Cisplatin-resistant Bladder Cancer Research Articles

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 ...

Biological characterization of cisplatin-resistant bladder cancer: Implications for second-line treatments?

Biological characterization of cisplatin-resistant bladder cancer: Implications for second-line treatments?

Expression of Class III Beta-tubulin Predicts Prognosis in Patients with Cisplatin-resistant Bladder Cancer Receiving Paclitaxel-based Second-line Chemotherapy.

Class III beta-tubulin (TUBB3) expression is recognized as a predictive marker for chemosensitivity to cisplatin- and taxane-based chemotherapies in various malignancies. The aim of this study was to clarify the predictive value of TUBB3 expression for the anticancer effects of first-line cisplatin-based chemotherapy and second-line paclitaxel-based chemotherapy in patients with urothelial cancer (UC). We reviewed 116 patients with UC (90 with bladder cancer and 27 with upper urinary tract cancer) treated with first-line cisplatin-based chemotherapy. Among them, 42 patients received a paclitaxel-based regimen as second-line chemotherapy for advanced cisplatin-resistant UC. TUBB3 expression was evaluated using immunohistochemistry, and survival analyses were performed using Kaplan-Meier survival curves and multivariate Cox proportional hazard analysis. TUBB3 was mainly detected in the cytoplasm of cancer cells, and 64 patients (55.2%) were judged as having positive TUBB3 expression. TUBB3 expression was significantly associated with tumour grade (p<0.001). TUBB3 expression was not associated with time to progression after first-line cisplatin-based chemotherapy. However, positive expression of TUBB3 was significantly associated with unfavourable overall survival in patients receiving second-line paclitaxel-based chemotherapy (p=0.021). In addition, a multivariate analysis model including T-stage and metastasis at the beginning of second-line therapy and regimen showed that TUBB3 expression was an independent predictor of poorer survival (hazard ratio(HR)=3.44, 95% confidential interval(CI)=1.15-10.33, p=0.027). TUBB3 expression was identified as a useful predictive factor for survival after second-line paclitaxel-based therapy in patients with cisplatin-resistant UC. Our results are useful for determining treatment strategies for such patients.

PKM2 Inhibitor Shikonin Overcomes the Cisplatin Resistance in Bladder Cancer by Inducing Necroptosis.

Cisplatin-based chemotherapy often results in the development of chemo-resistance when used to treat bladder cancer (BC), which is difficult to overcome. Recent data indicate that pyruvate kinase M2 (PKM2), a glycolytic enzyme for Warburg effect, is strongly upregulated in BC, and contributes to the cisplatin resistance in BC. However, the underlying mechanisms remain unclear. In this study, we also found that the expression level of PKM2 is also higher in cisplatin resistant BC cells and tumors. Down-regulation of PKM2 by siRNA or inhibition of PKM2 by shikonin re-sensitized the cisplatin resistant T24 cells. Shikonin and cisplatin together exhibit significantly greater killing effects than when used alone. Interestingly, we found shikonin kills the T24 cisplatin resistant cells by inducing necroptosis, as the cell death could not inhibited by apoptosis inhibitor, z-VAD, but compromised by RIP3 inhibitor, GSK872, or RIP3 siRNA. In contrast, shikonin induced apoptosis in T24 parental cells. We further investigate the underlying mechanism, and found that the dysregulation of Bcl-2 family proteins, including Bcl-2, PUMA, Bax, play an important role in deciding that shikonin kills the BC cells by necroptosis or apoptosis. Collectively, our results suggested that inducing necroptosis is an alternative way to overcome the apoptosis resistant in BC therapy, and orchestrating the regulation of Bcl-2, PUMA, and Bax in BC cisplatin resistant cells may improve the therapy effect of cisplatin in BC tumor.

Expression and Functions of Formyl Peptide Receptor 1 in Drug-Resistant Bladder Cancer.

