Development Of Bladder Tumors Research Articles

P282 Development of new bladder tumor following radical nephroureterectomy for Upper Tract Urothelial Carcinoma (UTUC)- a new disease or an old story?

P282 Development of new bladder tumor following radical nephroureterectomy for Upper Tract Urothelial Carcinoma (UTUC)- a new disease or an old story?

Latrophilin-3 as a downstream effector of the androgen receptor induces urothelial tumorigenesis.

Emerging evidence indicates that androgen receptor (AR) signaling plays a critical role in the pathogenesis of male-dominant urothelial cancer. Meanwhile, latrophilins (LPHNs), a group of the G-protein-coupled receptor to which a spider venom latrotoxin is known to bind, remain largely uncharacterized in neoplastic diseases. The present study aimed to determine the functional role of LPHN3 (encoded by the ADGRL3 gene), in association with AR signaling, in urothelial tumorigenesis. In human normal urothelial SVHUC cells, AR overexpression and androgen treatment considerably increased the expression levels of ADGRL3/LPHN3, while chromatin immunoprecipitation assay revealed the binding of AR to the promoter region of ADGRL3. In SVHUC or SVHUC-AR cells with exposure to a chemical carcinogen 3-methylcholanthrene, LPHN3 activation via ligand (e.g., α-latrotoxin, FLRT3) treatment during the process of the neoplastic/malignant transformation or LPHN3 knockdown via shRNA virus infection induced or reduced, respectively, the oncogenic activity. In N-butyl-N-(4-hydroxybutyl)nitrosamine-treated female mice, α-latrotoxin or FLRT3 injection accelerated the development of bladder tumors. Immunohistochemistry in surgical specimens further showed the significantly elevated expression of LPHN3 in non-muscle-invasive bladder tumors, compared with adjacent normal urothelial tissues, which was associated with a marginally (p = 0.051) higher risk of disease recurrence after transurethral resection. In addition, positivity of LPHN3 and AR in these tumors was strongly correlated. These findings indicate that LPHN3 functions as a downstream effector of AR and promotes urothelial tumorigenesis.

Type 2 innate lymphoid cells are not involved in mouse bladder tumor development.

Therapies for bladder cancer patients are limited by side effects and failures, highlighting the need for novel targets to improve disease management. Given the emerging evidence highlighting the key role of innate lymphoid cell subsets, especially type 2 innate lymphoid cells (ILC2s), in shaping the tumor microenvironment and immune responses, we investigated the contribution of ILC2s in bladder tumor development. Using the orthotopic murine MB49 bladder tumor model, we found a strong enrichment of ILC2s in the bladder under steady-state conditions, comparable to that in the lung. However, as tumors grew, we observed an increase in ILC1s but no changes in ILC2s. Targeting ILC2s by blocking IL-4/IL-13 signaling pathways, IL-5, or IL-33 receptor, or using IL-33-deficient or ILC2-deficient mice, did not affect mice survival following bladder tumor implantation. Overall, these results suggest that ILC2s do not contribute significantly to bladder tumor development, yet further investigations are required to confirm these results in bladder cancer patients.

Pilot study on quantifying the epithelial/mesenchymal hybrid state in the non-muscle invasive and muscle invasive bladder tumors: A promising marker of diagnosis and prognosis

BackgroundManifestation of epithelial-to-mesenchymal transition (EMT) program in tumor cells is associated with the occurrence of multiple intermediate phenotypic states namely epithelial (E), mesenchymal (M) and hybrid E/M across the epithelial-mesenchymal spectrum and these states exhibit different invasive properties. Understanding the cellular and molecular mechanisms defining the E/M hybrid state of the cells during bladder tumor development may significantly aid in the identification of novel diagnostic and prognostic markers. Materials and methodsThe present study is taken up to identify hybrid E/M score based on the immunohistochemical localization and surface expressions of epithelial proteins [E-cadherin and Beta-catenin] and mesenchymal marker proteins [N-cadherin and Vimentin] on formalin fixed paraffin embedded tumor sections of the prospective series of 99 non-muscle invasive bladder cancer (NMIBC) and 87 muscle invasive bladder cancer (MIBC) patients. E/M score was then statistically examined with patients’ demographics to assess its potential in the diagnosis and prognosis of UCB patients. ResultsAmong the E (E-cadherinhigh, β-cateninhigh), hybrid E/M (E-cadherinlow, β-cateninlow, N- cadherinhigh and Vimentinhigh) and M (N-cadherinhigh and Vimentinhigh) phenotypes, E/M phenotype was observed to be more prevalent in MIBC compared to E phenotype in NMIBC subtype. The current study reports the statistical association of tumor stage and tumor grade with the hybrid E/M state of urothelial tumor cells across both the subtypes. Hybrid E/M phenotype in MIBC patients was significantly shown to lower the overall survival time period compared to NMIBC patients. This supports the contribution of hybrid E/M state of tumor cells to the.aggressiveness of the disease. ConclusionsCharacterizing the hybrid E/M state instead of all or none phenotype becomes an imperative to understand the dynamics of EMT and MET in the tumor pathophysiology of NMIBC and MIBC subtypes, and could contribute to better patient stratification and therapeutic strategies.

