Abstract 5312: Next generation quantitative proteomic analysis of urothelial lesions reveals novel diagnostic biomarkers to distinguish pathologic pitfalls and protein-protein interactions

Abstract

Abstract There are several unmet needs remained to be solved in bladder urothelial lesions. Especially, the molecular biology of inverted urothelial papilloma (IUP) as a precursor disease of urothelial carcinoma is poorly understood. Also, muscle-invasive urothelial carcinoma (MIUC) of the bladder, showing a highly aggressive tumor behavior, has prompted the quest for robust biomarkers associated with invasion. A total of 265 tissue specimens from urinary bladder were enrolled for proteomic analysis and validation. Candidate biomarkers were selected through bioinformatic analysis, followed by validation. The latter comprised 2D and 3D invasion and migration assays, also a selection of external public datasets to evaluate mRNA expression and an in-house patient-derived tissue microarray (TMA) cohort to evaluate protein expression with immunohistochemistry (IHC). From the overall proteomic landscape, we found divergent ‘NU-like’ (low-risk) and ‘PUC-like’ (high-risk) signatures in IUP. The latter were characterized by altered metabolism, biosynthesis, and cell-cell interaction functions, indicating oncologic significance. Further machine learning-based analysis revealed SERPINH1, PKP2, and PYGB as potential diagnostic biomarkers discriminating IUP from PUC. The immunohistochemical validation confirmed PYGB as a specific biomarker to distinguish between IUP and PUC with inverted growth. For biomarkers predicting invasion, our multilayered platform-based analysis identified tubulin beta 6 class V (TUBB6) as a promising prognostic biomarker predicting MIUC of the bladder. The in vitro 2D and 3D migration and invasion assays consistently showed that inhibition of TUBB6 mRNA significantly reduced cell migration and invasion ability in two BUC cell lines with aggressive phenotype (TUBB6 migration, P = .0509 and P < .0001; invasion, P = .0002 and P = .0044; TGFBI migration, P = .0214 and P = .0026; invasion, P < .0001 and P = .0001; T24 and J82, respectively). Validation through multiple public datasets, including The Cancer Genome Atlas (TCGA) and selected GSE (Genomic Spatial Event) databases, confirmed TUBB6 as a potential biomarker predicting MIUC. Further protein-based validation with our TMA cohort revealed concordant results, highlighting the clinical implication of TUBB6 expression in BUC patients (overall survival: P < .001). We propose PYGB and TUBB6 as a novel IHC biomarker to predict IUP diagnosis or invasion and poor prognosis in routine practice, also select the optimal treatment in BUC patients. Citation Format: Han Suk Ryu, Soo Young Park, Da Sol Kim, Cheng Hyun Lee. Next generation quantitative proteomic analysis of urothelial lesions reveals novel diagnostic biomarkers to distinguish pathologic pitfalls and protein-protein interactions. [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 5312.