www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 16), pp: 26832-26844 Research Paper Functional characterisation of a novel ovarian cancer cell line, NUOC-1 Aiste McCormick1, Eleanor Earp1, Katherine Elliot1, Gavin Cuthbert2, Rachel O'Donnell1,3, Brian T. Wilson4, Ruth Sutton4, Charlotte Leeson1, Huw D. Thomas1, Helen Blair1, Sarah Fordham1, John Lunec1, James Allan1, Richard J. Edmondson5,6 1Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK 2Cancer Cytogenetics Department at Newcastle University, Newcastle upon Tyne, UK 3Northern Gynaecological Oncology Centre, Queen Elizabeth Hospital, Gateshead, UK 4Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK 5Division of Molecular and Clinical Cancer Sciences, Faculty of Biology, Medicine and Health, University of Manchester, St Mary’s Hospital, Manchester, UK 6Department of Obstetrics and Gynaecology, Manchester Academic Health Science Centre, St Mary’s Hospital, Central Manchester NHS Foundation Trust, Manchester, UK Correspondence to: Richard J. Edmondson, email: [email protected] Keywords: ovarian cancer, clear cell carcinoma, endometrioid carcinoma, mixed histology, cell line model Received: July 28, 2016 Accepted: February 20, 2017 Published: March 01, 2017 Copyright: McCormick et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Background: Cell lines provide a powerful model to study cancer and here we describe a new spontaneously immortalised epithelial ovarian cancer cell line (NUOC-1) derived from the ascites collected at a time of primary debulking surgery for a mixed endometrioid / clear cell / High Grade Serous (HGS) histology. Results: This spontaneously immortalised cell line was found to maintain morphology and epithelial markers throughout long-term culture. NUOC-1 cells grow as an adherent monolayer with a doubling time of 58 hours. The cells are TP53 wildtype, positive for PTEN, HER2 and HER3 expression but negative for oestrogen, progesterone and androgen receptor expression. NUOC-1 cells are competent in homologous recombination and non-homologous end joining, but base excision repair defective. Karyotype analysis demonstrated a complex tetraploid karyotype. SNP array analysis of parent and derived subpopulations (NUOC-1-A1 and NUOC-1-A2) cells demonstrated heterogeneous cell populations with numerous copy number alterations and a pro-amplification phenotype. The characteristics of this new cell line lends it to be an excellent model for investigation of a number of the identified targets. Materials and Methods: The cell line has been characterised for growth, drug sensitivity, expression of common ovarian markers and mutations, clonogenic potential and ability to form xenografts in SCID mice. Copy number changes and clonal evolution were assessed by SNP arrays. INTRODUCTION overall survival of 30–39% [2]. It has long been recognised by clinicians that ovarian cancer is a set of heterogeneous Epithelial ovarian cancer is often described as diseases but, despite this, ovarian carcinoma continues to the silent killer due to absence of symptoms and late be treated clinically as a single disease using a combination presentation. This combined with the lack of specific of debulking surgery and platinum-based chemotherapy. sensitive markers and techniques for screening leads to The observed variation in the clinical behaviour of ovarian diagnosis at late disease stage in more than 70% of patients. cancer alongside the growing data reporting molecular Ovarian cancer is the leading cause of gynaecological heterogeneity suggests that a heterogeneous in vitro model cancer mortality worldwide [1] and despite much research for the study of ovarian cancer is long overdue. into the treatment of ovarian cancer the overall mortality Established cell lines provide an invaluable tool for has changed little over the past 20 years with a 5-year studying biological functions at the molecular and cellular www.impactjournals.com/oncotarget 26832 Oncotarget level. Existing human ovarian cancer cell lines possess small bowel mesentery). Ascites was collected at the time the advantage of high proliferative capacity, clonogenicity of surgery for culture. Final histology confirmed FIGO and extended life span in culture. However, ovarian cancer Stage IIIc high grade mixed ovarian carcinoma of the cell lines have rarely been derived from chemotherapy- right ovary which was 80% endometrioid, 15% clear cell naive patients, many were established following viral and 5% high grade serous (HGS) carcinoma. Repeat CT transformation, such as with SV40 Large T antigen or imaging demonstrated new liver and peritoneal metastases