MP14-02 DEVELOPMENT OF A PORCINE MODEL OF BLADDER CANCER USING THE ONCOPIG

You have accessJournal of UrologyCME1 Apr 2023MP14-02 DEVELOPMENT OF A PORCINE MODEL OF BLADDER CANCER USING THE ONCOPIG Andreas Aulitzky, Rand Wilcox Vanden Berg, Kwanghee Kim, Hikmat Al-Ahmadie, Sebastien Monette, and Jonathan A. Coleman Andreas AulitzkyAndreas Aulitzky More articles by this author , Rand Wilcox Vanden BergRand Wilcox Vanden Berg More articles by this author , Kwanghee KimKwanghee Kim More articles by this author , Hikmat Al-AhmadieHikmat Al-Ahmadie More articles by this author , Sebastien MonetteSebastien Monette More articles by this author , and Jonathan A. ColemanJonathan A. Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Large animal cancer models have potential to improve and accelerate the development of new treatment modalities but few exist. Oncopigs (OPG) are particularly suitable due to their similarities to humans. We sought to develop a urothelial tumor model using the oncopig cancer model (OCM: University of Illinois Cancer Center). The OCM carries two mutations (KRASG12D and TP53R167H) which can be induced by exposure to a buffered Adeno Virus Cre solution (bAdCre). Three procedures (P1, P2, P3) were devised and investigated. METHODS: Eight OPGs underwent tumor inoculation. In P1, bAdCre with plant starch (n=3) was injected submucosally at 3 distinct locations in the bladder wall. In P2 the urothelium was denuded in 3 locations followed by bAdCre instillation (n=4). In P3 bAdCre was instilled combined with N-Dodecyl- β-D-Maltoside (DDM), an alkyl disaccharide and polar surfactant that enhances virus transfection in the bladder, with intact urothelium (n=1). Serial imaging, cystoscopies and blood draws were performed until sacrifice at 3 timepoints (days: 14, 21, 28). Tumor characteristics were assessed by necropsy and histopathological examination of the entire tumor tissue. RESULTS: In total, 7/8 (87.5%) OPGs grew bladder tumors. Histopathological examination revealed 2 different tumor types: neoplastic tumors (NT) and inflammatory tumors (IT). Using P1, 3 OPGs developed IT (3/3). 2 OPGs in 2 locations (2/3) and 1 in 1 location (1/3). In P2, 3/4 OPGs grew tumors. 1 OPG in all 3 locations, all 3 were NTs (3/3) (Figure 1 a), 1 OPG in 2 locations (2/3), both NTs and 1 OPG in 1 location (1/3), an IT. In P3 1 NT developed (1/1). All tumors showed muscle invasive growth. P3 showed the most superficial lesion after 21 days (pT2a). In P1, 2 pigs had pT3 tumors after 21 and 28 days. No complications were observed. Based on the histological (Figure 1 b: H&E), multiple immunohistochemical findings (e.g.: Figure 1 c: high molecular cytokeratins 34Be12; Figure 1 d: P53) and the growth pattern, NT were diagnosed as urothelial cell carcinomas, sarcomatoid variant. CONCLUSIONS: This is the first description of bladder tumor development in a porcine model using the OCM. We were able to create neoplastic tumors in a short period of time. The model is reproducible and neoplastic lesions show urothelial origin with sarcomatoid transformation. Source of Funding: MSKCC Sidney Kimmel Center for Urologic Cancers © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e182 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Andreas Aulitzky More articles by this author Rand Wilcox Vanden Berg More articles by this author Kwanghee Kim More articles by this author Hikmat Al-Ahmadie More articles by this author Sebastien Monette More articles by this author Jonathan A. Coleman More articles by this author Expand All Advertisement PDF downloadLoading ...

Clinical value of M1 macrophage-related genes identification in bladder urothelial carcinoma and in vitro validation.

Background: Tumor microenvironment (TME) takes a non-negligible role in the progression and metastasis of bladder urothelial carcinoma (BLCA) and tumor development could be inhibited by macrophage M1 in TME. The role of macrophage M1-related genes in BLCA adjuvant therapy has not been studied well. Methods: CIBERSOR algorithm was applied for identification tumor-infiltrating immune cells (TICs) subtypes of subjects from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) data sets. We identified potential modules of M1 macrophages by weighted gene co-expression network analysis (WGCNA). Nomogram was determined by one-way Cox regression and lasso regression analysis for M1 macrophage genes. The data from GEO are taken to verify the models externally. Kaplan-Meier and receiver operating characteristic (ROC) curves validated prognostic value of M1 macrophage genes. Finally, we divided patients into the low-risk group (LRG) and the high-risk group (HRG) based on the median risk score (RS), and the predictive value of RS in patients with BLCA immunotherapy and chemotherapy was investigated. Bladder cancer (T24, 5637, and BIU-87) and bladder uroepithelial cell line (SV-HUC-1) were used for in vitro validation. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to validate the associated genes mRNA level. Results: 111 macrophage M1-related genes were identified using WGCNA. RS model containing three prognostically significant M1 macrophage-associated genes (FBXO6, OAS1, and TMEM229B) was formed by multiple Cox analysis, and a polygenic risk model and a comprehensive prognostic line plot was developed. The calibration curve clarified RS was a good predictor of prognosis. Patients in the LRG were more suitable for programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte associate protein-4 (CTLA4) combination immunotherapy. Finally, chemotherapeutic drug models showed patients in the LRG were more sensitive to gemcitabine and mitomycin. RT-qPCR result elucidated the upregulation of FBXO6, TMEM229B, and downregulation of OAS1 in BLCA cell lines. Conclusion: A predictive model based on M1 macrophage-related genes can help guide us in the treatment of BLCA.

