Resistant Bladder Cancer Research Articles

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.

Abstract 97: RNA sequencing of bladder cancer patient-derived xenograft models identifies genes associated with chemoresistance

Abstract Background: Bladder cancer is among the top ten most common cancers, with about ~380,000 new cases and ~150,000 deaths per year worldwide. Platinum-based combination chemotherapy is commonly used to treat advanced bladder cancer. It has been shown that only ~50% of the patients with advanced bladder cancer respond to platinum-based therapy. Methods: We have utilized a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms that contribute to resistance of gemcitabine-cisplatin combination therapy in advanced bladder cancer. We have also identified key regulatory pathways in our PDX models that can be targeted to treat chemotherapy resistant bladder cancer using RNAseq analysis. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis, before and after a 21-day cisplatin/gemcitabine drug treatment regimen. Results: The RNA-seq data has indicated significant differences between the transcriptional profiles of drug-sensitive and drug-resistant tumors. PDXs retained morphology and shared 92-97% of genetic alterations of parental cancer cells. We identified 333 genes >2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Significantly up-regulated genes in drug resistant tumors analyzed include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane protein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment. Additional changes in gene expression based on RNA-seq data before and after drug treatment were also found. Conclusion: Chemoresistance to gemcitabine and cisplatin is associated with altered expression of several cell surface proteins and upregulation of the downstream signaling pathways. Targeting these cell surface proteins can possibly be harnessed to overcome chemoresistance. GPNMB is a type I transmembrane protein that has previously been shown to be up-regulated in many metastatic cancers including breast cancer. Potentially targeting GPNMB with glembatumumab vedotin, an antibody-drug-conjugate has shown promising results in treating several cancers including breast cancer and osteosarcoma. Further studies will elucidate whether targeting GPNMB is an effective strategy for the treatment of chemotherapy resistant bladder cancer. This study received funding by a developmental grant from the UCDCC. This work was conducted under the auspices of the USDOE by LLNL (DE-AC52-07NA27344). IM number: LLNL-688318 Citation Format: Kelly A. Martin, Nicholas R. Hum, Aimy Sebastian, Deepa K. Murugesh, Chong-Xian Pan, Ai-Hong Ma, Ralph de Vere White, Gaby Loots. RNA sequencing of bladder cancer patient-derived xenograft models identifies genes associated with chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 97. doi:10.1158/1538-7445.AM2017-97

Activity of RX-3117, an oral antimetabolite nucleoside, in subjects with metastatic bladder cancer resistant to gemcitabine: Preliminary results of a phase Ia/IIb study.

4544 Background: RX-3117 is an oral small molecule antimetabolite, cyclopentyl pyrimidyl nucleoside that is activated by uridine cytidine kinase 2. RX-3117 has shown efficacy in xenograft models of gemcitabine resistant pancreatic, bladder and colorectal cancer. Preliminary data from an analysis of a phase 1b/2a clinical study of RX3117 in metastatic bladder cancer is described. Methods: This phase 1b/2a study (NCT02030067) was designed to evaluate safety, tolerability and efficacy following treatment with 700 mg administered orally once-daily for 5 consecutive days with 2 days off per week for 3 weeks with 1 week off in each 4 week cycle in a 2-stage design. Eligible subjects (aged ≥ 18 years) were those with relapsed/refractory metastatic bladder cancer with any number of prior therapies. Prior therapy with platinum-based chemotherapy was required. The primary endpoint was to assess the efficacy and safety of RX-3117 in metastatic bladder cancer, with secondary aims of evaluating PFS and CBR. Results: With 9 subjects enrolled, median age was 66 years, ECOG PS was 0-1. All subjects had received gemcitabine/cisplatin in the perioperative or metastatic setting, and 4 subjects had received 3 or more prior therapies. The most frequent related adverse events were anemia, mild-moderate fatigue, vomiting and diarrhea. No dose limiting toxicities were observed. PFS and CBR will be presented at the meeting, as 5 subjects continue to receive therapy at the time of this submission. One subject continues on treatment at 139 days with persistent stable disease. Molecular profiling of his bladder tumor showed alterations in ARID1A, FBXW7, FGFR3, NF1, and TERT. The patient previously responded to an FGFR3 inhibitor but progressed after 9 months, with ctDNA assessments showing incurrence of TP53 alteration. Clinical benefit with RX-3117 was achieved in spite of incurrence of this alteration. Conclusions: RX-3117 demonstrated an excellent safety profile, and prolonged stable disease was seen in 1 subject who failed prior cisplatin/gemcitabine and FGFR3 inhibition. Activity persisted despite development a putative resistance alteration detected by ctDNA. Clinical trial information: NCT02030067.

