Cisplatin-resistant Bladder Cancer Cell Lines Research Articles

Triple Combination of Entinostat, a Bromodomain Inhibitor, and Cisplatin Is a Promising Treatment Option for Bladder Cancer.

Cisplatin is part of the first-line treatment of advanced urothelial carcinoma. Cisplatin resistance is a major problem but may be overcome by combination treatments such as targeting epigenetic aberrances. Here, we investigated the effect of the class I HDACi entinostat and bromodomain inhibitors (BETis) on the potency of cisplatin in two pairs of sensitive and cisplatin-resistant bladder cancer cell lines. Cisplatin-resistant J82cisR and T24 LTT were 3.8- and 24-fold more resistant to cisplatin compared to the native cell lines J82 and T24. In addition, a hybrid compound (compound 20) comprising structural features of an HDACi and a BETi was investigated. We found complete (J82cisR) or partial (T24 LTT) reversal of chemoresistance upon combination of entinostat, JQ1, and cisplatin. The same was found for the BETis JQ35 and OTX015, both in clinical trials, and for compound 20. The combinations were highly synergistic (Chou Talalay analysis) and increased caspase-mediated apoptosis accompanied by enhanced expression of p21, Bim, and FOXO1. Notably, the combinations were at least 4-fold less toxic in non-cancer cell lines HBLAK and HEK293. The triple combination of entinostat, a BETi, and cisplatin is highly synergistic, reverses cisplatin resistance, and may thus serve as a novel therapeutic approach for bladder cancer.

KLF1 Activates RAC3 to Mediate Fatty Acid Synthesis and Enhance Cisplatin Resistance in Bladder Cancer Cells.

While cisplatin remains a frontline treatment for bladder cancer (BCa), the onset of resistance greatly hampers its effectiveness. RAC3 is closely linked to chemoresistance in cancer cells, but its specific role in cisplatin resistance within BCa is still elusive. RAC3 expression in BCa was analyzed using bioinformatics and quantitative polymerase chain reaction (qPCR). The gene set enrichment analysis (GSEA) identified RAC3-enriched pathways and the correlation between RAC3 and fatty acid synthase (FASN), a gene involved in fatty acid synthesis. Potential upstream transcription factors of RAC3 were predicted and their interaction with RAC3 was confirmed via dual-luciferase and chromatin immunoprecipitation (ChIP) assays. T24/DDP, a cisplatin-resistant BCa cell line, was established to probe into the regulatory role of RAC3 in cisplatin resistance. Cell proliferation was evaluated by colony formation and the IC50 values after cisplatin treatment were determined using cell counting kit-8 (CCK-8). The levels of free fatty acids and triglycerides (TGs), as well as the expression of DGAT2 and FASN proteins, were measured to gauge the extent of fatty acid synthesis in cells. Elevated expression of RAC3 was observed in BCa and the cisplatin-resistant BCa cells (T24/DDP). The knockdown of RAC3 within T24/DDP cells was demonstrated to counteract cisplatin resistance. Subsequent analyses identified RAC3 as being notably enriched in the fatty acid synthesis pathway, with Kruppel-like factor 1 (KLF1) emerging as a key upstream regulator. The overexpression of RAC3 was correlated with increased cisplatin resistance in T24/DDP cells, an effect that was mitigated by the addition of the FASN inhibitor, Orlistat. Furthermore, the downregulation of KLF1 suppressed RAC3 expression, disrupted fatty acid synthesis, and attenuated cisplatin resistance in T24/DDP cells. Conversely, the co-overexpression of RAC3 counteracted the effects conferred by KLF1 knockdown. Our study has validated that KLF1 activates RAC3 to mediate fatty acid synthesis and promote cisplatin resistance in BCa, suggesting the KLF1/RAC3 axis as a potential target for combating cisplatin-resistant BCa.

Cabazitaxel as a promising therapy for cisplatin-resistant bladder cancer: a preliminary study.