Objective:To explore the correlation of formyl peptide receptor 1 expression with drug resistance and the functions of formyl peptide receptor 1 in drug-resistant bladder cancer.Methods:Expression of formyl peptide receptor 1 in T24 and T24/DDP cisplatin-resistant bladder cancer cell lines was tested by quantitative real-time Polymerase Chain Reaction and Western blotting. After incubation of T24/DDP with N-formyl-Met-Leu-Phe, the phosphor proteins were tested by Western blot analysis. We characterized the functions of formyl peptide receptor 1 in T24/DDP cells by assessing proliferation, migration, and changes of cell cycles.Results:Formyl peptide receptor 1 was expressed in both T24 and T24/DDP, and it was overexpressed in T24/DDP compared with T24. Formyl peptide receptor 1 activation promoted the expression of the messenger RNA of resistance-related proteins, such as multidrug resistance-associated protein 1 (MRP1) and lung resistance-related protein (LRP). The expression of 4 signal pathway proteins were upregulated: signal transducer and activator of transcription 3, Janus kinase 2, extracellular regulated protein kinases, and protein kinase B, while the expression of phosphatidylinositol 3-kinase was observed to be downregulated in drug-resistant bladder cancer cells. Formyl peptide receptor 1 activation also improved the expression of phospho-signal transducer and activator of transcription 3 and phospho-extracellular regulated protein kinases 1/2 and promoted the proliferation and migration of T24/DDP cells. In addition, formyl peptide receptor 1 inhibition led to the change in the cell cycle in T24/DDP.Conclusions:The overexpression of formyl peptide receptor 1 may be related to drug-resistant bladder cancer and promotes the deterioration of drug-resistant bladder cancer.

Rewiring of cisplatin-resistant bladder cancer cells through epigenetic regulation of genes involved in amino acid metabolism.

Alterations in DNA methylation are important epigenetic markers in bladder cancer (BC). These epigenome modifications may drive the mechanisms of aggressive chemo-resistant BC. Clinicopathological biomarkers that indicate chemotherapeutic resistance are critical for better assessing treatment strategies for individual patients. Thus, in this study, we aimed to determine whether DNA methylation of certain metabolic enzymes is significantly altered in cisplatin-resistant BC cells.Methods: To characterize CpG methylation and nucleosome accessibility in cisplatin-resistant BC cells, the Illumina Infinium HM450 DNA methylation assay was performed. Perturbed gene expression was found to be associated with cisplatin resistance, and the biological roles of spermidine/spermine N1-acetyltransferase (SAT1) and argininosuccinate synthase 1 (ASS1) were further studied using qRT-PCR analysis and various cell biology assays, including western blot.Results: ASS1 and SAT1, genes for amino acid and polyamine metabolism catalysts, respectively, were found to be vastly hypermethylated, resulting in greatly downregulated expression. ASS1 expression is of particular interest because prior studies have demonstrated its potential association with BC stage and recurrence. In regard to chemoresistance, we found that aberrant expression or induced stimulation of SAT1 restored cisplatin sensitivity in the cell culture system. We also found that the addition of exogenous arginine deiminase through administration of ADI-PEG 20 (pegylated arginine deiminase) increased ASS1 expression and enhanced cisplatin's apoptotic effects.Conclusions: Our study demonstrates a novel mechanistic link between the epigenetic perturbation of SAT1 and ASS1 and cancer metabolism in cisplatin-resistant bladder cancer cells. These findings suggest potential utility of SAT1 and ASS1 as predictive biomarkers in re-sensitizing bladder cancer to chemotherapy and personalizing therapy.

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.

Subclonal mutational heterogeneity and survival in cisplatin-resistant muscle-invasive bladder cancer.

