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. 2012 Mar 1;33(5):986–995. doi: 10.1093/carcin/bgs115

CLIC4 is a tumor suppressor for cutaneous squamous cell cancer

KStephen Suh 1,†, Mariam Malik 1,†, Anjali Shukla 1,†, Andrew Ryscavage 1, Lisa Wright 1, Kasey Jividen 1, John M Crutchley 1, Rebecca A Dumont 1, Ester Fernandez-Salas 2, Joshua D Webster 1, RMark Simpson 1, Stuart H Yuspa 1,*
PMCID: PMC3334517  PMID: 22387366

Abstract

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.

Introduction

Mammalian CLICs (chloride intracellular channels) comprise a family of six genes that are associated with intracellular anion channel activity with Cl- selectivity (1). CLICs are metamorphic proteins, transitioning between soluble and membrane associated states at least in part dependent on cellular redox (2,3). CLICs are structurally unrelated to canonical transmembrane ion channels and are more properly placed in the glutathione-S-transferase superfamily (4,5) consistent with sensitivity to redox changes.

Soluble CLIC proteins are primarily in the cytoplasm (6). CLIC proteins have multiple protein–protein interaction domains and phosphorylation sites as well as lipid modification sites and are reported to participate in a variety of specialized functions (7–9). CLIC1, CLIC3 and CLIC4 are expressed early in embryonic stem cells as a direct target gene of NANOG (SOX2 and NANOG for CLIC1; E2F4 and NANOG for CLIC4) (10). CLIC1, CLIC4 and CLIC5 are expressed in spermatozoa and bind to protein phosphatase 1 (11). CLIC3 interacts with extracellular signal-regulated kinase-7 in the nucleus of mammalian cells (12). Other CLICs participate in cell cycle progression, microglial phagocytosis of amyloid protein and cardiac muscle function among other activities (13–15). CLIC proteins are highly conserved through both vertebrates and invertebrates suggesting they have essential functions in morphogenesis and viability (16,17).

Among the CLIC proteins, the biology of CLIC4 has been studied most extensively. CLIC4 is abundant in the cytoplasm but has also been detected in mitochondrial and nuclear membranes and the endoplasmic reticulum (18–20). CLIC4 is a direct downstream target gene for p53 and c-Myc and is required for p53- and c-Myc-mediated apoptosis in several cell types (21,22). CLIC4 also contributes to tumor necrosis factor-α-mediated apoptosis independent of nuclear factor-kappaB (13). CLIC4 binds to components of the cytoskeleton (β-actin, ezrin and α-tubulin), chaperone proteins (AKAP350 and 14-3-3) and nuclear transporters (Ran, NFT2 and Importin-α) (19,23,24). CLIC4 is required for blood vessel lumen formation as endothelial cells undergo vascular tubulogenesis in vitro and in vivo (25,26) and participates in the maturation of keratinocytes and differentiation of adipocytes (27,28). A common property of cytoplasmic CLIC4 is its propensity to translocate to the nucleus under conditions of metabolic cell stress, growth inhibition or apoptosis. In fact, targeting CLIC4 to the nucleus of multiple cell types can cause growth arrest or apoptosis depending on the level of expression. Recent data indicate that nuclear translocation of CLIC4 under a variety of cell stress stimuli is mediated by NO-induced S-nitrosylation on critical cysteine residues that alter the redox-sensitive tertiary structure of CLIC4 increasing its association with nuclear import proteins (29). The association of nuclear CLIC4 and growth suppression in vitro parallels the presence of predominantly nuclear CLIC4 in growth arrested and differentiating cells of epithelial tissues in vivo (6,27). Nuclear CLIC4 enhances transforming growth factor-β (TGF-β) signaling by preventing the dephosphorylation of phospho-Smad 2/3, thus providing a pathway through which growth arrest and perhaps other cellular changes induced by CLIC4 may be mediated (30).

In human cancer tissues, CLIC4 protein is excluded from the nucleus of tumor cells and expression is reduced in tumor epithelial tissue (6). The extent of CLIC4 reduction in the tumor epithelium directly correlates with tumor progression (6). To model the participation of CLIC4 in cancer pathogenesis, we have evaluated changes in CLIC4 in the well-established skin carcinogenesis model in mice and extended the analysis to human skin cancer cell lines in vitro and cancer tissue in vivo. The results indicate that marked changes in expression and subcellular localization of CLIC4 occur early in tumorigenesis. By altering these changes experimentally, we show that CLIC4 is an important suppressor of squamous tumor development and progression and a mediator of TGF-β responsiveness in cancer cells.

