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The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H 2 O 2 as a ROS-inducing agent. In intact cells, H 2 O 2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H 2 O 2 -induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H 2 O 2 -treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells., Competing Interests: Conflict of interest Authors declare that there are no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The TGF-β-regulated Chloride Intracellular Channel 4 (CLIC4) is an essential participant in the formation of breast cancer stroma. Here, we used data available from the TCGA and METABRIC datasets to show that CLIC4 expression was higher in breast cancers from younger women and those with early-stage metastatic disease. Elevated CLIC4 predicted poor outcome in breast cancer patients and was linked to the TGF-β pathway. However, these associations did not reveal the underlying biological contribution of CLIC4 to breast cancer progression. Constitutive ablation of host Clic4 in two murine metastatic breast cancer models nearly eliminated lung metastases without reducing primary tumor weight, while tumor cells ablated of Clic4 retained metastatic capability in wildtype hosts. Thus, CLIC4 was required for host metastatic competence. Pre- and post-metastatic proteomic analysis identified circulating pro-metastatic soluble factors that differed in tumor-bearing CLIC4-deficient and wildtype hosts. Vascular abnormalities and necrosis increased in primary tumors from CLIC4-deficient hosts. Transcriptional profiles of both primary tumors and pre-metastatic lungs of tumor-bearing CLIC4-deficient hosts were consistent with a microenvironment where inflammatory pathways were elevated. Altogether, CLIC4 expression in human breast cancers may serve as a prognostic biomarker; therapeutic targeting of CLIC4 could reduce primary tumor viability and host metastatic competence., Competing Interests: The authors have declared that no competing interests exist.

CLIC4 (Chloride intracellular channel 4) belongs to a family of putative intracellular chloride channel proteins expressed ubiquitously in multiple tissues. CLIC4 is predominantly soluble and traffics between the cytoplasm and nucleus and participates in cell cycle control and differentiation. Transforming growth factor beta (TGF-β) elevates CLIC4, which enhances TGF-β signaling through CLIC4 mediated stabilization of phospho-Smad2/3. CLIC4 is essential for TGF-β induced conversion of fibroblasts to myofibroblasts and expression of matrix proteins, signaling via the p38MAPK pathway. Therefore, regulation of TGF-β signaling is a major mechanism by which CLIC4 modifies normal growth and differentiation. We now report that elevated CLIC4 alters Smad7 function, a feedback inhibitor of the TGF-β pathway. Overexpression of CLIC4 in keratinocytes, mouse embryonic fibroblasts and other mouse and human cell types increases the expression of Smad7Δ, a novel truncated form of Smad7. The alternatively spliced Smad7Δ variant is missing 94bp in exon 4 of Smad 7 and is conserved between mouse and human cells. The deletion is predicted to lack the TGF-β signaling inhibitory MH2 domain of Smad7. Treatment with exogenous TGF-β1 also enhances expression of Smad7Δ that is amplified in the presence of CLIC4. While Smad7 expression inhibits TGF-β signaling, exogenously expressed Smad7Δ does not inhibit TGF-β signaling as determined by TGF-β dependent proliferation, reporter assays and phosphorylation of Smad proteins. Instead, exogenous Smad7Δ acts as a dominant negative inhibitor of Smad7, thus increasing TGF-β signaling. This discovery adds another dimension to the myriad ways by which CLIC4 modifies TGF-β signaling., Competing Interests: The authors have declared that no competing interests exist.

