Bladder Cancer Patient Samples Research Articles

Glycoprotein nonmetastatic melanoma protein B impacts the malignant potential of bladder cancer cells through its hem-immunoreceptor tyrosine-based activation motif.

Bladder cancer is one of the most common cancers among men worldwide. Although multiple genomic mutations and epigenetic alterations have been identified, an efficacious molecularly targeted therapy has yet to be established. Therefore, a novel approach is anticipated. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that is highly expressed in various cancers. In this study, we evaluated bladder cancer patient samples and found that GPNMB protein abundance is associated with high-grade tumors, and both univariate and multivariate analyses showed that GPNMB is a prognostic factor. Furthermore, the prognosis of patients with high GPNMB levels was significantly poorer in those with nonmuscle invasive bladder cancer (NMIBC) than in those with muscle invasive bladder cancer (MIBC). We then demonstrated that knockdown of GPNMB in MIBC cell lines with high GPNMB inhibits cellular migration and invasion, whereas overexpression of GPNMB further enhances cellular migration and invasion in MIBC cell lines with originally low GPNMB. Therefore, we propose that GPNMB is one of multiple driver molecules in the acquisition of cellular migratory and invasive potential in bladder cancers. Moreover, we revealed that the tyrosine residue in the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) is required for GPNMB-induced cellular motility.

DOK7, a target of miR-299-5p, suppresses the progression of bladder cancer.

Bladder cancer (BLCA) is the 6th most common malignancy in males. microRNA (miRNAs) can function as tumor suppressors or oncogenic factors, which are of significance in the progression of BLCA. This study explored the mechanisms by which miR-299-5p modulates DOK7 (Docking Protein 7) expression and the functional role of DOK7 in the progression of BLCA. The expression of the DOK7 in BLCA patient samples was examined by RT-qPCR (Real-time quantitative polymerase chain reaction), Western blotting and Immunohistochemical (IHC) staining. The malignant phenotype of BLCA cells upon DOK7 overexpression or silencing was assessed by functional assays including cell count kit-9 (CCK8), colony formation and 5-ethynyl-2'-deoxyuridine (Edu) staining assays, as well as Transwell migration and invasion assays. The miRNA regulators of DOK7 were identified through bioinformatics prediction, and the biological role of miR-299-5p/DOK7 axis was validated by functional assays. The impact of miR-299-5p/DOK7 axis on Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway was further examined by Western blotting. DOX7 was significantly downregulated in BLCA tumor tissues compared with normal tissues. Ectopic DOK7 expression suppressed the proliferation, migration and invasion of BLCA cells. DOK7 overexpression also attenuated the tumorigenesis of BLCA cells in nude mice. miR-299-5p was a negative regulator of DOK7 expression in BLCA cells. miR-299-5p/DOK7 axis impaired the malignancy of BLCA cells through regulating the JAK signaling pathway. Our data indicate that miR-299-5p/DOK7 axis suppresses BLCA progression possibly by regulating the JAK signaling pathway.

IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

CD40 is a central costimulatory receptor implicated in productive antitumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway has demonstrated dose-limiting toxicities with minimal clinical activity, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe a role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of transpresented IL-15/IL-15Rα surface complexes, particularly by cross-presenting conventional type 1 DCs (Dendritic Cells), and associated enrichment of activated CD8 T cells. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, although they lack high levels of IL-15Rα at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Collectively, these data reveal an important role for IL-15 in mediating antitumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to develop combinations of these promising therapeutics for the treatment of patients with bladder cancer.

A new biomarker panel of ultraconserved long non-coding RNAs for bladder cancer prognosis by a machine learning based methodology