4512 Background: Biomarkers of survival and resistance in chemotherapy-resistant muscle-invasive bladder cancer (MIBC) are not well-characterized, but may inform management in this setting. Methods: Matched pre- and post-neoadjuvant cisplatin-based chemotherapy (NAC) tumor samples were obtained from 30 MIBC patients with gross residual disease (≥ pT2) at cystectomy, followed by whole exome sequencing of these “trios” (pre- and post-NAC tumor with matched germline samples). Phylogenetic analysis of matched tumor samples was performed to identify subclones, their associated mutations, and the corresponding enrichment in post-treatment tumors. Intratumoral heterogeneity was assessed by the proportion of mutations that were subclonal; the number of inferred subclones; and associated with overall survival using a Cox Proportional Hazards model. Results: Increased proportion of subclonal mutations in post-treatment tumors was associated with worse overall survival (HRR 1.86 [95% CI 1.12-3.06], p = 0.02), whereas pre-treatment proportion of subclonal mutations was only borderline statistically significant (HRR 1.48 [95% CI 0.99-2.20], p = 0.052). The total number of inferred tumor subclones in pre- or post-treatment tumor (or both) was associated with overall survival (HRR 1.60 [95% CI 1.05-2.43], p = 0.03), interpreted as a 60% increase in death rate per additional inferred subclone. While no single gene was statistically significantly enriched for new alterations in the post-chemotherapy resistant samples, we observed new post-treatment amplifications in cell-cycle genes ( E2F3, c-JUN), biallelic events in cell-cycle regulators ( FBXW7), and amplification of immune checkpoint genes ( PDL1/2). Conclusions: These results suggest that intratumoral heterogeneity (particularly post-therapy) predicts survival in a chemotherapy-resistant cohort. Further, alterations in cell cycle regulation may contribute to the mechanism of chemotherapy resistance. Finally, we observe evidence of immune checkpoint gene amplification post-treatment, suggesting that testing immune checkpoint blockade during NAC or, in high risk patients, following NAC may be warranted.

An Oncofetal Glycosaminoglycan Modification Provides Therapeutic Access to Cisplatin-resistant Bladder Cancer

BackgroundAlthough cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients with muscle-invasive bladder cancer (MIBC), only a minority responds to therapy and chemoresistance remains a major challenge in this disease setting. ObjectiveTo investigate the clinical significance of oncofetal chondroitin sulfate (ofCS) glycosaminoglycan chains in cisplatin-resistant MIBC and to evaluate these as targets for second-line therapy. Design, setting, and participantsAn ofCS-binding recombinant VAR2CSA protein derived from the malaria parasite Plasmodium falciparum (rVAR2) was used as an in situ, in vitro, and in vivo ofCS-targeting reagent in cisplatin-resistant MIBC. The ofCS expression landscape was analyzed in two independent cohorts of matched pre- and post-NAC–treated MIBC patients. InterventionAn rVAR2 protein armed with cytotoxic hemiasterlin compounds (rVAR2 drug conjugate [VDC] 886) was evaluated as a novel therapeutic strategy in a xenograft model of cisplatin-resistant MIBC. Outcome measurements and statistical analysisAntineoplastic effects of targeting ofCS. Results and limitationsIn situ, ofCS was significantly overexpressed in residual tumors after NAC in two independent patient cohorts (p<0.02). Global gene-expression profiling and biochemical analysis of primary tumors and cell lines revealed syndican-1 and chondroitin sulfate proteoglycan 4 as ofCS-modified proteoglycans in MIBC. In vitro, ofCS was expressed on all MIBC cell lines tested, and VDC886 eliminated these cells in the low-nanomolar IC50 concentration range. In vivo, VDC886 effectively retarded growth of chemoresistant orthotopic bladder cancer xenografts and prolonged survival (p=0.005). The use of cisplatin only for the generation of chemoresistant xenografts are limitations of our animal model design. ConclusionsTargeting ofCS provides a promising second-line treatment strategy in cisplatin-resistant MIBC. Patient summaryCisplatin-resistant bladder cancer overexpresses particular sugar chains compared with chemotherapy-naïve bladder cancer. Using a recombinant protein from the malaria parasite Plasmodium falciparum, we can target these sugar chains, and our results showed a significant antitumor effect in cisplatin-resistant bladder cancer. This novel treatment paradigm provides therapeutic access to bladder cancers not responding to cisplatin.

