Diagnostic Performance of Cortactin in the Diagnosis of Oral Squamous Cell Carcinoma


 Background: Cortactin gene was up-regulated in various human cancers. However, the role of cortactin in the diagnosis of oral squamous cell carcinoma (OSCC) remained unclear. The aim of this study was to investigate the diagnostic value of cortactin in OSCC patients.Methods: The relative mRNA expression levels of cortactin in OSCC tissues and adjacent normal oral mucosal tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Chi-square test was used to analyze the correlation between cortactin expression and clinical characteristics of patients. The diagnostic value of cortactin in OSCC patients was estimated via receiver operating characteristic (ROC) curve analysis.Results: Compared with the normal controls, cortactin mRNA expression was significantly increased in OSCC tissues (P<0.001). Importantly, notable correlations were found between cortactin expression and tumor size (P=0.040), TNM stage (P=0.018), lymph node metastasis (P=0.013) as well as recurrence (P=0.031). Furthermore, the result of ROC curve analysis showed that the area under the curve (AUC) was 0.867 with a sensitivity of 76.2% and a specificity of 86.9%. It revealed that the diagnostic value of cortactin was high in OSCC patients.Conclusions: Our data reveal that cortactin expression is up-regulated in OSCC and correlated with tumor progression. Cortactin may be a potential bio-marker for early diagnosis of OSCC.


Background
Oral squamous cell carcinoma (OSCC), a most common form of head and neck cancer worldwide, is originated from oral mucosal epithelial cells [1]. The incidence rate of OSCC is high and it becomes a health problem of human [2]. The potential risk factors including alcohol consumption and smoking, are involved in the initiation and development of OSCC [3]. The survival rate of OSCC patients is still low because of high metastasis, unfavorable prognosis and frequent relapse [4]. Early and effect diagnosis may reduce the death rate of OSCC patients [5]. The screening procedures and therapeutic strategies of OSCC patients mainly relied on standard histological evaluation, which focuses on the phenotypic alterations of cells and tissues [6]. Therefore, nding novel bio-markers for the diagnosis and prognosis of OSCC is an urgent need and may improve the therapeutic effect.
Cortactin gene, locating on human chromosome 11q13, encodes a cortical actin-binding protein, which belongs to activators of Arp2/3 complex and lamentous actin-binding proteins [7]. Cortactin protein also plays crucial roles in various cellular processes, such as cell invasion, migration and axon guidance [8]. It revealed that increased expression of cortactin was related to the ampli cation of 11q13, which participated in the development and prognosis of cancers [9]. A growing body of evidences had demonstrated that the aberrant expression of cortactin was con rmed in a variety types of human tumors [10,11]. In addition, knockdown of cortactin could inhibit tumor cell migration and invasion [12]. Recently, cortactin was found to be associated with the pathogenesis as well as progression, and served as a potential bio-marker for the prognosis of cancers [13]. Previous study had reported that protein expression level of cortactin was signi cantly higher in OSCC tissues than that in normal oral mucosal, and its over-expression could promote OSCC cell invasion [14]. However, the mRNA expression level and the diagnostic value of cortactin in OSCC remains poorly known.
In this study, we expected to detect the relative mRNA expression levels of cortactin in OSCC tissues and adjacent normal oral mucosal tissues, and analyze the correlation between cortactin expression and clinical characteristics of patients. We also estimated the diagnostic value of cortactin in OSCC patients.

Patients and samples
In this study, a total of 122 OSCC patients were enrolled from Chinese PLA General Hospital. Before this study, none patients had received any radio-or chemo-therapy treatment. This study was approved by the Ethical Committee of Chinese PLA General Hospital and the written informed consents were obtained from all patients.
OSCC tissues and adjacent normal oral mucosal tissues were collected from all patients by biopsy. These specimens were immediately frozen in liquid nitrogen and stored at -80℃ for use. The histopathological diagnosis of all tissue samples were con rmed by the pathologists with extensive experience. The detailed clinicopathologic characteristics of patients were listed in Table 1.
GAPDH served as internal control and the relative mRNA quanti cation of cortactin was calculated by 2 -∆∆Ct method. Each sample was examined in triplicate.

Statistical analysis
Statistical analyses were performed with SPSS 18.0 software and GraphPad Prism 5.0 software. The data were expressed as mean ± standard deviation (SD). Student's t-test was used to evaluate the difference between two groups. The relationship between cortactin expression and clinical characteristics of patients was analyzed via Chi-square test. Receiver operating characteristic (ROC) curve analysis was applied to estimate the diagnostic value of cortactin in OSCC. The difference was considered statistically signi cant when P<0.05.

Demographic information of the study subjects
In this study, we enrolled 44 female and 78 male patients with a mean age of 56.47±15.28 years (range, 46-77 years). There were 73 OSCC patients with smoking and 66 patients with alcohol consumption. 72 cases had T1-T2 tumor size and 50 cases had T3-T4 tumor size. 51 patients were checked in poor histological grade and 58 patients were diagnosed with -TNM stage. There were 46 cases of lymph node metastasis and 42 patients with recurrence. The detailed clinical information was listed in Table 1.

