Human Transitional Cell Carcinoma Research Articles

Assessment of Tumor-Associated Tissue Eosinophilia (TATE) and Tumor-Associated Macrophages (TAMs) in Canine Transitional Cell Carcinoma of the Urinary Bladder

Transitional cell carcinoma of the urinary bladder is a significant neoplasm in dogs, characterized by a poor prognosis and a high metastatic potential. These canine spontaneous tumors share many characteristics with human transitional cell carcinoma, making them an excellent comparative model. The role of inflammatory infiltration in tumor development and progression is frequently contradictory, especially concerning tumor-associated tissue eosinophils (TATE) and tumor-associated macrophages (TAMs). This study aims to analyze TATE and TAMs in canine transitional cell carcinoma of the urinary bladder. Congo Red staining was used to identify TATE, and immunohistochemistry was performed to detect TAMs in 34 cases of canine transitional cell carcinoma of the bladder carcinomas, categorized into low and high grades. Statistically significant differences were observed between the number of eosinophils and macrophages in the two groups of tumors. The number of TATE was higher in low-grade malignant tumors, but the number of TAMs was higher in high-grade tumors. Our findings suggest the importance of TATEs and TAMs in the aggressiveness of canine transitional cell carcinoma and propose their potential use as therapeutic targets.

Evaluation of the Small Heat Shock Protein Family Members HSPB2 and HSPB3 in Bladder Cancer Prognosis and Progression.

Bladder cancer (BlCa) represents the sixth most commonly diagnosed type of male malignancy. Due to the clinical heterogeneity of BlCa, novel markers would optimize treatment efficacy and improve prognosis. The small heat shock proteins (sHSP) family is one of the major groups of molecular chaperones responsible for the maintenance of proteome functionality and stability. However, the role of sHSPs in BlCa remains largely unknown. The present study aimed to examine the association between HSPB2 and HSPB3 expression and BlCa progression in patients, and to investigate their role in BlCa cells. For this purpose, a series of experiments including reverse transcription-quantitative PCR, Western blotting, MTT assay and flow cytometry were performed. Initial analyses revealed increased vs. human transitional carcinoma cells, expression levels of the HSPB2 and HSPB3 genes and proteins in high grade BlCa cell lines. Therefore, we then evaluated the clinical significance of the HSPB2 and HSPB3 genes expression levels in bladder tumor samples and matched adjusted normal bladder specimens. Total RNA from 100 bladder tumor samples and 49 paired non-cancerous bladder specimens were isolated, and an accurate SYBR-Green based real-time quantitative polymerase chain reaction (qPCR) protocol was developed to quantify HSPB2 and HSPB3 mRNA levels in the two cohorts of specimens. A significant downregulation of the HSPB2 and HSPB3 genes expression was observed in bladder tumors as compared to matched normal urothelium; yet, increased HSPB2 and HSPB3 levels were noted in muscle-invasive (T2-T4) vs. superficial tumors (TaT1), as well as in high-grade vs. low-grade tumors. Survival analyses highlighted the significantly higher risk for post-treatment disease relapse in TaT1 patients poorly expressing HSPB2 and HSPB3 genes; this effect tended to be inverted in advanced disease stages (muscle-invasive tumors) indicating the biphasic impact of HSPB2, HSPB3 genes in BlCa progression. The pro-survival role of HSPB2 and HSPB3 in advanced tumor cells was also evident by our finding that HSPB2, HSPB3 genes expression silencing in high grade BlCa cells enhanced doxorubicin toxicity. These findings indicate that the HSPB2, HSPB3 chaperone genes have a likely pro-survival role in advanced BlCa; thus, they can be targeted as novel molecular markers to optimize treatment efficacy in BlCa and to limit unnecessary interventions.

Reduction in mitochondrial oxidative stress mediates hypoxia-induced resistance to cisplatin in human transitional cell carcinoma cells

Tumor hypoxia is known to promote the acquisition of more aggressive phenotypes in human transitional cell carcinoma (TCC), including drug resistance. Accumulating evidence suggests that mitochondria play a central role in the chemoresistance of TCC. However, the role of mitochondria in the hypoxia-induced drug resistance in TCC remains elusive. The present study investigated the function of mitochondria in the drug resistance using a TCC cell line under hypoxic conditions. In vitro hypoxia (0.1% O2, 48 h) was achieved by incubating TCC cells in air chamber. Mitochondrial events involving hypoxia-induced drug resistance were assessed. Hypoxia significantly reduced the cisplatin-induced apoptosis of TCC cells. Additionally, hypoxia substantially decreased the level of mitochondrial reactive oxygen species (ROS) generated by cisplatin treatment. Analogously, elimination of mitochondrial ROS significantly rescued cells from cisplatin-induced apoptosis. Hypoxia enhanced mitochondrial hyperpolarization, which was not related to ATP production or the reversal of ATP synthase activity. The mitochondrial DNA (mtDNA) amplification efficiency data illustrated that hypoxia significantly prevented oxidative damage to the mitogenome. Moreover, transmission electron microscopy revealed that cisplatin-induced disruption of the mitochondrial ultrastructure was abated under hypoxic conditions. Notably, depletion of mtDNA by ethidium bromide abrogated hypoxia-induced resistance to cisplatin. Taken together, the present study demonstrated that TCC cells exposed to hypoxic conditions rendered mitochondria less sensitive to oxidative stress induced by cisplatin treatment, leading to enhanced drug resistance.

