Chemosensitivity Of Cell Lines Research Articles

Hh/IGF-1R/PI3K/Akt/MRP1 Pathway Induce Refractory Acute Myeloid Leukemia and Its Targeting Therpy

Hh/IGF-1R/PI3K/Akt/MRP1 pathway induce refractory acute myeloid leukemia Background: Although a great progress has made in the treatment of acute myeloid leukemia(AML), chemoresistance and relapse due to multidrug resistance (MDR) remains a therapeutic challenge. We have proven that LBH589 combined with Bortezomib had synergistic effects on proliferation, apoptosis and sensitivity to cytotoxic drugs via AKT and NF-κB pathways in chemoresistant HL60/ADR cells and refractory AML primary cells. Using gene chip analysis, we found that among those disregulated pathways, Hedgehog(Hh) signaling pathway was probably the upstream paths which could regulate others.Aims: The purpose of the study was to investigate the expression of Hh pathway in AML including primary cells and cell lines, analyze the relationship between Hh expression and clinical prognosis. Also investigate the biological effect of Hh pathway inhibitor NVP-LDE225 in reversing drug-resistance in HL60/adriamycin-resistant(ADR) cells and refractory AML primary cells and the underlying mechanisms. Besides, animal experiments were done to verify the results in vitro.Methods: Western blot assay were used to determine the protein expression of Ptch–Smo–Gli-1–Shh in HL60 and HL60/ADR cell lines and refractory or non-refractory primary AML cells. Kaplan–Meier curves were used to estimate Relapse-free survival(RFS) and Over-all survival(OS). HL60/ADR cells and refractory primary cells were treated with adriamycin or combined with NVP-LDE225 concentration among 0-10µM). Proliferation were evaluated by 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di-phenytetrazoliumromide (MTT) assay and cell apoptosis were analyzed by Annexin V-FITC/PI staining through flow-cytometry(FCM). Intercellular adriamycin accumulation(MFI) were analyzed by FCM. The changes in protein levels of Gli-1–IGF-1R–p-IGF-1R–IRS-1–Akt–p-Akt–MRP-1–Bcl-2 were detected by Western blot. Besides, we used a nude mouse xenograft model to verify the anti-proliferative effects in vivo.Results: We found that the refractory primary AML cells and HL60/ADR cells correlated with higher activation of the Hh pathway, however in non-refractory primary cells or chemosensitive cell lines HL60, such activation was less pronounced. Higher protein expression of this pathway was related to higher recurrence rate and associated with poor relapse free survival (RFS) and poor overall survival (OS)(P=0.002)(a). NVP-LDE225, a potent and selective Hh inhibitor, significantly reverted resistance of adriamycin(reversal fold was 3.75), induced cell apoptosis(P=0.007), increased the intracellular adriamycin accumulation(P=0.000), decreased protein expression of p-IGF-1R, IRS-1, p-Akt, Bcl-2, MRP1 also Gli-1(b). These effects were likely to be mediated via inhibition of IGF-1R/PI3K/Akt/MRP1 pathway. Besides, in a AML xenograft mouse model, NVP-LDE225 combined with ADM resulted in significant tumor regression(P=0.002) (c).Conclusions: These findings provided evidence that targeting the Hh pathway might be a therapeutic avenue for overcoming MDR resistance in myeloid leukemia.Keywords: Hedgehog signaling, acute myeloid leukemia, IGF-1R/PI3K/Akt signaling DisclosuresNo relevant conflicts of interest to declare.

Multidrug resistance-selective antiproliferative activity of Piper amide alkaloids and synthetic analogues

Twenty-five amide alkaloids (1–25) from Piper boehmeriifolium and 10 synthetic amide alkaloid derivatives (39–48) were evaluated for antiproliferative activity against eight human tumor cell lines, including chemosensitive and multidrug-resistant (MDR) cell lines. The results suggested tumor type-selectivity. 1-[7-(3,4,5-Trimethoxyphenyl)heptanoyl]piperidine (46) exhibited the best inhibitory activity (IC50=4.94μM) against the P-glycoprotein (P-gp)-overexpressing KBvin MDR sub-line, while it and all other tested compounds, except 9, were inactive (IC50 >40μM) against MDA-MB-231 and SK-BR-3. Structure–activity relationships (SARs) indicated that (i) 3,4,5-trimethoxy phenyl substitution is critical for selectivity against KBvin, (ii) the 4-methoxy group in this pattern is crucial for antiproliferative activity, (iii) double bonds in the side chain are not needed for activity, and (iv), in arylalkenylacyl amide alkaloids, replacement of an isobutylamino group with pyrrolidin-1-yl or piperidin-1-yl significantly improved activity. Further study on Piper amides is warranted, particularly whether side chain length affects the ability to overcome the MDR cancer phenotype.

