The notorious resistance of melanoma cells to radiation and chemotherapy has been a major obstacle to the treatment of tumors of this type. Studies performed in our laboratory over the past few years identified ATF2 as an important contributor to melanoma's resistance to radiation and chemical treatment. Our studies revealed that ATF2 is capable of altering death-signaling cascades in human melanoma cells. Accordingly, we sought to determine whether interfering with endogenous ATF2 transcriptional activities would alter the resistance of melanoma cells to radiation and chemotherapy. Forced expression of a peptide corresponding to amino acids 50-100 of ATF2 (ATF250-100) resulted in reduced resistance of late stage melanoma cells to radiation as well as to a series of chemotherapeutic agents. Such sensitization was enhanced when the expression of the ATF2 peptide was combined with the pharmacological inhibitor of stress kinases. Injection of ATF250-100 expressing human melanoma cells into nude mice led to an efficient decrease in tumor growth upon treatment with chemotherapeutic drugs or the pharmacological inhibitor of p38 MAPK. Forced expression in mouse melanoma models SW1 or B16F10 resulted in significant inhibition of tumor growth--to the point of complete regression and inhibition of metastasis depending on the mode of peptide administration. These findings point to the possible use of the ATF2 peptide for reducing melanoma tumorigenicity and metastasis. Mechanisms underlying the effect of the ATF2 peptide were shown to include decreased ATF2 and increased c-Jun transcriptional activities, which result in sensitization to apoptosis. Here we propose to extend these observations towards better assessment of the mechanisms underlying the effect of the ATF2 peptide and further explore, in the in vivo setting, ways to improve the ATF2 peptide's effect. To this end we propose to: (I) Identify the changes in ATF2 and c-Jun function and activities in the course of melanoma development and progression (gene mutation, RNA and protein expression and phosphorylation); (II) Determine the primary mechanism underlying the changes in ATF2 and c-Jun expression and activities in tumors that express the ATF2 peptide. (III) Assess whether a modified ATF2 peptide, either by mutations and truncations that affect phosphoacceptor sites or conformationally important domains, would elicit the biological changes in melanoma, in vitro and in vivo, that are seen with the 50 aa peptide; (IV) Assess the role of the immune response against the ATF2 peptide's expressing melanomas in vivo. Determine whether the ATF2 peptide expression in tumors elicits greater effects in vivo when combined with different treatments, including immune modulators and IL-12. (V) Identify the profile of genes that are commonly induced or suppressed in ATF2 peptide-expressing tumors on the basis of analysis of three tumor types in three mouse strains.
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