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Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 892-896

CLIC1 overexpression is associated with poor prognosis in pancreatic ductal adenocarcinomas

1 Department of Liver Disease, Shanxi Medical University Second Hospital, Taiyuan, People's Republic of China
2 Department of General Surgery, Shanxi Medical University Second Hospital, Taiyuan, People's Republic of China

Date of Web Publication25-Jul-2016

Correspondence Address:
Huanhu Zhang
Department of Liver Disease, Shanxi Medical University Second Hospital, No. 382 Wuyi Road, Taiyuan - 030001
People's Republic of China
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0973-1482.154057

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 > Abstract 

Background: Clinical significance of chloride intracellular channel 1 (CLIC1) in pancreatic ductal adenocarcinomas (PDAC) remains largely unknown. This study was performed to assess the expression of CLIC1 in benign and malignant pancreatic lesions, and to assess its clinicopathological significance.
Materials and Methods: Tissue samples from resected PDAC (n = 70) and their matched normal pairs were evaluated for CLIC1 expression by immunohistochemical staining. Their expression was correlated with different clinicopathological parameters.
Results: CLIC1 expression was significantly higher (67.1%) in PDAC than in adjacent control tissues (25.7%, P < 0.001). High CLIC1 levels were associated with the histological grade (P < 0.001) and tumor size (P < 0.001); but not with sex, age, tumor-node-metastasis (TNM) stage, tumor location, or lymph node metastasis (P < 0.05). Univariate Kaplan–Meier analysis showed that a positive CLIC1 expression was associated with a decreased overall survival (P < 0.01). Multivariate cox regression analysis showed that CLIC1 expression and lymph node metastasis were independent risk factors for disease-free survival.
Conclusion: The expression of CLIC1 might be closely related to the carcinogenesis, clinical biological behaviors, and prognosis of pancreatic ductal adenocarcinomas.

Keywords: Chloride intracellular channel 1, immunohistochemistry (IHC), Pancreatic ductal adenocarcinomas

How to cite this article:
Jia N, Dong S, Zhao G, Gao H, Li X, Zhang H. CLIC1 overexpression is associated with poor prognosis in pancreatic ductal adenocarcinomas. J Can Res Ther 2016;12:892-6

How to cite this URL:
Jia N, Dong S, Zhao G, Gao H, Li X, Zhang H. CLIC1 overexpression is associated with poor prognosis in pancreatic ductal adenocarcinomas. J Can Res Ther [serial online] 2016 [cited 2020 Oct 25];12:892-6. Available from: https://www.cancerjournal.net/text.asp?2016/12/2/892/154057

 > Introduction Top

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal solid tumor and is the fourth leading cause of cancer-related death, with an overall 5-year survival rate of <5%.[1],[2] PDAC occurs with a frequency of around nine patients per 100,000 individuals, making it the 11th most common cancer in the United States.[3] Owing to local invasion, early metastasis, and resistance to standard chemotherapy; the median survival time of PDAC patients is only 5–8 months, in spite of recent advances in surgical techniques and medical management.[4],[5]

Therefore, identification of clinical markers that can accurately predict tumor metastasis and prognosis, could allow clinicians to better choose appropriate treatment regimens.[6],[7] Studies have shown that chloride intracellular channel 1 (CLIC1) is highly expressed in malignant tumors, such as gastric, liver, lung, and colon cancers; suggesting that this protein may have important functions in the malignant transformation of these cell types.[8],[9],[10],[11] However, the significance of CLIC1 expression in the prognosis of PDAC has not been fully evaluated.

Using immunohistochemistry (IHC), here we investigated CLIC1 expression levels in benign and malignant lesions of the pancreas and studied the clinicopathological significance of its expression in the prognosis of PDAC.

 > Materials and Methods Top

Patients and clinicopathological data

The study was approved by the ethics committee of our hospital, and all patients provided informed consent. Samples from 70 patients with pathologically confirmed PDAC after surgery were collected from 2006 to 2013 at the Department of General Surgery in our hospital. None of the patients received either chemotherapy or radiotherapy before surgery. The patients comprised 40 male and 30 female patients with an average age of 69.8 years. According to the American Joint Committee on Cancer (AJCC 7th) staging system, nine (12.9%), 20 (28.6%), 21 (30.0%), and 20 (28.6%) patients were diagnosed with stage I, II, III, and IV disease, respectively. Additionally, 26 cases were diagnosed with lymph node metastases. All diagnoses of PDAC and lymph node metastasis were confirmed by histopathological examination, and all tissue samples were fixed in 4% formalin immediately after removal and embedded in paraffin for immunohistochemical staining.