Identification of Transmembrane Protein 98 as a Novel Chemoresistance-Conferring Gene in Hepatocellular Carcinoma

Chemoresistance is one of the major obstacles in systemic chemotherapy and targeted therapy for patients with advanced hepatocellular carcinoma. To identify novel chemoresistance-associated targets in hepatocellular carcinoma, chemoresistant hepatocellular carcinoma cell lines were established. By comparing the global gene expression profiles between chemoresistant and chemosensitive cell lines, eight novel chemoresistance-associated genes were identified to be significantly associated with the commonly augmented chemoresistance of hepatocellular carcinoma cells. One upregulated candidate named transmembrane protein 98 (TMEM98) was found to be overexpressed in 80 of 118 (67.80%) of patients with hepatocellular carcinoma. TMEM98 mRNA in tumor tissues was significantly higher than nontumor tissues of patients with hepatocellular carcinoma (P < 0.0001). Upregulation of TMEM98 was significantly correlated with advanced tumor stage (P = 0.048), high incidence of early tumor recurrence (P = 0.005), poor overall survival (P = 0.029), and poor disease-free survival (P = 0.011) of patients with hepatocellular carcinoma after hepatectomy. Importantly, upregulation of TMEM98 mRNA in patients with hepatocellular carcinoma who received transarterial chemoembolization (TACE) treatment was significantly higher than in patients without TACE treatment (P = 0.046). Moreover, patients with poor response to TACE treatment had higher degree of TMEM98 upregulation than the responsive patients. In vitro and in vivo studies showed that suppression of TMEM98 in chemoresistant hepatocellular carcinoma cells restored their chemosensitivity, while forced overexpression of TMEM98 enhanced their chemoresistance. The mechanism of TMEM98 in conferring chemoresistance of hepatocellular carcinoma might be possibly through activation of the AKT pathway and deactivation of p53. In conclusion, we identified a panel of novel common chemoresistance-associated genes and demonstrated that TMEM98 is a chemoresistance-conferring gene in hepatocellular carcinoma.

Synergism of Cyclin-Dependent Kinase Inhibitors with Camptothecin Derivatives in Small Cell Lung Cancer Cell Lines

Advanced small cell lung cancer (SCLC) has a dismal prognosis. Modulation of the camptothecin topotecan, approved for second-line therapy, may improve response. Our recent finding of synergistic enhancement of the cytotoxic activity of camptothecin (CPT) by cyclin-dependent kinase 4 inhibitors is extended here to a panel of camptothecin analogs comprising 10-hydroxy-CPT (HOCPT), topotecan (TPT; 9-[(dimethylamino)-methyl]-10-hydroxy-CPT), 9-amino-CPT (9AC), 9-nitrocamptothecin (rubitecan), SN38 (7-ethyl-10-hydroxycamptothecin) and 10-hydroxy-9-nitrocamptothecin (CPT109) in combination with PD0332991, CDK4I, roscovitine and olomoucine. SCLC cell lines employed are chemoresistant NCI-H417 and DMS153 and the chemosensitive SCLC26A line established at our institution. The CPT analogs exhibiting highest cytotoxicity towards the three SCLC lines tested were SN38 and 9AC, followed by rubitecan, HOCPT, TPT and CPT109. NCI-H417 and DMS153 revealed an approximately 25-fold and 7-fold higher resistance compared to the chemosensitive SCLC26A cell line. Whereas the CDK4/6 inhibitor PD0332991 proved less effective to chemosensitize SCLC cells to CPT analogs, the CDK inhibitors CDK4I, roscovitine and olomoucine gave comparable chemosensitization effects in combination with 9AC, SN38, rubitecan and to a lesser extent with TPT and CPT109, not directly related with topoisomerase mRNA expression. In conclusion, small chemical modifications of the parent CPT structure result in differing cytotoxicities and chemomodulatory effects in combination with CDKIs of the resulting analogs.

