Bladder Cancer (BC) represents a clinical and social challenge due to its high incidence and recurrence rates, as well as the limited advances in effective disease management. Currently, a combination of cytology and cystoscopy is the routinely used methodology for diagnosis, prognosis and disease surveillance. However, both the poor sensitivity of cytology tests as well as the high invasiveness and big variation in tumour stage and grade interpretation using cystoscopy, emphasizes the urgent need for improvements in BC clinical guidance. Liquid biopsy represents a new non-invasive approach that has been extensively studied over the last decade and holds great promise. Even though its clinical use is still compromised, multiple studies have recently focused on the potential application of biomarkers in liquid biopsies for BC, including circulating tumour cells and DNA, RNAs, proteins and peptides, metabolites and extracellular vesicles. In this review, we summarize the present knowledge on the different types of biomarkers, their potential use in liquid biopsy and clinical applications in BC.

Long non-coding RNAs (lncRNAs) have been claimed as key molecular players in gene expression regulation, being involved in diverse epigenetic processes. They are aberrantly expressed in various tumors, but their exact role in bladder cancer is still obscure. We have recently found a major role of the Polycomb repression complex in recurrence of non-muscle-invasive bladder cancer. Here, we report the xpression of Polycomb-related lncRNAs:antisense noncoding RNA in the INK4 locus (ANRIL) and HOX antisense intergenic RNA (HOTAIR) in these tumors. We studied a dataset of non-invasive bladder cancer samples by quantitative reverse transcription PCR (RT-qPCR) and analyzed also invasive bladder cancer samples using TCGA data. Our results showed that, while ANRIL seemed not to have a determining role, an increased HOTAIR expression appeared in recurrent and high-graded tumors associated with poor prognosis. In addition, through genome-wide transcriptome analyses, we observed that HOTAIR-EZH2-complex-regulated genes can efficiently discriminate between non-tumoral, recurrent, and non-recurrent bladder cancer samples. We also observed a significant correlation between EZH2 and HOTAIR expression levels. Using overexpression, knockdown, and pharmacological approaches in bladder cancer cell lines, we also observed that EZH2 regulates HOTAIR expression. Our findings indicate that HOTAIR expression has prognostic value for bladder cancer progression, recurrence, and survival and suggest that HOTAIR plays active roles in modulating the cancer epigenome, becoming an interesting candidate as a target for cancer diagnosis and therapy. The observed HOTAIR regulation by EZH2 and the possibility of modulating EZH2 activity with specific inhibitors open new possible paths to be explored in bladder cancer therapy.

Bladder Cancer (BC) represents a current clinical and social challenge. The recent studies aimed to describe the genomic landscape of BC have underscored the relevance of epigenetic alterations in the pathogenesis of these tumors. Among the epigenetic alterations, histone modifications occupied a central role not only in cancer, but also in normal organism homeostasis and development. EZH2 (Enhancer of Zeste Homolog 2) belongs to the Polycomb repressive complex 2 as its catalytic subunit, which through the trimethylation of H3 (Histone 3) on K27 (Lysine 27), produces gene silencing. EZH2 is frequently overexpressed in multiple tumor types, including BC, and plays multiple roles besides the well-recognized histone mark generation. In this review, we summarize the present knowledge on the oncogenic roles of EZH2 and its potential use as a therapeutic target, with special emphasis on BC pathogenesis and management.

Bladder cancer (BC) represents a clinical, social, and economic challenge due to tumor-intrinsic characteristics, limitations of diagnostic techniques and a lack of personalized treatments. In the last decade, the use of liquid biopsy has grown as a non-invasive approach to characterize tumors. Moreover, the emergence of omics has increased our knowledge of cancer biology and identified critical BC biomarkers. The rewiring between epigenetics and metabolism has been closely linked to tumor phenotype. Chromatin remodelers interact with each other to control gene silencing in BC, but also with stress-inducible factors or oncogenic signaling cascades to regulate metabolic reprogramming towards glycolysis, the pentose phosphate pathway, and lipogenesis. Concurrently, one-carbon metabolism supplies methyl groups to histone and DNA methyltransferases, leading to the hypermethylation and silencing of suppressor genes in BC. Conversely, α-KG and acetyl-CoA enhance the activity of histone demethylases and acetyl transferases, increasing gene expression, while succinate and fumarate have an inhibitory role. This review is the first to analyze the interplay between epigenome, metabolome and cell signaling pathways in BC, and shows how their regulation contributes to tumor development and progression. Moreover, it summarizes non-invasive biomarkers that could be applied in clinical practice to improve diagnosis, monitoring, prognosis and the therapeutic options in BC.

