Agent For Bladder Cancer Research Articles

Abstract 1583: Bcl-6 as a potential therapeutic target for bladder cancer

Abstract Introduction and Objectives: B-cell lymphoma 6 (Bcl-6) is known to be a nuclear protein and a master transcription factor for regulation of T follicular helper cells (TFH cells) proliferation. Previously, we reported on the increased angiogenic potential of human prostate cancer cells that overexpressed the Bcl-2. Comparative genomic profiling of the endothelial cells treated with/without conditioned media from Bcl-2-overexpressing human prostate cancer cells revealed approximately 250 differentially expressed genes. Interestingly, bioinformatics analysis identified Bcl-6 as one of several secreted proteins on the list. In this study, we first confirmed that Bcl-6 exists in liquid biopsy, and secondly evaluate the effect of Bcl-6 neutralization on angiogenesis and tumor growth in in vitro and in vivo models. Methods: We first analyzed Bcl-6 levels in serum and urine by ELISA. Then we tested the effect of Bcl-6 antibody for the following: ability to inhibit in vitro and in vivo angiogenesis, ability to inhibit cellular proliferation, and tumor growth in cell line-derived xenograft model. Results: First, we found that Bcl-6 exists in urine and serum. Interestingly, Bcl-6 level was significantly higher in cancer patients than healthy subjects. Next, we identified the effective clone tube formation assay. Then we further tested the effect of the antibody and exogenous Bcl-6 on cell proliferation, tube formation, and tube disruption in HUVEC. The exogenous Bcl-6 did not enhance cell proliferation of HUVEC. On the other hand, the Bcl-6 antibody significantly inhibited tube formation in HUVEC, but not tube disruption. Subsequently, we performed Matrigel plug assay to evaluate the effect on angiogenesis in vivo. The CD31 positive cells in Matrigel plug were significantly reduced by the Bcl-6 antibody treatment. To evaluate the effect on tumor growth, we first performed the cell proliferation assay to evaluate the effect on tumor growth in vitro. The Bcl-6 antibody significantly inhibited the cell proliferation of T24 and UM-UC-3 cell lines highly expressing Bcl-6. Finally, we tested the effect of the Bcl-6 antibody on tumor growth in an animal model. Intratumoral injection of Bcl-6 antibody (15 μg/tumor, twice weekly) significantly inhibited the tumor growth. Conclusions: This is the first time to identify that Bcl-6 may be a secreted protein and found that neutralization of Bcl-6 by antibody inhibits angiogenesis and tumor growth in both in vitro and in vivo. The results suggest that neutralizing Bcl-6 by antibody could be a promising therapeutic agent in bladder cancer. Citation Format: Hideki Furuya, Kaoru Murakami, Nari Kim, Charles J. Rosser. Bcl-6 as a potential therapeutic target for bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1583.

Tertiary lymphoid structure patterns aid in identification of tumor microenvironment infiltration and selection of therapeutic agents in bladder cancer.

Tertiary lymphoid structures (TLSs) are emerging as a potential predictor of prognosis and response to immunotherapy in some solid tumors. However, the comprehensive role of TLSs in bladder cancer remains unclear. Eighteen bladder cancer (BCa) datasets were downloaded from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), ArratyExpress and IMvigor210. Based on 39 validated TLS signature genes (TSGs), we evaluated the TLS patterns in all patients, and correlated the TLS patterns with prognosis and tumor microenvironment (TME) cell-infiltrating characteristics. The cox regression model and principal component analysis (PCA) algorithms were used to construct the TLS score, which helps to quantify the TLS pattern in individuals. The landscape of 39 validated TSGs in BCa was assessed first. Five distinct TLS patterns and four gene clusters were determined. TLS cluster C2 and gene cluster A were thought to be characterized by mature TLSs and showed better prognosis and higher immune cells infiltration than other clusters. The TLS score was discovered to be tightly correlated with the infiltration level of immune cells, and could predict the maturation status of TLSs to some extent. We found TLS score was an excellent predictor for prognosis in patients with BCa independent of tumor mutation burden (TMB), and low TLS score was related to better prognosis than high TLS score. Besides, low TLS score was correlated with a better response to immune checkpoint blockade (ICB) immunotherapy and commonly used chemotherapy drugs. Our work demonstrated the characteristics of TLSs in BCa. By using the TLS score, we could evaluate the TLS pattern in individuals. Better understanding of TLS pattern and the usage of TLS score could help instruct clinical strategy and precision medicine for BCa.

