The role of chloride intracellular channel 4 in tumors

Abstract

Tumors are among the most predominant health problems in the world, and the annual incidence of cancer is increasing globally; therefore, there is an urgent need to identify effective therapeutic targets. Chloride intracellular channel 4 (CLIC4) belongs to the family of chloride intracellular channels (CLICs), which are widely expressed in various tissues and organs, such as the brain, lung, pancreas, colorectum, and ovary, and play important roles in promoting apoptosis, promoting angiogenesis, maintaining normal proliferation of endothelial cells, and regulating the assembly and reconstruction of the cytoskeleton. The expression and function of CLIC4 in tumors varies. It has been reported that CLIC4 is low expressed in gastric cancer, skin cancer and prostate cancer, suggesting a tumor suppressor role. Interestingly, CLIC4 is overexpressed in pancreatic, ovarian and breast cancers, indicating a cancer-promoting role. CLIC4 expression is dysregulated in some solid tumors, which may be because CLIC4 is involved in the growth, migration or invasion of some cancer cells through various mechanisms. Regulation of CLIC4 expression may be a potential therapeutic strategy for some tumors. CLIC4 may be a promising therapeutic target and a biomarker for some cancers. In this study, we review the role of CLIC4 in several cancers and its value in the diagnosis and treatment of tumors.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-025-03737-7.

Introduction

Tumors are among the predominant health problems in the world [1]. Cancer is a collection of diseases caused by the interaction of genetic factors and environmental factors. Currently, several well-established risk factors for cancer development have been identified, including pathogenic infections, chronic alcohol consumption, tobacco smoking, and unhealthy dietary patterns [2, 3]. Common cancers include lung cancer, colorectal cancer, breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer, etc [4]. Based on research published in 2020, there will be approximately 19.3 million new cases of cancer and more than 10 million deaths from cancer worldwide [5]. The global incidence of cancer is increasing, and it is estimated that by 2040, the global cancer burden will reach 28.4 million cases [5]. To date, certain progress has been made in treatment, but cancer is still the second leading cause of death worldwide [6]. This compels us to further elucidate the genesis and developmental mechanisms of tumors and search for potential therapeutic targets.

Chloride ions are the most abundant anions in eukaryotes [7]. Chloride channels typically form transmembrane pore structures that facilitate the selective passage of chloride ions across cellular membranes [8]. Chloride ion channels play important roles in the stabilization of cell membrane potential, transepithelial transport, maintenance of intracellular pH, cell proliferation, fluid secretion, and regulation of the cell volume [9]. Intracellular chloride channel 4 (CLIC4) is a member of the intracellular chloride channel family (CLICs) [10], and the sequence of CLICs is highly conserved across multiple species, including bacteria and mammals [11]. Many studies have shown that CLIC4 is expressed in the brain [12], heart [13], lung [14], liver [14], pancreas [14], kidney [15], musculoskeletal system [16], and other tissues and organs. Studies have shown that CLIC4 is involved in promoting apoptosis [17], and angiogenesis [18], maintaining normal proliferation of endothelial cells [18], and regulating cytoskeleton assembly and remodeling [19], as well as participating in many pathophysiological processes. In recent years, an increasing number of studies have shown that CLIC4 is aberrantly expressed in many tumors in the human body, including tumors of the respiratory system [20], digestive system [21], urinary system [22], and reproductive system [23]. CLIC4 can regulate cancer cell growth, proliferation, invasion, metastasis, and tumor progression and may play an important role in the progression of several cancers (Table 1). In addition, CLIC4 may also serve as a biomarker for some tumors and may be a potential therapeutic target. This paper reviews the role of CLIC4 in some tumors and its potential clinical significance.

Table 1.

Functional characterization of CLIC4 in various tumors

Type of cancer	Functional roles	Mechanism	Refs.
Gliomas	Tumor growth(+)	The protein interacts with 14-3-3-ε and modulates key proteins involved in oxidative stress, mitochondrial stress, and ER stress	[45], [54]
Lung cancer	Not clear	The mechanism is unclear	[62], [20]
Gastric cancer	Tumor migration(-), invasion(-)	Inhibits the stem and EMT of cancer cells·	[67]
Pancreatic cancer	Not clear	The mechanism is unclear	[75]
Colorectal cancer	Tumor invasion(+), progression (+)	The mechanism is unclear, It may be associated with NR, VEGF, NRF2, MAPK, PI3K, AKT, miRNA-related	[21], [81]
Epithelial ovarian cancer	Tumor progression (+), proliferation (+), metastasis (+)	Acting as a downstream effector of TGF-β signal transduction, it can also promote the expression of angiogenic factors and also affect the microenvironment of ovarian cancer.	[23], [90]
Prostate cancer	Tumor proliferation(-), migration(-), invasion(-)	The mechanism is unclear.	[93]
Breast cancer	Tumor growth(+), migration (+), invasion (+)	TGF-β signal and ROS signaling transduction	[46], [100], [41]
Skin cancer	Not clear	Regulates TGF-β signaling	[37], [37, 38]
Head and neck squamous cell carcinoma	Tumor growth(+)	To regulate the proteins involved in mitochondrial and endoplasmic reticulum stress	[111]