Molecular Characteristics of Bladder Tumor: Increased Gene Expression of MAGE-A6 and MAGE-A11 with Decreased MicroRNA-34a and MicroRNA-125b.

Bladder cancer is recognized as one of the top ten most common cancers worldwide. Activation of oncogenes, inactivation of tumor suppressor genes, and dysregulation of androgen signaling pathways are three major pathophysiological causes in the development of bladder tumors. Discovering potential biomarkers is required for the management and immunotherapy of bladder cancer. Melanoma-associated antigen (MAGE)-A6 and MAGE-A11 are two cancer-testis antigens that are potential coregulators of androgen receptors. MicroRNAs, especially miR-34a and miR-125b are two important tumor suppressors that play a critical role in regulating different signaling pathways and inhibiting tumor development. Twenty-nine surgical tissue biopsies were collected from patients with no preoperative chemotherapy or radiotherapy (26 males and, 3 females, mean age±SD: 62.4±13.3 years). Seventeen adjacent uninvolved tissues with no abnormalities upon histological examination were considered normal controls (14 males and, 3 females, mean age±SD: 64.2±7.4 years) . Quantitative PCR was performed to evaluate the gene expression level of MAGE-A6, MAGE-A11, miR-34a, and miR-125b in bladder cancer biopsies. MAGE-A6 and MAGE-A11 expressions were significantly increased in bladder tumors compared with normal tissues. However, the expression levels of miR-34a and miR-125b were significantly downregulated in bladder tumor tissues.Interestingly, the expression level of all these genes was significantly associated with tumor grade, pathological stage (pT), and muscular invasion. MAGE-A6 and MAGE-A11 can be considered potential markers for the diagnosis and immunotherapy of bladder tumors. Furthermore, the modulation of miR-34a and miR-125b gene expression in association with increased MAGE-A6 and MAGE-A11 genes could open a new horizon in the improvement of bladder cancer.

Significance of tumor-associated macrophages in bladder cancer development

Bladder cancer is the 2 nd most common urological oncological disease in the worlds. Tumors can be muscle invasive and non-muscle invasive. Recently, tumor microenvironment (TME) became a focus of investigation in malignant tumors of the bladder. According to the currently available data, TME is a specific environment crating optimal conditions for carcinogenesis in the neoplastic lesion. The main parts of TME are extracellular matrix and stroma including vasculature, stromal, and immune cells. Additionally, TME includes cytokines, chemokines, and other compounds activating signal pathways necessary for tumor cells. Tumor-associated macrophages (TAMs) are being extensively studied as representatives of TME in solid tumors of varying locations. These macrophages can be classified into 2 phenotypes: M1 (pro-inflammatory and antitumor) and M2 (anti-inflammatory and protumor). The phenotypes perform different roles, and M2 macrophages regulate the most important processes of oncogenesis (invasion, proliferation, neoangiogenesis, etc.). In the context of bladder cancer, M2 macrophages are the most significant as they are the most numerous TAMs in TME. Aim . To study the role of tumor-associated macrophages in development of bladder tumors, as well as prognostic value of these macrophages.

Persistent γ-H2AX Formation and Expression of Stem Cell Markers in N-Butyl-N-(4-Hydroxybutyl)Nitrosamine-Induced Bladder Carcinogenesis in Rats.

We investigated γ-H2AX formation, a biomarker of DNA damage, and expression of stem cell markers (SCMs), including cytokeratin 14, aldehyde dehydrogenase 1A1 (ALDH1A1), and CD44, in the development of rat bladder tumors induced by short-term administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Histopathological examination showed that diffuse simple hyperplasia of the bladder urothelium induced by BBN recovered to the normal-appearing urothelium after withdrawal, whereas focal proliferative lesions were newly developed and subsequently progressed to benign papilloma and carcinoma. Immunohistochemical analysis revealed that BBN-induced γ-H2AX formation and ALDH1A1 and CD44 expression persisted at higher levels in the normal-appearing urothelium than those in the control group for long periods after withdrawal. Since persistent chronic inflammation was observed even after withdrawal, targeted gene expression analysis of inflammation-related factors revealed 101 genes, including Stat3 and Myc, that showed persistent high expression. Pathway analysis suggested that Stat3 and/or Myc activation may be associated with SCM expression. We focused on hepatocyte growth factor (Hgf), one of the genes predicted in relation to Stat3/Myc, and confirmed that HGF-positive cells increased by BBN persisted in the normal-appearing urothelium after withdrawal and colocalized with γ-H2AX and SCMs. These results suggested that the long-term persistence of γ-H2AX formation and SCM expression, which occurred during the early stages of bladder tumorigenesis, is not a transient response to exposure and might contribute to bladder tumorigenesis. Although further studies are needed, BBN-induced rat bladder tumors may originate from focal hyperplasia arising from SCM-positive cells via activation of the STAT3/MYC pathway after DNA damage involving γ-H2AX formation.

