Bladder Cancer Tissue Microarray Research Articles

PRDX2 regulates stemness contributing to cisplatin resistance and metastasis in bladder cancer.

Cisplatin (CDDP)-based chemotherapy has emerged as the primary treatment for muscle-invasive bladder cancer and metastatic bladder cancer. Nevertheless, a significant proportion of patients experience rapidly developed chemoresistance, leading to treatment ineffectiveness. Existing evidence suggests that chemoresistance is governed by various factors, including tumor stem cells, epithelial mesenchymal transition, and reactive oxygen species (ROS). However, limited research has been conducted on the role of PRDX2, a crucial ROS scavenger, in the modulation of chemoresistance in bladder cancer. Cisplatin-resistant cell lines were established using the concentration gradient overlay method, and differentially expressed genes in resistant cells were screened through RNA sequencing. The expression of PRDX2 in cells and tissues was assessed using RT-qPCR, Western Blot, and immunohistochemistry. The expression of PRDX2 in bladder cancer and adjacent tissues was evaluated using a bladder cancer tissue microarray. Furthermore, the impact of PRDX2 knockdown on tumor formation and metastasis was investigated in vivo by applying subcutaneous tumor xenografts tail vein metastasis assays. We demonstrated that PRDX2 is significantly upregulated in bladder tumors and cisplatin-resistant bladder tumor cell lines. Overexpression of PRDX2 can promote tumor proliferation, migration, and invasion both in vitro and in vivo. We have found that knockdown of PRDX2 expression can effectively reverse cell resistance to cisplatin. Mechanistically, our findings suggest that PRDX2 is involved in regulating tumor stemness and epithelial-mesenchymal transition (EMT). Knockdown of PRDX2 affects the PI3K-AKT and mTOR signaling pathways, thereby influencing tumor stemness and EMT, ultimately impacting the chemotherapy resistance of the tumor. This study provides a new insight into the regulation of chemotherapy resistance in bladder cancer by PRDX2. Targeting PRDX2 can serve as a potent therapeutic target for chemotherapy resistance.

Comprehensive analysis of PRPF19 immune infiltrates, DNA methylation, senescence-associated secretory phenotype and ceRNA network in bladder cancer.

Pre-mRNA processing factor 19 (PRPF19) is an E3 ligase that plays a crucial role in repairing tumor-damaged cells and promoting cell survival. However, the predictive value and biological function of PRPF19 in bladder urothelial carcinoma (BLCA) require further investigation. In this study, we utilized transcriptomic data and bladder cancer tissue microarrays to identify the high expression of PRPF19 in BLCA, suggesting its potential as a prognostic biomarker. To gain a better understanding of the role of PRPF19 in the immune microenvironment of BLCA, we performed single cell analysis and employed the LASSO method. Additionally, we examined the methylation profiles of PRPF19 using the SMART website. Our investigation confirmed the correlation between PRPF19 and BLCA cell senescence and stemness. Furthermore, we constructed a PRPF19-miR-125a-5p-LINC02693-MIR4435-2HG ceRNA network using the ENCORI and miRWALK databases. Our comprehensive analysis reveals that PRPF19 can serve as a prognostic marker for BLCA and is significantly associated with various immune-infiltrating cells in BLCA. Moreover, our findings suggest that PRPF19 influences cellular senescence through the regulation of stemness. Finally, we developed a ceRNA network that has the potential to predict the prognosis of BLCA patients. We confirmed the prognostic value and multiple biological functions of PRPF19 in BLCA. Furthermore, the specific ceRNA network can be used as a potential therapeutic target for BLCA.

Duality of the SVIL expression in bladder cancer and its correlation with immune infiltration

SVIL is a member of the villin/gelsolin superfamily and is responsible for encoding supervillin. It has been reported to be closely related to the occurrence and development of various tumors. However, the mechanism of SVIL in bladder cancer has not been reported yet. In this research, we evaluated the relationship between SVIL expression and bladder cancer in public dataset and examined the expression of SVIL in bladder cancer cell lines, tissue microarrays and patients in our cohort. Our work determined that the expression of SVIL in bladder cancer tissue was significantly lower than that in normal tissue. However, in bladder cancer tissues, the high expression of SVIL is significantly associated with poor prognosis. This kind of duality is very novel and has great research value. The expression level of SVIL can well predict the survival time of bladder cancer patients, and is an independent risk factor of bladder cancer patients. The expression of SVIL is also closely related to the immune tumor microenvironment of bladder cancer. Our research provides a basis for personalized therapeutic targets for bladder cancer.

STAG2 expression is associated with adverse survival outcomes and regulates cell phenotype in muscle-invasive bladder cancer.

STAG2 (Stromal Antigen 2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (n=119 bladder cancer clinical samples), we found several STAG2 mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (n=346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for MIBC patients. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth, whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy number variations, or cell cycle distribution. However, unbiased RNA sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion in vitro. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a non-canonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in STAG2-mutant MIBC.