CLIC4 Chloride intracellular channel 4, ER Endoplasmic reticulum, PAR1 Proteinase-activated receptor 1, TGF-β transforming growth factor-β, EMT epithelial-to-mesenchymal transition, NR nuclear receptor, VEGF vascular endothelial growth factor, NRF2 nuclear factor-erythroid 2-related factor, MAPK mitogen-activated protein kinase, PI3K phosphatidylinositol 3-kinase, AKT serine-threonine kinase protein B.ROS Reactive Oxygen Species

Structure and biological functions of CLIC4

Structure of CLIC4

CLIC4 has a molecular weight of approximately 28 kDa, and its gene is located on human chromosome 1 [24]. CLIC4, like other CLIC proteins, has two structural states: soluble and membrane-bound [11]. The soluble form of CLIC4 is highly homologous to the glutathione S-transferase protein (GST) superfamily, especially the GST-Omega class [25]. Li et al. [26] determined the crystal structure of the wild-type human soluble chloride ion channel CLIC4 (wCLIC4) with a resolution of 2.2 Å via X-ray crystallography. The structure is an asymmetric homologous trimer containing multiple α-helical and β-folds. These elements interact through hydrogen bond networks and hydrophobic contacts to form a relatively stable spherical or nearly spherical structure. CLIC4 consists of 253 amino acid residues with an N terminal that has a thioredoxin fold very similar to pentadidoxin and a C terminal that has an all-alpha helical structure [25]. The conformation of the N-terminal helical 2 (h2) region of wCLIC4 trimers is variable, and different conformations play key roles in mediating the oligomerization of CLIC4 or the interaction between CLIC4 and some structural elements on the bilayer membrane [26], which also reflects the conformational sensitivity of the N-terminal region. Interestingly, Littler et al. [25]accidentally constructed the fusion protein CLIC4 (ext), which is highly soluble and monomeric in solution. The water-soluble C-terminal region of CLIC4 (ext) can maintain a stable conformation in aqueous solution and participate in interactions with other water-soluble proteins or molecules [25]. Bioinformatic analysis has revealed the presence of a transmembrane domain (PTM) spanning amino acid residues Cys35 to Val57 proximal to the N-terminus when CLIC4 assumes its membrane-bound conformation [27] (Fig. 1). This transmembrane domain consists of two conventional α-helices and an irregular domain, The hydrophobic amino acids constituting this domain facilitate its integration into the membrane interior, thereby enabling stable membrane anchoring and subsequent modulation of ion diffusion processes [27, 28], including chloride ions [25].

Fig. 1.

Soluble form structure of CLIC4. It consists of three α-helices (h1-h3) located in the N domain and six α-helices (h4-h9) located in the C domain, where the N and C ends are located in the cytoplasm. There is also a hypothesized transmembrane domain (TMD) near the N-terminal.CLIC4 Chloride intracellular channel 4,TMD transmembrane domain

Biological functions of CLIC4

The biological function of CLIC4 is gradually being elucidated. Studies have shown that CLIC4 is regulated by redox reactions [25] and can switch between soluble forms and membrane-bound forms to play a variety of roles [29]. The soluble form of CLIC4 has pentodoxin-like enzyme activity, which helps maintain the redox environment in the cell [30]. When CLIC4 is in the membrane-bound state, it can play a role as a structural element of anion channels [29]. CLIC4 has been identified as a multimeric transmembrane protein that forms a pH-dependent, weakly selective pore structure upon membrane localization, enabling the transport of anions, including chloride ions across the membrane [25]. In addition, CLIC4 is involved in regulation of the intracellular and extracellular potassium ion balance [31]and the transport of other ions, such as sodium and calcium [32], thus maintaining cellular homeostasis.

CLIC4 can be localized to the Golgi, endoplasmic reticulum, nucleus [24], large and dense core vesicles [33], plasma membrane and mitochondria of mouse and human keratin-forming cells [32]. CLIC4 can localize to different subcellular organelles to perform different functions (Fig. 2). When CLIC4 is located in mitochondria, it helps maintain mitochondrial function and redox homeostasis and protects cells from damage [32]. When cells are subjected to various stresses, such as DNA damage, metabolic inhibition, and cytotoxicity, CLIC4 in the cytoplasm can translocate to the nucleus, thereby promoting apoptosis or cell cycle arrest [34]. This nuclear translocation of CLIC4 is achieved by the nitration of cysteine residues, which causes conformational changes in CLIC4, unfolding and extension of soluble proteins, and binding of nuclear localization signals (NLSs) hidden in soluble proteins to nuclear pore proteins (e.g., importin α and Ran) to cause the nuclear import of CLIC4 [25, 35]. The nuclear translocation of CLIC4 plays a pivotal role in multiple cellular processes, including tumor cell differentiation, stress-induced apoptosis, and transforming growth factor-β (TGF-β) signal transduction pathways [36, 37]. In addition, nuclear CLIC4 is required for keratinocyte differentiation [38]. CLIC4 also translocates from cytoplasmic lysates to the plasma membrane to regulate cell adhesion and migration [39]. Moreover, CLIC4 is an important regulator of intracellular reactive oxygen species (ROS) generation and helps maintain cell viability [40]. Surprisingly, the expression of CLIC4 was upregulated in activated macrophages [41], whereas the expression of CLIC4 was decreased in regulatory T cells [42], and based on the functions of macrophages and T cells in immunity and inflammation, it was hypothesized that CLIC4 may have important functions in immune regulation and the inflammatory response.