In recent decades, growing scientific evidence supports the role of ion channels in the development of different cancers. Both potassium selective pores and chloride permeabilities are considered the most active channels during tumorigenesis. High rate of proliferation, active migration, and invasiveness into non-neoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of chloride channel activity. In this context, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, this encouraging premise has not so far produced any valid new channel-targeted antitumoral molecule for cancer treatment. Problematic for drug design targeting ion channels is their vital role in normal cells for essential physiological functions. By targeting these membrane proteins involved in pathological conditions, it is inevitable to cause relevant side effects in healthy organs. In light of this, a new protein family, the chloride intracellular channels (CLICs), could be a promising class of therapeutic targets for its intrinsic individualities: CLIC1 and CLIC4, in particular, not only are overexpressed in specific tumor types or their corresponding stroma but also change localization and function from hydrophilic cytosolic to integral transmembrane proteins as active ionic channels or signal transducers during cell cycle progression in certain cases. These changes in intracellular localization, tissue compartments, and channel function, uniquely associated with malignant transformation, may offer a unique target for cancer therapy, likely able to spare normal cells. This article is part of a special issue itled "Membrane Channels and Transporters in Cancers.", (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Chloride Intracellular Channel 4 (CLIC4) is one of seven members in the closely related CLIC protein family. CLIC4 is involved in multiple cellular processes including apoptosis, cellular differentiation, inflammation and endothelial tubulogenesis. Despite over a decade of research, no comprehensive in situ expression analysis of CLIC4 in a living organism has been reported. In order to fulfill this goal, we generated a knock-in mouse to express Green Fluorescent Protein (GFP) from the CLIC4 locus, thus substituting the GFP coding region for CLIC4. We used GFP protein expression to eliminate cross reaction with other CLIC family members., Results: We analyzed CLIC4 expression during embryonic development and adult organs. During mid and late gestation, CLIC4 expression is modulated particularly in fetal brain, heart, thymus, liver and kidney as well as in developing brown adipose tissue and stratifying epidermis. In the adult mouse, CLIC4 is highly expressed globally in vascular endothelial cells as well as in liver, lung alveolar septae, pancreatic acini, spermatogonia, renal proximal tubules, cardiomyocytes and thymic epithelial cells. Neural expression included axonal tracks, olfactory bulb, Purkinje cell layer and dentate gyrus. Renal CLIC4 expression was most pronounced in proximal tubules, although altered renal function was not detected in the absence of CLIC4. Myeloid cells and B cells of the spleen are rich in CLIC4 expression as are CD4 and CD8 positive T cells., Conclusions: In a comprehensive study detailing CLIC4 expression in situ in a mouse model that excludes cross reaction with other family members, we were able to document previously unreported expression for CLIC4 in developing fetus, particularly the brain. In addition, compartmentalized expression of CLIC4 in specific adult tissues and cells provides a focus to explore potential functions of this protein not addressed previously.

Background: Chloride intracellular channel 4 (CLIC4) is highly expressed in the endothelium of remodeled pulmonary vessels and plexiform lesions of patients with pulmonary arterial hypertension. CLIC4 regulates vasculogenesis through endothelial tube formation. Aberrant CLIC4 expression may contribute to the vascular pathology of pulmonary arterial hypertension., Methods and Results: CLIC4 protein expression was increased in plasma and blood-derived endothelial cells from patients with idiopathic pulmonary arterial hypertension and in the pulmonary vascular endothelium of 3 rat models of pulmonary hypertension. CLIC4 gene deletion markedly attenuated the development of chronic hypoxia-induced pulmonary hypertension in mice. Adenoviral overexpression of CLIC4 in cultured human pulmonary artery endothelial cells compromised pulmonary endothelial barrier function and enhanced their survival and angiogenic capacity, whereas CLIC4 shRNA had an inhibitory effect. Similarly, inhibition of CLIC4 expression in blood-derived endothelial cells from patients with idiopathic pulmonary arterial hypertension attenuated the abnormal angiogenic behavior that characterizes these cells. The mechanism of CLIC4 effects involves p65-mediated activation of nuclear factor-κB, followed by stabilization of hypoxia-inducible factor-1α and increased downstream production of vascular endothelial growth factor and endothelin-1., Conclusion: Increased CLIC4 expression is an early manifestation and mediator of endothelial dysfunction in pulmonary hypertension.