Intravesical VAX014 Synergizes with PD-L1 Blockade to Enhance Local and Systemic Control of Bladder Cancer.

Emerging clinical evidence indicates that the combination of local administration of immunotherapy with systemic immune-checkpoint blockade targeting the PD-1/PD-L1 pathway improves response rates in select solid tumor indications; however, limited clinical experience with this approach exists in advanced bladder cancer patients. VAX014 is a novel bacterial minicell-based, integrin-targeted oncolytic agent undergoing clinical investigation for intravesical (IVE) treatment of nonmuscle-invasive bladder cancer. Here, we demonstrated that the antitumor activity of VAX014 following IVE administration was dependent upon CD4+ and CD8+ T cells in two syngeneic orthotopic bladder tumor models (MB49 and MBT-2). PD-L1 upregulation was found to be an acquired immune-resistance mechanism in the MB49 model, and the combination of VAX014 with systemic PD-L1 blockade resulted in a significant improvement in bladder tumor clearance rates and development of protective antitumor immunologic memory. Combination treatment also led to enhanced systemic antitumor immune responses capable of clearing distal intradermal tumors and controlling pulmonary metastasis. Distal tumors actively responding to combination therapy demonstrated a phenotypic shift from regulatory T cell to Th1 in intratumoral CD4+ T cells, which was accompanied by a higher percentage of activated CD8+ T cells and higher IFNγ. Finally, VAX014's target integrins α3β1 and α5β1 were overexpressed in tumor biopsies from advanced-stage bladder cancer patients, as well as in both the MB49 and MBT-2 orthotopic mouse models of bladder cancer. These collective findings provide a rationale for the clinical investigation of VAX014 and systemic PD-1/PD-L1 blockade in advanced-stage bladder cancer.

MP06-14 ROLE OF LATROPHILINS AS KEY DOWNSTREAM EFFECTORS OF THE ANDROGEN RECEPTOR IN MODULATING UROTHELIAL TUMORIGENESIS

You have accessJournal of UrologyCME1 May 2022MP06-14 ROLE OF LATROPHILINS AS KEY DOWNSTREAM EFFECTORS OF THE ANDROGEN RECEPTOR IN MODULATING UROTHELIAL TUMORIGENESIS Takuro Goto, Yuki Teramoto, Masato Yasui, Guiyang Jiang, Yujiro Nagata, George Netto, and Hiroshi Miyamoto Takuro GotoTakuro Goto More articles by this author , Yuki TeramotoYuki Teramoto More articles by this author , Masato YasuiMasato Yasui More articles by this author , Guiyang JiangGuiyang Jiang More articles by this author , Yujiro NagataYujiro Nagata More articles by this author , George NettoGeorge Netto More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002523.14AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We and others have indicated that androgen receptor (AR) signaling plays a critical role in urothelial cancer outgrowth. Meanwhile, our DNA microarray data suggest that latrophilin-3 (LPHN3), one of latrophilins (LPHNs) that are a group of the G-protein-coupled receptor where a spider venom latrotoxin (LTX) is known to bind, represents a downstream target of AR in bladder cancer cells. The present study aimed to determine the functional role of LPHNs, including LPHN1, LPHN2, and LPHN3, in the development of urothelial cancer. METHODS: We compared the levels of LPHN expression in 83 bladder tumors vs. paired normal bladder tissues (by immunohistochemistry) as well as in human normal urothelial SVHUC sublines stably expressing wild-type AR vs. vector only (by western blot). We then assessed the effects of ligand (e.g. LTX, FLRT3) treatment, as well as silencing of each LPHN via shRNA virus infection, on the neoplastic transformation of SVHUC-derived cells induced by a chemical carcinogen 3-methylcholanthrene (MCA). Bladder tumor development was also compared in C57BL/6 mice treated with a carcinogen BBN and LTX/FLRT3. RESULTS: The rates of immunoreactivity for LPHN1 (87% vs. 69%, P=0.012), LPHN2 (55% vs. 31%, P=0.002), and LPHN3 (51% vs. 37%, P=0.087) were significantly higher in bladder tumors than in non-neoplastic urothelial tissues. Moreover, LPHN3 positivity in non-muscle-invasive tumors was associated with the risk of disease recurrence after transurethral surgery (P=0.051). The expression levels of each LPHN were also higher in SVHUC-AR cells than in AR-negative SVHUC-vector cells, and androgen treatment in SVHUC-AR further increased them. Chromatin immunoprecipitation assay then revealed the binding of AR to the promoter region of each LPHN in SVHUC-derived cells. Meanwhile, induction in LPHN expression by LTX or FLRT3 treatment was seen in SVHUC cells. The in vitro transformation assay showed that LTX and FLRT3 induced the MCA-mediated oncogenic activity particularly in SVHUC-vector cells, while knockdown of LPHN1, LPHN2, or LPHN3 resulted in similar inhibition in that of MCA-SVHUC-AR cells. LTX (P=0.002) and FLRT3 (P=0.001) also accelerated the development of bladder tumors in BBN-treated female mice. CONCLUSIONS: These findings suggest that LPHNs, which are activated in bladder tumors as key downstream effectors of the AR, promote urothelial tumorigenesis. Accordingly, LPHN inhibition, along with AR inactivation, has the potential of being an effective chemopreventive approach for urothelial cancer. Source of Funding: Ferring Research Institute © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e82 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takuro Goto More articles by this author Yuki Teramoto More articles by this author Masato Yasui More articles by this author Guiyang Jiang More articles by this author Yujiro Nagata More articles by this author George Netto More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement PDF DownloadLoading ...