Materials and methods

Cell culture

Mouse keratinocytes and dermal fibroblasts were isolated from newborn Balb/c and Sencar mice and cultured as described previously (31) in Dulbecco's modified Eagle's medium containing 8% chelex-treated fetal bovine serum and supplemented with 0.05 mM calcium for keratinocytes and 1.4 mM calcium for fibroblasts. The mouse non-tumorigenic keratinocyte cell line S1, papilloma cell lines SP1 and 308 and Pam212 carcinoma cell lines (all derived in this laboratory) were maintained in the same medium as primary keratinocytes. Human foreskin-primary keratinocytes were maintained in keratinocyte serum-free medium (Epilife; Cascade) as described by the manufacturer. Human non-tumorigenic keratinocyte cell line HaCaT, papilloma cell line KC16-1 and squamous cell carcinoma (SCC) cell line SCC-13 (a kind gift from Dr James Rheinwald) were cultured in Dulbecco's modified Eagle's medium (Bio-Whittaker) containing 8% fetal bovine serum and 20 U/ml penicillin–streptomycin (both from Invitrogen). Genetically engineered SP1 cells with one flippase recognition target (FRT) site (SP1/F), SP1-FRT with the rTA transgene (SP1/F Tet-On) and SP1/F-Tet-On containing the Dox-inducible hemagglutinin epitope (HA)-CLIC4 transgene (SP1/F Tet-On CLIC4) (see below) were maintained in 0.05 mM Ca2+ medium. Keratinocytes isolated from the cytomegalovirus promoter-driven HA-CLIC4 transgenic FVB/N mice (Suh,K.S. and Yuspa,S.H, unpublished results) were maintained in 0.05 mM calcium medium and transduced with a replication-deficient v-rasHa retrovirus as described previously. Adenovirus encoding human p53 and vectors targeting CLIC4 to subcellular compartments had been reported previously (19).

Skin tumor induction studies and quantitative PCR analysis

Seven-week-old Sencar male mice or epidermal targeted K5-rTA-CLIC4-His-tagged double transgenic mice of mixed gender (doxycycline-inducible epidermal CLIC4) were initiated with 7, 12, dimethylbenz[a]anthracene and promoted with 12-O-tetradecanoyl-phorbol-13-acetate as described previously. Tumors were harvested at various times up to 52 weeks. Protein lysates were prepared from excised tumors as described previously. Studies on CLIC4 transcripts and phenotypic markers from squamous papillomas at high and low risk for progression to SCCs used complementary DNA isolated and verified in a previous study (32). For real-time PCR analysis, a Bio-Rad iQ iCycler and Bio-Rad iQ5-Standard Edition Version 2.0 from Bio-Rad were used to measure and analyze transcript expression levels. Complementary DNA (diluted 1:50 in the final volume reaction) was measured using iQ SYBR Green Supermix (Bio-Rad) and included experimental duplicate reactions. The primer sequences were as follows: mouse and human glyceraldehyde 3-phosphate dehydrogenase (F) tcc acc acc ctg ttg ctg ta, (R) acc aca gtc cat gcc atc ac and human CLIC4 (F) ttc ccc ttc att taa aca cct tt (R) tgc tat cta cat gca act ctg ga. Primers for mouse CLIC4, keratin 1 (K1) and keratin 13 (K13) were obtained from SABiosciences and p53 from Invitrogen. Relative standard curves were generated from log input (serial dilutions of pooled complementary DNA) versus the threshold cycle (Ct). The linear correlation coefficient (r2) was 0.99 for all three primer sets. The slope of the standard curve was used to determine the efficiency of target amplification (E) using the equation E = (10−1/slope−1) × 100. Similar high efficiency was obtained for all primers allowing for the comparative Ct method to be used. Relative quantitation was used to calculate the 2-(ΔΔCt) formula, where ΔΔCt represents the difference corrected for glyceraldehyde 3-phosphate dehydrogenase used as internal control. Electrophoresis analysis of amplified product from real-time PCR showed a single band.

Immunoblotting, thioredoxin reductase and growth assays

Cultured keratinocytes and cell lines were lysed in Cell Lysis Buffer (Cell Signaling) supplemented with Complete Mini tablets (Roche). Proteins were quantified by the Bradford method (Bio-Rad) and separated by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Monospecific polyclonal anti-N-terminus CLIC4 antibodies were used for immunoblots as described previously (18). Thioredoxin and thioredoxin reductase (TR1) antibodies were purchased from Abcam and used at a 1:1000 dilution. Whole-cell lysates were prepared using M-Per (Pierce) and TR1 activity was measured using a thioredoxin reductase assay kit from Cayman chemicals, Ann Arbor, MI, following manufacturer’s instructions. Briefly, reduction of 5,5′-dithio-bis(2-dinitrobenzoic acid) in presence of reduced nicotinamide adenine dinucleotide phosphate was measured in sample lysates from triplicate wells and TR-specific reductase activity was plotted after correcting for reductase activity in the presence of aurothiomalate, a TR-specific inhibitor. Biotin switch assays to quantify protein S-nitrosylation were performed as reported previously (29). Antibodies against p53 (clone DO-1; Abcam), β-actin antibody (MAB150; Chemicon), H-RAS (sc-520; Santa Cruz) and Cyclin-D1 (sc-246; Santa Cruz) were used at dilutions recommended by the manufacturer. Enhanced chemiluminescence SuperSignal (Pierce) system was used for detection. Thymidine incorporation, subcellular fractionation and TGF-β-dependent reporter assays were performed as described previously (30). For in vitro analyses of SCC-13 cell growth, cells were seeded in 24-well dishes at a concentration of 1.0 × 105 cells per well. After one day in culture, SCC-13 cells were infected in triplicate at 5 and 10 moi using null, CLIC4 and nuclear-targeted CLIC4 adenoviruses for 24 and 48 h as described previously (19). Cell growth was determined by 3-(4,5-dimethylthiazole-2-yl)-2,5-bdiphenyl tetrazolium bromide assay using a cell proliferation kit, the CellTiter 96 (Promega), as described by the manufacturer.