An Epigenomic Approach to Improving Response to Neoadjuvant Cisplatin Chemotherapy in Bladder Cancer.

Bladder cancer is among the five most common cancers diagnosed in the Western world and causes significant mortality and morbidity rates in affected patients. Therapeutic options to treat the disease in advanced muscle-invasive bladder cancer (MIBC) include cystectomy and chemotherapy. Neoadjuvant cisplatin-based combination chemotherapy is effective in MIBC; however, it has not been widely adopted by the community. One reason is that many patients do not respond to neoadjuvant chemotherapy, and no biomarker currently exists to identify these patients. It is also not clear whether a strategy to sensitize chemoresistant patients may exist. We sought to identify cisplatin-resistance patterns in preclinical models of bladder cancer, and test whether treatment with the epigenetic modifier decitabine is able to sensitize cisplatin-resistant bladder cancer cell lines. Using a screening approach in cisplatin-resistant bladder cancer cell lines, we identified dysregulated genes by RNA sequencing (RNAseq) and DNA methylation assays. DNA methylation analysis of tumors from 18 patients receiving cisplatin-based chemotherapy was used to confirm in vitro results. Cisplatin-resistant bladder cancer cells were treated with decitabine to investigate epigenetic sensitization of resistant cell lines. Our results show that HOXA9 promoter methylation status is associated with response to cisplatin-based chemotherapy in bladder cancer cell lines and in metastatic bladder cancer. Bladder cancer cells resistant to cisplatin chemotherapy can be sensitized to cisplatin by the DNA methylation inhibitor decitabine. Our data suggest that HOXA9 promoter methylation could serve as potential predictive biomarker and decitabine might sensitize resistant tumors in patients receiving cisplatin-based chemotherapy.

Cytotoxic effects of kazinol A derived from Broussonetia papyrifera on human bladder cancer cells, T24 and T24R2

BackgroundBroussonetia papyrifera (B. papyrifera), also known as paper mulberry, has been used as a traditional medicine for the treatment of several diseases, including ophthalmic disorders and impotency. However, the biological activity of kazinol A (1) among flavonols isolated from B. papyrifera has not been identified. PurposeWe identified a candidate metabolite for anti-human bladder cancer treatment from B. papyrifera and investigated the possible molecular mechanisms underlying its cytotoxic effects in T24 and cisplatin-resistant T24R2 human bladder cancer cells. MethodsT24 and T24R2 cells were treated with five flavonols from B. papyrifera and their cytotoxic effects were determined using MTT assay, cell cycle analysis, mitochondrial membrane potential, and propidium iodide staining. Autophagy rate was calculated by counting LC3-GFP dots in the cells. All related protein expressions were analyzed by immunoblotting. ResultsCompound 1 showed relatively higher cytotoxicity in the human bladder cancer cells, T24 and T24R2, rather than other tissues-originated cancer cells. Compound 1 significantly attenuated cell growth through G0/1 arrest mediated by a decrease in cyclin D1 and an increase of p21. Apoptosis and autophagy induced by compound 1 treatment was accompanied by a modulation of the AKT-BAD pathway and AMPK-mTOR pathway, respectively. ConclusionsOur results suggest that compound 1 induces cytotoxic effects in human bladder cancer cells, including the cisplatin-resistant T24R2. Compound 1 may be a candidate for the development of effective anti-cancer drug on human urinary bladder cancer.

