The Clinical Signification of Claudin-11 Promoter Hypermethylation for Laryngeal Squamous Cell Carcinoma.

BACKGROUND Claudin-11 (CLDN11) is frequently silenced by its promoter hypermethylation. Previous studies have shown that CLDN11 promoter hypermethylation is a potential biomarker for diagnosing various cancers. The aim of this study was to investigate CLDN11 promoter methylation and its potential relevance to clinicopathologic features and prognosis of patients with laryngeal squamous cell carcinoma (LSCC). MATERIAL AND METHODS Using the quantitative methylation-specific polymerase chain reaction (qMSP), CLDN11 promoter methylation was measured in 91 tumor tissues and their paired adjacent normal tissues, and the relationship between CLDN11 methylation and clinicopathologic features was evaluated. A receiver operating characteristic (ROC) curve was created to assess diagnostic values, and the Kaplan-Meier survival analysis was used to evaluate the association between CLDN11 methylation and prognosis of patients with LSCC. RESULTS Our results showed significantly elevated promoter methylation of CLDN11 in tumor tissues compared to their adjacent tissues (p=1.227E-16). CLDN11 promoter methylation also increased in patients with lymph node metastasis (p=0.009), advanced clinical stage (p=9.26E-06) and higher T classification (p=0.003). The area under the ROC curve (AUC) of CLDN11 was 0.884 (95% CI=0.835-0.932, p<0.01). The Kaplan-Meier analysis indicated that high CLDN11 promoter methylation levels were associated with poor overall survival of LSCC patients (log-rank test, p=0.007). CONCLUSIONS We demonstrated that CLDN11 promoter hypermethylation is a frequent event in LSCC, and contributes to metastasis and progression of LSCC. Thus, CLDN11 could be a potential biomarker for diagnosis and prognosis of LSCC patients.

Background

Laryngeal cancer is the second most common cancer among respiratory system tumors. According to a report by the American Cancer Society, 13,360 new laryngeal cancers and 3,660 new deaths were projected to occur in the USA in 2017 [1]. Laryngeal squamous cell carcinoma (LSCC) is the most common pathological type of laryngeal cancer [2]. In China, there has been an increasing incidence of laryngeal cancer in the younger population, especially in the northern region of China [3]. Despite remarkable advances in diagnostic methods and therapeutic strategies for LSCC, the 5-year survival rate has decreased over the past several decades in the USA, and the quality of life after surgical treatment is greatly impacted by pain, dry mouth, and speech disorders [1,4].

The pathogenesis of LSCC is a multifactorial process involving a variety of complicated factors, including epigenetic and genetic alterations, as well as environmental exposure to tobacco, alcohol, asbestos, and viral infection [5,6]. Studying epigenetics, especially aberrant promoter methylation of tumor suppressor genes (TSGs), which is the most common and best-characterized alteration, offers great potential for identifying biomarkers that can be used in the diagnosis and prognosis of LSCC patients [7]. There is a growing list of TSGs that are silenced or inactivated by promoter hypermethylation, which is considered to be a significant event in the initiation and progression of various cancers [8-11]. Claudin-11 (CLDN11) is a potential tumor suppressor gene located at chromosome 3 (3q26.2), and encodes a member of the claudin family, an integral membrane protein of the paracellular tight junction (TJ) [12,13]. TJs are often altered in human carcinomas, and the loss of TJs has been interpreted as an important step in cancer progression [14]. Moreover, TJs play a number of diverse roles in tumorigenesis [15±–18]. Previous studies have demonstrated that silencing of CLDN11 expression is associated with increased invasiveness in various cancers [19-21]. In addition, Soini et al. [22] reported that the poor outcome of patients with meningiomas was associated with reduced CLDN11 expression. CLDN11 acts as a candidate tumor suppressor gene whose expression is frequently diminished or silenced by its promoter hypermethylation in gastric cancer and malignant melanoma [19,23]. Moreover, a previous study reported that aberrant promoter methylation of CLDN11 could be associated with the malignant transformation of oral leukoplakia [24]. In addition, hypermethylation of CLDN11 frequently occurs in malignant melanoma, and can be used in a clinical setting to distinguish malignant melanocytic lesions from benign ones [23].