Characterizing the molecular and immune landscape of canine bladder cancer.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

Investigation of the Inhibitory Effect of Platycodin D in Human Transitional Cell Carcinoma Cell Line 5637

Platycodin D is an active component isolated from Chinese herb Platycodonis radix with various pharmacological activities, such as antitussive, expectorant, anti-inflammatory, and analgesic effects. Interestingly, platycodin D also exerts anticancer effects against several types of cancer. However, few studies on the anti-tumour effects of platycodin against urinary bladder cancer have been reported. In this study, we explored the anti-tumour effect of platycodin D against human bladder cancer and its mechanisms in vitro and in vivo. We found that platycodin D had significant anti-proliferative effects on four types of cancer cells, especially the 5637 bladder cancer cell line, and exerted these effects by preventing cell cycle progression from G0/G1 to S phase, down-regulating Ki-67 and cyclin D1 protein expression and up-regulating P21 protein expression. Furthermore, platycodin D inhibited 5637 cell migration by decreasing twist-related protein 1 (Twist1) and matrix metallopeptidase 2 (MMP2) expression and exerted significant tumour-suppressive effects in tumour-bearing nude mice. Platycodin D also increased caspase-9, caspase-8, caspase-3, and p53 expression and decreased Bcl-2 expression in tumour tissues. Taken together, our results provide a theoretical basis for application of platycodin D in treating urinary bladder cancer.

Abstract A02: Photodynamic therapy in combination with immunotherapy enhances the long-term cure of bladder cancer in tumored mice

Abstract Introduction and Objectives: To investigate the utility of combination approach for treating bladder cancer, mice bearing UMUC3 (human transitional cell carcinoma) and T24 (human urinary bladder) tumors were treated with HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a)-PDT alone and in combination with BCG. The HPPH, a multifunctional agent, was selected as a photosensitizer because of its higher tumor avidity, improved fluorescence imaging ability, and limited skin phototoxicity than Photofrin, a current clinical standard. Methods: Imaging: IVIS/Spectrum: A noninvasive in vivo, continuous wave fluorescence imaging apparatus used to assess the localization and distribution of the PS at variable time points. In vivo efficacy studies using UMUC3 and T24 tumors in SCID mice with PS HPPH in combination with BCG. Results: Imaging: Tumor uptake in UMUC3 and T24 tumors using IVIS Imaging. PS HPPH showed high tumor uptake and significant difference in their pharmacokinetic profiles in UMUC3 and T24 tumors implanted in SCID mice: The SCID mice bearing either UMUC3 or T24 tumors (4-5 mm size) were injected with the PS HPPH. The tumored mice (3 mice/group) at a dose of 0.47 u-mol/kg) and imaged at variable time points. The uptake of tumor, liver, and skin was measured by IVIS system and interesting pharmacokinetic profiles of the PS were observed. The optimal tumors uptake in UMUC3 and T24 were at 24 hours and 8-12 hours, respectively. In vivo efficacy studies: PS (HPPH) in combination with BCG shows enhanced PDT efficacy CR (Cures) Day 30 = 14/22 = 63.6% and RG (Re-Growth) 6/22 = 36.4% in bladder tumors (UMUC3). To investigate the utility of combination treatment approach for treating bladder cancer, mice bearing UMUC3 tumors were injected intravenously with HPPH (0.47 u-mol/kg) and the tumors were exposed to laser light (135 J/cm2, 75 mW/cm2) at 24 h post-injection. One hour after the PDT treatment, BCG was injected (1-1.5 × 106 CFU), SQ weekly x 3 doses, and at day 30 the results are very encouraging. Conclusions: The HPPH showed higher in vivo uptake (determined by fluorescence) in UMUC3 and T24 bladder cancer tumors with significant differences in the two tumor types. The treatment of HPPH-PDT in combination with BCG using SCID mice bearing UMUC3 tumors showed enhanced long-term tumor cure. Before initiating this approach to human phase I clinical trial, further studies are under way. Source of funding: Roswell Park Alliance Foundation. Citation Format: Farukh Durrani, Ahmed Aly, Khurshid Guru, Ravindra Pandey. Photodynamic therapy in combination with immunotherapy enhances the long-term cure of bladder cancer in tumored mice [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 A02.