Fig. 2.

Location and function of CLIC4 in cells and subcells. CLIC4 plays an ion transport role on the cell membrane. The endoplasmic reticulum regulates Ca2+ release and CLIC4 is involved in the regulation of endoplasmic reticulum stress under stress conditions such as hunger. When CLIC4 is located on the mitochondria, it helps maintain mitochondrial function and redox homeostasis. When cells are subjected to cytotoxicity, transforming growth factor-β (TGF-β), reactive oxygen species (ROS) and other stresses, CLIC4 in the cytoplasm can translocation the nucleus, which is achieved through S-nitration of cysteine residues, which leads to conformational changes in CLIC4, development and extension of soluble protein structures. Nuclear localization signal NLS plays a role, causing nuclear input of CLIC4, thereby promoting apoptosis or cell cycle arrest, and enhancing TGF-β signal transduction. CLIC4 Chloride intracellular channel 4, ROS reactive oxygen species, TGF-β transforming growth factor-β, NLS Nuclear localization sequence

CLIC4 can also interact with actin, tubulin and other cytoskeletal components to participate in the assembly and remodeling of the cytoskeleton [43]. In addition, CLIC4 can regulate autophagy by regulating the ionic permeability of lysosomes, affecting the function of lysosomes and the fusion of autophages [44]. CLIC4 can also affect the proliferation, migration, and angiogenesis of endothelial cells, playing important roles in vascular homeostasis and development [45]. Importantly, genetic ablation of CLIC4 in mice resulted in impaired angiogenesis [45] and compromised cutaneous wound healing [46]. However, in existing studies, the role of CLIC4 as a membrane protein with ion transport functions or other functions has not been fully clarified, and further studies are needed.

Cancer development and progression are complex processes involving the maintenance of cell proliferation, resistance to cell death, activation of invasion and metastasis, and immune escape [47]. In recent years, the expression and function of CLIC4 have been extensively studied in many cancers. CLIC4 expression is regulated by p53, c-Myc, and tumor necrosis factor-α (TNF-α), and these mediators are involved in the pathogenesis of a variety of tumors [48, 49]. CLIC4 has tumor-specific expression patterns and dual functional roles in carcinogenesis. Although downregulation of CLIC4 has been observed in certain malignancies, where it exhibits tumor-suppressive properties, its overexpression has been documented in various aggressive cancers, including pancreatic, ovarian, and breast carcinomas, where it appears to promote tumor progression [49]. In addition, the extent of CLIC4 upregulation in tumor mesenchymal cells is directly correlated with poor tumor prognosis [49]. In another study, CLIC4 in tumor mesenchymal cells was found to promote tumor cell migration and invasion by TGF-β signaling and promote epithelial‒mesenchymal transition (EMT), which facilitates tumor development [50]. Interestingly, decreased CLIC4 expression in cancer cells can downregulate matrix metalloproteinase 9 (MMP9) expression and inhibit cancer cell invasion, which provides a new approach for inhibiting cancer cell metastasis via photodynamic therapy [51]. In conclusion, CLIC4 plays an important role in the growth and development of tumors, and may be highly important for the treatment of some tumors.

CLIC4 in several cancers

Nervous system

Gliomas are the most common primary brain tumors, and their treatment has significantly improved in recent years; however, gliomas remain largely incurable [52]. Previous studies have shown that CLIC4 is abundantly expressed in oligodendrocytes and may be important in glial cell-associated diseases, especially gliomas [12]. Xu et al. [53] reported that the CLIC4 protein was upregulated in a dose-dependent manner during the injury of glioma cells (C6 cells) induced by different concentrations of H₂O₂, suggesting that CLIC4 is involved in the apoptosis of C6 cells induced by H₂O₂. Moreover, they reported that the ratios of Bcl-2-associated X protein (Bax) /B-cell lymphoma 2 (Bcl-2), cytochrome c and cleaved caspase-3 protein were also increased during this process. Notably, the Bax/ Bcl-2, cytochrome C and caspase-3 proteins are considered to be key molecules in cell apoptosis [54, 55]. Therefore, it was speculated that the downregulation of CLIC4 could promote H₂O₂-induced apoptosis in C6 glioma cells. To test their conjecture, they further inhibited CLIC4 expression through RNA interference (RNAi) technology and found that CLIC4-RNAi promoted the apoptosis of glioma C6 cells induced by H2O2 during cell injury [53]. These findings suggest that CLIC4 is a key protective factor in the apoptosis induced by oxidative stress. Furthermore, another study revealed that CLIC4 protein expression was upregulated and that its nuclear translocation was increased in U251 glioma cells under starvation conditions [44]. In addition, they reported that apoptosis was increased when CLIC4 was silenced. Further mechanistic analysis revealed that silencing CLIC4 triggered mitochondrial apoptosis via the Bcl-2/Bax and caspase-3 pathways and promoted endoplasmic reticulum stress-induced apoptosis via the proapoptotic transcription factor C/EBP homologous protein (CHOP) and caspase-4 [44]. In addition, when CLIC4 was silenced, the interaction of CLIC4 with 14-3-3ε was blocked, leading to an increase in Beclin 1, further increasing autophagy [44]. These results suggest that the inhibition of CLIC4 enhances the autophagy and apoptosis induced by oxidative stress, mitochondria, and the endoplasmic reticulum, which play key roles in the growth of gliomas. However, many experimental studies are needed to determine whether CLIC4 plays a role in the progression and prognosis of gliomas.