Mitochondria-Targeted Photodynamic Therapy with a Galactodendritic Chlorin to Enhance Cell Death in Resistant Bladder Cancer Cells.

Here, we report the rational design of a new third-generation photosensitizer (PS), a chlorin conjugated with galactodendritic units, ChlGal8, to improve the effectiveness of bladder cancer treatment. ChlGal8 shows better photochemical and photophysical properties than a recently reported homologous porphyrin, PorGal8. In addition to inheriting excellent photostability, the ability to generate singlet oxygen, and the ability to interact with the proteins galectin-1 and human serum albumin (HSA), ChlGal8 exhibits high absorption in the red region of the electromagnetic spectrum. In vitro studies of anticancer activity of ChlGal8 revealed that once this PS is taken up by UM-UC-3 bladder cancer cells, it induces high cytotoxicity after a single dose of light irradiation. In HT-1376 bladder cancer cells resistant to therapy, a second light irradiation treatment enhanced in vitro and in vivo photodynamic efficacy. The enhanced phototoxicity in HT-1376 cancer cells seems to be due to the ability of ChlGal8 to accumulate in the mitochondria, via facilitative glucose transporter 1 (GLUT1), in the period between single and repeated irradiation. A photodynamic therapy (PDT) regimen using an extra dose of light irradiation and ChlGal8 as PS represents a promising strategy in treating resistant cancers in a clinical setting.

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.

Expression of long non-coding RNA UCA1 and miR-34b in bladder cancer and analysis of clinic-pathologic correlation