MiADMSA ameliorate arsenic induced urinary bladder carcinogenesis in vivo and in vitro

Background and purposeArsenicosis is a major threat to public health and is a major cause of the development of urinary bladder cancer. Oxidative/ nitrosative stress is one of the key factors for these effects but the involvement of other associated factors is less known. There is a lack of data for the efficacy of chelator against urinary bladder carcinogenesis. The present study demonstrates the early signs of arsenic exposed urinary bladder carcinogenesis and its attenuation by Monoisoamyl dimercaptosuccinic acid (MiADMSA). MethodsMale rats were exposed to 50 ppm of sodium arsenite and dimethylarsinic acid (DMA) via drinking water for 18 weeks and treated with MiADMSA (50 mg/kg, orally once daily for 5 days) for 3 weeks with a gap one week between the two courses of treatments. We compared in vivo data with in vitro by co-exposing 100 nM of sodium arsenite and DMA to rat (NBT-II) as well as human transitional epithelial carcinoma (T-24) cells with 100 nM of MiADMSA. ResultsThe data showed that sodium arsenite and DMA exposure significantly increased the tissue arsenic contents, ROS, TBARS levels, catalase, SOD activities and significantly decreased GSH level which might be responsible for an increased 8−OHdG level. These changes might have increased pro-oncogenic biomarkers like MMP-9 and survivin in serum, bladder tissues, NBT-II, and T-24 cells. High cell migration and clonogenic potential in NBT-II and T-24 cells exposed to arsenic suggest pronounced carcinogenic potential. Significant recovery in these biomarkers was noted on treatment with MiADMSA. ConclusionEarly signs of urinary bladder carcinogenesis were observed in arsenic and DMA exposed rats which were linked to metal accumulation, oxidative/ nitrosative stress, 8-OHdG, MMP-9 and survivin which were reduced by MiADMSA possibly via its efficient chelation abilities in vivo and in vitro.

Characterizing the Molecular and Immune Landscape of Canine Bladder Cancer

Transitional cell carcinoma (TCC) is the most common bladder cancer in both humans and dogs. The majority of canine TCCs are invasive tumors of intermediate‐ to high‐grade at diagnosis and result in a median survival of less than a year. Approximately 20% of human TCCs are muscle‐invasive tumors, which frequently metastasize and have a poor prognosis. Human and canine TCCs share similarities in risk factors, histopathology, and sites of metastasis. Thus, naturally‐occurring canine TCC has the potential to serve as a model for the assessment of novel therapeutics which may inform treatment modalities in human TCC.In this study, we performed whole exome sequencing of 11 canine TCC tumors and three matched normal tissue samples, identifying 581 variants in known protein coding genes. Protein coding variants showed enrichment of KEGG pathways involved in PI3K‐Akt signaling, axon guidance, focal adhesion, and chemokine signaling. Variants were screened against the COSMIC Cancer Gene Census, identifying 37 variants in 27 cancer‐related genes. BRAF V to E missense mutations were identified in 4/11 samples and later confirmed via Sanger sequencing in 73% of samples (8/11). Other cancer genes mutated in at least 2 samples include LRP1B, CUL3, MSH2, and RNF213. Microarray analysis was utilized to characterize the transcriptome of canine TCC relative to normal bladder. 802 and 1,327 genes were up‐ and down‐regulated in tumor samples, respectively. Up‐regulated genes were enriched for cell cycle, p53 signaling, and metabolic KEGG pathways. Down‐regulated genes include members of focal adhesion, ECM‐receptor interaction, and PI3K‐Akt signaling pathways. Gene set enrichment analysis was performed, identifying four hallmark gene sets enriched in tumors: E2F targets, G2M checkpoint, mitotic spindle, and interferon alpha response.A major advantage of the dog model is the ability to evaluate novel immunotherapies in spontaneous tumors that develop under normal immunosurveilance. However, a basic understanding of the immune landscape of canine cancers, such as TCC, is required. Using immunohistochemical labeling, we quantified CD3+ T cells and MAC387+ myeloid cells in TCC tumors. CD3+ T cell counts were variable, exhibiting a bimodal distribution, ranging from 1 to 415 cells/mm2, suggesting immunologically “hot” and “cold” tumors. MAC387+ cell counts ranged from 3 to 613 cells/mm2 and did not correlate with CD3+ T cell counts. Clustering of tumors using expression of immune‐related genes (GO:0002376) revealed two distinct groups. Group I exhibited a strong type I interferon gene expression signature, showing enrichment of hallmark gene sets for interferon alpha and gamma responses. Group II exhibited enrichment of hallmark gene sets for G2M checkpoint and E2F targets. The type I interferon signature was applied to human muscle‐invasive bladder tumors in the TCGA database and was associated with a better disease‐free survival. Overall, the results of this study establish the utility of canine bladder cancer as a model for identifying novel therapeutics, specifically immunotherapies, for the treatment of human muscle‐invasive bladder cancer.Support or Funding InformationMorris Animal Foundation and Shipley Foundation

