CLIC4 is a highly conserved, multifunctional protein that causes growth arrest and terminal differentiation in skin keratinocytes and other cell types. We have reported that CLIC4 is reduced in virtually all major human cancer parenchyma and highly upregulated in virtually all human cancer stroma. Major insight into how CLIC4 functions in growth control came with our discovery that CLIC4 is a component of the TGFbeta pathway. In skin keratinocytes, TGFbeta causes CLIC4 to associate with Schnurri-2 and translocate to the nucleus. Nuclear CLIC4 prolongs TGFbeta signaling by inhibiting Smad2/3 dephosphorylation. TGFbeta is fundamental to the conversion of fibroblasts to myofibroblasts, an important event in skin wound healing, fibrosis and cancer development. We have now determined that CLIC4 participates in TGFbeta dependent myofibroblast conversion of dermal fibroblasts. Using primary dermal fibroblasts from CLIC4wt/wt and CLIC4fl/fl mice transduced with adenoviral Cre recombinase to ablate CLIC4 expression, we show that absence of CLIC4 inhibits myofibroblast conversion as measured by transcription and translation of alpha smooth muscle actin (alphaSMA). Expression of extracellular matrix components like the collagens, MMPs and thrombospondins that TGFbeta induces during fibroblast activation is reduced in TGFbeta treated adeno-Cre infected CLIC4 fl/fl fibroblasts. These cells also have higher cell motility compared to wild type controls. Fibroblasts deleted of CLIC4 have reduced activation of Smad2 and p38 upon TGFbeta stimulation. Smad2 siRNA and p38 kinase inhibitors prevented TGFbeta stimulation of myofibroblast conversion suggesting that CLIC4 might influence fibroblast differentiation through interaction with both Smad-dependent and Smad-independent TGFbeta pathways. Conversely, conditioned medium from fibroblasts that overexpress CLIC4 enhances p38 activation and migration of epithelial cells. Thus CLIC4 plays an important role in both epithelial and mesenchymal cell biology by altering TGFbeta signaling and is therefore an attractive target for modifying TGFbeta in both tissue compartments. Our previous studies have shown that the biological functions of CLIC4 relative to growth inhibition and cancer occur in the nucleus. CLIC4 has a functional nuclear localization signal but nuclear translocation of CLIC4 is defective in cancer cells. Thus it is important to understand the factors that control CLIC4 nuclear translocation. We have recently established that nitric oxide directly regulates nuclear translocation of CLIC4 by S-nitrosylation of specific cysteines in the protein. The modification induces a conformational change and results in enhanced association of the protein with nuclear import proteins Ran and importin alpha. Moreover, TNFalpha-induced nuclear translocation of CLIC4 is dependent on nitric oxide synthase (NOS)-activity. In macrophages iNOS (inducible nitric oxide synthase) induction and activity increased nuclear translocation and S-nitrosylation of CLIC4. Chemical inhibition or genetic ablation of iNOS decreases nuclear translocation and S-nitrosylation of CLIC4 in response to LPS and IFNgamma. Previous results showed that Ca2+-induced differentiation of keratinocytes enhances nitrosylation of CLIC4 as early as 3hrs. This is coincident with increased p-eNOS (S1176) that indicates enhanced eNOS activity. Chemical inhibition of eNOS activity using L-NAME inhibits Ca2+-induced nuclear translocation of CLIC4 and expression of K1 and K10. Expression of K10 can be increased under these conditions when an NO-donor is added back. We hypothesize that changes in redox potential in tumor cells are responsible for the altered nuclear localization of CLIC4. To that end, we have evaluated the expression and activity of thioredoxin reductase 1 (TR1) and thioredoxin in human and mouse tumor cell lines. Both human (SCC13 and HaCaT-Ras cells) and mouse (Pam212) carcinoma cells show high levels of TR1 protein and activity compared to normal cells. Our preliminary results suggest that pretreatment of Pam212 cells with auranofin, a TR1 inhibitor, followed by an NO-donor restores CLIC4 nuclear translocation. Indeed, Kras-transformed cells stably knocked down for Trx or TR1 show enhanced levels of CLIC4 translocation compared to the parental cell line. These results suggest that higher expression/activity of thioredoxin/thioredoxin reductase pathway may contribute to the aberrant translocation of CLIC4. The analysis of CLIC4 function in cancer pathogenesis should be facilitated by our recent production of CLIC4 knockout mice using recombineering technology. CLIC4 knockout mice are viable and fertile but develop skin erosions at high frequency after about 3 to 6 months. Knockout skin, fibroblasts and keratinocytes have TGFbeta signaling defects as evidenced by reduced Smad2 phosphorylation and increased c-Myc. Knockout keratinocytes also display defects in TGFbeta dependent transcript expression on PCR arrays. Knockout fibroblasts proliferate faster that WT cells and are less sensitive to TGFbeta induced growth arrest. Corneal epithelial wound healing is delayed in knockout mice suggesting the skin erosions might occur due to defects in wound healing. Currently additional mice are being generated targeting the CLIC4 knockout to epidermal cells and fibroblasts. Furthermore, a knockin mouse which would express Green Fluorescent Protein (GFP) under the control of CLIC4 promoter is undergoing construction. While these basic studies in tumor progression are ongoing we are also conducting studies to illuminate the mechanism of action of drugs that are designed to treat skin cancers. Ingenol-3-angelate (Ing3A), extracted from Euphorbia peplus, is currently in clinical trials for eradicating basal cell carcinoma (BCC), actinic keratosis and squamous cell carcinoma (SCC) in situ by topical application. Although structurally related to phorbol esters and a PKC activator, topical Ing3A, but not phorbol 12 myristate 13 acetate (PMA), inhibited the growth of subcutaneous tumors derived from PAM212 (mouse SCC) and B16 (mouse melanoma). Ing3A and PMA both induced acute neutrophilic inflammation on mouse skin, but only Ing3A caused subcutaneous hemorrhage and vascular damage. Both Ing3A and PMA activated Erk1/2 in epidermis, but Ing3A also activated Erk1/2 in skin dermal fibroblasts and endothelial cells. Pretreatment with topical cyclosporin A (CsA), verapamil or XR9576, modulators of P-glycoprotein (P-gp), prevented Ing3A induced hemorrhage but not neutrophil infiltration. CsA also impaired Ing3As anti cancer activity while the anti-inflammatory dexamethasone did not. In collaboration with Suresh Ambudkar we are examining the role of P-gp in the unusual properties of Ing3A. Ing3A, but not PMA, blocked photoaffinity labeling of human P-gp with [125I]-Iodoaryazidoprazosin and inhibited P-gp mediated drug resistance to HCT-15 cells. The intracellular levels of Ing3A were significantly lower in P-gp expressing cells and treatment with XR9576 increased the levels to those of cells that do not express P-gp, demonstrating that Ing3A binds to and is transported by P-gp. Taken together, our results suggest that P-gp mediated absorptive transport, dermal penetration and vascular damage contribute to the anti-cancer activity of Ing3A in vivo. These finding may serve as a paradigm for percutaneous absorption of other therapeutic agents and such studies are now in progress.
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