Objective To study the expression of long non-coding RNA (lncRNA)- urothelial carcinoma associated 1(UCA1) and miR-34b in bladder cancer and its correlation to the clinicopathologic features of bladder cancer. Methods Between January 2011 and October 2012 , the expression of UCA1 and miR-34b in 5 bladder cancer cell lines (T24, BIU-87, EJ, T24-MMC, T24-ADM)and 1 normal bladder cell lines (SV-HUC-1) were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). Meanwhile, the 56 bladder cancer specimens and paraneoplastic normal bladder tissues, which diagnosed by pathology were collected from bladder cancer patients undergoing radical resection of bladder. Among them, 41 cases were male and 15 cases were female. The mean age was (68.4±7.5)years old, range 52 to 78 years.43 cases were older than 65 years old, and 13 cases were less than 65 years old. The pathological classification included non muscle-invasive bladder cancer (NMIBC) 18 cases, muscle-invasive bladder cancer 38 cases; low grade papillary urothelial carcinoma 22 cases, high grade papillary urothelial carcinoma 34 cases; 12 cases were primary lesion, the other 44 cases were diagnosed as tumor recurrence. Real-time RT-PCR was performed to analyze the expression of UCA1 and miR-34b. Results The relative expression levels of UCA1 in the normal bladder cell lines (SV-HUC-1) and 5 bladder cancer cell lines (T24, BIU-87, EJ, T24-MMC and T24-ADM)were (0.0675±0.0133), (0.2934±0.0531), (0.4246±0.0650), (0.4206±0.0826), (0.6472±0.0875) and (0.7165±0.1032), respectively (P<0.05). Moreover, the expression levels of UCA1 were up-regulated in 2 drug resistant bladder cancer cells lines T24-MMC(0.6472±0.0875)and T24-ADM(0.7165±0.1032), as compared with the T24 bladder cancer lines (0.2934±0.0531) , respectively (P<0.05). However, the expression levels of miR-34b in 5 bladder cancer cell lines [T24(0.1600±0.0455), BIU-87(0.1720±0.0658), EJ(0.1150±0.0352), T24-MMC(0.0576±0.0087), T24-ADM(0.0510±0.0125)]were decreased(P<0.05), as compared with normal bladder cell lines SV-HUC-1(0.6384±0.1083). Moreover, the expression levels of miR-34b were down-regulated in 2 drug resistant bladder cancer cells lines T24-MMC(0.0576±0.0087)and T24-ADM(0.0510±0.0125), as compared with the T24 bladder cancer lines T24(0.1600±0.0455), respectively (P<0.05). The relative expression levels of UCA1 and miR-34b in bladder cancer tissues and paraneoplastic normal bladder tissues were (0.4225±0.0714)vs.(0.0532±0.0192)and(0.0340±0.0134)vs.(0.5643±0.0616), respectively (P<0.05). Statistical correlation analysis showed that UCA1 to be significantly negative correlated with miR-34b in bladder cancer specimens(r=-0.54, P<0.05). The high level of UCA1 and low level of miR-34b were significantly correlated with tumor malignant grade, invasiveness and recurrence. The 3-year overall survival rate (OS) in UCA1(+ )/miR-34b(-) group (27.6%) were significantly worse compared with non UCA1(+ )/miR-34b(-) group(73.7%). Conclusion High expression of UCA1 and low expression of miR-34b were associated with the occurrence and development of bladder cancer. Key words: Urinary bladder neoplasms; Long non-coding RNA; Urothelial carcinoma associated 1; MicoRNAs

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 2106: Transcriptome analysis of patient-derived bladder cancer xenografts identifies genes associated with chemoresistance

Abstract Background: Bladder cancer is among the ten most common cancers, with about ∼380,000 new cases and ∼150,000 deaths per year worldwide. Platinum-based combination chemotherapy is commonly used to treat advanced bladder cancer. It has been shown that only ∼50% of the patients with advanced bladder cancer respond to platinum-based therapy. Methods: We employed a patient-derived bladder cancer xenograft (PDX) platform to characterize the molecular mechanisms contributing to resistance to gemcitabine-cisplatin combination therapy in advanced bladder cancer and to identify novel candidates that can be targeted to treat chemotherapy resistant bladder cancer. Transcriptome profiling of P0 (passage 0) bladder cancer xenograft tumors from 4 PDX lines (2 gemcitabine-cisplatin resistant lines and 2 drug sensitive lines) was performed by RNA-Seq analysis. Results: PDXs retained the morphology fidelity and shared 92-97% of genetic alterations of parental cancer cells. The RNA-seq data suggested the presence of significant differences between the transcription profiles of drug-sensitive and drug-resistant tumors. We identified 333 genes &amp;gt;2 fold up or down regulated in the drug resistant tumors compared to the drug sensitive tumors. Genes down-regulated in drug resistant tumors include tight junction protein CLDN3 and regulators of G-protein signaling RGS2 and RGS3. Significantly up-regulated genes include metabolic enzymes ALDH2, ALDH3A1, ALDH4A1 and ALDH7A1, transporter proteins ABCA1, SLC1A4, SLC2A5, SLC30A1, SLC39A6, SLC7A5 and SLC9A3, Notch ligand JAG2, Growth hormone receptor GHR and transmembrane glycoprotein GPNMB. Consistent with the change of cell surface proteins such as GHR and GPNMB, the MAPK and the PI3K-AKT pathways were upregulated when PDXs became resistant to cisplatin treatment. Conclusion: Chemoresistance to gemcitabine and cisplatin is associated with altered expression of several cell surface proteins and upregulation of the downstream signaling pathways. Targeting these cell surface proteins can possibly be harnessed to overcome chemoresistance. GPNMB, a type I transmembrane protein highly up-regulated in the drug resistant tumors, has previously been shown to be over-expressed in various cancers. Targeting GPNMB with an antibody-drug-conjugate, glembatumumab vedotin, has shown promising results in treating several cancers including breast cancer and osteosarcoma. Further studies will elucidate whether targeting GPNMB is an effective strategy for the treatment of chemotherapy resistant bladder cancer. Citation Format: Aimy Sebastian, Kelly A. Martin, Chong-xian Pan, Ai-hong Ma, Ralph W. deVere White, Gabriela G. Loots. Transcriptome analysis of patient-derived bladder cancer xenografts identifies genes associated with chemoresistance. [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 2106.