3-Ketone-4,6-diene ceramide analogs exclusively induce apoptosis in chemo-resistant cancer cells

Multidrug-resistance is a major cause of cancer chemotherapy failure in clinical treatment. Evidence shows that multidrug-resistant cancer cells are as sensitive as corresponding regular cancer cells under the exposure to anticancer ceramide analogs. In this work we designed five new ceramide analogs with different backbones, in order to test the hypothesis that extending the conjugated system in ceramide analogs would lead to an increase of their anticancer activity and selectivity towards resistant cancer cells. The analogs with the 3-ketone-4,6-diene backbone show the highest apoptosis-inducing efficacy. The most potent compound, analog 406, possesses higher pro-apoptotic activity in chemo-resistant cell lines MCF-7TN-R and NCI/ADR-RES than the corresponding chemo-sensitive cell lines MCF-7 and OVCAR-8, respectively. However, this compound shows the same potency in inhibiting the growth of another pair of chemo-sensitive and chemo-resistant cancer cells, MCF-7 and MCF-7/Dox. Mechanism investigations indicate that analog 406 can induce apoptosis in chemo-resistant cancer cells through the mitochondrial pathway. Cellular glucosylceramide synthase assay shows that analog 406 does not interrupt glucosylceramide synthase in chemo-resistant cancer cell NCI/ADR-RES. These findings suggest that due to certain intrinsic properties, ceramide analogs’ pro-apoptotic activity is not disrupted by the normal drug-resistance mechanisms, leading to their potential use for overcoming cancer multidrug-resistance.

Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

BackgroundExportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. MethodsWe used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5' regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. ResultsThe first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. [Display omitted] ConclusionsOur data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures:Shacham:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

Monoclonal antibody-based immunotherapy of ovarian cancer: Targeting ovarian cancer cells with the B7-H3-specific mAb 376.96

ObjectiveThe high rate of relapse in patients with advanced ovarian cancer likely reflects the chemoresistance of cancer initiating cells (CICs). We evaluated the anti-tumor activity of monoclonal antibody (mAb) 376.96, which recognizes a B7-H3 epitope expressed on ovarian carcinoma cells (OCCs), in combination with the tyrosine kinase inhibitor Sunitinib and chemotherapy on chemosensitive and chemoresistant cells and CICs. MethodsEight ovarian cancer cell lines including platinum- and taxane-resistant cell lines were analyzed by flow cytometry to establish expression of the mAb 376.96-defined-B7-H3-epitope on differentiated ovarian cancer cells and CICs. Samples from 10 ovarian cancer patients were analyzed via immunohistochemistry for mAb 376.96-defined-B7-H3-epitope expression. In vitro studies assessed mAb 376.96 alone and in combination with Sunitinib on the growth of chemosensitive and chemoresistant cell lines and on the content of CICs. ResultsThe mAb-376.96-defined-B7-H3 epitope is expressed on both differentiated cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines and 10 patient derived ovarian cancer tumors. In vitro treatment of chemoresistant cell lines with mAb 376.96 resulted in decreased cell viability. mAb 376.96 enhanced the cytotoxicity of Sunitinib and reduced the content of CICs. ConclusionThe mAb-376.96-defined-B7-H3-epitope was found to be expressed on both differentiated ovarian cancer cells and CICs in chemosensitive and chemoresistant ovarian cancer cell lines. mAb 376.96 inhibited the in vitro growth of chemosensitive and chemoresistant OCCs and reduced the content of CICs when used with Sunitinib. Further studies examining B7-H3 as a potential target of mAb-based immunotherapy for this type of malignancy are warranted.

Germline single nucleotide polymorphisms associated with response of urothelial carcinoma to platinum-based therapy: the role of the host

BackgroundVariations in urothelial carcinoma (UC) response to platinum chemotherapy are common and frequently attributed to genetic and epigenetic variations of somatic DNA. We hypothesized that variations in germline DNA may contribute to UC chemosensitivity. Patients and methodsDNA from 210 UC patients treated with platinum-based chemotherapy was genotyped for 80 single nucleotide polymorphisms (SNPs). Logistic regression was used to examine the association between SNPs and response, and a multivariable predictive model was created. Significant SNPs were combined to form a SNP score predicting response. Eleven UC cell lines were genotyped as validation. ResultsSix SNPs were significantly associated with 101 complete or partial responses (48%). Four SNPs retained independence association and were incorporated into a response prediction model. Each additional risk allele was associated with a nearly 50% decrease in odds of response [odds ratio (OR) = 0.51, 95% confidence interval 0.39–0.65, P = 1.05 × 10-7). The bootstrap-adjusted area under the curves of this model was greater than clinical prognostic factors alone (0.78 versus 0.64). The SNP score showed a positive trend with chemosensitivity in cell lines (P = 0.115). ConclusionsGenetic variants associated with response of UC to platinum-based therapy were identified in germline DNA. A model using these genetic variants may predict response to chemotherapy better than clinical factors alone.

