Abstract 2467: Novel biomarkers of metastasis-prone breast cancer revealed by high-throughput proteomics assay

Abstract

Abstract Relatively high cost of gene expression-based prognostic tools in breast cancer (BC) hinders the public accessibility. Therefore, there is a need for development of robust prognostic biomarker for prediction of early and late metastasis. We designed high-throughput proteomics assay to compare protein expression profiles of BC with no metastasis (NM), late metastasis (LM), and early metastasis (M) on 29 patients with BC at Seoul National University Hospital (SNUH). We defined the LM as the development of metastasis after 5 years from the diagnosis. Mass spectrometry (MS) data were collected in data-dependent acquisition (DDA) mode and quantified by Perseus (version 1.5.8.5). ANOVA was used to determine differentially expressed proteins (DEP). Mutual information-based feature selection was used to pick features for metastasis. We used METABRIC dataset to validate prognostic significance. Cell migration and invasion assays were further performed using TNBC cell line after transfecting the cells with siRNA. Mean metastasis free survival time was 8.4 years with LM, and 1.6 years in the patients in M group. A total of 9,455 proteins were identified across the discovery set, and 6,639 proteins were quantified with label-free quantification intensities. 830 proteins were found to be differentially expressed among the groups with an FDR-adjusted p-value < 0.05. Mutual information selected TUBB as the most highly expressed in M group (median, 36.4), least in NM group (median, 36.0), and intermediate in LM groups (median, 36.1). The expression of VWA5A sequentially decreased from NM group (median, 29.6), LM group (median, 29.2), and M group (median, 28.2). In METABRIC cohort, we found that high TUBB expression is strong, significant poor prognostic factor for DFS in all cases with BC (p < 0.0001), ER+ subgroup (p = 0.0001), HER2+ subgroup (p = 0.0085), but not in TNBC (p = 0.1017). In similar manner, lower expression of VWA5A was significantly correlated with shorter DFS in all (p = 0.0003) and ER+ subtype (p < 0.0001), but not in HER2+ and TNBC subsets (p = 0.1350 and 0.2314, respectively). Discrepant prognostic significance of two candidate biomarkers in certain subtypes of BCs indicate that these molecules may behave differently according to intrinsic subtypes. Therefore, we performed functional experiments using TNBC cell lines. siRNA induced silencing of TUBB in MDA-MB-231 and Hs378T resulted in significantly decreased invasion and migration, while siVWA5A transfected BT20 and HCC70 showed stronger propensity toward invasion and migration. Though impacts on survival were not ascertained, our proteomics and in vitro analyses suggest TUBB and VWA5A as the feasible biomarkers representing the biological aggressiveness of BCs including TNBCs. Further investigations on TUBB and VWA5A are warranted to gather deeper understandings how these two molecules results in different biological behaviors of BCs. Citation Format: Jiwon Koh, Dabin Jeong, Dohyun Han, Sohyeon Yang, Sun Kim, Han Suk Ryu. Novel biomarkers of metastasis-prone breast cancer revealed by high-throughput proteomics assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2467.