The urothelial gene regulatory network: understanding biology to improve bladder cancer management.

The urothelium is a stratified epithelium composed of basal cells, one or more layers of intermediate cells, and an upper layer of differentiated umbrella cells. Most bladder cancers (BLCA) are urothelial carcinomas. Loss of urothelial lineage fidelity results in altered differentiation, highlighted by the taxonomic classification into basal and luminal tumors. There is a need to better understand the urothelial transcriptional networks. To systematically identify transcription factors (TFs) relevant for urothelial identity, we defined highly expressed TFs in normal human bladder using RNA-Seq data and inferred their genomic binding using ATAC-Seq data. To focus on epithelial TFs, we analyzed RNA-Seq data from patient-derived organoids recapitulating features of basal/luminal tumors. We classified TFs as "luminal-enriched", "basal-enriched" or "common" according to expression in organoids. We validated our classification by differential gene expression analysis in Luminal Papillary vs. Basal/Squamous tumors. Genomic analyses revealed well-known TFs associated with luminal (e.g., PPARG, GATA3, FOXA1) and basal (e.g., TP63, TFAP2) phenotypes and novel candidates to play a role in urothelial differentiation or BLCA (e.g., MECOM, TBX3). We also identified TF families (e.g., KLFs, AP1, circadian clock, sex hormone receptors) for which there is suggestive evidence of their involvement in urothelial differentiation and/or BLCA. Genomic alterations in these TFs are associated with BLCA. We uncover a TF network involved in urothelial cell identity and BLCA. We identify novel candidate TFs involved in differentiation and cancer that provide opportunities for a better understanding of the underlying biology and therapeutic intervention.

STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells.

Cohesin exists in two variants containing STAG1 or STAG2. STAG2 is one of the most mutated genes in cancer and a major bladder tumor suppressor. Little is known about how its inactivation contributes to tumorigenesis. Here, we analyze the genomic distribution of STAG1 and STAG2 and perform STAG2 loss-of-function experiments using RT112 bladder cancer cells; we then analyze the genomic effects by integrating gene expression and chromatin interaction data. Functional compartmentalization exists between the cohesin complexes: cohesin-STAG2 displays a distinctive genomic distribution and mediates short and mid-ranged interactions that engage genes at higher frequency than those established by cohesin-STAG1. STAG2 knockdown results in down-regulation of the luminal urothelial signature and up-regulation of the basal transcriptional program, mirroring differences between STAG2-high and STAG2-low human bladder tumors. This is accompanied by rewiring of DNA contacts within topological domains, while compartments and domain boundaries remain refractive. Contacts lost upon depletion of STAG2 are assortative, preferentially occur within silent chromatin domains, and are associated with de-repression of lineage-specifying genes. Our findings indicate that STAG2 participates in the DNA looping that keeps the basal transcriptional program silent and thus sustains the luminal program. This mechanism may contribute to the tumor suppressor function of STAG2 in the urothelium.

Essential Roles of Cohesin STAG2 in Mouse Embryonic Development and Adult Tissue Homeostasis.

Cohesin mediates sister chromatid cohesion and 3D genome folding. Two versions of the complex carrying STAG1 or STAG2 coexist in somatic vertebrate cells. STAG2 is commonly mutated in cancer, and germline mutations have been identified in cohesinopathy patients. To better understand the underlying pathogenic mechanisms, we report the consequences of Stag2 ablation in mice. STAG2 is largely dispensable in adults, and its tissue-wide inactivation does not lead to tumors but reduces fitness and affects both hematopoiesis and intestinal homeostasis. STAG2 is also dispensable for murine embryonic fibroblasts in vitro. In contrast, Stag2-null embryos die by mid-gestation and show global developmental delay and defective heart morphogenesis, most prominently in structures derived from secondary heart field progenitors. Both decreased proliferation and altered transcription of tissue-specific genes contribute to these defects. Our results provide compelling evidence on cell- and tissue-specific roles of different cohesin complexes and how their dysfunction contributes to disease.

Urothelial organoids originating from Cd49fhigh mouse stem cells display Notch-dependent differentiation capacity.

Understanding urothelial stem cell biology and differentiation has been limited by the lack of methods for their unlimited propagation. Here, we establish mouse urothelial organoids that can be maintained uninterruptedly for >1 year. Organoid growth is dependent on EGF and Wnt activators. High CD49f/ITGA6 expression features a subpopulation of organoid-forming cells expressing basal markers. Upon differentiation, multilayered organoids undergo reduced proliferation, decreased cell layer number, urothelial program activation, and acquisition of barrier function. Pharmacological modulation of PPARγ and EGFR promotes differentiation. RNA sequencing highlighted genesets enriched in proliferative organoids (i.e. ribosome) and transcriptional networks involved in differentiation, including expression of Wnt ligands and Notch components. Single-cell RNA sequencing (scRNA-Seq) analysis of the organoids revealed five clusters with distinct gene expression profiles. Together, with the use of γ-secretase inhibitors and scRNA-Seq, confirms that Notch signaling is required for differentiation. Urothelial organoids provide a powerful tool to study cell regeneration and differentiation.

Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy.

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.

Autophagic activity dictates the cellular response to oncogenic RAS.

RAS is frequently mutated in human cancers and has opposing effects on autophagy and tumorigenesis. Identifying determinants of the cellular responses to RAS is therefore vital in cancer research. Here, we show that autophagic activity dictates the cellular response to oncogenic RAS. N-terminal Apoptosis-stimulating of p53 protein 2 (ASPP2) mediates RAS-induced senescence and inhibits autophagy. Oncogenic RAS-expressing ASPP2((Δ3/Δ3)) mouse embryonic fibroblasts that escape senescence express a high level of ATG5/ATG12. Consistent with the notion that autophagy levels control the cellular response to oncogenic RAS, overexpressing ATG5, but not autophagy-deficient ATG5 mutant K130R, bypasses RAS-induced senescence, whereas ATG5 or ATG3 deficiency predisposes to it. Mechanistically, ASPP2 inhibits RAS-induced autophagy by competing with ATG16 to bind ATG5/ATG12 and preventing ATG16/ATG5/ATG12 formation. Hence, ASPP2 modulates oncogenic RAS-induced autophagic activity to dictate the cellular response to RAS: to proliferate or senesce.

The Lats2 tumor suppressor augments p53-mediated apoptosis by promoting the nuclear proapoptotic function of ASPP1.

Apoptosis is an important mechanism to eliminate potentially tumorigenic cells. The tumor suppressor p53 plays a pivotal role in this process. Many tumors harbor mutant p53, but others evade its tumor-suppressive effects by altering the expression of proteins that regulate the p53 pathway. ASPP1 (apoptosis-stimulating protein of p53-1) is a key mediator of the nuclear p53 apoptotic response. Under basal conditions, ASPP1 is cytoplasmic. We report that, in response to oncogenic stress, the tumor suppressor Lats2 (large tumor suppressor 2) phosphorylates ASPP1 and drives its translocation into the nucleus. Together, Lats2 and ASPP1 shunt p53 to proapoptotic promoters and promote the death of polyploid cells. These effects are overridden by the Yap1 (Yes-associated protein 1) oncoprotein, which disrupts Lats2-ASPP1 binding and antagonizes the tumor-suppressing function of the Lats2/ASPP1/p53 axis.

PML, YAP, and p73 are components of a proapoptotic autoregulatory feedback loop.

p73 has been identified as a structural and functional homolog of the tumor suppressor p53. The transcriptional coactivator Yes-associated protein (YAP) has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show the existence of a proapoptotic autoregulatory feedback loop between p73, YAP, and the promyelocytic leukemia (PML) tumor suppressor gene. We demonstrate that PML is a direct transcriptional target of p73/YAP, and we show that PML transcriptional activation by p73/YAP is under the negative control of the proto-oncogenic Akt/PKB kinase. Importantly, we find that PML and YAP physically interact through their PVPVY and WW domains, respectively, causing PML-mediated sumoylation and stabilization of YAP. Hence, we determine a mechanistic pathway in response to DNA damage that could have relevant implications for the treatment of human cancer.

Mutant p53 proteins: between loss and gain of function.

Cancer might result from both the aberrant activation of genes, whose physiological tuning is essential for the life of a normal cell, and the inactivation of tumor suppressor genes, whose main job is to preserve the integrity of cell genome. Among the latter, p53 is considered a key tumor suppressor gene that is inactivated mainly by missense mutations in half of human cancers. It is becoming increasingly clear that the resulting mutant p53 proteins gain oncogenic properties favoring the insurgence, the maintenance, and the spreading of malignant tumors. In this review, we mainly discuss the molecular mechanisms underlying gain of function of human tumor-derived p53 mutants, their impact on the chemoresistance and the prognosis of human tumors, with a special focus on head and neck cancers, and the perspectives of treating tumors through the manipulation of mutant p53 proteins.

The transcriptional coactivator Yes-associated protein drives p73 gene-target specificity in response to DNA Damage.

The transcriptional coactivator Yes-associated protein (YAP) has been shown to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here, we show that YAP requires the promyelocytic leukemia gene (PML) and nuclear body localization to coactivate p73. YAP imparts selectivity to p73 by promoting the activation of a subset of p53 and/or p73 target promoters. Endogenous p73, YAP, and p300 proteins are concomitantly recruited onto the regulatory regions of the apoptotic target gene p53AIP1 only when cells are exposed to apoptotic conditions. Silencing of YAP by specific siRNA impairs p300 recruitment and reduces histone acetylation on the p53AIP1 target gene, resulting in delayed or reduced apoptosis mediated by p73. We also found that YAP contributes to the DNA damage-induced accumulation of p73 and potentiates the p300-mediated acetylation of p73. Altogether, our findings identify YAP as a key determinant of p73 gene targeting in response to DNA damage.