Abstract TMEM-024: CHARACTERIZATION OF STEM CELLS IN HIGH GRADE SEROUS OVARIAN CARCINOMA CELLS
Authors: Maricela Gallardo, Alyse Huisken-Hill, Hanmin Wang, Christine Castanon, Juli J. Unternaehrer
Journal: Clinical Cancer Research (2017)

Abstract PURPOSE: The objectives of this study are to understand how transcription factors and miRNAs impact stemness in epithelial ovarian cancer (EOC). Our goal is to target EOC cancer stem cells (CSC) in order to arrest disease progression. Ovarian Cancer is the most fatal gynecological diagnosis for women. It is typically treated with surgery and chemotherapy, which is initially successful in that tumors shrink; however, most patients experience relapse. Cancer Stem Cells (CSCs) express a similar set of stem cell markers as pluripotent cells, and are thought to contribute to chemoresistance and relapse. Chemotherapy is effective on differentiated cells of the tumor, however cancer stem cells survive, and have the ability to repair and renew the tumor. Cancer cells can be re-sensitized to chemotherapy if stem cells are targeted. Several stem cell markers are expressed in advanced ovarian malignancies. We study the effect of the epithelial to mesenchymal transition (EMT) on stemness. During EMT, cells acquire mesenchymal characteristics and lose epithelial ones. EMT is caused by SNAI1 and other transcription factors that repress epithelial and adhesion molecules such as E-cadherin. EMT results in a stem cell-like phenotype in breast cancer. There is evidence that the EMT factor SNAI1 represses the miRNA let-7, which we hypothesize results in a higher proportion of CSC in EOC cells. Let-7's major function is to promote terminal differentiation and tumor suppression by targeting oncogenes and pluripotency factors. EXPERIMENTAL PROCEDURES: We performed flow cytometry for CD133, CD44, CD117, and aldehyde dehydrogenase to determine proportion of positive cells using published markers. q-RT-PCR was used to determine expression of pluripotency markers Oct4, Lin28 and Nanog. We used immunofluorescence microscopy to identify CSCs. Using a panel of biomarkers specific to CSCs we labeled for SNAI1, NANOG, LIN28A, OCT4, SSEA4, ALDH, TRA1-60, CD133, E-CAD, and N-CAD followed by fluorescent secondary antibodies and nuclear labeling with DAPI. We characterized three epithelial ovarian cell (EOC) lines (OVCAR8, OVSAHO, and COV 318) and patient-derived cells, with and without Snai1 manipulation. RESULTS: Cells were positive for stem cell markers to varying degrees. OVCAR-8 was positive for LIN28A, NANOG, CD44, and ALDH; OVSAHO for Lin 28a and ALDH; and COV318 for NANOG and ALDH. OVSAHO displayed most epithelial (E) characteristics, COV318 and patient samples were most mesenchymal (M), and OVCAR8 appeared to be a M/E hybrid. There was imperfect correlation between mRNA and protein expression of pluripotency markers. CONCLUSIONS: Higher expression of Snai1 correlates with a stem cell phenotype on a population level. On a single cell level, most Nanog-positive cells are Snail positive. However, no ovarian cancer cell perfectly mirrors the pluripotent marker expression seen in NCCIT, our positive control. SNAI1 is a potential target for therapy targeting ovarian cancer stem cells. Citation Format: Maricela Gallardo, Alyse Huisken-Hill, Hanmin Wang, Christine Castanon, Juli J. Unternaehrer. CHARACTERIZATION OF STEM CELLS IN HIGH GRADE SEROUS OVARIAN CARCINOMA CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-024.