Abstract 2858: Development of a first-in-class humanized antibody targeting CXCL1 in bladder cancer

Abstract Introduction and Objectives: Chemokine (C-X-C motif) ligand 1 (CXCL1) plays a crucial role in tumor initiation, promotion, and progression in bladder cancer (BCa) development. Previously, we reported on a monoclonal murine antibody (HL2401) towards CXCL1 which was capable of inhibiting growth in both in vitro and in vivo models. Herein, we have developed a first-in-class humanized monoclonal antibody neutralizing CXCL1 (NTC-001). This study is final step in pre-clinical stage. In this study, we aimed to evaluate efficacy and safety of the antibody to move into phase 1 clinical trials in near future. Methods: We humanized mouse anti-human CXCL1 antibody resulting in NTC-001, and then tested for the following: molecular weight and pH, specificity, binding affinity to ligand and to albumin, ability to inhibit in vitro angiogenesis, ability to inhibit cellular proliferation alone or with chemotherapy, and pharmacokinetics and pharmacodynamics. Results: NTC-001 had a molecular weight of 150kD, and was an acidic antibody (pH5.1). NTC-001 specifically bound human CXCL1 in both WB and ELISA. NTC-001 was bound to albumin and had minimal hepatic metabolism. NTC-001 significantly inhibited tube formation in HUVEC, and more efficiently with the formulation. NTC-001 significantly inhibited T24 cell proliferation (p<0.001). Inhibitory effect of NTC-001 on cell proliferation was correlated with the levels of CXCL1 expression in BCa cell lines. Compared to CXCL1 expression-low group (RT112 and UMUC-14), CXCL1 expression-high group (T24, 5637, 253J, 253J-BV) tended to show low IC50. Synergistic combination efficacy was observed in NTC-001 in combination with gemcitabine (combination index<1) though not with cisplatin. PK study demonstrated a half-life of 324 hours and 18 hours in CD-1 and NSG-SGM3, respectively. Conclusions: NTC-001 is a first in class humanized neutralizing monoclonal antibody towards CXCL1, and could be a promising therapeutic agent in BCa. We are going to proceed to complete our investigational new drug enabling studies in both small and large animals. Citation Format: Kaoru Murakami, Yuka Sasaki, Hideki Furuya, Charles Rosser. Development of a first-in-class humanized antibody targeting CXCL1 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2858.

Establishment of Three-Dimensional Bioprinted Bladder Cancer-on-a-Chip with a Microfluidic System Using Bacillus Calmette-Guérin.

Immunotherapy of bladder cancer is known to have favorable effects, although it is difficult to determine which patients will show a good response because of the different tumor microenvironments (TME). Here, we developed a bladder cancer-on-a-chip (BCOC) to mimic the TME using three-dimensional (3D) bioprinting and microfluidic technology. We fabricated a T24 and a 5637-cell line-based BCOC that also incorporated MRC-5, HUVEC, and THP-1 cells. We evaluated the effects of TME and assessed the immunologic reactions in response to different concentrations of Bacillus Calmette–Guérin (BCG) via live/dead assay and THP-1 monocytic migration, and concentrations of growth factors and cytokines. The results show that cell viability was maintained at 15% filling density in circle-shaped cell constructs at 20 μL/min microfluidic flow rate. A 3D co-culture increased the proliferation of BCOCs. We found that the appropriate time to evaluate the viability of BCOC, concentration of cytokines, and migration of monocytes was 6 h, 24 h, and three days after BGC treatment. Lastly, the immunotherapeutic effects of BCOC increased according to BCG dosage. To predict effects of immunotherapeutic agent in bladder cancer, we constructed a 3D bioprinted BCOC model. The BCOC was validated with BCG, which has been proven to be effective in the immunotherapy of bladder cancer.

