CONTROL OF EGFR GENE EXPRESSION IN HUMAN BREAST CANCER

人类乳腺癌中 EGFR 基因表达的控制

• 批准号: 2096790
• 负责人: SUSAN A CHRYSOGELOS
• 金额: $ 13.89万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2096790
• 关键词: DNA binding protein; DNA footprinting; breast neoplasms; epidermal growth factor; estrogen receptors; estrogens; gel electrophoresis; gene expression; genetic mapping; genetic promoter element; genetic regulatory element; genetic transcription; growth factor receptors; hormone related neoplasm /cancer; neoplastic cell; northern blottings; nucleic acid hybridization; nucleic acid sequence; polymerase chain reaction; posttranscriptional RNA processing; structural genes; transfection

The progression of human breast cancer from hormone-responsiveness to a more aggressive, estrogen-independent state may involve the development of the ability to constitutively produce growth factors or their receptors which permit a tumor to bypass the need for estrogen. Clinically, this more malignant phenotype is often characterized by the loss of estrogen receptor (ER) and the acquisition of epidermal growth factor receptor (EGFR). EGFR is an indicator of poor response to endocrine therapy, and its presence overrides the beneficial effects of ER in coexpressors. Because of its prognostic value and the strong transcriptional component to its expression, this project focuses on the regulatory mechanisms by which transcription of the EGFR gene is controlled in human breast cancer. Preliminary studies indicate that there are structural differences (as revealed by DNase I sensitivity) in the promoter, first exon, and intron I of the EGFR gene which correlate with its expression in human breast cancer cell lines. Specifically, a site around the exon I/intron I boundary disappears in high expressors, while a group of sites in intron I appears in these cell lines. Additionally, a region in the promoter shows .changes in both the level of sensitivity and the extent of the area which is susceptible. These regions will be further analyzed to precisely localize sites of protein-DNA interaction whose presence or absence are associated with EGFR expression. This will be done using human breast cancer cell lines (both ER+ and ER-) with a wide range of EGFR expression. Protein binding sites will be mapped sequentially by native genomic blotting gel retardation assays, and in vitro footprinting. In this way, the in vivo significance of the protein binding sites will be established first, before pinpointing them at single-nucleotide resolution. This strategy will also be used to identify the cis-regulatory elements involved in the induction of EGFR by estrogen in hormone-dependent cell lines. The functionality of the various sequence elements identified by these methods as protein binding sites will be assessed in transient transfection assays using CAT expression vectors transfected into human breast cancer cell lines expressing high and low levels of EGFR. In the long term, sequences which are shown to be involved in the regulation of EGFR levels will then be used to isolate the protein factors responsible for controlling EGFR expression. It is hoped that by understanding these regulatory mechanisms and the role they play in the evolution of more aggressive forms of human breast cancer, new opportunities will arise for developing effective treatments against the disease.

人类乳腺癌从激素反应到 更具侵略性的雌激素独立状态可能涉及发展 组成性产生增长因素或其其能力的能力 允许肿瘤绕过雌激素的受体。 临床上，这种更恶性的表型通常以 雌激素受体（ER）的丧失和表皮生长的获取 因子受体（EGFR）。 egfr是对 内分泌疗法及其存在覆盖了 ER在共表达器中。 因为它的预后价值和强大 其表达的转录组件，该项目的重点是 EGFR基因转录的调节机制是 在人类乳腺癌中控制。 初步研究表明 存在结构差异（如DNase I灵敏度所揭示） EGFR基因的启动子，第一个外显子和内含子I 它在人类乳腺癌细胞系中的表达。 具体而言， 外显子I/内含子I边界周围的位置在高表现器中消失， 而内含子中的一组位点出现在这些细胞系中。 此外，启动子中的一个区域显示了两个水平的变化 敏感性和易感性区域的程度。 这些 区域将进一步分析到精确本地化的位置 蛋白质-DNA相互作用的存在或不存在与 EGFR表达。 这将使用人类乳腺癌细胞系完成 （ER+和ER-）具有广泛的EGFR表达。 蛋白质结合 位点将通过天然基因组印迹凝胶顺序映射 延迟测定和体外足迹。 这样，体内 蛋白质结合位点的重要性将首先确定 在将它们定位为单核苷酸分辨率之前。 这个策略 还将用于识别涉及的顺式调节元素 通过雌激素诱导激素依赖性细胞系中的EGFR。 这 这些确定的各种序列元素的功能 将在瞬态评估蛋白质结合位点的方法 使用转染人的CAT表达矢量的转染测定法 乳腺癌细胞系高和低水平的EGFR。 在 长期，被证明与调节有关的序列 然后，EGFR水平将用于分离负责的蛋白质因子 用于控制EGFR表达。 希望通过理解这些 监管机制及其在进化中所起的作用 人类乳腺癌的积极形式，将会出现新的机会 开发针对该疾病的有效治疗方法。