Bladder cancer is a common malignancy worldwide, posing a substantial healthcare challenge. Current standard treatment regimens are primarily based on cisplatin, but their success is often limited by cisplatin resistance and associated toxicities. Therefore, there is an urgent need to develop effective and less toxic therapies as alternatives to cisplatin. We screened the activity of FDA-approved anti-cancer drugson a panel of cisplatin-resistant bladder cancer cell lines. Based on initial responses, cabazitaxel was selected for further evaluation of its inhibitory effects on the phenotypic properties of these cells. Cabazitaxel, primarily used for metastatic castration-resistant prostate cancer, demonstrated remarkable efficacy in inhibiting colony formation, proliferation, and migration of cisplatin-resistant bladder cancer cells. This study highlights the potential of drug repurposing as a cost-effective and efficient strategy to overcome drug resistance in bladder cancer.

CAB39 promotes cisplatin resistance in bladder cancer via the LKB1-AMPK-LC3 pathway

Systemic therapy for muscle-invasive bladder cancer (BC) remains dominated by cisplatin-based chemotherapy. However, resistance to cisplatin therapy greatly limits long-term survival. Resistance to cisplatin-based chemotherapy still needs to be addressed. In this study, we established three cisplatin-resistant BC cell lines by multiple cisplatin pulse treatments. Interestingly, after exposure to cisplatin, all cisplatin-resistant cell lines showed lower reactive oxygen species (ROS) levels than the corresponding parental cell lines. Using proteomic analysis, we identified 35 proteins that were upregulated in cisplatin-resistant BC cells. By knocking down eleven of these genes, we found that after CAB39 knockdown, BC cisplatin-resistant cells were more sensitive to cisplatin. Overexpression of CAB39 had the opposite effect. Then, the knockdown of six genes downstream of CAB39 revealed that CAB39 promoted cisplatin resistance in BC through LKB1. Moreover, a key cause of cisplatin-induced cell death is damage to mitochondria and increased ROS levels. In our study, cisplatin-resistant cells exhibited higher autophagic flux and healthier mitochondrial status after cisplatin exposure. We demonstrated that the CAB39-LKB1-AMPK-LC3 pathway plays a critical role in enhancing autophagy to maintain the health of mitochondria and reduce ROS levels. In addition, the autophagy inhibitor chloroquine (CQ) can significantly enhance the killing effect of cisplatin on BC cells. Compared with gemcitabine plus cisplatin (GC), GC plus CQ significantly reduced tumor burden in vivo. In conclusion, our study shows that CAB39 counteracts the killing of cisplatin by enhancing the autophagy of BC cells to damaged mitochondria and other organelles to alleviate the damage of cells caused by harmful substances such as ROS.

Abstract 3908: M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer

Abstract BACKGROUND: Chemotherapy resistance is recognized to occur not only through selection of pre-existing genetically resistant clones, but also through rapid phenotypic plasticity mechanisms. We previously reported that bladder cancer cells can rapidly transition to and from a chemo-resistant phenotype. Recently, N6-methyladenosine (m6A) RNA modifications were shown to dynamically and reversibly regulate mRNA processing, differentiation, and cell fate. Here, we tested the hypothesis that m6A modifications regulate phenotypic plasticity and transition to cisplatin resistance in bladder cancer (BC). METHODS: We utilized methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) and by RNA-seq to identify transcripts that were both differentially methylated and differentially expressed between cisplatin-sensitive and cisplatin-resistant BC cell lines. Candidate transcripts were annotated using Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA). Clinical relevance was evaluated using databases such as The Cancer Genome Atlas (TCGA) and the Oncology Research Information Network (ORIEN) avatar. Cancer-relevant genes were then validated in vitro by targeted immunoprecipitation and PCR (MeRIP-PCR) and qPCR. Candidate genes were then functionally validated in cell lines and in patient derived organoids (PDOs) by siRNA-mediated knockdown and cisplatin treatment, as well as by m6A measurement after short-term cisplatin treatment. RESULTS: MeRIP-seq and RNA-seq revealed that cisplatin-sensitive and cisplatin-resistant BC cells have distinct m6A profiles, with 130 transcripts that were both differentially methylated and differentially expressed. Using clinical and functional database tools, we filtered this list to 37 transcripts and ranked them based on clinical relevance in BC. Of the top 15 candidates, eight were successfully validated in vitro via repeat qPCR and MeRIP-PCR. In cisplatin-resistant cells, one candidate, SLC7A11, was found to have decreased m6A level, which was associated with increased RNA stability and elevated transcript and protein levels. Consistent with this, 48 hr cisplatin treatment of cisplatin-sensitive cells led to SLC7A11 hypomethylation, increased RNA stability, and elevated transcript and protein levels. Furthermore, depletion of SLC7A11 by siRNA-knockdown re-sensitized BC cell lines and PDOs to cisplatin. CONCLUSIONS: Using unbiased transcriptome-wide m6A profiling followed by targeted validation, we demonstrated that m6A modifications regulate expression of clinically relevant gene transcripts in BC, and that a subset of these modifications may promote resistance to chemotherapy Collectively, these results implicate epitranscriptomic plasticity as a driver in potentiating cisplatin resistance in bladder cancer. Citation Format: Emmanuelle Hodara, Aubree Mades, Tong Xu, Amir Goldkorn, Suhn Rhie. M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in 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 3908.

