TC2N抑制乳腺癌发生的功能及分子机制研究

In our previous study，TC2N has proven to be an important regulatory protein in tumor acting as an oncogene in lung cancer. Here, we found TC2N could be a candidate tumor suppressor in breast cancer because of its expression sharp decrease with the tumor development. Over-expression of TC2N in breast cancer cell could significantly restrain cell proliferation and tumor growth in vitro, which was further validated by xenografts in nude mice in vivo. GO enrichment analysis using TCGA BC dataset uncovered a probability that TC2N could regulate tumor growth by PI3K-AKT signaling pathway. And actually, TC2N could inhibit phosphorylation of p85 and AKT by western blot. Still, the regulatory mechanism and signaling pathway remain unclear. In this project, we mainly focused on the following issues: to further evaluate the inhibitory effect of TC2N on the development of breast cancer; to study the mechanism of TC2N inhibiting PI3K/Akt signaling pathway; and to analyze the relationship between TC2N gene and its clinical significance in breast cancer. This will help us to fully understand how TC2N inhibits the proliferation of breast cancer cell, to enrich our understanding of PI3K/AKT signaling pathway, and to provide theoretical support for clinical diagnosis and precise treatment in breast cancer.

在前期首次报道TC2N在肺癌发生过程中是一个新的癌基因基础上，我们通过数据库分析发现该基因在其它不同类型肿瘤中多为高表达，是典型癌基因；然而，仅在乳腺癌中，该基因表达显著下降，提示其在乳腺癌发生中具有特殊的作用和调控机制。实验分析发现TC2N高表达在体内外均能显著抑制乳腺癌细胞的增殖，是一个候选的抑癌基因。进一步研究发现，TC2N可通过抑制PI3K/AKT磷酸化抑制肿瘤生长。但相关抑癌作用和分子机制还需要系统研究和解析。本项目拟构建TC2N基因敲除动物化学致癌模型，评价TC2N在乳腺癌中的抑制作用；分析参与TC2N抑癌过程中PI3K/AKT信号通路下游的关键分子，阐明TC2N调控p85的机制。此外，解析TC2N在临床乳腺癌中的表达及临床意义分析。以期能系统阐述TC2N抑癌作用及分子机制，为临床乳腺癌诊断、治疗提供理论支持。

在该基金中，结合Oncomine公共数据库中乳腺癌RNA-seq数据集和组织芯片分析，我们发现TC2N在乳腺癌组织中普遍高表达，但随着肿瘤进展，TC2N表达量下降；乳腺癌患者临床样本病理特征分析提示TC2N高表达是乳腺癌患者良好预后因素，单变量Kaplan-Meier分析得到一致结果。这提示TC2N在乳腺癌肿瘤进展中发挥抑制作用。通过组织芯片人群样本资料分析，我们发现TC2N与肿瘤生长密切相关；体内外实验进一步验证TC2N能抑制肿瘤细胞生长。机制方面，通过数据库信号通路分析，我们发现TC2N与PI3K-AKT信号通路密切相关。利用CO-IP，Westernblot，抑制剂/基因干扰技术，我们发现TC2N能分别与ALK 和p55γ、EBP1竞争性结合，减少ALK-p55γ和EBP1-AKT复合体，进而抑制p55γ和AKT的磷酸化，发挥抑制乳腺癌肿瘤细胞生长功能。此外，我们发现TC2N具有抑制乳腺癌细胞侵袭与转移功能。TC2N差异表达，CTNNB1蛋白水平发生显著变化，提示TC2N抑制乳腺癌细胞侵袭与迁移的功能与CTNNB1信号通路密切相关。总之，在该基金中，我们从临床乳腺癌人群样本，细胞水平及动物水平初步确证了TC2N抑制乳腺癌进展的功能及分子机制。