Selective Susceptibility of Human Bladder Transitional Cell Carcinoma T24 Cells towards NBD Peptide

Bladder cancer is a recurrent disease that often develops chemoresistance and is on the rise in developing countries. After diagnosis surgery followed by immunotherapy and/or chemo- radiation therapy are the available treatments. These treatments do not provide protection against disease progression or recurrence thus highlighting the need to identify and develop drugs that are capable of inhibiting bladder cancer. T24 cell line (human bladder transitional cell carcinoma) was treated with NEMO Binding Domain (NBD) peptide and tested for its cytotoxicity, cell cycle inhibition, apoptosis and migration, in vitro. The cytotoxicity of NBD peptide was tested on non-cancerous cells (NIH-3T3-L1, CHO, Vero) and the peptide localization in T24 cells was confirmed using confocal microscopy. The effects of NBD peptide and the standard drugs (cisplatin, gemcitabine) were compared with untreated control. Peptide based chemotherapy for human bladder cells was tested that include combinations of NBD and standard drugs. Statistical significance was assessed between the different treatment groups. The viability of T24 cells after NBD peptide treatment was reduced to 50% at a dose of 18.5 µM after 48 h. The NBD peptide elicited selective cytotoxicity dose-dependently in T24 cells while being non-toxic on normal (non-cancer) cell lines. Sub G0-G1 accumulation of T24 cells was seen where they underwent necrosis and lost the ability to migrate when treated with 100 µM NBD. T24 cell death increased to ~78% when treated with a combination of NBD with cisplatin and gemcitabine (standard drugs) as opposed to ~50% when treated with NBD peptide alone. NBD peptide exhibits specific anti-cancer activity on T24 cells in vitro and found to be non-cytotoxic to select normal cell lines. Combining NBD with standard drugs demonstrated excellent inhibition of malignant growth than the individual anti-neoplastic agents.

The Effects of Orientin on Proliferation and Apoptosis of T24 Human Bladder Carcinoma Cells Occurs Through the Inhibition of Nuclear Factor-kappaB and the Hedgehog Signaling Pathway.

BackgroundOrientin is a flavone isolated from medicinal plants used in traditional Chinese medicine (TCM), which suppresses the growth of cancer cells in vitro. The effects of orientin in bladder cancer cells remains unknown. This study aimed to investigate the effect of orientin on proliferation and apoptosis of T24 human transitional cell bladder carcinoma cells in vitro in the presence of an agonist and an inhibitor of nuclear factor-kappaB (NF-κB).Material/MethodsT24 cells were cultured and divided into four study groups: an untreated control group; a group treated with 100 μM orientin; a group treated with 100 μM orientin with NF-κB agonist, phorbol 12-myristate 13-acetate (PMA); and a group treated with 100 μM orientin and the NF-κB inhibitor, IκBα. The MTT assay was performed to assess cell viability, and flow cytometry evaluated the cell cycle. The expression of proteins in the Hedgehog signaling pathway and inflammatory cytokines were determined by Western blot and enzyme-linked immunosorbent assay (ELISA).ResultsOrientin inhibited the proliferation of T24 cells, caused cell cycle arrest, reduced cell viability, and inhibited the expression of inflammatory mediators. Treatment of T24 cells with orientin inhibited the expression of NF-κB and components of the Hedgehog signaling pathway, and the NF-κB agonist, PMA, reversed these effects.ConclusionsTreatment of T24 human bladder carcinoma cells in vitro with orientin inhibited cell proliferation and promoted cell apoptosis by suppressing the Hedgehog signaling pathway and NF-κB.

Lipoxygenase-5 Expression in Canine Urinary Bladder: Normal Urothelium, Cystitis and Transitional Cell Carcinoma

Transitional cell carcinoma (TCC) is the most common canine urinary tract tumour and mimics human invasive TCC. Human TCCs overexpress lipoxygenase (LOX)-5 and the use of target inhibitors has proven effective in inhibiting neoplastic growth. In this study, we investigated the immunohistochemical expression of LOX-5 in normal canine urinary bladder, cystitis and TCC. The comparative expression of LOX-5, cyclo-oxygenase (COX)-1 and COX-2 among the three tissue groups was also examined. Biopsy samples from cases of cystitis and TCC were reviewed from 2012 to 2016; samples of histologically normal bladder were used as controls. Dogs were excluded if they had received glucocorticoids, non-steroidal anti-inflammatory drugs (NSAIDs) and/or chemotherapy prior to tissue collection. LOX-5 was expressed in 95% of TCCs, 23% of cases of cystitis and 10% of controls. LOX-5 and COX-2 immunohistochemistry scores were significantly (P<0.01) higher in TCCs versus cystitis and normal bladders. Results of this study support the rationale for further investigation of the use of NSAIDs with dual anti COX-2 and LOX-5 effect for the treatment of canine TCC.

