MASPIN抑制炎症诱导细胞恶性转化和肿瘤干细胞形成的分子机制研究

Tumor suppressive MASPIN is expressed in normal mammary epithelial cells but reduced or absent in breast carcinomas. Our and others studies founded that MASPIN functions as an endogenous inhibitor of histone deacetylase inhibitor 1, and epigenetically regulates tumor cell undergoing re-differentiation, and participates cell signaling transduction, and modifies multiple genes expression including inflammatory cytokines (Li, Cancer Research, 2006; Molecular Cancer Research, 2011). However, ectopic expression of MASPIN in cancer cell may not always prevent tumor growing in vivo mouse model (Teoh, 2004), which suggests that there is typically uncovered mechanism involved in its anti-tumor activity. Therefore, based on the theory that tumor is initiated from cancer stem cells with the characteristic of heterogeneity, we propose here that MASPIN could epigenetically inhabits inflammatory-induced EMT, which leads to further prevent cancer stem cell formation and tumor initiation. This study is going to continually focus on investigating how MASPIN down-regulates the expression and its activity of both interleukin 6 and Hypoxia-inducible factor 1-alpha in prostate and breast cancer cellular and mouse model, which cooperates with TGF signaling pathway to prevent tumor formation. The significant of this study is to explore and to uncover the inside molecular mechanism which will definitely benefit its further clinic application of MASPIN as a novel tumor suppressive protein, such as in early detection of tumor by reducing its expression, and in tumor prevention and therapy by developing an anti-tumor vaccine.

MASPIN（乳腺丝抑蛋白）是上皮细胞表达的一种肿瘤抑制分子。我们的研究发现：MASPIN抑制组蛋白去乙酰化酶活性，经表观遗传调控机制促进肿瘤细胞的再分化; 参与细胞内信号的传导和调节炎症因子等基因的表达（Cancer Research, 2006; Mol Cancer Res, 2011）。然而，MASPIN在肿瘤细胞中的重组（新）表达并没能阻止肿瘤的形成，提示可能存在有其它机制参与其抗肿瘤作用。基于肿瘤是由肿瘤干细胞发展形成和肿瘤组成的异质性理论，我们因此假设：MASPIN通过表观遗传调节机制抑制炎症诱导的细胞转化和肿瘤干细胞的形成，最终抑制肿瘤的发生。本课题拟在前期研究的基础上，以前列腺和乳腺肿瘤为模型，重点研究 MASPIN下调IL-6、HIF-1α表达和活性，抑制炎症和TGF相关分子诱导细胞恶性转化的分子机制，为MASPIN在预防和治疗肿瘤上的应用提供新的作用靶点和研究思路。

Maspin作为内源性HDAC1抑制分子的抗癌活性已经得到广泛证实，但其在前列腺癌（CaP）的发生中的作用和如何将其应用于临床尚不明确。本课题首先证明广谱HDAC抑制剂曲古霉素A（TSA）诱导肿瘤细胞表达maspin和p21同时，具有协助化疗药物（DTX）抑制CaP肿瘤细胞增殖和诱导细胞产生凋亡。采用了无血清悬浮培养技术，在体外建立CaP肿瘤干细胞样微球细胞培养模型。且获得的肿瘤干细胞样细胞表达系列干细胞分子标志物。经TSA处理，或经基因转染而使肿瘤细胞表达maspin均显著降低肿瘤细胞自发和经LPS诱导的干细胞形成。使用I类HDAC抑制剂MS-275结合雄激素受体拮抗剂药物Enzalutamide（恩杂鲁胺，MDV3100）处理肿瘤细胞后发现，虽然单独使用Enzalutamide处理肿瘤细胞不能诱导细胞凋亡，MS⁃275单独或联合enzalutamide处理均能明显抑制干细胞样肿瘤微球的形成、促进肿瘤细胞生长周期停滞和产生凋亡。小鼠体内实验也证实MS-275协同Enzalutamide的抗肿瘤活性；同时，发现Enzalutamide显著抑制表达maspin的细胞在小鼠体内的增殖与生长，并有效降低小鼠血清中PSA的水平。体内外研究发现，MS-275在增加maspin 表达的同时，降低雄激素受体AR， c-Myc和β-catenin 的蛋白水平，从而显著提高 Enzalutamide 的疗效。研究GEO数据也显示maspin与AR的表达在前列腺肿瘤的恶性化发展中呈负相关性。经siRNA 和shRNA 技术降低CaP 细胞maspin的表达后均发现有伴随AR以及AR调节的下游分子表达升高；ChIP实验显示maspin与AR启动子的部分序列相结合；体外研究显示，maspin有可能通过抑制c-Myc的表达和c-jun信号通路的激活而抑制炎症性IL-6处理和缺氧处理（CoCl2, 1% O2）诱导的细胞恶性转化和肿瘤干细胞样细胞的形成。我们的研究还发现， 检测尿液中的PCA3和PSA的核酸并计算其比值较tPSA能为临床提供更可靠和早期的诊断前列腺肿瘤的方法。结论：内源性HDAC1抑制分子maspin可通过抑制AR、c-Myc的表达和肿瘤细胞信号通路的激活，抑制细胞恶性转化和肿瘤干细胞形成，抑制肿瘤的发生而产生抗肿瘤作用，从而维持机体自稳和平衡、促进CaP的化疗和内分泌治疗作用