Identification and quantification of N-linked glycoproteins in ovarian cancer cells

Submission note: This report was submitted in total fulfilment of the requirements for the degree of Master of Science to the School of Molecular Science, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Insidious of all gynaecological malignancies, ovarian cancer persists to have a high mortality rate despite the low number of cases diagnosed per year. Currently, the only biomarker approved in the clinic for monitoring ovarian cancer is CA-125; however this marker is ineffective for early diagnosis and is expressed at high levels during pregnancy, endometriosis and menopause. This project aims to assess the glyco-proteomic profile of plasma membrane proteins in ovarian cancer cells, quantify antigen expression levels relative to the human ovarian surface epithelial cells (HOSE), and identify potential glycoproteins that may serve as diagnostic markers or therapeutic targets. Pilot proteomic studies on the A2780, OVCAR4 and HOSE cells led to the identification of 43 potential N-linked glycoproteins for HOSE, 60 for OVCAR4 and 24 for A2780. Antigen expression levels were then assessed in the ovarian cancer cell lines in comparison to the HOSE which led to the identification of 11 proteins in the OVCAR4 cell line with significant fold change values, notable were IMPAD1 and SYPL1. Known proteins associated with ovarian cancer, such as Prominin-1 and CD276 were also identified in this study. Further comprehensive analysis with the addition of OV90, SKOV3 and MCF7 cell lines led to the identification of 60 potential N-linked proteins for HOSE, 63 for A2780, 92 for OV90, 88 for OVCAR4 and 81 for MCF7. In addition, several proteins were identified that had either high or low fold change values relative to the HOSE and MCF7 cell lines. Of the list of proteins identified, three candidates were selected for further verification, namely, IMPAD1, SYPL1 and ASCT2, which although unknown for their roles in ovarian cancer, were consistently identified at high levels in the ovarian cancer cell lines. Verification through western blot and flow cytometry analysis confirmed the proteomics data. In conclusion, proteomics analysis led to the identification of many markers over expressed in ovarian cancer cells that could serve as potential markers warranting further clinical assessment.