Zhou Kaixia, Ma Xiaolu, Yan Tianqing, Hu Ling, Tian Yanan, Zheng Hui, Xie Suhong, Tong Ying, Wang Yanchun, Guo Lin, Lu Renquan
Department of Clinical Laboratory, Fudan University Shanghai Cancer Center, No. 270, Dong'an Road, Shanghai, 200032, PR China.
Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, PR China.
Cell Oncol (Dordr). 2025 Apr 14. doi: 10.1007/s13402-025-01057-6.
Ovarian cancer (OC) is the most lethal gynecological malignancy, with widespread metastasis and ascites being the leading causes of patient mortality. However, the mechanisms driving OC metastasis have not been sufficiently studied. This study aimed to investigate the mechanisms and key molecules promoting OC metastasis.
Public databases (StemChecker, GeneCards, GEO, and TCGA) were screened to identify metastasis-associated genes. Immunohistochemical staining and western blotting were employed to evaluate THEMIS2 expression and epithelial-mesenchymal transition (EMT) marker profiles across experimental groups. RNA sequencing coupled with pathway enrichment analysis revealed THEMIS2-regulated signaling pathways, while immunoprecipitation-mass spectrometry was utilized to identify THEMIS2 interaction partners. GST pull-down assays for active Rap1 quantified Rap1-GTP levels under varying THEMIS2 expression conditions. Wound healing and transwell invasion assays respectively assessed migratory and invasive capacities of OC cells following THEMIS2 expression perturbations in vitro. Abdominal cavity implantation metastasis model was established to evaluate OC cell colonization and invasive potential in vivo.
THEMIS2 expression is significantly elevated in OC tissues compared to normal ovarian tissues, and its high expression correlates with poor prognosis and malignant features. Experimental manipulation of THEMIS2 levels revealed that knockdown impended the migratory and invasive capacities of OC cells both in vitro and in vivo, while its overexpression exacerbated metastasis. THEMIS2 is involved in EMT and cytoskeleton rearrangement. RNA-seq analysis revealed that THEMIS2 positively correlates with Rap1 signaling pathway. Inhibition of Rap1 activity reversed the metastasis-promoting effects induced by THEMIS2 overexpression both in vitro and in vivo. Mechanistically, we uncovered that THEMIS2 functions as a molecular scaffold that recruits TBK1 (TANK Binding Kinase 1) to DOCK4 (Dedicator of Cytokinesis 4), facilitating site-specific phosphorylation at serine 1787 (S1787). This post-translational modification enables DOCK4 to engage with CRKII, subsequently triggering Rap1 signaling activation. These findings suggest that THEMIS2 promotes the metastatic potential of OC cells via DOCK4-mediated activation of Rap1 signaling.
THEMIS2 may serve as a predictive biomarker for OC prognosis, and targeting the Rap1 signaling pathway with specific inhibitors represents a promising therapeutic strategy for OC treatment.
卵巢癌（OC）是最致命的妇科恶性肿瘤，广泛转移和腹水是患者死亡的主要原因。然而，驱动OC转移的机制尚未得到充分研究。本研究旨在探讨促进OC转移的机制和关键分子。
筛选公共数据库（StemChecker、GeneCards、GEO和TCGA）以鉴定转移相关基因。采用免疫组织化学染色和蛋白质印迹法评估各实验组中THEMIS2的表达及上皮-间质转化（EMT）标志物谱。RNA测序结合通路富集分析揭示THEMIS2调控的信号通路，同时利用免疫沉淀-质谱法鉴定THEMIS2相互作用蛋白。通过GST下拉实验检测活性Rap1，定量不同THEMIS2表达条件下的Rap1-GTP水平。伤口愈合实验和Transwell侵袭实验分别评估体外干扰THEMIS2表达后OC细胞的迁移和侵袭能力。建立腹腔植入转移模型以评估OC细胞在体内的定植和侵袭潜能。
与正常卵巢组织相比，OC组织中THEMIS2表达显著升高，其高表达与不良预后和恶性特征相关。对THEMIS2水平进行实验性调控发现，敲低THEMIS2可在体外和体内抑制OC细胞的迁移和侵袭能力，而过表达则会加剧转移。THEMIS2参与EMT和细胞骨架重排。RNA测序分析表明THEMIS2与Rap1信号通路呈正相关。抑制Rap1活性可在体外和体内逆转THEMIS2过表达诱导的促转移作用。机制上，我们发现THEMIS2作为分子支架，将TBK1（TANK结合激酶1）招募至DOCK4（胞质分裂专用蛋白4），促进丝氨酸1787（S1787）位点特异性磷酸化。这种翻译后修饰使DOCK4能够与CRKII结合，随后触发Rap1信号激活。这些发现表明THEMIS2通过DOCK4介导的Rap1信号激活促进OC细胞的转移潜能。
THEMIS2可能作为OC预后的预测生物标志物，用特异性抑制剂靶向Rap1信号通路是一种有前景的OC治疗策略。