Independent Validation of a Model Using Cell Line Chemosensitivity to Predict Response to Therapy

Methods using cell line microarray and drug sensitivity data to predict patients' chemotherapy response are appealing, but groups may be reluctant to release details to preserve intellectual property. Here we describe a case study to validate predictions while treating the methods as a "black box." Medical Prognosis Institute (MPI) constructed cell-line-derived sensitivity scores (SSs) and combined scores (CSs) that incorporate clinical variables. MD Anderson researchers evaluated their predictions. We searched the Gene Expression Omnibus (GEO) to identify validation datasets, and we performed statistical evaluation of the agreement between prediction and clinical observation. We identified 3 suitable datasets: GSE16446 (n = 120; binary outcome), GSE17920 (n = 130; binary outcome), and GSE10255 (n = 161; continuous and time-to-event outcomes). The SS was statistically significantly associated with primary treatment responses for all studies (GSE16446: P = .02; GSE17920: P = .02; GSE10255: P = .02). Dichotomized SSs performed no better than chance for GSE16446 and GSE17920, and categorized SSs did not predict disease-free survival (GSE10255). SSs sometimes improved on predictions using clinical variables (GSE16446: P = .05; GSE17920: P = .31; GSE10255: P = .045), but gains were limited (95% confidence intervals for GSE16446 and GSE17920 include 0). The CS did not predict treatment response for GSE16446 (P = .55), but it did for GSE17920 (P < .001). Coefficients of clinical variables provided by MPI for CSs agree with estimates for GSE17920 better than estimates for GSE16446. Model predictions were better than chance in all three datasets. However, these scores added little to existing clinical predictors; statistically significant contributions were likely to be too small to change clinical practice. These findings suggest that discovering better predictors will require both cell line data and a clinical training dataset of patient samples.

Effects of hypoxia on human cancer cell line chemosensitivity

BackgroundEnvironment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia.MethodsA panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA).ResultsSorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs.ConclusionsA panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia.

β-Elemene enhances susceptibility to cisplatin in resistant ovarian carcinoma cells via downregulation of ERCC-1 and XIAP and inactivation of JNK

β-Elemene is a promising new plant-derived drug with broad-spectrum anticancer activity. It also increases cisplatin cytotoxicity and enhances cisplatin sensitivity in resistant human carcinoma cells. However, little is known about the mechanism of its action. To explore the potential therapeutic application of β-elemene as a drug-resistance modulator, this study investigated the underlying mechanism of β-elemene activity in cisplatin-resistant ovarian cancer cells. β-Elemene enhanced cisplatin sensitivity to a much greater extent in chemoresistant A2780/CP70 and MCAS human ovarian carcinoma cells compared to the chemosensitive parental cell line A2780. The dose-modifying factors for cisplatin were between 35 and 60 for A2780/CP70 cells and between 1.6 and 2.5 for A2780 cells. In the cisplatin-resistant ovarian carcinoma cells, β-elemene abrogated cisplatin-induced expression of excision repair cross-complementation group-1 (ERCC-1), a marker gene in the nucleotide excision repair pathway that repairs cisplatin-caused DNA damage. In addition, β-elemene not only reduced the level of X-linked inhibitor of apoptosis protein (XIAP), but also downregulated cisplatin-mediated XIAP expression in chemoresistant cells. Furthermore, β-elemene blocked the cisplatin-stimulated increase in the level of phosphorylated c-Jun NH2-terminal kinase (JNK) in these cells. These novel findings suggest that the β-elemene enhancement of cisplatin sensitivity in human chemoresistant ovarian cancer cells is mediated at least in part through the impairment of DNA repair activity and the activation of apoptotic signaling pathways, thereby making resistant ovarian cancer cells susceptible to cisplatin-induced cell death.