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.

A Potent Chemotherapeutic Strategy with Eg5 Inhibitor against Gemcitabine Resistant Bladder Cancer.

Development of resistance to gemcitabine is a major concern in bladder cancer therapy, and the mechanism remains unclear. Eg5 has been recently identified as an attractive target in cancer chemotherapy, so novel targeted chemotherapy with Eg5 inhibitor is expected to improve the anticancer effect in gemcitabine-resistant bladder cancer. In this research, RT112-Gr cells were 350-fold less sensitive to gemcitabine than the parental cell lines, while KU7-Gr cells were 15-fold less sensitive to gemcitabine than the parental cell lines. Human OneArray Microarray analysis was performed to obtain broad spectrum information about the genes differentially expressed in RT112 and RT112-Gr cells. The anti-proliferative activity of S(MeO)TLC, an Eg5 inhibitor, was analyzed in RT112-Gr cell lines using a cell viability assay. Furthermore, the inhibitory effect was evaluated in vivo using subcutaneous xenograft tumor model. According to the result of Human OneArray® GeneChip, RRM1 and RRM2 were up-regulated, while there was no significant change in Eg5. Trypan blue staining confirmed that in S(MeO)TLC and Gemcitabine combining S(MeO)TLC group cell viability were significantly decreased in RT112-Gr cells as compared with other groups. S(MeO)TLC and S(MeO)TLC+gemcitabine groups prominently suppressed tumor growth in comparison with other groups’ in vivo. There were no significant differences in S(MeO)TLC and gemcitabine+S(MeO)TLC group in the effect of inhibition of bladder cancer in vivo and in vitro. Our data collectively demonstrated that S(MeO)TLC represents a novel strategy for the treatment of gemcitabine resistant bladder cancer.

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

Abstract B21: Chemoresistant bladder cancer cells induces epithelial mesenchymal transition

Abstract Although chemotherapeutic treatments are efficacious in a number of cancers, some cancer cells ultimately will become resistant to chemotherapy. It has been suggested that chronic chemotherapy-resistance is linked to the induction of epithelial mesenchymal transition (EMT). EMT is associated with increased tumor invasiveness, development of angiogenesis, and the development of drug resistance and is linked to more aggressive cancer. The objective of this study is to determine if a link exists between mitomycin C (MMC)-resistant RT-4 bladder cancer cells and the induction of EMT. EMT is a process where cells acquire molecular alterations characterized by loss of cell-cell adhesion, repression of E-cadherin expression, and increased cell mobility. Clinical studies have shown that as bladder cancer grade increases, tumors typically exhibit a decreased expression of E-cadherin, a cell-cell adhesion molecule. In addition, there is an increase in N-cadherin expression in muscle invasive bladder tumors. We have shown that MMC-resistant human RT-4 bladder cancer cells demonstrate a decrease in cell-cell adhesion, a higher invasion potential, and a higher CD44(+)/CD24(-) ratio (96.4%) compared to parental human RT-4 bladder cancer cells. In addition, the invasion capacity of MMC-resistant RT-4 bladder cancer cells was higher than that of parental cells. The MMC-resistant RT-4 bladder cancer cells expressed higher levels of N-cadherin and numerous apoptotic related proteins including pSTAT3, RAD17, Bcl-X and catalase. Using an exon array we showed that the MMC-resistant RT-4 bladder cancer cells up-regulate several EMT markers including vimentin (77 times), transforming growth factor -beta (27 times), and Zinc finger E-box-binding homeobox 1 (33 times) and down-regulate E-cadherin (71 times). In summary, in our MMC-resistant RT4 bladder cancer cells, EMT was activated, and there was an increase in apoptotic related proteins as well as an increase N-cadherin expression. Our data suggest a link between chemotherapy-resistant bladder cancer cells and EMT. Deactivating EMT and down-regulating apoptotic related proteins may offer a novel strategy for overcoming bladder cancer drug resistance. Citation Format: Jingchun Liu, Darryl T. Martin, Marcia A. Wheeler, Robert M. Weiss. Chemoresistant bladder cancer cells induces epithelial mesenchymal transition. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B21.