BackgroundRecent studies have indicated that a special class of long non-coding RNAs (lncRNAs), namely Transcribed-Ultraconservative Regions are transcribed from specific DNA regions (T-UCRs), 100% conserved in human, mouse, and rat genomes. This is noticeable, as lncRNAs are usually poorly conserved. Despite their peculiarities, T-UCRs remain very understudied in many diseases, including cancer and, yet, it is known that dysregulation of T-UCRs is associated with cancer as well as with human neurological, cardiovascular, and developmental pathologies. We have recently reported the T-UCR uc.8+ as a potential prognostic biomarker in bladder cancer.ResultsThe aim of this work is to develop a methodology, based on machine learning techniques, for the selection of a predictive signature panel for bladder cancer onset. To this end, we analyzed the expression profiles of T-UCRs from surgically removed normal and bladder cancer tissues, by using custom expression microarray. Bladder tissue samples from 24 bladder cancer patients (12 Low Grade and 12 High Grade), with complete clinical data, and 17 control samples from normal bladder epithelium were analysed. After the selection of preferentially expressed and statistically significant T-UCRs, we adopted an ensemble of statistical and machine learning based approaches (i.e., logistic regression, Random Forest, XGBoost and LASSO) for ranking the most important diagnostic molecules. We identified a signature panel of 13 selected T-UCRs with altered expression profiles in cancer, able to efficiently discriminate between normal and bladder cancer patient samples. Also, using this signature panel, we classified bladder cancer patients in four groups, each characterized by a different survival extent. As expected, the group including only Low Grade bladder cancer patients had greater overall survival than patients with the majority of High Grade bladder cancer. However, a specific signature of deregulated T-UCRs identifies sub-types of bladder cancer patients with different prognosis regardless of the bladder cancer Grade.ConclusionsHere we present the results for the classification of bladder cancer (Low and High Grade) patient samples and normal bladder epithelium controls by using a machine learning application. The T-UCR’s panel can be used for learning an eXplainable Artificial Intelligent model and develop a robust decision support system for bladder cancer early diagnosis providing urinary T-UCRs data of new patients. The use of this system instead of the current methodology will result in a non-invasive approach, reducing uncomfortable procedures (such as cystoscopy) for the patients. Overall, these results raise the possibility of new automatic systems, which could help the RNA-based prognosis and/or the cancer therapy in bladder cancer patients, and demonstrate the successful application of Artificial Intelligence to the definition of an independent prognostic biomarker panel.

A tumor microenvironment preoperative nomogram for prediction of lymph node metastasis in bladder cancer.

Growing evidence suggests that tumor metastasis necessitates multi-step microenvironmental regulation. Lymph node metastasis (LNM) influences both pre- and post-operative bladder cancer (BLCA) treatment strategies. Given that current LNM diagnosis methods are still insufficient, we intend to investigate the microenvironmental changes in BLCA with and without LNM and develop a prediction model to confirm LNM status. "Estimation of Stromal and Immune cells in Malignant Tumors using Expression data" (ESTIMATE) algorithm was used to characterize the tumor microenvironment pattern of TCGA-BLCA cohort, and dimension reduction, feature selection, and StrLNM signature construction were accomplished using least absolute shrinkage and selection operator (LASSO) regression. StrLNM signature was combined with the genomic mutation to establish an LNM nomogram by using multivariable logistic regression. The performance of the nomogram was evaluated in terms of calibration, discrimination, and clinical utility. The testing set from the TCGA-BLCA cohort was used for internal validation. Moreover, three independent cohorts were used for external validation, and BLCA patients from our cohort were also used for further validation. The StrLNM signature, consisting of 22 selected features, could accurately predict LNM status in the TCGA-BLCA cohort and several independent cohorts. The nomogram performed well in discriminating LNM status, with the area under curve (AUC) of 75.1% and 65.4% in training and testing datasets from the TCGA-BLCA cohort. Furthermore, the StrLNM nomogram demonstrated good calibration with p >0.05 in the Hosmer-Lemeshow goodness of fit test. Decision curve analysis (DCA) revealed that the StrLNM nomogram had a high potential for clinical utility. Additionally, 14 of 22 stably expressed genes were identified by survival analysis and confirmed by qPCR in BLCA patient samples in our cohort. In summary, we developed a nomogram that included an StrLNM signature and facilitated the preoperative prediction of LNM status in BLCA patients.

Characterization of Cellular and Acellular Analytes from Pre-Cystectomy Liquid Biopsies in Patients Newly Diagnosed with Primary Bladder Cancer.