Identification of platinum resistance-related gene signature for prognosis and immune analysis in bladder cancer.

Purpose: Currently, there is limited knowledge about platinum resistance-related long non-coding RNAs (lncRNAs) in bladder cancer. We aim to identify platinum resistance-related lncRNAs and construct a risk model for accurate prognostic prediction of bladder cancer. Methods: Transcriptomic and clinical data were extracted from The Cancer Genome Atlas (TCGA) database, and platinum resistance-related genes were obtained from HGSOC-Platinum. The platinum resistance-related lncRNAs were obtained by the Spearman correlation analysis. Then, we constructed a risk score model through Cox regression analysis and the LASSO algorithm. The model was verified by analyzing the median risk score, Kaplan-Meier curve, receiver operating characteristic (ROC) curve, and heatmap. We also developed a nomogram and examined the relationship between the risk score model, immune landscape, and drug sensitivity. Lastly, we assessed the differential expression of PRR-lncRNAs in the cisplatin-resistant bladder cancer cell line and the normal bladder cancer cell line using qRT-PCR. Results: We developed and validated an eight-platinum resistance-related lncRNA risk model for bladder cancer. The risk model showed independent prognostic significance in univariate and multivariate Cox analyses. Based on multivariate analysis, we developed a nomogram. The modified model is both good predictive and clinically relevant after evaluation. Furthermore, immune-related and drug-sensitivity analyses also showed significant differential expression between high and low-risk groups. The qRT-PCR demonstrated that most of the lncRNAs were upregulated in cisplatin-resistance cancerous tissues than in control tissues. Conclusion: We have developed a predictive model based on eight platinum resistance-related lncRNAs, which could add meaningful information to clinical decision-making.

Vitamin D3 sensitizes resistant human bladder cancer cells to cisplatin by regulating Sirtuin 1 gene expression.

Cisplatin is a standard chemotherapeutic agent for advanced bladder cancer, but its efficacy is limited due to drug resistance. Vitamin D3 may reverse cancer multidrug resistance, but the potential molecular mechanisms are still only partially known. The purpose of this study was to explore the mechanism by which vitamin D3 reverses cisplatin resistance in bladder cancer to improve therapeutic efficacy and ameliorate the prognosis of cisplatin-resistant bladder cancer. The levels of vitamin D3 and sirtuin 1 protein were detected in cisplatin-resistant bladder cancer patients and cisplatin-sensitive patients. The cisplatin-resistant bladder cancer cell lines T24/DDP and UMUC3R were used as cell experimental models, and the migration, apoptosis, mitochondrial reactive oxygen species accumulation and autophagy of cells were assessed in the present study. Vitamin D3 levels were decreased, and sirtuin 1 protein levels were increased in cisplatin-resistant bladder cancer patients compared with cisplatin-sensitive bladder cancer patients. Vitamin D3 treatment markedly repressed sirtuin 1 expression, and overexpression of the sirtuin 1 gene led to mitochondrial reactive oxygen species generation, promoted the initiation of autophagosome formation and enhanced autophagic flux. Cisplatin treatment in the presence of vitamin D3 inhibited cell invasion and migration and induced apoptosis and enhancing the sirtuin 1 gene abolished the effect of vitamin D3 by regulating mitochondrial reactive oxygen species accumulation and autophagosome formation. These data support a mechanism wherein the sirtuin 1 gene plays a crucial role in vitamin D3 reversing cisplatin resistance in bladder cancer and may provide useful preventive and therapeutic applications in the future.