JAK-mediated autophagy regulates stemness and cell survival in cisplatin resistant bladder cancer cells

BackgroundAutophagy is a critical process in acquiring drug resistance in solid tumors. However, the mechanisms by which autophagy modulate resistance to chemotherapy in bladder cancer remains poorly understood. Material and methodsWe have established cisplatin resistant patient derived primary cultured cells as well as T24 bladder cancer cells. The autophagy flux as well as the effect of chemotherapeutic agents, gemcitabine (GC) and mitomycin (MM) were evaluated in these cells. Side population analysis, tumorisphere assay and expression of stemness genes were also studied to evaluate the role of autophagy during chemoresistance. The role of cytokines in autophagy induced chemoresistance was demonstrated. ResultsOur results show that resistant cells have higher basal autophagic flux. GC and MM led to increase in autophagy and combination of autophagy inhibitors synergistically inhibited bladder cancer cell growth. When autophagy was inhibited, only IFN-γ level was decreased in cytokine array. Blocking IFN-γ decreased JAK2 expression and reduced the autophagy. Inhibition of JAK2 decreased the proportion of cancer stem cell enriched side population, tumor sphere forming ability and led to decrease in expression of stemness genes. Combined treatment with inhibitors of JAK2 and autophagy led to inhibition in cell growth, reduced the levels of inflammatory cytokines and decreased the resistance gene expression. ConclusionGC and MM treatment increased autophagy process via IFN-γ mediated JAK2 and STAT3 pathway. General significanceThese findings support a key role of the autophagy in drug resistance. Thus, autophagy-based, “customized” combinatorial approaches may effectively manage resistance and relapse in bladder cancer.

Abstract 1260: Polymerase kappa determines the sensitivity of MTH1 inhibitors to cisplatin-resistant cell

Abstract Resistance is one the main reason for overall decrease in survival of cancer patient treated with cisplatin in different types of cancer. Cisplatin kills cancer cells by various mechanisms, but mainly through formation of inter- and intra stand crosslinks of DNA. Different types of translesion polymerase including Polymerase kappa (POLK) are involved in repair of DNA lesions. We observed high expression levels of POLK in cisplatin resistant bladder and ovarian cancer cells compared to parental cells. Due to its low proof-reading activity POLK can incorporate 8-oxo-dGTP into DNA. The MTH1 protein (Nudix hydrolase- NUDT1) sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Recently we have generated MTH1 inhibitors that damage the DNA and induce cancer specific cell death through incorporation of more oxidized dNTPs. We found cisplatin resistant bladder cancer cells (NTUB1/P) were more sensitive to MTH1 inhibitors in comparison to parental NTUB1 cells. As POLK is involved in incorporation of 8-oxo-dGTP into DNA, we hypothesized that high expression levels of POLK in cisplatin resistant cells make them more sensitive to MTH1 inhibitors as more 8-oxo-dGTP would be incorporated into DNA, resulting in more DNA damage and cell death in comparison to parental cells. Indeed, we observes higher induction of cleaved-PARP, γH2AX, cleaved-Caspase 3 and more annexin v positive cells in cisplatin resistant NTUB1/P cells in comparison to parental NTUB1 cells upon treatment with MTH1 inhibitors. MTH1 inhibitor also significantly delays the NTUB1/P xenograft tumor growth in comparison to vehicle treatment in immunosuppressive mice. Knocking down POLK in cisplatin resistant NTUB1/P cells by siRNA resulted in decreased incorporation of 8-oxo-dGTP and sensitivity to MTH1 inhibitors compared to non target control cells. Overexpression of POLK in NTUB1 and NTUB1/P cells results in further sensitization to MTH1 inhibitors. In conclusion elevated levels of POLK in cisplatin resistance cells determines increased sensitivity towards MTH1 inhibitors. Thus MTH1 inhibitors can be a potential promising therapy for the treatment of cisplatin resistant tumors in patients. Citation Format: Kumar Sanjiv, Helge Gad, Sean Rudd, Rachel Hurley, Patric Herr, José Manuel Calderón Montaño, Oliver Mortusewicz, Tobias Koolmeister, Sylvain Jaques, Estefanía Burgos Morón, Andreas Hoglund, Te-Chang Lee, Martin Scobie, Scott Kaufmann, John Weroha, Ulrika Warpman Berglund, Andrea Wahner Hendrickson, Thomas Helleday. Polymerase kappa determines the sensitivity of MTH1 inhibitors to cisplatin-resistant cell. [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 1260.