Generation of SP1 cells stably expressing an HA-CLIC4 transgene

Construction of CLIC4 plasmids was described elsewhere (19). HA-tagged CLIC4 (HA-CLIC4) was generated by PCR amplifying the full length mouse CLIC4 open reading frame using 5′-forward primer containing HA-tag (5′ cca tac gat gtt cca gat tac gct gcg ctg tcg atg ccc ctg aac ggg ctg aag gag gag gac aaa gag ccc ctc atc gag 3′; polyacrylamide gel electrophoresis purified; first round and 5′ aga tat gcg gcc gcc atg tac cca tac gat gtt cca gat tac gct gcg ctg tcg atg ccc ctg aac ggg ctg aag g 3′; polyacrylamide gel electrophoresis purified; second round) and 3′-reverse primer (gcg gcc gcc tac ctt ggt aag tct ctt ggc gac atc gct gta cgc gat ttc cac ctc ctt gtc gct ggg 3′; first and second round; polyacrylamide gel electrophoresis purified). The amplified DNA fragment was gel purified and ligated to pcDNA5/FRT/TO-TOPO (Invitrogen). Insert in-frame fusion integrity, orientation and expression were confirmed by DNA sequencing, restriction analysis, immunoblots and confocal microscopy (data not shown), respectively.

To generate SP1 cells that express and maintain exogenous HA-CLIC4 upon doxycycline (Dox) induction from a single FRT insertion site, SP1/F-Tet-On cells were transfected with the linearized plasmid encoding HA-CLIC4 under the regulation of the rTA promoter and selected with Blasticidin (2 μg/ml) and Hygromycin (2.5 μg/ml) as suggested by the manufacturer (Invitrogen). Antibiotic-resistant colonies were picked by Disk-Ring (Sigma), subcultured and analyzed for Dox-inducibility and overexpression of HA-CLIC4 by anti-CLIC4 and anti-HA immunoblotting and nuclear translocation confirmed upon treatment with apoptosis/stress inducers. Exogenous HA-CLIC4 (containing tTA promoter from Invitrogen) proteins were also induced by using Tet-Off adenovirus (10 moi; Clontech) without Dox induction.

Immunohistochemistry, immunofluorescence and tissue array staining

Human skin cancer tissue arrays were obtained from US Biomax and were used for CLIC4 expression and localization studies. Immunohistochemical staining (1:500 dilution; 5 mg/ml) used the N-terminus CLIC4 antibody (18) and procedures described by the array manufacturers. Mouse skin tumors were mounted on single slides. Anti-HA (Abcam) was used at a dilution of 1:800 and anti-phospho-Smad 2 (Cell Signaling) was used at a dilution of 1:100 on formalin-fixed sections. Citra Antigen Retrieval (Vector Labs) was performed in a microwave for 10 min. The stained slides were analyzed with bright field microscopy using Leitz-DMRB (Leica) and OpenLab (Improvision) software. Immunofluorescence staining (1:1000 dilutions of 5 mg/ml CLIC4 antibody) of cultured cells was performed as described previously (6).

Orthograft, xenograft and tumor development

Techniques for transducing primary keratinocytes with v-rasHa retrovirus, grafting tumor cell lines orthotopically to the back of nude mice and measuring tumor size were described previously (13,31). Mice were fed with doxycycline-containing feed (200 μg/g wt/wt; Bioserve) for cells modified for Tet-On induction and standard mouse feed (Purina) for Tet-Off induction. For grafting, 2 × 106 cells of v-rasHa-transformed newborn HA-CLIC4 transgenic mouse keratinocytes (constitutive expression of CLIC4) and 5 × 105 cells of SP1/F Tet-On CLIC4 (Dox-inducible CLIC4 cell line) were used per graft per mouse, respectively. For SCC-13 graft experiments, 2 × 106 SCC-13 cells were subcutaneously injected per graft per mouse. To consistently generate subcutaneous tumors in the nude mice, highly tumorigenic SCC-13 cells were selected by isolating the cells from a well-established subcutaneous tumor, followed by culture expansion and reintroduction into the subcutaneous environment. This in vivo passage was repeated for two cycles. Ten mice were used for each group (N = 10) in all grafting studies, and each grafting study was performed at least twice. Statistical significance of tumor sizes between the groups was analyzed using Two-Way analysis of variance with Bonferroni post-test comparison (SigmaStat). For expression of nuclear CLIC4, adenovirus equivalent to 5 × 108 particles in 100 μl of viral solution (Ad titer 1 × 1010 infectious Ad/ml in phosphate-buffered saline) was injected weekly directly into grafted tumors at three different sites.