RNF144A deficiency promotes PD-L1 protein stabilization and carcinogen-induced bladder tumorigenesis.

RNF144A is a DNA damage-induced E3 ubiquitin ligase that targets proteins involved in genome instability for degradation, e.g., DNA-PKcs and BMI1. RNF144A is frequently mutated or epigenetically silenced in cancer, providing the rationale to evaluate RNF144A loss of function in tumorigenesis. Here we report that RNF144A-deficient mice are more prone to the development of bladder tumors upon carcinogen exposure. In addition to DNA-PKcs and BMI1, we identify the immune checkpoint protein PD-L1 as a novel degradation target of RNF144A, since these proteins are expressed at higher levels in Rnf144a KO tumors. RNF144A interacts with PD-L1 in the plasma membrane and intracellular vesicles and promotes poly-ubiquitination and degradation of PD-L1. Therefore, Rnf144a KO stabilizes PD-L1 and leads to a reduction of tumor-infiltrating CD8+ T cell populations in the BBN-induced bladder tumors. The bladder tumors developed in WT and Rnf144a KO mice primarily express CK5 and CK14, markers of basal cancer subtype, as expected in BBN-induced bladder tumors. Intriguingly, the Rnf144a KO tumors also express GATA3, a marker for the luminal subtype, suggesting that RNF144A loss of function promotes features of cellular differentiation. Such differentiation features in Rnf144a KO tumors likely result from a decrease of EGFR expression, consistent with the reported role of RNF144A in maintaining EGFR expression. In summary, for the first time our study demonstrates the in vivo tumor suppressor activity of RNF144A upon carcinogenic insult. Loss of RNF144A promotes the expression of DNA-PKcs, BMI1 and PD-L1, likely contributing to the carcinogen-induced bladder tumorigenesis.

PD37-06 LATROPHILIN-3 AS A KEY DOWNSTREAM EFFECTOR OF THE ANDROGEN RECEPTOR INDUCES UROTHELIAL TUMORIGENESIS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-06 LATROPHILIN-3 AS A KEY DOWNSTREAM EFFECTOR OF THE ANDROGEN RECEPTOR INDUCES UROTHELIAL TUMORIGENESIS Takuro Goto, Yuki Teramoto, Yujiro Nagata, Guiyang Jiang, Taichi Mizushima, Satoshi Inoue, Hiroki Ide, George Netto, and Hiroshi Miyamoto Takuro GotoTakuro Goto More articles by this author , Yuki TeramotoYuki Teramoto More articles by this author , Yujiro NagataYujiro Nagata More articles by this author , Guiyang JiangGuiyang Jiang More articles by this author , Taichi MizushimaTaichi Mizushima More articles by this author , Satoshi InoueSatoshi Inoue More articles by this author , Hiroki IdeHiroki Ide More articles by this author , George NettoGeorge Netto More articles by this author , and Hiroshi MiyamotoHiroshi Miyamoto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: We and others have indicated that androgen receptor (AR) signaling plays a critical role in urothelial cancer outgrowth. Meanwhile, our DNA microarray data suggest that latrophilin-3 (LPHN3), one of latrophilins that are a group of the G-protein-coupled receptor where a spider venom latrotoxin (LTX) is known to bind, represents a downstream target of AR in bladder cancer cells. The present study aims to determine the functional role of LPHN3 in urothelial carcinogenesis. METHODS: We determined the expression of LPHN3 in 145 bladder tumors and paired normal bladder tissues (by immunohistochemistry) as well as in human normal urothelial SVHUC sublines stably expressing wild-type AR or vector only (by RT-PCR and western blot). We then assessed the effects of ligand (e.g. LTX, FLRT3) treatment on neoplastic/malignant transformation of SVHUC-derived cells, as well as bladder tumor development in C57BL/6 mice, induced by chemical carcinogens (e.g. MCA, BBN). RESULTS: Immunohistochemistry showed that LPHN3 was positive in 52% (47% 1+, 5% 2+) of tumors, which was significantly (P=0.049) higher than in non-neoplastic urothelial tissues [37% (all 1+)], and that LPHN3 positivity in non-muscle-invasive tumors was associated with the risk of tumor recurrence after transurethral surgery (P=0.051). The level of LPHN3 expression was higher in SVHUC-AR cells than in AR-negative SVHUC-vector cells. In addition, significant induction in the LPHN3 expression by LTX or FLRT3 treatment was seen in SVHUC, whereas LPHN3 knockdown via shRNA virus infection in SVHUC-AR resulted in considerable induction or reduction of the expression of the proteins positively (e.g. cleaved caspase-3) or negatively (e.g. Bcl-2, Bcl-xL), respectively, associated with apoptosis. Chromatin immunoprecipitation assay further revealed the binding of AR to the promoter region of LPHN3 in SVHUC-derived cells. The in vitro transformation assay showed that LTX and FLRT3 induced the MCA-mediated oncogenic activity in both SVHUC-AR and SVHUC-vector cells, while the inhibitory effect of LPHN3 knockdown on the neoplastic transformation was observed in MCA-SVHUC-AR cells. LTX/FLRT3 also significantly accelerated the development of bladder tumors in BBN-treated female mice. CONCLUSIONS: These findings suggest that LPHN3, which is activated in bladder tumors as a key downstream effector of the AR, promotes urothelial tumorigenesis. Accordingly, LPHN3 inhibition, along with AR inactivation, has the potential of being an effective chemopreventive approach for urothelial cancer. Source of Funding: Ferring Research Institute © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e657-e657 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takuro Goto More articles by this author Yuki Teramoto More articles by this author Yujiro Nagata More articles by this author Guiyang Jiang More articles by this author Taichi Mizushima More articles by this author Satoshi Inoue More articles by this author Hiroki Ide More articles by this author George Netto More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement Loading ...