Allyl-, Butyl- and Phenylethyl-Isothiocyanate Modulate Akt-mTOR and Cyclin-CDK Signaling in Gemcitabine- and Cisplatin-Resistant Bladder Cancer Cell Lines.

Combined cisplatin–gemcitabine treatment causes rapid resistance development in patients with advanced urothelial carcinoma. The present study investigated the potential of the natural isothiocyanates (ITCs) allyl-isothiocyanate (AITC), butyl-isothiocyanate (BITC), and phenylethyl-isothiocyanate (PEITC) to suppress growth and proliferation of gemcitabine- and cisplatin-resistant bladder cancer cells lines. Sensitive and gemcitabine- and cisplatin-resistant RT112, T24, and TCCSUP cells were treated with the ITCs, and tumor cell growth, proliferation, and clone formation were evaluated. Apoptosis induction and cell cycle progression were investigated as well. The molecular mode of action was investigated by evaluating cell cycle-regulating proteins (cyclin-dependent kinases (CDKs) and cyclins A and B) and the mechanistic target of the rapamycin (mTOR)-AKT signaling pathway. The ITCs significantly inhibited growth, proliferation and clone formation of all tumor cell lines (sensitive and resistant). Cells were arrested in the G2/M phase, independent of the type of resistance. Alterations of both the CDK–cyclin axis and the Akt–mTOR signaling pathway were observed in AITC-treated T24 cells with minor effects on apoptosis induction. In contrast, AITC de-activated Akt–mTOR signaling and induced apoptosis in RT112 cells, with only minor effects on CDK expression. It is concluded that AITC, BITC, and PEITC exert tumor-suppressive properties on cisplatin- and gemcitabine-resistant bladder cancer cells, whereby the molecular action may differ among the cell lines. The integration of these ITCs into the gemcitabine-/cisplatin-based treatment regimen might optimize bladder cancer therapy.

Histone Demethylase KDM7A Regulates Androgen Receptor Activity, and Its Chemical Inhibitor TC-E 5002 Overcomes Cisplatin-Resistance in Bladder Cancer Cells.

Histone demethylase KDM7A regulates many biological processes, including differentiation, development, and the growth of several cancer cells. Here, we have focused on the role of KDM7A in bladder cancer cells, especially under drug-resistant conditions. When the KDM7A gene was knocked down, bladder cancer cell lines showed impaired cell growth, increased cell death, and reduced rates of cell migration. Biochemical studies revealed that KDM7A knockdown in the bladder cancer cells repressed the activity of androgen receptor (AR) through epigenetic regulation. When we developed a cisplatin-resistant bladder cancer cell line, we found that AR expression was highly elevated. Upon treatment with TC-E 5002, a chemical inhibitor of KDM7A, the cisplatin-resistant bladder cancer cells, showed decreased cell proliferation. In the mouse xenograft model, KDM7A knockdown or treatment with its inhibitor reduced the growth of the bladder tumor. We also observed the upregulation of KDM7A expression in patients with bladder cancer. The findings suggest that histone demethylase KDM7A mediates the growth of bladder cancer. Moreover, our findings highlight the therapeutic potential of the KMD7A inhibitor, TC-E 5002, in patients with cisplatin-resistant bladder cancer.

Abstract B11: Multi-omic interrogation of gemcitabine and cisplatin-resistant bladder cancer cell lines identifies unique and shared mediators of chemosensitivity and resistance