Honokiol induces apoptotic cell death by oxidative burst and mitochondrial hyperpolarization of bladder cancer cells.

Bladder cancer is one of the most common types of malignant tumor worldwide. Current treatments, including chemo-/radiotherapy, only have limited efficacy on bladder cancer progression. Honokiol is an active component of Magnolia officinalis with multiple biological effects that may provide promising health benefits. In the present study, the anti-cancer properties of honokiol against bladder cancer cells were investigated by flow cytometric analysis. The results revealed that honokiol exhibited significant anti-proliferative effects on bladder cancer cell lines, particularly on BFTC-905 human transitional cell carcinoma cells. Furthermore, honokiol at low doses (≤25 µM) induced cell cycle arrest in G0/G1 phase, while it induced significant apoptotic cell death at high doses (≥50 µM; P<0.05). Furthermore, a significant accumulation of reactive oxygen species was identified in honokiol-treated cells. In addition, honokiol induced hyperpolarization of the mitochondrial membrane, which may lead to mitochondrial dysfunction. Finally, caspase-3/7 activation was identified in high-dose honokiol-treated bladder cancer cells. These results suggest that honokiol induces apoptosis via the mitochondrial pathway and honokiol-containing traditional herbal remedies may have a potential clinical application in the treatment of bladder cancer.

The Prognostic Value of some Epithelial-Mesenchymal Transition Markers and Metastasis-Related Markers in Human Transitional Cell Carcinoma of the Bladder

Recent reports provided evidence that epithelial to mesenchymal transition (EMT) and some matrix metalloproteinases (MMPs) contribute to the invasion and metastasis of cancer cells. This study investigated the expression pattern of some EMT markers (E-cadherin and Vimentin) and some MMPs (MMP-2 and MMP-9) in transitional cell carcinoma (TCC). Fifty five paraffin embedded biopsies were included in this study. Expression pattern of E-cadherin and Vimentin was evaluated by immunohistochemistry while cytoplasmic mRNA expression of both MMP-2 and MMP-9 were determined by in situ hybridization. The expression of all markers were significantly increased with the increase of patient's age (? 50 years), and furthermore an increase in men expression when compared to women. Interestingly, all healthy tissues showed positive E-cadherin expression while they did not show any expression of Vimentin, MMP-2 and MMP-9. E-cadherin expression decreased, whereas expression of Vimentin increased according to the grade and stage of the tumor. Similarly, both MMP-2 and MMP-9 expression were increased with the progression of TCC. The current study conclude that a decrease in E-cadherin together with increased Vimentin, MMP-2 and MMP-9 are significant markers that correlate with poor prognosis of TCC.

Abstract 885: Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells

Abstract Doxorubicin (Dox) is a chemotherapy drug widely used for treatment of several cancers including bladder cancer. However, cardiovascular side effects caused by doxorubicin-induced cardiotoxicity has necessitated the synthesis and validation of next generation chemotherapy drugs that are as effective but not cardiotoxic. AD 312 (N-Nitrosureidodaunorubicin; Daunomustine®, Paradox Pharmaceuticals, Inc.) and AD 198 (N- benzyladriamycin-14-valerate; Benzarubicin®, Paradox Pharmaceuticals, Inc.) are novel anthracyclines with confirmed non-cardiotoxicity in mice. Compared to Dox, AD 312 and AD 198 induce apoptosis in cells through distinct mechanisms. The tumor suppressor gene p53 is a key regulator of cell-cycle arrest and apoptosis. Dysregulation of p53 expression and function was identified in over 70% of the bladder cancers and reported as a potential target for therapy. As a result, small molecule compounds that can restore mutated p53 function to wild-type, including PRIMA-1, are under intensive investigation. In this study, we have compared the efficacy of AD 312 and AD 198 to Dox to induce apoptosis in human bladder transitional cell carcinoma (TCC) cells by MTT assay and assessed the possible mechanisms of anthracyclines-induced apoptosis in TCC cells expressing wild-type (wt) p53 (RT4, SW780) and mutated (mt) p53 (UM-UC-3, 5637, J82, and TCCSUP) protein. We have also investigated the effects of co-treatment of anthracyclines with PRIMA-1 in J82 cells, as a representative mt-p53 TCC cell line, and compared the co-treatment effects in RT4, as a representative wt-p53 cell line. Our results demonstrated that anthracyclines inhibited cell viability of all tested TCC cells in vitro, with AD 198 demonstrating highest efficacy. Furthermore, anthracyclines upregulated p53 protein expression in wt-p53 RT4 and SW780 cells, but not in tested mt-p53 UM-UC-3, 5637, J82, and TCCSUP cells. A strong correlation was observed between p53 mutation status and activation of caspase 3/7 and cleavage of PARP. The upregulated caspase 3/7 activity and PARP cleavage was observed in wt-p53, but not in mt-p53 TCC cells. AD198 blocked c-myc expression in mt-p53 TCC cells and no expression of c-myc was detected in wt-p53 TCC cells. Furthermore, sensitivity to AD 198 treatment in mt-p53 J82 cells was significantly improved by a co-treatment with PRIMA-1. However, PRIMA-1 had no effect on cell viability in RT4, the wt-p53 TCC cells. Overall, our results demonstrated that AD 312 and AD 198 are potent inhibitors of bladder cancer cells in vitro and are promising candidates for new therapeutic strategies to replace Dox for bladder cancer treatment. PRIMA-1 even further sensitized mt-p53 cells to AD 198 treatment and thereby this co-treatment improved the inhibition of bladder cancer in vitro. Citation Format: Sony Pandey, Leonard Lothstein, Maria Cekanova. Anthracycline-induced apoptosis through caspase3/7 activity is p53-dependent in bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 885.