Wogonoside exerts potential anti-tumor activity against bladder cancer in vivo and in vitro via regulation of GSK3β/ERK/AKT signaling pathway

Purpose: To explore the antitumor activity of wogonoside on bladder cancer, and its underlying mechanism of action.
 Methods: Methyl thiazolyl tetrazolium (MTT) assay was applied to determine the anti-proliferative activity of wogonoside (2 - 128 μM) on bladder cancer 5637 cell line at various times, and the halfmaximal inhibitory concentration (IC50) was measured. The antitumor activity of wogonoside (30 mg/kg, ip) against bladder cancer 5637 cell line was evaluated in nude mice bearing human bladder cancer 5637 cells. Additionally, western blotting and enzyme-linked immunosorbent assay (ELISA) were carried out to investigate the levels of the caspase-3, caspase-9, B cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X-protein (Bax), phosphorylated (p)-glycogen synthase kinase (GSK)-3β, p-extracellular signal-regulated kinases (p-ERK), and p-(protein kinase B) AKT.
 Results: The in vitro results revealed that wogonoside exerted anti-proliferative activity against bladder cancer 5637 cells with an IC50 of 20.59 μM (p < 0.01), in a concentration- and time-dependent manner. Furthermore, wogonoside treatment also significantly suppressed tumor volume in mice (p < 0.01). The potential mechanisms were mainly associated with apoptosis mediated by mitochondria via upregulation of caspase-3, caspase-9, and Bax levels and down-regulation of Bcl-2, p-GSK-3β, p-ERK, and p-AKT.
 Conclusion: The results reveal that wogonoside has remarkable anti-tumor potentials against bladder cancer. Further translational studies are warranted to test the clinical application of this medicinal agent in bladder cancer.

Current status and future directions of the use of novel immunotherapeutic agents in bladder cancer

To report the available information on the current status and future direction of the use of checkpoint inhibitors as novel immunotherapeutic agents in bladder cancer. In the past 3 years, five immunotherapies targeting programmed cell death 1 (Pembrolizumab and Durvalumab) or programmed cell death-ligand 1 (PD-L1) (Atezolizumab, nivolumab and Avelumab) pathways have been approved in second-line setting for patients who progressed during or after cisplatin-based chemotherapy. According to the most recent update, these patients should be PD-L1-positive to be eligible for immunotherapy. The use of novel checkpoint inhibitors was also very promising in other settings: Metastatic urothelial carcinoma without prior systemic treatment (IMvigor-130), as neoadjuvant treatment before radical cystectomy in patients with muscle invasive disease (PURE-01), and in Bacillus Calmette-Guérin (BCG) refractory nonmuscle invasive bladder cancer (KEYNOTE 057). Ongoing trials on the role of checkpoint inhibitors in bladder cancer may change our approach to different stages of bladder cancer. For metastatic urothelial carcinoma, the role of combined immune and chemotherapy may improve survival. For localized bladder cancer, immunotherapy as neoadjuvant therapy may be associated with less toxicity and better tolerability. Finally, in the setting of a BCG-refractory or BCG-naïve nonmuscle invasive disease checkpoint inhibitors may reduce/delay the risk of progression and subsequent cystectomy.

In vitro evaluation of cytotoxic properties of 5-Aminolevulinic acid (5-ALA) on bladder cancer cells.

5-Aminolevulinic (5-ALA) may be used as a photo diagnostic agent in bladder cancer. The aim of this study was to investigate the cytotoxic effect of 5-ALA on bladder cancer cells. T24 cells treated with various concentrations of mitomycin (MC), 5-ALA and an MC/5-ALA mixture were evaluated to determine the role of 5-ALA on MC cytotoxicity. Cell cycle analysis was conducted, and apoptosis was analyzed by flow cytometry. Caspase 3 enzyme and reactive oxygen species were measured. Our initial studies exploring the impact of combination therapy on cell viability demonstrated improved cytotoxic effects on T24 and RT cells with relatively low doses of 5-ALA/MC in conjunction with MC alone. Indicated no significant difference between the IC50 of MC and MC/5-ALA in T24 cells, while IC50 value was decreased by 25 % in RT4 cells in 5-ALA/MC in comparing with MC alone. However, examination of cell cycle phase arrests by flow cytometry revealed significant PreG1 apoptosis and cell growth arrest in G2/M in T24 cells treated with the MC/5-ALA mixture compared with MC treatment. In addition, caspase 3 enzyme was increased by 1.15-fold in T24 cells treated with MC/5-ALA in comparison with MC alone. These results suggest that 5-ALA might possess anti-cancer properties and is not only a photo diagnostic element.

Molecular Correlates of In Vitro Responses to Dacomitinib and Afatinib in Bladder Cancer.

