Characterization of lcmt in Animal Models of Cancer

描述（由申请人提供）：Ras基因在癌症中的突变频率高于任何其他癌基因。Ras蛋白通过一系列翻译后修饰与细胞膜结合，包括法尼基化、蛋白水解和羧基甲基化。Ras蛋白只有在与细胞膜结合时才具有生物活性。因此，Ras加工途径长期以来一直被认为是抗癌药物的有吸引力的靶标。我们已经克隆并表征了异戊二烯基半胱氨酸羧基甲基转移酶（Icmt），这是修饰Ras和其他CAAX蛋白的三种酶中的第三种。为了验证Icmt作为抗癌药物靶标，我们研究了由条件致癌K-Ras等位基因（LSL-KrasG 13 D）驱动的胰腺导管腺癌（PDA）的小鼠模型。在表达致癌K-Ras的相同胰腺细胞中消融Icmt（基因型Pdx-1-Cre;Icmtfl/fl;KrasLSL/+）。我们非常惊讶地观察到，Icmt缺乏显着加剧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剂的作用。我们提出在三个具体目标中阐明Icmt的肿瘤抑制剂样活性：目标1：LSL-KrasG 12 D驱动的PanIN病变恶化中Icmt缺陷的表征。我们将对在Pdx-1- Cre;Icmtfl+1;KrasLSL/+与Pdx-1-Cre;Icmtfl/fl;KrasLSL/+动物中观察到的PanIN损伤进行化学表征，我们将确定Icmt缺乏对Pdx-1-Cre; PanIN发展的KrasLSL/+模型需要导管上皮中的Icmt消融，我们将确定恶化的PanIN是否代表胰腺导管上皮细胞（PDEC）的细胞自主效应，我们将确定Icmt缺乏对雨蛙素诱导的胰腺炎症的影响。目的2：Icmt缺乏对肿瘤形成的交替小鼠模型的影响。我们将确定Icmt是否在K-Ras驱动的癌症的替代模型以及依赖于致癌H-Ras的模型中表现得像肿瘤抑制因子。目的3：培养细胞中Icmt缺陷的分子标志物。我们将研究分离的原发性PDEC，以发现Icmt缺陷的分子标志物，并确定哪种底物或哪种底物负责Icmt消融的生长促进作用。由于Notch 1缺陷表型模仿Pdx-1-Cre;KrasLSL/+模型中的Icmt缺陷，我们还将研究Notch信号传导中对Icmt的需求。我们相信，了解Icmt作为肿瘤抑制因子的机制不仅可以为Ras通路中的药物发现提供信息，还可以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.