xenograft passaged in immunocompromised mice [3–6]. and she died of progressive disease 52 days post-op after Very few cell lines are derived from mixed histology only 1 cycle of carboplatin. She did not have any known tumours and with this type of tumour being less common, relevant familial history for cancer predisposition. reliable models for the study of mixed histology tumours Bright light microscopy revealed a cobblestone are needed. Additionally, there is evidence to suggest morphology characteristic of epithelial cells which was that many cell lines contain significant misidentification, maintained during repeated passage (Figure 1A). The duplication, and loss of integrity [7] making new well growth of NUOC-1 was initially slow with a 128 hour characterised models desirable. doubling time, but with continued culture this stabilised In this study, we describe a new ovarian cancer cell to 58 hours. NUOC-1 formed colonies when grown on line derived from ascites of a chemotherapy-naïve patient. plastic at an efficiency of 2.2% +/− 0.6%. NUOC-1 cells The molecular and growth characteristics of this cell line stained positive for proteins characteristic of epithelial present unique features, thereby providing the research ovarian carcinoma (pancytokeritin, epithelial cell adhesion community with a new tool in the study of different molecule (EpCAM), epithelial related antigen MOC31 aspects of mixed histology ovarian cancer. and cancer antigen 125 (CA125)) and negative for germ cell related antigen D240 and Vimentin (Figure 1B–1F). RESULTS NUOC-1 cells did not express oestrogen, progesterone and androgen receptors (Figure 2A), but expressed the Molecular characterization of NUOC-1 cell line receptor tyrosine-protein kinase erbB-3 (HER-3) receptor and the receptor tyrosine-protein kinase erbB-2 (HER-2) The NUOC-1 cell line was derived from ascites of receptor at a higher level than LNCAP and SKOV-3 cell a chemotherapy naive patient. The female patient was line controls (Figure 2A). Sequencing of the entire coding of Caucasian background and 62 years old when she region of BRCA1, BRCA2 and phosphatase and tensin presented with disseminated intraabdominal malignancy. homologue deleted on chromosome 10 (PTEN) revealed no She underwent primary surgery with optimal cytoreduction germline or somatic mutations. Furthermore PTEN protein (with residual milliary disease over the diaphragms and expression was confirmed by western blotting (Figure 2B). Figure 1: Phenotypic appearances of NUOC-1 cells. (A) Brightfield demonstrating cobblestone monolayer (20x magnification); immunoflourescent images (40x magnification) with antibodies targeted against: (B) Alexafluor 596 anti-CA125; (C) FITC-anti- pancytokeratin; (D) Alexflour 488 anti-EpCAM; E( ) Alexafluor 596 anti-MOC 31; F( ) Alexaflour 596 anti-Vimentin. www.impactjournals.com/oncotarget 26833 Oncotarget P53 function assessment deemed HR competent with a 2.84 fold rise in RAD51 foci, compared to untreated controls (Figure 4A). Non Dysregulated tumour protein p53 is recognised as homologous end joining (NHEJ) function is assessed by a characteristic feature of HGS. To determine if the small the ability of cell extracts to rejoin incompatible vector proportion of the HGS within NUOC-1 immortalised, DNA ends correctly. NUOC-1 cells were found to be p53 status was determined. Sanger dideoxy sequencing NHEJ competent as evidenced by the ability to rejoin of TP53 Exons 3-9 detected no mutations. Consistently both compatible and incompatible DNA breaks correctly with functional p53, treatment with Mouse double (Figure 4B). Excess production of 8-OHdG inferred minute 2 homolog (MDM2)-p53 antagonist (Nutlin-3) the non-functioning of base excision repair (BER) and demonstrated accumulation of MDM2 in NUOC-1 cells was quantified by competitive ELISA in NUOC-1 cells concomitant with a dose dependent increase in p53 and (Figure 4C). AA8 (BER competent) with its derivative cyclin-dependent kinase inhibitor 1 (p21) protein levels EM9 cell lines (BER deficient due to XRCC1 mutation) (Figure 3A). In contrast, Nutlin-3 treatment of CP70 cells were used as positive and negative controls. NUOC-1 cell did not significantly induce p53 or p21, consistent with the 8-OHdG level was 4.0 nM +/- 0.64 nM, indicating that established TP53 mutation in this cell line. Nutlin-3 had a NUOC-1 cells were BER defective (Figure 4C). Massively greater growth inhibitory effect in TP53 wild-type NUOC-1 parallel sequencing identified heterozygous mutations in cells compared to TP53 mutant CP70 cells (GI50 0.7 µM +/- OGG1, WRN, NBN and NEIL3, and homozygous mutation 0.03