Profile Of Ovarian Cancer Tumor Fluid Using High-Throughput And Targeted Proteomic Analysis

Proteomic analysis of body fluids can be used to identify proteins associated with malignant transformation, and tumor fluid samples have emerged as a rich source for the identification of ovarian cancer in this context. To uncover differences among benign and malignant ovarian samples, we performed a high-throughput proteomic analysis consisting of albumin immunodepletion, isotope labeling with acrylamide, and in-depth proteomic profiling by LC-MS/MS in a pool of 10 samples of each histologic type (high-grade ovarian serous carcinoma and benign serous cystadenoma). After, we developed a panel for targeted proteomics using the multiple reaction monitoring (MRM) method for validation of 10 relevant proteins (ACTN4 (Alpha-actinin-4), GSTP1 (Glutathione S-transferase P), LDHA (L-lactate dehydrogenase A chain), LDHB (L-lactate dehydrogenase B chain), PRDX5 (Peroxiredoxin-5), TIMP1 (Metalloproteinase inhibitor 1), ACTG1 (Actin), ALDOA (Fructose-bisphosphate aldolase A), PKM (Pyruvate kinase PKM), and ENO1 (Alpha-enolase)). A total of 1135 proteins were identified in the high-throughput proteomic analysis, corresponding to 593 known genes, where 505 proteins (44%) were detected only in the malignant pool and 272 proteins (24%) were detected only in the benign pool. The protein-protein interaction analysis (STRING-DB) showed strong association of proteins detected in the malignant pool, according to biological process and cellular component, with response to stress, protein metabolic process, immune system process, positive regulation of cellular metabolic process, biogenesis, regulation of cellular organization, transport, protein trafficking, extracellular exosome, extracellular region part, and extracellular region. Differently, the set of proteins detected in the benign pool showed strong association with single organism signaling, cell communication, regulation of signal transduction, system development, response to stimulus, extracellular region part, extracellular exosome, cell periphery, cytosol, cell projection, endomembrane system, and cytoskeleton. Using targeted proteomic analysis, we verified that ACTN4, GSTP1, LDHB, PRDX5, TIMP1, ACTG1, ALDOA, LDHA, PKM, and ENO1 are significantly more abundant in malignant tumor fluid pool when compared to benign samples, confirming the association of proteins detected in the malignant pool with metabolic process, extracellular exosome, and extracellular region part. These results are in accordance with the fact that cancer cells must adjust cellular metabolism to support the demands of growth and remodel their cytoskeleton to allow migration and metastasis. Our findings contribute to show the differences among histologic types of ovarian tumor fluids. We emphasize the contribution of robust proteomic strategies to uncover relevant candidate tumor markers and consistent molecular signatures that can discriminate between benign and malignant tumors. Financial support: FAPESP (Proc. 2011-0947-1, 16/03809-3), CNPq (479934/2011-8, 454703/2014-7), CAPES, CISBi, NAP/USP (grant 12.1.17598.1.3) and Center for Cell Based Therapy - CTC-CEPID (Proc. FAPESP 2013/08135-2). Citation Format: Aline Poersch, Mariana L. Grassi, Camila S. Palma, Vinicius P. Carvalho, Guilherme P. Lanfredi, Helio H. A. Carrara, Francisco J. Candido dos Reis, Vitor M. Faca. Profile of ovarian cancer tumor fluid using high-throughput and targeted proteomic analysis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A191.