Human Cancer Biology

Reciprocal Modifications of CLIC4 in Tumor Epithelium and Stroma Mark Malignant Progression of Multiple Human Cancers Kwang S. Suh,1John M. Crutchley,1Arash Koochek,1Andrew Ryscavage,1Kiran Bhat,1Takemi Tanaka,1 Akira Oshima,2 Peter Fitzgerald,3 and Stuart H. Yuspa1

Abstract Purpose: CLIC4, a member of a family of intracellular chloride channels, is regulated by p53, c-Myc, and tumor necrosis factor-a. Regulation by factors involved in cancer pathogenesis, together with the previously shown proapoptotic activity of CLIC4, suggests that the may have a tumor suppressor function. To address this possibility, we characterized the expres- sion profile, subcellular localization, and 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 xeno- grafts 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 da- tabase 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 a-smooth muscle , suggesting that CLIC4 is up-regulated in myofibroblasts. Coculture of cancer cells and fibroblasts induced the expression of both CLIC4 and a-smooth muscle actin in fibroblasts adjacent to tumor nests. Introduction of CLIC4ornuclear 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.

CLIC4 belongs to the chloride intracellular channel (CLIC) homeostasis, and electroneutrality (1). CLIC members are family of with seven members (p64, CLIC1-CLIC5, localized in various cellular compartments, expressed in and parchorin) that may be involved with multiple cellular multiple tissue types, and have a major hydrophobic stretch functions, including regulating organellar volume, ionic that may be used to span the membrane. Soluble CLIC proteins reside in the cytoplasm but undergo a molecular rearrangement when localized to the cellular/organelle membrane and behave 1 Authors’ Affiliations: Laboratory of Cellular Carcinogenesis and Tumor as an anion channel or channel regulator (2). CLIC4 has Promotion, 2Laboratory of Cell Regulation and Carcinogenesis, and 3Genome Analysis Unit, Center for Cancer Research, National Cancer Institute, Bethesda, a putative Cl selective channel activity exhibiting a single Maryland channel conductance, and other family members (parchorin, Received 6/27/06; revised 9/8/06; accepted10/5/06. p64, CLIC1, and CLIC3) exhibit similar channel activities Grant support: Intramural Research Program of the Center for Cancer Research, (3–6). NIH, National Cancer Institute. CLIC4 is ubiquitously expressed in various tissue types with The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance high level of expression in skin, and up-regulation of CLIC4 is with 18 U.S.C. Section 1734 solely to indicate this fact. strongly associated with apoptosis in keratinocytes and other Requests for reprints: Stuart H.Yuspa, Laboratory of Cellular Carcinogenesis and cell types (7, 8). In specific cell types, CLIC4 is found in Tumor Promotion, Center for Cancer Research, National Cancer Institute, Bethesda, multiple subcellular compartments, including inner mitochon- MD 20892. Phone: 301-496-2162; Fax: 301-496-8709; E-mail: yuspas@ mail.nih.gov. drial membrane, trans-Golgi network, endoplasmic reticulum, F 2007 American Association for Cancer Research. large dense core vesicles, and actin cytoskeleton (3, 9–11). doi:10.1158/1078-0432.CCR-06-1562 Cytoplasmic CLIC4 translocates to the nucleus in cells