However, there are few studies that focus on the role of CLDN11 promoter methylation in LSCC. To the best of our knowledge, this is the first study to investigate the methylation status of CLDN11 in LSCC, and to correlate this methylation with clinical parameters, as well as overall survival of LSCC patients. We hypothesized that CLDN11 promoter hypermethylation is a potential biomarker for diagnosis and prognosis of this disease.

Material and Methods

Patients and collection of tissue samples

Tumor tissues and their paired adjacent normal tissues (dissected at >0.5 cm from the margin of the neoplastic lesion) were collected from 91 LSCC patients comprising 87 males and four females, all who had undergone surgical operation at the Department of Otolaryngology of Ningbo Medical Center Lihuili Hospital, Zhejiang, China. The age of the patients ranged from 40 to 86 years, and the median age was 60 years. The majority of patients (80%) were cigarette smokers. No chemotherapy or radiotherapy had been administered to these patients prior to surgical intervention, and none of the patients had a prior history of cancer. Histological confirmation of tumors was performed by two pathologists according to the Union for International Cancer Control (UICC) classification (TNM 2002). There were 78 well or moderately differentiated cases and 13 poorly differentiated cases. Furthermore, there were 44 stage I/II and 47 stage III/IV cases. According to the postoperative pathology, there were 32 cases with lymph node metastasis and 59 cases without lymph node metastasis. All samples were frozen immediately in liquid nitrogen and stored at −80°C until required. All participants signed an informed consent form. The study received approval from the local Ethical Committee at the Department of Otolaryngology at Ningbo Medical Center Lihuili Hospital, Zhejiang, China.

DNA extraction and bisulfite modification

Genomic DNA was extracted from tissue samples using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA quality and quantity were assessed using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific Co. Ltd., Wilmington, USA). Eluted DNA was subjected to bisulfite modification using a Methylamp DNA modification kit with ZYMO EZ DNA Methylation-Gold Kit according to the manufacturer’s instructions (Zymo Research, Orange, CA, USA).

Quantitative methylation-specific polymerase chain reaction (qMSP)

qMSP was used to determine the DNA methylation status of the CLDN11 promoter. The bisulfite-treated DNA was amplified using a LightCycler 480 (Roche Diagnostics, Mannheim, Germany) according to the instrument guidelines. The primer sequences used in this experiment were CLDN11-Forward 5′-ATAAGTTGATAGGAGAATCGAATCG-3′, CLDN11-Reverse 5′-AACGAAAATAACCCTAAACACGTA-3′. ACTB-Forward 5′-TGGTGATGGAGGAGGTTTAGTAAGT-3′, ACTB-Reverse 5′-AACCAATAAAACCTACTCCTCCCTTAA-3′. The polymerase chain reaction (PyroMark PCR Kit; Qiagen) amplification consisted of 50 cycles (of 95°C for 20 seconds, 60°C for 30 seconds and 72°C for 30 seconds) after an initial denaturation step (at 95°C for 10 minutes). The Bisulfite-Converted Universal Methylated Human DNA Standard (ZYMO Research Company) was used as a positive control. To correct for differences in both quality and quantity between samples, ACTB was used as an internal control. Levels of methylation were calculated by percentage of methylated reference (PMR), PMR=2−ΔΔCt×100%, in which ΔΔCt= (Ctsample–Ctinternal control)–(Ctpositive control–Ctinternal control).

Statistical analyses

All statistical analyses were performed using the SPSS software v18.0 (SPSS Inc., Chicago, IL, USA). Paired sample t-tests were used to compare CLDN11 promoter methylation levels between tumor tissues and their paired adjacent normal tissues. Analysis of variance (ANOVA) was used to evaluate the association between CLDN11 methylation and clinicopathologic features, including gender, age, smoking behavior, histological classification, T classification, lymph node metastasis, and clinical stage. Overall survival curves were calculated by Kaplan-Meier analysis and the differences between curves were evaluated using the log-rank test. A p value <0.05 was considered to be statistically significant. All figures were created using the GraphPad Prism 6 software (GraphPad, San Diego, CA, USA).