Respiratory system

Lung cancer is the most common form of cancer and the leading cause of cancer-related death worldwide [56]. Currently, lung cancer treatment includes surgical treatment, chemotherapy and targeted drug therapy, but the recurrence rate is still high, and the treatment effect is poor; therefore, it is necessary to explore the mechanisms of lung cancer development and progression [57, 58]. Okudela et al. [20] reported that CILC4 protein levels were reduced in human primary lung cancer and several lung cancer cell lines (A549, TKB14, and H2087) and reported that knocking down CLIC4 in immunodeficient noncancer cell lines (NHBE-T) increased cell growth activity. The recovery of CLIC4 in lung cancer cell lines with low CLIC4 expression impaired its growth activity, suggesting that CILC4 may act as a tumor suppressor. In addition, the same study reported that the level of CLIC4 decreased with increasing histological grade of lung adenocarcinoma, and the recurrence rate in patients with low CLIC4 expression was slightly greater than that of patients with high CLIC4 expression [20]. These results suggest that CLIC4 may be an important tumor suppressor in the progression of lung cancer. In endothelial cells, CLIC4 has been demonstrated to localize at the plasma membrane, where it facilitates thrombin-induced rapid activation of RhoA and subsequent phosphorylation of ezrin/radixin/moesin (ERM) proteins [59]. Upon phosphorylation, ERM proteins mediate membrane-cytoskeletal reorganization, a critical process in endothelial barrier dysfunction [60]. Thrombin activates protease-activated ceceptor 1 (PAR1), a G-protein-coupled receptor [61], triggering mucosal inflammation and endothelial barrier disruption [62]. Mechanistically, CLIC4 has been shown to compromise endothelial integrity through the ERM/PAR1 signaling pathway [59]. This barrier dysfunction increases vascular permeability, facilitating tumor cell intravasation into the bloodstream and ultimately promoting cancer metastasis [63]. These studies suggest that CLIC4 may play a cancer-promoting role in lung cancer. Combined with the research results of the above experimental teams, it appears that CLIC4 has opposite effects on lung cancer (promoting or suppressing cancer). Based on experimental methods, experimental samples, and the dynamic regulation of CLIC4 in vivo, and given that the experimental research object of Okudela et al. [20] was a specific part of lung adenocarcinoma, it may not be able to explain all the cases. In the future, many experiments are needed to clarify the specific role and mechanism of CLIC4 in lung cancer.

Digestive system

Malignant tumors of the digestive system account for one-third of global cancer-related deaths and have become some of the deadliest cancers [64]. Despite many advances in diagnosis and treatment in recent years, the diagnosis and precise treatment of some digestive tract tumors remains a great challenge [65]. As a widely distributed chloride channel protein, CLIC4 is involved in the occurrence and development of various digestive system tumors.

Gastric cancer

Gastric cancer (GC) is one of the most prevalent cancers in the world; it has the fifth highest incidence rate and fourth highest mortality rate [5]. Wang et al. [66] reported that CLIC4 expression was lower in gastric cancer tissues than in normal gastric tissues and that the CLIC4 expression level was negatively correlated with clinical stage. To further evaluate the role of CLIC4 in tumor development, they used real-time polymerase chain reaction (PCR) and western blot analysis to show that CLIC4 expression was negatively correlated with the tumorigenic potential of three different GC cell lines, MKN45, N87 and AGS cells, which suggests that CLIC4 may be a tumor suppressor. Moreover, the results of a scratch assay of N87 and AGS GC cells demonstrated the inhibitory effect of CLIC4 on GC tumor cell migration [66]. They further refined the nude mouse tumorigenic assay to assess the effect of CLIC4 on tumor growth and reported that CLIC4 overexpression inhibited tumor growth whereas CLIC4 knockdown promoted tumor growth [66]. Further analysis revealed that CLIC4 overexpression inhibited the expression of CD44 and OCT4, thereby suppressing the migration, invasion and epithelial-mesenchymal transition (EMT) of GC cells [66]. Notably, CD44 and OCT4 are GC stem cell markers that are strongly associated with a poor prognosis [67–69]. In addition, EMT is a major mechanism for generating cancer stem cells that can promote GC progression and metastasis [70]. Thus, CLIC4 acts as a tumor suppressor in GC, and CLIC4 overexpression inhibits cancer cell stemness and EMT, thus suppressing tumor growth and progression. These findings also provide new insights into the pathogenesis of GC, suggesting that CLIC4 may be a potential therapeutic target for GC. Interestingly, in CLIC4 overexpressing GC cells, MKN45 cells are more sensitive to 5-fluorouracil and etoposide treatment [66], which opens new possibilities for the development of novel targeted therapies for GC.

