Elevated ZNF703 Protein Expression Is an Independent Unfavorable Prognostic Factor for Survival of the Patients with Head and Neck Squamous Cell Carcinoma

Aim. Data from The Cancer Genome Atlas (TCGA) show that the ZNF703 gene amplifies and overexpresses in head and neck squamous cell carcinomas (HNSCC). However, the clinical relevance of this observation in HNSCC is unclear. The purpose of this study was to clarify the expression of ZNF703 protein and its prognostic effect on HNSCC. Methods. Two hundred ten HNSCC patients from Sun Yat-Sen University Cancer Center with complete survival follow-up were included in this study. Tumor samples from primary sites were collected. The expression of the ZNF703 protein was tested by immunohistochemistry (IHC). Results. The high expression of ZNF703 in HNSCC tumor tissues was significantly higher than that of the matched noncancerous tissues (48.6% versus 11.6%, P < 0.001). ZNF703 overexpression was correlated with tumor position (laryngeal carcinoma) and recurrence (all P < 0.05). Multivariate analysis revealed that ZNF703 protein overexpression was an independent prognostic factor (P = 0.022, hazard ratio = 1.635, 95% CI 1.073–2.493) in HNSCC patients. Conclusion. ZNF703 overexpression is associated with adverse prognosis in HNSCC, which might be a novel biomarker of HNSCC.


Introduction
Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common malignancy in the world, with approximately 600,000 newly diagnosed cases per year [1,2]. HNSCC is a group of fairly heterogeneous tumors that can occur from the base of the cranium to the clavicles. On the basis of anatomic sites, HNSCC can be divided into various types; the paranasal sinuses, nasal cavities, nasopharynx, orbits, oral cavity, oropharynx, hypopharynx, and larynx make up 90% of HNSCC and the 5-year survival rate of all HNSCC is approximately 50% [3]. Patients diagnosed at an early stage can have a superior quality of life and life expectancy with surgery alone [3]. The majority of patients are diagnosed at an advanced stage, whose prognosis is extremely poor [4]. For patients in stage IV, the long-term survival of HNSCC patients is only approximately 15 months [5]; the high recurrence rate highlights the need for a cure. Despite advances in diagnosis and treatment, the survival rates for many types of HNSCC have improved little over the past forty years [2,3]. Therefore, a deeper understanding of HNSCC pathogenesis is needed to promote the development of improved therapeutic approaches.
In recent years, the development of high-throughput sequencing technology provides the best opportunity for exploring fundamental tumorigenic mechanisms. According to The Cancer Genome Atlas (TCGA) database, which included 4000 cases and more than 20 tumor types, ZNF703 is one of the most frequently altered genes in the pan-cancer group. In HNSCC, the incidence of an increased ZNF703 copy number was 28%, and the high-level of gene amplification accounted for 7.0% [http://www.cbioportal.org/publicportal/].
The zinc finger protein 703 (also known as ZPO1, ZEPPO1) gene is located on chromosome arm 8p12. The ZNF703 protein belongs to the NET (Noc/Nlz, Elbow, and Tlp-1) family, which plays an important role in the embryonic development of zebrafish [6] and Drosophila [7]. It has been shown that ZNF703 gene amplification stimulates migration and proliferation while reducing cell to cell adhesion [8][9][10] and is speculated to be associated with poorer outcomes [9][10][11][12] in breast cancer. Two studies from China also discovered that ZNF703 acts as an oncogene that accelerates malignant progression in gastric cancer [13] and is associated with worse prognosis in colorectal cancer patients [14]. As a consequence, we propose that ZNF703 may also play an important role in the pathogenesis of HNSCC. However, we are not aware of any reports about ZNF703 in HNSCC until now. Thus, we conducted this retrospective study to explore the expression of ZNF703 protein and its clinical relevance in HNSCC.