Background:The HER family of proteins (EGFR, HER2, HER3 and HER4) have long been thought to be therapeutic targets for bladder cancer, but previous clinical trials targeting these proteins have been disappointing. Second generation agents may be more effective.Objective:The aim of this study was to evaluate responses to two second-generation irreversible tyrosine kinase inhibitors, dacomitinib and afatinib, in bladder cancer cell lines.Methods:Cell lines were characterized by targeted next generation DNA sequencing, RNA sequencing, western blotting and flow cytometry. Cell survival responses to dacomitinib or afatinib were determined using (3-[4,5-dimethylthioazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) or [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosylfate (PMS) cell survival assays.Results:Only two cell lines of 12 tested were sensitive to afatinib. Sensitivity to afatinib was significantly associated with mutation in either HER2 or HER3 (p < 0.05). The two cell lines sensitive to afatinib were also responsive to dacomitinib ralong with an additional 4 other cell lines out of 16 tested. No characteristic was associated with dacomitinib sensitivity. Molecular profiling demonstrated that only two genes were high in both afatinib and dacomitinib sensitive cells. Further rhigher expression of RAS pathway genes was noted for dacomitinib responsive cells.Conclusions:This study confirms that cell line screening can be useful in pre-clinical evaluation of targeted small molecule inhibitors and suggests that compounds with similar structure(s) and target(s) may have distinct sensitivity profiles. Further rcombinational targeting of additional molecularly relevant pathways may be important in enhancing responses to HER targeted agents in bladder cancer.

Ethanol extract of loquat fruit skin inhibits the proliferation and metastatic potential of EJ human bladder carcinoma cells

ABSTRACTAlthough studies on biological properties of loquat plant have been performed, research on the molecular mechanism of action in loquat fruit skin is very limited. Therefore, we investigated the molecular mechanisms underlying the inhibitory activity of ethanol extract from loquat fruit skin (EEFS) against the proliferation and metastatic potential of EJ bladder carcinoma cells. Treatment of EJ cells with EEFS significantly inhibited their proliferation by inducing cell-cycle arrest at the G1 phase. Expression of CDK2, CDK4, cyclin D1, and cyclin E was altered by EEFS treatment. Interestingly, p27KIP1, but not p21WAF1 or p53, was identified as a candidate CDK inhibitor (CDKI) associated with the EEFS-mediated inhibition of proliferation. Among mitogen-activated protein kinases, ERK1/2 phosphorylation was significantly elevated but not that of p38 and JNK. In addition, phospho-AKT (protein kinase B; PKB) was not affected by EEFS treatment. Aggressive phenotypes, including migratory and invasive potentials of EJ cells, were similarly diminished by EEFS. Gelatin zymography showed that the enzymatic activity of matrix metalloproteinase (MMP)-9 was markedly inhibited by treatment with EEFS. Results from electrophoretic mobility shift assays demonstrated that MMP-9 inhibition was associated with reduced binding activities of transcription factors NF-κB, activator protein-1, and specificity protein 1 to its promoter region. Taken together, our data clearly indicate that EEFS inhibits the proliferation of EJ cells through p27KIP1, which is associated with G1 arrest and increased extracellular signal-regulated kinase phosphorylation. Metastatic potential represented by MMP-9 activity was also remarkably inhibited by EEFS, suggesting that EEFS is a novel chemotherapeutic agent for bladder cancers.

Abstract SY36-01: Optimizing neoadjuvant therapy prior to surgery: Biomarker and target discovery

Abstract SY36-01: Optimizing neoadjuvant therapy prior to surgery: Biomarker and target discovery

DNA Repair Pathway Alterations in Bladder Cancer.

Most bladder tumors have complex genomes characterized by a high mutation burden as well as frequent copy number alterations and chromosomal rearrangements. Alterations in DNA repair pathways—including the double-strand break (DSB) and nucleotide excision repair (NER) pathways—are present in bladder tumors and may contribute to genomic instability and drive the tumor phenotype. DNA damaging such as cisplatin, mitomycin C, and radiation are commonly used in the treatment of muscle-invasive or metastatic bladder cancer, and several recent studies have linked specific DNA repair pathway defects with sensitivity to DNA damaging-based therapy. In addition, tumor DNA repair defects have important implications for use of immunotherapy and other targeted agents in bladder cancer. Therefore, efforts to further understand the landscape of DNA repair alterations in bladder cancer will be critical in advancing treatment for bladder cancer. This review summarizes the current understanding of the role of DNA repair pathway alterations in bladder tumor biology and response to therapy.