MiR-193a-3p promotes the multi-chemoresistance of bladder cancer by targeting the HOXC9 gene

Chemoresistance prevents the curative cancer chemotherapy and presents a formidable challenge for both cancer researchers and clinicians. We have previously shown that miR-193a-3p promotes the multi-chemoresistance of bladder cancer cells via repressing its three target genes: SRSF2, PLAU and HIC2. Here, we showed that as a new direct target, the homeobox C9 (HOXC9) gene also executes the promoting effect of miR-193a-3p on the bladder cancer chemoresistance from a systematic study of multi-chemosensitive (5637) and resistant (H-bc) bladder cancer cell lines in both cell culture and tumor-xenograft/nude mice system. Paralleled with the changes in the drug-triggered cell death, the activities of both DNA damage response and oxidative stress pathways were drastically altered by a forced reversal of miR-193a-3p or HOXC9 levels in bladder cancer cells. In addition to a new mechanistic insight, our results provide a set of the essential genes in the miR-193a-3p/HOXC9/DNA damage response/oxidative stress pathway axis as the diagnostic targets for the guided anti-bladder cancer chemotherapy.

Abstract 605: ErbB receptors and cisplatin resistance: the case for bladder cancer

Abstract Nearly 30% of superficial bladder cancer (BC) cases develop into muscle invasive (MI) cases. With an initial discovery rate of 30% of MI cases, it is clear that treatment of MI disease should be a priority. Currently radical cystectomy is used in MI cases with neoadjuvant platinum based chemotherapy (PBC) highly recommended in the literature. With 1% of MI BC patients receiving this neoadjuvant therapy, metastases are common and PBC is the current treatment for these patients. However, cisplatin resistance is common in patients with MI BC leading to an average survival of only 15 months with a 5 year survival of 20%. Increased expression of epidermal growth factor receptor (EGFR) and related receptors ErbB2 and ErbB3 in BC is well documented. These receptors control cell growth and survival and several EGFR ligands are present in normal human urine. Thus we hypothesized that these receptors may play a role in BC resistance to PBC. EGFR family inhibition (EGFRi) has been investigated in conjunction with PBC in clinical trials. However, these trials did not show and increased benefit due to many of these patients having previously failed PBC therapy, EGFR/ErbB status not being part of the inclusion criteria, and frequent resistance to EGFRi in this patient population. We wanted to determine a mechanism for cisplatin and EGFRi resistance with the hypothesis that this could lead to novel therapeutics for MI BC. The BC cell lines T24, TCCSUP, J82 and RT4 were assayed for cisplatin sensitivity by flow cytometry. The TCCSUP and J82 cells were deemed cisplatin sensitive due to increased apoptosis when treated with cisplatin. The T24 and RT4 cells showed no apoptosis increase and were deemed to be cisplatin resistant. MTT and clonogenic assay determined that cisplatin resistant cells were also resistant to EGFRi. Similarly, cisplatin sensitive cells were sensitive to EGFRi. Additionally, it was determined that treating all cell lines with cisplatin and dacomitinib had a synergistic effect on reducing cell proliferation. Activation of EGFR, ErbB2 and ErbB3 was assayed by western blotting. Cisplatin sensitive cells underwent EGFR driven ErbB activation while cisplatin resistant cells underwent ErbB3 driven ErbB activation. Additionally, activation of EGFR Y845, Y1045, and Y1068 was increased by cisplatin in resistant cells. These results indicate that EGFRi and cisplatin, while working in parallel pathways, can influence one another. EGFR binding may be a prognostic marker for response to EGFRi. If EGFR binds primarily with ErbB2, the cells would likely respond to EGFRi; but, if EGFR binds primarily with ErbB3, the cells would likely be EGFRi resistant. Activation of EGFR Y845 (a Src binding site) may determine the preferential ErbB binding. EGFR Y845 activation may be blocked by EGFRi thus abrogating the effect of cisplatin and returning the cells to a cisplatin sensitive state. Citation Format: Benjamin Asher Mooso, Duanna Challenger, Paramita Ghosh. ErbB receptors and cisplatin resistance: the case for bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2014-605