TEAD4 functions as a prognostic biomarker and triggers EMT via PI3K/AKT pathway in bladder cancer

BackgroundThe distant metastasis is the primary cause of cancer morbidity and mortality for bladder cancer (BLCA) paitents. All the recommended therapy for it largely depends on how far the cancer has invaded. It has been confirmed that epithelial to mesenchymal transition (EMT) is the leading reason for the BLCA metastasis which makes BLCA difficult to cure. The aim of the present study is to identify the BLCA-related genes that can be used as the new prognostic biomarker and treatment target, and to investigate the functional mechanisms of TEAD4 in EMT dysregulation.MethodsThe "limma" R package was used to identify the differentially expressed genes (DEGs) between the normal and the tumor samples from TCGA BLCA and GTEx databases. Kaplan–Meier and UniCox analysis were used to filter DEGs with prognostic value in BLCA. Step muti-Cox analysis was used to construct a prognostic risk score model based on clinical phenotype characters. Gene set enrichment analysis (GSEA) was performed to explore the possible molecular mechanisms affecting the prognosis in BLCA. Unsupervised hierarchical clustering analysis was performed to evaluate the effects of EMT process on the prognosis. Single-sample GSEA (ssGSEA) was used to calculate the correlation betweeen the expression of DEGs and EMT enrichment scores. TEAD4 expression and its association with pathological grading and survival were appraised in samples from TCGA dataset and BLCA tissue microarray. Colony formation assays and CCK8 assays were performed to study the changes in BLCA cell proliferation when the TEAD4 levels was down- or up-regulated in BLCA cells. Transwell and wound healing assays were utilized to analyze the impact of TEAD4 on the invasion and metastasis of the BLCA cells. Western Blot was carried out to detect the changes of EMT-related markers and the active molecules involved in PI3K/AKT signaling in BLCA cells. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted on the genes related to TEAD4 expression. 740Y-P (activator of PI3K/AKT pathway) and LY294002 (inhibitor of PI3K/AKT pathway) were applied to evaluate the contribution of PI3K/AKT signaling pathway in the EMT of BLCA cells. To examine the in vivo effect of TEAD4 on tumor metastasis, we designed a metastatic nude-mouse model by tail vein injection of TEAD4-knockdown BLCA cells. And PET/CT imaging was used to assess the extent of lung metastases.ResultsA total of 1592 DEGs were recognized, among which 4 DEGs have been identified as independent prognostic factors for BLCA, such as FASN, IGFL2, PLOD1 and TEAD4. TCGA BLCA samples were divided into significantly different low- and high-risk groups according to the median risk score; GSEA analysis showed that HALLMARK EMT pathway was the top enriched gene signature when compared high-risk and low-risk groups, which was also verified by unsupervised cluster analysis. EMT signature-derived ssGSEA scores demonstrated that TEAD4 had the most positive correlation with EMT process. In addition, TEAD4 expression was upregulated in TCGA BLCA samples and correlated with pT stage, tumor stage and tumor grade. Functional studies showed that TEAD4 knockdown via lentiviral TEAD4 shRNA inhibited cell migration and invasion in vitro and in vivo, with the reduced expression of EMT related markers in BLCA cell lines; the migration and invasion of TEAD4 knockdown cells could be restored by ectopic expression of TEAD4. Meanwhile, KEGG enrichment analysis of genes related to TEAD4 expression showed that enrichment was significantly related to PI3K/AKT pathway. The pathway inhibitor LY294002 blocked the TEAD4-induced enhancement of migration and invasion as well as the expression EMT-related markers, whereas the agonist 740Y-P rescued the decreased migration, invasion and EMT induced by TEAD4 knockdown.ConclusionsTEAD4 is closely correlated with poor prognosis in BLCA and mediates its metastasis through regulating EMT via PI3K/AKT pathway, proving that TEAD4 is not only an effective biomarker for predicting the prognosis but also a great potential target for treatment of metastatic BLCA.

TROP2 Expression Across Molecular Subtypes of Urothelial Carcinoma and Enfortumab Vedotin-resistant Cells

Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) targeting TROP2, which has recently been approved for treatment-refractory metastatic urothelial cancer (UC). However, the variability of TROP2 expression across different bladder cancer (BC) subtypes, as well as after enfortumab vedotin (EV) exposure, remains unknown. Using gene expression data from four clinical cohorts with >1400 patient samples of muscle-invasive BC and a BC tissue microarray, we found that TROP2 mRNA and protein are highly expressed across basal, luminal, and stroma-rich subtypes, but depleted in the neuroendocrine subtype. In addition, TROP2 mRNA levels are correlated with NECTIN4 mRNA but are more highly expressed than NECTIN4 mRNA in patient cohorts and BC cell lines. Moreover, CRISPR/Cas9-mediated knockdown of TROP2 demonstrates that its expression is one factor governing SG sensitivity. After prolonged EV exposure, cells can downregulate NECTIN4, leading to EV resistance, but retain TROP2 expression and remain sensitive to SG, suggesting nonoverlapping resistance mechanisms to these ADCs. While our findings warrant further validation, they have significant implications for biomarker development, patient selection, and treatment sequencing in the clinic as well as clinical trial design and stratification for metastatic BC patients. Patient summaryIn this report, we investigated the expression levels of the drug target TROP2 across different molecular subtypes of bladder cancer in multiple patient cohorts and cell lines. We found high levels of TROP2 in most subtypes except in the neuroendocrine subtype. Overall, TROP2 gene expression is higher than NECTIN4 gene expression, and cells resistant to enfortumab vedotin (EV), a NECTIN4-targeting antibody-drug conjugate, remain sensitive to sacituzumab govitecan (SG). Our findings suggest that SG may be effective across most bladder cancer subtypes, including the bladder cancers previously treated with EV.

