Abstract
Abstract Background: Mutations and inactivation of the tumor suppressor gene TSC1 are recurrent (6-10%) events in invasive bladder cancer, but the mechanism by which TSC1 loss promotes bladder cancer development beyond mTORC1 activation is uncertain. The purpose of this study was to assess the functional effects of TSC1 loss in bladder cancer and uncover new therapeutic targets for patients with TSC1 mutations. Materials and methods: We performed differential gene expression and pathway analyses using The Cancer Genome Atlas (TCGA) expression data for TSC1-mutated bladder cancer (TSC1mt-BLCA, n=26) and TSC1 wild-type tumors (TSC1wt-BLCA, n=382) and compared them to an internal cohort of TSC1/TSC2-mutant tumors (n=48). Transcriptomic, epigenetic, and functional analyses, including RNA-seq, H3K27ac ChIP-seq, TFE3 CUT&RUN, ATAC-seq and TFE3 knock-out using CRISPR/Cas9 were conducted on TSC1-mt/wt BLCA cell lines. Results: Comparison of TSC1mt-BLCA and TSC1wt-BLCA TCGA tumors identified 26 genes upregulated in TSC1mt-BLCA out of 60 genes commonly upregulated in Tuberous Sclerosis Complex (TSC) syndromic tumors. Among them, GPNMB showed the greatest increase in expression (median fold change 9.8). GSEA and DESeq2 analyses implicated both mTORC1 hyperactivation and increased lysosomal gene expression in TSC1mt-BLCA. Expression differences in several genes, including GPNMB, and in pS6 were confirmed by immunohistochemistry (IHC) of 5 independent BWH TSC1mt-BLCA and TSC1wt-BLCA specimens each. TFE3, a key regulator of lysosomal gene expression, was elevated in expression and localized to the nucleus in TSC1mt-BLCA by IHC. Molecular studies of TSC1-mt/wt BLCA isogenic cell lines (HCV29, RT4) recapitulated the phenotype found in human tumors. TFE3 was both post-translationally modified (assessed by immunoblot) and predominantly nuclear in TSC1-null cell lines compared to TSC1wt cells, and the localization was partially reversed by rapamycin treatment leading to reduced expression of lysosomal genes. TSC1mt HCV29 cells showed higher expression of multiple genes increased in TSC1mt-BLCA tumors (GPNMB, SQSTM1, GNPDA1). TFE3 knock-out HCV29 clones showed markedly reduced growth, which was not seen with TFE3 KO in TSC1-addback HCV29 cells. GSEA of RNA-seq data indicated that TFE3 KO led to significant reduction in expression of lysosome genes, as well as those involved in ribosome synthesis, DNA replication and DNA excision repair processes. TFE3 CUT&RUN confirmed that TFE3 binds to promoter regions of those genes, thereby directly regulating these pathways. In contrast, TFE3 KO led to an increase in inflammatory and cellular stress genes that could impede TSC1mt cell growth. Conclusions: Our findings show that TSC1mt-BLCA tumors and cell lines have a transcriptional signature similar to that of TSC syndromic tumors and show nuclear localization and transcriptional activation of TFE3. Aberrant TFE3 activation likely contributes to TSC1mt-BLCA development and may therefore be amenable to targeted therapy. Citation Format: Magdalena Losko, Clemens K Probst, Elio Adib, Heng Du, Michelle Hirsch, Krinio Giannikou, David J Kwiatkowski. TSC1 mutant bladder cancer is characterized by a TSC-associated gene expression signature due to TFE3 transcriptional activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A116.
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