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比任何其他癌基因都要频繁。RAS蛋白通过一系列翻译后修饰与膜结合，包括法尼化、蛋白分解和羧甲基化。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一个有吸引力的靶点。我们克隆并鉴定了异丙基半胱氨酸羧甲基转移酶(ICMT)，它是修饰RAS和其他CAAX蛋白的三种酶中的第三种。为了验证ICMT作为抗癌药物靶点的有效性，我们研究了条件致癌K-RAS等位基因(LSL-KrasG13D)驱动的小鼠胰腺导管腺癌(PDA)模型。ICMT在表达致癌K-RAS的同一胰腺细胞中被消融(Pdx-1-Cre；Icmtfl/fl；KrasLSL/+)。我们非常惊讶地观察到ICMT缺乏明显加剧了K-RAS诱导的胰腺上皮内瘤变(Panin)。这一惊人的结果不仅表明ICMT可能不适合用于药物靶向，而且它还表明这种酶以及由此推断的一种或多种底物在某些情况下起到了肿瘤抑制作用。我们建议从三个特定的目标来阐明ICMT的肿瘤抑制样活性：目的1：在LSL-KrasG12D驱动的Panin皮损恶化过程中ICMT缺陷的特征。我们将对Pdx-1-Cre；Icmtfl+L；KrasLSL/+vs Pdx-1-Cre；Icmtfl/fl；KrasLSL/+动物观察到的Panin损伤的免疫组织化学特征，我们将确定ICMT缺乏对PDx-1-Cre的影响；KrasLSL/+模型的Panin发育需要去除导管上皮细胞中的ICMT，我们将确定加重的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缺乏在诱导胰腺炎症中的作用。目的2：ICMT缺乏对小鼠肿瘤模型的影响。我们将确定ICMT的作用是否类似于K-RAS驱动的癌症的替代模型以及依赖于致癌的H-RAS的模型中的肿瘤抑制因子。目的3：培养细胞ICMT缺陷症的分子标记。我们将对分离的原代PDECs进行研究，以发现ICMT缺乏症的分子标记，并确定哪种或哪些底物对ICMT的促进生长作用负责。由于Notch1缺乏在PDX-1-CRE；KrasLSL/+模型中表现为ICMT缺乏，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作用机制，不仅将为RAS途径中的药物发现提供信息，还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.