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. To model this skin rash in a mouse model, EGFR was ablated in the epidermis in the litters derived from Keratin 5 driven Cre recombinase transgenic mice crossed with EGFR floxed mice. 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. Macrophages influence the differentiation of the skin and control the infiltration of mast cells within the EGFR ablated skin. Recruitment of a macrophage cell line, RAW , in migration assays is greater in the presence of culture supernatants from keratinocytes isolated from EGFR null mice when compared to similar culture supernatants from EGFR expressing mice. We tested the effects of minocycline, a drug in clinical use for treating the cutaneous rash of anti EGFR treated patients, in chemotaxis assays with the macrophage cell line. Minocycline reduces the migration induced by EGFR null supernatants in a dose dependent manner independent of its antibiotic properties. In vivo, subcutaneous administration of minocycline improves skin xerosis and whisker morphology of EGFR null mice. Supernatants of cultured EGFR wildtype and null keratinocytes induce different levels of inflammatory markers in macrophages and cause the differential phosphorylation of the kinase FAK and p38. In contrast S6 kinase, ERK1/2, JNK1/2 and AKT phosphorylation levels are not different upon stimulation with wildtype and null supernatants. Mass Spectrometry tests on the supernatants of EGFR expressing and EGFR null keratinocytes are in progress to identify the chemotactic factors that could be possibly targeted in migration assays and in vivo. Bone marrow derived macrophages isolated from 2 week old mice wiltype and nullfor epidermal EGFR display a different colony forming ability and have different mRNA expression levels of inflammatory markers. Thus the systemic disease emanating from cytokines and chemokines released from the skin of the skin targeted EGFR null mice influences progenitor cells in the bone marrow. ctivation of MET signaling is associated with multiple cancers but its function in the process of carcinogenesis has not been elucidated. The multistage induction of tumors on mouse skin was used to address this question in collaboration with Glenn Merlino. Double transgenic mice were generated by crossing K5-PKCalpha mice that overexpress PKCalpha in basal keratinocytes and are tumor promotion sensitive with MT-HGF mice that overexpress the MET ligand HGF under a metallothionein 1 promoter to create MT-HGF/ K5-PKCalpha (DT) mice and their respective controls. DT animals developed six fold more squamous tumors than other genotypes when exposed to a single tumor initiating application of dimethylbenz(a)anthracene followed by six applications of tumor promoting 12-O-tetradecanoyl-13-phorbol acetate (TPA). Both DT and MT-HGF keratinocytes in vitro displayed MET-mediated autonomous conversion to a morphological and biochemical phenotype characteristic of initiation by oncogenic H-Ras. Enhanced MET signaling activated EGFR through specific upregulation of EGFR ligands, a common pathway downstream from oncogenic Ras initiation. Inhibition of EGFR activity reversed many of the biochemical changes resulting from enhanced MET signaling. In vitro and in vivo, MT-HGF keratinocytes and mice responded to TPA like wildtype keratinocytes while DT keratinocytes and mice responded to TPA like promotion sensitive K5-PKCalpha keratinocytes. Thus, enhanced MET signaling alone did not influence the response to a tumor promoter. In vivo without chemical initiation, DT mice developed multiple squamous papillomas when promoted by TPA while none of the three other genotypes developed tumors. Mutations were not detected in any Ras allele in DT tumors. Together these results indicate that enhanced keratinocyte MET activity can initiate skin tumor formation through autocrine activation of EGFR, and the initiating activity of MET in epidermal keratinocytes can substitute for RAS mutations to produce skin tumors in response to a strong promoting stimulus. 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. To explore this effect 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 by inhibiting downstream oncogenic pathways. Thus, upon identifying the high-risk population developing ras-driven NMSC, clinical application of rapamycin as a chemopreventive and therapeutic agent for the population including those receiving Braf inhibitor is warranted. S100 proteins are frequently upregulated in cancer from multiple sites. This laboratory has been particularly interested in S100A7 and its murine homologue S100A7/15 because of its high expression in psoriasis, a cutaneous inflammatory disease with resistance to skin cancer development. We developed an epidermal targeted conditional S100A7/15 transgenic mouse that has many of the skin changes and inflammatory characteristics of human psoriasis. We used these mice to induce cutaneous cancers by initiating with 25ug of DMBA and promoting for 20 weeks with 5ug of TPA. During the 10 months of observation, we noted that benign tumors developing on the S100A7/15 mice were smaller than tumors in control groups and failed to progress to cancer. Currently analysis of the inflammatory infiltrates that might influence progression is underway.
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