www.aacrjournals.org 121 Clin Cancer Res 2007;13(1) January 1, 2007 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology undergoing growth arrest or apoptosis in response to p53, DNA immunohistochemical staining as described by the array manufac- damage, and metabolic stress, and this nuclear trafficking turers, and the stained slides were analyzed with bright-field micros- is mediated by the nuclear localization signal in the CLIC4 copy using Leitz-DMRB (Leica, Heidelberg, Germany) and OpenLab sequence and the cellular nuclear import machinery (12). (Improvision, Coventry, United Kingdom) software. Fluorescent immunostaining of tissue arrays was done similarly to Furthermore, targeting CLIC4 to the nucleus of several cell the immunohistochemical staining except that fluorescent-labeled types with a nuclear targeting vector accelerates apoptosis. The secondary antibodies (FITC and Texas Red, Vector Lab, Burlingame, discovery that CLIC4 expression level and nuclear translocation CA) were used and mounted with 4¶,6-diamidino-2-phenylindole– may be important in cellular stress/apoptotic signaling sug- containing VectaMount (Vector Lab). a-Smooth muscle actin (aSMA) gested that CLIC4 expression and localization could be altered antibodies were purchased from Abcam (Cambridge, MA). in human cancers. This possibility is reinforced by data For nonradioactive in situ hybridization/staining, a specific region of showing regulation of CLIC4 expression by both p53 and mouse and human CLIC4 cDNA was chosen by combination analyses c-Myc, two mediators of cancer pathogenesis in multiple tumor of sequence alignment to avoid detecting other CLIC members and sites (8, 13). This study was undertaken to evaluate changes genomic structure (exon/intron junction) inspection to avoid detecting in CLIC4 gene integrity, transcript and protein expression, and the genomic sequence. PCR primers were designed to cross exon 5and exon 6 (3¶ untranslated region) to generate f200-bp fragments from subcellular localization in a series of human tumors and tumor both mouse and human cDNAs. cDNAs encoding 3¶ untranslated cell lines. The results indicate that modification of CLIC4 region sequences were generated by reverse transcription-PCR (RT-PCR) expression, subcellular localization, and tissue compartment is from a human brain cDNA library and used as a template for PCR. frequent and common to many human tumor types, suggesting The PCR fragment was cloned into pGEM-Teasy (mouse) or pCRII that CLIC4 is an important participant in human cancer (human), and SP6 or T7 primers were used to generate digoxigenin- development. labeled sense and antisense strands with 200-bp length. The labeled strands and the protocols described by Basset-Sequin et al. (15) and Materials and Methods DIG-High Primer Detection kit from Roche (Indianapolis, IN) were used for visualization of the stained tissue. Cell culture Human breast cancer cell line MB231 was maintained in DMEM Mutation analyses (BioWhittaker, Walkersville, MD) containing 8% fetal bovine serum Bioinformatics. The open reading frame (ORF) sequences of the CLIC1, CLIC2, CLIC3, CLIC4, CLIC5, CLIC5B (Gemini) and 20 units/mL penicillin/streptomycin (Invitrogen, Carls- seven CLIC ( , and CLIC6 bad, CA). Mouse dermal fibroblasts were isolated from newborn mice ) were compared with the human expressed sequence tag and maintained in DMEM as described previously (14). MCF10CA1a database (updated December 10, 2004), using the tblastn program e cells (human breast cancer cell line) were grown in DMEM/F12 (version 2.2.10) with an value cutoff of 0.00001. The expressed supplemented with 5% horse serum (DMEM/F12, Invitrogen). sequence tag sequences that showed similarity were sorted into readily MCF10CA1a breast epithelial cells and fibroblasts were cocultured by identifiable cancer families based on the tissue type classification of the seeding 0.5 106 of each type together in 100-mm tissue culture dishes database entries. Additionally, whereas each positive database hit with sterile coverslips in DMEM/F12 medium. NCI60 tumor cell lines typically matched more than one CLIC family member, the bit scores e were cultured at the Developmental Therapeutics Program, National and values were used to assign each hit to a specific CLIC protein that Cancer Institute (Frederick, MD). represented the ‘‘best match.’’ RT-PCR of NCI60 human tumor cell lines. Total RNA samples of Microarray blots 60 different human tumor cell lines were prepared by National Cancer Human ‘‘matched’’ tumor cDNA microarray (Clontech, Mountain Institute Developmental Therapeutics Program. First-strand cDNA was View, CA) was processed as described by the manufacturer using 33P generated from 1 Ag total RNA per cell type by Superscript-III (ICN Biomedicals, Irvine, CA)–labeled human CLIC4 and human (Invitrogen) at 50jC according to the manufacturer’s instruction. A ubiquitin DNA probes. portion of the cDNA generated was then used for RT-PCR using CLIC4 gene – specific primer sets (set-A: forward, 5¶-CAGTCCCGGAGCA- Immunoblots and tumor lysate array-blot analyses GAAGCAGCAGC-3¶; reverse, 5¶-GGAGTAGCCTGTTAGGAAAAGCG-3¶ ¶ ¶ ¶ Monospecific polyclonal anti–NH2-terminal CLIC4 antibodies were andset-B:forward,5-CAGCAGCCCTCGCCGTTC-3 ; reverse, 5 - generated and used for immunoblots as described previously (7). A GACATCTCTTTTACAAACGC-3¶). Set-A primers were used in RT-PCR primary antibody against actin (Calbiochem, San Diego, CA) was used for 20 cycles to generate a larger-size PCR fragment (899 bp) of CLIC4, at dilutions suggested by the manufacturer. Anti-mouse, anti-rabbit, and then a portion of the first RT-PCR product and primer set-B was and anti-goat secondary antibodies conjugated to horseradish peroxi- used for PCR to generate only the ORF of CLIC4 (812 bp). High-fidelity dase (GE Healthcare Bio-Sciences Corp., Piscataway, NJ) and Super- PCR enzyme (Roche) was used for all PCR reactions. The final PCR Signal chemiluminescent substrate (Pierce, Rockford, IL) were used for product was purified by using the PCR purification kit (Qiagen, the antigen detection. Human ‘‘matched’’ CancerScreen 200 (Protea Valencia, CA) and sequenced at the National Cancer Institute DNA Biosciences, Morgantown, WV/Clinomics, Watervliet, NY) protein- sequencing facility. DNA sequences were analyzed by Sequencher lysate microarrays were processed as described by the manufacturer. (Gene Codes Corp., Ann Arbor, MI) and Pairwise Blast (National Center for Biotechnology Information) followed by aligning the Immunohistochemistry, immunofluorescence, and sequences with ClustalW Multiple Sequence Alignment program tissue microarray staining (National Center for Biotechnology Information). Human tissue microarrays were obtained from multiple sources: TARP tissue arrays (National Cancer Institute), ‘‘matched’’ human Xenograft and adenovirus injection tumor tissue arrays (Imgenex, San Diego, CA), Food and Drug MB231 cells (2 106) or a mixture of MCF10CA1a (4 105) and Administration–standard normal and tumor tissue arrays (Biochain, mouse fibroblasts (wild type or transgenic; 3 105) were s.c. injected Hayward, CA) and human cancer screen tissue arrays (Clinomics), into the back of athymic mice. Xenograft studies were repeated at least prostate tumor arrays (Baylor Specialized Programs of Research twice with 10 mice per study group. For i.t. adenovirus injection studies, Excellence), and human tissue arrays (Accumax) were used for CLIC4 the tumor-bearing mice were injected twice a week with 200 AL PBS expression and localization studies. The slides were subjected to containing recombinant adenovirus (1 109 particles per injection)

Clin Cancer Res 2007;13(1) January 1, 2007 122 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. CLIC4 Expression Is Altered in Epithelial Cancers encoding green fluorescent protein (GFP), wild-type CLIC4 (Cyt- that spot intensity varies even among samples from normal CLIC4), or nuclear-targeted CLIC4 (Nuc-CLIC4). Typically, three sites tissues of different patients (Fig. 1A), suggesting that CLIC4 were injected per treatment. Production of CLIC4 recombinant expression is not uniform in different tissue compartments or adenovirus was described previously (12). Efficiency of recombinant homeostatic states. In contrast to CLIC4 hybridization, blotting adenovirus delivery was qualitatively assessed by fluorescent microsco- the membrane with an ubiquitin probe shows uniform py of the frozen sections of MB231 tumors that were injected with GFP adenovirus. hybridization in all of the cDNA spots (Fig. 1A), showing that equal amounts of cDNA are spotted on the array membrane. Results When the Oncomine database (Cancer Bioinformatics, Com- prehensive Cancer Center, University of Michigan) was used CLIC4 expression is altered in specific human cancers. To as an independent bioinformatics approach, mean CLIC4 examine the expression pattern of CLIC4 transcripts in a variety transcript level was reduced in selected studies, including lung of human cancers, a membrane containing matched cDNAs (Fig. 1B), prostate (Fig. 1B), breast, sarcoma, and brain (data derived from normal and tumor tissues of the same patient not shown). (Human Tumor Array, Clontech) was probed with 32P-labeled To determine if changes in CLIC4 detected in CLIC4 cDNA. The array data show that CLIC4 transcript level is the cDNA microarray correspond with the protein expression, frequently down-regulated in several human tumor types, a membrane containing human tissue lysates derived from especially in breast, ovary, and kidney (Fig. 1A). However, matched normal and tumor tissue was probed with CLIC4 CLIC4 transcripts are up-regulated in some tumors, suggesting antibody (Fig. 1C). Similar to the cDNA array data, CLIC4 that suppression may be specific to a particular cell type within protein expression varied among the normal samples of the the tumor tissue. In some tissues (e.g., stomach), CLIC4 same tissue type, although all spots represent an equal amount expression is low in both normal and the matched tumor, of protein. Furthermore, CLIC4 protein levels also varied in suggesting that basal CLIC4 expression varies among tissues as tumors when compared with the matching normal tissues we have reported previously (7). Examination of the medical (Fig. 1C), implying that altered CLIC4 expression in tumors information associated with the tumor samples indicated that may be dependent on the abundance of cell types that comprise reduction of CLIC4 expression measured by array methodology the major portion of the tumors analyzed. From the limited is not correlated to patient age and tumor stage. It can be noted samples analyzed, CLIC4 protein may be reduced in kidney

