High Throughput Screen for Small Molecule Inhibitors of Colorectal Cancer Cell Pr

描述（由申请人提供）：结直肠癌（CRC）是美国癌症死亡率和发病率的主要原因之一。研究表明，结直肠癌是由具有重要细胞功能的关键基因的逐步改变（突变）引起的。这些基因包括肿瘤抑制基因（TSGs）和致癌基因。例如，tsg如腺瘤性息肉病大肠（APC）和p53的体细胞或种系失活；KRAS和BRAF的致癌激活在结直肠癌的发病机制中至关重要。然而，尽管有这些知识，针对CRC中改变的信号通路的特定成分的治疗仍处于相对早期的阶段。我们的研究小组先前已经证明锌指转录因子KLF5在调节肠上皮细胞增殖中起重要作用。KLF5主要表达于肠上皮的增殖隐窝细胞区。KLF5在转染细胞中的异位表达导致增殖率增加，并导致锚定独立生长。此外，NIH3T3和IEC6细胞中HRAS和KRAS的致癌激活分别导致转化，同时KLF5水平升高。重要的是，通过遗传或药理学手段减少KLF5导致致癌ras转化细胞的增殖和不依赖锚定生长速率降低。此外，KRAS活化的结直肠癌含有高水平的KLF5。这些结果表明，KLF5是激活KRAS的促增殖和转化活性的关键介质，迄今为止，KRAS在约50%的CRC中发生突变。降低这类结直肠癌中KLF5的表达可能为结直肠癌的治疗提供一种新的治疗方法。本研究项目的长期目标是了解CRC中调节KLF5表达的信号通路。我们的中心假设是KLF5是含有活化KRAS的CRC增殖的关键介质。我们的目的是通过高通量筛选（HTS）鉴定结直肠癌细胞中KLF5表达的小分子抑制剂，从而更好地了解KLF5在调节结直肠癌增殖中的生物学功能，并开发潜在的治疗结直肠癌的药物。利用这一R03授权机制，我们提出了2个特定目标：(1)使用基于细胞的荧光素酶报告基因检测对KLF5的小分子抑制剂进行HTS，(2)进行二级和反筛选试验，以验证在特定目标1中鉴定的活性化合物。虽然超出了R03授权的范围，但我们也将与MLSCN中心合作，尝试制定进一步测试的策略，以提供活性化合物的结构和功能的最终细化。在该项目结束时，我们将能够鉴定出几种高活性和特异性的KLF5抑制剂，从而进一步研究KLF5在介导结直肠癌增殖中的生物学功能。这些化合物的鉴定也可能有助于开发新的结直肠癌治疗方法。

DESCRIPTION (provided by applicant): Colorectal cancer (CRC) is one of the leading causes of cancer mortality and morbidity in the United States. Studies indicate that CRC results from stepwise changes (mutations) in key genes with important cellular functions. These genes include tumor suppressor genes (TSGs) and oncogenes. For example, somatic or germline inactivation of TSGs such as adenomatous polyposis coli (APC) and p53; and oncogenic activation of KRAS and BRAF are crucial in the pathogenesis of CRC. However, despite this knowledge, therapies targeting to specific components of the altered signaling pathways in CRC remain at a relatively early stage. Our group previously demonstrated that a member of the Kr¿ppel-like factor (KLF) family of zinc finger transcription factors, KLF5, plays important roles in regulating proliferation of intestinal epithelial cells. KLF5 is predominantly expressed in the proliferating crypt cell compartment of the intestinal epithelium. Ectopic expression of KLF5 in transfected cells results in increased rates of proliferation and leads to anchorage independent growth. In addition, oncogenic activation of HRAS and KRAS in NIH3T3 and IEC6 cells, respectively, leads to transformation with a concomitant increase in KLF5 levels. Importantly, reduction of KLF5 by genetic or pharmacological means results in reduced rates of proliferation and anchorage-independent growth in oncogenic RAS-transformed cells. Moreover, CRC with activated KRAS are shown to contain high levels of KLF5. These results indicate that KLF5 is a key mediator for the pro-proliferative and transforming activities of activated KRAS, heretofore mutated in approximately 50% of CRC. Reduction of KLF5 expression in such CRC may offer a novel therapeutic approach in the treatment of CRC. The long-term GOAL of this research project is to understand the signaling pathways that modulate KLF5 expression in CRC. Our CENTRAL HYPOTHESIS is that KLF5 is a key mediator of proliferation of CRC containing activated KRAS. Our OBJECTIVE is to identify small molecule inhibitors of KLF5 expression in CRC cells using high throughput screening (HTS), with which to better understand the biological functions of KLF5 in modulating CRC proliferation and to develop potential therapeutic agents in the treatment of CRC. Using this R03 grant mechanism, we propose 2 SPECIFIC AIMS: (1) To perform HTS for small molecule inhibitors of KLF5 using cell-based luciferase reporter assays, and (2) To perform secondary and counter screening assays with which to validate the active compounds identified in specific aim 1. Although beyond the scope of this R03 grant, we will also attempt to develop strategies for further testing, in collaboration with the MLSCN center, to provide a final refinement of the structure and function of the active compounds. At the conclusion of the proposed project, we will be able to identify several highly active and specific inhibitors of KLF5 with which to further investigate the biological functions of KLF5 in mediating CRC proliferation. The identification of these compounds may also aid in the development of novel therapeutic approaches for CRC.