Cutaneous wound healing is a complex process involving blood clotting, inflammation, migration of keratinocytes, angiogenesis, and, ultimately, tissue remodeling and wound closure. Many of these processes involve transforming growth factor-β (TGF-β) signaling, and mice lacking components of the TGF-β signaling pathway are defective in wound healing. We show herein that CLIC4, an integral component of the TGF-β pathway, is highly up-regulated in skin wounds. We genetically deleted murine CLIC4 and generated a colony on a C57Bl/6 background. CLIC4(NULL) mice were viable and fertile but had smaller litters than did wild-type mice. After 6 months of age, up to 40% of null mice developed spontaneous skin erosions. Reepithelialization of induced full-thickness skin wounds and superficial corneal wounds was delayed in CLIC4(NULL) mice, resolution of inflammation was delayed, and expression of β4 integrin and p21 was reduced in lysates of constitutive and wounded CLIC4(NULL) skin. The induced level of phosphorylated Smad2 in response to TGF-β was reduced in cultured CLIC4(NULL) keratinocytes relative to in wild-type cells, and CLIC4(NULL) keratinocytes migrated slower than did wild-type keratinocytes and did not increase migration in response to TGF-β. CLIC4(NULL) keratinocytes were also less adherent on plates coated with matrix secreted by wild-type keratinocytes. These results indicate that CLIC4 participates in skin healing and corneal wound reepithelialization through enhancement of epithelial migration by a mechanism that may involve a compromised TGF-β pathway., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Chloride intracellular channel (CLIC) 4 is a member of a redox-regulated, metamorphic multifunctional protein family, first characterized as intracellular chloride channels. Current knowledge indicates that CLICs participate in signaling, cytoskeleton integrity and differentiation functions of multiple tissues. In metabolically stressed skin keratinocytes, cytoplasmic CLIC4 is S-nitrosylated and translocates to the nucleus where it enhances transforming growth factor-β (TGF-β) signaling by protecting phospho-Smad 2 and 3 from dephosphorylation. CLIC4 expression is diminished in multiple human epithelial cancers, and the protein is excluded from the nucleus. We now show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines, and the protein is excluded from the nucleus. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear translocation of CLIC4. Adenoviral-mediated reconstitution of nuclear CLIC4 in squamous cancer cells enhances TGF-β-dependent transcriptional activity and inhibits growth. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts inhibits tumor growth, whereas elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth and enhances TGF-β signaling. These results indicate that CLIC4 suppresses the growth of squamous cancers, that reduced CLIC4 expression and nuclear residence detected in cancer cells is associated with the altered redox state of tumor cells and the absence of detectable nuclear CLIC4 in cancers contributes to TGF-β resistance and enhances tumor development.

Nuclear translocation of cytosolic CLIC4 is an essential feature of its proapoptotic and prodifferentiation functions. Here we demonstrate that CLIC4 is induced concurrently with inducible nitric oxide synthase (iNOS) and S-nitrosylated in proinflammatory peritoneal macrophages. Chemical inhibition or genetic ablation of iNOS inhibits S-nitrosylation and nuclear translocation of CLIC4. In macrophages, iNOS-induced nuclear CLIC4 coincides with the pro- to anti-inflammatory transition of the cells because IL-1β and CXCL1 mRNA remain elevated in CLIC4 and iNOS knockout macrophages at late time points, whereas TNFα mRNA is elevated only in the iNOS knockout macrophages. Active IL-1β remains elevated in CLIC4 knockout macrophages and in macrophages in which CLIC4 nuclear translocation is prevented by the NOS inhibitor l-NAME. Moreover, overexpression of nuclear-targeted CLIC4 down-regulates IL-1β in stimulated macrophages. In mice, genetically null for CLIC4, the number of phagocytosing macrophages stimulated by LPS is reduced. Thus, iNOS-induced nuclear CLIC4 is an essential part of the macrophage deactivation program.

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca(2+)-induced differentiation, stress-induced apoptosis, and modulating TGF-beta signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin alpha and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor alpha-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor alpha-induced nitrosylation and association with importin alpha and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.

CLIC4 is a highly conserved, multifunctional member of the chloride intracellular channel family of proteins. The protein is largely cytoplasmic but translocates to the nucleus upon a variety of stimuli including TGF-beta, TNF-alpha and etoposide. Nuclear resident CLIC4 causes growth arrest, terminal differentiation and apoptosis. Recently, it was discovered that TGF-beta causes CLIC4 to associate with Schnurri-2 and together they translocate to the nucleus and dissociate thereafter. The nuclear function of CLIC4 was further illuminated by the discovery that CLIC4 enhances TGF-beta signaling by associating with phospho-Smad2 and 3 and preventing their dephosphorylation. Enhanced TGF-beta dependent gene expression and growth inhibition are downstream consequences of this activity of CLIC4. In this article, we speculate on other consequences of the CLIC4 relation to TGF-beta signaling and the potential for CLIC4 to participate in other cellular functions related to normal homeostasis and disease.

