Cristina Segovia

Analysis of the Polycomb-related lncRNAs HOTAIR and ANRIL in bladder cancer

BackgroundLong 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.FindingsWe 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.ConclusionsOur 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.

A Polycomb-mir200 loop regulates clinical outcome in bladder cancer

Bladder cancer (BC) is a highly prevalent disease, ranking fifth in the most common cancers worldwide. Various miRNAs have recently emerged as potential prognostic biomarkers in cancer. The miR-200 family, which repressed the epithelial-to-mesenchymal transition (EMT), is repressed in multiple advanced cancers. However, its expression and function in BC is still poorly understood. Here we show that miR-200 family displays increased expression, probably due to the activation of specific oncogenic signaling pathways, and reduced promoter methylation, in BC compared to normal bladder samples. Furthermore, we show that the expression of these miRNAs is decreased in high grade and stage tumors, and the down-regulation is associated with patients poor clinical outcome. Our data indicate that the miR-200 family plays distinct roles in Non-Muscle (NMIBC) and Muscle-Invasive BC (MIBC). In MIBC, miR-200 expression post transcriptionally regulates EMT-promoting transcription factors ZEB1 and ZEB2, whereas suppresses BMI1 expression in NMIBC. Interestingly, we show that increased EZH2 and/or BMI1 expression repress the expression of miR-200 family members. Collectively, these findings support a model of BC progression through a coordinated action between the Polycomb Repression Complex (PRC) members repressing the miR-200 expression, which ultimately favors invasive BC development. Since pharmacological inhibition of EZH2 in BC cell lines lead to increased miR-200 expression, our findings may support new therapeutic strategies for BC clinical management.

BMP4 induces M2 macrophage polarization and favors tumor progression in bladder cancer.

Purpose: Bladder cancer is a current clinical and social problem. At diagnosis, most patients present with nonmuscle-invasive tumors, characterized by a high recurrence rate, which could progress to muscle-invasive disease and metastasis. Bone morphogenetic protein (BMP)–dependent signaling arising from stromal bladder tissue mediates urothelial homeostasis by promoting urothelial cell differentiation. However, the possible role of BMP ligands in bladder cancer is still unclear. Experimental Design: Tumor and normal tissue from 68 patients with urothelial cancer were prospectively collected and analyzed for expression of BMP and macrophage markers. The mechanism of action was assessed in vitro by experiments with bladder cancer cell lines and peripheral blood monocyte–derived macrophages. Results: We observed BMP4 expression is associated and favored type II macrophage differentiation. In vitro experiments showed that both recombinant BMP4 and BMP4-containing conditioned media from bladder cancer cell lines favored monocyte/macrophage polarization toward M2 phenotype macrophages, as shown by the expression and secretion of IL10. Using a series of human bladder cancer patient samples, we also observed increased expression of BMP4 in advanced and undifferentiated tumors in close correlation with epithelial–mesenchymal transition (EMT). However, the p-Smad 1,5,8 staining in tumors showing EMT signs was reduced, due to the increased miR-21 expression leading to reduced BMPR2 expression. Conclusions: These findings suggest that BMP4 secretion by bladder cancer cells provides the M2 signal necessary for a protumoral immune environment. In addition, the repression of BMPR2 by miR-21 makes the tumor cells refractory to the prodifferentiating actions mediated by BMP ligands, favoring tumor growth. Clin Cancer Res; 23(23); 7388–99. ©2017 AACR.

Opposing roles of PIK3CA gene alterations to EZH2 signaling in non-muscle invasive bladder cancer.

The high rates of tumor recurrence and progression represent a major clinical problem in non-muscle invasive bladder cancer. Previous data showed that EZH2-dependent signaling mediates these processes, whereas the frequent alterations of PIK3CA gene (copy gains and mutations) are predictive of reduced recurrence. Here we show, using clinical samples and bladder cancer cell lines, a functional interaction between EZH2- and PIK3CA-dependent signaling pathways. PIK3CA alterations mediated, on the one hand, the increased expression of two miRNAs, miR-101 and miR-138, which posttranscriptionally downregulate EZH2 expression. On the other hand, PIK3CA alterations facilitate the activation of Akt which phosphorylates EZH2 on Ser21, precluding the trimethylation of histone H3 in K27. Remarkably the increased expression of miR101 or miR138 and the expression of Ser21-phosphorylated EZH2 are good prognostic factors regarding non-muscle invasive bladder cancer recurrence and progression. Collectively, this study provides molecular evidences indicating that the gene expression rewiring occurring in primary bladder tumors, associated with increased EZH2 expression and activity and mediating the increased recurrence and progression risk, are prevented by PIK3CA-dependent signaling. This molecular process may have deep implications in the management of bladder cancer patients and in the design of novel molecularly targeted therapeutic approaches.

Liquid Biopsy Biomarkers in Bladder Cancer: A Current Need for Patient Diagnosis and Monitoring

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.

Epigenetic mutations and cancer therapy Effectiven(EZH2)

Cancer arises as a consequence of accumulating genomic alterations, which affect primarily oncogenes and tumor suppressor genes (TSGs). This condition creates a particular landmark that can be exploited in therapy. Not surprisingly, many pharma companies launched programs aimed to inhibit the gain of function activities caused by oncogene mutation and/or amplification, and many of these inhibitors are currently in the clinics. The situation is not so simple in the case of TSGs, where a common loss of function precludes, in most cases, their actual use as therapeutic targets.

Abstract PR01: Therapeutic targeting of cdk4 in bladder cancer

Bladder cancer (BC) is a current clinical and social problem. At diagnosis, 70% of the patients display non-muscle invasive tumor (NMIBC), a relatively indolent disease treated by transurethral resection followed by local instillation in some cases. Unfortunately, NMIBC displays an extremely high recurrence rate, and these recurrent tumors may display tumor progression showing muscle invasive (MIBC) characteristics. 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. 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. Since RB1 mutation occurs in only 20-30% of BC, we hypothesized the potential use of Palbociclib for BC management. A series of BC cell lines of known genomic characteristics and differing in their RB1 status, were tested for their sensitivity to Palbociclib. 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 NMIBC clinical samples. Future studies, using various NMBIC and metastatic MIBC transgenic 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. This abstract is also being presented as Poster A04. Citation Format: Carolina Rubio, Fernando Lopez-Calderon, Cristina Segovia, Marta Duenas, Monica Martinez-Fernandez, Irene Otero, Federico de la Rosa, Felipe Villacampa, Daniel Castellano, Jesus M. Paramio. Therapeutic targeting of cdk4 in bladder cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr PR01.