Efficacy of EGFR Inhibitors and NSAIDs Against Basal Bladder Cancers in a Rat Model: Daily vs. Weekly Dosing, Combining EGFR Inhibitors with Naproxen, and Effects on RNA Expression.

There are few effective treatments specifically aimed at basal bladder cancer. Female F344 rats administered N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) develop large invasive bladder cancers. We determined the efficacy of daily vs weekly dosing of EGFR inhibitors, determined the efficacy of naproxen combined with an EGFR inhibitor, and performed RNA analysis of bladder tumors treated for 5 days with EGFR inhibitors or NO-naproxen to identify pharmacodynamic biomarkers. Erlotinib (6 mg/Kg BW daily or 21 or 42 mg/Kg BW weekly), lapatinib (25 or 75 mg/Kg BW daily or 263 or 525 mg/Kg BW weekly) and/or naproxen (30 mg/Kg BW daily) were administered to OH-BBN-treated rats beginning 2-12 weeks post OH-BBN. Rats were sacrificed 28 weeks after the final OH-BBN treatment to determine the effects of the EGFR inhibitors + naproxen on bladder weights and tumor development. In a separate study, rats were treated with OH-BBN. When palpable tumors developed, rats were treated with erlotinib, lapatinib, gefitinib, or the NSAID NO-naproxen for 5 days. RNA analysis was performed on the tumors. Daily or weekly dosing of erlotinib or lapatinib and daily dosing of naproxen reduced large tumor formation up to 70%, while combining daily lapatinib and naproxen reduced tumors 100%. RNA Analysis: All EGFR inhibitors strongly reduced cell proliferation and chromosome replication pathways, while NO-naproxen altered the G protein receptor, oxygen homeostasis and immune function pathways. While daily and weekly dosing with EGFR inhibitors and naproxen were effective, combining lapatinib and naproxen yielded no tumors. This might encourage its clinical use in an adjuvant setting with superficial basal tumors, and perhaps even in a more advanced setting. Furthermore, RNA analysis identified specific pathways that might be potential pharmacodynamic biomarkers in clinical trials.

Relevance of iNOS expression in tumor growth and maintenance of cancer stem cells in a bladder cancer model.

The expression of inducible nitric oxide (NO) synthase (iNOS) in human bladder cancer (BC) is a poor prognostic factor associated with invasion and tumor recurrence. Here, we evaluated the relevance of iNOS expression in BC progression and in cancer stem cell (CSC) maintenance in a murine BC model. Also, iNOS expression and CSC markers were analyzed in human BC samples. iNOS inhibitors (L-NAME or 1400W) or shRNA were used on murine BC model with different iNOS expressions and invasiveness grades: MB49 (iNOS+, non-muscle invasive (NMI)) and MB49-I (iNOS++, muscle invasive (MI)), in order to analyzed cell proliferation, tumor growth, angiogenesis, number of CSC, and pluripotential marker expression. iNOS, SOX2, Oct4, and Nanog expressions were also analyzed in human BC samples by qPCR and immunohistochemistry. iNOS inhibtion reduced parameters associated with tumor progression and reduced the number of CSC, wich resulted higher in MB49-I than in MB49, in concordance with the higher expression of SOX2, Oct4, and Nanog. The expression of SOX2 was notoriously diminished, when iNOS was inhibited only in the MI cell line. Similar results were observed in human samples, where MI tumors expressed higher levels of iNOS and pluripotential genes, in comparison to NMI tumors with a positive correlation between those and iNOS, suggesting that iNOS expression is associated with CSC. iNOS plays an important role in BC progression and CSC maintenance. Its inhibition could be a potential therapeutic target to eradicate CSC, responsible fortumor recurrences. KEY MESSAGES: • iNOS expression is involved in bladder tumor development, growth, and angiogenesis. • iNOS expression is involved in bladder cancer stem cell generation and maintenance, playing an important role regulating their self-renewal capacity, especially in muscle invasive murine bladder cancer cells. • iNOS expression is higher in human muscle invasive tumors, in association with a high expression of pluripotential genes, especially of SOX2.