Abstract Neoadjuvant platinum-based chemotherapy (NAC) followed by radical cystectomy is the preferred first-line option for treating patients diagnosed with muscle-invasive bladder cancer (MIBC). One of the two most commonly utilized chemotherapy regimens for MIBC consists of combination therapy with gemcitabine and cisplatin (GC). Importantly, cisplatin-based chemotherapy leads to complete response (pT0) in approximately 25% of patients, while the remaining 75% are left with residual disease. For MIBC patients, the presence of residual disease at cystectomy is associated with significantly poorer prognosis. Previous efforts to screen tumor-derived specimens have identified a number of mutational biomarkers associated with NAC response; however, to date these efforts have been limited by the number of patients profiled and the molecular and mutational heterogeneity intrinsic to bladder cancers. Moreover, these efforts have primarily relied on a single technology (e.g., whole-exome sequencing or transcriptome profiling), and were not designed to look at GC-resistance patterns in a way that integrates these data types. Therefore, we sought to systematically define the molecular phenotypes associated with chemoresistant bladder cancer cells, and to functionally interrogate these features to better define essential genes and pathways that govern chemoresponsiveness. To accomplish this, we have extensively characterized a panel of six bladder cancer cell lines (KU-19-19, T24, TCCSUP, 253J, 5637, and RT112), each of which has three derivative cell lines with acquired resistance to gemcitabine, cisplatin, and GC combination. For all of these cell lines we have performed whole-transcriptome mRNA sequencing, whole-exome sequencing, and reverse phase protein microarray. Additionally, we have performed functional genomic screening using whole-genome CRISPR knockout libraries, in the combination GC-resistant derivative cell lines. Using these data we identified multiple important chemoresistance genes and pathways, both previously known and novel. Interestingly, even though we found heterogeneous expression patterns seen across different –omic platforms, the functional genomic screens in GC-resistant cell lines converged on many genes and pathways that are essential to DNA damage response. These data suggest that functional genomic screens are perhaps more likely to identify genes and pathways that are directly linked to each of these drugs' specific mechanisms of action, as opposed to context-specific molecular differences seen across cell lines. Importantly, these results could enable improved prediction and personalization of therapy through identifying biomarkers that predict chemosensitivity and resistance. This study also suggests that identifying more generalizable strategies to overcome chemoresistance could increase the number of bladder cancer patients experiencing deeper and more complete responses to these common chemotherapeutic drugs. Citation Format: Robert T. Jones, Tahlita C. M. Zuiverloon, Hedvig Vekony, Andrew Goodspeed, Teemu D. Laajala, Molishree Joshi, Colin Sempeck, Annie Jean, Megan Tu, Julia D. Wulfkuhle, Emanuel F. Petricoin, James C. Costello, Dan Theodorescu. Multi-omic interrogation of gemcitabine and cisplatin-resistant bladder cancer cell lines identifies unique and shared mediators of chemosensitivity and resistance [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 B11.

CD40 is Positively Correlated with the Expression of Nucleophosmin in Cisplatin-Resistant Bladder Cancer.

Objective To verify and evaluate the value of CD40 as a noninvasive biomarker of cisplatin-resistant bladder cancer, we studied the expression of CD40 and the correlation between nucleophosmin (NPM1) and CD40 in cisplatin-resistant bladder cancer. Methods Three cisplatin-resistant bladder cancer cell lines (T24/0.8DDP, BIU87/0.3DDP, and PUMC-91/0.6DDP) were studied, and lentivirus was used to silence NPM1 expression. The expression of CD40 and NPM1 in three NPM1 silencing bladder cancer cell lines were detected by fluorescence microscopy and Western Blot. The effects and proteomic bioinformatics of NPM1 gene knockout on cisplatin-resistant bladder cancer cells were analyzed by liquid chromatography-mass spectrometry (LC-MS) and gene ontology analysis (GO analysis). Results The NPM1 gene was successfully silenced in three drug-resistant bladder cancer cell lines by lentivirus infection. The knockdown efficiency was 70%. After NPM1 gene knockout, 492 differential proteins were detected by LC-MS, whose fold change was more than 1.5 (p < 0.05). A total of 57022 peptides, 54347 unique peptides, and 6686 protein groups were identified in all proteins of the tested cells (FDR < 0.01). Hierarchical clustering and principal component analysis showed that 264 functional proteins were downregulated and 228 functional proteins were upregulated in the gene silencing group compared with those of the negative controls. By GO analysis, the proteins affected by NPM1 cover a large number of proteins with biological functions, which may play an important role in the development of tumor in 492 differential proteins. The CD40 was the most significantly downregulated protein after NPM1 silencing, with a difference of 2.6-fold change in abundance. Conclusions There is a positive correlation between CD40 protein and NPM1 protein in drug-resistant bladder cancer. Because NPM1 can reflect the characteristics of bladder cancer, CD40 may be a more sensitive marker for monitoring the prognosis of bladder cancer.

Myeloid-derived suppressor cells are essential partners for immune checkpoint inhibitors in the treatment of cisplatin-resistant bladder cancer.

