干细胞因子Sall4在顺铂治疗卵巢癌疗效监测和耐药复发中的临床价值和机制研究

Ovarian cancer is burdened by the highest mortality rate among gynecological cancers and approximately 85% of the patients will experience relapse after treatment. The chromosomal region, 20q12-13, is commonly amplified in ovarian cancer and we hypothesize that human Sall4 gene, located on chromosome 20q13.2, is an important candidate gene that plays an essential role in ovarian cancer initiation. We have found that Sall4 over-expression is seen in more than 20% of ovarian cancer and it is significantly increased in the middle stage of malignant transformation model. Down-regulation of Sall4 expression by shRNA leads to apoptosis and growth retardation. However, whether Sall4 is involved in the resistance of cisplatin, one of the first-line chemotherapies for ovarian cancer, or can be a potential monitoring target for cisplatin efficacy have not been reported yet. In this study, we will assess the level of Sall4 in newly diagnosed and relapsed patients by analyzing their peripheral blood and tumor tissue. The clinical value of Sall4 in monitoring cisplatin efficacy and the risk of relapse will be further elucidated by the well-established circulating tumor cells detecting platform in our lab. In addition, we will dissect the mechanism of Sall4 in the cisplatin resistance in cell lines and mouse models by both gain of function and loss of function studies, combined with cytotoxicity assays, and explore the association of Sall4 with Notch pathway. We will also verify the potential of Sall4 as a therapeutic target by using a specific Sall4 peptide in the treatment of ovarian cancer.

卵巢癌致死率在妇科肿瘤中高居首位，约85%的患者治疗后经历复发。染色体20q12-q13区域扩增常见于卵巢癌，而干细胞因子Sall4可能是该区域扩增的靶基因。我们的前期研究表明Sall4过表达于超20%的卵巢癌患者，在卵巢癌恶性转化中期即显著升高，下调Sall4表达水平导致细胞凋亡和生长阻滞。但Sall4是否参与到卵巢癌化疗药物顺铂的耐药及能否作为其疗效监测靶点尚未见报道。我们拟从临床角度，检测初诊和复发患者外周血和肿瘤组织中Sall4的表达，并借助自身建立的CTCs分离鉴定平台动态研究CTCs中Sall4表达在卵巢癌顺铂疗效和耐药复发中的监测价值，以期寻找新的监测指标；进一步，在细胞和动物水平通过Sall4功能缺失和获得性实验，结合细胞毒性实验和分子生物学技术，探究Sall4在卵巢癌顺铂耐药中的作用机制；最后通过制备靶向Sall4的多肽探究Sall4作为顺铂耐药卵巢癌治疗靶点的可行性。

卵巢癌致死率在妇科肿瘤中高居首位，治疗后耐药是影响卵巢癌预后生存的重大难题，这与治疗后卵巢癌干细胞残留可能有关。经典的干细胞通路分子NOTCH3在卵巢癌发生、肿瘤干细胞维持以及铂类药物抵抗中起到了关键性的作用，本课题探索SALL4是否受到NOTCH3的调控并参与了顺铂耐药。在研究过程中，未发现足够证据支持NOTCH3对SALL4的直接调控作用，而发现miR-1299和lncRNA TUG1在卵巢癌中的功能及其对NOTCH3的调控。研究表明，miR-1299在卵巢癌中是NOTCH3的负调控因子，在卵巢癌组织中显著下调，miR-1299的上调显著抑制了卵巢癌细胞增殖、集落形成和DNA合成，并诱导肿瘤细胞发生G0/G1期周期停滞。lncRNA TUG1可充当miR-1299的海绵，作为内源性RNA竞争性吸附miR-1299而上调NOTCH3的表达，从而促进卵巢癌恶性增殖。lncRNA TUG1同时是NOTCH3的潜在下游靶标，在卵巢癌的进展中形成miR-1299/NOTCH3/TUG1的反馈环路。前期研究发现在RNA水平上卵巢癌组织SALL4远高于正常组织，进而本课题从转录水平抑制的角度上选取了BRD4抑制剂JQ1，对其在卵巢癌中靶向SALL4的治疗作用进行了研究，发现JQ1可在RNA和蛋白水平上快速降解SALL4，该作用呈时间和剂量效应。JQ1处理内源性高表达SALL4细胞出现与SALL4敲低相似的效应，提示SALL4是JQ1作用靶点之一。内源性高表达SALL4细胞对JQ1更敏感，提示JQ1对SALL4阳性卵巢癌有选择性杀伤作用，SALL4有望成为治疗卵巢癌的潜在靶点。将CTC-SALL4检测平台用于妇科妊娠滋养细胞疾病（GTN）中，恶性GTN、良性GTN和健康孕妇相比较总CTC、E/M-CTC和M-CTC的数量存在显著差异，进行ROC分析以评估CTC表型在区分GTN患者和健康孕妇方面的效能，AUC为0.826。Cutoff值为8.5个/4 mL时Youden指数最佳，此时灵敏度和特异性分别为53.66%和100%。对检出CTC数量高于cutoff值的患者进一步检测CTC-SALL4，SALL4高表达CTC（>2个信号点）出现在66.67%（10/15）的恶性GTN患者中，而在良性患者中未见（0/5）。CTC-SALL4是诊断GTN的潜在标志物。