Fig. 1. Expression of CLIC4 is altered in several human cancer types. A, tumor array representing cDNAs derived from the ‘‘matched’’ normal (N)andtumor(T) tissues (paired horizontally) of multiple human cancer types is probed with radioactively labeled CLIC4 or ubiquitin (inset) DNA probes. B, oncomine database (University of Michigan Comprehensive Cancer Center) was searched with ‘‘CLIC4’’ as a query.Two examples of statistically significant changes (prostate/Lapointe/ Stanford University/P =1.8 10 21 and lung/Bhattacharjee/Harvard Medical School/P =3 109). Blue columns, normal tissues; red columns, cancerous tissues. Brackets, 1.5 times interquartile range; dots, outliers; line within box, median; box boundaries, upper and lower quartiles. Two independent studies. C, tumor lysate dot blot representing 200 tumor samples (600 ng per spot) is probed with NH2-terminal CLIC4 antibody. ‘‘Matched’’ normal (solid gray arrowhead) and tumor (solid black arrowhead) tissue samples are spotted vertically as a pair. Open circles are marker spots for an orientation. The experiment was repeated twice, and independent experiments with 10-fold less amount of lysate loaded per spot showed similar results.

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Fig. 2. CLIC4 expression and subcellular localization are altered in human tumors. Human normal and matched tumor (roman numerals, tumor stage) tissue sections representing (A) esophagus, (B)kidney,and(C)colon from the tissue microarrays are immunostained with anti-CLIC4 antibody and visualized by bright-field microscopy under 40 magnification. Inset, lower magnification (10). Black arrows, CLIC4 localized to the nucleus in the normal tissues. Similar staining patterns of CLIC4 were found in multiple human tumor types. Representative immunohistochemical stainings.

cancer, but a particular expression pattern was not detected tumor stroma (Fig. 2A-C). Specifically in kidney, CLIC4 is also in other tumors (Fig. 1C). Consistent with the cDNA data, the located in the apical portion of distal tubular cells and their pathology reports associated with the tissue samples on the nuclei (Fig. 2B). Most notably, total CLIC4 in tumor cells array indicate that altered CLIC4 expression was not dependent decreases with increasing tumor grade as shown in Fig. 3 for on patient age and tumor stage. colon cancer but seen in most other cancers studied. CLIC4 expression is diminished in tumor cells and up-regulated Conversely, total stromal CLIC4 increases with tumor grade in tumor stroma. The variable expression patterns for CLIC4 in (Fig. 3A-D). These results indicate that highly proliferative lysed whole tumors prompted us to examine CLIC4 in human cancer cells first exclude or reduce CLIC4 in the nucleus and tumor material in situ using matched tissue arrays that are then suppress CLIC4 in the entire cell mass. To test if restoring immunostained with a specific CLIC4 antibody. In vivo, CLIC4 CLIC4 levels in tumor cells would influence tumor growth, is nuclear in multiple normal epithelial tissues (e.g., esophagus, xenografts of the highly malignant breast cancer cell line kidney, and colon), consistent with the slow cycling of most MB231 were established in nude mice. Wild-type CLIC4 and epithelial cells (12). In general, stromal CLIC4 expression is nuclear-targeted CLIC4 were introduced into established very low. In tumors from the great majority of multiple human tumors by i.t. injection of recombinant adenovirus or a control tissues, three distinct and common changes can be observed. viral vector expressing GFP (Fig. 4A and B). Immunofluorescent Nuclear CLIC4 is lost; CLIC4 levels are reduced in the tumor microscopy of frozen tissue sections indicate that many cells epithelium; and CLIC4 expression is markedly up-regulated in from xenograft tumors are efficiently infected by the GFP

Clin Cancer Res 2007;13(1) January 1, 2007 124 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. CLIC4 Expression Is Altered in Epithelial Cancers recombinant virus (Fig. 4A). In the same setting, the expression within the ORF region. Multiple sequence alignment data show of either wild-type or nuclear-targeted CLIC4 retards the growth that CLIC1 contained a variant at position 168, where the of xenograft tumors significantly compared with the GFP leucine in the sequence KFLDG changes to serine, and this control vector. In addition, targeting CLIC4 to the nucleus is mutation (T ! C) was occasionally (12 of 552 cases) found in more effective for tumor inhibition than raising the cytoplasmic cancer types examined. However, no mutations were found in level of CLIC4 in the tumor. CLIC4 (29 ORF) and other CLIC members when sequences CLIC4 is not deleted or mutated in human tumors. Trans- were examined for frequent or consistent differences between membrane and nuclear localization signal motifs regulate the the original CLIC genes and the cancer-derived expressed subcellular localization of CLIC4 (12). Blast analysis of the sequence tag sequences. To verify the bioinformatics data by sequences of the seven CLIC proteins against the entire human an independent method, human CLIC4 ORF was amplified by expressed sequence tag database revealed 2,049 sequences that RT-PCR using the total RNA extracted from NCI-60 tumor cell showed significant similarity to CLIC genes. Of these 2,049 lines, and the amplified DNA fragment was then sequenced for sequences, 602 were further grouped into 23 distinct cancer analysis. When DNA sequences were examined by multiple types based on tumor and tissue annotations associated with sequence alignment, mutations were not detected in CLIC4 each entry (Table 1). Within each cancer group, the matched ORFregioninanyof60tumorcelllines.Thesetwo sequences were further subdivided into each CLIC family independent mutation analyses showed that the altered member, and DNA sequences were examined for mutations subcellular localization or diminished expression of CLIC4

Fig. 3. CLIC4 down-regulation in the epithelium and up-regulation in the stroma increases with higher tumor grade. Matched normal and tumor tissues were immunostained with CLIC4 antibody.Tissues from rectum representing normal (A), tumor grade 1 (B, T-I ), grade 2 (C, T-II), and grade 3 (D, T-III ). CLIC4 localized to the nucleus in normal epithelium and grade 1tumors (blue arrow) and the interstitial or stromal compartment of normal and tumor tissues (red arrow). Left, lower magnification (10) of the tissues. Dotted boxes, origin of right.