Myeloid-derived suppressor cells (MDSCs) are one of the key players that contribute to immune evasion. The purpose of the present study was to investigate whether MDSCs could be a novel target for the treatment of cisplatin-resistant bladder cancer. We established cisplatin-resistant bladder cancer cell lines (MB49R, MBT-2R, and T24R) and evaluated chemokine expression and MDSC expansion. We also assessed the antitumor effect by depleting MDSCs with or without a α-PD-L1 antibody using MB49R xenograft models. The chemokine expression of CXCL1, CXCL2, and CCL2 increased in cisplatin-resistant cells compared to those in their parent strains. Monocytic MDSCs (Mo-MDSCs) were observed more frequently compared to polymorphonuclear MDSCs (PMN-MDSCs) in MB49R tumors. The immunosuppressive genes arginase 1 and iNOS were comparably expressed in each MDSC subtype. In vivo, combination therapy targeting both PMN- and Mo-MDSCs using α-Gr1 and α-Ly6C antibodies significantly reduced tumor volume with increased infiltration of CD8 T cells in the tumor. Finally, co-targeting pan-MDSCs and PD-L1 remarkably reduced the tumor growth. These findings suggest that targeting MDSCs might enhance the therapeutic effect of immune checkpoint inhibitors in cisplatin-resistant bladder cancers.

Distinctive mutational spectrum and karyotype disruption in long-term cisplatin-treated urothelial carcinoma cell lines

The DNA-damaging compound cisplatin is broadly employed for cancer chemotherapy. The mutagenic effects of cisplatin on cancer cell genomes are poorly studied and might even contribute to drug resistance. We have therefore analyzed mutations and chromosomal alterations in four cisplatin-resistant bladder cancer cell lines (LTTs) by whole-exome-sequencing and array-CGH. 720–7479 genes in the LTTs contained point mutations, with a characteristic mutational signature. Only 53 genes were mutated in all LTTs, including the presumed cisplatin exporter ATP7B. Chromosomal alterations were characterized by segmented deletions and gains leading to severely altered karyotypes. The few chromosomal changes shared among LTTs included gains involving the anti-apoptotic BCL2L1 gene and losses involving the NRF2 regulator KEAP1. Overall, the extent of genomic changes paralleled cisplatin treatment concentrations. In conclusion, bladder cancer cell lines selected for cisplatin-resistance contain abundant and characteristic drug-induced genomic changes. Cisplatin treatment may therefore generate novel tumor genomes during patient treatment.

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.

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.

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.

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.

Abstract A10: MicroRNA 27a is downreguated in cisplatin resistant bladder cancer cells, and contributes to resistance through the targeting of xCT, a cystine transporter involved with glutathione production