Pancreatic cancer

Pancreatic cancer is the fourth most common cause of cancer-related death in Western countries, and despite advances in treatment, the prognosis is still poor [71], with a 5-year survival rate of approximately 12% [72]. Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent type of pancreatic cancer [73]. Zou et al. [74] reported that CLIC4 expression was greater in PDAC tissue than in paracancerous tissue and benign lesions. Moreover, in patients with PDAC, high CLIC4 expression is often closely associated with a poor prognosis and short survival time [74]. Another study also showed that CLIC4 was an adverse factor in terms of the median survival of PDAC patients and may be a potential prognostic biomarker [75]. In addition, CLIC4 is overexpressed in pancreatic circulating tumor cells (CTCs) compared with peripheral blood mononuclear cells [76]. Notably, CTCs have been shown to be valuable in the prediction of prognosis and as risk factors for disease recurrence in patients with PDAC [77]. These findings also suggest that CLIC4 may play an important role in prognosis prediction and as a novel treatment target for PDAC.

Colorectal cancer

In recent years, there has been a significant rise in both the incidence and mortality rates of rectal cancer (CRC) in China [78]. This disease presents a substantial threat to public health and imposes a considerable economic burden [79]. Studies have shown that recurrence and metastasis are the leading causes of death in paients with CRC [80]. Recent studies have shown that CLIC4 is strongly associated with CRC stage and prognosis. Yokoyama et al. [81] reported that the expression level of CLIC4 was greater in malignant epithelial tissue cells from patients with early-stage CRC lesions than in normal colorectal mucosal tissues; however, as CRC progressed, the amount of CLIC4 decreased significantly or tended to decrease. Furthermore, in advanced CRC, the expression of CLIC4 in the stromal cells of CRC-adjacent colorectal tissues was lower than that in tumor stromal cells [81]. CLIC4 expression has been reported to be a marker for CRC stem cells and is closely associated with the invasive potential of CRC stem cell-like cells and poor prognosis of the disease [82]. In addition, in CRC stromal cells, higher expression of CLIC4 is associated with a higher pathological grade of the tumor [49]. These studies show that the expression of CLIC4 is an important predictor of histopathological status and prognosis in CRC patients. In addition, analysis of transcriptomic changes between human CRC DLD-1 cells with CLIC4 knockdown, overexpression cells and control cells revealed that 9 CRC-related signaling pathways, including the nuclear receptor (NR), vascular endothelial growth factor (VEGF), adhesion, nuclear factor-erythroid 2-related factor 2 (NRF2), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/serine-threonine kinase protein B (AKT), insulin, interleukin (IL)-18 and miRNA regulatory pathways, overlap with the CLIC4 signaling pathway [21]. This finding also suggested that CLIC4 is closely related to CRC; however, the specific mechanism underlying the role of CLIC4 in CRC development is currently unknown, and further studies are needed.

Urogenital system and breast cancer tumors

The diagnosis and treatment of urogenital tumors and breast cancer have improved, but morbidity and mortality rates remain high [6, 83]. CLIC4 has been shown to play an important role in epithelial ovarian cancer, prostate cancer, and breast cancer.

Epithelial ovarian cancer

In the United States, epithelial ovarian cancer (EOC) causes the greatest number of deaths among tumors of the female reproductive system [84]. Yao et al. [85] reported that CLIC4 was highly expressed in the mesenchyme of EOCs and was associated with the upregulation of the myofibroblast marker SMA. Notably, tumor-associated fibroblasts can promote cancer progression [86]. An in vitro study revealed that CLIC4 expression was upregulated during the TGF-β1-induced transformation of ovarian cancer fibroblasts to myofibroblasts, whereas the use of CLIC4 inhibitors attenuated the expression of proangiogenic factors associated with myofibroblast differentiation [85]. Further mechanistic analysis revealed that TGF-β1 stimulates cells to produce ROS, leading to the upregulation of CLIC4, which in turn leads to myofibroblast transdifferentiation, thereby promoting tumor progression [85]. These findings suggest that CLIC4 is an important regulator of myofibroblast transdifferentiation and plays a key role in myofibroblast-dependent tumor progression, which also provides new ideas for preventing myofibroblast differentiation and myofibroblast-dependent tumor progression. Carbohydrate antigen 125 (CA125) is known to play an important role in ovarian cancer screening. Approximately 80% of patients with EOC have elevated CA125 levels at the time of initial diagnosis, and CA125 is considered a reliable diagnostic biomarker for ovarian cancer [87, 88]. CLIC4 serum levels are significantly elevated in patients with EOC [89], and the combination of CLIC4 and CA125 has been suggested as a potential serologic biomarker for the diagnosis of EOC [23]. CLIC4 is also present in the exosomes of ovarian cancer cells [9], and exosomes promote ovarian cancer progression by affecting the microenvironment of ovarian cancer [90], which also suggests that CLIC4 is a biomarker for ovarian cancer. In addition, Singha et al. [23] reported that the inhibition of CLIC4 in EOC cell lines suppressed cell proliferation and metastasis. The same study also revealed that when CLIC4 expression was increased in patient tumor cells, the patient survival rate decreased [23]. These studies suggest that CLIC4 overexpression in patients with epithelial ovarian cancer is associated with a poor prognosis. Therefore, CLIC4 could be a potential therapeutic target for epithelial ovarian cancer and is important for the development of drugs targeting epithelial ovarian cancer.

