Reciprocal modifications of CLIC4 in tumor epithelium and stroma mark malignant progression of multiple human cancers

Laboratory of Cellular Carcinogenesis and Tumor Promotion, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA.
Clinical Cancer Research (Impact Factor: 8.19). 02/2007; 13(1):121-31. DOI: 10.1158/1078-0432.CCR-06-1562
Source: PubMed

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

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    • "The chloride channel (CLIC1-5) except CLIC4 became overexpressed in cancer cells. CLIC4 expression reduced in tumour cells [115, 116] and ion channels used to inhibit cancer cell growth [117]. The flow of potassium ions plays important functions, such as cell proliferation, angiogenesis or cell migration, which have also recently been assessed [118, 119]. "
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    • "CLIC4 (chloride intracellular channel 4) is a downstream effector of TGF signaling pathway, regulating transition of normal fibroblasts to activated pro-metastatic myo-fibroblasts through p38 signaling. Renal, ovarian, and breast cancers showed increased production of CLIC4, which should be considered as a new target of anti-tumor therapy (Suh et al., 2007; Shukla et al., 2013). TGF is also responsible for tumor recurrence through IL-8-depend-ent activation of cancer stem-like cells, as was shown in patients with breast cancer (Bhola et al., 2013). "
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    • "CLIC4 belongs to the chloride intracellular channel (CLIC) family of proteins, the most studied of the seven highly homologous members [14], [15]. Reports on the subcellular localization of CLIC4 in vitro still do not form a coherent pattern; CLIC4 seems localized in the cytoplasm, mitochondria [16], ER membrane, in large dense core vesicles in neurons, and in the nucleus [14]. "
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