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. CLIC4 is a redox sensitive protein. Collaboration with the Hatfield laboratory indicates that selenoprotein antioxidants have a marked effect on keratinocyte viability and skin integrity. We are addressing the role of CLIC4 in selenoprotein function. CLIC4 null keratinocytes are less responsive to TGF-beta induced migration in vitro and adhere with less efficiency on plates coated with matrix secreted by wild type keratinocytes. Skin tumor induction with chemical carcinogens did not demonstrate differences in benign tumor formation between CLIC4 null and intact mice, and results for malignant conversion are pending. Currently, mice lacking CLIC4 in fibroblasts are being generated by crossing with FSP1 Cre mice. CLIC4-GFP knockin mice have been generated to study the expression of CLIC4 during embryonic development and in adult tissues and cell types. CLIC4 expression is high in hair follicles, and CLIC4 is highly expressed in the renal cortex as opposed to the medulla. CLIC4 is also highly expressed in spermatozoa and a diminishing gradient is seen in spermatocytes and spermatogonia. CLIC4 is highly expressed in endothelial cells in blood vessels of all organs studied including cardiac blood vessels, but myocardial expression is low. CLIC4 mRNA expression is particularly high in macrophages upon stimulation with IFNgamma and LPS. In the absence of CLIC4, macrophages are defective in phagocytosis. Furthermore, the expression of IL-1beta, iNOS and CXCL1 is prolonged in null compared to wildtype macrophages suggesting attenuation of these immunomodulators is misregulated in the absence of CLIC4. TGF-beta regulates the dissipation of macrophage activity, and TGF-beta signaling is lower in CLIC4 null macrophages, with lower Smad4 levels. Overexpression of nuclear targeted CLIC4 or HA-CLIC4 in stimulated macrophages downregulates active IL-1beta protein suggesting CLIC4 is essential for TGF-beta-mediated downregulation of the immune response. In the past year, we have furthered our study of CLIC4 and pSmad2 protein expression in cancer. In separate collaborations with Dr. Carter Van Waes and Dr. Addie Alkhas we have shown that CLIC4 and nuclear CLIC4 are reduced in HNSCC and SCC of the cervix. Collaborations with Mark Simpson and Joshua Webster have demonstrated that expression of exogenous CLIC4 in skin SCC orthografts causes growth inhibition and activation of TGF-beta signaling in tumor cells. Furthermore, conditional overexpression of exogenous CLIC4 in mouse skin reduces the de novo tumor yield in initiation-promotion studies. While we have focused attention on CLIC4 in tumor cells, we are also cognizant of the role of CLIC4 and TGF-beta in the tumor stroma. CLIC4 is essential for TGF-beta to induce myofibroblast conversion in the formation of a cancer stroma. Conditioned media from tumor cells induces CLIC4 expression in fibroblasts via TGF-beta signaling. Tumor cells secrete several folds greater amounts of TGF-beta compared to normal cells. Conditioned media from fibroblasts overexpressing CLIC4 enhances migration and invasion of epithelial cells via TGF-beta signaling. These conditioned media secrete more TGF-beta than control fibroblasts. Thus CLIC4 enhances crosstalk between tumor cells and the tumor microenvironment through TGF-beta signaling. Myofibroblast conversion via a CLIC4 pathway is Smad and p38 dependent. p38alpha is the major isoform responsible for myofibroblast conversion and contrary to a previous report, changes in ROS do not alter conversion in dermal fibroblasts. Absence of CLIC4 enhances interaction of phospho p38 with PPM1a, its specific phosphatase. PPM1a interacts with CLIC4, and it is likely that this interaction interrupts PPM1a association with its other substrates thereby explaining how CLIC4 could potentially enhance both pSmad and p38 activation. We have continued to study the contribution of active transdermal transport of drugs through keratinocyte P-gp in collaboration with the Blumberg and Ambudkar laboratories. Cyclosporin A is a well-known P-gp substrate. Cyclosporin A is detected in plasma of nude and haired mice after topical application of cyclosporin A at 16 mg/kg. A stable level of plasma cyclosporin A (110-140 nmol/L) is detected when cyclosporin A is applied daily for 7 consecutive days to the dorsal skin of nude mice at 16 mg/kg. XR9576, a P-gp inhibitor, partially blocks skin P-gp mediated cyclosporin A absorption. We used an efflux assay and a P-gp mediated drug toxicity resistance assay to identify P-gp substrates among a number of phorbol ester derivates and other natural compounds that activate protein kinase C. Phorbol 12,13-dihexanoate, phorbol 12,13-dibutyrate and phorbol 12,13-dibenzoate are substrates of P-gp. However, when topically applied to the dorsal skin of nude mice, all three phorbol ester derivates failed to induce hemorrhage presumeably because of their weak activity as a protein kinase C activator. Our cancer studies have also extended into understanding the activity of RasGRP3 in melanoma and prostate cancer in collaboration with the Blumberg laboratory. RasGRP3, a Ras activator, is elevated in these tumors from patients. Experimentally, overexpression of rasGRP3 in tumor cells enhances prostate or melanoma xenografts while ablation of rasGRP3 in these same cells inhibits tumor formation.

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Mascia, Francesca; Schloemann, Derek T; Cataisson, Christophe et al. (2016) Cell autonomous or systemic EGFR blockade alters the immune-environment in squamous cell carcinomas. Int J Cancer 139:2593-7
Kelsey, Jessica S; Cataisson, Christophe; Chen, Jinqiu et al. (2016) Biological activity of the bryostatin analog Merle 23 on mouse epidermal cells and mouse skin. Mol Carcinog 55:2183-2195
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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Shukla, A; Edwards, R; Yang, Y et al. (2014) CLIC4 regulates TGF-?-dependent myofibroblast differentiation to produce a cancer stroma. Oncogene 33:842-50
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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
Wright, Lisa Nolan; Ryscavage, Andrew; Merlino, Glenn et al. (2012) Modeling the transcriptional consequences of epidermal growth factor receptor ablation in Ras-initiated squamous cancer. Clin Cancer Res 18:170-83

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