Eukaryotic Translation Elongation Factor 1-Alpha 1 Inhibits p53 and p73 Dependent Apoptosis and Chemotherapy Sensitivity

The p53 family of transcription factors is a key regulator of cell proliferation and death. In this report we identify the eukaryotic translation elongation factor 1-alpha 1 (eEF1A1) to be a novel p53 and p73 interacting protein. Previous studies have demonstrated that eEF1A1 has translation-independent roles in cancer. We report that overexpression of eEF1A1 specifically inhibits p53-, p73- and chemotherapy-induced apoptosis resulting in chemoresistance. Short-interfering RNA-mediated silencing of eEF1A1 increases chemosensitivity in cell lines bearing wild type p53, but not in p53 null cells. Furthermore, silencing of eEF1A1 partially rescues the chemoresistance observed in response to p53 or p73 knockdown, suggesting that eEF1A1 is a negative regulator of the pro-apoptotic function of p53 and p73. Thus, in the context of p53-family signaling, eEF1A1 has anti-apoptotic properties. These findings identify a novel mechanism of regulation of the p53 family of proteins by eEF1A1 providing additional insight into potential targets to sensitize tumors to chemotherapy.

Abstract 4027: Pathway analysis of chemoresistance in ovarian cancer cell lines.

Abstract Introduction: Ovarian Cancer is the most deadly female reproductive cancer and fifth most common cause of death from cancer among women. Limited characterization of the chemoresistant population demonstrates the need for improved therapy for ovarian cancer. Pathway analysis of microarray data of paired chemosensitive and chemoresistant ovarian cancer cell lines may help to elucidate important contributors to chemoresistance, and paired analyses can eliminate the significant heterogeneity among patient samples. Significant genes may serve as potential targets for novel drug therapies. Some genes likely contribute to resistance of multiple chemotherapies and represent common pathways of resistance. Methods: Microarray data of three pairs of chemosensitive and chemoresistant ovarian cancer cell lines were analyzed using GeneSpring Single Experiment Analysis of 517 pathways. These pairs include SKOV3ip1 and SKOV3TRip2 (taxane-resistant), HeyA8 and HeyA8MDR (multi-drug resistant), and A2780cp20 and A780cp55 (increasing cisplatin resistance). Significant genes within significant pathways were compared between types of drug resistance. Results: Between SKOV3ip1 and SKOV3TRip2, 8 pathways were significantly (p&amp;lt;0.001) modulated, dominated by the selenium pathway and folate/B12 metabolism. Between HeyA8 and HeyA8MDR, 9 pathways were significantly modified, led by DNA regulation and cell cycle progression. These differences between the SKOV3 model were interesting, given that both chemoresistant lines were developed in the same way. In A2780cp20 and A2780cp55, 7 pathways were significantly affected, highlighted by members of the Electron Transport Chain and controls on oxidative phosphorylation and autophagy. Validation of individual genes as important to chemoresistance will follow from this analysis. Conclusions: Multiple pathways are implicated in chemoresistance, even when selected by the same chemotherapeutic agent. Pathways contributing to metabolism, DNA repair, cell cycle control, and stress responses dominated. The high variability in pathway overexpression highlights the importance of patient-specific approaches to overcoming chemoresistance in ovarian cancer. Citation Format: William P. Jackson, Ashwini A. Katre, Zachary A. Dobbin, Adam D. Steg, Charles N. Landen. Pathway analysis of chemoresistance in ovarian cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4027. doi:10.1158/1538-7445.AM2013-4027

Abstract 548: CXCR2 controls breast cancer metastasis and chemoresistance through PI3K/AKT and COX-2 signalings.