Simple SummaryThe standard of care for patients diagnosed with localized bladder cancer (BCa) is cystectomy. However, emerging evidence shows that the patients receiving surgical intervention often experience a return of their cancer. Examining patient blood samples for rare events, such as circulating tumor cells (CTCs) and large extracellular vesicles (LEVs), may reveal biomarkers indicative of the presence of cancer and provide an insight into disease progression. Through computational methodologies, each event in the blood was characterized to determine its rarity within the sample and then compared to events found in normal donors. We demonstrate that a wide range of CTCs and LEVs are found in significantly higher proportions among BCa patients compared to normal donors. This result suggests that the blood liquid biopsy is a proficient analyte for detecting BCa to ultimately guide clinical decisions to improve patient treatment outcomes.Urinary bladder cancer (BCa) is the 10th most frequent cancer in the world, most commonly found among the elderly population, and becomes highly lethal once cells have spread from the primary tumor to surrounding tissues and distant organs. Cystectomy, alone or with other treatments, is used to treat most BCa patients, as it offers the best chance of cure. However, even with curative intent, 29% of patients experience relapse of the cancer, 50% of which occur within the first year of surgery. This study aims to use the liquid biopsy to noninvasively detect disease and discover prognostic markers for disease progression. Using the third generation high-definition single cell assay (HDSCA3.0), 50 bladder cancer patient samples and 50 normal donor (ND) samples were analyzed for circulating rare events in the peripheral blood (PB), including circulating tumor cells (CTCs) and large extracellular vesicles (LEVs). Here, we show that (i) CTCs and LEVs are detected in the PB of BCa patients prior to cystectomy, (ii) there is a high heterogeneity of CTCs, and (iii) liquid biopsy analytes correlate with clinical data elements. We observed a significant difference in the incidence of rare cells and LEVs between BCa and ND samples (median of 74.61 cells/mL and 30.91 LEVs/mL vs. 34.46 cells/mL and 3.34 LEVs/mL, respectively). Furthermore, using classification models for the liquid biopsy data, we achieved a sensitivity of 78% and specificity of 92% for the identification of BCa patient samples. Taken together, these data support the clinical utility of the liquid biopsy in detecting BCa, as well as the potential for predicting cancer recurrence and survival post-cystectomy to better inform treatment decisions in BCa care.

Abstract 1194: MEK inhibition in combination with PI3K/AKT/MTOR is a promising therapeutic in basal bladder cancer

Abstract There are diverse resources available for data relevant to the drug screening of bladder cancer cell lines in 2D culture, such as the Cancer Cell Line Encyclopedia (CCLE) and Genomics of Drug Sensitivity in Cancer (GDSC) databases; however, 3D culture is more representative of in vivo response in patients. Moreover, drug screens in 3D reveal differential drug response and expression of genes and proteins that indicate the potential of more effective selection of drugs for specific patients: the basic tenet of personalized oncology. Therefore, we screened bladder cancer cell lines in 3D culture using a library of curated small-molecule inhibitors to identify compounds and classes of compounds with efficacy in bladder cancer. Genomic and transcriptomic data for these bladder cancer cell lines were used to correlate genomic molecular parameters with drug response with the aim of discerning biomarkers of response or to suggest plausible drug combinations. This allowed us to identify groups of tumors that are vulnerable to specific drugs or classes of drugs. We found that MEK inhibitors were a promising target among basal bladder cancers, an RNA-based molecular subtype of bladder cancer, especially aggressive and exhibiting poor prognosis. Furthermore, trametinib was found to be a “very active” MEK inhibitor in basal cancers, classified using drug sensitivity 3 (DSS3) scoring. PI3K/AKT/MTOR inhibitors target a parallel signaling pathway to MEK inhibition; therefore, we tested PI3K/AKT/MTOR combinations with trametinib in basal bladder cancer to query for synergistic properties. Additionally, we tested PI3K/AKT/MTOR combinations with trametinib using ex vivo organoids of bladder cancer patient samples collected directly at operation. Taken together, MEK inhibition is a promising therapeutic strategy in the basal subtype of bladder cancer, particularly in combination with parallel pathway inhibition. Moving forward, we plan to test PI3K/AKT/MTOR combinations with trametinib in trametinib-resistant basal bladder cancer cell lines to investigate how this affects signaling and re-sensitization to trametinib. Citation Format: Athena M. Apfel, Nathan M. Merrill, Nathalie M. Vandecan, Liwei Bao, Xu Cheng, Lluis A. Lopez-Barcons, Kathleen C. Day, Phillip L. Palmbos, Mark L. Day, Aaron M. Udager, Matthew B. Soellner, Sofia D. Merajver. MEK inhibition in combination with PI3K/AKT/MTOR is a promising therapeutic in basal bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1194.