Prostate cancer

Prostate cancer is the second most common tumor in men worldwide [91]. Considerable progress has recently been made in surgical and hormonal treatments, but the mortality rate associated with prostate cancer metastasis remains high [92]. There is growing evidence that epithelial–mesenchymal transition (EMT) promotes prostate cancer metastasis and progression [93]. CLIC4 overexpression has been reported to inhibit tumor progression by decreasing the expression of the tumor stem cell markers CD44 and OCT4, which further inhibits cell migration, invasion and EMT [66]. Studies have shown that both the mRNA and protein levels of CLIC4 are lower in prostate cancer cells than in normal prostate epithelial cells [49, 94], and that its downregulation is associated with a poor prognosis in prostate cancer patients [95]. The level of dead-end 1 (DND1), which is thought to play a key role in the inhibition of proliferation and promotion of apoptosis in tumors [96], is negatively correlated with the level of CLIC4 [94]. Zhang et al. [94] reported that silencing DND1 increased the mRNA level of CLIC4, which further inhibited proliferation, invasion and EMT in prostate cancer. In addition, Zou et al. [95] showed that CLIC4 may be regulated by epigenetic modifications of the fat mass and obesity-associated(FTO) protein, which plays a key role in cancer [97]. In further experiments, these authors demonstrated that knockdown of the FTO protein increased the N6-methyladenosine (m6A) level of CLIC4, leading to a decrease in CLIC4 mRNA levels, which promoted prostate cancer proliferation and metastasis [95]. These studies demonstrated the involvement of CLIC4 in the migration, invasion, and EMT of prostate cancer cells through mRNA, providing new insights into the pathogenesis of prostate cancer.

Breast cancer

In recent years, breast cancer has become the most common cause of cancer-related death in women [98]. Considerable progress has been made in the diagnosis and treatment of breast cancer, but this disease remains a public health problem worldwide [99]. Therefore, exploring the molecular mechanisms underlying breast cancer is essential. Sanchez et al. [100] reported that CLIC4 was highly expressed in young breast cancer patients with aggressive disease, patients with localized metastases, and patients with a poor prognosis. This finding is consistent with previous findings suggesting that CLIC4 is an important marker of poor prognosis in patients with breast cancer [9, 100]. Further mechanistic analyses revealed that CLIC4 is a downstream effector of TGF-β signaling in breast cancer patients [100]. TGF-β plays an important role in the migration and invasion of breast cancer cells, which ultimately leads to tumor progression [101]. Furthermore, when breast cancer cells were injected into wild-type (WT) and CLIC4 knockout female mice, both developed primary tumors. Interestingly, CLIC4 knockout mice had almost no lung metastasis, whereas WT mice developed significant lung metastasis [100], suggesting that the ability to metastasize to the lungs requires host CLIC4 in these breast cancer models. Furthermore, CLIC4 deficiency was not compensated for by other CLIC4 paralogous homologs [100]. Shukla et al. [50] demonstrated that CLIC4 is upregulated in cancer-associated fibroblasts (CAFs) and modulated by TGF-β signaling. During TGF-β-mediated fibroblast activation, CLIC4 potentiates p38 MAPK signaling through the inhibition of p38 dephosphorylation, thereby promoting cellular differentiation. Furthermore, CLIC4 enhances breast cancer invasion and migration via a TGF-β-dependent mechanism. These findings implicate CLIC4 as a crucial mediator in breast cancer metastasis, primarily through the TGF-β signaling pathway. However, the potential involvement of additional signaling cascades requires further investigation to establish a comprehensive understanding of the role of CLIC4 in metastatic progression.

In vitro studies by Al et al. [40] demonstrated that CLIC4 knockout in mouse 6DT1 breast tumor cells significantly enhanced ROS accumulation and increased cellular sensitivity to H₂O₂-induced apoptosis. This finding aligns with previous reports identifying the glutathione reductase-like enzymatic activity and antioxidant function of CLIC4 [30], suggesting its crucial role in maintaining cellular redox homeostasis. Notably, CLIC4 deletion resulted in increased mitochondrial activity, increased membrane potential, reduced Bcl-2 expression, degradation of mitochondrial uncoupling protein 2, and ultimately increased apoptosis, collectively contributing to suppressed 6DT1 tumor growth [40].