Results

CLIC4 expression is reduced in mouse and human skin cancer cell lines and in chemically induced mouse skin tumors

To investigate the expression of CLIC4, protein samples from multiple mouse and human skin tumor cell lines were probed with mono-specific N-terminal CLIC4 antibody (Figure 1A). CLIC4 protein is decreased in mouse and human immortalized and skin tumor cell lines relative to primary keratinocytes derived from Balb/c and Sencar mouse strains or human foreskin. In the lines studied, CLIC4 downregulation is generally associated with the reduction of p53 expression (or mutational inactivation in the case of HaCaT cells). By quantitative PCR, CLIC4 transcript expression correlated with the protein level suggesting the silencing of CLIC4 in skin tumor cells is transcriptionally regulated (Figure 1B). CLIC4 levels also decrease as chemically induced skin tumors progress from benign papillomas to SCCs, indicating CLIC4 expression is tumor-stage dependent in this model (Figure 1C). Furthermore, CLIC4 transcripts tend to decrease with premalignant progression as detected by quantitative PCR analysis of individual papillomas at low risk (K1 high and K13 low) and high risk (K1 low and K13 high) for malignant conversion although the CLIC4 differences do not reach statistical significance (Figure 1D) (32). The reduction in CLIC4 levels in skin tumors is associated with diminished expression of p53 transcripts in these tissues (Supplementary Figure 1A is available at Carcinogenesis Online) although exogenous expression of p53 via adenoviral vector failed to upregulate CLIC4 protein expression in tumor cell lines (Supplementary Figure 1B is available at Carcinogenesis Online). Examination of mouse skin and skin tumors in situ by immunohistochemistry (Figure 1E) reveals that CLIC4 is largely nuclear in normal skin, whereas CLIC4 is lost from the nuclear compartment in cancers. Quantitative analysis of human skin cancer tissue microarrays confirms that nuclear localization of CLIC4 and overall CLIC4 expression is reduced in many primary (SCC and basal cell carcinoma) and some secondary epithelial tumors of the skin (Supplementary Figure 2 is available at Carcinogenesis Online). Simply elevating the proliferation rate in normal keratinocytes by stimulating with keratinocyte growth factor does not alter CLIC4 expression (Supplementary Figure 3 is available at Carcinogenesis Online).

Fig. 1.

Fig. 1.

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CLIC4 expression is reduced in mouse and human tumor cell lines and mouse skin tumors. (A) Immunoblots for CLIC4 and p53 were performed with protein samples from primary mouse keratinocytes (P) from Balb/c and Sencar strains and cell lines representing non-tumorigenic immortalized (S1), papilloma (308 and SP-1) and carcinoma (Pam212) or human foreskin keratinocytes (P) and cell lines representing non-tumorigenic immortalized (HaCaT), papilloma (KC16-1) and SCC-13. Ponceau-Red stain is used as a loading control. (B) The same cell lines were used to determine CLIC4 transcript levels using quantitative PCR. (C) Protein samples generated from 7, 12, dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoyl-phorbol-13-acetate-promoted skin tumors or adjacent normal skin were probed for CLIC4 by immunoblot. Each lane represents an individual normal skin or tumor sample. Actin is used as the loading control. (D) Quantitative PCR analysis of CLIC4, keratin 1 or keratin 13 transcript levels from four independent samples (each bar) of normal mouse skin or skin papillomas generated to be at low risk (K1 high and K13 low) or high risk for malignant conversion (K1 low and K13 high). Each sample is run in duplicate represented by error bars. (E) Immunostaining for CLIC4 is detected in sample tissue sections of normal mouse skin (A), papilloma (B) and carcinoma (C).

Translocation of CLIC4 to the nucleus is abrogated by neoplastic transformation of keratinocytes

Oncogenic transformation of primary mouse keratinocytes by v-rasHa abrogates CLIC4 nuclear translocation induced in normal primary keratinocytes by senescence (Figure 2A), tumor necrosis factor-α or etoposide-induced stress (Figure 2B). This change persists for at least 32 h after treatment (data not shown). This suggests that changes in CLIC4 trafficking occur early in transformation. CLIC4 nuclear exclusion is also seen in etoposide-challenged mouse (Pam212) and human (SCC-13) cutaneous SCC cell lines (Figures 2C and D). Nevertheless, the potential for growth inhibition in response to nuclear CLIC4 persists in tumor cells, since elevating CLIC4 by infection with adenovirus-expressing wild-type (Cyt-CLIC4) or nuclear-targeted CLIC4 (Nuc-CLIC4) (Figure 2E, left panel) retarded the growth of SCC-13 cells (Figure 2E, right panel) when compared with a null adenovirus. Nuclear-targeted CLIC4 was more effective. The localization of the targeted CLIC4 in SCC13 cells is shown in Figure 2F. These results suggest that given proper expression and subcellular residence, CLIC4 could have antitumor activity.

