Am J Transl Res 2020;12(9):5924-5939 www.ajtr.org /ISSN:1943-8141/AJTR0111977 Original Article Ceramide synthase 6 predicts poor prognosis and activates the AKT/mTOR/4EBP1 pathway in high-grade serous ovarian cancer Yinong Shi1*, Chen Zhou2*, Huan Lu1, Xiaoxiao Cui1, Jun Li3, Shuheng Jiang3, Hao Zhang4, Rong Zhang1 1Fengxian District Center Hospital Graduate Student Training Base, Jinzhou Medical University, Shanghai 201499, China; 2Department of Obstetrics and Gynecology, The Affiliated Changzhou No. 2 People’s Hospital of Nanjing Medical University, Changzhou 213000, Jiangsu, China; 3State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China; 4Department of Obstetrics and Gynecology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai 200011, China. *Equal contributors. Received April 5, 2020; Accepted July 26, 2020; Epub September 15, 2020; Published September 30, 2020 Abstract: Objective: Ovarian cancer is one of the most common gynecological malignancies worldwide, and its mor- tality rate ranks first among gynecologic cancers. Ceramide synthases are closely related to cancer development. In this study, we investigated the role of ceramide synthase 6 (CerS6) in the development of serous ovarian cancer. Methods: Expression of CerS6 in cancerous and healthy ovarian tissue was assessed by database analysis and im- munohistochemistry. The biological role of CerS6 in serous ovarian cancer cells was assessed by CerS6 knockdown followed by cell counting, colony formation, transwell migration, wound healing, and flow cytometry assays and mea- surement of tumor proliferation in nude mice. Signaling pathway components were analyzed by Western blotting. Gene enrichment was analyzed by GSEA and R, and RNA sequencing was used to compare the transcriptomes of serous ovarian cancer cells with and without CerS6 knockdown. Results: High CerS6 expression in ovarian cancer tissues was closely related to poor prognosis. Knockdown of CerS6 inhibited serous ovarian cancer cell proliferation, invasion, and metastasis and promoted their apoptosis. In addition, CerS6 knockdown increased the proportion of serous ovarian cancer cells in G2/M phase. CerS6 regulates cell cycle through the AKT/mTOR/4EBP1 signaling pathway, which affects cell proliferation and metastasis. The GSEA, R, and RNA sequencing analyses showed that knocking down CerS6 significantly affects cell cycle in serous ovarian cancer cells. Conclusions: CerS6 may have an oncogenic role in ovarian cancer and may represent a new prognostic marker and therapeutic target for serous ovarian cancer. Keywords: High-grade serous ovarian cancer, CerS6, cell cycle, AKT/mTOR/4EBP1 signaling pathway Introduction 22,500 ovarian cancer-related deaths occurr- ed in 2015 [4]. A recent study reported that Ovarian cancer is one of the three malignant the 5-year survival rate of patients with stage I gynecological tumors. According to the latest ovarian cancer is 90%, whereas the 5-year global cancer statistics, ovarian cancer has the survival rate of patients with stage III and IV highest mortality rate among gynecological ovarian cancer is less than 10% [3]. Although tumors worldwide [1]. Ovarian cancer cases the pathogenesis of ovarian cancer is com- have increased worldwide in recent years [2] plex and unclear, epidemiological and genetic and currently account for 2.5% of all malignant analyses have linked ovarian cancer occur- tumors in women each year [3]. In the United rence with specific genetic mutations [5]. The States, approximately 22,530 new cases of most common mutations associated with ovar- ovarian cancer and 13,980 ovarian cancer- ian cancer, such as those in BRCA1, BRCA2, related deaths occurred in 2019 [1]. In China, and p53, are related to defective homologous 52,100 new cases of ovarian cancer and recombinant DNA repair [6-9] and are accom- Ceramide synthase 6 in high-grade serous ovarian cancer panied by changes in the PI3K/AKT/mTOR and OV15, IGROV1, and CAOV-3 were obtained from RAS/RAF/MEK signaling pathways [10]. the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and the cell lines One of the six identifying hallmarks of tumor OVCAR3, OVCAR8, and HEK293T were obtain- cells is abnormal metabolism [11]. Tumor tis- ed as gifts from Shanghai Cancer Institute sues meet the biological energy, biosynthesis, (Shanghai, China). All cells were cultured ac- and redox requirements for rapid growth th- cording to American Type Culture Collection rough metabolic reprogramming [12]. Cerami- (ATCC) instructions. An anti-CerS6 antibody de synthases, key enzymes in eukaryotic lipid (ab115539, Abcam, Cambridge, UK) was used metabolism that produce ceramide [13], have for Western blotting and immunohistochemis- recently