Abstract Background: Breast cancer is the chief culprit leading to woman's death. The major cause is the chemoresistance and distant metastasis before or after surgery and standard treatment. Recent studies suggest that chemokines and their receptors may play important roles in breast cancer metastasis and chemoresistance. Purpose: The chemokine receptor CXCR2 is associated with tumor development in many cancers. Although IL-8 and Gro-alpha as the major ligands contribute to breast cancer progression, the function of their receptor CXCR2 in breast cancer is elusive. In this study, we investigated the role of CXCR2 in breast cancer metastasis and chemoresistance. Experimental design:Human breast cancer cell lines including MCF-7 and SKBR-3 (low metastasis), MDA-MB-231 (medium metastasis), MDA-MB-231HM (high lung metastasis), MDA-MB-231BO (high bone metastasis), and MDA-MB-231/Gem (Gemcitabine-resistant) were used to investigate cell proliferation, migration, invasion, colony formation, apoptosis, and tumorigenesis by MTT, scratch-wound, transwell, soft agar, flow cytometery, and animal assays after CXCR2 or its associated proteins were over expressed or silenced/inhibited. Protein expression in cell lines and tissues was examined by either Western blot, ELISA, immunofluorescence, or immunohistochemical staining. Results: CXCR2 expression was higher in chemoresistant breast cancer cell lines, high-metastatic cell lines and breast cancer tissues than in chemosensitive cell lines, low-metastatic cell lines, and breast cancer tissues. Overexpression of CXCR2 promoted cell proliferation, migration, invasion, cell anti-apoptosis, tumor formation, metastasis, and chemoresistance, but silencing of CXCR2 inhibited those activities both in vitro and in vivo. Mechanism studies revealed that CXCR2 suppresses AKT1 to promote breast cancer metastasis and chemoresistance through cyclooxygenase-2 (COX-2; PTGS2) to regulate caveolin-1, MMP2, MMP9, E-cardherin, and beta-catenin, which can be inversed by knock down of CXCR2 or COX-2, and over expression of AKT1 and P85alpha. Additionally, over expression of CXCR2 and COX-2, or silencing of PI3K/AKT1 inhibits p53 phosphorylation (Serine 15) and E2F1 expression, but activates MDM2, Bcl-xL and Bcl-2, leading to increased anti-apoptosis. Tissue microarray analysis from 300 breast cancer patients also indicated that over expression of CXCR2 is associated with enhanced COX2 expression and reduced AKT1 expression in metastatic and chemoresistant breast carcinomas (P &amp;lt; 0.001), which may be used to predict breast cancer development. Conclusions: Our results suggest that CXCR2 controls breast cancer metastasis and chemoresistance through regulating COX-2 and AKT1. Thus, antagonists of CXCR2 and COX-2 may be used synergistically to treat breast cancer patients particularly with high metastasis and chemoresistance. Citation Format: Han Xu, Fengjuan Lin, Ziliang Wang, Jiao Meng, Huizhen Sun, Zihao Qi, Yan Wang, Zhouluo Ou, Zhimin Shao, Genhong Di, Gong Yang. CXCR2 controls breast cancer metastasis and chemoresistance through PI3K/AKT and COX-2 signalings. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 548. doi:10.1158/1538-7445.AM2013-548 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 3423: GSI-1 synergizes with LDE225 in ovarian cancer cells by inhibiting the proteasome.

Abstract Background: Cancer stem cells (CSCs) have been implicated in the development and recurrence of chemoresistant tumors in multiple malignancies. Previous research has suggested that targeting stem cell pathways, such as Notch and Hedgehog, may aid in overcoming the chemoresistant properties of these cells. However, attempts to target the Notch and Hedgehog pathways synergistically have had mixed results using different inhibitors, and the mechanism through which these inhibitors act is not fully known. Objective: To explore the potential synergy of concurrent targeting of the Notch and Hedgehog pathways and the mechanism through which this may occur. Methods: The Notch pathway was targeted with GSI-1 and GSI-XXI, and the Hedgehog pathway was targeted with the Smoothened small molecule inhibitor LDE225. The proteasome inhibitor Bortezomib was used alone and in combination with LDE225. Effects of these drugs on three pairs of chemosensitive and chemoresistant ovarian cancer cell lines were examined: SKOV3ip1 and SKOV3TRip2 (taxane-resistant), HeyA8 and HeyA8MDR (multi-drug resistant), and A2780ip2/A2780cp20 (platinum resistant). Cell viability was determined by the MTT assay. PI staining and PARP cleavage were used to assess cell cycle arrest and apoptosis. Proteasome activity was determined by the Proteasome-glo chymotrypsin-like cell-based assay. Results: GSI-1 significantly decreased cell viability alone and synergized with LDE225 in SKOV3TRip2 cells. However, GSI-XXI did not reduce cell viability or exhibit synergy with LDE225. In addition, specific inhibition of Notch via siRNA-mediated downregulation did not result in synergy with LDE225, suggesting a Notch-independent mechanism of synergy with GSI-1. To elucidate differences between GSI-1 and GSI-XXI, we examined effects of these drugs on the proteasome. GSI-1 inhibited the proteasome to the same extent as the conventional proteasome inhibitor bortezomib, while GSI-XXI did not affect proteasome activity. Combination treatment with bortezomib and LDE225 also resulted in synergy in SKOV3TRip2 cells. Monotherapy with both GSI-1 and bortezomib decreased transcription of the hedgehog target genes Patched, Gli1, and Gli2. This effect was enhanced when these drugs were combined with LDE225. Combination therapy also resulted in arrest of cells in the G2/M and S phases (60% of cells in S/G2/M with combination therapy vs. 38% of control, p&amp;lt;0.001), as well as an increase in apoptosis and PARP cleavage. Conclusion: The Notch inhibitor GSI-1 acts synergistically with Hedgehog inhibition through a Notch-independent decrease in proteasome activity. Similar synergy was noted with the proteasome inhibitor bortezomib. This synergistic combination appears to be working through S/G2/M phase arrest. Combined Hedgehog and proteasome inhibition offers a promising approach to targeting chemoresistant cells in ovarian malignancies. Citation Format: Mata R. Burke, Adam Steg, Dae Hoon Jeong, Zachary C. Dobbin, Charles N. Landen. GSI-1 synergizes with LDE225 in ovarian cancer cells by inhibiting the proteasome. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3423. doi:10.1158/1538-7445.AM2013-3423

