EIF5A2 controls ovarian tumor growth and metastasis by promoting epithelial to mesenchymal transition via the Title TGFβ pathway Zhao, Guannan; Zhang, Wenjing; Dong, Peixin; Watari, Hidemichi; Guo, Yuqi; Pfeffer, Lawrence M; Tigyi, Gabor; Author(s) Yue, Junming Cell & Bioscience, 11, 70 Citation https://doi.org/10.1186/s13578-021-00578-5 Issue Date 2021-04-07 Doc URL http://hdl.handle.net/2115/80967 Rights(URL) https://creativecommons.org/licenses/by/4.0/ Type article File Information Yue EIF5A2 paper.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP Zhao et al. Cell Biosci (2021) 11:70 https://doi.org/10.1186/s13578-021-00578-5 Cell & Bioscience RESEARCH Open Access EIF5A2 controls ovarian tumor growth and metastasis by promoting epithelial to mesenchymal transition via the TGFβ pathway Guannan Zhao1,2, Wenjing Zhang3, Peixin Dong4, Hidemichi Watari4, Yuqi Guo5*, Lawrence M. Pfefer1,2, Gabor Tigyi6 and Junming Yue1,2* Abstract Background: Epithelial to mesenchymal transition (EMT) contributes to tumor metastasis and chemoresistance. Eukaryotic initiation factor 5A2 (EIF5A2) is highly expressed in a variety of human cancers but rarely expressed in normal tissues. While EIF5A2 has oncogenic activity in several cancers and contributes to tumor metastasis, its role in ovarian cancer is unknown. In this study, we investigate whether EIF5A2 contributes to ovarian tumor metastasis by promoting EMT. Methods: To investigate the role of EIF5A2, we knocked out (KO) EIF5A2 using lentiviral CRISPR/Cas9 nickase in high invasive SKOV3 and OVCAR8 cells and overexpressed EIF5A2 in low invasive OVCAR3 cells using lentiviral vector. Cell proliferation, migration and invasion was examined in vitro ovarian cancer cells and tumor metastasis was evaluated in vivo using orthotopic ovarian cancer mouse models. Results: Here we report that EIF5A2 is highly expressed in ovarian cancers and associated with patient poor survival. Lentiviral CRISPR/Cas9 nickase vector mediated knockout (KO) of EIF5A2 inhibits epithelial to mesenchymal transi- tion (EMT) in SKOV3 and OVCAR8 ovarian cancer cells that express high levels of EIF5A2. In contrast, overexpression of EIF5A2 promotes EMT in OVCAR3 epithelial adenocarcinoma cells that express relatively low EIF5A2 levels. KO of EIF5A2 in SKOV3 and OVCAR8 cells inhibits ovarian cancer cell migration and invasion, while its overexpression pro- motes cell migration and invasion in OVCAR3 adenocarcinoma cells. We further demonstrate that EIF5A2 promotes EMT by activating the TGFβ pathway and KO of EIF5A2 inhibits ovarian tumor growth and metastasis in orthotopic ovarian cancer mouse models. Conclusion: Our results indicate that EIF5A2 is an important controller of ovarian tumor growth and metastasis by promoting EMT and activating the TGFβ pathway. Keywords: EIF5A2, CRISPR, Cas9 nickase, Lentiviral vector, Ovarian cancer, Epithelial to mesenchymal transition, Orthotopic ovarian cancer mouse model Background *Correspondence: [email protected]; [email protected] 1 Department of Pathology and Laboratory Medicine, College Ovarian cancer (OC) has the highest mortality rate of Medicine, The University of Tennessee Health Science Center, among gynecological malignancies [1]. Early stage OC Memphis, TN 38163, USA patients have no obvious symptoms and are often diag- 5 People′s Hospital of Zhengzhou University, Zhengzhou, Henan, China Full list of author information is available at the end of the article nosed only at later stages III and IV, when tumors have © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhao et al. Cell Biosci (2021) 11:70 Page 2 of 12 already metastasized to the peritoneal cavity or other In the present study we provide evidence that EIF5A2 abdominal organs. Early stage OC patients respond contributes to ovarian tumor growth and metastasis by to chemotherapy, but eventually become resistant to promoting EMT via activation of the TGFβ pathway. chemotherapy. Although multi-modality treatment approaches applied in OC therapy include debulking surgery, chemotherapy, targeted therapy, and immuno- Materials and methods therapy, the fve-year survival rate remains poor at 35 Cell culture to 40% [2–5]. Te molecular mechanisms driving OC Ovarian cancer cell line