Genetics of RAS, PTEN, BRAF and CDKN2A in Melanoma

描述（由申请人提供）：RAS基因对黑色素瘤的发生至关重要。 它仅在10%的病例中发生突变，但在人类和小鼠黑色素瘤中与CDKN 2A中的突变合作。 RAS蛋白通过几种下游途径介导其生物化学作用，但最重要的是通过促分裂原活化蛋白激酶（MAPK）级联，或通过磷酸肌醇-3-激酶（PI 3 K）控制蛋白激酶B/Akt。 最近已经证明，在黑色素瘤中，这两种途径都受到高突变率的影响。 Akt通路上的突变涉及PTEN。 PTEN是一种肿瘤抑制因子，与RAS有几个共同的特征。 它是一种蛋白酪氨酸磷酸酶，但也具有脂质磷酸酶活性。 因此，它是PI 3 K的负调节剂和通过PKB/Akt的细胞凋亡的效应物。 我们已经证明，黑色素瘤中的PTEN丢失是一个常见的事件，发生在约30%的标本检查。 最近，平行通路也被证明与突变有关。 BRAF位于MAPK级联上RAS的下游。 最近在超过60%的黑色素瘤中发现了BRAF突变。 这里提供的数据表明，大多数这些BRAF突变发生与PTEN异常或其他Akt通路畸变。 反过来，BRAF/PTEN突变1）与RAS突变相互作用，2）与RAS一样，与CDKN 2A丢失一致。 这表明RAS在黑色素瘤发展中的显著生化功能被这两种癌症基因的突变所涵盖的假设。 我们提出了四个具体目标来验证这一点。首先，我们将在小鼠中模拟我们的遗传观察结果，并预测小鼠中Braf激活和Pten缺失与Cdkn 2a缺陷将导致黑色素瘤的发展。 我们将在p16和p53缺陷的背景下产生携带Pten和Braf改变的小鼠。第二，我们将使用遗传和药理学策略分别和一起检查转基因小鼠系、黑素细胞、黑色素瘤和其他细胞类型中RAS或BRAF和PTEN的改变。 第三，由于这些基因的改变发生在p16功能被废除的背景下，我们预测PTEN和BRAF，像RAS一样，将对p16表达施加控制，并将使用鼠敲除株来评估这些蛋白对p16的控制。 第四，我们将开发使用RNA干扰的遗传技术，以测试黑色素瘤抑制和药物治疗的策略。

DESCRIPTION (provided by applicant): The RAS gene is centrally important to the development of melanoma. It is mutated in only 10% of cases, but cooperates with mutations in CDKN2A in both human and murine melanomas. RAS proteins mediate their effects biochemically through several downstream pathways, but most importantly through the mitogenactivated protein kinase (MAPK) cascade, or through the control of protein kinase B/Akt via phosphoinositol-3-kinase (PI3K). Recently it has been demonstrated that in melanoma both of these pathways are affected by a high incidence of mutation. Mutations on the Akt pathway involve PTEN. PTEN is a tumor suppressor that shares several characteristics with RAS. It is a protein tyrosine phosphatase, but also has lipid phosphatase activity. Thus it is a negative regulator of PI3K and an effector of apoptosis through PKB/Akt. We have demonstrated that PTEN loss in melanoma is a frequent event, occurring in about 30% of specimens examined. And recently, the parallel pathway was also shown to be involved by mutation. BRAF lies immediately downstream of RAS on the MAPK cascade. Mutations in BRAF have been recently found in over 60% of melanomas. Data presented here demonstrate that most of these BRAF mutations occur with PTEN abnormalities or other Akt-pathway aberrations. In turn, mutations in BRAF/PTEN occur 1) reciprocally with RAS mutations, and 2) like RAS, in concert with CDKN2A loss. This suggests the hypothesis that the salient biochemical functions of RAS in melanoma development are encompassed by mutations in these two cancer genes. We propose four specific aims to test this. First, we will model our genetic observations in the mouse, and predict Braf activation and Pten loss with Cdkn2a deficiency in the mouse will engender the development of melanoma. We will generate mice carrying Pten and Braf alterations in the background of p16 and p53 deficiency. Second, we will examine RAS or BRAF and PTEN alterations separately and together in the transgenic murine lines, melanocytes, melanoma and other cell types using genetic and pharmacologic strategies. Third, since alterations in these genes occur in the background of abrogation of p16 function, we predict PTEN and BRAF, like RAS, will exert control over p16 expression, and will assess the control of p16 by these proteins using murine knockout lines. And fourth, we will develop genetic techniques using RNA interference to test strategies for melanoma inhibition and pharmacological therapy.