Non-melanoma skin cancer is an extensive and growing problem in the United States and around the world. Of particular concern is the dramatically increased prevalence of non-melanoma skin cancer in therapeutically immunosuppressed patients. The most studied population concerning this problem is solid organ transplant recipients. The immunosuppressive therapy they receive to prevent organ rejection has been implicated in this problem of increased skin cancer. However, the specific mechanisms by which these agents cause increases in skin cancer are not well understood. We used the Skh-1 hairless mouse model of UVB induced skin carcinogenesis to examine the effects of two immunosuppressive agents, cyclosporine A (CsA) and sirolimus (SRL). These two agents have very different mechanisms of action and as it turns out very different effects on UVB induced inflammation and carcinogenesis. We also tested the hypothesis that using both drugs in combination would result in further reduced inflammation and therefore reduced tumor development and progression. By examining key stages of the carcinogenesis process we have found important differences in the effects of each of these agents alone and in combination on UVB induced inflammation and skin carcinogenesis.
The first step was to determine if these agents caused differences in the acute UVB response. We examined markers of post-UVB inflammation such as edema, myeloperoxidase (MPO), and dermal neutrophil infiltration in dorsal skin of Skh-1 mice at the peak of the post-UVB inflammatory response. We found an increase in markers of inflammation caused by treatment with CsA, and a decrease in markers of inflammation with treatments of SRL and CsA+SRL. The next step was to examine continuously immunosuppressed mice concurrently exposed to UVB for 13 weeks, the point right before tumors begin to emerge. Again we found very similar changes in the skin. CsA treatment overall caused increases in markers of inflammation while SRL and CsA+SRL decreased them. To quantify the effect of these changes on tumor development, this same experiment was repeated, however, after 12 weeks of concurrent immunosuppression and UVB exposure all treatments were stopped and tumors were allowed to progress. We found that CsA caused larger and more aggressive tumors to develop while SRL did not significantly affect tumor size burden or grade and CsA+SRL resulted in reduced tumor burden. The CsA+SRL treatment group also developed the least number of malignant tumors. Finally, to determine the effects of these agents on the progression of tumors, mice were exposed to UVB for 15 weeks to induce tumor development then UVB was stopped and immunosuppression was begun. As in the previous experiments, CsA caused larger and more aggressive tumors to develop. This was associated with increases in innate immune mediators in the tumor microenvironment, such as mast cells and the cytokine TGFβ1, as well as increased DNA damage. Treatment with either SRL or CsA+SRL caused smaller less aggressive tumors to develop in association with a concomitant reduction in the same innate immune mediators.
In light of research in our laboratory regarding the efficacy of celecoxib (CX) as an inhibitor of UVB induced inflammation and carcinogenesis, topical celecoxib was administered in combination with CsA+SRL to determine if a further reduction in inflammation and tumor development could be achieved. All four of the same experimental designs previously used were examined. We found that CX administered concurrently with CsA+SRL was able to further reduce markers of inflammation as well as tumor development but was not able to affect tumor progression as measured by histological grading.
A preliminary experiment was conducted to determine the likelihood of a much newer immunosuppressive agent, leflunomide, having anti-inflammatory effects on acute UVB induced inflammation. We found that we could effectively obtain consistent and adequate levels of the active metabolite of leflunomide in the serum of our mice, however, other outcomes were quite varied and no definitive conclusions could be drawn concerning its effects on UVB-induced inflammation.
Finally, the efficacy of topical treatment with a DNA repair enzyme, OGG1, was tested. We found that this liposome encapsulated enzyme could penetrate into the epidermis of our mice and reduce the levels of its DNA damage target, 8-hydroxy-deoxyguanine. This also translated into a reduction in tumor development.
In conclusion the data in this thesis demonstrate that CsA and SRL differentially alter the acute and chronic inflammatory responses to UVB, as well as the cutaneous tumor microenvironment, which in turn contribute to enhanced and inhibited development of UVB-induced skin cancer, respectively, in a murine model of skin carcinogenesis. Furthermore, these data reveal that co-administration of CsA and SRL can lead to a reduced UVB-induced inflammatory response contributing to reduced skin cancer development and progression compared to either immunosuppressant alone. Further studies with these and other immunosuppressants need be conducted in order to develop immunosuppressive regimens that maintain strong graft function and minimize the risk of post transplant malignancies.