Expression and prognostic value of SULT1A2 in bladder cancer.

Sulfotransferase Family 1A Member 2 (SULT1A2) is a protein coding gene. Several studies have reported that SULT1A2 may have a chemical carcinogenic effect if expressed as a functional protein. The present study aimed to investigate the expression and potential role of SULT1A2 in bladder cancer (BC). Data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases were used to analyze SULT1A2 expression in BC. In addition, reverse transcription-quantitative PCR and western blot analyses were performed to detect SULT1A2 expression in BC cells and tissues. Immunohistochemistry analysis was performed on 100 formalin-fixed, paraffin-embedded BC tissues and corresponding adjacent normal bladder tissues (ANBTs) to verify SULT1A2 expression and determine the clinical significance of SULT1A2 in BC. Gene set enrichment analysis (GSEA) was performed to determine the potential biological processes and internal molecular mechanisms. The results demonstrated that SULT1A2 was highly expressed in BC tissues compared with ANBTs. Furthermore, high SULT1A2 expression was significantly associated with the staging of BC. Analyses of TCGA datasets and BC tissue microarray indicated that high SULT1A2 expression was significantly associated with a favorable overall survival in patients with BC. In addition, GSEA revealed pathways, diseases and biological processes associated with SULT1A2. Taken together, the results of the present study suggest that SULT1A2 acts as an oncogene in BC, and thus may serve as a biomarker for tumor staging and prognosis in patients with BC.

ROC1 promotes the malignant progression of bladder cancer by regulating p-I\u03baB\u03b1/NF-\u03baB signaling

BackgroundRegulator of cullins 1 (ROC1) is an important catalytic subunit of cullin–RING E3 ligase. Nuclear factor-kappa B (NF-κB) signaling is closely related to tumor invasion and metastasis. Earlier, we reported that ROC1 was associated with a poor prognosis in patients with bladder cancer (BCa). However, it is unclear whether ROC1 is involved in the NF-κB signaling associated with malignant BCa progression.MethodsThe expression of ROC1 and p65 in bladder cancer and paracancerous tissues were detected by immunohistochemistry (IHC). Pearson correlation was used to assess correlation between ROC1 and p65 protein expressions. The wound-healing and transwell assays were used to monitor cell invasion and migration. The effect of ROC1 on the expression of key proteins in the NF-κB signaling was determined by immunofluorescence and western blot (WB). Cycloheximide (CHX), MG132 and immunoprecipitation assays were used to evaluate the effect of ROC1 on the ubiquitination of phosphorylated inhibitor of kappa B alpha (p-IκBα). A lung metastasis mouse model was generated to detect the role of ROC1 in tumor metastasis.ResultsWe found that ROC1 was up-regulated in BCa tissues and cell lines, and high ROC1 levels were positively correlated with higher tumour grade, lymph node metastasis, distant metastasis and poor prognosis. Linear-regression analysis showed significant a Pearson correlation between ROC1 and nuclear p65 expression in BCa tissue microarray (TMA) samples. Functional studies demonstrated that ROC1 promoted BCa cell invasion and migration. In vitro and in vivo experiments showed that ROC1 activated NF-κB signaling by enhancing the ubiquitination of p-IκBα, which caused p65 nuclear translocation and promoted the transcription of some metastasis-related target genes, such as urokinase-type plasminogen activator receptor (uPAR), intracellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), and matrix metalloproteinase 9 (MMP9), resulting in promoting BCa metastasis.ConclusionROC1 plays an important role in the progression of BCa and serves as a potential diagnostic and therapeutic target for patients with BCa.