Abstract 3798: A glycan-binding malaria protein provides therapeutic access to cisplatin-resistant bladder cancer

Abstract Bladder cancer is a disease of compelling morbidity and mortality mainly due to its high recurrence rate. Cisplatin-based chemotherapy is an integral part of muscle invasive bladder cancer (MIBC) treatment. Although responses are common, a significant number of patients develop resistance to cisplatin and second line treatment options are not well established. No improvement of the MIBC treatment strategy has been achieved in the past two decades and therefore; new approaches to systemic therapy are urgently needed. The Plasmodium falciparum host-cell anchor protein VAR2CSA has been evolutionarily optimized to bind distinctly modified chondroitin sulfate (CS) glycosaminoglycan (GAG) chains expressed exclusively in the mammalian placenta. This is the underlying key event behind pregnancy-associated malaria outbreaks in endemic regions of the world. We have recently discovered that placental-type CS chains are re-expressed in the malignant compartment as a secondary oncofetal CS (ofCS) modification (Salanti et al. 2015, Cancer Cell). In the present study, we have analyzed the expression and role of ofCS in cisplatin-resistant bladder cancer and evaluated the potential of a VAR2-drug conjugate (VDC) to target cisplatin-resistant tumors. Using recombinant VAR2CSA protein (rVAR2) as an ofCS detection reagent, we analyzed a tissue microarray of 52 chemotherapy-naïve MIBC samples, with 36 matched post-chemotherapy cystectomy specimens from patients receiving neoadjuvant gemcitabine/cisplatin. In chemoresistant tumor specimens, of-CS expression was significantly upregulated in residual patient tumors after neoadjuvant chemotherapy (p = 0.001) and it was associated with advanced tumor stage (ypT3/4, p = 0.005) and poor overall survival (p = 0.04). Microarray analysis of primary human bladder tumors and subsequent in situ proximity ligation assay validation identified S100A9 and CD44 as the major ofCS-modified proteoglycans in chemoresistant bladder cancer. Binding of rVAR2 to ofCS chains on bladder cancer cells facilitated rapid internalization of the protein. Moreover, a rVAR2-drug conjugate (VDC) efficiently killed all bladder cancer cells in the low nanomolar IC50 concentration range in vitro and retarded growth of chemoresistant orthotopic MIBC xenografts in vivo. In summary, we demonstrate how a glycan-binding malaria protein can be utilized to gain therapeutic access to cisplatin-resistant bladder cancer. Thus, we provide a method to target cancer-specific glycan modifications for therapeutic intervention as a second line treatment in MIBC, not responding to cisplatin. Citation Format: HTOO ZARNI OO, Roland Seiler, Sherry S. Lee, Davide Tortora, Gunjan Kumar, Chris Wang, Thomas M. Clausen, Mette Ø. Agerbæk, Jamie R. Rich, John S. Babcook, Peter C. Black, Ali Salanti, Mads Daugaard. A glycan-binding malaria protein provides therapeutic access to cisplatin-resistant bladder cancer. [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 3798.

301 - Regulation of AMPK-dependent MDR1 expression by resveratrol in cisplatin-resistant human bladder cancer T24 cells via attenuating NF-κB and CRE transcriptional activity

301 - Regulation of AMPK-dependent MDR1 expression by resveratrol in cisplatin-resistant human bladder cancer T24 cells via attenuating NF-κB and CRE transcriptional activity

Repression of the miR-93-enhanced sensitivity of bladder carcinoma to chemotherapy involves the regulation of LASS2.

