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Supplementary Information from In Vivo Disruption of an Rb–E2F–Ezh2 Signaling Loop Causes Bladder Cancer

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posted on 2023-03-30, 22:47 authored by Mirentxu Santos, Mónica Martínez-Fernández, Marta Dueñas, Ramón García-Escudero, Begoña Alfaya, Felipe Villacampa, Cristina Saiz-Ladera, Clotilde Costa, Marta Oteo, José Duarte, Victor Martínez, Ma José Gómez-Rodriguez, Ma Luisa Martín, Manoli Fernández, Patrick Viatour, Miguel A. Morcillo, Julien Sage, Daniel Castellano, Jose L. Rodriguez-Peralto, Federico de la Rosa, Jesús M Paramio

Supplementary Information. Supplementary Methods Supplementary Figures 1-6. Supp Fig. 1: Identification of bladder tumors by Computerized tomography (CT). Supp Fig 2: Determination of effective elimination of pRb and p130 in bladder tumors. Supp Fig 3: Expression of E2F1, E2F2 and E2F3a genes in mouse bladder tumors. Supp Fig 4: Polycomb-bound genes associate with early recurrence in external datasets of bladder cancer. Supp Fig. 5: Expression of SUZ12 and EED genes in human NMIBC. Supp Fig 6: Expression of E2F1, 2 and 3b genes in human NMIBC. Supplementary Tables 1, 7, 8, 9, 13 and 14. Supp Table 1: Sequence of the Oligonucleotides used in PCR analysis. Supp Table 7: Gene Set Enrichment Analysis of the deregulated genes in mouse bladder tumors. Supp Table 8: Overlap between the downregulated genes in mouse bladder tumors and human bladder cancer datasets from Oncomine database. Supp Table 9: Overlap between the upregulated genes in mouse bladder tumors and human bladder dataset from Oncomine database. Supp Table 13: Gene Set Enrichment Analysis of the deregulated genes in human recurrent bladder tumors. Supp Table 14: Overlap between the upregulated genes in our series of human recurrent bladder tumors and human bladder cancer datasets from Oncomine database. Supplementary References

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ARTICLE ABSTRACT

Bladder cancer is a highly prevalent human disease in which retinoblastoma (Rb) pathway inactivation and epigenetic alterations are common events. However, the connection between these two processes is still poorly understood. Here, we show that the in vivo inactivation of all Rb family genes in the mouse urothelium is sufficient to initiate bladder cancer development. The characterization of the mouse tumors revealed multiple molecular features of human bladder cancer, including the activation of E2F transcription factor and subsequent Ezh2 expression and the activation of several signaling pathways previously identified as highly relevant in urothelial tumors. These mice represent a genetically defined model for human high-grade superficial bladder cancer. Whole transcriptional characterizations of mouse and human bladder tumors revealed a significant overlap and confirmed the predominant role for Ezh2 in the downregulation of gene expression programs. Importantly, the increased tumor recurrence and progression in human patients with superficial bladder cancer is associated with increased E2F and Ezh2 expression and Ezh2-mediated gene expression repression. Collectively, our studies provide a genetically defined model for human high-grade superficial bladder cancer and demonstrate the existence of an Rb–E2F–Ezh2 axis in bladder whose disruption can promote tumor development. Cancer Res; 74(22); 6565–77. ©2014 AACR.