Malignant melanoma is a very aggressive disease with a high propensity for metastatic spread and chemotherapeutic resistance. Expression of mutated ras has been observed in 15-25 percent of melanomas and premalignant lesions. However the frequency of ras mutations is likely an underestimate of the contribution of aberrant signaling through ras since alterations in anyone of the numerous positive and negative effectors of Ras can result in a similar phenotype as activated ras. Past studies have shown that an expression of activated Ha-ras is necessary for the development of melanoma in several transgenic mouse models. As a key regulator of growth factor and stress signals, ras leads to activation of multiple effector pathways such as Raf/MEK/Erk, phosphoinositide 3-kinase/PDK/Akt, and Ra1GDS. The hypothesis to be explored in this project is that activation of the PI3-OH kinase/PDK/Akt pathway is critical for the malignant progression and metastatic spread of melanoma. To address this hypothesis, we will introduce ras mutants that discriminate between effector pathways. Mutations in the effector binding loop eliminate the ability of ras to bind and specifically activate certain downstream components of the ras network. Ras constructs will be introduced into melanocytes and each pathway will be examined for its effects on the transformed phenotype, activation of downstream effector functions, and the development and progression of melanoma both in cell culture, transplanted cells, and in transgenic mice.
Specific aims to address this hypothesis are: 1) to characterize the effect of activated PI 3- kinase pathway melanocyte transformation in cell cultures; 2) to develop and characterize transgenic mice in which we have targeted expression of the ras loss-of-function mutants, C40 and S35, and activated Akt and Raf to melanocytes and; 3) to study the in vivo effect of activation of PI-3 kinase pathway on melanoma progression and metastatic disease. We will study effect of an activated PI3 kinase pathway on histopathology of the tumors, on regulating the tumor response to stress signals, a hypoxic environment and UV exposure, and on the development of metastatic disease. Comparisons will be made with single transgenic mice and double transgenic mice on an INK4a-/- background (a melanoma susceptibility marker) and by exposing the transgenic mice on a C3H background to the carcinogen, DMBA. These studies will provide new models for studying the cancer biology of melanoma and provide new models that will be used to evaluate of new, potential therapeutic agents and identify new molecular targets for intervention.