Sulforaphane for the chemoprevention of bladder cancer: molecular mechanism targeted approach.

The clinical course for both early and late stage Bladder Cancer (BC) continues to be characterized by significant patient burden due to numerous occurrences and recurrences requiring frequent surveillance strategies, intravesical drug therapies, and even more aggressive treatments in patients with locally advanced or metastatic disease. For these reasons, BC is also the most expensive cancer to treat. Fortunately, BC offers an excellent platform for chemoprevention interventions with potential to optimize the systemic and local exposure of promising agents to the bladder mucosa. However, other than smoking cessation, there is a paucity of research that systematically examines agents for chemoprevention of bladder cancers. Adopting a systematic, molecular-mechanism based approach, the goal of this review is to summarize epidemiological, in vitro, and preclinical studies, including data regarding the safety, bioavailability, and efficacy of agents evaluated for bladder cancer chemoprevention. Based on the available studies, phytochemicals, specifically isothiocyanates such as sulforaphane, present in Brassicaceae or “cruciferous” vegetables in the precursor form of glucoraphanin are: (a) available in standardized formulations; (b) bioavailable- both systemically and in the bladder; (c) observed to be potent inhibitors of BC carcinogenesis through multiple mechanisms; and (d) without toxicities at these doses. Based on available evidence from epidemiological, in vitro, preclinical, and early phase trials, phytochemicals, specifically isothiocyanates (ITCs) such as sulforaphane (SFN) represent a promising potential chemopreventitive agent in bladder cancer.

A new HSP90 inhibitor as therapeutic agent for bladder cancer.

416 Background: Hsp90 represents one of the most promising biological targets for the treatment of cancer, including bladder cancer. A number of Hsp90 inhibitors that target the N-terminal ATP-binding pocket have demonstrated potent antiproliferative effects. However, a major drawback is that they induce a prosurvival heat shock response (HSR). We demonstrate the effects of a novel Hsp90 beta selective inhibitor on bladder cancer cells, which shows potent antiproliferative effects without inducing HSR. Methods: Cell Titer-Glo luminescent anti-proliferative assay was used to determine the IC50 numbers in UC3 cells. Trypan Blue Cytotoxicity assay was performed for 24h treatment with increasing concentrations of the inhibitor. Effects of the cmpound on Hsp90’s client protein degradation were investigated by Western Blot. Results: This new compound exhibits potent anti-proliferative in bladder cancer cells. IC50 number is determined as 0.30 µM for UC3 cancer cells. The toxicity assay was also performed over UC3 cells at 24h.1uM KU new compound has the similar effects on UC3 cells as 10 uM 17AAG: inhibit the cancer cells growth to half, but maintain over 60% viability of the cells. The western blot were also performed over UC3 cells, and some new target proteins such as FGFR3 and PKM2 were investigated. The data showed that, this new compound would not induce the heat shock response like 17AAG (Hsp27), and did cause some Hsp90β related protein degradation (CXCR4). FGFR3, PKM2, Her2, Hsf-1and B-raf all show degradation to different extent. Conclusions: A novel Hsp90 inhibitor, exhibits potent anti-proliferative and cytotoxic activity along with client protein degradation, without induction of HSR in bladder cancer cell lines. The reduction of Hsp90 beta related client protein caused by this compound suggests the potential to develop isoform specific inhibitors of Hsp90 for better antitumor therapies.

Immunotherapy in Bladder Cancer.

With the advent of Bacille Calmette Guerin (BCG), bladder cancer was one of the earliest cancers where the concept of immunotherapy was utilized. While this is true, recent advances in the use of immunotherapy are enabling oncologists to expand the armamentarium for the treatment of bladder cancer. Unacceptable side effects and failure to produce a durable response with the use of chemotherapeutic agents in bladder cancer has led to the evaluation of more targeted and personalized approaches. Increased understanding of the underlying carcinogenesis of bladder cancer, coupled with the ability to engineer targeted agents implicated in bladder cancer associated pathways has provided new avenues for the management of this disease. Newer immunotherapeutic approaches have generated a great deal of interest in bladder cancer along with other diseases. In this article we will focus on various forms of immunotherapies that may have a therapeutic potential in bladder cancer. We will briefly review the current status of "non-targeted" immunotherapeutic agents like BCG, interferons and interleukins in bladder cancer. But the main focus of this article is to discuss the emerging role of "targeted" immunotherapeutic agents like cytotoxic T cell lymphocyte associated protein-4 blocking antibody and programmed death pathway blocking antibodies in localized or metastatic bladder cancer.