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CLIC4 in infiltrating ductal carcinoma of human breast showed similar staining patterns as seen with the immunofluorescence methods (Fig. 5E). To investigate whether aSMA expression is a consequential outcome of CLIC4 expression, primary human fibroblasts were infected with the recombinant CLIC4 adeno- virus; aSMA protein level was significantly up-regulated upon CLIC4 overexpression (Fig. 5F). In situ hybridization of kidney tumor sections indicates that up-regulation of CLIC4 in the tumor stroma is transcriptionally mediated, whereas transcripts for CLIC4 are rare in the tumor cells (Fig. 6A). To determine if the induction of stromal CLIC4 and aSMA involve an interaction with tumor cells, normal mouse fibroblasts were cocultured with human breast tumor cell line MCF10CA1a or normal breast epithelial cells and monitored for CLIC4 and aSMA expression. Figure 6B reveals that adjacent to tumor islands, fibroblasts coexpress aSMA and CLIC4, whereas the proteins are undetectable in tumor cells. This response was not elicited in coculture with normal breast epithelial cells. With increasing distance from tumor islands, only cytoplasmic CLIC4 is detected in fibroblasts, and with even greater distance, neither marker is detected. This interaction was confirmed in an in vivo xenograft model where human dermal fibroblasts were genetically modified by infection with retrovirus-encoding GFP. When grafted together with MCF10CA1a cells, the GFP-marked fibroblasts were detected in the stroma surrounding tumor nests where they expressed high levels of CLIC4 (Fig. 6C). To Fig. 4. Expression of CLIC4 and nuclear-targeted CLIC4 retards tumor growth further verify the immunostaining and the in vitro data, mouse in xenografts. A, xenograft tumors established from breast carcinoma cell line fibroblasts derived from a CLIC4-overexpressing transgenic MB231alone (without fibroblasts) in nude mice were injected i.t. with recombinant 4 adenovirus expressing GFP (rGFP). Fluorescent microscopy detects GFP mouse line or wild-type mouse fibroblasts were mixed expression in multiple tumor nests. Inset, 4¶,6-diamidino-2-phenylindole (DAPI) with MCF10CA1a tumor cells and used in xenograft studies staining for orientation. B, MB231xenograft tumors were allowed to establish, (Fig. 6D-F). Approximately 3-fold more CLIC4 is expressed by and recombinant adenovirus expressing wild-type (Cyt-CLIC4) or nuclear-targeted CLIC4 (Nuc-CLIC4) was injected i.t. twice per week. Each group represents transgenic fibroblasts relative to wild-type fibroblasts in vitro 10 mice, and xenograft experiments were repeated twice with similar results. (Fig. 6D). In a xenograft model, tumors retaining the fibro- Points, mean tumor measurements; bars, SD. blasts overexpressing CLIC4 had a growth advantage over the wild-type fibroblast control, and both mixed xenograft models frequently observed in tumor tissue samples was not due to produced larger tumors than the MCF10CA1a tumor cells alone an alteration in the CLIC4 gene, but most likely due to (Fig. 6E). Injection of wild-type control or transgenic fibroblast transcriptional or pretranslational or posttranslational modifi- alone did not produced tumors (data not shown). CLIC4 is also cation. up-regulated in the stroma surrounding the tumor nests in the CLIC4 colocalizes with a-SMA in stroma of human tumors. xenograft tumors most prominently in tumors grafted with Strong up-regulation of CLIC4 in tumor stroma was a transgenic fibroblasts (Fig. 6F, inset). Double immunostaining consistent finding in many human cancer types and suggested of the xenograft tumors with CLIC4 and aSMA antibodies an important function for CLIC4 in the tumor microenviron- further suggested that aSMA and CLIC4 are coexpressed in the ment. A previous report suggested that CLIC4 is up-regulated tumor stroma formed by the grafted fibroblasts (Fig. 6F). in myofibroblastic differentiation (16). The up-regulation of Together, these data indicate that the induction of CLIC4 and aSMA during myofibroblast differentiation is a characteristic of aSMA in fibroblasts results from a tumor-stromal interaction; stromal reaction in cancer (17). To determine if CLIC4 is each is induced independently, and Figs. 5F and 6B together coexpressed with aSMA in normal tissues or cancers, a number suggest that CLIC4 induction likely precedes the induction of of normal and cancerous tissues were analyzed by double aSMA in myofibroblast differentiation. immunostaining on tissue arrays (Fig. 5A-C). As seen in the immunohistochemical studies, most normal tissues express Discussion CLIC4 in epithelial cells, whereas CLIC4 expression is down- regulated in tumor cells. In the normal tissues, aSMA is Maintenance of intracellular chloride is an essential cellular primarily detected in the vascular walls where CLIC4 is also function to control volume, pH, and electrogenic balance, and expressed (see, e.g., Fig. 5B and C). Previous studies have this is critical to cell viability. Five families of chloride channels indicated that endothelial CLIC4 also participates in vascular regulate chloride flux in mammals (1). However, only a few tubulogenesis (18). Both aSMA and CLIC4 are markedly reports have examined changes in chloride channels in cancer up-regulated and colocalized in the stroma of selected tumor cells (19). Voltage-gated chloride channels CLC-2, CLC-3, and types (Fig. 5D), suggesting that CLIC4 is a common marker for myofibroblast differentiation and likely participates in the formation of myofibroblasts. Immunohistochemical staining of 4 Suh et al., unpublished data.

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Table 1. Mutations of CLIC family members in human cancers

Cancer type Total CLIC1 CLIC2 CLIC3 CLIC4 CLIC5 CLIC5B CLIC6

Adenocarcinoma 136 129 1 6 Ascites 33 33 Astrocytoma 8 8 Chondrosarcoma 30 28 2 Choriocarcinoma 8 2 6 Epitheliod carcinoma 44 44 Fibrosarcoma 44 Glioblastoma 20 20 Hepatocellular carcinoma 16 15 1 Insulinoma 12 9 3 Large cell carcinoma 22 19 1 2 Leiomyosarcoma 38 28 1 9 Leukemia 17 16 1 Lymphoma 13 11 1 1 Melanoma 107 102 3 2 Myeloma 15 15 Neuroblastoma 26 26 Papillary carcinoma 7 7 Retinoblastoma 3 3 Rhabdomyosarcoma 5 3 2 Small cell carcinoma 11 11 Squamous cell carcinoma 18 17 1 Teratocarcinoma 9 5 Total 602 552 6 9 29 0 0 6 Mutations found 25 12 0 0 0 0 0 0

NOTE: The DNA sequences of the seven CLIC family members were compared with the human expressed sequence tag database, and the expressed sequence tag sequences (total 602) that showed similarity were sorted into readily identifiable cancer types and then further subdivided into CLIC isoforms. From 25 total mutations, only the mutations found in the ORF of CLIC isoforms are shown at the bottom.

