DNA-binding polyamides as Hedgehog inhibitors in human carcinoma

DNA 结合聚酰胺作为人类癌症中的 Hedgehog 抑制剂

• 批准号: 8055214
• 负责人: Amanda E Hargrove
• 金额: $ 4.84万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8055214
• 关键词: Adverse effects; Affect; Affinity; Apoptosis; Apoptotic; BCL2 gene; Basal cell carcinoma; Base Pair Mismatch; Behavior; Binding; Binding Sites; Biological Assay; Brain; Carcinoma; Cell Culture Techniques; Cell Line; Cell Nucleus; Cell Proliferation; Cell Separation; Cell Surface Receptors; Cell Survival; Cell model; Cells; Cessation of life; Characteristics; Chemistry; Chromatin; Cultured Tumor Cells; DNA; DNA Binding; DNA Minor Groove Binding; DU145; Development; Down-Regulation; Electrophoretic Mobility Shift Assay; Erinaceidae; Event; Gene Expression; Gene Expression Regulation; Gene Targeting; Generic Drugs; Genes; Genetic Transcription; Genome; Gli2 protein; Growth; Human; Imidazole; In Vitro; Individual; Investigation; Knock-out; Libraries; Ligand Binding; Ligands; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Measures; Messenger RNA; Methods; Microarray Analysis; Microscopy; Minor Groove; Modeling; Neoplasm Metastasis; Nylons; Pancreas; Pathway interactions; Phase; Process; Prostate; Public Health; Pyrroles; Quantitative Reverse Transcriptase PCR; Recombinants; Reporting; Research; Research Personnel; Response Elements; Role; Scientist; Series; Signal Transduction; Skin; Solid; Specificity; Structure; Testing; Therapeutic; Titrations; Transcription Coactivator; Tumor Cell Line; base; cancer cell; cancer therapy; cell type; chromatin immunoprecipitation; cyclopamine; ds-DNA; extracellular; genome-wide; in vitro activity; inhibitor/antagonist; insight; mRNA Expression; malignant breast neoplasm; neoplastic cell; novel; promoter; research study; small molecule; smoothened signaling pathway; therapeutic development; tool; transcription factor; tumor progression

DESCRIPTION (provided by applicant): Cancer remains an important concern for public health, with nearly 1.5 million new cases and 500,000 deaths estimated for 2009. Recently, overactivity of the Hedgehog (HH) signaling pathway has been reported in a large number of human carcinomas, including breast, prostate, and lung cancer. In addition, several studies support the requirement of an active HH pathway for the proliferation, survival and metastasis of malignant cells. HH signaling is therefore an attractive target for therapeutic development and merits further mechanistic investigation in cell culture models to evaluate its role in the development and progression of cancer. In normal HH signaling, exogenous HH ligands bind to cell surface receptors, resulting in a signaling cascade that culminates in the activation of the Gli2 transcription factor. Gli2 promotes transcription of over 130 genes, several with anti-apoptotic and proliferation-promoting activity. Selective regulation of these genes would allow researchers to carefully probe the downstream effects of overactive HH signaling. DNA-binding polyamides are small molecules that represent uniquely effective tools for the investigation of gene-specific transcriptional inhibition. Through a programmable combination of heterocyclic and aliphatic residues, polyamide structures bind the minor groove of DNA with high sequence selectivity and affinity. Upon exogenous addition, polyamides have been shown to regulate gene expression in cancer cell cultures by interfering with transcription factor-DNA interactions in the nucleus. It is thus proposed that the development of a small library of polyamides targeted to genes regulated by Gli2 will allow for the role of HH signaling in cancer to be studied at the gene, genome-wide, and cellular level. The specific aims of this proposal are: 1) to develop a small library of polyamides that interrupt Gli2- DNA interactions in a sequence-specific manner; 2) to investigate the ability of these compounds to alter mRNA expression levels in healthy and tumor cell cultures; and 3) to assess the effect of polyamide transcriptional inhibitors on global genome expression, cell proliferation and cell viability. To begin, we will synthesize six polyamides targeted to cancer-relevant genes and measure their DNA-binding affinity as well as their ability to displace Gli2 from the promoter sequences of these genes in vitro. We will then study the effects of these compounds on the mRNA levels of their target genes, as well as their ability to bind to chromatin- bound DNA, in healthy cells as well as basal cell carcinoma and prostate cancer cell models. The genome wide effects of polyamide transcriptional inhibition will be analyzed using standard microarrays, and the gross cell effects will be measured through microscopy, commercial proliferation assays, and cell sorting. These results will be compared to similar studies with a generic HH pathway inhibitor. The development of Gli2- inhibiting polyamides will provide novel tools for the investigation of HH signaling in cancer and lend insight into the suitability of this pathway for gene-targeted cancer therapy. PUBLIC HEALTH RELEVANCE: Scientists have recently discovered a process necessary for the growth and proliferation of cancer cells. This research will develop a method to study how this process affects cancer cells and healthy cells and whether stopping this process could provide a new way to treat cancer. More specifically, this research will give insight into the utility of "gene-targeted" cancer therapies, which have the potential to be highly effective cancer treatments with fewer side effects than current therapies.