Differentially Expressed Extracellular Vesicle-Contained microRNAs before and after Transurethral Resection of Bladder Tumors.

Bladder cancer (BC) is currently diagnosed and monitored by cystoscopy, a costly and invasive procedure. Potential biomarkers in urine, blood, and, more recently, extracellular vesicles (EVs), have been explored as non-invasive alternatives for diagnosis and surveillance of BC. EVs are nanovesicles secreted by most cell types containing diverse molecular cargo, including different types of small RNAs, such as microRNA (miRNA). In this study, we performed next-generation sequencing of EV-contained miRNA isolated from urine and serum of 41 patients with non-muscle invasive BC (27 stage Ta, 14 stage T1) and 15 non-cancer patients (NCP) with benign cystoscopy findings. MiRNA sequencing was also performed on serum supernatant samples for T1 patients. To identify potential BC-specific biomarkers, expression levels of miRNA in presurgery samples were compared to those at postsurgery check-ups, and to NCPs. Results showed that two miRNAs, urinary EV-contained miR-451a and miR-486-5p, were significantly upregulated in presurgery samples from T1 patients compared to postsurgery check-up samples. This was confirmed in a replica EV/RNA isolation and sequencing run of 10 T1 patients from the primary run; however, analyses revealed no differential expression of miRNAs in serum EVs, serum supernatant, or when comparing BC patients to NCPs. This is the first study to investigate EV-containing miRNA sequencing in pre- and postsurgery BC patient samples and our findings suggest that urinary EV-contained miR-451a and miR-486-5p may be potential biomarkers for recurrence-free survival of BC patients with stage T1 disease.

Abstract 4846: A quantitative proteo-genomic analysis for targeted therapies in the management of bladder cancer survival

Abstract Bladder cancer is the most common type of urothelial carcinoma (UC), and it represents about 5% of all new cancers in the United States. The diagnostics, treatment, and five-year survival rates of this cancer have seen no significant improvements for nearly last three decades. Due to the lack of development of new treatment options, bladder cancer poses a bigger threat than many other cancers. In this current study, we have analyzed over 400 patients' proteo-genomic sequencing and expression data from The Cancer Genome Atlas (TCGA) project using the cBioportal platform. We found two genes, TP53 and CDKN2A, which were significantly altered in the bladder cancer patient samples. The mutations and copy number alteration (CNA) of TP53 were present in more than 50% of patient samples, while CDKN2A was deleted in approximately 40% of patient samples. However, we found that patients with CDKN2A deletion significantly contributed to patient survival compared to TP53 mutational inactivation. Based on this finding, we further analyzed the survival associated genetic alterations in only those patient samples that have the CDK2NA deletion. Analyses indicated that bladder cancer patients with the CDKN2A gene deletion have differential expression of PIK3CA, RPS6KB1, CCND1, and TFRC and worse prognosis. Next, we analyzed protein level expression data using MD Anderson cell line project (MCLP portal) database and found that phospho-RPS6KB1 (p70-S6K_pT389) and CCND1 (Cyclin D1) were the least expressed proteins in bladder cancer cell lines compared to over fifteen different types of cancer cell lines. Additionally, data analyses of The Cancer Therapeutics Response Portal (CTRP) and MCLP database, we found two drugs- hyperforin and CD437 which have the highest Spearman's Rho correlation with CCND1 and RPS6KB1, respectively, with significantly low p-values. Importantly, these drugs sensitize the cell lines that have low CCND1 and RPS6KB1, respectively, and would likely be beneficial for patient survival. Next, we validated our findings in three bladder cancer cell lines, T24, 5637, and HT1376. We monitored the expression of the above proteins in these bladder cancer cell-lines and compared them with other cancer cell lines. Experimental data found a strong agreement with our high throughput cell-line and patient data analysis. Besides, both hyperforin and CD437 drugs exhibit a strong cytotoxic effect on all three bladder cell lines. Furthermore, hyperforin and CD437 were found to increase the expression of CDKN2A in addition to increasing CCND1 and RPS6KB1, respectively. In summary, we characterized four different genes PIK3CA, RPS6KB1, CCND1, and TFRC, which are associated with CDKN2A mediated survival in bladder cancer. Our findings also provide a novel therapeutic approach for bladder cancer patients. Funding: This work was supported by the DoD grant to Dr. Srivastava (DAMD17-03-1-0107) and by CHIRP funding (CHIRP ID# IAA-A-HL-14-007). Disclaimers: The contents of this publication are the sole responsibility of the author(s) and do not necessarily reflect the views, opinions or policies of The Uniformed Services University of the Health Sciences (USUHS), The Henry M. Jackson Foundation (HJF), the Department of Defense (DoD), the Departments of the Army, Navy, or Air Force. *Correspondence: meera.srivastava@usuhs.edu OR alakesh.bera.ctr@usuhs.edu. Citation Format: Sreejato Chatterjee, John Karaian, Madhan Subramanian, Eric Russ, Michael Eklund, Harvey B. Pollard, Meera Srivastava, Alakesh Bera. A quantitative proteo-genomic analysis for targeted therapies in the management of bladder cancer survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4846.