SKOV3 was purchased from metastasis and chemoresistance remain unclear. Tus, ATCC and cultured in Dulbecco’s Modifed Eagle it is of great importance to identify new predictive bio- Medium (DMEM) supplemented with 10% FBS (Hyclone; markers for early diagnosis and develop new drugs to Logan, UT), 100 U/ml penicillin/streptomycin (Invit- improve OC therapy. rogen; Carlsbad, CA). OVCAR3 and OVCAR8 cell lines Eukaryotic initiation factor 5A (EIF5A) is a eukaryotic were purchased from National Cancer Institute and cul- translation initiation factor that participates in the initia- tured in RPMI 1640 with 10% FBS (Hyclone; Logan, UT), tion and elongation process in protein synthesis. EIF5A 1% penicillin/streptomycin (Invitrogen; Carlsbad, CA). is the only known protein that undergoes hypusination All cell lines were grown at 37 °C with 5% CO2. Cell lines through posttranslational modifcation. Deoxyhypusine were authenticated using Short Tandem Repeat (STR) synthase (DHPS) cleaves the polyamine spermidine and analysis by ATCC and tested negative for mycoplasma the 4-aminobutyl group is transferred to lysine residue 50 using the luciferase assay (Lonza, Allendale, NJ). of EIF5A, which is subsequently hydroxylated by deoxy- hypusine hydroxylase (DOHH) to facilitate EIF5A matu- ration [6–9]. Tere are two isoforms of EIF5A, EIF5A1 Lentiviral vector production and EIF5A2, which share sequence similarity of 84% in Te lentiviral CRISPR/Cas9 nickase-mediated EIF5A2 mRNA and 94% protein [10]. EIF5A1 is expressed in the gene editing vectors were constructed by annealing two majority of cell types and required for embryonic devel- gRNA oligonucleotide pairs and subcloning them into opment, while EIF5A2 is expressed only in specifc cell BsmII site of lentiviral vector Lentiguide-puro vector types and is not required for embryonic development (#52,963, Addgene), and gRNAs were driven by human [11, 12]. Interestingly, EIF5A2 is aberrantly amplifed or U6 promoter. Two gRNA sequences, 5′ AAC GGC TTC upregulated in various cancers including ovarian can- GTG GTG CTC AA and 5′ CGC AAG GCC GAG CAC cer, lung, pancreatic cancer, and hepatocellular carci- TGC AT were designed to target exon 1 of EIF5A2 gene. noma, and contributes to tumor growth and metastasis TGFβR2 Knockdown (KD) CRISPR/Cas9 nickase vectors [7, 10, 13, 14]. Terefore, EIF5A2 is an attractive drug were constructed as described previously [28]. EIF5A2 target for cancer therapy based on its aberrant expres- lentiviral overexpression vector was purchased from sion in various cancer types. Although EIF5A2 is upregu- Applied Biological Materials Inc. (Richmond, Canada). lated in ovarian cancer, its functional role has not been Lentivirus was produced by packaging in 293FT cells as characterized at the mechanistic level. Previous stud- we reported previously [29]. EIF5A2 KO and TGFβR2 KD ies demonstrated that EIF5A2 contributed to epithelial stable cell lines were established by transducing SKOV3 to mesenchymal transition (EMT) in colorectal, gastric, and OVCAR8 ovarian cancer cells with the lentiviral and breast cancer [15–17]. It is well known that EMT CRISPR/Cas9 nickase vector and selected with 2 μg/ml contributes to tumor initiation, progression, invasion, puromycin and 10 μg/ml blasticidin. LentiCas9-blast was metastasis, EMT is regulated by multiple signaling path- used as the control vector without gRNAs. Te EIF5A2 ways including ERK1/2, AKT, WNT in diferent cancers stable expression and control cells were established by [18–20]. Although EMT contributes to tumor metastasis, transducing OVCAR3 with lentiviral EIF5A2 and empty the role of EMT in ovarian cancer is somewhat contro- control vectors and selected with 2 μg/ml puromycin. versial due to the same expression levels of E-cadherin in the ovary and other distant metastatic organs [21]. How- ever, accumulating evidence indicates that EMT plays MTT cell proliferation assay an important role in ovarian tumor metastasis [22–26], SKOV3, OVCAR8 EIF5A2 KO or OVCAR3 expressing Our previous studies showed that TGFβ promoted EMT and corresponding control cells (3000/well) were plated in ovarian cancer cells [27], and BIRC5 (survivin) expres- into 96-well plates. Cell proliferation was measured at 24, sion