LC−MS/MS Analysis of Ovarian Cancer Metastasis-Related Proteins Using a Nude Mouse Model: 14-3-3 Zeta as a Candidate Biomarker

Peritoneal implantation is the most common metastatic pattern of epithelial ovarian cancer, and the five-year survival rate of patients is dramatically decreased when large-scale peritoneal metastasis occurs. This study aimed to determine serum proteins that could be used to detect early peritoneal metastasis of ovarian cancer. The secreted (or shed) proteins of the ovarian cancer cell line SKOV-3 were analyzed using LC−MS/MS, and 97 proteins were identified in the SKOV-3 culture supernatant. After the SKOV-3 cells were xenografted into the peritoneal cavities of nude mice, 3 of the 97 proteins were detected in animal sera. Following enzyme-linked immunosorbent assay (ELISA)-based screening of clinical blood samples, one of the three proteins, 14-3-3 zeta, was identified as a candidate biomarker. The average serum levels of 14-3-3 zeta in patients with epithelial ovarian cancer and benign gynecological diseases were significantly different. The expression of 14-3-3 zeta was associated with the degree of cancer peritoneal metastasis, the emergence of ascites, bilateral involvement, and the clinical stage and substage. Using 14-3-3 zeta, the overall diagnostic accuracy for ovarian cancer was greatly improved. Furthermore, siRNA-based experiments demonstrated that 14-3-3 zeta was responsible for approximately 62, 65, and 30% of the migratory, invasive, and implantation abilities of SKOV-3 cells, respectively. The present results demonstrated that the nude mouse xenograft model is an efficient system for performing function-oriented biomarker discovery, which can be used for a variety of research tasks in future molecular diagnoses, targeted therapies, and ovarian cancer vaccine development.

腹膜种植是上皮性卵巢癌最常见的转移模式，当发生大规模腹膜转移时，患者的五年生存率会显著降低。本研究旨在筛选可用于检测卵巢癌早期腹膜转移的血清蛋白。研究通过液相色谱-串联质谱（LC−MS/MS）分析卵巢癌SKOV-3细胞系的分泌（或脱落）蛋白，在SKOV-3细胞培养上清液中鉴定出97种蛋白。将SKOV-3细胞异种移植至裸鼠腹腔后，在动物血清中检测到上述97种蛋白中的3种。经基于酶联免疫吸附试验（ELISA）的临床血液样本筛选后，这3种蛋白中的14-3-3ζ（14-3-3 zeta）被鉴定为候选生物标志物。上皮性卵巢癌患者与良性妇科疾病患者的血清14-3-3ζ平均水平存在显著差异。14-3-3ζ的表达与癌症腹膜转移程度、腹水产生、双侧受累情况以及临床分期和亚分期均相关。采用14-3-3ζ可大幅提升卵巢癌的整体诊断准确率。此外，小干扰RNA（siRNA）实验证实，14-3-3ζ分别介导了SKOV-3细胞约62%、65%和30%的迁移、侵袭及种植能力。本研究结果表明，裸鼠异种移植模型是一种高效的功能导向生物标志物发现体系，可用于未来分子诊断、靶向治疗及卵巢癌疫苗研发等多项研究工作。