External Replication of Urinary Bladder Cancer Prognostic Polymorphisms in the UK Biobank.

Introduction: Multiple studies have reported genetic associations with prognostic outcomes of urinary bladder cancer. However, the lack of replication of these associations prohibits establishing further evidence-based research directions. Moreover, there is a lack of independent bladder cancer patient samples that contain prognostic measures, making genetic replication analyses even more challenging.Materials and Methods: We have identified 1,534 eligible patients and used data on Hospital Episode Statistics in the UK Biobank to model variables of otherwise non-collected events on bladder cancer recurrence and progression. Data on survival was extracted from the Death Registry. We have used SNPTEST software to replicate previously reported genetic associations with bladder cancer recurrence (N = 69), progression (N = 23), survival (N = 53), and age at the time of diagnosis (N = 20).Results: Using our algorithm, we have identified 618 recurrence and 58 UBC progression events. In total, there were 209 deaths (106 UBC-specific). In replication analyses, eight SNPs have reached nominal statistical significance (p < 0.05). Rs2042329 (CWC27) for UBC recurrence; rs804256, rs4639, and rs804276 (in/close to NEIL2) for NMIBC recurrence; rs2293347 (EGFR) for UBC OS; rs3756712 (PDCD6) for NMIBC OS; rs2344673 (RGS5) for MIBC OS, and rs2297518 (NOS2) for UBC progression. However, none have remained significant after adjustments for multiple comparisons.Discussion: External replication in genetic epidemiology is an essential step to identify credible findings. In our study, we identify potential genetic targets of higher interest for UBC prognosis. In addition, we propose an algorithm for identifying UBC recurrence and progression using routinely-collected data on patient interventions.

Urinary microbiota - a potential biomarker and therapeutic target for bladder cancer.

Purpose. To identify potential biomarkers and/or therapeutic targets for bladder cancer we characterized and analysed the composition of the urinary microbiota from bladder cancer and non-cancer patients.Methodology. In this study, we collected urine samples from 29 bladder cancer patients and 26 non-cancer patients. To avoid contamination and the impact of antibiotics, urine specimens were collected in a clean manner prior to antibiotic administration. Using the amplicon-based next-generation sequencing approach, the potential determinant bacteria were estimated in a between-group comparison. The results illustrated the differences in microbiota abundance among cancer and non-cancer patients and the overall number of cases carrying these bacteria.Results. We found that the urine samples contained a conserved microbiota with four phyla (Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes), which accounted for 94.4 % of bacteria in all cases. Comparing the microbiota between the bladder cancer and control group, five genera of bacteria (Streptococcus, Bifidobacterium, Lactobacillus, Veillonella and Actinomyces) existed in all samples, but with significant intergroup differences (P<0.05). The bladder cancer patients presented with a higher abundance of Actinomyces, while the other strains were enriched in the control group. A higher abundance of Actinomyces europaeus was also observed in the bladder cancer group compared to the control group.Conclusion. The samples collected from the bladder cancer patients displayed a significantly different pattern relative to those from the control group. The higher abundance of A. europaeus observed in bladder cancer patient samples also suggests that the strain may be indicative of bladder cancer. The urinary microbiota may be a potential biomarker and therapeutic target for bladder cancer.