Fig. 2.

Fig. 2.

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Neoplastic keratinocytes exclude CLIC4 from the nucleus. (A) Cultured primary mouse keratinocytes were transduced with v-rasHa (+) and maintained in culture for 6 days to allow senescence (A) or treated with tumor necrosis factor-α (TNF-α) (25 ng/ml) or etoposide (100 μM) for the indicated times (B). CLIC4 (green) subcellular localization was monitored by immunofluorescence and confocal microscopy. (C) Primary mouse and (D) primary human keratinocytes or SCC cells (PAM212 or SCC-13) were treated with etoposide for the indicated times and monitored by immunofluorescence for CLIC4 (red) nuclear translocation. Hoechst stain (blue) was used as the nuclear marker in all immunostaining. (E) SCC-13 cells (Parental) were transduced with empty vector (N) or native CLIC4 (Cyt) or nuclear-targeted CLIC4 (Nuc) adenoviruses. Left panel is an immunoblot of the exogenous proteins. The right panel measures growth response at two time-points in triplicate using the CellTiter 96 proliferation assay kit. *P = 0.015 between Null and Cyt-CLIC4 and **P = 0.0008 between Null and Nuc-CLIC4 at 48 h of Ad virus treatment (T-test). (F) Localization of CLIC4 in SCC13 cells in response to each targeting vector detected by subcellular fractionation and immunoblotting for CLIC4. Cells were treated for 22 h with 5 moi for each vector. N = null adeno vector.

Reconstitution of nuclear CLIC4 in tumor cells enhances TGF-β signaling

Diminished responsiveness to TGF-β signaling is commonly encountered in epithelial tumor cells (33). Since CLIC4 is an integral component of TGF-β signaling, we asked if reduced levels of CLIC4 in tumor cells could contribute to TGF-β resistance. To this end, we reconstituted tumor cell lines with CLIC4 or nuclear CLIC4 by adenoviral transduction and assayed TGF-β-dependent transcriptional activity using a reporter assay (Figure 3A). Nuclear CLIC4 stimulated TGF-β-dependent transcriptional activity in the absence of exogenous recombinant TGF-β in all the three mouse lines, and in the presence of nuclear CLIC4, transcriptional activity in response to exogenous TGF-β was the highest in all lines. SCC-13 cells were less responsive to all stimuli although nuclear CLIC4 tended to increase transcriptional activity above other treatments. Similarly, nuclear CLIC4 alone or together with TGF-β caused growth inhibition in all cell lines that were resistant to growth inhibition by recombinant TGF-β in the absence of exogenous CLIC4 (Figure 3B).

Fig. 3.

Fig. 3.

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Nuclear CLIC4 enhances TGF-β signaling in tumor cells. Non-tumorigenic mouse (S1) and tumorigenic mouse (308 and Pam212) and human (SCC-13) cell lines were used. (A) Cells were transfected with the TGF-β-inducible luciferase reporter plasmid p3TP-lux and transfection control plasmid pRLTK for 24 h and then transduced with vector (null) and native CLIC4 or nuclear-targeted CLIC4 (Nuc-CLIC4) adenoviral vectors and treated where indicated with 100 pg/ml of TGF-β1 for 16 h. p3TP luciferase activity was normalized to pRLTK activity and data presented as fold change relative to empty vector without TGF-β1 treatment. (B) CLIC4-transduced cells were treated where indicated with 100 pg/ml of TGF-β1 for 24 h and pulsed with H3-thymidine for 2 h, lysed and counted. Data are presented as (A) fold change or (B) percentage relative to empty vector (null) without TGF-β1 treatment. (A) and (B) represent results of triplicate determinations, and each experiment was repeated three times with similar results. Statistical analysis in (A) and (B) is shown relative to respective empty vector with or without TGF-β1 treatment. *P < 0.05, **P < 0.005.