However, in existing studies, the molecular mechanism of ROS in breast cancer growth and metastasis is still unclear. In vivo experimental evidence revealed that CLIC4-deficient 6DT1 cell-derived xenografts exhibit significantly reduced tumor masses and extensive necrotic areas highlighting the therapeutic potential of CLIC4 inhibition in breast cancer management [40]. CLIC4 potentially drives breast cancer progression through two distinct mechanisms: facilitating metastasis via TGF-β signaling pathway activation and promoting tumor growth through ROS-mediated processes, both of which are strongly associated with adverse clinical outcomes in breast cancer patients. These dual oncogenic functions position CLIC4 as a promising molecular target for therapeutic intervention in breast cancer management. Notably, Chiang et al. [51] demonstrated that photodynamic therapy (PDT) treatment of MDA-MB-231 breast cancer cells resulted in significant downregulation of both CLIC4 expression and matrix metalloproteinase 9 (MMP9) activity, consequently suppressing cancer cell invasion. These findings implicate CLIC4 as a crucial mediator in the PDT-induced inhibition of cancer cell invasiveness. This discovery not only elucidates a novel mechanism underlying the anti-invasive effects of PDT but also reinforces the potential of CLIC4 as a therapeutic target in breast cancer treatment strategies.

The role of CLIC4 in other cancers

Skin cancer

Skin cancer is the fifth most common cancer worldwide, affecting millions of people each year [102, 103]. Skin cancer is curable in its early stages, but its annual incidence remains high, drug therapy and clinical diagnosis represent other challenges, and skin cancer has become a growing global burden [104]. Keratinocytes are the dominant cell type in the skin [105]. CLIC4 is localized in the cytoplasm and mitochondria of human and mouse skin keratinocytes [106, 107]. CLIC4 is also associated with skin stem cells [108]. These findings suggest that CLIC4 may play an important role in the skin. Suh et al. [36] reported that in mouse and human cutaneous squamous cell carcinoma, as well as in chemically induced mouse squamous cell carcinoma models, the expression of CLIC4 was notably downregulated, accompanied by a clear phenomenon of nuclear exclusion. This decrease in CLIC4 expression is closely correlated with the malignant progression of cutaneous squamous cell carcinoma, strongly implying its potential function as a tumor suppressor in skin cancer. In vivo experiments showed that an increase in CLIC4 inhibited the growth of squamous cell carcinoma in mouse skin. Adenovirus-mediated nuclear translocation of CLIC4 has been shown to effectively suppress squamous tumor growth in both murine and human systems. Mechanistic studies has revealed that cellular stress induces S-nitrosylation of cytoplasmic CLIC4, enhancing its binding affinity for nuclear import proteins and facilitating its nuclear translocation, ultimately leading to cell cycle arrest and growth inhibition [35]. Furthermore, nuclear-localized CLIC4 potentiates TGF-β signaling by stabilizing phosphorylated Smad2/3 and preventing its dephosphorylation, thereby amplifying its growth-inhibitory effects [36, 37]. These findings collectively establish CLIC4 as a tumor suppressor in cutaneous squamous cell carcinoma. However, the molecular mechanisms underlying CLIC4 downregulation during skin cancer progression remain elusive, with the potential involvement of genetic alterations such as mutations or deletions in the CLIC4 gene requiring further investigation [49].

Head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck and includes squamous cell carcinoma of the oral cavity, pharynx and larynx [109]. CLIC4 may play a role in the pathogenesis of HNSCC. Previous studies have shown that CLIC4 is more highly expressed in oral squamous cells (OSCCs) than in normal gingival tissues [110]. Xerez et al. [111] further evaluated the expression of CLIC4 in OSCC and reported that CLIC4 exhibited cytoplasmic immunostaining or simultaneous staining in the nucleus and cytoplasm of tumor epithelial cells. Interestingly, they reported increased CLIC4 immunostaining in the OSCC stroma [111]. In addition, Xue et al. [110] reported that ATP-induced apoptosis was increased when CLIC4 was specifically knocked down in HN4 head and neck squamous cells. Further mechanistic analysis revealed that knockdown of CLIC4 with or without ATP treatment increased the expression levels of Bax and active Caspase-3, the proapoptotic transcription factor C/EBP homologous protein (CHOP), and active cysteine asparaginase 4 and downregulated the expression of Bcl-2. Notably, BAX, along with Bcl-2 and Caspase-3, are key constituents of mitochondrial apoptosis [112]. CHOP and active cysteine asparaginase 4 are considered endoplasmic reticulum stress-associated proteins [113], suggesting that CLIC4 knockdown exerts proapoptotic effects through the mitochondrial and endoplasmic reticulum stress pathways. In contrast, another study found that the expression of CLIC4 in the tumor parenchyma gradually decreased during the progression of head and neck squamous cell carcinoma [114]. MiR-142-3p is a direct target of CLIC4 and is responsible for downregulating CLIC4 in the tumor epithelium [114]. It has been reported that miR-142-3p plays an oncogenic or pro-oncogenic role in cancer [115]. In a pathological state, the level of miR-142-3p is elevated, indicating that miR-142-3p is a cancer biomarker [116]. However, current studies has not yet clarified the mechanism by which miR-142-3p affects CLIC4 and whether it has a paracrine effect or an effect on gene expression within immune cells, and further studies are still needed. Interestingly, the results reported by the two research teams mentioned above are not consistent with the finding that CLIC4 affects the progression of head and neck squamous cell carcinoma. Owing to the differences in experimental study designs, experimental methods, etc., and in combination with the dynamic regulation of CLIC4 in vivo, the in vitro experiments of Xue et al. [110] may not be able to account for all circumstances, and further in-depth studies are still needed.