The Prognostic Value of some Epithelial-Mesenchymal Transition Markers and Metastasis-Related Markers in Human Transitional Cell Carcinoma of the Bladder

Recent reports provided evidence that epithelial to mesenchymal transition (EMT) and some matrix metalloproteinases (MMPs) contribute to the invasion and metastasis of cancer cells. This study investigated the expression pattern of some EMT markers (E-cadherin and Vimentin) and some MMPs (MMP-2 and MMP-9) in transitional cell carcinoma (TCC). Fifty five paraffin embedded biopsies were included in this study. Expression pattern of E-cadherin and Vimentin was evaluated by immunohistochemistry while cytoplasmic mRNA expression of both MMP-2 and MMP-9 were determined by in situ hybridization. The expression of all markers were significantly increased with the increase of patient's age (? 50 years), and furthermore an increase in men expression when compared to women. Interestingly, all healthy tissues showed positive E-cadherin expression while they did not show any expression of Vimentin, MMP-2 and MMP-9. E-cadherin expression decreased, whereas expression of Vimentin increased according to the grade and stage of the tumor. Similarly, both MMP-2 and MMP-9 expression were increased with the progression of TCC. The current study conclude that a decrease in E-cadherin together with increased Vimentin, MMP-2 and MMP-9 are significant markers that correlate with poor prognosis of TCC.

Diagnostic performance of the urinary canine calgranulins in dogs with lower urinary or urogenital tract carcinoma

BackgroundOnset of canine transitional cell carcinoma (TCC) and prostatic carcinoma (PCA) is usually insidious with dogs presenting at an advanced stage of the disease. A biomarker that can facilitate early detection of TCC/PCA and improve patient survival would be useful. S100A8/A9 (calgranulin A/B or calprotectin) and S100A12 (calgranulin C) are expressed by cells of the innate immune system and are associated with several inflammatory disorders. S100A8/A9 is also expressed by epithelial cells after malignant transformation and is involved in the regulation of cell proliferation and metastasis. S100A8/A9 is up-regulated in human PCA and TCC, whereas the results for S100A12 have been ambiguous. Also, the urine S100A8/A9-to-S100A12 ratio (uCalR) may have potential as a marker for canine TCC/PCA. Aim of the study was to evaluate the diagnostic accuracy of the urinary S100/calgranulins to detect TCC/PCA in dogs by using data and urine samples from 164 dogs with TCC/PCA, non-neoplastic urinary tract disease, other neoplasms, or urinary tract infections, and 75 healthy controls (nested case-control study). Urine S100A8/A9 and S100A12 (measured by species-specific radioimmunoassays and normalized against urine specific gravity [S100A8/A9USG; S100A12USG], urine creatinine concentration, and urine protein concentration and the uCalR were compared among the groups of dogs.ResultsS100A8/A9USG had the highest sensitivity (96%) and specificity (66%) to detect TCC/PCA, with specificity reaching 75% after excluding dogs with a urinary tract infection. The uCalR best distinguished dogs with TCC/PCA from dogs with a urinary tract infection (sensitivity: 91%, specificity: 60%). Using a S100A8/A9USG ≥ 109.9 to screen dogs ≥6 years of age for TCC/PCA yielded a negative predictive value of 100%.ConclusionsS100A8/A9USG and uCalR may have utility for diagnosing TCC/PCA in dogs, and S100A8/A9USG may be a good screening test for canine TCC/PCA.