Redox status determines nitrosylation and subcellular localization of CLIC4 in tumor cells

Our previous studies, as well as the work of others, have revealed the critical role of redox status in the regulation of CLIC4 conformation and nuclear translocation (3,29). We wondered whether altered redox status of tumor cells could influence the subcellular localization of CLIC4. In general, cancer cells produce high levels of reactive oxygen species and are in a potential state of oxidative stress. To compensate, the antioxidant defense systems are highly engaged in cancer cells with particular elevations of glutathione levels that facilitate evasion from reactive oxygen species-induced senescence (34). Antioxidant defenses are regulated centrally through Nrf-2 and other transcription factors and peripherally through the thioredoxin (Trx)–Tr-1 pathway. These key antioxidant molecules are generally superelevated in cancer cells and are considered targets for therapy (35,36). Tr-1 protein levels are elevated in the human SCC cell lines studied relative to normal keratinocytes (Figure 4A, lower panel) but not consistently in mouse tumor cells (Figure 4A, upper panel). Nevertheless, Tr-1 enzymatic activity is higher and decay of enzymatic activity is reduced in tumor cell lysates from both species (Figure 4B). Auranofin is an inhibitor of Tr-1 and has been shown to have antitumor activity in vitro and in vivo (37). Auranofin treatment enhances S-nitrosylation of exogenous HA-tagged CLIC4 (SNO-HA) in v-rasHa-transformed primary keratinocytes but not normal primary keratinocytes (Mock) as determined by biotin switch assay that quantifies protein S-nitrosylation (Figure 4C). Auranofin also facilitates nuclear translocation of CLIC4 in response to NO-generating agents, S-nitrosoglutathione and diethylenetriamine NONOate (DETA/NO), in PAM212 cancer cells (Figure 4 D). Together these results suggest that the excessively antioxidative state of cancer cells may inhibit the CLIC4 modifications required for nuclear translocation. Alteration of this redox environment to a more oxidative state by agents such as Auranofin together with increasing expression of CLIC4 could allow CLIC4 to manifest an antitumor activity.

Fig. 4.

Fig. 4.

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Inhibition of TR-1 activity in tumor cells modifies CLIC4 nitrosylation and subcellular localization. Primary mouse and human keratinocytes and tumor cell lines or human HaCaT cells and tumorigenic derivative (HaCaT-R) were (A) subjected to immunoblotting for TR-1 levels. (B) Cell lysates were assayed for TR-1 activity. Each point represents results from triplicate determinations and activity over time in tumor lines versus normal keratinocytes or HaCaT cells, respectively is significant P < 0.01 using one-way analysis of variance with Bonferroni post-test comparison. (C) Normal keratinocytes or v-rasHa-transformed keratinocytes were infected with adenovirus to overexpress HA-CLIC4 followed by treatment with 0.1 μM Auranofin for 3.5 h. Lysates were used to perform a biotin switch assay (29) to detect the level of S-nitrosylation on CLIC4 (SNO-HA), and the biotin pulldown was immunoblotted with HA-antibody. Five percent of lysates was used as input controls. (D) Pam212 cells were pretreated with 1 μM Auranofin for 1.5 h followed by treatment with NO donors (100 μM S-Nitrosoglutathione or 500 μM DETA/NO) for 1 h. Cells were fixed and immunostained for CLIC4 (fluorescein isothiocyanate) and the nuclei counterstained for 4′,6-diamidino-2-phenylindole followed by confocal microscopy.

Reconstitution of CLIC4 in tumor cells retards tumor growth

Studies were then designed to test if increasing CLIC4 in tumors in vivo could influence tumor development and growth. SP1 HA-CLIC4 Tet-On cells respond to doxycycline in culture with a 2- to 3-fold increase in CLIC4 although the system is leaky (Supplementary Figure 4A is available at Carcinogenesis Online). These cells were grafted onto nude mice orthotopically, and mice were fed with doxycycline-containing mouse chow at various times after grafting (Figure 5A). Tumor growth was inhibited by the addition of dietary doxycycline, with maximal inhibitory effect (80%) resulting from the earliest induction of CLIC4 expression. Even late induction of CLIC4 reduced the rate of tumor growth, but the requirement to terminate the tumor study precluded an analysis of tumor regression. A random subset of these tumors was immunostained for HA and phospho-Smad 2 (Figure 6A). The percent of viable tumor nuclei positive for phospho-Smad 2 was correlated to the expression of exogenous CLIC4 detected in each tumor immunoblot (Supplementary Figure 4B is available at Carcinogenesis Online, upper CLIC4 band; Figure 6B) revealing a correlation coefficient of 0.91 with the exclusion of a few outliers. It also appears that overexpressing CLIC4 in vivo by this mechanism results in nuclear residence of the exogenous protein in many tumor cells. In a second independent in vivo tumor study, keratinocytes derived from CLIC4 transgenic mice (cytomegalovirus-driven) were transduced by a v-rasHa retrovirus and grafted to nude mice (Figure 5B). At the time of grafting, the CLIC4 transgene in the donor cells was expressed 5.3-fold over the endogenous CLIC4 (Supplementary Figure 4C is available at Carcinogenesis Online). The CLIC4 transgene markedly retarded tumor growth in vivo. In a third in vivo tumor study, human SCC-13 cells were grafted subcutaneously into the flank of nude mice. Wild-type or nuclear-targeted CLIC4 was delivered by weekly intratumoral injections of recombinant adenovirus (Figure 5C). Both CLIC4 adenoviral constructs inhibited tumor growth when compared with injection of adeno-β-gal but delivering CLIC4 to the nucleus was most effective. In a final test, we asked if elevated CLIC4 expression also influenced de novo tumor induction. As shown in Figure 5D, Keratin 5 (K5) rTA Tet-O-CLIC4 FVB/N double transgenic mice or single transgenic littermates were placed on a doxycycline diet and subjected to a 7, 12, dimethylbenz[a]anthracene–12-O-tetradecanoyl-phorbol-13-acetate tumor induction protocol. Overexpression of CLIC4 in targeted keratinocytes reduced the tumor yield by 50% over that of single transgenic K5-rTA littermates. Because the number of mice in this study was small, statistical significance could not be confirmed.

