Characterization of lcmt in Animal Models of Cancer

描述（由申请人提供）：Ras基因在癌症中比任何其他癌基因更频繁地发生突变。Ras蛋白通过一系列翻译后修饰（包括法尼基化、蛋白水解和羧基甲基化）与细胞膜结合。Ras蛋白只有在与细胞膜结合时才具有生物活性。因此，Ras加工途径一直被认为是抗癌药物的一个有吸引力的靶点。我们克隆并鉴定了异戊酰半胱氨酸羧甲基转移酶（Icmt），这是三种修饰Ras和其他CAAX蛋白的酶中的第三种。为了验证Icmt作为抗癌药物靶点，我们研究了由条件致癌K-Ras等位基因（LSL-KrasG13D）驱动的胰腺导管腺癌（PDA）小鼠模型。在表达致癌K-Ras的胰腺细胞（基因型Pdx-1-Cre、Icmtfl/fl、KrasLSL/+）中消融Icmt。我们非常惊讶地发现，Icmt缺乏显著加剧了K-Ras驱动的胰腺上皮内瘤变（PanIN）。这一惊人的结果不仅表明Icmt可能不适合用于药物靶向，而且还表明该酶及其一种或多种底物在某些情况下起肿瘤抑制作用。我们提出在三个特定目的中阐明Icmt的肿瘤抑制样活性：目的1：表征Icmt缺乏在LSL-KrasG12D驱动的PanIN病变加重中的作用。我们将用免疫组织化学方法对Pdx-1- Cre、Icmtfl+ 1、KrasLSL/+ vs Pdx-1-Cre;Icmtfl/fl；KrasLSL/+动物，我们将确定Icmt缺乏对Pdx-1-Cre的影响；KrasLSL/+模型的PanIN发展需要导管上皮的Icmt消融，我们将确定加剧的PanINs是否代表胰腺导管上皮细胞（PDECs）的细胞自主效应，我们将确定Icmt缺乏对小蛋白诱导的胰腺炎症的影响。目的2:Icmt缺乏对小鼠肿瘤模型的影响。我们将确定Icmt在K-Ras驱动癌症的替代模型以及依赖致癌H-Ras的模型中是否表现得像肿瘤抑制因子。目的3：培养细胞中Icmt缺乏的分子标记。我们将研究分离的初级PDECs，以发现Icmt缺乏的分子标记，并确定哪种底物或底物对Icmt消融的生长促进作用负责。因为Notch1缺陷表现为Pdx-1-Cre缺陷；KrasLSL/+模型，我们还将研究Notch信号中对Icmt的要求。我们相信，了解Icmt作为肿瘤抑制因子的机制不仅将为Ras通路的药物发现提供信息，而且将揭示癌症生物学的新的重要方面。

DESCRIPTION (provided by applicant): Ras genes are mutated in cancer more frequently than any other oncogene. Ras proteins associate with membranes by virtue of a series of post-translational modifications that include farnesylation, proteolysis and carboxyl methylation. Ras proteins are biologically active only when associated with cellular membranes. Accordingly, the Ras processing pathway has long been considered an attractive target for anti-cancer drugs. We have cloned and characterized isoprenylcysteine carboxyl methyltransferase (Icmt), the third of the three enzymes that modifies Ras and other CAAX proteins. To validate Icmt as an anti-cancer drug target we studied a mouse model of pancreatic ductal adenocarcinoma (PDA) driven by a conditional oncogenic K-Ras allele (LSL-KrasG13D). Icmt was ablated in the same pancreatic cells in which oncogenic K-Ras was expressed (genotype Pdx-1-Cre;Icmtfl/fl;KrasLSL/+). We were extremely surprised to observe that Icmt deficiency markedly exacerbated K-Ras driven pancreatic intraepithelial neoplasia (PanIN). This striking result not only suggests that Icmt may not be suitable for drug targeting but it also suggests that the enzyme, and by inference one or more of its substrates, acts as a tumor suppressor in some contexts. We propose to elucidate the tumor suppressor-like activity of Icmt in three specific aims: Aim 1: Characterization of Icmt deficiency in the exacerbation of LSL-KrasG12D driven PanIN lesions. We will characterize immunohistochemically the PanIN lesions observed in Pdx-1- Cre;Icmtfl+l;KrasLSL/+ vs Pdx-1-Cre;Icmtfl/fl;KrasLSL/+ animals, we will determine if the effect of Icmt deficiency on the Pdx-1-Cre;KrasLSL/+ model of PanIN development requires ablation of Icmt in ductal epithelium, we will determine if the exacerbated PanINs represent a cell autonomous effect of pancreatic ductal epithelial cells (PDECs), and we will determine the effect of Icmt deficiency on caerulein-induced pancreatic inflammation. Aim 2: Effect of Icmt deficiency on alternate mouse models of neoplasia. We will determine if Icmt behaves like a tumor suppressor in an alternate model of K-Ras driven cancer as well as a model that depends on oncogenic H-Ras. Aim 3: Molecular markers of Icmt deficiency in cultured cells. We will study isolated primary PDECs to discover molecular markers of Icmt deficiency and to determine which substrate, or substrates, is responsible for the growth promoting effect of Icmt ablation. Because Notch1 deficiency phenocopies Icmt deficiency in the Pdx-1-Cre;KrasLSL/+ model we will also study the requirement for Icmt in Notch signaling. We believe that understanding the mechanism through which Icmt behaves like a tumor suppressor will not only inform drug discovery in the Ras pathway but will reveal new and important aspects of cancer biology.