Targeted inhibition of phosphatidyl inositol-3-kinase p110β, but not p110α, enhances apoptosis and sensitivity to paclitaxel in chemoresistant ovarian cancers

The phosphatidylinositol 3-kinase (PI3K) pathway is one of the critical signaling cascades playing important roles in the chemoresistance of human cancer cells, including ovarian cancer. In this study, we investigated the potential of targeting the PI3K p110β-isoform as a novel approach to overcome the chemoresistance in ovarian cancer. The effects on apoptosis, cell viability, proliferation and migration in chemoresistant ovarian cancer cell were determined following targeted p110β inhibition by small interfering RNA (siRNA). Seven paclitaxel (PTX)-resistant sublines (SKpacs and A2780pac) were produced from SKOV3 and A2780 ovarian cancer cell lines. We, first, evaluated the expression of PI3K p110 isoforms in chemosensitive and chemoresistant ovarian cancer cell lines and patient specimens, and found that p110β-isoform was significantly overexpressed both in a panel of ovarian cancer samples, and in PTX-resistant sublines compared with their parent cell lines. RNA interference-mediated p110β silencing augmented PTX-mediated apoptosis (31.15 ± 13.88 %) and reduced cell viability (67 %) in PTX-resistant cells, whereas targeting p110α did not show a significant change in cell viability and apoptosis. In addition, p110β silencing impaired cell proliferation (60 %) in PTX-resistant SKpac cells. We also found the combined treatment group with p110β siRNA and PTX showed a significant inhibition of tumor growth of SKpac cells compared to the PTX-only treated group in a xenograft nude mouse model. Thus, the siRNA-mediated silencing of PI3K p110β resensitizes PTX-resistant ovarian cancer cells, and may be a useful therapeutic strategy for PTX-resistant ovarian cancers.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-013-0807-9) contains supplementary material, which is available to authorized users.

Chromatin H3K27me3/H3K4me3 histone marks define gene sets in high-grade serous ovarian cancer that distinguish malignant, tumour-sustaining and chemo-resistant ovarian tumour cells

In embryonic stem (ES) cells, bivalent chromatin domains containing H3K4me3 and H3K27me3 marks silence developmental genes, while keeping them poised for activation following differentiation. We have identified gene sets associated with H3K27me3 and H3K4me3 marks at transcription start sites in a high-grade ovarian serous tumour and examined their association with epigenetic silencing and malignant progression. This revealed novel silenced bivalent marked genes, not described previously for ES cells, which are significantly enriched for the PI3K (P<10(-7)) and TGF-β signalling pathways (P<10(-5)). We matched histone marked gene sets to gene expression sets of eight normal fallopian tubes and 499 high-grade serous malignant ovarian samples. This revealed a significant decrease in gene expression for the H3K27me3 and bivalent gene sets in malignant tissue. We then correlated H3K27me3 and bivalent gene sets to gene expression data of ovarian tumour 'stem cell-like' sustaining cells versus non-sustaining cells. This showed a significantly lower expression for the H3K27me3 and bivalent gene sets in the tumour-sustaining cells. Similarly, comparison of matched chemo-sensitive and chemo-resistant ovarian cell lines showed a significantly lower expression of H3K27me3/bivalent marked genes in the chemo-resistant compared with the chemo-sensitive cell line. Our analysis supports the hypothesis that bivalent marks are associated with epigenetic silencing in ovarian cancer. However it also suggests that additional tumour specific bivalent marks, to those known in ES cells, are present in tumours and may potentially influence the subsequent development of drug resistance and tumour progression.