Fig. 5.

Fig. 5.

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CLIC4 expression inhibits growth of mouse and human squamous tumor orthografts. (A) SP-1/F-Tet-On CLIC4-HA cells (doxycycline-inducible CLIC4-HA) were placed as orthografts on a prepared skin bed on nude mice, and mice were started on a doxycycline diet at various times after grafting. Tumor measurements were made weekly with calipers in three dimensions and recorded as volume. (B) Keratinocytes were isolated from wild-type FVB/N or cytomegalovirus-CLIC4-HA (FVB/N) transgenic mouse skin and transduced with retrovirus-encoding v-rasHa in culture. After 8 days in culture, cells were placed as orthografts on nude mice and tumor size was measured weekly. (C) SCC-13 human squamous cancer cells were placed subcutaneously on the flank of nude mice. When tumors were palpable at 4 weeks, adenoviruses encoding Beta-gal, native CLIC4 or nuclear-targeted CLIC4 were injected into the tumors at three sites each week. Tumors were measured weekly and reported as the fold increase in size relative to the starting size at week 4. Each group in grafting experiments consisted of 10 mice. Statistical evaluation used Two-Way analysis of variance with Bonferroni post-test comparison. *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001. (D) Six to seven-week-old FVB/N single transgenic K5-rTA or K5-rTA-Tet-O-CLIC4 double transgenic mice with doxycycline-inducible CLIC4 targeted to the epidermis by the keratin 5 (K5) promoter were initated once with 7, 12, dimethylbenz[a]anthracene, placed on a doxycycline diet 24 h later and promoted with 12-O-tetradecanoyl-phorbol-13-acetate for 20 weeks. Tumor multiplicity was monitored each week for 30 weeks. Each group consisted of six mice and error bars are standard error of the mean. Because of the small number of mice in the study, statistical significance could not be achieved. One mouse with tumors in the CLIC4 group expired at week 20.

Fig. 6.

Fig. 6.

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Induction of CLIC4 in tumors correlates with active TGF-β signaling. (A) A subset of the tumors from Figure 5A shown as immunoblots in Supplementary Figure 4B, available at Carcinogenesis Online, was immunostained with anti-HA antibody to detect expression of exogenous CLIC4 and anti-phospho-Smad 2 to detect activation of TGF-β signaling. Shown are two independent tumors from the never doxycycline group (left panels) and the always doxycycline group (right panels) for SP-1/F-Tet-On CLIC4-HA tumor orthografts (doxycycline-inducible CLIC4-HA). (B) Correlation plot of phospho-Smad 2 positive nuclei versus exogenous CLIC4-HA quantified by densitometry from the upper band of immunoblots of SP-1/F-Tet-On CLIC4-HA tumor lysates shown in Supplementary Figure 4B, available at Carcinogenesis Online. Nuclear p-Smad2 was quantified using Aperio Genie pattern recognition image analysis software in combination with Aperio’s nuclear quantification software. Only the epithelial component of tumor tissue in each section was examined and necrotic or keratinizing areas were excluded. After training to identify keratinocyte nuclei, a minimum of 10 000 nuclei per slide were evaluated and the % of diaminobenzidene-labeled nuclei identified. A positive correlation coefficient of 0.91 was obtained without inclusion of outliers as shown. Only a single section for each tumor was studied and sampling errors cannot be excluded in outlying samples.

Discussion

Changes in CLIC4 expression under a variety of biological situations and various cell types are increasingly recognized with the application of array techniques. CLIC4 expression is a marker for cutaneous stem cells (38) and is upregulated in skin when mice are subjected to the stress of dietary arsenic (39). CLIC4 is highly upregulated in activated fibroblasts from cancer stroma and from fibroblasts treated with TGF-β (27,40). Elevated expression of CLIC4 is also seen in damaged retinas from mice undergoing prolonged exposure to fluorescent light (41). CLIC4 is a marker for uterine fibroids (42), Hep2G cells transduced with an HCV core protein effector (43) and in advanced stage malignant melanoma undergoing epithelial to mesenchymal transition (44). Thus, elevation of CLIC4 may be common in mesenchymal pathology. CLIC4 is essential for endothelial tube formation in vasculogenesis (25,26), a function that may be particularly important in tumor growth. CLIC4 expression is reduced in T cells cocultured with regulatory T cells (45) and highly upregulated in activated macrophages (46), suggesting it may also participate in immune function.