TAp73 expression and P1 promoter methylation, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle-invasive bladder cancer (MIBC)

ABSTRACTIntrinsic and/or acquired resistance to cisplatin is a significant obstacle in the treatment of muscle-invasive bladder cancer. p73, a p53 homolog and determinant of chemosensitivity, is rarely mutated in bladder cancer (BC). However p73 expression and therefore function can be repressed through epigenetic changes. In this study, we sought to identify DNA methylation status of p73, expression of TAp73 isoform, and their role in cisplatin sensitivity in BC. Primary tumor samples from 338 bladder cancer patients showed decreased TAp73 expression in MIBC compared to superficial BC. Low TAp73 protein expression was associated with shorter overall survival. To investigate if the loss of expression was methylation dependent, we utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples. We found 12 distinct CpGs in the p73 gene locus that were hypermethylated in tumors compared to adjacent normal tissues. Patients with high p73 promoter methylation specifically at CpG site cg07382920 had worse survival. In vitro, treatment with a DNA demethylating agent, decitabine (DAC), decreased TAp73 methylation and upregulated expression in both CR-T24 (cisplatin resistant T24 cells) and wild type T24 cells. Furthermore, treatment with DAC increased cisplatin response in wild type T24 and CR-T24. Our studies indicate that TAp73 expression and P1 promoter methylation, specifically at the cg073892920 site, may have prognostic and diagnostic value in MIBC. In the setting of P1 promoter hypermethylation, DAC could be used as a potentiating agent of cisplatin-based chemotherapy.

Dynamic m6A mRNA methylation reveals the role of METTL3-m6A-CDCP1 signaling axis in chemical carcinogenesis

N6-methyladenosine (m6A) is the most abundant internal modification in mammalian mRNAs. Despite its functional importance in various physiological events, the role of m6A in chemical carcinogenesis remains largely unknown. Here we profiled the dynamic m6A mRNA modification during cellular transformation induced by chemical carcinogens and identified a subset of cell transformation-related, concordantly modulated m6A sites. Notably, the increased m6A in 3′-UTR mRNA of oncogene CDCP1 was found in malignant transformed cells. Mechanistically, the m6A methyltransferase METTL3 and demethylases ALKBH5 mediate the m6A modification in 3′-UTR of CDCP1 mRNA. METTL3 and m6A reader YTHDF1 preferentially recognize m6A residues on CPCP1 3′-UTR and promote CDCP1 translation. We further showed that METTL3 and CDCP1 are upregulated in the bladder cancer patient samples and the expression of METTL3 and CDCP1 is correlated with the progression status of the bladder cancers. Inhibition of the METTL3-m6A-CDCP1 axis resulted in decreased growth and progression of chemical-transformed cells and bladder cancer cells. Most importantly, METTL3-m6A-CDCP1 axis has synergistic effect with chemical carcinogens in promoting malignant transformation of uroepithelial cells and bladder cancer tumorigenesis in vitro and in vivo. Taken together, our results identify dynamic m6A modification in chemical-induced malignant transformation and provide insight into critical roles of the METTL3-m6A-CDCP1 axis in chemical carcinogenesis.

2371 Validation of a novel PD-L1 assay for bladder cancer circulating tumor cells

OBJECTIVES/SPECIFIC AIMS: Bladder cancer patients being considered for immune checkpoint blockade are often judged on immunohistochemical staining for the checkpoint target protein PD-L1 in the original surgery or biopsy sample. However, sampling error or the clinical evolution of most patients’ cancer can render the original PD-L1 assessment no longer accurate. In contrast, circulating tumor cells (CTCs) allow serial noninvasive sampling of the current tumor status throughout a patient’s clinical course including those with the highest metastatic potential. We therefore sought to develop a method for quantifying PD-L1 expression in CTCs towards addressing inherent limitations of current UC management. METHODS/STUDY POPULATION: This work utilizes both cancer cell lines as well as patient samples. Positive and negative control cancer cell lines were assessed via “industry standard” antibodies for PD-L1 expression via Western blots and immunofluorescence, and a threshold-based method was developed for reliable quantification. PDL-1 expression was additionally verified via interferon-mediated up-regulation. CTCs isolated from bladder cancer patient samples via a density centrifugation method were then assessed for PD-L1 via the same antibodies. RESULTS/ANTICIPATED RESULTS: We will show preliminary preclinical and clinical data that validates the sensitivity and specificity of our assay. A case study will be presented that illustrate the potential useful of the novel approach we describe and which should be complementary to current clinical practices. In a patient with metastatic bladder cancer, this method effectively detected the PD-L1 expression in CTCs taken at a time coincident to when the patient derived an excellent response to the PD-L1 checkpoint inhibitor Pembrolizumab. DISCUSSION/SIGNIFICANCE OF IMPACT: This work highlights the potential utility of CTCs in the management of bladder cancer. It may be the case that this assay in conjunction with current methods of patient selection for immunotherapy may allow for better response prediction than either method alone.

