Molecular mechanisms of transcriptional repression of PTEN in human breast cancer

项目描述（申请人提供）：本项目旨在研究人类乳腺癌中PTEN被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，导致异常的PI 3 K通路活性和随后的不受控制的细胞生长。在目标1中，将在正常乳腺上皮中定义PTEN的转录谱;这是理解在癌症中观察到的PTEN转录抑制的关键的第一步。为了实现这一目标，将纯化正常乳腺上皮，并将使用RNA-seq来对源自PTEN和假基因PTENP 1的不同RNA种类进行编目。将使用定量实时PCR（qRT-PCR）测量相对丰度。将研究鉴定的每个转录本之间的关系及其与PTEN蛋白表达的关联。此外，将确定乳腺上皮细胞的各种亚型之间的PTEN转录谱的差异，因为这对于PTEN缺陷导致乳腺癌的机制可能是重要的。 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。通过比较正常上皮与乳腺癌活检和乳腺癌细胞系中的RNA-seq和qRT-PCR实验的结果，将确定与乳腺癌特异性相关的PTEN和PTENP 1转录谱的变化。非编码RNA将被研究为通过募集抑制复合物到PTEN或通过直接作用的PTEN抑制的分子原因。microRNA可通过与3 'UTR中的种子匹配物结合而引起新生PTEN mRNA的降解;因此，这将作为PTEN转录物下调的替代模式进行研究。为了将候选阻遏物复合物牵连到PTEN阻遏中，将采用药理学抑制、染色质免疫沉淀（ChIP）和所鉴定的候选物的瞬时敲低。考虑到PTEN/PI 3 K通路在许多癌症以及其他疾病中失调，理解PTEN的详细调节可以阐明以PTEN缺陷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.