Elevated expression of chloride intracellular channel 1 is correlated with poor prognosis in human gliomas

Human gliomas represent the most common primary brain tumors in both children and adults. According to histopathological and clinical criteria established by the World Health Organization (WHO), this dismal disease can be classified as well-differentiated low grade astrocytomas [World Health Organization (WHO) grade I~II], anaplastic astrocytomas (WHO grade III) and glioblastoma multiforme (GBM, WHO grade IV) [1]. Despite recent therapeutic advances, the survival of patient with glioma is still poor. The median overall survival of patients with malignant gliomas is no more than one year and local recurrence occurs in more than 90% of patients [2]. Recent studies have indicated that patients’ age, Karnofsky performance status (KPS) score, histologic grade, and tumor necrosis are important prognostic factors for gliomas [3]. However, the prognosis of both high- and low-grade tumors remains heterogeneous. The median survival time of patients with high-grade gliomas range from 5 to 59 months and some patients with low-grade tumors also present poor outcome [4]. Similar with other human solid tumors, the predominant features of gliomas are extensive local tumor invasion and metastasis, in which multiple molecular events are involved. Focusing on these genetic background and molecular pathogenic processes is necessary to identify novel diagnostic and prognostic markers for improving the clinical outcome of patients with gliomas.

In mammals, the chloride intracellular channel (CLIC) gene family has six members, including CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, and CLIC6 [5]. This family is defined by a conserved, approximately 230 amino acid core sequence which comprises the C-termini of all known CLICs. CLIC1 is a newly discovered member of the CLIC family [6]. In 1997, it was originally cloned from a human monocytic cell line activated by the phorbol ester, phorbol 12-myristate 13 acetate [7]. CLIC1 is expressed ubiquitously in human tissues and is usually localized in the cytoplasm and nucleoplasm with a soluble form. It has been demonstrated to be involved in the regulation of cell cycle, cell proliferation and differentiation [8]. In the G2/M phase, CLIC1 is detected on the plasma membranes of cells, and the inhibition of CLIC1 function prolongs the mean time of the cell cycle in cell culture [9]. Recent studies have found that CLIC1 is over-expressed in malignant tumors, such as hepatocellular carcinoma [10], gallbladder carcinoma [11], gastric carcinoma [12], and colorectal cancer [13, 14]. CLIC1 has been considered as a sensor and an effector during oxidative stress, which may lead cells through all the phases of the cell cycle [15]. Thus, CLIC1 is hyperactivated in cancer cells which are in a highly proliferative state, and plays important roles in tumor invasion and metastasis.

In central nervous system, several lines of evidence support that CLIC1 plays a fundamental role in activated microglia and is involved in the pathophysiology of several neurodegenerative diseases [16]. Additionally, Kang et al. [17] found that small cell populations of GBM2 cancer stem cells (CSCs) were resistant to chemotherapeutic agent BCNU and highly expressed CLIC1. They further demonstrated that CLIC1 was involved in the resistance of BCNU-resistant CSCs. However, the clinicopathological significance and prognostic value of CLIC1 in clinical glioma specimens are still unclear. To address this problem, CLIC1 expression in human gliomas and nonneoplastic brain tissues were measured by immunohistochemistry. The association of CLIC1 immunostaining with clinicopathological factors or prognosis of glioma patients was statistically analyzed.

Similar with other human solid tumor cells, the glioma cells do not only have limitless replicative potential but also readily invade surrounding brain tissues and metastasize to other tissues, which make complete surgical resection practically impossible and lead to poor prognosis. Therefore, molecules involved in the aggressive process are potential prognostic and therapeutic markers. In the present study, our data shown for the first time that the up-regulation of CLIC1 at both mRNA and protein levels in glioma tissues compared with its expression in non-neoplastic brain tissues. Additionally, highly CLIC1 protein expression was significantly correlated with advanced WHO stage and low KPS scores, suggesting that this protein might be of clinical relevance in the aggressiveness of gliomas. Together with these findings, we also demonstrated that CLIC1 expression was a statistically significant risk factor affecting overall survival of patients with glioma and was an independent risk factor predicting short overall survival.

As a member of the CLIC family, CLIC1 functions as a real chloride channel in plasma and nuclear membranes [19]. The distribution of intracellular CLIC1 expression varies depending on the cell types, from intracellular vesicular pattern to intranuclear distribution. Its physiological roles include ion homeostasis modulation, cell volume regulation, transepithelial transport, and regulation of electrical excitability [20]. Accumulating number of studies have reported that CLIC1 is up-regulated in many tumor cells, such as hepatocellular carcinoma [10], gallbladder carcinoma [11], gastric cancer [12], colon cancer [13], nasopharyngeal carcinoma [21], and breast cancer [22], and plays important roles in tumor progression by modifying cell cycle, apoptosis, cell adhesion, and promoting tumor metastasis. For example, Chen et al. [12] found that the high levels of CLIC1 expression in gastric cancer significantly correlated with lymph node metastasis, lymphatic vessels and surrounding tissues infiltration, pathological staging, and survival time of patients; Wang et al. [23] shown that CLIC1 expression in lung adenocarcinoma was positively correlated with the T staging of the tumor and was negatively correlated with the shorter postoperative survival time of patients; Similarly, overexpression of CLIC1 was detected in gallbladder carcinoma and also found to significantly increase cell motility and invasion of the poorly metastatic gallbladder carcinoma cell line [11]. These results strongly imply that CLIC1 plays an important role in tumor advancement. However, its connection with human glioma has remained unknown. Our current study provided the evidence in support of such a connection using a cohort of 128 archived clinical glioma specimens. We first detected high expression of CLIC1 in glioma tissues compared with non-neoplastic brain tissues. Further support for a possible role of CLIC1 in glioma pathogenesis derived from the analysis that revealed a strong correlation of CLIC1 expression with the histopathological staging and inversely, with the survival of the disease. These findings are consistent with the previous reports which indicated that overexpression of CLIC1 is a potential prognostic marker for hepatocellular carcinoma [9], gallbladder carcinoma [10], gastric cancer [11], and lung adenocarcinoma [23].

In summary, our data provide the first evidence that CLIC1 expression might play an important role in the regulation of aggressiveness in human gliomas. The elevated expression of CLIC1 might represent a valuable prognostic marker for this disease. This study adds to the current realization on the involvement of CLIC1 in tumorigenesis and progression of human malignant tumors.