Abstract A18: Targeting epigenetic dysregulation in bladder cancer through inhibition of EZH2

Abstract Tumor genomic profiling has demonstrated that chromatin modifiers are frequently mutated in urothelial bladder cancers. The initial description of the mutational landscape of human bladder tumors indicated that 89% of cases had at least one alteration in histone-modifying genes and 64% had alterations in components of the SWI/SNF chromatin remodeling complex, suggesting that epigenetic dysregulation may play an important role in development and progression of bladder tumors. Among chromatin modifiers, ARID1A, a key component of the SWI/SNF remodeling complex, shows mutation rates of 25% in muscle-invasive bladder cancers. The chromatin modifier EZH2 functions in the suppression of gene expression. EZH2 is a histone lysine methyltransferase and the catalytic subunit of the PRC2 complex, which methylates histone H3 at lysine 27 (H3K27) to generate the transcriptionally repressive trimethylated modification state H3K27me3. EZH2 is frequently overexpressed in a wide variety of cancers, where it represses key tumor suppressors and genes involved in apoptosis and differentiation and, thus, often correlates with poor prognosis and survival. Given the role of EZH2 in cancer, we are currently developing the EZH2 inhibitor CPI-1205 in patients with castration-resistant prostate cancer. Recent publications have suggested that mutations in ARID1A create hypersensitive contexts for EZH2 inhibition in ovarian clear cell carcinomas. Given the high frequency of ARID1A mutations in bladder cancer, we were interested in whether a similar hypersensitive context exists for EZH2 inhibition in this indication. Long-term phenotypic growth assays in a panel of 21 bladder cancer cell lines treated with our second-generation EZH2 inhibitor CPI-0209 demonstrated preferential sensitivity in cell lines harboring ARID1A mutations. Sensitive cell lines showed increased subG1 populations compared to nonresponsive lines, indicating increased cell death in response to long-term EZH2 inhibition. Transcriptional profiling after CPI-0209 treatment in a panel of bladder cancer cell lines with and without ARID1A mutations showed widespread activation of EZH2 target gene expression, consistent with EZH2's transcriptionally repressive functions. These effects also translated to efficacy in vivo, where CPI-0209 treatment in bladder cell-line derived xenografts harboring ARID1A mutations led to dose-dependent tumor growth inhibition and tumor regression. Further, cotreatment of CPI-0209 with the chemotherapeutic agent cisplatin demonstrated combinatorial effects on cell viability in vitro and on in vivo tumor growth, suggesting EZH2 inhibition is also beneficial in the context of current chemotherapeutic agents in use for bladder cancers. These data support further investigation of CPI-0209 in clinical trials in bladder cancers with ARID1A mutations. Citation Format: Patricia J. Keller, Rosana Meyer, Emily Greenwald, Xinwei Han, Andrew R. Conery, Jennifer A. Mertz, Patrick Trojer. Targeting epigenetic dysregulation in bladder cancer through inhibition of EZH2 [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A18.

Abstract A04: DNA damage repair gene mutations and associated tumor immune landscape as biomarkers of response to immunotherapy in muscle-invasive urothelial cancer

Abstract Molecular subtyping of urothelial cancer (UC) has significantly advanced the understanding of bladder tumor heterogeneity and development of prognostic and predictive biomarkers. Evolving evidence across cancers strongly suggests that tumor immunoediting has a profound impact on the behavior of cancer cells and their adaptation to the coevolving microenvironment and response to treatment. In alignment with this concept, recent immune checkpoint blockade (ICB) therapies in UC have demonstrated the predictive potential of mutations in the DNA damage repair (DDR). A comprehensive understanding of DDR mutation-associated expression of immune regulatory genes could thus aid in expansion of current immunotherapies and predictive biomarkers for the design of patient-tailored combination treatments. We thus investigated the pretreatment tumor transcriptomic profiles of the five recently described molecular subtypes of muscle-invasive urothelial cancer (MIUC; n=408) from the Genomic Data Commons, to determine subtype-specific immune cell abundance, expression of 67 immune regulatory genes, and association with DDR gene mutation profiles. Analysis using CIBERSORT immune cell abundance determination tool showed significant differences in immune cell profiles and abundance between MIUC subtypes. Expression patterns of a selected panel of 67 genes including both immune-stimulatory and -inhibitory genes showed significant associations with subtypes and DDR mutation status. Findings from our study provide compelling evidence for coexpression of multiple immune checkpoint genes including PD-1, PD-L1, IDO1, TIGIT, TIM-3, TGFB1, LAG3, and others, that potentially contribute to compensatory immune evasion in bladder tumors. Our findings also emphasize the urgent need for biomarker discovery approaches that combine molecular subtype, DDR gene mutation status, tumor immune landscape classification, and immune checkpoint gene expression to increase the number of patients responding to immunotherapies. Citation Format: Thiago Vidotto, Sarah Nersesian, Charles Graham, David Robert Siemens, Madhuri Koti. DNA damage repair gene mutations and associated tumor immune landscape as biomarkers of response to immunotherapy in muscle-invasive urothelial cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A04.

Predictors of Intravesical Recurrence After Radical Nephroureterectomy and Prognosis in Patients with Upper Tract Urothelial Carcinoma.