Patients and Tissue Specimens.
A total of 210 HNSCC patients (160 male and 50 female) between January 2001 and December 2008 with complete survival follow-up documents and available tumor samples were enrolled in our study. All patients had undergone surgery at Sun Yat-Sen University Cancer Center, Guangzhou. The exclusion criteria were as follows: (i) accepted neoadjuvant radiotherapy and/or chemotherapy before surgery, (ii) having distant metastasis at the time of diagnosis (stage IVc), (iii) incomplete records, (iv) having other malignant tumors at any time during the treatment and follow-up, (v) with severe complications before treatment. The TNM stage was reclassified on the basis of the American Joint Committee TNM Staging system (7th ed., 2010). All patients were followed up until April 2014. Two hundred ten paraffin-embedded specimen blocks and 43 matched adjacent noncancerous tissue blocks (as control group) were obtained from the pathology department of Sun Yat-Sen University Cancer Center. All the slides were reviewed to reconfirm the diagnosis by two independent pathologists. All works were conducted in accordance with the Declaration of Helsinki (1964). Written and informed consent was obtained from every patient, and the study was approved by the ethics review board of Sun Yat-Sen University Cancer Center. Local or distant metastasis was confirmed by biopsy or radiological examination.

Immunohistochemistry (IHC) and
Assessment. The paraffin sections (4 mm thick) were dewaxed and rehydrated, and endogenous peroxidase was first blocked with 0.3% H 2 O 2 methanol. For antigen retrieval, the tissue slides were blocked in the 0.01 M citrate buffer (pH 6.0) in a microwave oven for 30 min. Nonspecific binding was inhibited with normal goat serum for 30 min. The primary ZNF703 antibody (ab155210, 1 : 400, Abcam, Cambridge, MA, USA) was used, and slides were put in a moist chamber overnight at 4 ∘ C. Subsequently, the slides were incubated with biotinylated secondary antibody for 30 min at 37 ∘ C. Horseradish peroxidase was subsequently applied. Finally, Meyer's hematoxylin was used for nucleus counterstaining. The negative control was obtained by omitting the primary antibody.
All immunostaining slides were judged by two independent pathologists without knowledge of the clinic-pathologic information. The specific intensities of staining were identified as follows: 0 = none; 1 = weak; 2 = moderate; 3 = strong. The proportion scores were as follows: 0 = none; 1 = 1-10%; 2 = 11-50%; 3 = 51-80%; 4 = >81%. The ZNF703 immunoreactive score for each tissue was calculated by multiplying the score of the staining intensity by that of the percentage of positive cells. If the discrepancy between the two pathologists' immunoreactive score was larger than 6, the tissue section was reevaluated by the two pathologists face to face.

Statistical
Analysis. The SPSS 19.0 software was used for statistical analyses. A receiver operating characteristic (ROC) curve and the median was used to determine the cut-off value to distinguish high or low ZNF703 expression. The relationship between clinic-pathological characteristics and ZNF703 expression was assessed by a 2 -test. Overall survival time was defined as the duration between the time of disease diagnosis and the time of death. The Kaplan-Meier method was used for survival analysis. The Log-Rank test was conducted for differential survival analysis. Additionally, the Cox test was performed for multivariate analysis. The statistical tests were two-sided probability tests. Statistical significance was defined as less than 0.05.

The Relationship between ZNF703 Expression and Clinicopathological Factors.
The ZNF703 protein was located in the cytoplasm and nucleus, but mainly in the nucleus (Figure 2). The overexpression rate of ZNF703 protein was 48.6% in HNSCC tumor cells, it was significantly higher than that of the adjacent noncancerous squamous epithelium cells (Figure 3, 48.6% versus 11.6%, < 0.001). The overexpression of ZNF703 was related to recurrence and primary tumor site but not related to T stage, N stage, age, gender, BMI, differentiation, and complications (Table 1).  Figure 4). It was also observed that male patients, patients who consumed tobacco and alcohol, and patients with advanced stage disease, complications, or poorer differentiation had an apparently shorter OS. Meanwhile, poorer differentiation, advanced stage disease, without neck dissection or adjuvant radiotherapy and laryngeal cancer were all associated with shorter DFS (

