Felipe Villacampa

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.

In Vivo Disruption of an Rb–E2F–Ezh2 Signaling Loop Causes Bladder Cancer

Bladder cancer is a highly prevalent human disease in which retinoblastoma (Rb) pathway inactivation and epigenetic alterations are common events. However, the connection between these two processes is still poorly understood. Here, we show that the in vivo inactivation of all Rb family genes in the mouse urothelium is sufficient to initiate bladder cancer development. The characterization of the mouse tumors revealed multiple molecular features of human bladder cancer, including the activation of E2F transcription factor and subsequent Ezh2 expression and the activation of several signaling pathways previously identified as highly relevant in urothelial tumors. These mice represent a genetically defined model for human high-grade superficial bladder cancer. Whole transcriptional characterizations of mouse and human bladder tumors revealed a significant overlap and confirmed the predominant role for Ezh2 in the downregulation of gene expression programs. Importantly, the increased tumor recurrence and progression in human patients with superficial bladder cancer is associated with increased E2F and Ezh2 expression and Ezh2-mediated gene expression repression. Collectively, our studies provide a genetically defined model for human high-grade superficial bladder cancer and demonstrate the existence of an Rb-E2F-Ezh2 axis in bladder whose disruption can promote tumor development.

PIK3CA gene alterations in bladder cancer are frequent and associate with reduced recurrence in non-muscle invasive tumors

Bladder cancer (BC) is the fifth most common cancer in the world, being the non‐muscle invasive tumors (NMIBC) the most frequent. NMIBC shows a very high frequency of recurrence and, in certain cases, tumor progression. The phosphatidylinositol 3‐kinase (PI3K) pathway, which controls cell growth, tumorigenesis, cell invasion and drug response, is frequently activated in numerous human cancers, including BC, in part through alterations of PIK3CA gene. However, the significance of PIK3CA gene alterations with respect to clinicopathological characteristics, and in particular tumor recurrence and progression, remains elusive. Here, we analyzed the presence of mutations in FGFR3 and PIK3CA genes and copy number alterations of PIK3CA gene in bladder tumor and their correspondent paired normal samples from 87 patients. We observed an extremely high frequency of PIK3CA gene alterations (mutations, copy gains, or both) in tumor samples, affecting primarily T1 and T2 tumors. A significant number of normal tissues also showed mutations and copy gains, being coincident with those found in the corresponding tumor sample. In low‐grade tumors PIK3CA mutations associated with FGFR3 mutations. Alterations in PIK3CA gene resulted in increased Akt activity in tumors. Interestingly, the presence of PIK3CA gene alterations, and in particular gene mutations, is significantly associated with reduced recurrence of NMIBC patients. Importantly, the presence of FGFR3 mutations may influence the clinical outcome of patients bearing alterations in PIK3CA gene, and increased recurrence was associated to FGFR3 mutated, PIK3CA wt tumors. These findings may have high relevance in terms of using PI3K‐targeted therapies for BC treatment.

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.

Recurrent Aortitis Associated With IgG4-Related Disease

rent IgG4-RD aortitis successfully treated with azathioprine. The patient was a 46-year-old man with a history of nonmetastatic clear-cell renal carcinoma (pT1aN0M0) treated with right radical nephrectomy 5 months previously. On admission, he reported progressive lumbar pain, night sweats, fatigue ascular involvement as the clinical presentation of IgG4-related disease (IgG4-RD) is rare. Here, we report a case of aortic involvement with recurrence after initial response to steroids, subsequently treated with azathioprine. To our knowledge, this is the first case of recurV

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.

Genetic polymorphisms and sunitinib outcome in metastatic renal-cell carcinoma: A prospective observational study and validation.

403 Background: Sunitinib (SU) is an oral small-molecule, multi-targeted receptor tyrosine kinase inhibitor, which is approved as first-line treatment for metastatic renal cell carcinoma (RCC). In a previous study, using a commercially available DNA microarray genotyping system, we identified a group of single nucleotide polymorphisms (SNPs) associated with survival and toxicity in RCC patients, treated with SU. In this study, we validated our previous data using an independent series (García-Donas J, et al. Lancet Oncol 2011). METHODS 27 metastatic RCC treatment-naive patients, recruited prospectively from January 2010 to May 2011. All the patients received SU standard treatment. A total of 92 of single nucleotide polymorphisms (SNPs) in 34 genes involved in the pharmacokinetic and pharmacodynamic pathways of drugs, were analyzed using Drug inCode pharmacogenetic service. For validation we performed genotyping in 83 samples using the KASPar SNP genotyping system. RESULTS In patients with CYP1A2*1F and CYP2C19 *2 and *4 polymorphisms, no statistically significant associations were observed, among drug metabolizing genes and toxicity or survival. Catechol-O-methyltransferase(COMT) is involved in the inactivation of several substances suchs as cathecolamines and estrogens and Val(158)Met polymorphism which were associated with PFS and OS, it was observed in our initial study. Met/Met and Val/Met carriers had statistical significant difference in PFS and OS (p = 0.0001 and p = 0.0001, respectively) compared to Val/Val carriers. In the validation series, we were able to confirm the effect on PFS (p = 0.0102). CONCLUSIONS Our preliminary analysis suggested that CYP1A2*1F and CYP2C19*2 and *4polymorphism may be associated with SU toxicity in RCC patients, but findings were not validated in an independent series. However, we could confirm an association between COMT VAl(158)Met polymorphisms and PFS. To our knowledge this is the first study to report COMT polymorphism to be associated with RCC survival.

Analysis of molecular profiling of renal cell carcinoma: Identification of a 4-microRNA signature as a prognostic value in patients with stage I-II disease.

395 Background: Renal-cell carcinoma (RCC) accounts for 3% of all cancers and produces over 12,000 deaths every year in the EE.UU. Currently, there is a need to identify stage I/II RCC patients with high risk of relapse following nephrectomy, given that these patients do not receive adjuvant treatment and ≈ 25% of them relapse. MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNA and playing an important role as regulators of gene expression during tumorigenesis. Our willing is to define a miRNA expression profile associated with a high risk of relapse in early RCC. METHODS We analyzed 113 pts. with RCC stage I-II of a local data-base who had undergone nephrectomy from 2000 to 2008. RNA was extracted from FFPE samples using RecoverAll (Ambion). RNA samples were hybridized to Human miRNA Microarray Release 14.0, 8x15K (Agilent Technologies. Data were normalized using Quantile Normalization. Only 396 miRNAs with detectable signal in at least 10% of the hybridized samples were considered for further analysis. Identification of miRNAs related with recurrence risk and subsequent developing and validation of miRNA expression-based prediction models of recurrence risk were performed in BRB-ArrayTools v4.2.1. RESULTS We identified a 4-miRNA expression signature that distinguishes early stage RCC patients with low and high recurrence risk (p value = 0.0013; HR = 4.68 [1.82-12.0]). Distant recurrence free survival rate at five years was 97.4 and 81.2 for the low and high recurrence risk groups respectively. High levels of miR-424 were related with a high recurrence risk (p = 0.023). CONCLUSIONS The TNM staging system lacks accuracy to identify prognostic markers of survival in early RCC. Our results suggest that specific miRNAs are involved in the recurrence of early disease. We have found a miRNA expression signature that identifies patients with high risk of developing distant metastasis using the FFPE sample of primary tumors. High expression levels of miR-424 were related with a high recurrence risk. MiR-424 is a hypoxia induced miRNA whose expression has been related with HIF-1α and HIF-2α stabilization and angiogenesis induction.