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. Additional studies on the immunologic basis of the autoimmune phenotype through TGFbeta and T-reg function are being conducted in collaboration with John O'Shea. Since CLIC4 is released from myofibroblasts, we are designing studies to determine if circulating CLIC4 might serve as a tumor biomarker. 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. We examined the plasma of patients before and after treatment with gefitinib and documented changes in chemokines and leukocyte counts associated with the extent of rash or the presence of pruritus. 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. Bone marrow derived macrophages from EGFR null mice differ from wildtype mice in colony formation and expression of transcripts for inflammatory mediators. Thus, the systemic disease emanating from cytokines and chemokines released from the skin of the epidermal targeted EGFR null mice influences progenitor cells in the bone marrow. Braf inhibitors are clinically important agents for the treatment of advanced melanoma, however secondary cutaneous tumors are a common side effect; non-melanoma skin cancers (NMSCs) arise in 15-30% of patients on Braf inhibitor therapy and 60% of these harbor ras mutations. NMSC also occurs in organ transplant patients receiving immunosuppressive therapy, and switching from a calcineurin inhibitor-containing regimen to rapamycin reduces the incidence. To examine the effect of rapamycin on ras-driven epidermal squamous cell tumors, in collaboration with Phillip Dennis at Johns Hopkins Cancer Center, we treated mutant K-RasLA2 mice with rapamycin or vehicle by intraperitoneal injection and found that rapamycin prevented the development of squamous skin tumors and also rapidly reduced the tumor size. We employed a syngeneic xenograft model using H-Ras mutant primary murine keratinocytes to determine if rapamycin could hamper the ability of a Braf inhibitor to enhance squamous skin tumor growth. As seen in the K-RasLA2 model, rapamycin alone diminished the growth of tumors and it decreased the volume even more in the presence of a Braf inhibitor. Furthermore, treatment of established tumors with rapamycin resulted in significant tumor shrinkage even in the continuous presence of a Braf inhibitor. In vitro, Braf inhibition enhanced mutant H-Ras-induced activation of the Raf-ERK and mTOR pathways in keratinocytes, while rapamycin addition blocked the activation of signaling pathways and decreased cell proliferation. Taken together, rapamycin prevents murine skin tumor development arising from oncogenic mutations in two distinct ras gene alleles, and reduced the tumor size and inhibited downstream oncogenic pathways. Thus, upon identifying the high-risk population developing ras-driven NMSC, administration of rapamycin as a chemopreventive and therapeutic agent, including those receiving Braf inhibitors, may be considered.
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