MP39-05 SYNERGISTIC ANTITUMOR EFFECT OF SAHA AND CISPLATIN IN CISPLATIN RESISTANT BLADDER CANCER CELL LINE

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2014MP39-05 SYNERGISTIC ANTITUMOR EFFECT OF SAHA AND CISPLATIN IN CISPLATIN RESISTANT BLADDER CANCER CELL LINE Ha Rim Kook, Young Ju Lee, Jae Seung Yeon, Han Sol Lee, Choong Hee Noh, Kwang Mo Kim, Ki Beom Kim, Jung Keun Lee, Sangchul Lee, Seong Jin Jeong, Sung Kyu Hong, Seok-Soo Byun, Eunsik Lee, and Sang Eun Lee Ha Rim KookHa Rim Kook More articles by this author , Young Ju LeeYoung Ju Lee More articles by this author , Jae Seung YeonJae Seung Yeon More articles by this author , Han Sol LeeHan Sol Lee More articles by this author , Choong Hee NohChoong Hee Noh More articles by this author , Kwang Mo KimKwang Mo Kim More articles by this author , Ki Beom KimKi Beom Kim More articles by this author , Jung Keun LeeJung Keun Lee More articles by this author , Sangchul LeeSangchul Lee More articles by this author , Seong Jin JeongSeong Jin Jeong More articles by this author , Sung Kyu HongSung Kyu Hong More articles by this author , Seok-Soo ByunSeok-Soo Byun More articles by this author , Eunsik LeeEunsik Lee More articles by this author , and Sang Eun LeeSang Eun Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1320AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cisplatin-based chemotherapy remains first-line treatment for advanced bladder cancer. No standard chemotherapeutic agent has been established for patients with cisplatin resistant bladder cancer. We investigated the synergistic antitumor effect of of suberoylanilide hydroxamic acid (SAHA) and cisplatin in cisplatin resistant bladder cancer cells (T24R2). METHODS The cisplatin resistant human bladder cancer cell line (T24R2) was treated with cisplatin and/or SAHA. Tumor cell proliferation was assessed by cell counting kit-8 assay and clonogenic assay. Synergism was determined by combination index. Changes in cell cycle were determined by flow cytometry. Expression of caspase-3, 8 and 9, PARP, cytochrome c, p21, Bcl-2, Bad, p27, cyclin A, cyclin D1, and cyclin E were analyzed by Western blotting. RESULTS Synergistic antitumor effect between cisplatin and SAHA was observed by cell counting kit-8 assay, clonogenic assay and confirmed with combination index less than 1.0. The underlying mechanism could be synergistic cell cycle arrest, activation of apoptotic pathway including capase-3, -8, -9 and fragmented PARP or decreased expression in anti-apoptotic Bcl-2 and increased expression in pro-apoptotic BAD. CONCLUSIONS SAHA may synergistically enhancethe antitumor effect of cisplatin and resensitize cisplatin resistant bladder cancer cells. These findings suggest the potential use of SAHA as a combination agent to enhance the antitumor effect of cisplatin in patients with advanced bladder cancer. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e427-e428 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Ha Rim Kook More articles by this author Young Ju Lee More articles by this author Jae Seung Yeon More articles by this author Han Sol Lee More articles by this author Choong Hee Noh More articles by this author Kwang Mo Kim More articles by this author Ki Beom Kim More articles by this author Jung Keun Lee More articles by this author Sangchul Lee More articles by this author Seong Jin Jeong More articles by this author Sung Kyu Hong More articles by this author Seok-Soo Byun More articles by this author Eunsik Lee More articles by this author Sang Eun Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP34-11 