Abstract 3071: The role of Apela in high-grade serous ovarian cancer
Authors: Daily Laura R. , Ganguly Debolina , Hayes D Neil , Wang Yinan , Sims Michelle M. , Cheng Jinjun , ElNaggar Adam C. , Pfeffer Lawrence M. 
Published: August 2018

Abstract
Objective: To assess the expression of Apela and evaluate its co-expression with cancer stem cell markers and angiogenesis markers in high-grade serous ovarian cancer and further elucidate the role of this poorly characterized gene.
Background: High-grade serous ovarian cancer (HGSOC) presents a major therapeutic challenge due to advanced stage at diagnosis, aggressive metastatic behavior, and therapeutic resistance. A critical barrier to progress in early diagnosis and treatment is the lack of understanding of the mechanisms that underlie ovarian cancer tumorigenesis. Apela (Apelin early ligand A), also called Toddler and Elabela, is a novel ligand for the G-coupled protein receptor Apelin-R, which has been implicated in cancer tumorigenesis and angiogenesis through activation of the PIK3-AKT (phosphatidylinositol-4,5-bisphosphate 3-kinase and protein-kinase B) pathway. This signaling pathway plays an important role in embryonic stem cell growth and self-renewal. Current research indicate that Apela may be involved in cancer stem cells and angiogenesis pathways as well. Markers of angioneogenesis (Angptl4 [angiopoietin-like protein 4]) and cancer cell stemness (Nestin and CD133/PROM1), were selected for study with Apela because of known expression in HGSOC and other studies showing co-expression in other high-grade malignancies with Apela.
Methods: RNA was extracted from established ovarian cancer cell lines (OVCAR3, OVCAR8, CAOV4, OV90, and SKOV3) and expression of Apela, Apelin (another ligand for the Apelin-R), Apelin-R and ANGPTL-4 was determined by qPCR and normalized to actin expression. Formalin-fixed paraffin-embedded (FFPE) slides of HGSOC tissue and normal adjacent ovarian tissue were subjected to RNAscope Multiplex Fluorescent in situ hybridization (ISH) using individual gene probes for Angptl4, Nestin, CD133 and Apela to detect expression at the mRNA level. Immunohistochemistry (IHC) was then performed to confirm Angptl4, Nestin, CD133 and Apela co-expression at the protein level in the FFPE HCSOC tissue. All images were analyzed by confocal microscopy. The TCGA database was analyzed to determine levels of upregulation of Apela in ovarian cancer. An ovarian cancer microarray using 10 paired matched controls was utilized to further validate the presence of the
Results: Apela expression can be detected in FFPE tissue at the RNA level and the same pattern of protein expression is seen with IHC. Apela and Angptl4 co-localize with the cancer stem cell markers nestin and CD133. Apela is upregulated in HGSOC when examining the TCGA database compared to other forms of cancer.
Conclusions: Apela is expressed at relatively low levels in established ovarian cancer cell lines. However, Apela is highly localized in ovarian cancer tissue, and is co-expressed with markers of angiogenesis and stem cells, possibly representing a stem cell niche that promotes tumorigenesis.
Citation Format: Laura R. Daily, Debolina Ganguly, D Neil Hayes, Yinan Wang, Michelle M. Sims, Jinjun Cheng, Adam C. ElNaggar, Lawrence M. Pfeffer. The role of Apela in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3071.