CLIC4 (chloride intracellular channel 4), a multifunctional protein that traffics between the cytoplasm and nucleus, interacts with Schnurri-2, a transcription factor in the bone morphogenetic protein (BMP) signalling pathway. Here we show that transforming growth factor beta (TGF-beta) promotes the expression of CLIC4 and Schnurri-2 as well as their association in the cytoplasm and their translocation to the nucleus. In the absence of CLIC4 or Schnurri-2, TGF-beta signalling is abrogated. Direct nuclear targeting of CLIC4 enhances TGF-beta signalling and removes the requirement for Schnurri-2. Nuclear CLIC4 associates with phospho (p)-Smad2 and p-Smad3, protecting them from dephosphorylation by nuclear phosphatases. An intact TGF-beta signalling pathway is essential for CLIC4-mediated growth-arrest. These results newly identify Schnurri-2 and CLIC4 as modifiers of TGF-beta signalling through their stabilization of p-Smad2 and 3 in the nucleus.

Chloride intracellular channel 4 (CLIC4) is a putative chloride channel for intracellular organelles. CLIC4 has biological activities in addition to or because of its channel activity. In keratinocytes, CLIC4 resides in the mitochondria and cytoplasm, and CLIC4 gene expression is regulated by p53, TNF-alpha, and c-Myc. Cytoplasmic CLIC4 translocates to the nucleus in response to cellular stress conditions including DNA damage, metabolic inhibition, senescence, and exposure to certain trophic factors such as TNF-alpha and LPS. Nuclear translocation is associated with growth arrest or apoptosis, depending on the level of expression. In the nucleus CLIC4 interacts with several nuclear proteins as demonstrated by yeast two-hybrid screening and co-immunoprecipitation. Nuclear CLIC4 appears to act on the TGF-beta pathway, and TGF-beta also causes CLIC4 nuclear translocation. In human and mouse cancer cell lines, CLIC4 levels are reduced, and CLIC4 is excluded from the nucleus. CLIC4 soluble or membrane-inserted status is dependent on redox state, and redox alterations in cancer cells could underly the defect in nuclear translocation. CLIC4 is reduced and excluded from the nucleus of many human epithelial neoplasms. Paradoxically, CLIC4 is reciprocally upregulated in tumor stroma in conjunction with the expression of alpha-smooth muscle actin in the fibroblast to myofibroblast transition. Overexpression of CLIC4 in cancer cells inhibits tumor growth in vivo. Conversely, overexpression of CLIC4 in tumor stromal cells stimulates tumor growth in vivo. Thus, CLIC4 participates in normal and pathological processes and may serve as a useful target for therapies in disturbances of homeostasis and neoplastic transformation.

Keratinocyte differentiation requires integrating signaling among intracellular ionic changes, kinase cascades, sequential gene expression, cell cycle arrest, and programmed cell death. We now show that Cl(-) intracellular channel 4 (CLIC4) expression is increased in both mouse and human keratinocytes undergoing differentiation induced by Ca(2+), serum and the protein kinase C (PKC)-activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Elevation of CLIC4 is associated with signaling by PKCdelta, and knockdown of CLIC4 protein by antisense or shRNA prevents Ca(2+)-induced keratin 1, keratin 10 and filaggrin expression and cell cycle arrest in differentiating keratinocytes. CLIC4 is cytoplasmic in actively proliferating keratinocytes in vitro, but the cytoplasmic CLIC4 translocates to the nucleus in keratinocytes undergoing growth arrest by differentiation, senescence or transforming growth factor beta (TGFbeta) treatment. Targeting CLIC4 to the nucleus of keratinocytes via adenoviral transduction increases nuclear Cl(-) content and enhances expression of differentiation markers in the absence of elevated Ca(2+). In vivo, CLIC4 is localized to the epidermis in mouse and human skin, where it is predominantly nuclear in quiescent cells. These results suggest that CLIC4 participates in epidermal homeostasis through both alterations in the level of expression and subcellular localization. Nuclear CLIC4, possibly by altering the Cl(-) and pH of the nucleus, contributes to cell cycle arrest and the specific gene expression program associated with keratinocyte terminal differentiation.

