The cancer genome sequencing projects have unveiled a myriad of novel genes mutated in human tumors, a fraction of which are recurrently altered. For many of these genes, a relevant challenge is understanding their function in normal cells and in tumors as well as revealing how mutations can be leveraged from a translational standpoint. To understand the involvement of novel bladder cancer genes in urothelial carcinogenesis, we have taken a three-way approach: 1) assessing the association of mutations with clinical-pathologic characteristics of the tumor, 2) generating conditional mouse models to recapitulate carcinogenesis in vivo, and 3) harnessing the potential of organoids as a model to understand their biology and function during malignant transformation. We have focused on two genes, STAG2 and RBM10. STAG2 codes for a component of cohesin, whose major role is chromosome segregation. However, the molecular pathology studies have indicated that mutations are mainly associated with bladder cancers of low-grade and stage lacking aneuploidy: mutations are present in 30-40% of low-grade papillary tumors and 12-15% of muscle-invasive tumors. We have generated a novel conditional Stag2-knockout mouse and have shown that STAG2 has unique functions during embryo development and that it is largely dispensable in the adult under the conditions tested. In the urothelium, Stag2 deletion is not sufficient to give rise to tumors; experiments are ongoing testing its cooperation with other bladder cancer oncogenes and tumor suppressor genes. RBM10 is involved in pre-mRNA splicing, and tumor-specific alterations involving NUMB mRNA exon inclusion have been demonstrated in lung cancer. RBM10 is mutated in 5-8% of bladder cancers, regardless of stage and grade, with a predominance among tumors with urothelial differentiation. We have generated a novel conditional Rbm10 knockout mouse, which has revealed that RBM10 is also required for normal embryo development and that it is largely dispensable in the adult. In the urothelium, Rbm10 deletion in the urothelium is not sufficient to give rise to tumors and experiments are ongoing testing its cooperation with other bladder cancer oncogenes and tumor suppressor genes. To facilitate the study of the role of these genes in bladder tumorigenesis, we have developed methods to consistently establish normal mouse urothelial organoids. These organoids can be maintained in culture uninterruptedly for more than one year, they are strictly dependent on EGF and Wnt signals, and they can be induced to differentiate upon growth factor depletion. PPAR gamma agonists and EGFR inhibitors further support the urothelial program. Differentiated organoids show downregulated expression of basal cell markers, upregulation of uroplakins and adhesion molecules, and acquisition of barrier function. RNA-Seq analyses (both bulk and single cell) have revealed novel pathways involved in normal urothelial differentiation. These organoids are powerful tools to accelerate the discovery of the functions of bladder cancer genes and facilitate drug screening. Overall, I will show how our group and our collaborators are able to exploit complementary features of genetic mouse models and organoids to accelerate research on the biology of these tumor suppressor genes and its translation. This work has been possible thanks to the collaboration of the following groups: N. Malats (CNIO, Madrid), A. Losada (CNIO, Madrid), J. Valcárcel (CRG, Barcelona), M. Hoglund (U. Lund, Lund), L. Dyrskjot (U. Aarhus, Aarhus), A. Hartmann (U. Erlangen, Germany), among others.
Citation Format: Francisco X. Real. Genetic mouse models and organoids: Synergy for functional genomics of bladder cancer genes [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr IA16.