ITPR3 facilitates tumor growth, metastasis and stemness by inducing the NF-\u0138B/CD44 pathway in urinary bladder carcinoma

BackgroundBladder carcinoma is one of the most common urological cancers. ITPR3, as a ubiquitous endoplasmic reticulum calcium channel protein, was reported to be involved in the development and progression of various types of cancer. However, the potential roles and molecular mechanism of ITPR3 in bladder cancer are still unclear. Herein, we elucidated a novel role of ITPR3 in regulating the proliferation, metastasis, and stemness of bladder cancer cells.MethodsThe expression of ITPR3 in bladder cancer was analyzed using public databases and bladder cancer tissue microarrays. To demonstrate the role of ITPR3 in regulating the NF-ĸB/CD44 pathway and the progression of bladder cancer, a series of molecular biology and biochemistry methods was performed on clinical tissues, along with in vivo and in vitro experiments. The methods used included western blot assay, quantitative RT-PCR assay, immunofluorescence assay, immunohistochemistry (IHC) assays, wound healing assay, Transwell assay, colony formation assay, tumorsphere formation assay, cell flow cytometry analysis, EdU assay, MTT assay, cell transfection, bisulfite sequencing PCR (BSP), a xenograft tumor model and a tail vein cancer metastasis model.ResultsHigher ITPR3 expression was found in bladder cancer tissues and bladder cancer cells compared with the corresponding normal peritumor tissues and SV-HUC-1 cells, which was attributed to demethylation in the ITPR3 promoter region. ITPR3 promoted the proliferation of bladder cancer by accelerating cell cycle transformation and promoted local invasion and distant metastasis by inducing epithelial-to-mesenchymal transition (EMT). Meanwhile, ITPR3 maintained the cancer stemness phenotype by regulating CD44 expression. NF-κB, which is upstream of CD44, also played a critical role in this process.ConclusionsOur study clarifies that ITPR3 serves as an oncogene in bladder cancer cells and represents a novel candidate for bladder cancer diagnosis and treatment.

Prognostic markers in pT3 bladder cancer: A study from the international bladder cancer tissue microarray project

PurposeWe evaluated the prognostic value of 10 putative tumor markers by immunohistochemistry in a large multi-institutional cohort of patients with locally advanced urothelial cancer of the bladder (UCB) with the aim to validate their clinical value and to harmonize protocols for their evaluation. Materials and MethodsPrimary tumor specimens from 576 patients with pathologic (p)T3 UCB were collected from 24 institutions in North America and Europe. Three replicate 0.6-mm core diameter samples were collected for the construction of a tissue microarray (TMA). Immunohistochemistry (IHC) for 10 previously described tumor markers was performed and scored at 3 laboratories independently according to a standardized protocol. Associations between marker positivity and freedom from recurrence (FFR) or overall survival (OS) were analyzed separately for each individual laboratory using Cox regression analysis. ResultsThe overall agreement of the IHC scoring among laboratories was poor. Correlation among the 3 laboratories varied across the 10 markers. There was generally a lack of association between the individual markers and FFR or OS. The number of altered cell cycle regulators (p53, Rb, and p21) was associated with increased risk of cancer recurrence (P < 0.032). There was no clear pattern in the relationship between the percentage of markers altered in an 8-marker panel and FFR or OS. ConclusionsThis large international TMA of locally advanced (pT3) UCB suggests that altered expression of p53, Rb, and p21 is associated with worse outcome. However this study also highlights limitations in the reproducibility of IHC even in the most expert hands.

Abstract 2071: Identification of FOXO1 as a tumor suppressor modulated by androgen receptor/estrogen receptor-βsignals: Implications for bladder cancer promotion and chemoresistance

Abstract Introduction and Objective: The androgen receptor (AR) and estrogen receptor-β (ERβ) pathways have been implicated in urothelial tumor outgrowth, as well as chemoresistance, for which underlying mechanisms are poorly understood. Therefore, the present study aims to identify downstream effectors of AR and ERβ signals and further investigate their role in bladder cancer promotion and chemosensitivity. Methods: We first performed a transcription factor (TF) profiling assay in an AR/ERβ-positive and ERα-negative bladder cancer line, UMUC3. We then performed in vitro and in vivo assays, using cell line and mouse models, as well as immunohistochemical staining in two sets of bladder cancer tissue microarray. Results: The TF array data in UMUC3 cells treated with mock versus synthetic androgen R1881 displayed that 14 out of 96 were down-regulated. Specifically, the expression levels of FOXC1, STAT4, and FOXO1 were reduced by &amp;gt;60%. Subsequent RT-PCR showed that dihydrotestosterone and estradiol treatment significantly down-regulated the expression of FOXO1 (58% and 28% decreases), but not that of STAT4 (22% and 2% decreases) or FOXC1 (10% and 2% decreases), respectively. Of note, our online search identified 6 and 3 putative binding sites of AR and ERβ, respectively, in the FOXO1 promoter region. In chemical carcinogen-induced models, FOXO1 knockdown via shRNA virus infection or inhibitor treatment resulted in considerable induction of the neoplastic transformation of urothelial cells or bladder cancer development in mice. Similarly, FOXO1 inhibition significantly induced the viability/migration/invasion of bladder cancer cells and reduced the cytotoxic effects of cisplatin. Finally, immunohistochemistry in surgical specimens showed down-regulation and up-regulation of the expression of FOXO1 and its phosphorylated inactive form (p-FOXO1), respectively, in bladder cancer tissues, which was further associated with patient outcomes. In a separate tissue microarray consisting of muscle-invasive bladder cancer specimens from patients who received cisplatin-based neoadjuvant chemotherapy, p-FOXO1 expression tended to correlate with chemoresistance (i.e. 41% of responders vs. 69% of non-responders; P=0.068). Conclusions: FOXO1 is found to be a potential tumor suppressor modulated by both AR and ERβ signals. FOXO1 signaling may thus represent a novel chemopreventive and/or therapeutic target for bladder cancer. Its inactivation may also be useful for overcoming chemoresistance. Citation Format: Hiroki Ide, Satoshi Inoue, Taichi Mizushima, Guiyang Jiang, Takuro Goto, Yujiro Nagata, Yuki Teramoto, George J. Netto, Mototsugu Oya, Hiroshi Miyamoto. Identification of FOXO1 as a tumor suppressor modulated by androgen receptor/estrogen receptor-βsignals: Implications for bladder cancer promotion and chemoresistance [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 2071.