CLC-5are localized to various intracellular organelles and up-regulation of CLIC4 in myofibroblast transformation of expressed at high levels in patient biopsies from low- and high- tumor stroma precedes the expression of aSMA and is induced grade malignant gliomas (20). Glycine-gated chloride channels by factors released from tumor cells. Enhancement in growth of in endothelial cells inhibit the stimulatory effect of vascular xenograft tumors with fibroblasts overexpressing CLIC4 versus endothelialgrowthfactoronbloodvesselgrowth(21). control suggests that up-regulation of CLIC4 in tumor stroma Calcium-activated chloride channels CLCA1 and CLCA2 are participates in tumor progression. These reciprocal changes of significantly down-regulated in f80% of colorectal carcino- CLIC4 expression in tumor epithelium and stroma explain why mas and >90% of highly proliferating tumor cells (22, 23). tumor lysate studies did not show a consistent expression Reconstitution of CLCA2 expression in highly malignant cell pattern and suggest that whole tumor lysates should be lines reduced Matrigel invasion in vitro and prevented the evaluated cautiously when used to identify a particular marker. growth or metastasis of tumor cells transplanted s.c. in nude Previous studies have indicated that CLIC4 transcripts are mice (24, 25). Expression and function of the KCl cotrans- up-regulated substantially in breast cancer myofibroblasts in porter is essential for growth and invasion of cervical cancer response to transforming growth factor-h (16). Furthermore, cells, and inhibition of KCl transporter activity prevents the growth of breast cancer cells in vitro is enhanced when growth of cervical cancer cells in vivo (26). Together, these cocultured with serum-stimulated fibroblasts that have a gene results suggest that the regulation of chloride transport can expression profile of myofibroblasts, including marked up- influence tumor development and progression, but the changes regulation of CLIC4 (27). The exact role played by CLIC4 in may be specific to particular tumors and particular chloride stromal fibroblasts remains to be determined. Our studies channel families. indicate that up-regulation of stromal CLIC4 is common to We now report a more generalized pattern of cancer- many tumors and correlates with increasing malignant behav- associated changes for CLIC4. In a study of multiple human ior of the cancer cells. Myofibroblasts contribute essential solid cancers, we find that CLIC4 expression is commonly functions in tumor growth under experimental testing, includ- reduced in the tumor epithelium, is excluded from the nucleus ing secretion of matrix metalloproteinases (28), promotion of cancer cells (in contrast to its location in the nucleus of non- of angiogenesis (29), promotion of tumor cell growth and cancerous differentiated epithelial cells), and is highly up- invasion (27, 29), and chemokine release (30). A recent report regulated in the tumor stroma, associated with myofibroblast suggests that secretion of stromal cell–derived factor 1 by differentiation. The extent of these changes in general correlates tumor myofibroblasts recruits endothelial cells and stimulates directly with the stage of tumor progression in selected tumor tumor growth (29). Stimulation of CLIC4 by this factor would types studied. Furthermore, our studies suggest that restoration also be of consequence because CLIC4 and aSMA also of CLIC4 levels, and particularly CLIC4 in the nucleus of tumor colocalize in the muscularis of vascular structures in both cells, inhibits tumor growth. Coculture studies suggest that the normal and tumor tissue, and CLIC4 has a shown role in

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Fig. 5. CLIC4 and aSMA are coexpressed and colocalized in tumor stroma. Human tissue arrays with normal (top)and matching tumor (C, bottom) sections were immunostained with CLIC4 (inset, green, right)andaSMA (inset, red, left) antibodies, and stained images were merged (yellow). Selected tissue types are (A) normal kidney and clear cell carcinoma (CCC), (B) normal liver and hepatocellular carcinoma grade 2 (HCCII), and (C)normal pancreas and adenocarcinoma grade 2 (ACII). D, multiple malignant human tumors were double immunostained and analyzed as described above. E, infiltrating ductal carcinoma (IDC)andnormal(inset)from breast were immunostained with anti-CLIC4 antibody. F, primary human dermal fibroblasts were infected with the recombinant CLIC4 or null adenovirus, and the lysates from the treated cells were immunoblotted with anti-CLIC4 or anti-aSMA. Fold increase in CLIC4 and aSMA was determined by the ImageMaster densitometry program and normalized against the actin signal.

tubular morphogenesis of blood vessels (18). The relationship In tumor lysates, a reduction in CLIC4 expression was of CLIC4 and aSMA then seems to be of functional common to many tumor types but with substantial variability consequence in several physiologic and pathologic situations. among and within tumor specimens. Analysis of tumor sections Previously, CLIC4 was reported to interact with components in situ revealed that variability in lysates was due to the of the cytoskeleton in a complex with , , opposing changes of loss of expression in tumor epithelium h-actin, and 14-3-3 proteins (9), suggesting that CLIC4 is and gain of expression in tumor stroma. The loss of CLIC4 in involved in cytoskeleton function in addition to its channel tumor epithelium was pervasive across almost all major cancer activity. This characteristic of CLIC4 may explain its importance types. For example, systematic immunohistochemical analyses in tumor stroma because overexpression of recombinant of tumors with various grades in the format of tissue micro- CLIC4 in fibroblasts reduces cell motility (16), a property that arrays (270 kidney cores from 190 cases, 189 esophagus cores may enhance the interaction of myofibroblasts with adjacent from 66 cases, 189 colon cores from 67 cases, and 189 rectum tumor cells. cores from 63 cases) showed a loss of CLIC4 in f80% of the