Abstract 3068: Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in bladder cancer model

Abstract Purpose: Paclitaxel is an effective anti-neoplastic agent in many solid tumors including bladder cancer. With the development of new drug delivery strategies, novel formulations of Cremophor EL-free paclitaxel have been developed. Here, we investigated the in vitro and in vivo anti-tumor effect of a mucoadhesive oral formulation of paclitaxel (DHP107) in comparison with iv paclitaxel in a human bladder cancer model. Methods: Four human bladder cancer cell lines (RT4, T24, J82, HT1376) and one murine bladder cancer cell line (MBT-2) were used in this study. In vitro antitumor activity and underlying mechanisms were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, cell-cycle analysis and apoptosis assay. In the in vivo study, RT4 cell line was implanted subcutaneously in the flank of nu/nu mice, and treated with iv paclitaxel and 2 doses of DHP107. The effects were assessed by the tumor growth, body weight and Ki-67 staining in tumor tissues. Results: The IC50 values of DHP107 ranged from 4.15 to 34.72 μg/ml with MTT assay. In RT4 cell line, both drugs induced similar G2-M arrest and apoptosis. The xenograft experiments showed that both DHP107 (per os, 50 mg/kg, equivalent of paclitaxel 10 mg/kg) and iv paclitaxel (intraperitoneal, 10 mg/kg) attenuated the growth of RT4 tumors in mice, equivalently. The higher dose (per os, 100 mg/kg) of DHP107 resulted in increased efficacy compared with the lower dose (50 mg/kg) without weight loss in mice. However, significant weight loss was observed in iv paclitaxel (-20% at Day 11) treated group. We observed significantly decreased proliferation with Ki-67 staining in DHP107 (100 mg/kg) treated group than in DHP107 (50 mg/kg) or iv paclitaxel (10 mg/kg) treated groups. Conclusion: Our findings provide the evidence that DHP107 showed comparable antitumor effect with iv paclitaxel and is a promising anti-tumor agent in bladder cancer. Clinical studies in patients with bladder cancer warrant further investigation. Citation Format: Se Hyun Kim, Jung Min Kim, Tae Soo Kim, Won Suk Lee, Woo Sun Kwon, Seung Tak Lim, Joong Bae Ahn, Se-Kyu Kim, Yeong Woo Jo, Sun Young Rha. Anti-tumor effect of DHP107, a mucoadhesive lipid formulae of oral paclitaxel, in bladder cancer 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 3068.

Tasquinimod modulates tumor-infiltrating myeloid cells and improves the antitumor immune response to PD-L1 blockade in bladder cancer

ABSTRACTThe infiltration of myeloid cells helps tumors to overcome immune surveillance and imparts resistance to cancer immunotherapy. Thus, strategies to modulate the effects of these immune cells may offer a potential therapeutic benefit. We report here that tasquinimod, a novel immunotherapy which targets S100A9 signaling, reduces the immunosuppressive properties of myeloid cells in preclinical models of bladder cancer (BCa). As single anticancer agent, tasquinimod treatment was effective in preventing early stage tumor growth, but did not achieve a clear antitumor effect in advanced tumors. Investigations of this response revealed that tasquinimod induces an increase in the expression of a negative regulator of T cell activation, Programmed-death-ligand 1 (PD-L1). This markedly weakens its antitumor immunity, yet provokes an “inflamed” milieu rendering tumors more prone to T cell-mediated immune attack by PD-L1 blockade. Interestingly, the combination of tasquinimod with an Anti-PD-L1 antibody enhanced the antitumor immune response in bladder tumors. This combination synergistically modulated tumor-infiltrating myeloid cells, thereby strongly affecting proliferation and activation of effector T cells. Together, our data provide insight into the rational combination of therapies that activate both innate and adaptive immune system, such as the association of S100A9-targeting agents with immune checkpoints inhibitors, to improve the response to cancer immunotherapeutic agents in BCa.