Immunohistochemical analysis and evaluation of CLIC1 expression

Immunohistochemical staining was performed using the standard immunoperoxidase staining procedure, and CLIC1 expression in the specimens was evaluated according to the methods described by Li et al.[12] The sections were semiquantitatively scored according to the extent of immunore activity as follows: 0, 0% immunoreactive cells; 1, < 5% immunoreactive cells; 2, 5–50% immunoreactive cells; and 3, > 50% immunoreactive cells. Additionally, the staining intensity was scored semiquantitatively as follows: 0, negative; 1, weak; 2, intermediate; and 3, strong. The final immunoreaction score was defined as the sum of both parameters (extension and intensity) and the samples were classified as negative (0), weakly stained (1–2), moderately stained (3), and strongly stained (4–6). For statistical purposes, only moderate and strong final immunoreaction scores were considered positive; the other final scores were considered negative.

Statistical analysis

The data were analyzed using Statistical Package for the Social Sciences version 18.0 (SPSS 18.0). The interrelationship of CLIC1 expression with histology or clinical factors was analyzed using the χ2 independence or Fisher's exact test. The Kaplan–Meier test was used for univariate survival analysis. The Cox proportional hazard model was used for multivariate analysis and for determining the 95% confidence interval. P < 0.05 was considered statistically significant.

 > Results Top

CLIC1 expression in benign and malignant lesions of the pancreas

In many types of tumors, CLIC1 has a critical role in cancer development and progression.[13] To determine the potential role of CLIC1 in PDAC progression, we evaluated CLIC1 expression in PDAC by IHC [Figure 1]a,[Figure 1]b,[Figure 1]c,[Figure 1]d,[Figure 1]e. CLIC1 was mainly located in the cytoplasm and cell membrane of cancer cells. Approximately 67.1% (47/70) of the PDAC cases had positive CLIC1 staining in the tumor cells. In contrast, only 25.7% (18/70) of the cases had positive staining in the adjacent control tissues (P < 0.001) [Figure 1]d.
Figure 1: The enhanced expression of CLIC1 in PDAC and negative association with prognosis. CLIC1 was predominantly localized in the cell cytoplasm and membrane (black arrow, positive staining; scale bar, 100 μm). (a) Negative staining in normal pancreatic tissues. (b) Weak staining in the well-differentiated adenocarcinoma (TNM stage I). (c) Strong staining in the poorly-differentiated adenocarcinoma (TNM stage III). (d) Moderate staining in perineural invasion (invaded nervous, red arrow). (e) The average staining scores of CLIC1 expression in PDAC tissues and their matched normal pairs. (f) Kaplan–Meier plots of overall survival in PDAC patients with positive and negative CLIC1 expression scores. CLIC1 = Chloride intracellular channel 1, PDAC = pancreatic ductal adenocarcinomas, TNM = tumor, necrosis, and metastasis

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The association of CLIC1 expression with clinicopathological characteristics of pancreatic adenocarcinoma

As shown in [Table 1], theCLIC1 overexpression was found to be significantly correlated with the histological grade (P < 0.001) and the tumor size (P < 0.001), indicating a potential role of CLIC1 expression in promoting aggressive phenotypes in PDAC. However, CLIC1 expression exhibited no significant association with the presence of lymph node metastasis or other clinicopathological characteristics, such as gender, age, tumor location, or the TNM (tumor, necrosis, and metastasis) stage (P > 0.05).
Table 1: Association of CLIC1 expression with the clinicopathological characteristics of PDAC

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Correlation between CLIC1 expression and survival in patients with pancreatic cancer

The survival information of 70 patients was obtained through phone calls. Twenty-nine patients survived over 1 year and 41 patients survived less than 1 year, with an average survival time of 9.4 months. The patients that survived less than 1 year were found to have significantly higher CLIC1 expression than patients that survived over 1 year [Table 2]. The Kaplan–Meier survival analysis revealed that the histological grade (P < 0.001), TNM stage (P < 0.05), presence of lymph node metastasis (P < 0.05), and tumor size (P < 0.01) were significantly associated with the average survival time. The average survival time for CLIC1-negative patients was significantly higher than that of patients with positive CLIC1 expression (P < 0.001) [Figure 1]f and [Table 3]. To obtain a more precise estimate about prognosis, the Cox proportional hazard regression model was applied. Results confirmed that the CLIC1 expression, as well as the presence of lymph node metastasis, was negatively correlated with postoperative survival, suggesting that high CLIC1 expression is a risk factor [Table 4].
Table 2: Correlation between patient survival time and the expression of CLIC1

