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SIX1 Promotes Bladder Cancer Stemness by Repressing DHRS2-Mediated lysosomal degradation of CD44

T. Xie^1,2, M. Zheng³, G. Xu², Y. Li³, H. Guo^1,2, M. Ding^1,2, J. Zhuang^1,2

¹Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Department of Urology, Nanjing, China
²Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Department of Urology, Nanjing, China
³Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine, Department of Urology, Nanjing, China

Introduction:

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.

Material and methods:

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.

Results:

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.