A recent study by Neyret-Kahn et al. sought to characterize these changes. The authors first conducted chromatin immunoprecipitation sequencing (ChIP-seq) to detect active versus repressive histone marks in human primary tumors (MIBC n=13, NMIBC n=2), bladder cancer cell lines (n = 7), and patient-derived normal human urothelium in proliferation (NHU, n = 2). ChIP-seq and RNA-sequencing (RNA-seq) data were used to classify samples into molecular subtypes. This led to an integrated epigenetic map of chromatin regions that were active promoters and enhancers versus repressors in the different subtypes. Gene set enrichment analysis (GSEA) revealed that active chromatin was strongly enriched in basal clusters at genes involved in reduced luminal differentiation and increased tumor aggressiveness, stemness, and epithelial-mesenchymal transition. FOXA1 was associated with active chromatin in luminal samples and NMIBC and NHU samples.
In contrast, KRT6A was found in most basal/squamous samples and NHU. Using SE regions alone, samples could be divided into molecular subtypes successfully. To integrate enhancers with gene expression, SEs were then assigned to nearby putative transcriptional targets. Luminal-specific SEs were linked to transcription factors (TFs) that drive the luminal phenotype, namely GATA3, PPARg, and FOXA1, in addition to TGF-beta signaling genes. SE regions in basal tumors were associated with genes contributing to the basal phenotype, such as EGFR, inflammation, and FOXO signaling. TF networks identified in luminal and basal subtypes were subsequently confirmed in a separate single-cell RNA-seq dataset.
The role of FOXA1 in regulating bladder cancer SEs was confirmed through ChIP-seq of luminal and basal cell lines. Importantly, this revealed that FOXA1 could regulate both subtypes, as its binding overlapped with 87% of bladder SEs and was associated with genes involved in regulating urothelial differentiation. To further understand the role of FOXA1, short-term knockdown was conducted in luminal and basal subtypes. This resulted in decreased proliferation and reduced cell viability in both subtypes, in addition to downregulating genes involved in the cell cycle and checkpoints and upregulation in genes involved in inflammation and interferon response. Importantly, the immune checkpoint modulator CD274 (PD-L1) was among these upregulated genes. There was a slight shift from the luminal to the basal subtype. However, long-term CRISPR knockdown of FOXA1 caused a substantial shift from the luminal to the basal phenotype. Interestingly, overexpression of ZBED2, a putative master TF in basal subtypes, led to the downregulation of FOXA1. This overexpression also enhanced the expression of cell cycle progression genes and lowered interferon response genes.
This atlas of enhancers, repressors, TFs, histone marks, and target genes in different subtypes of bladder cancer is highly valuable and may help uncover novel therapeutic targets. For instance, enhancer rewiring plays a role in treatment response, and targeting SEs has proven efficacious in leukemia and lymphoma. Moreover, many of the molecular players identified in this study were linked to inflammation, a key modulator of response to immune checkpoint inhibitor therapy.
Written by: Bishoy M. Faltas, MD, Director of Bladder Cancer Research, Englander Institute for Precision Medicine, Weill Cornell Medicine
References:
- Neyret-Kahn, H, Fontugne, J, Meng, XY, et al. Epigenomic Mapping Identifies An Enhancer Repertoire That Regulates Cell Identity In Bladder Cancer Through Distinct Transcription Factor Networks. Oncogene. 2023.