LncRNA-ANCR介导的组蛋白甲基化酶EZH2稳定性在乳腺癌转移中的作用和机制

Metastases are the main cause of breast cancer patients. Epithelial-Mesenchymal Transition (EMT) plays a pivotal role in cancer invasion and metastasis. Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. However, the role of lncRNA in EMT and breast cancer invasion and metastasis remains largely unknown. Our pilot data showed that the expression of lncRNA ANCR was low in many breast cancer cell lines and breast cancer tissue samples, and knockdown of ANCR led to epithelia-to-mesenchymal transition (EMT) in MCF10A cells whereas overexpression of ANCR reversed EMT in MDA-MB-231 cells. We also showed that ANCR can bind to and destabilize histone methyltransferase EZH2. Because EZH2 is often over-expressed in breast and other cancers and promotes tumorigenesis and metastasis, we hypothesize that ANCR may inhibit breast cancer metastasis by decreasing the stability of EZH2. In this study, we will delineate the molecular mechanism via which ANCR binds to EZH2 in EMT and mediates breast cancer metastasis using in vitro cancer cell line and in vivo nude mouse models. Meanwhile, we will investigate the relationship between ANCR-mediated EZH2 stability and its phosphorylation and glycosylation modification. We will also analyze breast cancer clinical samples to unfold the correlation of the expression of ANCR and EZH2 with patients' survival. Our research may result in novel therapeutic interventions of breast cancer.

乳腺癌转移是乳腺癌患者死亡的最主要原因。上皮-间质细胞转化（EMT）在癌症浸润和转移过程中发挥重要作用。长链非编码RNA（lncRNA）是多种不同生物过程的重要调节子。但有关lncRNA介导EMT，参与乳腺癌转移的研究还较少。我们的前期工作发现: lncRNA-ANCR在多种乳腺癌细胞系和乳腺癌组织中低表达；下调ANCR的表达能引起乳腺上皮细胞发生EMT；过表达ANCR可以逆转乳腺癌细胞间质表型；ANCR能够降低组蛋白甲基化酶EZH2蛋白的稳定性。鉴于EZH2与乳腺癌转移关系密切，我们推断ANCR可能通过影响EZH2的稳定性抑制乳腺癌转移。本研究将在细胞水平、裸鼠动物模型水平和人乳腺癌样本水平上，探讨ANCR介导EZH2在乳腺癌细胞EMT及乳腺癌转移中的作用；阐明ANCR影响EZH2稳定性的分子机制；揭示ANCR和EZH2的表达与乳腺癌预后的相关性，为寻找新的乳腺癌治疗靶点提供实验依据。

EZH2作为重要的表观遗传调控因子和EMT诱导因子参与多种癌症的转移。EZH2的稳定性受多种蛋白质翻译后修饰的调控。长链非编码RNA（lncRNAs）通过调节蛋白质的稳定性参与多种癌症的进程。到目前为止，EZH2蛋白的稳定性是否受lncRNAs的调控还未见报道。本项研究我们发现在乳腺癌细胞中，lncRNA ANCR 能够和EZH2结合，并且促进CDK1对EZH2磷酸化（T345和T487），导致EZH2泛素化降解，最终降低其蛋白稳定性。对lncRNA ANCR功能的研究显示，ANCR在乳腺癌癌组织和乳腺癌细胞中均是低表达；敲低ANCR可以诱发MCF10A乳腺上皮细胞发生EMT，且能够促进MCF10A和MCF7乳腺癌细胞侵袭迁移。在ANCR低表达的MDA-MB-231乳腺癌细胞中，过表达ANCR能够部分逆转EMT现象，且能够抑制其侵袭迁移能力。过表达ANCR通过降低EZH2的稳定性抑制E-cadherin、HOXA10和DAB2IP等基因的表达。此外，在同时过表达ANCR和EZH2的回复实验中，进一步确认ANCR在乳腺癌中的功能主要是通过抑制EZH2蛋白的稳定性来实现的。通过裸鼠皮下和尾静脉注射检测ANCR对乳腺癌细胞体内成瘤和肺转移能力的影响，发现ANCR在体内同样能够抑制乳腺癌细胞的成瘤和肺转移。总之，我们的研究发现ANCR是一个能够抑制EZH2稳定性的lncRNA，提出了EZH2稳定性调节的一个新机制；同时 发现ANCR通过调控EZH2蛋白的稳定性抑制乳腺癌细胞EMT和乳腺癌的转移。这一新的发现预示着ANCR可能是一个乳腺癌潜在的诊断和治疗的新靶标，为乳腺癌的诊断和治疗提供新的思路。