Abstract A09: Photoimmunotherapy (PIT) for treatment of bladder cancer

Abstract Introduction: The use of light as a means of therapy for bladder cancer (BC) has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of phototherapy called photoimmunotherapy (PIT). To begin with, anti-EGFR antibody panitumumab (pan) was labeled with the photo-absorber (PA), IRDye 700Dx (IR700), to create pan IR700, an antibody-PA conjugate that is activated by near-infrared radiation (NIR). However, PIT may be less effective when a tumor lacks “overwhelming” expression of a single target such as EGFR. Hence, a combinatorial PIT approach for targeting BC expressing EGFR and HER2, using PA-labeled pan and trastuzumab (tra), respectively. Methods: Human BC tissue microarray (TMAs) and BC cell lines were analyzed for expression of EGFR and HER2. Mechanism of PIT-induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive and singlet oxygen species (ROS/SOS). Finally, efficacy of PA-labeled antibodies singly and in combination was analyzed in vitro and in vivo. Results: Most BC tissues have some expression of EGFR, but squamous cell carcinoma (SCC) of bladder has maximum EGFR expression. About 45% of BC tissues stain for both EGFR and HER2. Pan IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Pan alone, pan IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species (SOS) had a substantially larger contribution towards cell death than reactive oxygen species (ROS). NIR-PIT with pan IR700 demonstrated reduced growth compared to only pan IR700-treated UMUC-5 xenograft tumors. In non-SCC BCs we used a combinatorial PIT approach using pan IR700 and tra IR700 to target EGFR and HER2, respectively. In vitro, PIT in the presence of combination pan IR700 and tra IR700 was more efficacious than either agent alone with lethal dose 50 (LD50) of 28.66 J/cm2 for combination PIT compared to 71.55 J/cm2 for the PanIR700 alone. In vivo, tumor xenografts treated with combination PIT showed significant tumor growth retardation compared to single-agent therapy. Conclusion: PIT is a new targeted treatment for BC. Our data demonstrate that pan IR700-induced PIT selectively kills EGFR-expressing bladder cancer. Combination PIT is a promising approach for treating BC by selectively inducing death in BC cells with low/moderate expression of surface receptors. Citation Format: Reema Railkar, Mohammad Rashid Siddiqui, Thomas Sanford, Hisataka Kobayashi, Peter L. Choyke, Piyush K. Agarwal. Photoimmunotherapy (PIT) for treatment of bladder 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 A09.

Suppressed OGT expression inhibits cell proliferation while inducing cell apoptosis in bladder cancer

BackgroundThis study aimed to explore hyper-O-linked N-acetylglucosaminylation (O-GlcNAcylation) with an elevation of the expression of O-linked-β-N-acetylglucosamine transferase (OGT) in human bladder cancer.MethodsImmunohistochemical staining for OGT and O-GlcNAcylation was performed in 20 paired human bladder cancer and adjacent normal tissues, as well as in human bladder cancer tissue microarrays (N = 169). The expression level of OGT and O-GlcNAcylation in cell lines were detected using the Western blot analysis. The effects of O-GlcNAcylation on the cell proliferation of bladder cancer were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clone formation assays. Cell apoptosis and cell cycle analysis were detected using flow cytometry. The autophagy of bladder cancer cells was investigated using the Western blot analysis, and GFP–LC3 plasmid was used to detect the autophagic flux. MTT assay was performed to detect the sensitivity of bladder cancer cells to cisplatin after OGT knockdown.ResultsThe expression of OGT and the O-GlcNAcylation were upregulated in bladder cancer tissues and cell lines. O-GlcNAcylation and OGT were observed in nucleus and cytoplasm and found to be higher in muscle-invasive bladder cancer (MIBC) than in non-muscle-invasive bladder cancer (NMIBC). Reducing hyper-O-GlcNAcylation by OGT knockdown inhibited the proliferation of bladder cancer cells in vitro and xenograft tumor growth in vivo, triggered apoptosis, as well as led to cell cycle arrest. It also increased autophagy in bladder cancer cells. This study demonstrated increased autophagy pro-survival, but not pro-death. Reducing hyper-O-GlcNAcylation by OGT knockdown facilitated the chemosensitivity of bladder cancer cells to cis-platinum.ConclusionsThe data indicated that hyper-O-GlcNAcylation enhanced oncogenic phenotypes and was involved in DNA damage response in bladder cancer.

