Molecular mechanisms of transcriptional repression of PTEN in human breast cancer

人乳腺癌中 PTEN 转录抑制的分子机制

• 批准号: 8645950
• 负责人: Kyrie Jean Pappas
• 金额: $ 4.27万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-03-01 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8645950
• 关键词: 3' Untranslated Regions; Binding; Biological Assay; Biopsy; Breast Cancer Cell; Breast Cancer Treatment; Breast Epithelial Cells; Cancer cell line; Cataloging; Catalogs; Chromatin; Complex; Disease; Down-Regulation; Drug Targeting; Epithelium; Genetic Transcription; Goals; Human; Knowledge; Luciferases; Malignant Neoplasms; Mammary Neoplasms; Maps; Measures; Messenger RNA; MicroRNAs; Molecular; Mutate; Mutation; PTEN gene; Pathway interactions; Primary Neoplasm; Protein phosphatase; Pseudogenes; RNA; Regulation; Relative (related person); Reporter; Repression; Role; Sampling; Seeds; Source; Techniques; Testing; Therapeutic; Time; Transcript; Tumor Suppressor Proteins; Untranslated RNA; Variant; base; chromatin immunoprecipitation; design; gene repression; histone modification; mRNA Stability; malignant breast neoplasm; mammary epithelium; novel diagnostics; novel therapeutics; outcome forecast; prevent; promoter; protein expression; public health relevance; research study; transcriptome sequencing; uncontrolled cell growth

DESCRIPTION (provided by applicant): This project aims to investigate the molecular mechanism by which PTEN is transcriptionally repressed in human breast cancer. PTEN is mutated in roughly 40% of breast cancers, but even in the absence of mutations, PTEN is often transcriptionally repressed, leading to aberrant PI3K pathway activity and subsequent uncontrolled cell growth. In aim 1, the transcriptional profile of PTEN will be defined in normal mammary epithelium; a critical first step for understanding the transcriptional repression of PTEN observed in cancer. To accomplish this goal, normal mammary epithelium will be purified and RNA-seq will be employed to catalog distinct RNA species originating from PTEN and pseudogene PTENP1. Relative abundance will be measured using quantitative real-time PCR (qRT-PCR). The relationship between each of the transcripts identified, and their association with PTEN protein expression will be investigated. Moreover, the difference in PTEN transcriptional profile between the various subtypes of mammary epithelial cells will be determined, as this may be important for the mechanism by which PTEN deficiency contributes to breast cancer. In aim 2, we will identify (1) cancer-specific changes in PTEN transcription and (2) the hypothesized chromatin-based mechanism by which these changes occur. By comparing the results of the RNA-seq and qRT-PCR experiments in normal epithelium to breast cancer biopsies and breast cancer cell lines, changes in the PTEN and PTENP1 transcriptional profile that are specifically associated with breast cancer will be determined. Noncoding RNAs will be investigated as a molecular cause of PTEN repression through the recruitment of repressive complexes to PTEN or by direct action. microRNAs may cause degradation of the nascent PTEN mRNA by binding to seed matches in the 3'UTR; therefore, this will be investigated as an alternative mode of downregulation of PTEN transcript. To implicate candidate repressor complexes in PTEN repression, pharmacological inhibition, chromatin immunoprecipitation (ChIP), and transient knockdowns of identified candidates will be employed. Given that the PTEN/PI3K pathway is dysregulated in many cancers among other diseases, understanding the detailed regulations of PTEN may illuminate a paradigm for diseases characterized by PTEN deficiency and open the door for novel diagnostic and/or therapeutic approaches.

描述(申请人提供)：该项目旨在研究PTEN在人类乳腺癌中转录抑制的分子机制。PTEN在大约40%的乳腺癌中发生突变，但即使在没有突变的情况下，PTEN也经常被转录抑制，导致PI3K途径的异常活性和随后的细胞生长失控。在目标1中，将定义正常乳腺上皮中PTEN的转录图谱；这是了解在癌症中观察到的PTEN转录抑制的关键第一步。为了实现这一目标，将提纯正常乳腺上皮，并将使用RNA-SEQ来分类起源于PTEN和假基因PTENP1的不同RNA物种。相对丰度将使用定量实时聚合酶链式反应(qRT-PCR)进行测量。我们将研究鉴定出的每个转录本之间的关系，以及它们与PTEN蛋白表达的关系。此外，将确定不同亚型乳腺上皮细胞之间PTEN转录图谱的差异，因为这可能对于PTEN缺陷导致乳腺癌的机制很重要。在目标2中，我们将确定(1)PTEN转录的癌症特异性变化和(2)这些变化发生的基于染色质的假想机制。通过比较正常上皮、乳腺癌活检组织和乳腺癌细胞系的RNA-seq和qRT-PCR实验结果，将确定与乳腺癌特异相关的PTEN和PTENP1转录图谱的变化。非编码RNA将作为PTEN抑制的分子原因，通过向PTEN募集抑制性复合体或通过直接作用进行研究。MicroRNAs可能通过与3‘端非编码区中的种子匹配结合而导致新生PTEN mRNA的降解，因此，这将被作为PTEN转录下调的另一种方式进行研究。为了在PTEN抑制中涉及候选阻遏复合体，将采用药物抑制、染色质免疫沉淀(CHIP)和识别的候选抑制物的瞬时击倒。考虑到PTEN/PI3K通路在许多癌症和其他疾病中都是异常调节的，了解PTEN的详细调控可能会为以PTEN缺乏为特征的疾病提供一个范例，并为新的诊断和/或治疗方法打开大门。