Abstract Cisplatin resistance represents a serious obstacle to the chemotherapeutic treatment of many tumors. The purpose of this study was to create a cell culture model of cisplatin resistant bladder cancer (CRBC), then use this model to identify changes in the expression of microRNAs (miRs) that may influence resistance. Single cell clones resistant to cisplatin were generated from parental bladder cancer cell lines by continuous culture in increasing concentrations of cisplatin for several months. The resultant CRBC cell lines were able to tolerate high doses of cisplatin (typically &amp;gt; 10 μM). MicroRNA expression in parental and CRBC cell lines was measured using q-pcr based low-density tiling arrays and a panel of miRs which were consistently altered in all CRBC cell lines was established. In addition the induction and repair of specific cisplatin-DNA adducts in parental and CRBC cell lines was assessed using antibodies against the guanine–guanine (Pt-[GG]) intrastrand crosslink. Four fold more cisplatin was required to produce the equivalent damage in CRBC cell lines compared to the parental control, while the rate of repair, as assessed by measurement of the removal of cisplatin-DNA adducts over time, remained broadly similar between parental and resistant lines. This demonstrated that resistance was due to reduced adduct formation rather than any defect in cross-link repair. Glutathione (GSH) inactivates cisplatin by binding with it irreversibly to form Pt(SG)2 adducts, thus preventing the drug from forming cytotoxic adducts with DNA. One cause of decreased adduct formation is therefore deregulation of the GSH biosynthesis pathway. Upon examination intracellular levels of GSH and its oxidized form glutathione disulfide (GSSG) were found to be increased in resistant cells. The expression of a number of genes involved with the synthesis of GSH was then measured by rt-pcr and western blotting, and several key proteins involved with GSH biosynthesis were found to be upregulated in resistant cells, suggesting increased levels of intracelluar GSH could be responsible for the observed decrease in the levels of cisplatin-DNA adducts observed in resistant cells. The 3′ untranslated regions (3′ UTRs) of these mRNAs were examined using TargetScan, an online miR target prediction program, to find potential sites of miR regulation. The most upregulated protein, xCT (SLC7A11), responsible for cystine import (known to be the rate-limiting step in GSH synthesis) was found to contain target sites for three miRs which had been shown to be significantly downregulated in our model of CRBC, namely miRs 25, 27a and 32. Upregulation of miRs 25, 27a and 32 by transfection of resistant cells with the pri-miR precursor molecules revealed that restoring expression of miR-27a resensitized CRBC cells to the cytotoxic effects of cisplatin. Restoration of miR-27a expression in CRBC cells was also shown to result in a downregulation of the expression of xCT at both the protein and mRNA level. In summary, we have identified a panel of microRNAs that are consistently dysregulated in CRBC, and implicated one of these (miR-27a) in the modulation of cisplatin resistance via increased glutathione production. We also provide evidence that reversal of this change in miR expression results in a reversal of the effects on downstream target, and critically, a reversal of the cisplatin resistant phenotype. The contribution made by miR-27a to the development of cisplatin resistance might represent a potential site for therapeutic intervention in the treatment of CRBC. Measurement of miR-27a expression in tumor material or urine may also serve as a predictive biomarker for patients likely response to cisplatin chemotherapy. Citation Format: Ross M. Drayton, Ewa Dudziec, James W.F. Catto, Helen E. Bryant. MicroRNA 27a is downreguated in cisplatin resistant bladder cancer cells, and contributes to resistance through the targeting of xCT, a cystine transporter involved with glutathione production [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A10.

Enhanced S100 calcium‐binding protein P expression sensitizes human bladder cancer cells to cisplatin

• To investigate the role of S100 calcium-binding protein P (S100P) in the gain of cis-diamminedichloroplatinum (II) (cisplatin) resistance in bladder cancer, having previously found, with cDNA microarrays using two pairs of parental (T24, KK47) and their cisplatin-resistant bladder cancer cell lines (T24/DDP10, KK47/DDP20), that S100P mRNA expression was significantly reduced in cisplatin-resistant cells. • S100P mRNA and protein expression levels were investigated by northern and western blot analyses, respectively. • Intracellular S100P localization was examined by immunocytochemistry and immunohistochemistry. • S100P over-expression, obtained by transfection with S100P expression plasmid, was used to investigate whether or not S100P affected cellular resistance to cisplatin. • S100P mRNA showed increased expression by cisplatin stimulation in parental cell lines. • On the other hand, S100P mRNA and protein expression levels were markedly reduced in cisplatin-resistant cells. • The over-expression of S100P in resistant cells resulted in an increased sensitivity to cisplatin. • In bladder cancer cells, S100P was expressed and localized mainly in the nucleus. • S100P expression was also involved in cisplatin sensitivity. • S100P might thus represent a molecular marker predicting cisplatin sensitivity and a molecular therapeutic target for cisplatin-based chemotherapy.

Inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1) modulates the acquisition of cisplatin resistance in bladder cancer cell lines

To investigate the molecules that regulate the acquisition of cis-diamminedichloroplatinum (II) (cisplatin) resistance, we performed cDNA microarrays using two pairs of parental and cisplatin-resistant bladder cancer cell lines. We found a markedly reduced expression of inositol 1,4,5-trisphosphate (IP3) receptor type1 (IP3R1), endoplasmic reticulum membrane protein, in cisplatin-resistant cells. The suppression of IP3R1 expression using small interfering RNA in parental cells prevented apoptosis and resulted in decreased sensitivity to cisplatin. Contrarily, overexpression of IP3R1 in resistant cells induced apoptosis and increased sensitivity to cisplatin. These results suggest that cisplatin-induced downregulation of IP3R1 expression was closely associated with the acquisition of cisplatin resistance in bladder cancer cells.