Nmi通过负调控人端粒酶逆转录酶抑制乳腺癌发生发展的作用机制和临床意义

The aberrant activation of hTERT is closely associated with tumorigenesis and development of breast cancer. It is very important to investigate and identify the specific transcriptional regulator of hTERT in breast cancer. In our preliminary studies, Nmi was found as a new hTERT promoter-binding protein in breast cancer cells based on magnetic-biopanning and proteomic methods. Nmi inhibited hTERT activity, was lowly expressed in breast cancer-stem like cells and affected the stemness of stem-like cells in breast cancer. However, it is still unclear about the precise molecular mechanisms and clinical significance of Nmi in tumorigenesis and development in breast cancer via regulates hTERT. This project plans to knock down or overexpress Nmi in breast cancer cell lines or animal models, and further investigate its effects on hTERT transcription, telomerase activity, telomere length, stemness of cancer stem cells, and tumor growth and development. We also plan to simultaneously knock down or overexpress Nmi and hTERT to analyze the reversibility of hTERT on the above biological function changes mediated by Nmi. Moreover, we will also investigate the correlation between Nmi and hTERT expression and their relationship with tumor classification, stage, prognosis and recurrence in clinical tissue samples from patients with breast cancer, thereby clarifying its clinical significance. This project aims to provide theoretical and experimental evidence for developing Nmi/hTERT as new targets in breast cancer diagnosis and treatment.

端粒酶亚单位hTERT的异常活化与乳腺癌发生发展密切相关，发现和确定乳腺癌特异性的hTERT转录调控因子意义重大。前期研究我们采用磁生物淘洗和蛋白质组学方法，在乳腺癌细胞中垂钓和鉴定出新型的hTERT启动子结合蛋白Nmi，发现其抑制hTERT转录，在乳腺癌干细胞中低表达并影响乳腺癌干细胞的干性，但Nmi通过调控hTERT表达进而参与乳腺癌发生发展的具体分子机制和临床意义，尚不明确。本课题拟在乳腺癌细胞和动物模型中敲低或过表达Nmi，研究其对hTERT表达，端粒酶活性，端粒长度，肿瘤干细胞干性以及肿瘤生长和转移的影响，并在此基础上敲低或过表达hTERT，研究其对Nmi介导的上述功能的逆转性；同时利用组织芯片，结合临床资料，分析乳腺癌患者组织中二者的表达相关性，及其与肿瘤分型、分期、预后和复发的联系，明确其临床意义。本课题旨在为确立Nmi/hTERT为乳腺癌诊断和治疗新靶标提供理论和实验依据。

N-myc and STAT interactor (NMI)被证明可作为转录共活化子与不同的转录因子结合，调节多种信号转导机制，包括DNA损伤、细胞周期和上皮间充质转化。尽管如此，NMI在肿瘤干细胞(CSCs)调控中的作用及其相关的分子机制仍不清楚。在本课题中，我们研究了NMI对乳腺癌CSCs性状的调控，并揭示了其潜在的分子作用机制。我们发现NMI在乳腺癌干细胞(BCSCs)富集群中表达较低。NMI的敲低表达促进了CSCs的性状，而其过表达抑制了CSCs的性状，包括CSCs相关标记物的表达、CD44+CD24-细胞群的数量和乳腺肿瘤球形成的能力。我们还发现NMI介导的对BCSCs性状的调控至少部分是通过对hTERT信号的调节实现的。NMI敲低表达可上调乳腺癌细胞中hTERT的表达，而过表达则下调hTERT的表达，NMI介导的CSCs性状和细胞侵袭能力的改变可被hTERT逆转。小鼠模型研究也证实NMI敲低表达可通过上调hTERT信号通路促进乳腺癌的进程。对乳腺癌患者临床组织样本的进一步分析表明，NMI表达与hTERT表达呈负相关，低NMI高hTERT表达与乳腺癌患者更差的临床TNM分期相关。此外，我们还证实了乳腺癌细胞中YY1蛋白通过与NMI相互作用参与了NMI介导的hTERT的转录调控。进一步地，我们发现乳腺癌细胞中EZH2可以结合在NMI启动子区，介导H3K27的三甲基化进而抑制NMI的转录。EZH2对乳腺癌细胞的转移和干性维持的促进作用可以被NMI的过表达所逆转，反之亦然。同样地，乳腺癌患者临床组织样本中，EZH2表达与NMI表达呈负相关，且患者的临床TNM分期越高，EZH2表达越高，NMI表达越低。总之，我们的研究结果表明在EZH2的转录调控下，NMI通过与YY1互作共同靶向hTERT继而抑制乳腺癌的干性维持和转移，从而为理解BCSCs的衍变机制提供了新的见解，并提示EZH2-NMI-YY1-hTERT信号轴有望成为乳腺癌诊断和治疗的新靶标。