Slfn5/JARID2/组蛋白甲基化抑制乳腺癌发生EMT

Epithelial-mesenchymal transition (EMT) is an important initial stage of invasion and metastasis in breast cancer, hence it is an important approach to research new molecular which can inhibit EMT for anti-cancer drug research and development. We recently reaearched and found that: (1) the expression of SLFN5 in breast cancer cell line with high invasion abitilities was significantly lower than that in non-invasive cancer cell line using real-time PCR analysis; (2) knockdown SLFN5 with its specific siRNA transfection resulted in the decreased expression in E-cadherin compared with negative siRNA control, suggesting that SLFN5 could inhibit EMT; (3) using mass spectrometry combined with co-immunoprecipitation analysis, SLFN5 protein was found to bind Jarid2 protein; and (4) using Histone methyltransferase inhibitor to treat MCF7 cells with SLFN5 knockdown by specific siRNA, no significant change was detected in the expression of E-cadherin. From the above raw data we speculate that SLFN5 may play roles in the inhibition of EMT via competitive combinding to Jarid2 protein resulting in the inhibition of Histone methylation, and promote the expression of E-cadherin, thereby inhibiting EMT iniation. We plan to explore the role of SLFN5 in breast cancer EMT and its molecular mechanisms. Multiple levels including animal models, clinical cancer tissue specimens and molecular biological means will be employed to elucidate SLFN5 inhibitory effects on EMT and its molecular mechanisms. This study is beneficial for research and development of target molecules for anti-breast cancer drugs.

上皮间质化（EMT）是乳腺癌细胞浸润转移的重要启动阶段，寻找EMT抑制分子是抗癌转移新药研发的重要途径。预实验首次发现：侵袭性乳腺癌细胞系SLFN5表达明显低于非侵袭性细胞系，其siRNA能下调E-钙粘蛋白表达，提示SLFN5能抑制EMT，质谱及免疫共沉淀显示SLFN5与组蛋白甲基转移酶的募集分子JARID2（J2）结合，转移酶抑制剂处理SLFN5siRNA转染的细胞发现E-钙粘蛋白没有变化，由此推测SLFN5是乳腺癌EMT抑制分子，通过竞争性结合募集分子J2抑制组蛋白甲基转移酶，促进E-钙粘蛋白表达从而抑制EMT。拟从动物模型、临床癌组织标本、高/低转移细胞，运用分子生物学、表观遗传学、分子互作等方法，研究SLFN5对乳腺癌EMT的作用，SLFN5与J2分子互作，对组蛋白甲基转移酶的抑制，以及抑制基因启动子区组蛋白赖氨酸甲基化，上调E-钙粘蛋白的表达，为抗癌药物研发提供新的靶点。

乳腺癌是女性发病率最高的癌症，严重威胁女性健康。癌细胞的浸润转移是癌症致死的主要原因。我们提出Schlafen 5 (SLFN5)可能是乳腺癌侵袭的抑制性基因，抑制侵袭转移的初始阶段——EMT的发生。SLFN5在乳腺癌(BRCA)中的作用尚未确定。我们利用TCGA、临床标本和人乳腺癌细胞系，显示SLFN5表达与BRCA转移呈负相关。SLFN5敲低可诱导BRCA细胞系上皮间充质转化(EMT)并增强侵袭转移能力。过表达导致间充质上皮转化(MET)并抑制侵袭转移能力。SLFN5能抑制ZEB1的表达，但不抑制ZEB2、SNAI1、SNAI2、TWIST1、TWIST2、microRNA (miR)-200家族的表达。ZEB1基因的敲低和过表达实验证实它是SLFN5表型和侵袭改变的中间介导分子。此外，ChIP-Seq结果显示在ZEB2、SNAI1、SNAI2、TWIST1、TWIST2、E-cadherin (CDH1) microRNA (miR)-200家族的启动子中没有明显的结合峰，而在ZEB1启动子区有明显的结合峰。荧光素酶报告基因启动子活性证实SLFN5通过直接结合ZEB1启动子上的SLFN5结合基序抑制了ZEB1的转录，而SLFN5的C-末端缺失突变体则没有这种结合作用。SLFN5通过ZEB1影响细胞增殖和凋亡，抑制乳腺癌嘌呤代谢途径。ZEB1敲低和过表达分别阻断了SLFN5敲低和过表达引起的PTEN和AKT通路以及菌落形成能力的变化。荧光素酶报告基因检测表明，ZEB1可以通过结合PTEN启动子中的基序抑制MCF7细胞中PTEN启动子的活性。代谢组学分析表明，SLFN5影响许多代谢途径，特别是减少嘌呤代谢物，包括肌苷、黄嘌呤和次黄嘌呤。另外SLFN5通过AKT/GSK-3β/β-catenin通路抑制MT1-MMP表达抑制癌细胞迁移和侵袭。综上所述，SLFN5调控可逆上皮和间充质转化，通过抑制ZEB1转录抑制BRCA转移，并且可以抑制乳腺癌细胞的增殖，促进凋亡，调控代谢，提示SLFN5可能是BRCA治疗的潜在靶点。