The aberrant expression of miRNA has an important function in bladder cancer (BC). Previous studies indicate that LASS2 is involved in the development of sensitivity to chemotherapy in cancer cells. In the present study, the miRNAs related to LASS2 were selected by using miRNA profiling to distinguish chemo-resistant and chemo-sensitive tumor specimens from patients. Higher levels of miR-93 were observed in the cisplatin-resistant BC cell line RT4, compared to the cell line T24. The role of miR-93 in chemo-sensitivity was demonstrated both in cell culture and mouse tumor xenograft models. We found that inhibiting miR-93 promoted cisplatin-induced apoptosis due to the accumulation of DNA damage. A reporter gene assay was performed, and the results showed miR-93 was not a target of the 3′ untranslated region of LASS2, but had an altered protein expression level. Inhibitors of miR-93 could also enhance the chemo-sensitivity of tumor cells transfected with si-LASS2, but the effect was very slight. These findings suggest that miR-93 plays an important role in the chemo-sensitivity of BC, and may be involved in regulating the LASS2 gene.

Upregulated expression of BCL2, MCM7, and CCNE1 indicate cisplatin-resistance in the set of two human bladder cancer cell lines: T24 cisplatin sensitive and T24R2 cisplatin resistant bladder cancer cell lines.

PurposeThe mechanism of resistance to cisplatin during treatment of bladder cancer (BC) has been a subject of intense investigation in clinical research. This study aims to identify candidate genes associated with resistance to cisplatin, in order to understand the resistance mechanism of BC cells to the drug, by combining the use of microarray profiling, quantitative reverse transcription-polymerase chain reaction (RT-PCR), and Western blot analyses.Materials and MethodsThe cisplatin sensitive human BC cell line (T24) and the cisplatin resistant BC cell line, T24R2, were used for microarray analysis to determine the differential expression of genes that are significant in cisplatin resistance. Candidate upregulated genes belonging to three well-known cancer-related KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways (p53 tumor suppressor, apoptosis, and cell cycle) were selected from the microarray data. These candidate genes, differentially expressed in T24 and T24R2, were then confirmed by quantitative RT-PCR and western blot. A fold change ≥2 with a p-value <0.05 was considered significant.ResultsA total of 18 significantly upregulated genes were detected in the three selected cancer-related pathways in both microarray and RT-PCR analyses. These genes were PRKAR2A, PRKAR2B, CYCS, BCL2, BIRC3, DFFB, CASP6, CDK6, CCNE1, STEAP3, MCM7, ORC2, ORC5, ANAPC1, and ANAPC7, CDC7, CDC27, and SKP1. Western blot analyses also confirmed the upregulation of BCL2, MCM7, and CCNE1 at the protein level, indicating their crucial association with cisplatin resistance.ConclusionsThe BCL2, MCM7, and CCNE1 genes might play distinctive roles in cisplatin resistance in BC.

Establishment of cisplatin-resistant human bladder cancer cell line and investigation of its resistant mechanism

Objective To establish the cisplatin (CDDP)-resistant cell line from human bladder cancer cell line T24 and to investigate its resistant mechanism to CDDP. Methods A CDDP-resistant bladder cancer cell line T24/CDDP was established by gradually increasing dose of cisplatin and high-dose stimulation.The IC50and resistance index were estimated with cell counting Kit-8 (CCK-8). Cell growth curve, doubling time, and cell cycle phase distribution were measured. The expressions of protein kinase Cα(PKCα) and P- glycoprotein (P-gp) of T24 cells and T24/CDDP cells were measured with Western blot. Results The CDDP-resistant cell line T24/CDDP was established after 11 months, with a stable resistance to cisplatin and a resistant index of 4.17. The doubling time of T24 cells and T24/CDDP cells was(33.61±0.41)h and (39.94±0.63) h, with a significant difference (t=14.59, P=0.00). Compared to the S phase of T24 cells[(30.63±2.74)%], the S-phase of T24/CDDP cells was increased[(35.38±3.31)%], without significant difference (P>0.05). The G0/G1 phase of T24/CDDP cells[(60.42±3.25)%]was significantly more than that of T24 cells[(47.25±4.17)%], and the G2/M phase of T24/CDDP cells[(8.95±2.81)%]was significantly less than that of T24 cells[(17.37±3.46)%](t=4.31, P=0.006; t=3.27, P=0.015). Western blot showed that expressions of PKCα and P-gp in T24/CDDP cells were significantly higher than T24 cells[(3.12±0.11) vs (1.37±0.06), t=24.19, P=0.00; (1.98±0.08) vs (0.47±0.03), t=30.61, P=0.00]. Conclusions T24/CDDP cell line showed a typical resistant phenotype and biological characteristics, which may be related to the overexpression of PKCα and P-gp protein. Key words: Urinary bladder neoplasms/DT; Cisplatin/TU; Drug resistance, neoplasm