Tetrahydroporphyrin-tetratosylat (THPTS): A near-infrared photosensitizer for targeted and efficient photodynamic therapy (PDT) of human bladder carcinoma. An in vitro study

BackgroundEfficacy of PDT in muscle-invasive bladder cancer is hampered by low tissue penetration of most photosensitizers by short excitation wavelength. THPTS is excitable at near-infrared (760nm) allowing tissue penetration up to 15mm. We examined the cellular effects of THPTS-PDT in human bladder cancer cells. Material and methodsWe used four human transitional carcinoma cell lines, epithelial bladder progenitors (HBLAK) and bladder smooth muscle cells (HBSMC). We used flow cytometry to examine pharmacokinetics of THPTS, confocal laser scanning microscopy to analyze subcellular localization and production of reactive oxidative species (ROS), examined cytotoxicity and cell death pathways (qRT-PCR). ResultsTotal uptake varied between cell lines and was significantly high in HBLAK and HBSMC. Lysosomal localization was mainly seen in cancer cells and HBLAK, while THPTS was distributed throughout the cytoplasm in HBSMC. Significant ROS production was detected 30min after THPTS-PDT. Growth arrest occurred within 4h and resulted in apoptotic and necrotic cytotoxicity after 24h. Cytotoxicity was dose-dependent and specifically high in cancer cells and HBLAK and significantly low in HBSMC. ConclusionTHPTS-PDT induces cellular mechanisms leading to cellular growth arrest, apoptosis and necrosis in human bladder cancer cells. These effects are only partly dependent on the total amount of THPTS uptake and rather dependent on its subcellular compartmentalization. HBSMC are hardly affected by THPTS-PDT confirming tumor specificity and safety. THPTS is a promising new photosensitizer with the unique advantage of deep tissue penetration allowing the treatment of solid tumors and warranting further animal studies.

Abstract 4739: The upregulation of cyclooxygenase-2 protein expression by receptor tyrosine kinase inhibitors in bladder cancer in vitro

Abstract Several types of cancer, including transitional cell carcinoma (TCC) overexpress several receptor tyrosine kinases (RTKs), including the platelet-derived growth factor receptor (PDGFR), c-kit receptor, epidermal growth factor receptor (EGFR), as well as the vascular endothelial growth factor receptor (VEGFR). Receptor tyrosine kinase inhibitors (RTKIs) are used as targeted therapies for patients diagnosed with cancer with high expression of RTKs. Cyclooxygenase-2 (COX-2) is highly expressed in bladder cancer as well other types of cancers and is one of the key proteins during tumorigenesis. In this study, we validated the effects of RTKIs, Masitinib® (AB1010) and Axitinib on cell proliferation of human (UMUC-3 and T24) and canine (K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) bladder TCC cells. Using WB analysis, tested human and canine TCC cells, except T24 cells, expressed PDGFR and c-kit receptors. There was no detectable EGFR and VEGFR expressions in any of tested cells by WB analysis. The K9TCC#1Lillie and K9TCC#5Lilly cells had a high expression of COX-2, however K9TCC#2Dakota and human T24 had moderate expression of COX-2, and no COX-2 expression was detected in UMUC3 cells by WB analysis. Both RTKIs, Masitinib® and Axitinib inhibited cell proliferation of the tested human and canine bladder TCC cells in a dose-dependent manner by MTS and apoptosis assays. Interestingly, both tested RTKIs, Masitinib® and Axitinib increased COX-2 protein expressions in tested canine TCC cells. Combined treatment of RTKIs with COX-2 inhibitors decreased cell proliferation of tested TCC cells more effectively as either treatments alone. Our results indicate a possible role of COX-2 signaling pathway in developed RTKIs-resistance in bladder cancer cells in vitro. Co-treatment of RTKIs with COX-2 inhibitors might indicate better clinical outcomes in treatments of patients diagnosed with bladder cancer. Citation Format: Jennifer Bourn, Maria Cekanova. The upregulation of cyclooxygenase-2 protein expression by receptor tyrosine kinase inhibitors in bladder cancer in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4739.

Abstract 4409: Assessment of sonic hedgehog pathway inhibition in a novel orthotopic xenograft bladder cancer murine model