Low Frequency of Intratumor Heterogeneity in Bladder Cancer Tissue Microarrays

Background:Intratumoral heterogeneity (ITH) is associated with clinical challenges such as possible differences in response to treatment and difficulties in classifying the tumor. Previously, ITH has been described in bladder cancer using detailed genetic analyses. However, in this disease, it is not known to what extent ITH actually occurs, or if it involves molecular subtyping, when assessment is achieved by immunohistochemistry (IHC) on the protein level using tissue microarrays (TMAs), the method most widely applied when analyzing large sample numbers.Objective:We aimed to investigate ITH by IHC in bladder cancer TMAs.Methods:Staining for eleven immunohistochemical markers (CK5, Cyclin D1, E-Cadherin, EGFR, FGFR, GATA3, HER2, p16, p63, P-Cadherin and RB1) was performed, and differences in staining patterns were assessed both within 1981 individual tissue-cores and by comparing two cores from the same tumor in 948 cases according to our pre-specified criteria. Presence of ITH was associated with clinicopathological data such as stage, grade, molecular subtype and survival.Results:Intracore ITH in one or several markers was associated with grade 3, stage T1 and the genomically unstable molecular subtype. ITH in three or more markers was found in 5% between cores (intercore heterogeneity) and in 2% within cores (intracore heterogeneity). No association with survival was found for any of the ITH groups.Conclusions:We observed ITH in a small proportion of the tumors, suggesting that ITH has only a limited impact on TMA bladder cancer studies.

MP54-08 PROSTAGLANDIN RECEPTORS INDUCE UROTHELIAL TUMOURIGENESIS AS WELL AS BLADDER CANCER PROGRESSION AND CISPLATIN RESISTANCE PRESUMABLY VIA MODULATING PTEN EXPRESSION

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2018MP54-08 PROSTAGLANDIN RECEPTORS INDUCE UROTHELIAL TUMOURIGENESIS AS WELL AS BLADDER CANCER PROGRESSION AND CISPLATIN RESISTANCE PRESUMABLY VIA MODULATING PTEN EXPRESSION Eiji Kashiwagi, Satoshi Inoue, Taichi Mizushima, Jinbo Chen, Hiroki Ide, Takashi Kawahara, Leonardo Reis, Alexander Baras, George Netto, and Hiroshi Miyamoto Eiji KashiwagiEiji Kashiwagi More articles by this author , Satoshi InoueSatoshi Inoue More articles by this author , Taichi MizushimaTaichi Mizushima More articles by this author , Jinbo ChenJinbo Chen More articles by this author , Hiroki IdeHiroki Ide More articles by this author , Takashi KawaharaTakashi Kawahara More articles by this author , Leonardo ReisLeonardo Reis More articles by this author , Alexander BarasAlexander Baras 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/10.1016/j.juro.2018.02.1699AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostaglandin E2 (PGE2) has been shown to play a central role in not only inflammation but also cell proliferation as an antiapoptotic molecule. Accordingly, inhibition of PGE2 is considered a possible anticancer strategy. Meanwhile, PGE2 exerts its effects via G-protein-coupled receptors (GPCRs), including PGE2 receptor 2 (EP2) and EP4. Nonetheless, it remains to be resolved whether and how GPCR signaling is involved in bladder cancer (BC) initiation and outgrowth. The present study aimed to investigate the functional role of prostaglandin receptors, as well as the efficacy of a selective cyclooxygenase-2 inhibitor celecoxib, in urothelial tumorigenesis and cancer progression. METHODS We performed immunohistochemistry in bladder cancer tissue microarrays consisting of 129 urothelial neoplasms and separate 37 muscle-invasive BCs from patients who subsequently received cisplatin-based neoadjuvant chemotherapy, in vitro transformation assay in a normal urothelial SVHUC line, and western blot/RTPCR/cell growth assays in BC lines. RESULTS EP2/EP4 expression was elevated in BCs compared with non-neoplastic urothelial tissues and in BCs from those who were resistant to neoadjuvant chemotherapy. Strong positivity of EP2/EP4 in non-muscle-invasive tumors or positivity of EP2/EP4 in muscle-invasive tumors strongly correlated with disease progression or disease-specific mortality, respectively. In SVHUC cells, exposure to a chemical carcinogen 3-methylcholanthrene (MCA) considerably increased and decreased the expression of EP2/EP4 and PTEN, respectively. Treatment with selective EP2/EP4 antagonist or celecoxib also resulted in prevention in MCA-induced neoplastic transformation of SVHUC cells. In BC lines, EP2/EP4 antagonists and celecoxib effectively inhibited cell viability and migration, as well as augmented PTEN expression. Furthermore, these drugs enhanced the cytotoxic activity of cisplatin in BC cells. EP2/EP4 and PTEN were also elevated and reduced, respectively, in cisplatin-resistant BC sublines. CONCLUSIONS EP2 and EP4 play an important role in inducing urothelial tumorigenesis, bladder tumor progression, and chemoresisance in BC cells, via downregulating PTEN expression. Thus, EP2/EP4 inactivation, using available inhibitors, has the potential of being not only a chemopreventive and therapeutic option for urothelial cancer but also a means of chemosensitization particularly in patients with EP2/EP4-positive tumors. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e714-e715 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Eiji Kashiwagi More articles by this author Satoshi Inoue More articles by this author Taichi Mizushima More articles by this author Jinbo Chen More articles by this author Hiroki Ide More articles by this author Takashi Kawahara More articles by this author Leonardo Reis More articles by this author Alexander Baras More articles by this author George Netto More articles by this author Hiroshi Miyamoto More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Prostaglandin receptors induce urothelial tumourigenesis as well as bladder cancer progression and cisplatin resistance presumably via modulating PTEN expression