Clin Cancer Res 2007;13(1) January 1, 2007 128 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. CLIC4 Expression Is Altered in Epithelial Cancers cases (P < 0.05; 0.94-1.0 of 95% confidence interval with a An analysis of the human genomic database has revealed probability for true negative 0.7-0.87; by VassarStat software) as that several cancer-related genetic aberrations are within close cancers progress. Similar data were also obtained from breast, proximity of CLIC4. However, sequencing of CLIC4 cDNA lung, prostate, and other tumor types (368 tumor tissue cores from the NCI60 tumor cell lines and human expressed from 225cases) by immunofluorescence analysis using anti- sequence tag sequence analysis indicate that the CLIC4 gene CLIC4 and anti-aSMA antibodies. Together, these data suggest is not deleted, and the CLIC4 sequence is highly preserved in that the loss of CLIC4 in tumor epithelium and the reciprocal human cancers. Moreover, neither the nuclear localization increase in the tumor stroma are common events during signal nor the transmembrane domains of CLIC4 was mutated. tumor progression. Based on the cDNA array results, particu- The lack of deletions or mutations in the CLIC4 gene suggests larly in breast, ovary, and kidney, and in situ hybridization, that CLIC4 at some level is essential for cell viability. reduced expression in tumor cells is transcriptionally regulated. Nevertheless, we cannot rule out that aberrations near the The underlying mechanisms that suppress CLIC4 expression are CLIC4 genetic locus may affect regulation of CLIC4 expression not known at this time. and alter the protein level; however, another mechanism of We considered that intrinsic alterations in CLIC4 through gene silencing seems more likely. If the mutations or genomic rearrangements could alter expression. function of CLIC4 is its essential contribution to normal CLIC4 is localized to the tip region of the p-arm of physiology, then other CLIC family members or other chloride 1 at 1p36.11 (4D2.3 region in mouse) where channel families may compensate and maintain the intracellu- DNA aberrations are frequent in multiple human cancer types. lar ionic homeostasis in tumors. However, little is published on

Fig. 6. Induction of CLIC4 and aSMA in fibroblasts results from a tumor stromal interaction. A, CLIC4 transcripts (black arrow) in the tumor stroma of a kidney tumor (clear cell carcinoma) in tissue arrays were detected by nonradiolabeled in situ hybridization. Inset, lower magnification (10). B, malignant breast cancer cell line MCF10CA1a or normal breast cell line MCF10A (inset , control) were cocultured with primary fibroblasts for 2 d and immunostained with CLIC4 (red)andaSMA (green) antibodies.White dotted line, colonies of cancer cells and normal breast epithelial cells. C, human dermal fibroblasts were transduced with a retrovirus encoding GFP, mixed with MCF10CA1a cells and xenografted to nude mice. Tumor sections were immunostained for GFP and CLIC4. D, lysates of cultured primary dermal fibroblasts from CLIC4 transgenic (TR) and wild-type (WT)micewere immunoblotted with anti-CLIC4 and anti-actin antibodies. E, fold increase in CLIC4 was determined by the ImageMaster densitometry program and normalized against the actin signal. MCF10CA1a cancer cells alone or mixed with primary fibroblasts (transgenic or wild type) were s.c. injected into nude mice, and the xenograft tumors were measured weekly. Each group represents 10 mice. Points, mean tumor measurements; bars, SD. F, tumors from (E) were excised, sectioned, and immunostained for CLIC4 and aSMA.

www.aacrjournals.org 12 9 Clin Cancer Res 2007;13(1) January 1, 2007 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Human Cancer Biology chloride regulation in cancer where changes in cell volume, pH, cells but in the cytoplasm in tumor cells. Other proteins with and oxygenation suggest that the physiologic environment may altered localization in tumor cells include h-catenin (membrane require specific modifications in ion transport to maintain to nucleus/cytoplasm; refs. 46, 47), cadherins (membrane growth and viability (31). to nucleus; ref. 48), BCL10 (cytoplasm to nucleus; ref. 49), Our immunostaining studies revealed predominant nuclear fibroblast growth factor receptor-3 (cytoplasm to nucleus; localization of CLIC4 in the epithelium of multiple normal ref. 50), and breast tumor kinase (nucleus to cytoplasm; tissues. In vitro, targeting CLIC4 to the nucleus by genetic or ref. 51). Changes in localization of such important proteins pharmacologic methods is associated with an apoptotic are likely to cause a loss/gain of function in essential activities response in many cell types, particularly when CLIC4 levels and interaction with alternative substrates or binding partners. are also increased (12). However, most normal epithelia in vivo Such changes could have substantial effects on cell behavior and are in a G0 resting state, and we have found a number of contribute to the malignant phenotype. situations in vitro (differentiation, senescence, and growth From our studies, we conclude that CLIC4 undergoes unique inhibition) where endogenous nuclear CLIC4 is associated with and paradoxical changes during cancer pathogenesis that are cell cycle arrest.4 CLIC4 therefore may contribute to the control common to many tumor types. An obligate reduction in of cell cycle, either through an influence on the ionic milieu of expression in tumor cells and induced expression in stromal the nucleus or an independent function. An cells in association with aSMA and myofibroblast conversion independent function of CLIC4 in the nucleus is supported by must be advantageous to tumor growth as these changes the recent finding that CLIC4 localizes at the centrosome and increase with malignant progression. As aSMA up-regulation midbody of mitotic cells (32). Furthermore, our prior work and myofibroblast differentiation in reactive stroma are revealed that nuclear CLIC4 is predominantly in the nucleo- currently used for clinical grading of certain tumors, including plasm (12), where it would not be expected to participate in ion breast, liver, lung, colon, stomach, prostate, and pancreas transport. Whatever specific functions are carried out by nuclear (52–54), CLIC4 expression may be a useful addition to the CLIC4 are obviously detrimental to cancer cells as CLIC4 is diagnostic criteria. Our studies also suggest that CLIC4 uniformly excluded from the nucleus of tumor cells even at reduction in the tumor mass is a consequence of epigenetic early stages of malignant progression. Precisely how CLIC4 is factors and therefore has the potential to be reversed. excluded from the nucleus of cancer cells in the absence of Adenoviral transduction studies, although not exceedingly alterations in the nuclear localization signal or cotransporters efficient, provide support for the principle that modifying must await an understanding of factors that control nuclear CLIC4 expression in cancer cells can reduce tumor growth. translocation under physiologic situations. Both CLIC1 and Likewise, modifying stromal CLIC4 could alter myofibroblast CLIC4 are subject to redox regulation with major structural activity. Together, these results add CLIC4 to a growing list of rearrangements and functional activities dependent on redox novel targets for cancer therapy. state (33, 34). Because cancer cells frequently exhibit altered redox regulation (35, 36), this control of CLIC4 subcellular localization should be considered. Acknowledgments Subcellular translocation of important signaling molecules is We thank Dr. Susan L. Holbeck [National Cancer Institute (NCI) Developmental a new paradigm in sorting out changes that are relevant to Therapeutics Program] for providing RNA and cells from the NCI60 collection, malignant transformation. For instance, immunohistochemical Dr. Mark Miller (DNA Sequencing MiniCore Facility) for sequencing PCR products, studies showed that cell cycle regulatory or proapoptotic Susan Garfield and Stephen Winchovich (NCI Confocal Core Facility) for guidance CIP1 KIP1 in immunofluorescent studies, Dr. Narayan Bhat (ScienceApplications International proteins p21 (37) and p27 (38, 39), KLF4/GKLF (40), Corporation, Inc., Frederick, NCI) for adenoviral amplification, Drs. SteveTurner and Apaf-1 (41), cRel (42), p33ING1 (43), supressin (44), and Pat Muraso (Clinomics Bioscience/Cytomix) for providing tumor lysate arrays, and nucleophosmin (45) were localized in the nucleus of normal Joanna Anders for assistance with references.