attracted increasing attention within try. Additional Western blotting antibodies rec- the tumor field. The ceramide synthase family ognizing AKT (#4685), p-AKT (#4060), mTOR includes six genes (CerS1-6) [14] that are (#2972), p-mTOR (#2971), 4EBP1 (#9644), and encoded on different chromosomes and syn- p-4EBP1 (#9451) were obtained from Cell thesize fatty acyl-CoAs of different chain Signaling Technology (Danvers, MA, USA), and a lengths [13]. Among them, CerS6, also known primary anti-GAPDH antibody (M1210-2) was as LASS6, mainly synthesizes C16: 0-cera- obtained from Sigma (St. Louis, MO, USA). mide [15], which promotes cell migration and Secondary antibodies were purchased from metastasis and is an important mediator of Jackson ImmunoResearch (West Grove, PA, apoptosis [16, 17]. Many recent studies have USA). shown that CerS6 is involved in the occurren- ce and progression of a variety of tumors. For Clinical samples and database example, CerS6 is highly expressed in breast cancer [18] and non-small-cell lung cancer [17] Human ovarian cancer tissue and normal ovar- cells. Knockdown of CerS6 in non-small-cell ian tissue used in this study were obtained lung cancer cells attenuates lung metastasis from the Department of Obstetrics and Gy- [17], whereas overexpression of CerS6 in hu- necology, Fengxian District Center Hospital man head and neck squamous cell carcinoma Graduate Student Training Base, Jinzhou xenografts leads to their rapid growth [19]. Medical University and the Department of However, few studies have investigated the Obstetrics and Gynecology, The Affiliated mechanistic role of CerS6 in the development Changzhou No. 2 People’s Hospital of Nanjing of ovarian cancer. Medical University. None of the patients re- ceived radiotherapy, chemotherapy, or other In the early stage of this study, we found that related antitumor therapies before surgery. All CerS6 is highly expressed in serous ovarian human tissues were obtained with informed cancer cells and is associated with poor prog- consent, and the study was approved by the nosis. Immunohistochemistry results con- Research Ethics Committee of Fengxian Dis- firmed that high CerS6 expression is closely trict Center Hospital Graduate Student Training related to high-grade and advanced-stage Base, Jinzhou Medical University. We down- serous ovarian cancer. Therefore, in the later loaded and analyzed ovarian cancer cohort stage of this study, we investigated the effect data from the Cancer Genome Atlas (TCGA, of CerS6 on the biological functions of serous https://cancergenome.nih.gov/) and the Gene ovarian cancer cells in vivo and in vitro and Expression Omnibus (GEO, https://www.ncbi. explored the possible mechanism for its role nlm.nih.gov/geo/). in ovarian cancer. Our results indicate that CerS6 may be a new molecular biomarker for Immunohistochemical staining the prognosis of serous ovarian cancer and may represent a new therapeutic target for the Immunohistochemistry was performed as pre- treatment of this common malignancy. viously described in [20] with an anti-CerS6 antibody (1:200, ab115539, Abcam). The in- Materials and methods tensity of CerS6 staining was scored using the following criteria: 0-5% staining was scored as Cell culture and reagents 0; 6-35% staining as 1; 36-70% staining as 2; and > 70% staining as 3. A total score < 2 was The human epithelial ovarian cancer cell lines considered to represent negative expression, HO8910PM, OVCAR5, OVCAR8, ES-2, SKOV-3, and a score ≥ 2 was considered to represent 5925 Am J Transl Res 2020;12(9):5924-5939 Ceramide synthase 6 in high-grade serous ovarian cancer positive expression. The scoring was perfor- Western blotting med in a blinded manner by two senior pathologists. Cells were lysed and cell proteins extract- ed with protein extraction buffer (Sangon, Cell transfection and RNA interference Shanghai, China). Western blotting analysis was performed as described previously [20]. Cells were plated at 70% confluence and trans- fected with CerS6-specific small interfering Cell proliferation assay (CCK-8 assay) RNA (siRNA) or with nontargeted siRNA (Gene- Pharma, Shanghai, China) using Lipofecta- Cell viability was measured using the Cell mine® RNAiMAX (13778150, Invitrogen, Wal- Counting Kit-8 (CCK-8, Dojindo, Japan) accord- tham, MA, USA) following the manufacturer’s ing to the manufacturer’s instructions. Cells instructions. The CerS6 siRNA sequences with the indicated treatments were grown in were as follows: siRNA1 sense, 5’-GCAGGC- 96-well plates at 4,000 cells per well. CCK-8 UGAGGACCUCUAUTT-3’, anti-sense, 5’-AUAGA- was added to each well and incubated for 1 h GGUCCUCAGCCUGCTT-3’; siRNA2 sense, 5’- at 37°C. Absorbance