SIRT1亚细胞定位与卵巢癌顺铂耐药研究

Ovarian carcinoma is the most lethal gynecologic malignancy, primarily owing to high rate of drug resistance and tumor recurrence although the underlying mechanisms are largely unclear. SIRT1 belongs to a NAD+-dependent histone/protein deacetylase. It modulates multiple signaling and plays a role in autophagy and apoptosis. In our recent study, we had found that only nuclear location of SIRT1 was associated with worse response to chemotherapy and shorter disease-free survival duration, and was an independent factor for tumor recurrence by multivariate analysis. The nuclear expression of SIRT1 and the autophagy induced by cisplatin in cisplatin-resistant human ovarian cancer cell line showed much higher level of expression than that in the parental sensitive cell lines. The inhibition of SIRT1 promoted the sensitivity of cancer cells to cisplatin, whereas the activation of SIRT1 facilitated autophagy. These data suggest that SIRT1 subcellular localization may mediate cisplatin resistance via cell autophagy and apoptosis. Therefore, in the current study, we propose to determine the mechanism on how SIRT1 subcellular localization affects cisplatin-resistance via autophagy and apoptosis, using multiple approaches of cellular and molecular biology (e.g. quantitative analysis of autophagy-related protein's acetylation). Further, we propose to perform the preclinical studies of SIRT1-related additional chemotherapy in ovarian cancer stem cells and patient-derived xenograft model. Our combinated approaches are expected to offer novel approaches to targeting the chemoresistant cell population in ovarian cancer.

卵巢癌致死率居妇科恶性肿瘤之首，主要归因于肿瘤的高耐药性及复发率，具体机制尚不清楚。SIRT1是NAD+依赖的组蛋白/蛋白去乙酰化酶，调控多种信号途径，参与细胞自噬及凋亡。我们前期研究发现：卵巢癌组织SIRT1仅细胞核定位的患者对化疗不敏感、无瘤生存时间缩短，是肿瘤复发的独立预后因素，顺铂耐药株细胞核SIRT1表达、顺铂诱发的自噬活性均较配对敏感株增高，SIRT1抑制剂促进癌细胞对顺铂敏感，而激动剂促进癌细胞自噬，提示SIRT1亚细胞定位可能通过细胞自噬及凋亡影响卵巢癌顺铂耐药。因此，本课题拟采用自噬相关蛋白乙酰化定量分析等细胞及分子生物学方法，探讨SIRT1亚细胞定位—细胞自噬及凋亡—肿瘤耐药及其具体机制，阐明SIRT1亚细胞定位与肿瘤顺铂耐药的关系，并在此基础上，利用卵巢癌肿瘤干细胞及患者衍生的移植瘤模型进行SIRT1相关性辅助化疗的临床前期实验，为靶向卵巢癌顺铂耐药提供新的治疗策略。

本课题围绕SIRT1亚细胞定位对卵巢癌细胞迁徙和侵袭、顺铂耐药的影响及其具体机制展开研究。通过对453例卵巢癌患者肿瘤组织进行免疫组织化学染色，发现SIRT1胞质高表达与患者较好的预后相关。免疫电镜观察显示SIRT1分布于胞核、胞质基质、粗面内质网和线粒体内。过表达野生型SIRT1在胞核和胞质内均有分布，过表达核输出序列（NESs）突变型SIRT1（SIRT1NESmt）主要位于胞核，过表达核定位序列（NLSs）突变型SIRT1（SIRT1NLSmt）主要分布于胞质，但NESs突变导致外源性SIRT1丧失去乙酰化酶活性。SIRT1细胞裸鼠移植瘤的生长速度低于SIRT1NLSmt细胞，SIRT1细胞移植瘤SIRT1表达主要位于胞核，SIRT1NLSmt细胞移植瘤SIRT1表达主要位于胞质。SIRT1NLSmt细胞较SIRT1细胞的迁移和侵袭能力显著降低，乙酰化蛋白组学定量分析显示SIRT1NLSmt细胞的间叶标志物（vimentin和fibronectin）显著下调、上皮标志物（细胞角蛋白/CK、桥粒相关蛋白和紧密连接相关蛋白）显著上调，并伴随vimentin、CK-18和desmoplakin乙酰化水平的改变，提示胞质定位的SIRT1通过阻遏上皮-间质转化（EMT）抑制细胞迁移和侵袭。SIRT1NLSmt细胞较SIRT1细胞对顺铂显著耐药、细胞凋亡明显减少；经顺铂处理后，SIRT1NLSmt细胞vimentin表达升高、E-cadherin表达下降，提示顺铂引发SIRT1NLSmt细胞EMT过程，促进肿瘤细胞顺铂耐药。IGROV1、HEY细胞株存在TP53功能差异；过表达SIRT1和SIRT1NLSmt均造成细胞内p62累积和Beclin1减少、自噬流减弱；过表达SIRT1上调Mn-SOD的表达，但过表达SIRT1NLSmt缺乏此作用，SIRT1能够与Mn-SOD结合，但对其K68乙酰化水平无显著影响，提示p53、自噬和Mn-SOD途径不能合理解释耐药机制。有趣的是，我们还发现化疗后SIRT1NLSmt细胞较SIRT1细胞的多倍体肿瘤巨细胞（PGCCs）数量增多，且肿瘤干细胞标记物ALDH1A3和EpCAM增高，提示SIRT1亚细胞定位还可能通过PGCCs途径促进肿瘤干性形成、影响卵巢癌耐药，但具体机制有待后续深入研究。