Abstract Purpose of the study: To develop a reliable and reproducible bladder cancer murine model for the evaluation of the SHH pathway. Introduction: The sonic hedgehog (SHH) signaling pathway has been shown to play an integral role in the maintenance and progression of bladder cancer. Smoothened inhibitors are currently used in the clinic for treatment of some skin cancers, however they have failed to be effective in bladder cancers in vitro. Blocking alternative downstream molecules of the SHH pathway may be an efficacious strategy for bladder cancer treatment. In vitro assays possess a number of limitations such as environmental differences, loss of heterogenicity and vascularization and artificial levels of growth factors and cytokines within the cell culture media. To overcome this animal models are needed to facilitate the study of carcinogenesis mechanisms. A new bladder cancer model is now required to evaluate experimental therapeutics as previous animal models have utilized the mistakenly identified cervical cancer cell line KU7. Methods: A novel orthotopic murine bladder cancer model was developed to assess the role of the SHH signalling pathway in tumor growth and disease progression. The model comprises the instillation of luciferase-expressing UM-UC3 human transitional cell carcinoma cells into the bladder of athymic nude mice following a poly-L-lysine irrigation of the bladder. Tumors are measured by bioluminescent imaging after intra-peritoneal injection of luciferin. Antisense oligonucleotides (ASO) targeted to the SHH pathway transcription factors, Gli family zinc fingers 1 and 2 (Gli1, Gli2), were assessed in this model. Immunohistochemistry (IHC) of proteins in the SHH pathway were evaluated and compared to our in-house tissue microarray (TMA) of clinical bladder cancer samples. Results: Tumors in this model grew in an average period of time of 40 days with high rates of engraftment and reproducibility. In vitro analysis of SHH pathway inhibition showed a decrease in bladder cancer cell viability and increase in apoptosis following Gli ASO treatment. This effect bypasses smoothened protein regulation which was found to be ineffective in some cell lines. Gli ASO treatment reduced tumor size, and prevented further growth, of UM-UC3 bladder cancers in this mouse model. IHC confirmed Gli2 knock-down and showed increased apoptosis and decreased proliferation in the in vivo tumors. Comparison of modelled tumors to clinical samples showed an increased and diffused Gli2 staining throughout the tissue in both cases. Conclusions: A murine intravesical model consisting of human tumors provides a robust and effective in vivo system for the assessment of cancer inhibiting treatments. Gli2 ASO proved to be a promising treatment for bladder cancer by inhibiting tumor growth and disease progression. Citation Format: Peter A. Raven, Sebastian Frees, Betty Zhou, Claudia Chavez-Munoz, Michael Cox, Alan So. Assessment of sonic hedgehog pathway inhibition in a novel orthotopic xenograft bladder cancer murine model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4409.

Abstract 617: Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib

Abstract Transitional cell carcinoma (TCC) is the most common bladder cancer in humans and their canine companions. Canine TCCs are usually papillary infiltrative TCCs of intermediate to high grade. Genetic defects identified in human TCCs aid in the diagnosis and therapy of human bladder cancer. We utilized whole exome sequencing to screen canine TCCs for gene mutations that contribute to pathogenesis. Genomic DNA was isolated from 11 canine TCCs, 3 matched normal tissue samples, and 2 canine TCC cell lines. Whole exome capture was conducted using the Canine Agilent Sure-select in-solution capture system, and captured fragments were sequenced using an Illumina HiSeq2000 platform. The sequences were mapped to the CanFam3.1 canine reference genome and somatic mutations were identified using Freebayes. Somatic mutations were characterized and compared to the Cancer Gene Census (COSMIC). Nonsense, missense, and insertion/deletion mutations were identified in 126 genes shown to be drivers or repressors in human cancer. Missense mutations were further screened using SIFT to identify alterations deleterious to protein function. The genes exhibiting in/dels, nonsense, or missense mutations with SIFT scores &amp;lt; 0.45 in at least 2 sites or 2 samples were: BRAF, RPL5, RANBP2, EWSR1, NONO, PTPRB, LYL1, JAK1, MSH2, PER1, PIM1, and WRN. In fact, an activating BRAF V to E mutation was identified in 5 of the tumors and both cell lines. The RPL5 and RANBP2 mutations were also hotspot mutations identified in 5 and 3 of the samples, respectively. Mutations were confirmed using Sanger sequencing of amplified genomic DNA. Interestingly, deleterious RANBP2 mutations were only observed in non-BRAF mutant samples. Drug sensitivity assays using the BRAF V600E targeting drug, Vemurafenib, were conducted in the BRAF mutant canine cell lines as well as the BRAF mutant human A375 melanoma cell line. As previously described, the IC50 for the sensitive A375 line was approximately 100 nM, while each of the BRAF mutant canine lines had an IC50 value ≥ 10 μM. Reverse transcriptase PCR was used to amplify the coding sequence for BRAF from the Bliley canine TCC cell line. The amplified transcript (2125 bp) was sequenced, confirming the heterozygous expression of the V548 to E mutant form of BRAF in this cell line. The sequence also indicated that the predicted expressed protein (AA 10 - 715 of XP_013975364.1, corresponding to predicted exons 1 through 20 of XM_014119889.1) exhibited 99% homology to human BRAF AA53 - 763 (NP_004324.2). BRAF protein expression in these cell lines was confirmed by Western blot analysis. Thus, insensitivity to Vemurafenib is not due to differences in canine BRAF, reduced expression, or alternative splicing of the expressed transcript as previously observed in some Vemurafenib resistant human melanomas. These data indicate that although constitutively active BRAF is expressed in canine TCC, other factors may contribute to pathogenesis. Citation Format: Belen Hernandez, Kathryn Cronise, James C. Costello, Rodney Page, Susan Lana, Kenneth L. Jones, Dawn L. Duval. Canine transitional cell carcinoma of the bladder expresses activated BRAF, but is not sensitive to vemurafenib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 617.