Results

In the present study, we measured the CLDN11 promoter methylation levels in 91 LSCC tissues and their adjacent non-tumor tissues using qMSP. Results indicated that methylation levels of CLDN11 in tumor tissues were significantly higher than in the paired tissues (p=1.227E-16, Figure 1).

Figure 1

Comparison of methylation levels of CLDN11 between tumor tissues and paired adjacent normal tissues. There was a significant difference in methylation of the CLDN11 promoter in LSCC tissues compared to normal tissues (N=91).

Previous studies have shown that abnormal promoter methylation correlates with clinicopathological characteristics in numerous cancers, such as colorectal cancer [25], oral cancer [26], bladder cancer [27], and gastric cancer [28]. We therefore investigated the correlation of CLDN11 methylation with the clinicopathological features of LSCC patients, such as gender, age, smoking behavior, histological classification, T classification, lymph node metastasis, and clinical stage. As shown in Table 1, methylation levels of CLDN11 in patients with lymph node metastasis were significantly higher than in patients without lymph node metastasis (p=0.009). When stratified for TNM stages, CLDN11 methylation in patients with advanced stages of LSCC was remarkably elevated compared to early stages (p=9.26E-06). In addition, we found a marked increase in CLDN11 methylation in patients with T3+4 compared to T1+2 (p=0.003). However, there was no statistically significant association between CLDN11 methylation and other clinicopathological features, such as age, smoking behavior, or histological classification.

Table 1

Clinicopathologic features of patients with LSCC, and correlation between the methylation status of CLDN11 and these features.

Features	Variables	No.	Mean ±SD	P value
Gender	Female	4	0.164±0.101	0.393
	Male	87	0.260±0.223
Age	<60 y	46	0.254±0.203	0.917
	≥60 y	45	0.259±0.237
Smoking behavior	No	18	0.259±0.203	0.955
	Yes	73	0.256±0.224
Histological classification	Well/moderately	78	0.258±0.219	0.824
	Poorly	13	0.244±0.229
Clinical stage	Stage I+II	44	0.155±0.099	9.26E-06
	Stage III+IV	47	0.351±0.257
T classification	T1+2	55	0.196±0.168	0.003
	T3+4	36	0.348±0.256
Lymph metastasis	No	59	0.213±0.193	0.009
	Yes	32	0.337±0.245

To further evaluate the diagnostic significance of CLDN11, we then constructed a ROC curve by plotting sensitivity versus specificity (Figure 2). Results showed that the AUC of CLDN11 was up to 0.884 (95% CI=0.835–0.932, p<0.01). Based on the maximum Youden index, distinguishing between patients with LSCC from controls, the cutoff point of methylation frequency was 0.571, at which the sensitivity and specificity for predicting LSCC were 92.30% and 73.60%, respectively. Our results demonstrated that the methylation level of the majority of tumor tissues was less than the cutoff value. In addition, we performed Kaplan-Meier analysis to evaluate the association of methylated CLDN11 with overall survival. We used the mean methylation level of tumor tissues to classify patients into two groups: 30 patients were placed into the hypermethylation group, and the remaining patients were categorized as the hypomethylation group [29]. Kaplan-Meier analysis indicated that patients with higher methylation frequency tended to have a poorer overall survival compared to patients with lower methylation (log-rank test, p=0.007, Figure 3).

Figure 2

Receiver operating characteristic (ROC) analyses of the curve (N=91). The cutoff point was defined as the maximum Youden index, which is highlighted by the arrow.

Figure 3

Kaplan-Meier curves of overall survival of LSCC patients in the CLDN11 hypermethylation group and the CLDN11 hypomethylation group. The log-rank test result showed that patients with CLDN11 hypermethylation had a poor overall survival (p=0.007).

Discussion

Loss of claudins may reduce cell-cell adhesion, which offers a selective advantage to developing tumor cells [30]. Previous studies have shown that claudin-11 is downregulated in a variety of cancers [19-21]. In mouse skin tumorigenesis, expression of CLDN11 also decreased during tumor progression [31]. In the current study, we investigated the association of CLDN11 with LSCC.