References 1. Jentsch TJ, SteinV,Weinreich F, Zdebik AA. Molecu- the aqueous phase into phospholipid membranes, 11. Edwards JC, Tulk B, Schlesinger PH. Functional lar structure and physiological function of chloride where it functions as an anion channel. Am J Physiol expression of p64, an intracellular chloride channel channels. Physiol Rev 2002;82:503 ^ 68. Cell Physiol 2002;282:C1103 ^ 12. protein. J Membr Biol 1998;163:119^ 27. 2. Harrop SJ, DeMaere MZ, Fairlie WD, et al. Crystal 7. Fernandez-Salas E, Sagar M, Cheng C, Yuspa SH, 12 . Suh KS, Mutoh M, Nagashima K, et al. The organ- structure of a soluble form of the intracellular chloride Weinberg WC. p53 and tumor necrosis factor a regu- ellular chloride channel protein CLIC4/mtCLIC trans- ion channel CLIC1 (NCC27) at 1.4-A resolution. J Biol late the expression of a mitochondrial chloride channel locates to the nucleus in response to cellular stress Chem 2001;276:44993^5000. protein. J Biol Chem1999;274:36488^97. and accelerates apoptosis. J Biol Chem 2004;279: 3. Duncan RR,Westwood PK, Boyd A, Ashley RH. Rat 8. Fernandez-Salas E, Suh KS, Speransky VV, et al. 4632^41. brain p64H1, expression of a new member of the p64 mtCLIC/CLIC4, an organellular chloride channel pro- 13. Shiio Y, Suh KS, Lee H, Yuspa SH, Eisenman RN, chloride channel protein family in endoplasmic reticu- tein, is increased by DNA damage and participates in Aebersold R. Quantitative proteomic analysis of lum. J Biol Chem 1997;272:23880 ^ 6. the apoptotic response to p53. Mol Cell Biol 2002;22: myc-induced apoptosis: a direct role for Myc induc- 4. Nishizawa T, Nagao T, Iwatsubo T, Forte JG, 3610^20. tion of the mitochondrial chloride ion channel, Urushidani T. Molecular cloning and characterization 9. Suginta W, Karoulias N, Aitken A, Ashley RH. Chlo- mtCLIC/CLIC4. J Biol Chem 2006;281:2750 ^ 6. of a novel chloride intracellular channel-related protein, ride intracellular channel protein CLIC4 (p64H1)binds 14. Flanders KC, Sullivan CD, Fujii M, et al. Mice lacking parchorin, expressed in water-secreting cells. J Biol directly to brain dynamin I in a complex containing Smad3 are protected against cutaneous injury in- Chem 2000;275:11164 ^ 73. actin, tubulin and 14^ 3-3 isoforms. Biochem J 2001; duced by ionizing radiation. Am J Pathol 2002;160: 5. Qian Z, Okuhara D, Abe MK, Rosner MR. Molecular 359:55^64. 10 57 ^ 6 8. cloning and characterization of a mitogen-activated 10. Chuang JZ, Milner TA, Zhu M, Sung CH. A 29 kDa 15. Basset-Seguin N, Escot C, Moles JP, Blanchard JM, protein kinase-associated intracellular chloride chan- intracellular chloride channel p64H1is associated with Kerai C, Guilhou JJ. C-fos and c-jun proto-oncogene nel. J Biol Chem 1999;274:1621 ^ 7. large dense-core vesicles in rat hippocampal neurons. expression is decreased in psoriasis: an in situ quanti- 6. Tulk BM, Kapadia S, Edwards JC. CLIC1inserts from J Neurosci 1999;19:2919^ 28. tative analysis. J Invest Dermatol 1991;97:672 ^ 8.

Clin Cancer Res 2007;13(1) January 1, 2007 130 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. CLIC4 Expression Is Altered in Epithelial Cancers

