CLIC4 is a metamorphic, multifunctional, redox regulated protein that is lost from cancer cells during tumor progression. To evaluate the function of CLIC4 in vivo we have generated CLIC4 floxed mice and ablated the gene en toto or in skin keratinocytes. The null mice have spontaneous skin wounds and reduced skin wound healing in full thickness and abrasion experiments. Further, they have defective corneal wound healing. The basis for these wound healing defects could be related to the integral function of CLIC4 in TGFbeta signaling. Keratinocytes from CLIC4 null mice (KO) have reduced TGFbeta responses with lower induction of pSmads after stimulation with TGFbeta. They also have reduced p21 expression. Mapping of CLIC4 expression using a mouse model where GFP has been inserted downstream of the CLIC4 promoter reveals differential expression of CLIC4 during mouse brain development and high expression is kidney cortex, bone marrow and testes in adult mice. CLIC4 KO mice have enlarged spleens and extramedullary hematopoiesis and a blood cell phenotype suggestive of an autoimmune disease. CLIC4 is highly expressed in macrophages where CLIC4 nuclear translocation in proinflammatory macrophages is important for deactivation of the cells, including downregulating IL-1beta and iNOS. Nuclear translocation of CLIC4 in these cells is dependent on iNOS activity. We are now working to establish the pathway and mechanism that is responsible for this prolonged pro-inflammatory response in CLIC4 knockout macrophages. TGFbeta signaling, as measured by Smad4 and p-Smad3 levels, as well as p-p38 levels is altered in CLIC4 null macrophages compared to wildtype macrophages. Both of these pathways have been implicated in deactivation of macrophages, and moreover are modulated by CLIC4 in TGFbeta mediated growth arrest in keratinocytes and TGFbeta mediated myofibroblast conversion, respectively. CLIC4 modifies carcinogenesis in a TGFbeta context dependent manner. Similar to the dual role of TGFbeta signaling, CLIC4 is a tumor suppressor in the epithelium and a tumor promoter in the stromal compartment. Tumor cells express low levels of nuclear CLIC4 and are refractory to TGFbeta dependent growth inhibition. Restoring nuclear CLIC4 in these cells reinstates TGFbeta transcriptional activity and growth inhibition. Overexpression of exogenous CLIC4 in tumor orthografts enhances TGFbeta signaling and inhibits tumor growth. Our studies indicate that reduced CLIC4 expression and its exclusion from the nucleus in cancers contribute to TGFbeta resistance and enhance tumor development. In contrast to the tumor, CLIC4 expression is highly enhanced in tumor stroma. Xenografts using fibroblasts overexpressing CLIC4 form larger tumors that express greater amounts of alpha smooth muscle actin, a hallmark of activated fibroblasts. Genetic ablation of CLIC4 in primary fibroblasts prevents or reduces constitutive or TGFbeta induced expression of alpha smooth muscle actin, and extracellular matrix components that are markers of myofibroblasts. Stromal CLIC4 enhances tumor cell migration and invasion in a TGFbeta dependent manner. CLIC4 is significantly involved in the development of a nurturing tumor microenvironment by enhancing TGFbeta signaling and thus acts as a tumor promoter in this compartment. Based on the above, CLIC4 may be an attractive target both in cancer stages where TGFbeta signal augmentation or inhibition is required due to responses in separate tissue compartments. Targeting CLIC4 would also be a more specific approach in therapy and mitigate some of the severe side effects of global targeting of the TGFbeta pathway. Abrogation of CLIC4 nuclear translocation in human and mouse cancer cells may be a consequence of enhanced antioxidant pathways in the transformed cells, namely the thioredoxin/thioredoxin reductase pathway. Nuclear CLIC4 can be restored by inhibiting this pathway and by treating the cells with nitric oxide (NO) donors to induce translocation. We are collaborating with Larry Keefer using NO-releasing prodrugs like PABA-NO and JSK to inhibit antioxidant pathways and in the process release NO. We are working to delineate whether CLIC4 is a target of these drugs in human squamous carcinoma cells. Several years ago we identified ingenol-3-angelate, a PKC activator now in the clinic for topical treatment of actinic keratosis, basal cell carcinoma and squamous cell cancer in situ, as a substrate for cutaneous P-glycoprotein ( P-gp,ABCB1). The unique character of this drug?s activity is by its ability to penetrate below the epidermis by absorptive transport to damage tumor blood vessels. In collaboration with Dr John Wood at Colorado State University, we evaluated synthetic ingenol derivatives for their effect on P-gp mediated efflux and PKC activation. All compounds are able to block P-gp mediated efflux and activate PKC in mouse keratinocytes. In order to do a wider screen of natural products for PKC activators, we established mouse keratinocyte cell lines that stably express an AP-1 reporter gene. Since ingenol-3-angelate is only about 50-60% effective in causing regression of skin lesions, we hope to find a more effective topical anti-skin cancer agent that is transported by absorptive transfer and active against tumor blood vessels.

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Shukla, Anjali; Yang, Yihan; Madanikia, Sara et al. (2016) Elevating CLIC4 in Multiple Cell Types Reveals a TGF- Dependent Induction of a Dominant Negative Smad7 Splice Variant. PLoS One 11:e0161410
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Sengupta, Aniruddha; Lichti, Ulrike F; Carlson, Bradley A et al. (2013) Targeted disruption of glutathione peroxidase 4 in mouse skin epithelial cells impairs postnatal hair follicle morphogenesis that is partially rescued through inhibition of COX-2. J Invest Dermatol 133:1731-41
Ansbro, Megan R; Shukla, Suneet; Ambudkar, Suresh V et al. (2013) Screening compounds with a novel high-throughput ABCB1-mediated efflux assay identifies drugs with known therapeutic targets at risk for multidrug resistance interference. PLoS One 8:e60334
Mascia, Francesca; Lam, Gary; Keith, Christopher et al. (2013) Genetic ablation of epidermal EGFR reveals the dynamic origin of adverse effects of anti-EGFR therapy. Sci Transl Med 5:199ra110
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