Discussion
A variety of genetic changes have been described previously in HNSCC, including frequent chromosome 8p amplification [16][17][18][19][20][21]. It has also been identified that amplifications at 8p12 occurred frequently in multiple cancer types. This was suggestive of the presence of an important oncogene in this region, and numerous studies have been carried out to identify the driver gene on 8p12.
In the TCGA database of HNSCC, the amplification rate of the ZNF703 gene is 7% [http://www.cbioportal.org/publicportal]. Our results also showed that the expression level of ZNF703 protein was significantly higher in HNSCC than in the adjacent noncancerous squamous epithelial cells, the overexpression rates were 48.6% and 11.6%, respectively ( < 0.001). Furthermore, ZNF703 overexpression was observed more frequently in larynx cancer and was correlated with higher risk of recurrence. The 5-year OS rate was 58.9% and 68.2% in the high and low expression groups, respectively. All these results indicated that ZNF703 may play a role in the tumorigenesis and metastasis of HNSCC and act as an oncogene.
Overexpression of ZNF703 was also found in other cancers, such as breast cancer, gastric cancer, and colorectal cancer. Zhang et al. reported that the positive expression rate of ZNF703 in early stage breast cancer patient was 91.3% [12].  Yang et al. found that the protein was also upregulated in invasive gastric carcinoma tissues [13] and colorectal cancer samples [14]. Interestingly, further studies reported that patients with higher N stage and high risk of tumor relapse and death had a higher level of ZNF703 expression ( < 0.05).
This finding prompted researchers to consider whether the ZNF703 protein is a candidate factor for promoting HNSCC growth, progress and metastasis.
Although there is still no direct report about the pathogenic mechanism of ZNF703 in HNSCC, reports in other cancer types may help to understand its mechanism.
Earlier studies also showed that ZNF703 stimulated mammary epithelial cells toward immortalization rather than differentiation and enhanced the invasive potential of breast cancer cells [9,10]. The possible mechanism is thought to be involved in the EMT. The ZNF703 could downregulate E-cadherin and promote metastasis-associated p120-catenin isoform 1 expression. Subsequent data demonstrated that ZNF703 overexpression increased lung metastases in a mouse breast cancer model [8,30]. It may also play a role in the activation of stem cell-related gene expression, leading to an increase in cancer stem cells [10]. On the other hand, it was reported to be involved in the activation of the Akt/mTOR signaling pathway, downregulated ERalpha, and the reduction of the antitumor effect of tamoxifen in breast cancer in vitro [12]. Based on these results we suggest that ZNF703 may also play an important role in HNSCC formation and progression.
In our study, we only detected ZNF703 protein expression with IHC, so there is no final conclusion about the status of ZNF703 gene amplification or mRNA levels in HNSCC. However, according to the TCGA database of patient tumor samples and the CCLE database of cell lines and other literature [9], the copy number and gene expression are highly positively correlated, = 0.72 [http:// www.cbioportal.org/public-portal].
On the basis of these results, we speculate that ZNF703 could be a valuable independent factor in HNSCC for predicting prognosis as well as a promising biomarker to help design optimal individual treatments.

Conclusion
On the one hand, our findings suggested that the overexpression of ZNF703 is common in patients with HNSCC and is related to the aggressive clinical course and poor prognosis. Therefore, more aggressive therapy and closer follow-up should be adopted in patients with ZNF703 overexpression. On the other hand, our findings also provide new clues for further exploration of new treatments in HNSCC. To the best of our knowledge, this is the first report about the expression of ZNF703 in HNSCC. However, due to the retrospective research and small sample size, further prospective clinical study and genomic, cellular function and mechanistic studies are needed to prove our conclusions.

Conflict of Interests
The authors declare no conflict of interests regarding the publication of this paper.

Authors' Contribution
The first authors Hang Yang and Wen-Qi Jiang, contributed equally to this paper.