Melanoma: Microenvironment and Oncogene Interactions

恶性黑色素瘤是一种侵袭性很强的疾病，具有很高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突变的ras的表达。然而，ras突变的频率可能低估了ras异常信号的作用，因为ras众多正负效应因子中的任何一个的改变都可以导致与激活的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。作为生长因子和应激信号的关键调节因子，ras可激活多种效应通路，如Raf/MEK/Erk、肌醇磷脂3-激酶/PDK/Akt和Ra1GDS。这个项目中要探索的假设是，PI3-OH激酶/PDK/Akt通路的激活在黑色素瘤的恶性进展和转移扩散中起关键作用。为了解决这一假设，我们将引入区分效应器途径的ras突变体。效应器结合环中的突变消除了RAS结合并特异性激活RAS网络的某些下游组件的能力。RAS结构将被引入黑素细胞，每条途径都将被检测其对转化表型、下游效应功能的激活以及黑色素瘤在细胞培养、移植细胞和转基因小鼠中的发展和进展的影响。针对这一假说的具体目的是：1)表征激活的PI 3-激酶途径在细胞培养中转化黑素细胞的作用；2)建立和鉴定靶向表达ras功能缺失突变体C40和S35并激活Akt和Raf到黑素细胞的转基因小鼠；3)研究PI-3途径激活在黑色素瘤进展和转移疾病中的体内作用。我们将研究激活的PI3激酶通路在肿瘤组织病理学中的作用，在调节肿瘤对应激信号、低氧环境和紫外线照射的反应以及在转移疾病发展中的作用。将与INK4a-/-背景(黑色素瘤易感标记物)的单转基因小鼠和双转基因小鼠进行比较，并将C3H背景的转基因小鼠暴露于致癌物质DMBA。这些研究将为黑色素瘤的癌症生物学研究提供新的模型，并提供新的模型，用于评估新的潜在治疗药物和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.