MARKEDLY INCREASED OCT-4 AND NANOG EXPRESSION CORRELATES WITH MULTIDURG RESISTANCE (MDR) PROCESS IN BLADDER CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2014MP34-11 MARKEDLY INCREASED OCT-4 AND NANOG EXPRESSION CORRELATES WITH MULTIDURG RESISTANCE (MDR) PROCESS IN BLADDER CARCINOMA Yi Sun, Liang Liang, Yule Chen, Yongyi Cheng, Yonggang Xu, and Dalin He Yi SunYi Sun More articles by this author , Liang LiangLiang Liang More articles by this author , Yule ChenYule Chen More articles by this author , Yongyi ChengYongyi Cheng More articles by this author , Yonggang XuYonggang Xu More articles by this author , and Dalin HeDalin He More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1024AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer (BCa) is the most prevalent and easy recurrent cancer worldwide, high propensity for recurrence and progression are still serious problems leading to high costs and life-treatment. Multidrug resistant always induced metastasis and treatment failure. Cancer stem cells (CSC) model theoretically contribute to BCa metastasis and mulitidrug resistantce. In this study, we screened BCa stemness specific marker and further detected the potential roles during multidrug resistant process. METHODS 65 pairs of primary and relapsed chemo-resistant tumors were collected during past 10 years from shaanxi provincial people¡¯s hospital, differential expression profile of stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2) were screened by IHC staining and qPCR assay. The specific stemness markers were further confirmed with newly constructed multidrug resistant (253J-ADM) and their parental cell by WB, qPCR ,in vitro and in vivo tumorigenesis assay. We finally evaluated the self-renewal ability and chemosensitivity change after knocking down specific markers by siRNA in multidrug resistant BCa cell line. RESULTS Highly expressed the stemness markers (Nanog, Oct4, Bmi1, CD117, CD133, and ABCG2) was detected in relapsed chemo-resistant tumors vs primary tumor (*p<0.05 for Bmi1, CD117, CD133, and ABCG2). Oct4 and Nanog expression was significantly observed in chemo-resistant patients (**p< 0.01), which also showed correlation to prognosis. Similar results obtain by qPCR array. Furthermore, Oct4 and Nanog expression were also upregulated in multidrug resistant (253J-ADM) cell compared with parental cell, the higher tumorigenesis ability were also observed by in vitro and in vivo tumorigenesis assay(*p<0.05). Knocking down of Oct4 or Nanog in multidrug resistant BCa cell line dramatically suppressed cell renew ability (*p<0.05) and enhanced chemosensitivity to chemo-drugs (mitomycin/ pirarubicin) (*p<0.05). CONCLUSIONS These data indicate that cancer stem-like properties were expanded during the acquisition of multidrug resistance in BCa. Clinically, BCa stemness markers (Oct4 and nanog) overexpression may promote the BCa recurrence to resist multidrugs, which could be a potential therapeutic target for chemoresistant BCa treatment. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e365-e366 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yi Sun More articles by this author Liang Liang More articles by this author Yule Chen More articles by this author Yongyi Cheng More articles by this author Yonggang Xu More articles by this author Dalin He More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Intercellular Transfer of P-Glycoprotein from the Drug Resistant Human Bladder Cancer Cell Line BIU-87 Does Not Require Cell-to-Cell Contact

The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process.