Abstract 5475: Cancer exosome promotes cisplatin resistance In bladder cancer and inhibition of exosome sensitizes bladder cancer cells to cisplatin chemotherapy

Abstract Exsosomes are nano-vesicles secreted by most types of cells, and substantially increased secretion in cancer cells. Their role in tumorigenesis/cancer progression have been extensively studied but their role in regulating therapy response have been suggested without in depth investigation. We are particularly interested in exosome's roles in cisplatin resistant, since cisplatin is one of the most widely used chemodrugs for many cancer types. To investigate the role of exosomes in cisplatin resistance in bladder cancer, we examined the cisplatin sensitivity among five bladder cancer cell lines. The expression levels of exosome associated genes that are involved in exosome biogenesis and secretion/internalization were examined by quantative PCR. Interestingly, cisplatin resistant bladder cancer cells express relative high level of exosome associated genes as compared to cisplatin sensitive cells. More importantly, cisplatin treatment induces the expression of exosome associated genes’ expression in cisplatin resistant cells, but not in sensitive cell. Among all those genes, we are particularly interested in Rab gene family, and mechanistic study of targeting those Rab proteins is undergoing. The correlative exosome genes profiles with cisplatin resistance suggested exosome production might facilitate cancer cells escaping from cisplatin-induced cell death; thereby, inhibition of exosome might sensitize cancer cells response to cisplatin. To test this, we have screened several compounds that are known to inhibit exosome biogenesis and uptake, and. found that inhibition of exosomes by dynasore + NSC23766, indeed can sensitize bladder cancer cells to cisplatin. In summary, we concluded that cancer exosome play a critical role of promoting cisplatin resistance in bladder cancer, and inhibition of exosomes sensitizes bladder cancer cell response to cisplain. The exosome related profiles potentially can serve as therapy biomarkers Citation Format: Chiahao Wu, Chris Silvers, Elizabeths Guancial, Jong-Wei Hsu, Edward Messing, Yi-Fen Lee. Cancer exosome promotes cisplatin resistance In bladder cancer and inhibition of exosome sensitizes bladder cancer cells to cisplatin chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2015-5475

Protein expression profile related to cisplatin resistance in bladder cancer cell lines detected by two-dimensional gel electrophoresis.

We used a proteomic approach to compare the differentially regulated protein expression profiles of cisplatin-naïve and cisplatin-resistant bladder cancer cell lines to screen candidate molecules related to cisplatin resistance. The cisplatin-resistant cell line T24 was established by the stepwise exposure of T24 cells to up to 40 μM of cisplatin. We performed a comprehensive study of protein expression in bladder cancer cell lines that included cisplatin-naïve (T24) and cisplatin-resistant cells (T24CDDPR) by means of agarose two-dimensional gel electrophoresis followed by analysis of liquid chromatography tandem mass spectroscopy. We identified 25 obviously different spots for T24 and T24 CDDPR. Seven spots had increased expression and 18 spots had decreased expression in T24CDDPR compared to those in T24. Cytoskeletal proteins and enzyme modulators were prominent among differential proteins. Of the 25 proteins, we selected HNRNPA3, PCK2, PPL, PGK1, TKT, SERPINB2, GOT2, and EIF3A for further validation by Western blot. HNRNPA3, PGK1, TKT, and SERPINB2 had more than 1.5-times incremental expression in T24CDDPR compared to that in T24. PCK2 and PPL expressions were decreased less than 20% in T24CDDPR compared to that in T24. The results of 25 new proteins in this study could be valuable and could lead to the development of a new molecular marker.