted individual data and a unified detection method are required to achieve a more persuasive conclusion.
In conclusion, our meta-analysis demonstrated that over-expressed SOX9, as evaluated by IHC, is positively related to poor OS and DFS in human solid tumor patients. Over-expressed SOX9 could be served as a potential biomarker for unfavorable clinicopathological prognostic factors in patients with various solid tumors, suggesting that directly targeting SOX9 could be promising therapeutic approaches for solid malignancies.

Literature search strategy
This systematic review and meta-analysis is reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement [53]. We performed a thorough search of PubMed, Embase and Web of Science databases for studies measuring expression of SOX9 and survival in patients with solid tumors from 1997 to August 2017.
The search terms included the following key words in various combinations: SOX9, prognosis, prognostic, survival, and overall survival. The hits were restricted to human studies of solid tumors and those published in English. The references list of review and bibliographies were further sifted to identify additional potentially relevant studies to avoid omission due to the electronic search approach.

Study inclusion and exclusion criteria
The collected studies included in this meta-analysis had to meet the following criteria: (1) a pathological diagnosis of cancer was made; (2) SOX9 expression in patients with any type of tumor was measured via immunohistochemistry; (3) associations of SOX9 expression with OS, DFS or clinicopathological features were described; (4) HRs and 95% confidence intervals (CIs) were reported or could be calculated (based on the information in the paper); and (5) when the same author reported repeated results from the same population, the most complete report was included. The exclusion criteria for this meta-analysis were as follows: (1) unpublished papers; (2) laboratory articles, reviews and letters; (3) non-English language articles; (4) overlapping articles or ones with duplicate data; (5) articles with only animal experiments; (6) studies without information about survival curves; and (7) SOX9 expression in patients with any type of tumor was analyzed only using RT-PCR method.

Data extraction and quality assessment
All data were extracted independently by two investigators (Haihua Ruan and Xichuan Li). For each eligible study, the following characteristics were extracted: first author's name, publication year, region, type of cancer, number of patients, patients' ages, followup times, detection methods, cut-off values, survival data (including OS and DFS) and clinicopathological parameters, such as gender, tumor differentiation, tumor size, lymph node metastasis, distant metastasis and clinical stage. For studies that presented only Kaplan-Meier curves was used to extract the survival data. The cut-off values of SOX9 expression were differently indicated among the included studies. Briefly, the percentage scoring (PS) of immunoreactive tumor cells was calculated as follows: 0 (0 %), 1 (1-25 %), 2 (26-50 %), 3 (51-75 %) and 4 (76-100 %). The staining intensity was visually scored and stratified as follows: 0 (negative); 1 (weak); 2 (moderate); and 3 (strong). The immunoreactivity score (IRS) was obtained in some studies by multiplying the percentage and the intensity score.

Statistical analysis
This meta-analysis was performed using Stata 12.0 (Stata Corporation, College Station, TX, USA) software. Pooled estimates of HRs and their 95% CIs were used to estimate the association between SOX9 expression and patients' survival. The chisquared test (Cochrane' s Q test) and I-squared statistical test were used to analyze the heterogeneity between studies. When the result of a Q-test (I 2 > 50% or P < 0.05) indicated heterogeneity, the randomeffects model was used for the meta-analysis. Otherwise, a fixed-effects model was used. HR with its 95% CI over 1.0 indicated poor prognosis patients with increased SOX9 expression. Funnel plots were used to graphically represent the publication bias. Begg's (rank correlation) test was adopted to confirm the publication bias. Begg's (rank correlation) and Egger's (regression asymmetry) tests were adopted to confirm the publication bias.

Author contributions
Haihua Ruan and Xichuan Li conceived and carried out the analysis. Haihua Ruan and Xichuan Li performed literature search, study selection and data extraction. Hongyu Zhang and Shuangyan Hu conducted statistical analysis and quality assessment. Xiaoting Li and Xiaobo Li prepared tables and figures. Haihua Ruan wrote the manuscript. Gang Du revised the manuscript. All authors reviewed the manuscript.