Clinical, prognostic, and therapeutic significance of heat shock protein 27 in bladder cancer.

Heat shock protein 27 (HSP27) is highly expressed in many cancers, and its prognostic and predictive value has been reported. HSP27 knockdown using siRNA or OGX-427 (an anti-sense oligonucleotide sequence targeting HSP27) is reported to have anti-cancer effects and to enhance chemosensitivity of cancer cells to chemotherapeutic agents. However, conflicting results have been reported regarding the clinical significance of HSP27 in bladder cancer (BC). Furthermore, long-term suppression of HSP27 has not been investigated in BC. In this study, we investigated the association between HSP27 expression and BC characteristics in 132 BC patient samples, as well as its prognostic value to determine the potential of HSP27 as a clinical biomarker. Additionally, we applied five different shRNAs targeting HSP27 in three invasive BC cell lines to analyze the long-term knockdown effects of HSP27. Our study revealed a significant association between HSP27 expression and adverse pathological characteristics such as high-stage and -grade BC. However, HSP27 expression was not associated with clinical outcomes such as tumor recurrence, progression, and patient survival. Interestingly, although our shRNAs had obvious knockdown effects on HSP27 in BC cells, we did not find consistent effects on apoptosis of BC cells or chemotherapeutic sensitivity of BC cells to cisplatin. Therefore, although HSP27 may be a predictor of adverse pathological characteristics in BC, its role as a prognostic biomarker and therapeutic target seems to be limited.

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.

Involvement of cancer-derived IgG in the proliferation, migration and invasion of bladder cancer cells.

It is widely accepted that immunoglobulin (Ig), the classical immune molecule, is extensively expressed in many cell types other than B-cells (non-B-IgG), including some malignant cells. The expression of Ig in malignant cells has been associated with a poor prognosis. In the present study, immunohistochemical analysis detected strong positive staining of IgG in three bladder cancer cell lines, the cancer cells in 77 bladder cancer patient samples and the cells in 3 cystitis glandularis tissue samples, while negative staining was observed in 4 specimens of normal transitional epithelial tissues. Importantly, functional transcripts of IgG with unique VHDJH rearrangement patterns were also found in bladder cancer cells. The knockdown of IgG in bladder cancer cell lines using small interfering RNA significantly inhibited the proliferation, migration and invasion of the cells. Notably, high IgG expression, as determined by immunostaining, was significantly correlated with a high histological grade and recurrence. The results of the present study suggested that IgG expression is involved in the malignant biological behavior and poor prognosis of bladder cancer. Therefore, IgG may serve as a novel target for bladder cancer therapy.

Hypoxia enhances the malignant nature of bladder cancer cells and concomitantly antagonizes protein O-glycosylation extension.

Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may offer potential to treat tumour hypoxic niches harbouring more malignant cells.