PurposeWe investigate factors that may contribute individually to bladder recurrence and find out the potential candidate to receive postoperative single dose intravesical chemotherapy.Materials and MethodsA total of 217 patients who were diagnosed with upper tract urothelial carcinoma (UTUC) underwent radical nephroureterectomy (RNU) between 2012 and 2016 in a single hospital. The possible risk factors that may contribute to development of bladder recurrence and overall survival were analysed. In order to find out the relationship between 1st bladder recurrence timing and outcome, we divided the 54 of 56 patients (2 patients with prophylactic intravesical chemotherapy excluded) with bladder recurrence after RNU into 2 groups, using the median time of 1st bladder recurrence and confirmed with the “minimum P-value” approach. The primary endpoint was the development of relapsing high-risk non-muscle invasive bladder cancer (NMIBC). The predictive factors of early recurrence and prognostic factors of survival were also analysed.ResultsAmong 217 patients with UTUC under RNU, intravesical recurrence occurred in 56 (25.8%) patients after a median follow-up of 35.2 (1.18–83.34) months. On multivariable analysis, the preoperative ureter manipulation (p=0.009) was a significant predictor for the development of bladder tumours. As for overall survival, renal rein invasion (p=0.017), neutrophil to lymphocyte ratio (p=0.021), and main tumour size (p=0.015) were significant predictors. For 54 patients who developed bladder recurrence, the optimal cut-off point of early recurrence was determined to be 10 months after surgery (p=0.042). Preoperative ureter manipulation (p=0.005) and tumour located both pelvicalyceally and ureterically (P=0.042) were identified as independent factors associated with early recurrence. An end-stage renal disease history and surgical margin positive patient has more late bladder recurrence.ConclusionBladder recurrence was common in UTUC after RNU. Early bladder recurrence was associated with more relapsing high-risk NMIBC and preoperative ureter manipulation was identified as an independent factor associated with early recurrence.

DNA damage repair gene mutations and their association with tumor immune regulatory gene expression in muscle invasive bladder cancer subtypes

BackgroundMolecular subtyping of urothelial cancer (UC) has significantly advanced the understanding of bladder tumor heterogeneity and development of prognostic and predictive biomarkers. Evolving evidence across cancers strongly suggests that tumor immunoediting has a profound impact on the behaviour of cancer cells and their adaptation to the co-evolving microenvironment and response to treatment. In alignment with these concepts, recent immune checkpoint blockade (ICB) therapies in UC have demonstrated the predictive potential of mutations in the DNA damage repair (DDR) genes. A comprehensive understanding of DDR gene inactivation associated expression of immune regulatory genes could thus aid in expansion of current immunotherapies and predictive biomarkers for the design of patient-tailored combination treatments.MethodsWe investigated pre-treatment tumor transcriptomic profiles of the five recently described molecular subtypes of muscle invasive urothelial cancer (MIUC; n = 408) from The Cancer Genome Atlas, to determine subtype specific immune cell abundance, expression of 67 immune regulatory genes, and association with DDR gene inactivation (via mutation, copy number alteration) profiles.ResultsAnalysis using CIBERSORT immune cell abundance determination tool showed significant differences in immune cell profiles and abundance between MIUC subtypes. Expression patterns of a selected panel of 67 genes including both immune stimulatory and inhibitory genes, showed significant associations with subtypes, and DDR gene mutation status.ConclusionFindings from our study provide compelling evidence for co-expression of multiple immune checkpoint genes including, PD-1, PD-L1, IDO1, TIGIT, TIM-3, TGFB1, LAG3, and others, that potentially contribute to compensatory immune evasion in bladder tumors. Our findings also emphasize the urgent need for biomarker discovery approaches that combine molecular subtype, DDR gene mutation status, tumor immune landscape classification, and immune checkpoint gene expression to increase the number of patients responding to immunotherapies.

Systemic transduction of p16INK4a antitumor peptide inhibits lung metastasis of the MBT-2 bladder tumor cell line in mice.

p16INK4a (p16) is a key molecule in bladder tumor (BT) development. We previously reported that a p16 antitumor peptide inhibited the growth of subcutaneous BT grafts in mice through restoration of p16 function using a Wr-T peptide transporter system. In the present study, the efficacy of mouse p16 peptide administration in a mouse lung metastasis model for BT and also the toxicity of peptides by cardiac peptide injection were evaluated. Mouse lung metastases were developed by tail vein injection of a p16-deficient MBT-2 cell line. Six-week-old C3H/He female mice were divided into three groups: A control group (n=12) receiving no treatment; a group treated once on the 3rd experimental day (n=12); and a group treated three times on the 3rd, 5th and 7th experimental days (n=10) with an injection of a mixture of 80 nmol mouse p16 peptide and 50 nmol Wr-T into the tail vein. At the 14th experimental day, the lung metastases were histologically evaluated. Lung metastases were observed in 100% (12/12), 41.7% (5/12) and 30% (3/10) of the aforementioned three groups, respectively. The number and area of metastatic lung tumors were significantly different between control and treatment groups (control vs. triple treatment group for the number and area, P=0.0029 and P=0.0296, respectively). Immunohistochemistry demonstrated that phosphorylated retinoblastoma (Rb) protein was decreased in lung tumors of the treatment groups, compared with the control group. The toxicity of p16 peptide transduction was evaluated by using low-dose treatment (three dosages) and high-dose treatment (two dosages) on three male and three female C3H/He mice in early and late experimental phases. In low and high dose groups, no notable change was determined in body weight or blood analyses in early or late phases following mouse p16 peptide administration. In addition, no notable change was observed histologically in bone marrow of treatment groups. To conclude, systemic p16 peptide administration decreased lung tumor development in a mouse metastatic BT model without severe adverse events, as assessed by blood analyses and histological evaluation.