Cancer stem cells play a critical role in the initiation and progression of bladder cancer. Identifying key transcription factors that maintain cancer stemness features is essential for developing targeted therapies.

Single-cell trajectory analysis using Monocle2, combined with SCENIC-based transcription factor regulatory analysis, identified key transcription factors involved in maintaining stemness in bladder cancer. Functional assays, both in vitro and in vivo, were used to validate the oncogenic role of the transcription factor SIX1. To investigate downstream targets, RNA sequencing and CUT&Tag sequencing analyses were performed. Co-immunoprecipitation and immunofluorescence microscopy were employed to assess protein interactions.

SIX1 was identified as a crucial transcription factor for bladder cancer stemness. It was upregulated in bladder cancer tissues and associated with adverse clinical characteristics and poorer prognosis. SIX1 enhanced tumor progression and stemness both in vitro and in vivo. Further analyses demonstrated that SIX1 directly represses DHRS2 transcription by binding to its promoter region. Disrupting this binding reversed the enhanced stemness phenotype in bladder cancer cells. Mechanistically, DHRS2 interacts with the stemness marker CD44, promoting its endocytosis and subsequent degradation via the ubiquitin-lysosomal pathway. Inhibition of DHRS2 increased CD44 expression, reversing the tumorigenic effects driven by SIX1.

This study highlights the SIX1-DHRS2-CD44 regulatory axis as a critical pathway for bladder cancer progression and stemness maintenance. Targeting this axis presents a promising therapeutic strategy for controlling cancer stemness in bladder cancer.