There are few typical clinical symptoms of the early stage of laryngeal cancer, especially in supraglottic cancer [32]. Patients with laryngeal cancer are usually diagnosed by computerized tomography (CT), magnetic resonance imaging (MRI) and endoscopic biopsy [33]. However, the sensitivity and specificity of imaging techniques are unsatisfactory [34,35], and very few patients with laryngeal cancer are diagnosed through conventional imaging techniques [3]. Biopsy remains the gold standard for diagnosing laryngeal cancer [36]. This approach, however, is limited due to its invasiveness, and is not suitable for large-scale screening. Therefore, there is an urgent need to further investigate molecular mechanisms and to develop potential biomarkers to improve early diagnosis of this disease. Accumulating studies have reported other biomarkers in LSCC, including CD133 [37], P53, Bcl-2, and caspase-3 [38]. Here, we provided another biomarker (CLDN11) in LSCC.

Abnormal promoter methylation of TSGs may be an early event in tumorigenesis, and thus a warning signal for the onset of LSCC [39]. In recent years, CLDN11 methylation has been reported to be important in tumor occurrence and development. Agarwal et al. [19] found that CLDN11 methylation was elevated in gastric cancer tissues compared to their matching gastric mucosal tissues, and promoter hypermethylation of CLDN11 has been shown to decrease gene expression, resulting in increased cell motility and invasion. In addition, Walesch et al. [23] reported a significant increase in the methylation level of CLDN11 in malignant melanoma metastases compared to primary malignant melanoma and nevus cell nevi. Furthermore, Abe et al. [24] hold the view that aberrant promoter methylation of CLDN11 is associated with high-risk oral leukoplakia. In our study, a significant increase in CLDN11 promoter methylation was found in LSCC tissues compared to non-cancerous tissues, suggesting that CLDN11 methylation is a frequent tumor-specific event in LSCC, and could serve as a potential biomarker for the detection of LSCC.

Lymph metastasis, clinical stage, and T classification are important prognostic indicators in patients with squamous cell carcinoma of the head and neck, which influence survival rates and determine the treatment modality [39-42]. Moreover, patients with advanced stage laryngeal cancer who receive total laryngectomy followed by chemoradiation therapy experience many kinds of discomfort [43,44]. Our study also found that CLDN11 methylation was remarkably higher in patients with lymph node metastasis, advanced clinical stage, and higher T classification, in agreement with previous studies, suggesting that CLDN11 could play a crucial role in disease progression and metastasis.

Regarding head and neck cancers, several tumor markers have been evaluated in an attempt to improve diagnosis and follow-up, including carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA-M), squamous cell carcinoma antigen (SCCA) and cytokeratin 19 fragments (CYFRA 21-1). Eleftheriadou et al. [45] reported that the sensitivity of CEA, TPA-M, SCCA, and CYFRA 21-1 in the diagnosis of laryngeal cancer was from 16.5% to 46.8%. In our study, CLDN11 had a higher sensitivity and specificity in the screening of laryngeal cancer, with an AUC of 0.884, sensitivity of 92.30%, and specificity of 73.60%, suggesting that CLDN11 methylation could have a higher diagnostic value in distinguishing patients with LSCC from controls.

Previous quantitative reviews have shown that the expression of CLDN11 significantly correlated with survival of breast cancer patients [46]. Multiple factors regulate gene transcription, and aberrant DNA methylation is one of the most important mechanisms [47]. In our study, methylation frequencies were found to be negatively related to the prognosis of LSCC, suggesting that aberrant methylated CLDN11 could be a novel biomarker for predicting the prognosis of patients with LSCC, resulting in a massive impact on clinical decision making.

However, there are some limitations of this research. First, our cohort consisted of only 91 patients with LSCC; a greater number of samples are needed to identify patients with a higher predictive capability. Second, due to the limited amount of tissues, we were unable to have matched gene expression. The expression of CLDN11 should be further investigated, along with the association between CLDN11 promoter methylation and expression.

Conclusions

Our study provided evidence that CLDN11 promoter methylation occurred frequently in LSCC, and was significantly correlated with lymph node metastasis, advanced clinical stage, and higher T classification. In addition, patients with CLDN11 hypermethylation have poor overall survival. These results suggest that CLDN11 hypermethylation could be a potential biomarker for diagnosis and prognosis of LSCC.