Secreted frizzled-related protein 4 expression is positively associated with responsiveness to Cisplatin of ovarian cancer cell lines in vitro and with lower tumour grade in mucinous ovarian cancers

BackgroundOvarian cancer is one of the most lethal malignancies in women, as it is frequently detected at an advanced stage, and cancers often become refractory to chemotherapy. Evidence suggests that dysregulation of pro-apoptotic genes plays a key role in the onset of chemoresistance. The secreted Frizzled-Related Protein (sFRP) family is pro-apoptotic and also a negative modulator of the Wnt signalling cascade. Studies have demonstrated that the re-expression of sFRPs, in particular sFRP4, is associated with a better prognosis, and that experimentally induced expression results in cell death.ResultsIn vitro experimental models determined that sFRP4 was differentially expressed in chemosensitive (A2780) and chemoresistant (A2780 ADR and A2780 Cis) ovarian cell lines, with chemosensitive cells expressing significantly higher levels of sFRP4. Transfection of the chemoresistant cell lines with sFRP4 significantly increased their sensitivity to chemotherapy. Conversely, silencing of sFRP4 expression in the chemosensitive cell line resulted in a corresponding increase in chemoresistance. Comparison of sFRP4 expression in tumour biopsies revealed a positive trend between sFRP4 expression and tumour grade, with mucinous cyst adenocarcinomas exhibiting significantly decreased sFRP4 levels compared to mucinous borderline tumours.ConclusionsThis study indicates a role for sFRP4 as a predictive marker of chemosensitivity in ovarian cancer and suggests that this pathway may be worth exploiting for novel therapies.

MiRNAs associated with chemo-sensitivity in cell lines and in advanced bladder cancer

BackgroundMicroRNA is a naturally occurring class of non-coding RNA molecules that mediate posttranscriptional gene regulation and are strongly implicated in cellular processes such as cell proliferation, carcinogenesis, cell survival and apoptosis. Consequently there is increasing focus on miRNA expression as prognostic factors for outcome and chemotherapy response. Only approximately 50% of patients with bladder cancer respond to chemotherapy. Therefore, predictive markers, such as miRNAs, that can identify subgroups of patients who will benefit from chemotherapy will have great value for treatment guidance.MethodsWe profiled the expression of 671 miRNAs in formalin fixed paraffin embedded tumors from patients with advanced bladder cancer treated with cisplatin based chemotherapy. We delineated differentially expressed miRNAs in tumors from patients with complete response vs. patients with progressive disease and in tumors form patients with short and long overall survival time. Furthermore, we studied the effect of up- and down regulation of key miRNAs on the cisplatin sensitivity in eight bladder cancer cell lines with different sensitivities to cisplatin.ResultsmiRNA expression profiling identified 15 miRNAs that correlated with response to chemotherapy and 5 miRNAs that correlated with survival time. Three miRNAs were associated with both response and survival (886-3p, 923, 944). By changing the cellular level of the response-identified miRNAs in eight bladder cell lines with different cisplatin sensitivity we found that down-regulation of miR-27a, miR296-5p and miR-642 generally reduced the cell viability, whereas up-regulation of miR-138 and miR-886-3p reduced the viability of more than half of the cell lines. Decreasing miR-138 increased the cisplatin sensitivity in half of the cell lines and increasing miR-27a and miR-642 generally increased cisplatin sensitivity.ConclusionsMiRNAs seem to be involved in cisplatin based chemo response and may form a new target for therapy and serve as biomarkers for treatment response.

Altered Death Receptor Signaling Promotes Epithelial-to-Mesenchymal Transition and Acquired Chemoresistance

Altered death receptor signaling and resistance to subsequent apoptosis is an important clinical resistance mechanism. Here, we investigated the role of death receptor resistance in breast cancer progression. Resistance of the estrogen receptor alpha (ER)-positive, chemosensitive MCF7 breast cancer cell line to tumor necrosis factor (TNF) was associated with loss of ER expression and a multi-drug resistant phenotype. Changes in three major pathways were involved in this transition to a multidrug resistance phenotype: ER, Death Receptor and epithelial to mesenchymal transition (EMT). Resistant cells exhibited altered ER signaling, resulting in decreased ER target gene expression. The death receptor pathway was significantly altered, blocking extrinsic apoptosis and increasing NF-kappaB survival signaling. TNF resistance promoted EMT changes, resulting in a more aggressive phenotype. This first report identifying specific mechanisms underlying acquired resistance to TNF could lead to a better understanding of the progression of breast cancer in response to chemotherapy treatment.