Conclusions

Increasing evidence suggests that the abnormal expression of CLIC4 plays a significant role in the development of various tumors, including glioma, gastric cancer, pancreatic cancer, colorectal cancer, epithelial ovarian cancer, breast cancer, prostate cancer, skin cancer, and head and neck squamous cell carcinoma. Numerous studies have demonstrated that CLIC4 plays a dual role in tumorigenesis, functioning as a tumor suppressor when expressed at low levels in tumor cells, whereas its overexpression in various malignancies, including pancreatic, ovarian, and breast cancers, has been shown to promote tumor progression in vivo. Furthermore, emerging evidence suggests that CLIC4 expression levels are significantly correlated with clinical outcomes, establishing its potential as a valuable prognostic biomarker in several cancer types. In addition, CLIC4 is closely associated with tumor prognosis and survival, making it a potential biomarker for diagnosis and prognosis. Interestingly, CLIC4 has also been linked to adverse outcomes in certain cancer patients. Its role appears to vary across tumor types: in digestive system tumors, for example, CLIC4 acts as a tumor suppressor in gastric cancer but promotes cancer progression in colon cancer. Similarly, studies on the role of CLIC4 in head and neck squamous cell carcinoma have yielded inconsistent results, likely due to differences in experimental design and methodology, as well as the dynamic regulation of CLIC4 in vivo. For example, the findings of Xue et al. [110] were based on in vitro experiments, which may not fully capture the complexities of in vivo conditions. Further research is needed to clarify these seemingly contradictory effects. Although the expression of CLIC4 is clearly dysregulated in many solid cancers, the underlying mechanisms remain poorly understood. Therefore, extensive studies are needed to elucidate the precise pathogenesis of CLIC4 in tumor biology. Notably, CLIC4 appears to have paradoxical effects on lung cancer, demonstrating both tumor-promoting and tumor-suppressing activities. This apparent contradiction may be attributed to several factors, including methodological variation in experimental approaches, differences in sample characteristics, and the complex dynamic regulation of CLIC4 in vivo. Specifically, the study by Okudela et al. [20], which focused on a particular subtype of lung adenocarcinoma, may not fully represent the diverse biological behaviors of CLIC4 across different lung cancer subtypes. Therefore, comprehensive investigations incorporating larger sample sizes, standardized methodologies, and subtype-specific analyses are warranted to elucidate the precise role and underlying molecular mechanisms of CLIC4 in lung carcinogenesis. This review summarizes the role of CLIC4 in some tumors, which is highly important for the diagnosis and treatment of CLIC4 expression-related tumors.

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Abbreviations

CLIC4

Chloride intracellular channel 4

CLICs

Chloride intracellular channels

GST

Glutathione S-transferase

ROS

Reactive oxygen species

TNF-α

Tumor necrosis factor-α

TGF-β

Transforming growth factor-β

EMT

Epithelial-to-mesenchymal transition

MMP9

Matrix metalloproteinase 9

Bcl-2

B-cell lymphoma-2

Bax

Bcl-2 Associated protein X

PAR1

Proteinase-activated receptor 1

GC

Gastric cancer

PDAC

Pancreatic ductal adenocarcinoma

CTCs

Circulating tumor cells

CRC

Colorectal cancer

VEGF

Vascular endothelial growth factor

NR

Nuclear receptor

NRF2

Nuclear factor-erythroid 2-related factor 2

MAPK

Mitogen-activated protein kinase

PI3K

Phosphatidylinositol 3-kinase

AKT

Serine-threonine kinase protein B

IL-18

Interleukin-18

EOC

Epithelial ovarian cancer

CA125

Carbohydrate antigen 125

DND1

Dead-End 1

FTO

the fat mass and obesity-associated

NLSs

Nuclear localization signals

WT

Wild-type

HNSCC

Head and neck squamous cell carcinoma

OSCCs

Oral squamous cells

CHOP

C/EBP homologous protein

ERM

Ezrin/Radixin/Moesin

PDT

photodynamic therapy

RNAi

RNA interference

Author contributions

XL and YW drafted the manuscript and contributed equally. MR, QL, JL, LZ, SY, and LT participated in the literature search and analysis of the data to be included in the review. GW and JA were involved in the design of the study and assisted in the preparation of the figures and tables. HJ and BT edited and revised the manuscript. All authors have read and approved the final version of the manuscript. Data authentication is not applicable.

Funding

The present study was supported by grants from the National Natural Science Foundation of China (grant nos. 81960507, 82073087 and 82160112), the Collaborative Innovation Center of the Chinese Ministry of Education (2020-39), the Science and Technology Bureau fund of Zunyi City [grant no. ZUN SHI KE HE HZ ZI (2019)93Hao] and the Science and Technology Plan Project of Guizhou Province [grant nos. QIAN KE HE JI CHUZK (202) YI BAN 323].

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.