Melanoma: Microenvironment and Oncogene Interactions

恶性黑色素瘤是一种非常具有侵袭性的疾病，具有高度的转移扩散倾向和化疗耐药性。在15- 25%的黑色素瘤和癌前病变中观察到ras突变的表达。然而，ras突变的频率可能低估了通过ras的异常信号的贡献，因为ras的众多阳性和阴性效应中的任何一种的改变都可能导致与激活的ras相似的表型。过去的研究表明，在几种转基因小鼠模型中，活化的Ha-ras的表达对于黑色素瘤的发展是必要的。ras作为生长因子和应激信号的关键调控因子，可激活Raf/MEK/Erk、磷酸肌苷3-激酶/PDK/Akt、Ra1GDS等多种效应通路。本项目拟探讨的假设是PI3-OH激酶/PDK/Akt通路的激活对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，我们将介绍区分效应通路的ras突变体。效应结合环的突变消除了ras结合和特异性激活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，每个途径将被检查其对转化表型的影响，下游效应功能的激活，以及在细胞培养、移植细胞和转基因小鼠中黑色素瘤的发生和进展。解决这一假设的具体目的是：1)在细胞培养中表征活化的PI 3-激酶途径黑素细胞转化的影响；2)开发和表征转基因小鼠，我们可以靶向表达ras功能缺失突变体C40和S35，并激活Akt和Raf到黑色素细胞和；3)研究PI-3激酶通路激活对黑色素瘤进展及转移的体内影响。我们将研究激活的PI3激酶通路对肿瘤组织病理学的影响，对调节肿瘤对应激信号、缺氧环境和紫外线暴露的反应以及转移性疾病发展的影响。将在INK4a-/-（黑色素瘤易感性标记）背景下对单转基因小鼠和双转基因小鼠进行比较，并将C3H背景下的转基因小鼠暴露于致癌物DMBA中。这些研究将为研究黑色素瘤的癌症生物学提供新的模型，并为评估新的、潜在的治疗药物和确定新的干预分子靶点提供新的模型。

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.