Potential therapeutic roles of tanshinone IIA in human bladder cancer cells.

Tanshinone IIA (Tan-IIA), one of the major lipophilic components isolated from the root of Salviae Miltiorrhizae, has been found to exhibit anticancer activity in various cancer cells. We have demonstrated that Tan-IIA induces apoptosis in several human cancer cells through caspase- and mitochondria-dependent pathways. Here we explored the anticancer effect of Tan-IIA in human bladder cancer cell lines. Our results showed that Tan-IIA caused bladder cancer cell death in a time- and dose-dependent manner. Tan-IIA induced apoptosis through the mitochondria-dependent pathway in these bladder cancer cells. Tan-IIA also suppressed the migration of bladder cancer cells as revealed by the wound healing and transwell assays. Finally, combination therapy of Tan-IIA with a lower dose of cisplatin successfully killed bladder cancer cells, suggesting that Tan-IIA can serve as a potential anti-cancer agent in bladder cancer.

Anthocyans as tertiary chemopreventive agents in bladder cancer: anti-oxidant mechanisms and interaction with mitomycin C.

Bladder cancer is associated with high rates of recurrence making tertiary chemoprevention an attractive intervention strategy. Anthocyanins have been shown to possess chemopreventive properties and are detectable in urine after oral ingestion, with higher concentrations achievable via intravesical administration alongside current chemotherapeutic regimens. Yet their apparent ability to protect against certain DNA damage may in turn interfere with cancer treatments. Our aim was therefore to determine the potential of anthocyanins as chemopreventive agents in bladder cancer, their mode of action and effects, both alone and in combination with mitomycin C (MMC). In this study we showed that mirtoselect, a standardised mixture of anthocyanins, possesses significant anti-proliferative activity, causing growth inhibition and apoptosis in bladder cancer cell lines. The anti-oxidative potential of mirtoselect was examined and revealed significantly fewer H2O2-induced DNA strand breaks, as well as oxidised DNA bases in pre-treated cells. In contrast, endogenous levels of oxidised DNA bases were unaltered. Investigations into the possible protective mechanisms associated with these anti-oxidant properties revealed that mirtoselect chelates metal ions. In mirtoselect/MMC combination studies, no adverse effects on measures of DNA damage were observed compared to treatment with MMC alone and there was evidence of enhanced cell death. Consistent with this, significantly more DNA crosslinks were formed in cells treated with the combination. These results show that mirtoselect exerts effects consistent with chemopreventive properties in bladder cancer cell lines and most importantly does so without adversely affecting the effects of drugs used in current treatment regimens. We also provide evidence that mirtoselect's anti-oxidative mechanism of action is via metal ion chelation. Overall these results suggest that mirtoselect could be an effective chemopreventive agent in bladder cancer and provides the necessary pre-clinical data for future in vivo animal studies and clinical trials.

Dual targeting of heat shock proteins 90 and 70 promotes cell death and enhances the anticancer effect of chemotherapeutic agents in bladder cancer

Heat shock proteins (HSPs), which are molecular chaperones that stabilize numerous vital proteins, may be attractive targets for cancer therapy. The aim of the present study was to investigate the possible anticancer effect of single or dual targeting of HSP90 and HSP70 and the combination treatment with HSP inhibitors and chemotherapeutic agents in bladder cancer cells. The expression of HSP90 and the anticancer effect of the HSP90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) coupled with cisplatin, docetaxel, or gemcitabine were examined using immunohistochemistry, quantitative real-time PCR, cell growth, flow cytometry, immunoblots and caspase-3/7 assays. The expression of HSP70 under HSP90 inhibition and the additive effect of HSP70 inhibitor pifithrin-μ (PFT-μ) were examined by the same assays and transmission electron microscopy. HSP90 was highly expressed in bladder cancer tissues and cell lines. 17-AAG enhanced the antiproliferative and apoptotic effects of each chemotherapeutic agent. 17-AAG also suppressed Akt activity but induced the upregulation of HSP70. PFT-μ enhanced the effect of 17-AAG or chemotherapeutic agents; the triple combination of 17-AAG, PFT-μ and a chemotherapeutic agent showed the most significant anticancer effect on the T24 cell line. The combination of 17-AAG and PFT-μ markedly suppressed Akt and Bad activities. With HSP90 suppression, HSP70 overexpression possibly contributes to the avoidance of cell death and HSP70 may be a key molecule for overcoming resistance to the HSP90 inhibitor. The dual targeting of these two chaperones and the combination with conventional anticancer drugs could be a promising therapeutic option for patients with advanced bladder cancer.