描述（由申请人提供）：癌症仍然是公共卫生的一个重要问题，2009年估计有近150万新发病例和50万例死亡。最近，Hedgehog（HH）信号通路的过度活跃已被报道在大量的人类癌症，包括乳腺癌，前列腺癌和肺癌。此外，一些研究支持恶性细胞增殖、存活和转移需要活性HH途径。因此，HH信号传导是治疗开发的有吸引力的靶标，并且值得在细胞培养模型中进行进一步的机制研究，以评估其在癌症发展和进展中的作用。 在正常的HH信号传导中，外源性HH配体与细胞表面受体结合，导致信号传导级联，最终激活Gli 2转录因子。Gli 2促进超过130个基因的转录，其中一些具有抗凋亡和增殖促进活性。这些基因的选择性调节将使研究人员能够仔细探测过度活跃的HH信号传导的下游效应。DNA结合聚酰胺是一种小分子，代表了研究基因特异性转录抑制的独特有效工具。通过杂环和脂肪族残基的可编程组合，聚酰胺结构以高序列选择性和亲和力结合DNA的小沟。在外源性添加后，已显示聚酰胺通过干扰细胞核中的转录因子-DNA相互作用来调节癌细胞培养物中的基因表达。因此，建议开发针对Gli 2调控基因的小型聚酰胺文库，将允许在基因、全基因组和细胞水平上研究HH信号传导在癌症中的作用。 该提案的具体目标是：1）开发一个以序列特异性方式中断Gli 2- DNA相互作用的小型聚酰胺库; 2）研究这些化合物改变健康和肿瘤细胞培养物中mRNA表达水平的能力; 3）评估聚酰胺转录抑制剂对整体基因组表达、细胞增殖和细胞活力的影响。开始，我们将合成六种针对癌症相关基因的聚酰胺，并测量它们的DNA结合亲和力以及它们在体外从这些基因的启动子序列中置换Gli 2的能力。然后，我们将研究这些化合物对其靶基因的mRNA水平的影响，以及它们在健康细胞以及基底细胞癌和前列腺癌细胞模型中与染色质结合DNA结合的能力。将使用标准微阵列分析聚酰胺转录抑制的全基因组效应，并通过显微镜、商业增殖试验和细胞分选测量总细胞效应。将这些结果与使用通用HH通路抑制剂的类似研究进行比较。Gli 2抑制聚酰胺的开发将为研究癌症中HH信号传导提供新的工具，并深入了解该途径对基因靶向癌症治疗的适用性。 公共卫生相关性：科学家最近发现了癌细胞生长和增殖所必需的过程。这项研究将开发一种方法来研究这一过程如何影响癌细胞和健康细胞，以及停止这一过程是否可以提供一种治疗癌症的新方法。更具体地说，这项研究将深入了解“基因靶向”癌症疗法的效用，这种疗法有可能成为高效的癌症治疗方法，副作用比目前的疗法少。