Oncogenic Activation of Pak1-Dependent Pathway of Macropinocytosis Determines BCG Entry into Bladder Cancer Cells

Bacille Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that is used widely as a vaccine for tuberculosis and is used as an effective treatment for superficial bladder carcinoma. Despite being the most successful cancer biotherapy, its mechanism of action and response determinants remain obscure. Here, we establish a model system to analyze BCG interaction with bladder cancer cells, using it to show that these cells vary dramatically in their susceptibility to BCG infection. Unexpectedly, the uptake of BCG by bladder cancer cells occurs by macropinocytosis rather than phagocytosis. BCG entry into bladder cancer cells relied upon Rac1, Cdc42, and their effector kinase Pak1. The difference in susceptibility between BCG-permissive and -resistant bladder cancer cells was due to oncogenic activation of signaling pathways that activate macropinocytosis, with phosphoinositide 3-kinase inhibitor activation stimulating BCG uptake independently of Akt. Similarly, activated Ras strongly activated Pak1-dependent uptake of BCG. These results reveal that oncogenic activation of macropinocytosis determines BCG uptake by bladder cancer cells, implying that tumor responsiveness to BCG may be governed by the specific mutations present in the treated cancer cell.

905 Hypoxia is independently associated with poor outcome in urothelial bladder cancer patients treated with radical cystectomy

INTRODUCTION AND OBJECTIVES: Cisplatin-based chemotherapy is the first-line standard treatment in metastatic bladder cancer (BC), with response rates of only 50% but significant toxicity. Clinical and pathological parameters are unable to distinguish between cisplatin sensitive and resistant BCs. Therefore, predictive markers are urgently needed to avoid unnecessary toxicity. Cisplatin-based chemotherapeutic agents exhibit their cytotoxic effect by cross-linking DNA preventing DNA-duplication for mitosis in dividing cells. This process leads ultimately to apoptosis of these cells. ERCC1 has been suggested to be involved in the elimination of cisplatin-DNA adducts decreasing efficacy of the therapy. More recently, in vitro studies in various tumors demonstrated that MMP-7 plays a causal role in the development of cisplatin resistance by inhibiting chemotherapy-induced apoptosis. However, its chemotherapy predicting value in BC has not been evaluated yet. Therefore, we assessed the predictive value of MMP-7 and ERCC1 expressions in patients with metastatic BC treated with cisplatin-based chemotherapy. METHODS: Protein expressions of ERCC1 and MMP-7 were analyzed by immunostaining in chemo-naive tumor specimens of 72 patients treated with cisplatin-based chemotherapy (GC or MVAC) because of a metastatic BC. The expression levels were correlated with the clinical follow-up data. RESULTS: High ERCC1 and MMP-7 levels were associated with significantly shorter patientsâ€TM survival (p 0.025 and p 0.026 respectively). The highest predictive significance (p 0.017) could be reached when ERCC1 and MMP-7 were combined; the median survival time was 13.6 months in cases where both ERCC1 and MMP-7 levels were low, 10.2 months if one of these marker level were high and 5.7 months when both ERCC1 and MMP-7 levels were elevated. CONCLUSIONS: Our data reveals for the first time MMP-7 as a predictive factor in BC patients treated with cisplatin-based chemotherapy. The combination of MMP-7 with ERCC1 provides the highest predictive accuracy in this patient group. These data together suggest that ERCC1 and MMP-7 may help to select patients who will not benefit from a cisplatin-based therapy. These patients might be optimal candidates for studies with new chemotherapeutic agents. Furthermore, specific inhibition of these molecular factors may help to overcome chemotherapy resistance.

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.