MP21-10 DEVELOPMENT OF AN EFFICIENT BLADDER TUMOR MODEL BY RECOMBINING WITH MESENCHYMAL CELLS

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2014MP21-10 DEVELOPMENT OF AN EFFICIENT BLADDER TUMOR MODEL BY RECOMBINING WITH MESENCHYMAL CELLS Kenichiro Ishii, Takeshi Sasaki, Manabu Kato, Kenjiro Ito, Motomu Sakuragi, Nobuyuki Oda, Hideki Kanda, Yasushi Yamada, Kiminobu Arima, Taizo Shiraishi, and Yoshiki Sugimura Kenichiro IshiiKenichiro Ishii More articles by this author , Takeshi SasakiTakeshi Sasaki More articles by this author , Manabu KatoManabu Kato More articles by this author , Kenjiro ItoKenjiro Ito More articles by this author , Motomu SakuragiMotomu Sakuragi More articles by this author , Nobuyuki OdaNobuyuki Oda More articles by this author , Hideki KandaHideki Kanda More articles by this author , Yasushi YamadaYasushi Yamada More articles by this author , Kiminobu ArimaKiminobu Arima More articles by this author , Taizo ShiraishiTaizo Shiraishi More articles by this author , and Yoshiki SugimuraYoshiki Sugimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.838AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Aberrant activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. Many solid tumors are composed of carcinoma cells and surrounding stromal cells named as carcinoma-associated fibroblasts (CAFs). They secrete a number of growth factors such as EGF family, FGFs, HGF, and IGFs, which are also produced by rat mesenchymal cells (Ishii et al., Endocr Relate Cancer, 2009). Bladder cancer is the second most common genitourinary malignancy, with transitional cell carcinoma representing 90% of the cases. Unfortunately, bladder cancer has the high recurrence rate after treatment, even when superficial tumors are completely removed by a laser or electrical force. We hypothesize that the aberrant activation between stromal cells and bladder cancer cells is probably involved in the high recurrence rate of bladder cancer. The development of efficient tumor models that mimic human disease would benefit to investigate new cancer therapeutic agents. Here we have developed a new model of bladder tumors by recombining rat mesenchymal cells with T24 human bladder cancer cells. METHODS Rat mesenchymal cells were prepared from 18-day SD rat fetuses. Briefly, bladder, urogenital sinuses, and stomach were dissected from the fetuses and separated into epithelial and mesenchymal components. Recombinants were prepared by mixing 5 x 105 T24 cells and 1 x 105 rat mesenchymal cells in suspension. Pelleted cells were resuspended in neutralized type I rat tail collagen gels, and then grafted beneath the renal capsule of adult male nude mice. RESULTS As generally known, T24 tumors without rat mesenchymal cells were not well grown in vivo. In contrast, T24 tumors that formed with rat mesenchymal cells derived from urogenital sinus (UGM) and bladder (BLM) but not stomach (STM) showed accelerated cell proliferation and angiogenesis. In in vitro cell culture experiments, several growth factors such as EGF, TGF-alpha, and IL-6 stimulated cell proliferation of T24 cells. Only IL-6 treatment activated cellular PI3/Akt pathway in T24 cells, whereas IL-6 treatment reduced VEGF secretion from T24 cells. Cell motility of T24 cells was significantly increased by EGF and TGF-alpha treatment. CONCLUSIONS In this study, we demonstrate that our recombination model using mesenchymal cells may be useful to understand the tumor-stromal interactions in bladder cancer. Identifying mechanisms underlying the aberrant activation between stromal cells and bladder cancer cells is an initial step toward developing new cancer therapeutic agents in bladder cancer. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e229 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Kenichiro Ishii More articles by this author Takeshi Sasaki More articles by this author Manabu Kato More articles by this author Kenjiro Ito More articles by this author Motomu Sakuragi More articles by this author Nobuyuki Oda More articles by this author Hideki Kanda More articles by this author Yasushi Yamada More articles by this author Kiminobu Arima More articles by this author Taizo Shiraishi More articles by this author Yoshiki Sugimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...