自噬相关基因ULK1在乳腺癌演进中的分子调控机制及其临床意义研究

Autophagy is a catabolic process that widely involves in the whole process of human cancers.ULK1 is the furthest upstream kinase involved in autophagosome formation. However, the function and expression of ULK1 in human breast cancer is still scarcely explored. In our prelimilary study, we found that the mRNA and protein levels of ULK1 decreased in breast cancer cells and fresh breast carcinoma tissues, compared with the immortalized breast epithelial cell line (MCF-10A) and matched normal tissues. Gradual decreased expression of ULK1 was associated with breast cancer progression from ductal hyperplasia (DH), to atypical ductal hyperplasia (ADH), and then to ductal carcinoma in situ (DCIS), and finally to invasive ductal carcinomas (IDC). As patients with lower level of ULK1 had higher metastatic potential and poorer prognosis, ULK1 might be used as a novel prognostic biomarker for breast cancer patients. We also found that the mRNA level of ULK1 increased obviously in breast cancer cells after treatment with 5-aza-2'-deoxycytidine. Additionally, overexpression ULK1 inhibited MDA-MB-231 cells invasive ability and suppressed MDA-MB-231 xenograft tumor growth. In this proposal, we further extend our research to detect the aberrent CpG island methylation in promoter region of ULK1 gene , and demonstrate the effect of ULK1 on the breast cancer cells genomic instability, proliferation , the ability to form mice xenograft, invasion and migration of breast cancer cells, and the carcinogenesis transformation of MCF-10A under 3-D culture in vitro. We will also explore the molecular mechanisms of ULK1 affecting breast tumorigenesis through regulation of autophagy and other new signaling pathways, and clarify the significance of over-expressing of ULK1 to breast cancer therapy. The study will provide a comprehensive understanding of relationship between autophagy and breast cancer, introduce a new molecular platform for molecular diagnosis , prognostic evaluation, of breast cancer, and reveal a new valuable pathway for breast cancer therapy.

自噬贯穿于肿瘤演进过程中。ULK1是自噬途径的核心蛋白，但其在乳腺癌中的表达和意义尙不清楚。我们研究发现ULK1在乳腺癌细胞株及癌组织中低表达，随着乳腺癌的演进其表达呈逐渐减少的趋势，是一个独立的预后因素；甲基化抑制剂处理后的乳腺癌细胞其ULK1 mRNA表达增高；而过表达ULK1减弱MDA-MB-231细胞的侵袭能力及体内成瘤能力。本课题拟深入研究乳腺癌演进中ULK1启动子区CpG岛的异常甲基化与其低表达的关系；研究ULK1对乳腺癌细胞的基因组稳定性、增殖、成瘤及侵袭转移能力的影响，在体外三维培养模型下研究干扰ULK1对MCF-10A的恶性转化作用；探讨ULK1通过调控自噬及其它新的生物学通路影响乳腺癌恶性进展的分子机制；阐明过表达ULK1能否增强乳腺癌细胞对化疗的敏感性。本研究有助于深入理解自噬在乳腺癌中的作用，为乳腺癌的分子诊断、预后评估等提供新的分子平台，并为乳腺癌的治疗提供新思路

自噬贯穿于肿瘤演进过程中。ULK1 是自噬途径的核心蛋白，但其在乳腺癌中的表达和意义尙不清楚。在本项目中我们重点探讨了ULK1在乳腺癌演进中的作用和分子调控机制，及其对乳腺癌治疗的意义。重要结果及关键数据如下：① 明确乳腺癌演进中ULK1表达失调的原因：发现乳腺癌从良性导管增生性病变→重度不典型增生→导管内癌→浸润性癌演变过程中，ULK1 蛋白的表达呈逐渐减少的趋势；且ULK1低表达的患者预后差，易发生转移，可作为乳腺癌患者新的独立的预后因子。阐明ERK激酶参与了β-TRCP 介导的ULK1泛素化蛋白酶体降解，这是导致ULK1在乳腺癌中表达下调的重要原因。② 明确ULK1 的抑癌功能及其分子调控机制：发现ULK1低表达的患者易发生远处转移；发现在低氧培养条件下ULK1低表达显著促进乳腺癌细胞的侵袭迁移能力，且易导致小鼠骨转移的发生；揭示在低氧环境下ULK1的低表达使乳腺癌细胞线粒体自噬能力显著下降，而这种线粒体自噬能力的缺失，使细胞ROS的产生增多并促使炎症因子的分泌，从而显著促进乳腺癌细胞的侵袭迁移能力，并诱导破骨细胞的成熟，导致骨转移病灶的形成。③ 明确ULK1表达对乳腺癌治疗的重要意义：发现ULK1低表达的患者对阿霉素耐药；细胞学和动物实验结果进一步证实ULK1缺失易导致乳腺癌细胞对阿霉素不敏感，回复ULK1可以逆转这一现象。以上结果说明过表达ULK1能增强乳腺癌细胞对化疗药物的敏感性。本研究有助于深入理解自噬在乳腺癌发生发展中的作用，为乳腺癌的分子诊断、预后评估、复发转移等提供新的分子平台，并为乳腺癌的治疗提供新思路。.受该基金资助，我们发现年轻的乳腺癌患者行保乳手术治疗后，同行改良根治术相比，局部复发率相对较高，但远处转移和总生存率无明显差异，提出年轻乳腺癌患者行保乳治疗是可行的。发现靶向AKT的抗癌化合物Excisanin A能通过抑制integrin β1/FAK/PI3K/AKT/β-catenin通路，抑制乳腺癌细胞的侵袭转移能力。发现2-嘧啶-5-酰胺基噻唑类化合物DC120 通过抑制AKT 及其下游信号通路抑制肿瘤干细胞样SP 细胞的增殖、悬浮球囊形成能力，并能增加CDDP对肿瘤干细胞样SP 细胞的化疗敏感性。.