Abstract Background/Aim: The acquisition of stem-like properties and increased cellular plasticity is thought to drive tumor progression and therapeutic resistance, but grade-specific molecular trajectories across the epigenetic landscape in bladder cancer remain poorly defined. This study examined the dynamic rewiring of signaling networks and proteomic landscapes during reprogramming of low-grade (HTB-2) and high-grade (HTB-5) bladder cancer cells, and during their subsequent differentiation into embryoid bodies. Materials and Methods: Six experimental models were analyzed, including the parental HTB-2 and HTB-5 cells, their Sendai virus-reprogrammed counterparts (rep HTB-2 and rep HTB-5) and embryoid bodies generated from reprogrammed derivatives (rep HTB-2 EB and rep HTB-5 EB), with SV-HUC-1 uroepithelial cells used as a control. Phosphoproteomic and integrated proteomic analyses were performed to define grade-specific signaling architectures and shared plasticity-associated signatures, followed by clinical validation using pan-cancer datasets. Results: Phosphoproteomic reconstruction revealed grade-dependent kinase network architectures associated with stem-like induction. These findings indicate that reprogramming induced cell-line-specific signaling rewiring, with HTB-2 cells showing enhanced MAPK/Src-family-associated phosphorylation and HTB-5 cells showing increased AKT/PRAS40 and STAT1/STAT3 phosphorylation together with reduced ERK1/2-MSK1/2 signaling. During differentiation, low-grade cells underwent metabolic reprogramming, while high-grade cells favored cytoskeletal remodeling and extracellular matrix organization. Integrated proteomics defined a shared plasticity signature, with reprogrammed models recapitulated key bladder cancer features and clinically relevant outcomes. Conclusion: These findings support a hierarchical model of bladder cancer progression where reprogramming induces a transient intermediate state that enables invasive features upon re-differentiation. The study reveals grade-specific signaling and proteomic adaptations, identifying differentiation as a critical window for uncovering prognostic biomarkers and therapeutic targets. Together, these results suggest that reprogrammed bladder cancer models provide a biologically relevant platform to study tumor plasticity, progression and therapeutic vulnerability.
İskender et al. (Mon,) studied this question.