Molecular mechanisms of transcriptional repression of PTEN in human breast cancer

项目描述（申请人提供）：本项目旨在研究PTEN在人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通路活性异常，随后细胞生长不受控制。在目标1中，PTEN的转录谱将在正常乳腺上皮中定义；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，正常乳腺上皮将被纯化，RNA-seq将被用于分类来自PTEN和假基因PTENP1的不同RNA种类。相对丰度将采用实时荧光定量PCR （qRT-PCR）测定。每个转录本之间的关系，以及它们与PTEN蛋白表达的关系将被研究。此外，PTEN转录谱在乳腺上皮细胞不同亚型之间的差异将被确定，因为这可能对PTEN缺乏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)这些变化发生的假设的基于染色质的机制。通过将正常上皮细胞与乳腺癌活检组织和乳腺癌细胞系的RNA-seq和qRT-PCR实验结果进行比较，确定与乳腺癌特异性相关的PTEN和PTENP1转录谱的变化。非编码rna将通过募集PTEN抑制复合物或通过直接作用作为PTEN抑制的分子原因进行研究。microRNAs可能通过与3'UTR中的种子匹配体结合而导致新生PTEN mRNA的降解；因此，这将作为PTEN转录下调的另一种模式进行研究。为了使候选抑制因子复合物参与PTEN抑制，将采用药物抑制、染色质免疫沉淀（ChIP）和已确定候选抑制因子的瞬时敲除。鉴于PTEN/PI3K通路在许多癌症和其他疾病中失调，了解PTEN的详细调控可能阐明以PTEN缺乏为特征的疾病的范例，并为新的诊断和/或治疗方法打开大门。

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.