Premalignant progression and malignant conversion are multistage processes and in the skin carcinogenesis model this progression occurs with predictable sequential expression of markers and genetic/epigenetic changes that define stages of progression. For example, a subset of skin papillomas is marked for progression as they erupt (high risk tumors), and they have a specific phenotypic and genetic signature. CLIC4, a p53 and TNFalpha regulated gene, is a metamorphic, multifunctional, redox regulated protein that is lost from cancer cells and highly expressed in cancer stroma during tumor progression. CLIC4 translocates to the nucleus where it is an integral component of the TGFbeta signaling pathway. Nitrosylation of critical cysteines in CLIC4 determine nuclear translocation, and altered NO production in tumor cells may reduce nuclear translocation. In previous studies we have shown that overexpressing CLIC4 in tumor cells reduces tumor growth and increases tumor response to growth suppression by TGFbeta. Additional studies indicate that a contrasting role for CLIC4 exists in tumor stroma where it is required for TGFbeta induced myofibroblast conversion. Overexpression of CLIC4 in stromal cells enhances tumor growth in vivo and invasion in vitro. The interaction of CLIC4 with the phosphatase PPM1a is essential to its TGFbeta regulation by preventing dephosphorylation of p-Smads and p-p38 after activation of the TGFbeta receptor. CLIC4 null mice have an autoimmune phenotype with spontaneous skin erosions and enlarged spleens with massive extramedullary hematopoiesis possibly related to high circulating G-CSF. CLIC4 contributes to deactivation of macrophages, and bone marrow derived macrophages from CLIC4 null mice produce supra-normal levels of TNFalpha, IL-6, IL1-beta and iNOS in response to LPS. LPS treatment increases the phosphorylation of p38 in these cells, and inhibition of p38 decreases the levels of IL-6 in CLIC4 null macrophages. We are evaluating CLIC4 as a biomarker for cancer development and recurrence. . Ovarian cancer cells and ovarian cancer stroma and breast cancer cells and breast cancer stroma release CLIC4 into exosomes. In mouse models the level of circulating CLIC4 in exosomes corresponds directly to increasing tumor growth and metastatic development. Using breast cancer cells deleted of CLIC4 by CRSPR, indicates CLIC4 does not modify primary tumor growth or metastasis and CLIC4 in circulating exosomes is derived from the tumor cells. Furthermore, CLIC4 deficient mice present reduced metastatic burden, indicating that CLIC4 from the host may be involved in mechanisms such as formation of the metastatic niche, extravasation of tumor cells, and angiogenesis. The mechanism for the loss of CLIC4 expression in the tumor epithelial cells of squamous cancers in multiple tissue sites remains unexplained. Current studies indicate that methylation of the CLIC4 gene is not altered and suggest the involvement of microRNA in regulating CLIC4 transcription/translation. We have identified several microRNAs that directly target the CLIC4 3'UTR leading to protein downregulation. One microRNA currently under investigation is also upregulated in the circulation of patients with head and neck squamous cell carcinoma, where CLIC4 is often downregulated. CLIC4 has redox enzymatic activity that is not lost bysignificantly affected by phosphorylation by PKC CKII oandr PKC-alphaCDK5. The subcellular localization of CLIC4 can be modified in response to oxidative stress and seems to depend on the levels of catalase and the reduced tripeptide glutathione (GSH) content in cells. With the development of targeted based cancer therapy, the skin has evolved as a primary target for adverse events leading to treatment failure. Our long experience combining skin and cancer biology place us in a unique position to evaluate these unexpected relationships. A subset of patients with NSCLC, HNSCC, mCRC and pancreatic cancer are responding to therapy by several agents directed against the epidermal growth factor receptor (EGFR). Uniformly patients develop a papulopustular follicutis often accompanied by alopecia, xeroderma and changes in nails and eyelashes. The discomfort and pruritis can be so severe that treatment may be terminated. To model this skin rash in a mouse, EGFR was ablated in the epidermis using cre-lox technology. The skin of double transgenic mice (EGFR null) reproduced the hallmarks of the skin lesions of patients undergoing chemotherapy with anti-EGFR agents: inflammation, pruritis, dry skin with neutrophilic pustules and infiltration of mast cells, macrophages and lymphocytes. We also documented changes in plasma cytokine/chemokine levels emanating from the skin lacking the EGFR. The mouse studies suggest that macrophages or mast cells may be fundamentally causative in the rash phenotype, and human adverse skin response to anti-EGFR drugs may be predicted based on circulating chemokine/cytokine levels before treatment begins. Macrophages play a pathogenic role in the development of EGFR depleted skin lesions as treatment of null mice with clodronate to eliminate macrophages reduces the severity of the phenotype. The intense cutaneous inflammation elicited by systemic inhibition of EGFR activity suggested that the immune microenvironment in the tumor mass may also be modulated by these drugs. To test this, wildtype or EGFR null mouse keratinocytes were transformed by oncogenic H-ras and grafted to syngeneic immune competent hosts where they formed squamous cell carcinomas. After cancers formed, mice bearing wildtype cancers were treated for one week with gefitinib to inhibit EGFR systemically. Both tumor cell autonomous and systemic loss of EGFR activity indicated significant changes in T-reg infiltration into the tumor microenvironment and other immune mediated alterations suggesting that local immune modulation by anti-EGFR drugs might contribute to their anti-tumor activity.

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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
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
Padmakumar, Vc; Masiuk, Katelyn E; Luger, Dror et al. (2014) Detection of differential fetal and adult expression of chloride intracellular channel 4 (CLIC4) protein by analysis of a green fluorescent protein knock-in mouse line. BMC Dev Biol 14:24
Shukla, A; Edwards, R; Yang, Y et al. (2014) CLIC4 regulates TGF-?-dependent myofibroblast differentiation to produce a cancer stroma. Oncogene 33:842-50
Wojciak-Stothard, Beata; Abdul-Salam, Vahitha B; Lao, Ka Hou et al. (2014) Aberrant chloride intracellular channel 4 expression contributes to endothelial dysfunction in pulmonary arterial hypertension. Circulation 129:1770-80
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
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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