Trihydrophobin 1促进雄激素受体降解及其在乳腺癌进展过程中作用的研究

【作者】 邹惟莹；

【导师】 顾建新； 谢建辉；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 雄激素信号通路在前列腺的发育、前列腺增生以及前列腺癌的发生发展中起极其重要的作用。本实验室之前的研究发现trihydrophobin 1 (TH1)蛋白在前列腺高表达。在体内体外实验中TH1均可以与雄激素受体相互作用,并且抑制雄激素受体的转录活性和内源性前列腺特异性抗原的表达。TH1还能够与雄激素受体一同与具有雄激素反应性的前列腺特异性抗原启动子相互结合,表明TH1参与了雄激素受体转录调控其下游基因的过程。在本论文中,我们发现在TH1抑制雄激素信号通路的过程中TH1能够诱导雄激素受体蛋白水平的降低,促进了雄激素受体的泛素化降解,但是TH1不影响雄激素受体的入核。而雄激素受体的稳定性受它N/C端的相互作用影响,TH1可以抑制雄激素受体的N/C端相互作用,但是并不单独参与雄激素受体N端或C端的降解。基于以上实验结果,我们提出TH1能够通过促进雄激素受体的泛素化降解来抑制雄激素信号的传导。这些结果还提示TH1可能直接参与雄激素相关的一些发育以及生理病理过程。TH1是负性转录延长因子复合体中的一个成员,与该复合体的其他亚基一同参与转录暂停。尽管有研究发现负性转录延长因子复合体可以抑制雌激素受体α介导的转录,但是没有研究报导TH1与肿瘤的发生发展的关系。在本论文中,我们发现TH1的蛋白水平与人类乳腺癌的进展呈负相关。免疫组化实验显示TH1蛋白在乳腺癌临床Ⅲ-Ⅳ期患者的乳腺癌组织中的表达明显低于乳腺癌临床Ⅰ-Ⅱ期的患者(P<0.01)。而在发生3个以上转移淋巴结转移患者的乳腺癌组织中TH1的蛋白水平明显低于3个及以下淋巴结转移患者的乳腺癌组织(P<0.001)。为了进一步研究TH1在乳腺癌发生发展中的分子机制,我们在MDA-MB-231细胞中制作了TH1过表达的稳转株,并且用RNA干扰的方法制作了在MCF-7细胞中TH1持续性下调的稳转株。我们可以观察到TH1可以抑制乳腺癌细胞的生长增殖,但是对细胞增殖的抑制作用不大。TH1还能够降低细胞周期蛋白D1的蛋白水平,上调CDK抑制子p21的蛋白水平,使乳腺癌细胞发生G0/G1期的细胞周期阻滞。TH1还可以降低β-catenin的水平并且抑制ERK的活性。此外,TH1可以抑制乳腺癌细胞的迁移和运动,在划痕实验中,TH1抑制了伤痕的修复,抑制了上皮间质化分子标记的相应改变,提示TH1可能抑制乳腺癌细胞的侵袭能力。以上结果显示TH1能够抑制乳腺癌细胞的增殖和侵袭,其蛋白水平的下调参与了乳腺癌的进展过程并且为乳腺癌的诊断及治疗提供了一个新的线索。更多还原

【Abstract】 The androgen-signaling pathway plays critical roles in normal prostate development, benign prostatic hyperplasia, established prostate cancer, and in prostate carcinogenesis. In this study, we report that trihydrophobin 1 (TH1) is a potent negative regulator to attenuate the androgen signal-transduction cascade through promoting androgen receptor (AR) degradation. TH1 interacts with AR both in vitro and in vivo, decreases the stability of AR, and promotes AR ubiquitination in a ligand-independent manner. TH1 also associates with AR at the active androgen-responsive prostate-specific antigen (PSA) promoter in the nucleus of LNCaP cells. Decrease of endogenous AR protein by TH1 interferes with androgen-induced luciferase reporter expression and reduces endogenous PSA expression. Taken together, these results indicate that TH1 is a novel regulator to control the duration and magnitude of androgen signal transduction and might be directly involved in androgen-related developmental, physiological, and pathological processes. Trihydrophobin 1 (TH1) is a member of negative elongation factors (NELF) complex, which is involved in transcriptional pausing. Although NELF complex attenuates the estrogen receptor a-mediated transcription, little is known about the relationship between TH1 and tumor progression. Here, we reported that protein level of TH1 was negatively correlated with the malignancy of human breast cancer. Immunohistochemical analysis revealed that TH1 expression in clinical stage III-IV primary breast cancer tissues was statistically significantly lower than that in stage I-II breast tissues (P<0.01), and especially inversely associated with lymph node metastasis (P<0.001). Furthermore, we demonstrated that overexpression of TH1 in MDA-MB-231 cells inhibited and knockdown of TH1 in MCF-7 cells enhanced cell migratory ability of breast cancer cells. Moreover, upregulation of TH1 in MDA-MB-231 cells resulted in the decrease of cell cycle protein cyclin D1,β-catenin and ERK activity, and the increase of cyclin-dependent kinase inhibitor p21. In contrast, knockdown of TH1 in MCF-7 cells enhanced the expression of cyclin D1 andβ-catenin, increased the activity of ERK, and downregulated the expression of p21. Additionally, overexpression of TH1 in MDA-MB-231 cells prevented and, however, knockdown of TH1 in MCF-7 cells induced epithelial-mesenchymal transition (EMT) in breast cancer cells. Taken together, our results suggest that TH1 might play an important role in regulation of proliferation and invasion in human breast cancer, and thereby might be a potential target for human breast cancer treatment.更多还原