**2.5 Transcriptomics and clinical data of ovarian cancer patients extracted from TCGA database**

Level 3 RNA-seq V2 transcriptomics data of 411 OC patients were extracted from The Cancer Genome Atlas (TCGA) data portal (http://cancergenome.nih.gov/) with the corresponding clinical data, including cancer status (with tumor or tumor-free), clinical stage (stages IIA, IIB, IIC, IIIA, IIIB, IIIC, and IV), neoplasm histologic grade (G1, G2, G3, G4, and GX), anatomic neoplasm subdivision (right, left, and bilateral), age at initial pathologic diagnosis (aged from 30 to 87),

Its anticancer effect has been shown by many *in vitro* and *in vivo* experiments in multiple cancers, including ovarian cancer, breast cancer, triple-negative breast cancer, cervical cancer, lung cancer, gastric cancer, colon cancer, glioblastoma, melanoma, and leukemia [4, 6], with a wide safe and clinically reachable drug concentration of anticancer according to its pharmacokinetic range in treatment of a parasite-infected patient [7]. It offers a promising opportunity to develop a new anticancer drug via drug repositioning of this existing compound with confirmed

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

Ovarian cancer, a very common cancer with high mortality and poor survival in

This book chapter discussed the anti-cancer effects of ivermectin on ovarian cancer in the following aspects: (i) ivermectin inhibited cell proliferation and growth, blocked cell cycle progression, and promoted cell apoptosis in ovarian cancer [4, 21]; (ii) ivermectin inhibited ovarian cancer growth through molecular networks to target the key molecules in energy metabolism pathways, including glycolysis, Kreb's cycle, oxidative phosphorylation, and lactate shuttle pathways [21]; (iii) Integrated omics revealed that ivermectin mediated lncRNA-EIF4A3 mRNA axes in ovarian cancer to exert its anticancer capability [4, 13]; and (iv) lasso regression identified the prognostic model of ivermectin-related three-lncRNA signature (ZNRF3-AS1, SOS1-IT1, and LINC00565) that is significantly related to overall survival and clinicopathologic characteristics of ovarian cancers [4].

**2.1 Ovarian cancer cell biological behaviors affected by ivermectin**

The normal ovarian cells IOSE80 and ovarian cancer cells TOV-21 and SKOV3 were treated with ivermectin to measure ivermectin-mediated ovarian cancer cell biological behavior changes. (i) IOSE80, TOV-21G, and SKOV3 were treated with ivermectin (0–60 μM) for 24 h, followed by the use of CCK8 to measure the IC50 of ivermectin in each cell. (ii) TOV-21G and SKOV3 were treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) for 24 h, followed by the use of EdU assay to measure DNA synthesis in each cell. (iii) TOV-21G and SKOV3 were treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) for 48 h, followed by clonogenic

women [9, 10], are involved in multiple signaling pathway network changes [11, 12]. Many intracellular molecules and signaling pathways would be the targets of ivermectin [13]. Ivermectin have shown a potential addition role for ovarian cancer treatment. For example, ivermectin can improve the chemosensitivity of overran cancer via targeting Akt/mTOR signaling pathway [14], and can inhibit PAK1-dependent growth of ovarian cancer cells via blocking the oncogenic kinase PAK1 [15]. Ivermectin also acts as a PAK1 inhibitor to induce autophagy in breast cancer [16]. Ivermectin can enhance p53 expression and cytochrome C release, and reduce the expression levels of CDK2, CDK4, CDK6, Bcl-2, cyclin E, and cyclin D1 in glioblastoma, those promoted the cancer cell apoptosis [17]. Ivermectin can inhibit cancer cell proliferation via decreasing YAP1 protein expression in the Hippo pathway [18]. Ivermectin represses WNT-TCF pathway in WNT-TCF-dependent disease [19]. Ivermectin can promote TFE3 (Ser321) dephosphorylation to block the binding between TFE3 and 14-3-3, and induce TFE3 accumulation in the nucleus of human melanoma cells [20]. Moreover, ivermectin also affects other signaling pathway network in human cancers, including oxidative stress, mitochondrial dysfunction, angiogenesis, epithelial-mesenchymal transition, drug resistance, and stemness in tumor [6]. Thereby, ivermectin demonstrates the potential therapeutic

clinical safety [8].

**2. Methods**

**204**

efficiency in multiple malignant tumors.

lymphatic invasion (yes/no), primary therapy outcome success (complete remission/response, partial remission/response, progressive disease, and stable disease), additional radiation therapy (yes/no), survival time (days), tumor residual disease (no macroscopic disease, 1–10 mm, 11–20 mm, and > 20 mm), survival status (0 = alive, and 1 = dead), and PANCAN (Pan-Cancer Atlas). TANRIC (http://ibl.mdanderson.org/tanric/design/basic/index.html) was used for survival analysis of lncRNAs in ovarian cancer. The large-scale CLIP-Seq data with starBasev 2.0 (http://starbase.sysu.edu.cn/mirCircRNA.php) was used to predict the EIF4A3 binding mRNAs. The Kaplan–Meier method relative to the log-rank test was used for survival analysis of mRNAs in ovarian cancers. Statistical significance was set as p value <0.05. GenCLiP 3 (http://ci.smu.edu.cn/genclip3/analysis.php) was used for pathway enrichment analysis of the association of EIF4A3-binding mRNAs and patient survival rates. The detailed procedure was described previously [4].
