Quantitative proteomics identifies TUBB6 as a biomarker of muscle‐invasion and poor prognosis in bladder cancer

Muscle‐invasive urothelial carcinoma (MIUC) of the bladder shows highly aggressive tumor behavior, which has prompted the quest for robust biomarkers predicting invasion. To discover such biomarkers, we first employed high‐throughput proteomic method and analyzed tissue biopsy cohorts from patients with bladder urothelial carcinoma (BUC), stratifying them according to their pT stage. 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). 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