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Table 3: Univariate analyses showing the overall survival rate for patients with PDAC

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Table 4: Multivariate analysis the overall survival rate for patients with PDAC

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 > Discussion Top

PDAC involves a highly invasive, rapidly proliferating tumor.[14] Unfortunately, the most aggressive conventional treatments for this tumor are still associated with poor survival. Therefore, it is necessary to identify prognostic biomarkers that are independently correlated with tumor aggressiveness. This study documents CLIC1 immunostaining in PDAC for the first time, and shows that high CLIC1 expression is found in approximately 67.1% of cases.

CLIC1 is a newly discovered member of the p64 chloride channel protein family and locates on human chromosome 6p21.3.[15] CLIC1 plays important roles in many physiological processes, such as the regulation of cell volume and membrane potential, acidification of organelles, cell cycle regulation, and cell proliferation and differentiation.[16],[17],[18] Consistent with these, high CLIC1 expression has been reported in many types of cancers.[13] In this study, we also found that significantly increased CLIC1 expression was detected in the vast majority of PDAC tissues when compared with adjacent control tissues.

CLIC1 has been characterized as an oncogene as it plays important roles in promoting cancer cell survival, proliferation, invasion, and migration in several malignancies.[9],[19] Chen et al.,[20] have performed differential proteomics studies on gastric cancer tissues and adjacent normal tissues and have shown that CLIC1 expression was 1.95-fold higher (range, 0.01–6.19-fold) in tumors than in noncancerous mucosa. Ding et al.,[6] also found that gallbladder cancer tissues have significantly high levels of CLIC1 expression, and that the expression of CLIC1 was significantly higher in the plasma of individuals with early gallbladder cancer than in the plasma of normal individuals, suggesting that this protein can be used as a potential hematological marker for early gallbladder cancer. Consistent with these, our results suggest that CLIC1 may play an important role in the progression of PDAC.

Invasion and metastasis are the predominant characteristics of malignant diseases.[21] In multiple studies it has been shown that elevated levels of CLIC1 in different types of tumors correlate with metastatic potential, increased malignancy; and thus, an unfavorable prognosis for patients.[9],[20] In lung adenocarcinoma, Wang et al.,[10] found that the upregulation of CLIC1 significantly promoted cell migration and invasion. Furthermore, in gastric cancer,[20] elevated CLIC1 expression was strongly correlated with lymph node metastasis, lymphatic invasion, perineural invasion, and pathological staging. Here we also found that upregulated CLIC1 expression was strongly correlated with the degree of tumor differentiation and the tumor size in PDAC. Further survival analysis showed that after surgery, the survival time of cases with CLIC1 positive expression was significantly lower than that of cases with negative expression. Cox multivariate analysis showed that CLIC1 expression was negatively correlated with the postoperative survival ratio, and was the evaluation factor for independent poor prognosis. These findings indicate its potential role in promoting aggressive phenotypes of PDAC, and a potential utility of CLIC1 expression levels as a prognostic biomarker of PDAC.

 > Conclusion Top

CLIC1 overexpression is present in a substantial proportion of PDAC and is significantly correlated with clinical biological behaviors. More importantly, CLIC1 overexpression, along with the presence of lymph node metastasis, represents a significant prognostic for total postoperative survival. CLIC1 may be closely associated with the occurrence and development of PDAC, and CLIC1 overexpression may be used as an effective predictor for the prognosis of this disease.

 > Acknowledgements Top

This work was supported by the Natural Science Foundation of Shanxi Province (No. 20110313013-3 and 2014021037-3).

 > References Top

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Wang P, Zeng Y, Liu T, Zhang C, Yu PW, Hao YX, et al. Chloride intracellular channel 1 regulates colon cancer cell migration and invasion through ROS/ERK pathway. World J Gastroenterol 2014;20:2071-8.  Back to cited text no. 11
Li M, Lu J, Zhang F, Li H, Zhang B, Wu X, et al. Yes-associated protein 1 (YAP1) promotes human gallbladder tumor growth via activation of the AXL/MAPK pathway. Cancer Lett 2014;355:201-9.  Back to cited text no. 12
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Li M, Zhang S, Wang Z, Zhang B, Wu X, Weng H, et al. Prognostic significance of nemo-like kinase (NLK) expression in patients with gallbladder cancer. Tumour Biol 2013;34:3995-4000.  Back to cited text no. 21


  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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