Cortactin expression was up-regulated in OSCC patients
The relative mRNA expression of cortactin in OSCC tissues and adjacent normal tissues was detected by qRT-PCR. The results showed that cortactin expression was signi cantly higher in OSCC tissues than that in adjacent normal tissues at mRNA level (P<0.001, Figure 1).

Relationship between cortactin expression and clinical features of OSCC patients
According to the mean value of cortactin expression, 122 OSCC patients were divided into high cortactin expression group and low cortactin expression group. We analyzed the correlation of cortactin expression with clinical characteristics of patients. As shown in Table 1, elevated expression of cortactin was closely associated with tumor size (P=0.040), TNM stage (P=0.018), lymph node metastasis (P=0.013) and recurrence (P=0.031). However, there was no obvious correlation between cortactin expression and age, gender, smoking, alcohol consumption or histological grade (P>0.05, Table 1).

The diagnostic value of cortactin in OSCC patients
The ROC curve was established to assess the diagnostic value of cortactin in OSCC patients. The results indicated that the area under the curve (AUC) was 0.867 (95%CI=0.823-0.912, P<0.001) with a sensitivity of 76.2% and a speci city of 86.9% (Figure 2). The optimal cutoff value of cortactin expression was 1.65. Cortactin expression could be an effective bio-marker for the diagnosis of OSCC patients.

Discussion
OSCC is a common oral cancer and metastasis and recurrence are two characteristics of OSCC. Despite advances in treatment and diagnostics the incidence and mortality in patients with OSCC are increasing. The tumorigenesis of OSCC is a complex multi-step process which associated with the dysregulation of oncogenes and tumor suppressor genes [15]. In addition, most OSCC patients are diagnosed at advanced stages, and the routine therapeutic methods are not effective for them [16]. Early diagnosis may improve the prognosis and treatments of OSCC. Therefore, there is an urgent requirement to identify novel biomarkers for the diagnosis, therapy and prognosis of OSCC.
Cortactin protein possess a multi-domain structure consisting of an acidic domain at the amino terminus, a proline-rich helical region and an Src homology SH3 domain located at the carboxyl terminus [17,18]. Cortactin protein was initially considered as a substrate of Src protein kinase and a cytoskeletal protein that plays a potential role in the regulation of the actin cytoskeleton, actin assembly and adhesion [19]. Besides, cortactin protein was involved in the pathogenesis and development of some human tumors. In the study of Zhao et al., increased expression of cortactin was observed in hepatoma carcinoma (HCC) cells, and it regulated the invasion and migration of HCC cells [20]. Ni et al. mentioned that cortactin was higher in colon cancer tissues than that in adjacent non-tumor tissues, and over-expression of cortactin enhanced cell colony formation and tumor growth [21].
In the present study, the qRT-PCR results showed that the relative expression of cortactin in OSCC tissues was signi cantly up-regulated compared with that in adjacent normal tissues at mRNA level, which was similar to previous studies [20,21]. It revealed that cortactin gene might play an oncogenic role and exhibit tissue-speci c in OSCC. Moreover, increased expression of cortactin was more frequently occurred in patients with larger tumor size, advanced TNM stage, positive lymph node metastasis and recurrence. According to these results, we hypothesized that cortactin expression might be involved in the development of OSCC. Our ndings were consistent with the previous studies, for example, Lu et al.
reported that the mRNA expression level of cortactin gene was over-expressed in esophageal squamous cell carcinoma and associated with tumor stage as well as lymph node metastasis of patients [22]. In head and neck squamous cell carcinoma, patients with positive cortactin expression tended to have recurrence or metastasis [23]. Nevertheless, a study carried out by Tsai et al. showed that the immunostaining scores of cortactin was obviously linked with histological grade of pancreatic and ampulla of Vater adenocarcinoma patients, which was inconsistent with our result [24]. The functions of cortactin in different cancers might be varied, and the sample size, its source as well as expression pattern might also cause the difference.
A large number of researches had demonstrated that cortactin was correlated with the prognosis of various tumors, including OSCC [25][26][27]. In the study of Li et al., they reported that the negative expression of cortactin was an independent prognostic factor and had a survival advantage in epithelial ovarian cancer [28]. Cortactin was demonstrated to be an important indicator of the malignancy and metastasis of liver cancer and might have predictive value in the prognosis of HCC [27,29]. However, little is known about the diagnostic value of cortactin in OSCC. In this study, the result of ROC curve analysis indicated that cortactin mRNA expression could be a potential bio-marker for the diagnosis of OSCC patients with high values of AUC, sensitivity and speci city.

Conclusions
In summary, cortactin is up-regulated in OSCC and correlated with the progression of this tumor. What's more, cortactin may be a potential bio-marker for the diagnosis of OSCC. However, the precise molecular mechanism of cortactin in OSCC remains poorly studied, further researches are required. The subjects had been informed the objective. Certainly, written consents were signed by every subject in this study.

Consent for publication
We obtaining permission from participants to publish their data.
Availability of data and materialsThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Competing interestsThe authors declare that they have no competing interests.   Figure 1 The relative mRNA expression of cortactin in OSCC tissues and adjacent normal oral mucosal tissues. Compared with normal tissues, cortactin mRNA expression was obviously up-regulated in OSCC tissues (***, P<0.001).