16. Ronnov-Jessen L, Villadsen R, Edwards JC, promote tumor growth and angiogenesis through Jr.,Yang S. Comparative expression of NFnBproteins Petersen OW. Differential expression of a chloride elevated SDF-1/CXCL12 secretion. Cell 2005;121: in melanocytes of normal skin vs. benign intradermal intracellular channel gene, CLIC4, in transforming 335^48. naevus and human metastatic melanoma biopsies. growth factor-h1-mediated conversion of fibroblasts 30. Allinen M, Beroukhim R, Cai L, et al. Molecular Pigment Cell Res 2004;17:173 ^ 80. to myofibroblasts. Am J Pathol 2002;161:471 ^ 80. characterization of the tumor microenvironment in 43.Vieyra D, Senger DL,ToyamaT, et al. Altered subcel- 17. Chaponnier C, Gabbiani G. Pathological situations breast cancer. Cancer Cell 2004;6:17^32. lular localization and low frequency of mutations of characterized by altered actin isoform expression. 31. Lang F, Ritter M, Gamper N, et al. Cell volume in the ING1in human brain tumors. Clin Cancer Res 2003;9: J Pathol 2004;204:386 ^ 95. regulation of cell proliferation and apoptotic cell death. 5952^61. 18. Bohman S, Matsumoto T, Suh K, et al. Proteomic Cell Physiol Biochem 2000;10:417^ 28. 44. Manne U, Gary BD, Oelschlager DK, Weiss HL, analysis of vascular endothelial growth factor-induced 32. Berryman MA, Goldenring JR. CLIC4 is enriched Frost AR, Grizzle WE. Altered subcellular localiza- endothelial cell differentiation reveals a role for chlo- at cell-cell junctions and colocalizes with AKAP350 tion of suppressin, a novel inhibitor of cell-cycle ride intracellular channel 4 (CLIC4) in tubular morpho- at the centrosome and midbody of cultured mam- entry, is an independent prognostic factor in colo- genesis. J Biol Chem 2005;280:42397 ^ 404. malian cells. Cell Motil Cytoskeleton 2003;56: rectal adenocarcinomas. Clin Cancer Res 2001;7: 19. Suh KS, Yuspa SH. Intracellular chloride channels: 15 9 ^ 72. 3495^503. critical mediators of cell viability and potential tar- 33. Littler DR, Harrop SJ, Fairlie WD, et al. The intracel- 45. Falini B, Mecucci C, Tiacci E, et al. Cytoplasmic gets for cancer therapy. Curr Pharm Des 2005;11: lular chloride ion channel protein CLIC1undergoes a nucleophosmin in acute myelogenous leukemia 2753 ^ 64. redox-controlled structural transition. J Biol Chem with a normal karyotype. N Engl J Med 2005; 20. OlsenML,SchadeS,LyonsSA,AmaralMD, 2004;279:9298 ^ 305. 352:254^66. Sontheimer H. Expression of voltage-gated chloride 34. Littler DR, Assaad NN, Harrop SJ, et al. Crystal 46. Kimura Y, FuruhataT, Mukaiya M, et al. Frequent h- channels in human glioma cells. J Neurosci 2003;23: structure of the soluble form of the redox-regulated catenin alteration in gallbladder carcinomas. JExp Clin 5572 ^ 82. chloride ion channel protein CLIC4. FEBS J 2005; Cancer Res 2003;22:321 ^ 8. 21. Yamashina S, Konno A, Wheeler MD, et al. Endothe- 272:4996 ^5007. 47. Aust DE,Terdiman JP,Willenbucher RF, et al. Altered lial cells contain a glycine-gated chloride channel. Nutr 35. Liu J, Hinkhouse MM, Sun W, et al. Redox regula- distribution of h-catenin, and its binding proteins E- Cancer 2001;40:197 ^ 204. tion of pancreatic cancer cell growth: role of glutathi- cadherin and APC, in ulcerative colitis-related colorec- 22. BustinSA,LiSR,PhillipsS,DorudiS.Expressionof one peroxidase in the suppression of the malignant tal cancers. Mod Pathol 2001;14:29 ^ 39. HLA class II in colorectal cancer: evidence for en- phenotype. Hum GeneTher 2004;15:239 ^ 50. 48. Han AC, SolerAP,Tang CK, Knudsen KA, Salazar H. hanced immunogenicity of microsatellite-instability- 36. Laurent A, Nicco C, Chereau C, et al. Controlling Nuclear localization of E-cadherin expression in Mer- positive tumours.Tumour Biol 2001;22:294 ^8. tumor growth by modulating endogenous production kel cell carcinoma. Arch Pathol Lab Med 2000;124: 23. Bustin SA, Li SR, Dorudi S. Expression of the Ca2+- of reactive oxygen species. Cancer Res 2005;65: 114 7 ^ 51. activated chloride channel genes CLCA1and CLCA2 is 948^56. 49.YeH,DoganA,KarranL,etal.BCL10expressionin downregulated in human colorectal cancer. DNA Cell 37. Wang Z, Kishimoto H, Bhat-Nakshatri P, Crean C, normal and neoplastic lymphoid tissue. Nuclear local- Biol 2001;20:331 ^ 8. Nakshatri H. TNFa resistance in MCF-7 breast cancer ization in MALT lymphoma. Am J Pathol 2000;157: 24. Pauli BU, Abdel-Ghany M, Cheng HC, Gruber AD, cells is associated with altered subcellular localization 1147 ^ 54. Archibald HA, Elble RC. Molecular characteristics and of p21CIP1and p27KIP1. Cell Death Differ 2005;12: 50. Zammit C, Barnard R, Gomm J, et al. Altered in- functional diversity of CLCA family members. Clin Exp 98^100. tracellular localization of fibroblast growth factor re- Pharmacol Physiol 2000;27:901 ^ 5. 38. MinYH, Cheong JW, Kim JY, et al. Cytoplasmic mis- ceptor 3 in human breast cancer. J Pathol 2001;194: 25. Elble RC, Pauli BU. Tumor suppression by a proa- localization of p27Kip1protein is associated with con- 27^ 34. poptotic calcium-activated chloride channel in mam- stitutive phosphorylation of Akt or protein kinase B 51. DerryJJ, Prins GS, RayV,TynerAL. Altered localiza- mary epithelium. J Biol Chem 2001;276:40510^ 7. and poor prognosis in acute myelogenous leukemia. tion and activity of the intracellular tyrosine kinase 26. ShenMR,ChouCY,HsuKF,etal.KClcotransportis Cancer Res 2004;64:5225^ 31. BRK/Sik in prostate tumor cells. Oncogene 2003;22: an important modulator of human cervical cancer 39. Sgambato A, Ratto C, Faraglia B, et al. Reduced 4212^ 20. growth and invasion. J Biol Chem 2003;278: expression and altered subcellular localization of the 52. Seemayer TA, Lagace R, Schurch W, Tremblay G. 39941^50. cyclin-dependent kinase inhibitor p27(Kip1) in human Myofibroblasts in the stroma of invasive and meta- 27. Samoszuk M,Tan J, Chorn G. Clonogenic growth of colon cancer. Mol Carcinog 1999;26:172 ^ 9. static carcinoma: a possible host response to neopla- human breast cancer cells co-cultured in direct con- 40. Pandya AY,Talley LI, Frost AR, et al. Nuclear locali- sia. Am J Surg Pathol 1979;3:525^ 33. tact with serum-activated fibroblasts. Breast Cancer zation of KLF4 is associated with an aggressive phe- 53. Martin M, Pujuguet P, Martin F. Role of stromal Res 2005;7:R274 ^ 83. notype in early-stage breast cancer. Clin Cancer Res myofibroblasts infiltrating colon cancer in tumor inva- 28. Stuelten CH, DaCosta BS, Arany PR, Karpova TS, 2004;10:2709 ^ 19. sion. Pathol Res Pract 1996;192:712^ 7. Stetler-Stevenson WG, Roberts AB. Breast cancer 41. Besse B, Cande C, Spano JP, et al. Nuclear locali- 54. Ronnov-Jessen L, Petersen OW, Koteliansky VE, cells induce stromal fibroblasts to express MMP-9 via zation of apoptosis protease activating factor-1 pre- Bissell MJ. The origin of the myofibroblasts in breast secretion of TNF-a and TGF-h. J Cell Sci 2005;118: dicts survival after tumor resection in early-stage cancer. Recapitulation of tumor environment in culture 2143 ^ 53. non-small cell lung cancer. Clin Cancer Res 2004; unravels diversity and implicates converted fibroblasts 29. Orimo A, Gupta PB, Sgroi DC, et al. Stromal fibro- 10:5665^9. and recruited smooth muscle cells. J Clin Invest 1995; blasts present in invasive human breast carcinomas 42. McNulty SE, del Rosario R, Cen D, Meyskens FL, 95:859^73.

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Kwang S. Suh, John M. Crutchley, Arash Koochek, et al.

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