E16011 Background: MIBC is treated, in most cases, by cystectomy and platinum-based chemotherapy. Nonetheless, the metastatic spreading occurs very often with fatal consequences. Various targeted therapies are being clinically and preclinically tested for BC management. MIBC have been associated with loss of cell cycle control. Defects in the RB pathway are a major mechanisms of cell cycle deregulation in MIBC. RB1 mutations were reported in 13% of TCGA samples, although significant mutations also were noted in CDKN1A, CDKN2A, and CCND3. The CDK 4/6 complex combines with cyclin D1 to inhibit RB activity. Therapeutic targeting of this complex improves outcome in ER positive advanced breast cancer. Methods: Palbociclib (PD-0332991) is a cdk4/6 inhibitor currently tested for the treatment of other malignancies characterized by the presence of a functional RB1 gene. A series of BC cell lines of known genomic characteristics and differing in their RB1 status, were tested for their sensitivity to Palbociclib. Results: unexpectedly, we observed a similar response to Palbociclib in pRb wt and pRb mutant cell lines in vitro and in xenografts in vivo, although with different biochemical and cell cycle effects. Genomic characterization of these treated cells shows strong gene expression divergence as a consequence of Palbociclib treatment in pRb wt and mutant cells. Nonetheless, bioinformatic analyses revealed FoxM1 as a possible common regulator of some downregulated genes in both cases. Importantly, FoxM1 has been demonstrated a bona fide cdk4 substrate and may confer cis-platinum resistance. We observed reduced FoxM1 phosphorylation upon Palbociclib treatment in all cell lines tested, and also increased sensitivity to cis-platinum. Remarkably, we found that phosphorylated FoxM1 is a potential poor prognosis factor in human BC clinical samples. Conclusions: in our study we observed that the activity of Palbociclib in BC cell lines is independent on the RB1 status and showed synergistic activity with cisplatin. Future studies, using mouse models based on the genetic inactivation of Rb1 with other tumor suppressor genes, will precede the possible development of appropriate clinical trials testing the use of Palbociclib in the management of BC patients.

Bladder cancer (BC) remains a major clinical challenge owing to its high recurrence, limited treatment options, and molecular heterogeneity. Despite recent therapeutic advances, prognosis remains poor, and resistance is frequent, underscoring the need for improved experimental models to study tumorigenesis and therapeutic response. A key limitation of advanced BC research is the scarcity of in vivo models that accurately reflect invasive disease, with even fewer capturing the complexity of metastasis. To investigate how the cell-of-origin and specific combinations of driver mutations influence in bladder tumorigenesis, we developed and characterized four genetically engineered mouse models of advanced BC by targeting two combinations of tumor suppressor genes ( Pten and Trp53 , or Pten , Trp53 , Rb1 , and Rbl1 ) in basal or suprabasal urothelial cells through intravesical of Cre-adenovirus delivery. Loss of the retinoblastoma family reduced cancer-specific survival and was associated with more differentiated carcinomas. In both genetic backgrounds, luminal-derived tumors developed earlier but showed fewer metastatic events. Histopathological and transcriptomic analyses revealed that these tumors resemble human basal-squamous and stroma-rich subtypes, sharing regulatory networks and activated signaling pathways with human invasive tumors. Notably, tumors lacking retinoblastoma family genes exhibited increased immune infiltration, reinforcing the value of these models for diverse preclinical applications. To overcome detection and latency limitations, we established tumor-derived cell lines and generated syngeneic graft models. These were validated as preclinical platforms, exhibiting therapeutic responses to CDK4/6 inhibition and anti-PD-L1 immunotherapy. Our findings highlight the value of these novel models for studying BC progression and evaluating emerging therapeutic strategies in immunocompetent settings.