Characterization of Pathways Controlling Cancer at the Level of Gene Regulation

癌症是一种主要的疾病负担，改变这种状况的一个希望是通过更好地了解这种疾病来开发新的治疗方法。最近关于哺乳动物短RNA活动的发现可能会为癌症提供新的见解和新的治疗方法。该计划的一个共同目标是研究短RNA，如microRNAs，在正常细胞和癌细胞基因调控中的作用。 有强有力且迅速增长的证据表明，miRNA调控的变化与恶性转化有关，实际上可能是致癌转化中的关键事件。在人类癌症的一个子集中经常过度表达/扩增的mir-17-92-1簇的功能将是 通过在小鼠癌症模型中创造这些微小RNA的特定突变来进行研究。 利用微珠阵列技术和新的克隆技术，将分析正常细胞和处于相同发育状态的肿瘤细胞中microRNA群体的变化。将开发具有短发夹状RNA调控表达的载体，该短发夹状RNA可产生用于沉默基因的特定siRNA 用于转基因途径的分析。此外，还将测试筛选shRNA-慢病毒载体小库的方法，以确定当沉默时抑制或刺激肿瘤发展的基因。此外，表达shRNAs的逆转录病毒载体文库将用于筛选(A)调节pRb缺陷细胞的增殖和/或生存的基因，(B) 调节K-ras驱动的肺癌模型的发展速度，以及(C)对ES细胞分化至关重要的基因。短RNA在转录沉默中的潜在作用将在胚胎干细胞中进行研究。这些过程可能是重要的表观遗传沉默和基因组稳定性 癌细胞。还将研究胚胎干细胞中miRNAs在发育和增殖中的作用。将使用需要少量RNAi的克隆技术来表征T细胞发育过程中microRNAs和siRNAs谱的变化。Arf启动子的激活是一种早期信号 致癌转化。该启动子在正常情况下被E2F3B蛋白沉默，将p19Arf-MDM2-P53途径连接到p16INK4a-CyCD/CDK4-PRB-EDF途径。E2F3B复合体和其他E2F因子在Arf启动子调控中的作用将被研究。

Cancer is a major disease burden and one hope of changing this is to develop new treatments through a better understanding of the disease. Recent discoveries concerning the activities of short RNAs in mammals may provide both new insights and new treatments of cancer. A common goal of this Program is to investigate the roles of short RNAs, such as microRNAs, in regulation of genes in normal and cancer cells. There is strong and rapidly growing evidence suggesting that changes in miRNA regulation are related to malignant transformation and in fact could be a critical event in oncogenic transformation. The function of the mir-17-92-1 cluster which is frequently overexpressed/amplified in a subset of human cancers will be investigated by creation of specific mutations of these microRNAs in the context of mouse models of cancer. Changes in microRNA populations in normal cells and tumor cells of the same developmental state will be analyzed using both bead-array technology as well as new cloning technology. Vectors with regulated expression of a short hairpin RNA which generates a specific siRNA for silencing a gene will be developed for transgenic analysis of pathways. Additionally, methods will be tested for screening of small libraries of shRNA-lentiviral vectors to identify genes which, when silenced, either inhibit or stimulate tumor development. Furthermore, libraries of retro viral vectors expressing shRNAs will be used in screens to identify (a) genes that modulate the proliferation and/or survival of pRB-deficient cells , (b) genes that modulate the rate of development of a K-ras-driven lung cancer model, and (c) genes important for the differentiation of ES cells. The potential role of short RNAs in transcriptional silencing will be investigated in embryonic stem cells. These processes could be important for epigenetic silencing and genomic stability of cancer cells. ES cells will also be studied for the role of miRNAs in development and proliferation. Changes in the spectrum of microRNAs and siRNAs during T-cell development will be characterized using a cloning technology which requires small amounts of RNAi. Activation of the Arf promoter is an early signal in oncogenic transformation. This promoter is silenced under normal conditions by the E2F3B protein, linking the p19Arf-mdm2-p53 pathway to the p16INK4a-cycD/cdk4-pRB-EdF pathway. The role of E2F3B complexes and other E2F factors in regulation of the Arf promoter will be studied.