Background:We investigated the role of prostaglandin receptors (e.g. prostaglandin E2 receptor 2 (EP2), EP4) and the efficacy of celecoxib in urothelial tumourigenesis and cancer progression.Methods:We performed immunohistochemistry in bladder cancer (BC) tissue microarrays, in vitro transformation assay in a normal urothelial SVHUC line, and western blot/reverse transcription–polymerase chain reaction/cell growth assays in BC lines.Results:EP2/EP4 expression was elevated in BCs compared with non-neoplastic urothelial tissues and in BCs from those who were resistant to cisplatin-based neoadjuvant chemotherapy. Strong positivity of EP2/EP4 in non-muscle-invasive tumours or positivity of EP2/EP4 in muscle-invasive tumours strongly correlated with disease progression or disease-specific mortality, respectively. In SVHUC cells, exposure to a chemical carcinogen 3-methylcholanthrene considerably increased and decreased the expression of EP2/EP4 and phosphatase and tensin homologue (PTEN), respectively. Treatment with selective EP2/EP4 antagonist or celecoxib also resulted in prevention in 3-methylcholanthrene-induced neoplastic transformation of SVHUC cells. In BC lines, EP2/EP4 antagonists and celecoxib effectively inhibited cell viability and migration, as well as augmented PTEN expression. Furthermore, these drugs enhanced the cytotoxic activity of cisplatin in BC cells. EP2/EP4 and PTEN were also elevated and reduced, respectively, in cisplatin-resistant BC sublines.Conclusions:EP2/EP4 activation correlates with induction of urothelial cancer initiation and outgrowth, as well as chemoresistance, presumably via downregulating PTEN expression.

Epidermal Growth Factor Receptor (EGFR)-targeted Photoimmunotherapy (PIT) for the Treatment of EGFR-expressing Bladder Cancer.

The use of light as a means of therapy for bladder cancer has a long history but has been hampered by a lack of tumor specificity and therefore, damage to the normal bladder mucosa. Here, we describe a targeted form of phototherapy called photoimmunotherapy (PIT), which targets EGFR-expressing bladder cancer. Anti-EGFR antibody panitumumab was labeled with the photoabsorber (PA), IRDye 700Dx (IR700), to create a panitumumab-IR700 antibody-PA conjugate that is activated by near-infrared radiation (NIR). Bladder cancer tissue microarray (TMA) and bladder cancer cell lines were analyzed for expression of EGFR. Mechanism of PIT-induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive oxygen species. Finally, the in vivo effect was studied in xenografts. EGFR staining of TMAs showed that while most bladder cancers have expression of EGFR to a varying degree, squamous cell carcinomas (SCC) have the highest expression of EGFR. Panitumumab-IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Panitumumab alone, panitumumab-IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species contributed toward cell death. NIR-PIT with panitumumab-IR700 reduced growth compared with only panitumumab-IR700-treated UMUC-5 xenograft tumors. PIT is a new targeted treatment for bladder cancer. Panitumumab-IR700-induced PIT selectively kills EGFR-expressing bladder cancer cells in vitro and in vivo and therefore warrants further therapeutic studies in orthotopic xenografts of bladder cancer and ultimately in patients. Mol Cancer Ther; 16(10); 2201-14. ©2017 AACR.

Garlic extract in bladder cancer prevention: Evidence from T24 bladder cancer cell xenograft model, tissue microarray, and gene network analysis.