Purpose: CLIC4, a member of a family of intracellular chloride channels, is regulated by p53, c-Myc, and tumor necrosis factor-alpha. Regulation by factors involved in cancer pathogenesis, together with the previously shown proapoptotic activity of CLIC4, suggests that the protein may have a tumor suppressor function. To address this possibility, we characterized the expression profile, subcellular localization, and gene integrity of CLIC4 in human cancers and determined the functional consequences of CLIC4 expression in tumor epithelium and stromal cells., Experimental Design: CLIC4 expression profiles were analyzed by genomics, proteomics, bioinformatics, and tissue microarrays. CLIC4 expression, as a consequence of crosstalk between stroma and epithelium, was tested in vitro by coculture of breast epithelial tumor cells and normal fibroblasts, and the functional consequences of CLIC4 expression was tested in vivo in xenografts of human breast tumor cell lines reconstituted with CLIC4 or mixed with fibroblasts that overexpress CLIC4 transgenically., Results: In cDNA arrays of matched human normal and tumor tissues, CLIC4 expression was reduced in renal, ovarian, and breast cancers. However, CLIC4 protein levels were variable in tumor lysate arrays. Transcript sequences of CLIC4 from the human expressed sequence tag database and manual sequencing of cDNA from 60 human cancer cell lines (NCI60) failed to reveal deletion or mutations in the CLIC4 gene. On matched tissue arrays, CLIC4 was predominantly nuclear in normal human epithelial tissues but not cancers. With advancing malignant progression, CLIC4 staining became undetectable in tumor cells, but expression increased in stromal cells coincident with up-regulation of alpha-smooth muscle actin, suggesting that CLIC4 is up-regulated in myofibroblasts. Coculture of cancer cells and fibroblasts induced the expression of both CLIC4 and alpha-smooth muscle actin in fibroblasts adjacent to tumor nests. Introduction of CLIC4 or nuclear targeted CLIC4 via adenovirus into human breast cancer xenografts inhibited tumor growth, whereas overexpression of CLIC4 in stromal cells of xenografts enhanced tumor growth., Conclusion: Loss of CLIC4 in tumor cells and gain in tumor stroma is common to many human cancers and marks malignant progression. Up-regulation of CLIC4 in tumor stroma is coincident with myofibroblast conversion, generally a poor prognostic indicator. Reactivation and restoration of CLIC4 in tumor cells or the converse in tumor stromal cells could provide a novel approach to inhibit tumor growth.

Myc is a key regulatory protein in higher eukaryotes controlling important cellular functions such as proliferation, differentiation, and apoptosis. Myc is profoundly involved in the genesis of many human and animal cancers, and the abrogation of Myc-induced apoptosis is a critical event in cancer progression. Because the mechanisms that mediate Myc-induced apoptosis are largely unknown, we analyzed protein expression during Myc-induced apoptosis using an isotope-coded affinity tag quantitative proteomics approach and identified that a proapoptotic mitochondrial chloride ion channel, mtCLIC/CLIC4, is induced by Myc. Myc binds to the mtCLIC gene promoter and activates its transcription. Suppression of mtCLIC expression by RNA interference inhibited Myc-induced apoptosis in response to different stress conditions and abolished the cooperative induction of apoptosis by Myc and Bax. We also found that Myc reduces the expression of Bcl-2 and Bcl-xL and that the apoptosis-inducing stimuli up-regulate Bax expression. These results suggest that up-regulation of mtCLIC, together with a reduction in Bcl-2 and Bcl-xL, sensitizes Myc-expressing cells to the proapoptotic action of Bax.

Chloride intracellular channel (CLIC)4 is a p53- and tumor necrosis factor alpha (TNFalpha)-regulated chloride channel protein that is localized to the mitochondria and cytoplasm of mouse and human keratinocytes. CLIC4 protein increases in differentiating keratinocytes and in keratinocytes exposed to DNA-damaging agents and metabolic inhibitors. Increasing CLIC4 levels by transduction of recombinant CLIC4 causes apoptosis. CLIC4 translocates to the nucleus under a variety of conditions of cell stress, and nuclear CLIC4 is associated with cell cycle arrest and accelerated apoptosis. Reduction of CLIC4 and several other CLIC family members by expressing a doxycycline-regulated CLIC4 antisense also causes apoptosis in squamous cancer cell lines. Expressing antisense CLIC4 in tumors derived from transplanting these cells into nude mice inhibits tumor growth, increases tumor apoptosis, and reduces tumor cell proliferation. Co-administration of TNFalpha intraperitoneally enhances the tumor-inhibitory influence of CLIC4 antisense expression. Together, these results suggest that CLIC4 is important for keratinocyte viability and may be a novel target for anti-cancer therapy.