The mechanism by which CLIC4 expression diminishes during epithelial cancer development is not clear. Evidence for mutation, deletion or rearrangement of the CLIC4 gene was not found (6). Our studies indicate the regulation of CLIC4 is transcriptional. The last 400 bases of the CLIC4 promoter region and into the first exon reveal a high frequency of CpG islands, suggesting the possibility of promoter silencing by methylation (19). However, methylation-specific PCR and combined bisulfite modification and restriction analysis failed to provide any evidence for promoter methylation at these sites (data not shown), and treatment of cancer cell lines with 5-azacytidine could not increase CLIC4 expression. Restoring expression of p53 does not increase CLIC4 expression in tumor lines. Thus, at this time the mechanism underlying reduced CLIC4 expression in tumors remains unknown.

The translocation of cytoplasmic CLIC4 to the nucleus appears to be a critical step in the growth arrest and proapoptotic functions of the protein that is lost in tumor cells (19). CLIC4 has a functional nuclear localization signal, binds to nuclear import proteins and readily translocates to the nucleus under conditions of cell stress (19,47). The absence of nuclear residence or induced translocation of endogenous CLIC4 early in tumor formation when cytoplasmic CLIC4 protein is still abundant is enigmatic. CLIC4 protein folding is known to be influenced by oxidation state (3,4), and many of the metabolic stresses that induce nuclear translocation are pro-oxidant. Furthermore, recent studies indicate that nitric oxide synthase-mediated nitrosylation of critical cysteines in CLIC4 is required for association with nuclear import proteins and nuclear translocation (29). Tumor cells commonly have elevated reactive oxygen levels and increases in nitric oxide synthase activity but compensate by elevating antioxidant pathways (34,37). Transformation by a RAS oncogene is sufficient to increase intracellular reactive oxygen but this is compensated by an increase in antioxidant defense particularly through glutathione, TR-1 and catalase (48,49). These conditions could determine the tertiary structure of CLIC4 and thus the exposure of its nuclear localization signal. This is not without precedent. Redox state is reported to regulate the nuclear import of transcription factors in osteoclast differentiation (50). By inhibiting TR-1 with Auranofin, we restored nitrosylation of CLIC4 in ras-transformed mouse keratinocytes, suggesting that the redox environment in tumor cells does influence this modification of CLIC4. In Pam212 cells, Auranofin also enhanced nuclear translocation of CLIC4, but it remains to be determined if this is a general response in tumor cells with sufficient CLIC4 expression. From the data in Figure 6, it is clear that if the level of CLIC4 is elevated sufficiently in tumors, CLIC4 can be found in the nucleus of tumor cells and this is associated with inhibition of tumor growth. Pharmacological modification of tumor cell oxidant state is an intriguing approach to cancer cell therapy since it is likely to have broad application among multiple tumor types. Whether CLIC4 contributes to the growth inhibitory or lethal effects of such therapy remains to be established.

The discovery that nuclear CLIC4 functions in the TGF-β pathway to preserve nuclear phospho-Smad 2/3 and enhance TGF-β signaling provides mechanistic clarity for how this protein causes growth arrest and perhaps other aspects of it broad activities (30). We now show that nuclear CLIC4 can enhance TGF-β responsiveness in cancer cells that were refractory to TGF-β growth inhibition and have diminished TGF-β-dependent transcriptional signaling. Since TGF-β is abundant in the tumor microenvironment, restoring the expression of CLIC4 and its nuclear translocation in tumor cells could enhance response to the antitumor functions of TGF-β. We present evidence that this occurs in vivo under conditions of artificially enhanced CLIC4 expression. To date we have not been able to elevate endogenous CLIC4 in tumors. In normal keratinocytes treated with TGF-β, CLIC4 translocates to the nucleus in the company of a chaperone protein, Schnurri-2 (30). The status of Schnurri-2 in tumors is not known. The current findings suggest that understanding CLIC4 regulation in tumor cells and how to modulate that regulation could reveal novel approaches to inhibit tumor growth.

Supplementary material

Supplementary Figures 1–4 can be found at http://carcin.oxfordjournals.org/.

Funding

This work was supported by the Intramural research program of the Center for Cancer Research, National Cancer Institute, USA.

Supplementary Material

Supplementary Data
supp_33_5_986__index.html (1.3KB, html)

Acknowledgments

Conflict of Interest Statement: None declared.

Glossary

Abbreviations

CLIC

Chloride intracellular channel

FRT

flippase recognition target

HA

hemagglutinin epitope

SCC

squamous cell carcinoma

TGF-β

transforming growth factor-β

TR

thioredoxin reductase

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Supplementary Materials

Supplementary Data
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