There is a growing interest in the use of naturally occurring agents in cancer prevention. This study investigated the garlic extract affects in bladder cancer(BC) prevention. The effect of garlic extract in cancer prevention was evaluated using the T24 BC BALB/C-nude mouse xenograft model. Microarray analysis of tissues was performed to identify differences in gene expression between garlic extract intake and control diet, and gene network analysis was performed to assess candidate mechanisms of action. Furthermore, we investigated the expression value of selected genes in the data of 165BCpatients. Compared to the control group, significant differences in tumor volume and tumor weight were observed in the groups fed 20mg/kg (p<0.05), 200mg/kg, and 1000mg/kg of garlic extract (p<0.01). Genes (645) were identified as cancer prevention-related genes (fold change >2 and p<0.05) by tissue microarray analysis. A gene network analysis of 279 of these genes (p<0.01) was performed using Cytoscape/ClueGo software: 36genes and 37gene ontologies were mapped to gene networks. Protein kinaseA (PKA) signaling pathway including AKAP12, RDX, and RAB13 genes were identified as potential mechanisms for the activity of garlic extract in cancer prevention. In BC patients, AKAP12 and RDX were decreased but, RAB13 was increased. Oral garlic extract has strong cancer prevention activity invivo and an acceptable safety profile. PKA signaling process, especially increasing AKAP12 and RDX and decreasing RAB13, are candidate pathways that may mediate this prevention effect.

SOX10 is over-expressed in bladder cancer and contributes to the malignant bladder cancer cell behaviors.

To detect the expression level and significance of SOX10 in human bladder cancer. Immunohistochemical analyses were performed to assess SOX10 protein level using a bladder cancer tissue microarray (including 59 spots of cancer tissues and 46 spots of paired normal tissues) and 31 specimens and to define the relationship between SOX10 and clinicopathological bladder cancer characteristics in patients. SOX10 protein and mRNA levels in bladder cancer cell lines (T24, 5637, BIU87, EJ) and transitional cell papilloma cell line (RT4) were tested by western blotting and quantitative real-time PCR (q-PCR), respectively. Cell Counting Kit-8 (CCK-8) and colony formation assays were performed to investigate bladder cancer cell proliferation after SOX10 knockdown. The effect of SOX10 on cell migration and invasion was analyzed by Transwell and Matrigel assays. Kaplan-Meier survival curves and Cox regression analyses were used to evaluate SOX10 prognostic significance for bladder cancer patients. The mechanisms by which SOX10 promote bladder cancer progression were examined by western blotting. SOX10 protein was upregulated in 74.4% of bladder cancer tissues compared with adjacent normal tissues (32.6%). SOX10 protein was also upregulated in malignant cell lines. In addition, high SOX10 expression was related with clinical stage (P=0.008), T stage (P=0.004), histological grade (P=0.002) and lymph node metastasis (P=0.006). Kaplan-Meier survival curves and Cox regression analyses showed that SOX10 functioned as an independent prognostic factor for overall survival. SOX10 knockdown in bladder cancer cells significantly impacted proliferation, migration and invasion, and SOX10 might promote bladder cancer progression by altering β-catenin and Met expression. SOX10 was over-expressed in bladder cancer and promoted malignant bladder cancer cell behaviors. SOX10 has potential as a molecular target for bladder cancer treatment.

Epidermal growth factor receptor (EGFR) targeted photoimmunotherapy (PIT) for the treatment of EGFR expressing bladder cancer.

291 Background: There are limited treatment options for patients with non-muscle invasive bladder cancer (NMIBC) who fail intravesical bacillus Calmette-Guerin (BCG). The use of light as a means of therapy for bladder cancer (BC) has a long history but has been hampered by a lack of tumor specificity and toxicity. Monoclonal antibody (mAb)-conjugates represent an emerging therapeutic approach for malignancies that improves upon tumor specificity. The use of a mAb-photo-absorber (PA) conjugate is a more selective method of delivering light therapy and has been termed “photoimmunotherapy” (PIT). In this report, PIT is used to target the epidermal growth factor receptor (EGFR). EGFR is a good tumor-specific target as it is enriched in the basal molecular subtype of BC. In the present study, anti-EGFR antibody panitumumab (pan) was labeled with the PA, IRDye 700Dx (IR700), to create a pan IR700 mAb-PA conjugate which is activated by near-infrared radiation (NIR). Methods: BC tissue microarray (TMA) and BC cell lines were analyzed for expression of EGFR. Mechanism of PIT induced cell death was studied using proliferation assays, transmission electron microscopy (TEM), and production of reactive oxygen species. Finally, the in vivo effect was studied in xenografts. Results: EGFR staining of TMAs showed that while most BCs have expression of EGFR to some degree, squamous cell carcinomas (SCC) have the highest expression of EGFR. Pan IR700 activated by NIR light rapidly killed UMUC-5 cells, a bladder SCC line. Pan alone, pan IR700 without NIR, or NIR alone had no effect on cells. TEM demonstrated that cell death is due to necrosis. Singlet oxygen species contributed towards cell death. NIR-PIT with pan IR700 reduced growth compared to only pan IR700 treated UMUC-5 xenograft tumors. Conclusions: PIT is a new targeted treatment for BC. Our data demonstrate that pan IR700-induced PIT selectively kills EGFR-expressing bladder cancer cells in vitro and in vivo and hence warrants further studies in orthotopic models and in patients. We suspect that EGFR-directed therapy with PIT is a viable strategy for the basal molecular subtype of BC and will be testing this hypothesis in the future.