Abstract LB-158: P73, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle invasive bladder cancer (MIBC)

Abstract Intrinsic and/or acquired resistance to cisplatin is a major obstacle in the treatment of advanced bladder cancer (BC). It is important to find markers that predict tumor responsiveness to cisplatin-based chemotherapy. P73, a p53 homologue, plays an important role in cisplatin sensitivity. TAp73, a major isoform of p73, utilizes an extrinsic promoter (P1) and is pro-apoptotic. We utilized Illumina 450K methylation arrays to interrogate over 150 BC patient samples and found 9 distinct CpGs in the P1 promoter region that were hypermethylated in tumors compared to adjacent normal tissues (p&amp;lt;.05). To determine the functional link between promoter methylation and transcriptional regulation of TAp73, we treated BC cell lines (253J, HT1376, and T24) with DNA hypomethylating agent decitabine (DAC) at various doses (0,1,5 μM). We found that a 10-20% decrease in methylation across 5 interrogated CpG sites resulted in a two fold increase in TAp73 expression. We hypothesize that demethylation of the P1 promoter increases TAp73 expression, sensitizing BC cells to cisplatin. To test this, T24 cells were pretreated with DAC (2μM), followed by cisplatin treatment (0.5μg/mL). Clonogenicity of T24 was measured, as well as changes in p73 methylation and expression. Results show a decrease in clonogenic potential following combination therapy. T24 treated with DAC alone or in combination with cisplatin showed a 15% decrease in P1 promoter methylation, while the same was not seen in cells treated with cisplatin alone. Treatment with DAC and cisplatin did not have a clear effect on TAp73 mRNA expression. We are investigating whether TAp73 protein expression reflects the effects of DAC. To determine the clinical value of TAp73 protein expression we utilized tissue microarrays (TMAs) of 354 BC patients. Immunohistochemical staining was assigned H-score indicating intensity of TAp73 expression and percentage of positive nuclei. Results showed lower TAp73 expression to be associated with patient samples of higher tumor grade and staging (p&amp;lt;.001), suggesting that TAp73 expression is lost as the disease progresses from superficial to advanced BC. Consequently, low TAp73 protein expression was associated with shorter overall survival (p&amp;lt;.05). To further investigate clinical utility of TAp73 promoter methylation and expression, we analyzed tumor samples from 24 patients treated with cisplatin in a neoadjuvant setting. Results showed hypermethylation in tumor compared with non-tumor samples (p&amp;lt;.05), but no significant difference in mRNA expression was observed. Studies continue to focus on understanding the mechanism of cisplatin resistance in the context of epigenetic dysregulation and utilization of DAC as a potentiating agent of cispatin-based chemotherapy in muscle invasive BC. Supported by NCI grants CA067267 and CA016056. Citation Format: Rebecca D. Greenspan, Nithya Krishnan, Carl Morrison, Angela Omilian, Wiam Bshara, Amber Worral, Kristopher Attwood, Donald L. Trump, Anna Woloszynska-Read, Candace S. Johnson. P73, a potential marker for chemoresponsiveness to cisplatin therapy and survival in muscle invasive bladder cancer (MIBC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-158. doi:10.1158/1538-7445.AM2015-LB-158

Abstract 1359: Identification of a novel hypermethylated tumor suppressor gene in human bladder cancer

Abstract Bladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Although bladder cancer patients have a low mortality rate, long term follow-up with repeated cystoscopy is required due to the high recurrence nature of the tumor. Therefore, a non-invasive detection assay is urgently required for bladder cancer patients. Aberrant promoter hypermethylation which is considered as a hallmark of cancer for over a decade, plays an important role in controlling cancer progression. Nevertheless, detection of promoter hypermethylation in bodily fluid has been implicated as a non-invasive and sensitive tool for cancer diagnosis. We aim to identify novel markers that are hypermethylated in bladder cancer. In this regard, by using illumina 27K CpG island methylation array, we identified several novel targets that are hypermethylated in 7 bladder cancer patient samples but not in primary normal human urothelim. Combined bisulfite restriction analysis (COBRA) and bisulphite pyrosequencing confirmed the array result in bladder cancer cell lines. Further, bisulphite pyrosequencing demonstrated that a significantly higher methylation level of those targets in cancer samples with higher histological grade (P &amp;lt; 0.05) and pathological stage (P &amp;lt; 0.05). The potential of non-invasive diagnosis of bladder cancer detection by using the methylation of these targets in voided urine samples is under-way. The tumor suppressive function of these methylated targets in bladder cancer deserves further investigation. Citation Format: Chia-Ming Yeh, Pi-Che Chen, Wen-Yu Huang, Cheng-Huang Shen, Cheng-Da Hsu, Michael Wy Chan. Identification of a novel hypermethylated tumor suppressor gene in human bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1359. doi:10.1158/1538-7445.AM2014-1359