mtCLIC/CLIC4 is a p53 and tumor necrosis factor alpha (TNFalpha) regulated intracellular chloride channel protein that localizes to cytoplasm and organelles and induces apoptosis when overexpressed in several cell types of mouse and human origin. CLIC4 is elevated during TNFalpha-induced apoptosis in human osteosarcoma cell lines. In contrast, inhibition of NFkappaB results in an increase in TNFalpha-mediated apoptosis with a decrease in CLIC4 protein levels. Cell lines expressing an inducible CLIC4-antisense construct that also reduces the expression of several other chloride intracellular channel (CLIC) family proteins were established in the human osteosarcoma lines SaOS and U2OS cells and a malignant derivative of the mouse squamous papilloma line SP1. Reduction of CLIC family proteins by antisense expression caused apoptosis in these cells. Moreover, CLIC4-antisense induction increased TNFalpha-mediated apoptosis in both the SaOS and U2OS derivative cell lines without altering TNFalpha-induced NFkappaB activity. Reducing CLIC proteins in tumor grafts of SP1 cells expressing a tetracycline-regulated CLIC4-antisense substantially inhibited tumor growth and induced tumor apoptosis. Administration of TNFalpha i.p. modestly enhanced the antitumor effect of CLIC reduction in vivo. These results suggest that CLIC proteins could serve as drug targets for cancer therapy, and reduction of CLIC proteins could enhance the activity of other anticancer drugs.

The passage of ions to form and maintain electrochemical gradients is a key element for regulating cellular activities and is dependent on specific channel proteins or complexes. Certain ion channels have been the targets of pharmaceuticals that have had impact on a variety of cardiovascular and neurological diseases. Chloride channels regulate the movement of a major cellular anion, and in so doing they in part determine cell membrane potential, modify transepithelial transport, and maintain intracellular pH and cell volume. There are multiple families of chloride channel proteins, and respiratory, neuromuscular, and renal dysfunction may result from mutations in specific family members. Interest in chloride channels related to cancer first arose when the multidrug resistance protein (MDR/P-glycoprotein) was linked to volume-activated chloride channel activity in cancer cells from patients undergoing chemotherapy. More recently, CLC, CLIC, and CLCA intracellular chloride channels have been recognized for their contributions in modifying cell cycle, apoptosis, cell adhesion, and cell motility. Moreover, advances in structural biology and high-throughput screening provide a platform to identify chemical compounds that modulate the activities of intracellular chloride channels thereby influencing chloride ion transport and altering cell behavior. This review will focus on several chloride channel families that may contribute to the cancer phenotype and suggest how they may serve as novel targets for primary cancer therapy.

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.

mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.

Keratinocyte differentiation requires integrating signaling among intracellular ionic changes, kinase cascades, sequential gene expression, cell cycle arrest, and programmed cell death. We now show that Cl¯ intracellular channel 4 (CLIC4) expression is increased in both mouse and human keratinocytes undergoing differentiation induced by Ca2+, serum and the protein kinase C (PKC)-activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Elevation of CLIC4 is associated with signaling by PKCδ, and knockdown of CLIC4 protein by antisense or shRNA prevents Ca2+-induced keratin 1, keratin 10 and filaggrin expression and cell cycle arrest in differentiating keratinocytes. CLIC4 is cytoplasmic in actively proliferating keratinocytes in vitro, but the cytoplasmic CLIC4 translocates to the nucleus in keratinocytes undergoing growth arrest by differentiation, senescence or transforming growth factor β (TGFβ) treatment. Targeting CLIC4 to the nucleus of keratinocytes via adenoviral transduction increases nuclear Cl¯ content and enhances expression of differentiation markers in the absence of elevated Ca2+. In vivo, CLIC4 is localized to the epidermis in mouse and human skin, where it is predominantly nuclear in quiescent cells. These results suggest that CLIC4 participates in epidermal homeostasis through both alterations in the level of expression and subcellular localization. Nuclear CLIC4, possibly by altering the Cl¯and pH of the nucleus, contributes to cell cycle arrest and the specific gene expression program associated with keratinocyte terminal differentiation. [ABSTRACT FROM AUTHOR]