Melanoma: Microenvironment and Oncogene Interactions

恶性黑色素瘤是一种非常具有侵袭性的疾病，具有高转移扩散和化疗耐药性的倾向。 在15- 25%的黑色素瘤和癌前病变中观察到突变ras的表达。 然而，ras突变的频率可能低估了通过ras的异常信号传导的贡献，因为Ras的许多阳性和阴性效应子中的任何一个的改变都可以导致与活化的ras相似的表型。 过去的研究表明，激活的Ha-ras的表达是必要的黑色素瘤在几个转基因小鼠模型的发展。 作为生长因子和应激信号的关键调节因子，ras导致多个效应子途径的激活，例如Raf/MEK/Erk、磷酸肌醇3-激酶/PDK/Akt和Ra 1GDS。 本项目中要探索的假设是PI 3-OH激酶/PDK/Akt通路的激活对于黑色素瘤的恶性进展和转移扩散至关重要。 为了解决这一假设，我们将介绍ras突变体，区分效应途径。 效应物结合环中的突变消除了ras结合并特异性激活ras网络的某些下游组分的能力。 Ras构建体将被引入黑素细胞中，并将检查每种途径对转化表型、下游效应子功能的激活以及细胞培养物、移植细胞和转基因小鼠中黑素瘤的发展和进展的影响。 解决这一假设的具体目标是：1）表征细胞培养物中活化的PI 3-激酶途径黑素细胞转化的作用; 2）开发和表征转基因小鼠，其中我们靶向表达ras功能丧失突变体C40和S35，并活化Akt和Raf至黑素细胞; 3）研究PI-3激酶通路的激活对黑色素瘤进展和转移性疾病的体内作用。 我们将研究活化的PI 3激酶通路对肿瘤组织病理学的影响，调节肿瘤对应激信号、低氧环境和紫外线暴露的反应，以及转移性疾病的发展。 将在INK 4a-/-背景（黑色素瘤易感性标志物）下对单转基因小鼠和双转基因小鼠进行比较，并将C3 H背景下的转基因小鼠暴露于致癌物DMBA。这些研究将为研究黑色素瘤的癌症生物学提供新的模型，并提供新的模型，用于评估新的潜在治疗药物，并确定新的干预分子靶点。

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.