Resistant Sublines Research Articles

1,25 dihydroxyvitamin D3 inhibits the proliferation of thyroid cancer stem-like cells via cell cycle arrest

ABSTRACTBackground: An anti-proliferative effect of vitamin D has been reported in different carcinomas, including thyroid cancer. Cancer stem cells (CSCs), a very small fraction of cancer cells, are widely believed to be responsible for cancer initiation, relapse and metastasis. Objectives: We addressed the question as to whether CSCs derived from the anaplastic thyroid carcinoma cell lines SW1736, C643, HTh74 and its doxorubicin- resistant subline HTh74R are also a target of vitamin D action.Methods: The effect of calcitriol on growth of HTh74, HTh74R, SW1736 and C643 cell lines was investigated by cell viability assays. In stem-enriched cells derived from thyro-spheres cell cycle analysis and apoptotic assays were performed. Furthermore, the role of calcitriol in the formation of cancer thyro-spheres and its putative differentiation-inducing effect were analysed. Results: CSCs isolated as thyro-spheres from all the four anaplastic thyroid carcinoma cells expressed vitamin D receptors as did their parental cells. Calcitriol inhibited proliferation of anaplastic thyroid carcinoma cells with a more pronounced effect on doxorubicin-resistant HTh74R cells, and it significantly reduced the capacity to form stem cell-derived spheres and decreased the size of these spheres that consist of CSCs and their progenitor cells. As revealed by cell cycle analysis, calcitriol induced G2/M phase arrest in thyro-sphere cells derived cells from HTh74, HTh74R and C643 but did not affect apoptosis. Finally, calcitriol altered morphology of CSCs. Conclusion: Calcitriol inhibited the growth of CSCs derived from anaplastic thyroid cancer cells. It may also exert a pro-differentiation effect in thyroid CSCs.

Oncogene swap as a novel mechanism of acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitor in lung cancer.

Mutant selective epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs), such as rociletinib and AZD9291, are effective for tumors with T790M secondary mutation that become refractory to first‐generation EGFR‐TKI. However, acquired resistance to these prospective drugs is anticipated considering the high adaptability of cancer cells and the mechanisms remain largely obscure. Here, CNX‐2006 (tool compound of rociletinib) resistant sublines were established by chronic exposure of HCC827EPR cells harboring exon 19 deletion and T790M to CNX‐2006. Through the analyses of these resistant subclones, we identified two resistant mechanisms accompanied by MET amplification. One was bypass signaling by MET amplification in addition to T790M, which was inhibited by the combination of CNX‐2006 and MET‐TKI. Another was loss of amplified EGFR mutant allele including T790M while acquiring MET amplification. Interestingly, MET‐TKI alone was able to overcome this resistance, suggesting that oncogenic dependence completely shifted from EGFR to MET. We propose describing this phenomenon as an “oncogene swap.” Furthermore, we analyzed multiple lesions from a patient who died of acquired resistance to gefitinib, then found a clinical example of an oncogene swap in which the EGFR mutation was lost and a MET gene copy was gained. In conclusion, an “oncogene swap” from EGFR to MET is a novel resistant mechanism to the EGFR‐TKI. This novel mechanism should be considered in order to avoid futile inhibition of the original oncogene.

MP84-03 EXPRESSION OF ANDROGEN RECEPTOR SPLICE VARIANTS IN A NOVEL CASTRATION RESISTANT PROSTATE CANCER XENOGRAFT MODEL

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-03 EXPRESSION OF ANDROGEN RECEPTOR SPLICE VARIANTS IN A NOVEL CASTRATION RESISTANT PROSTATE CANCER XENOGRAFT MODEL Takahiro Kimura, Mariko Honda, Kojiro Tashiro, Shigehiro Bando, Hiroshi Sasaki, Yuko Kamata, Jun Miki, and Shin Egawa Takahiro KimuraTakahiro Kimura More articles by this author , Mariko HondaMariko Honda More articles by this author , Kojiro TashiroKojiro Tashiro More articles by this author , Shigehiro BandoShigehiro Bando More articles by this author , Hiroshi SasakiHiroshi Sasaki More articles by this author , Yuko KamataYuko Kamata More articles by this author , Jun MikiJun Miki More articles by this author , and Shin EgawaShin Egawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2227AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Androgen receptor (AR) splice variants have been reported as key players in the development of castration resistant prostate cancer (PCa). AR splice variant 7 (AR-V7) is a form of them encoding a truncated AR protein that lacks the ligand-binding domain and remains constitutively active as a transcription factor without androgen. AR-V7 in circulating tumor cells from patients with metastatic castration-resistant PCa was recently demonstrated to be associated with resistance to abiraterone and enzalutamide. We have established a mouse xenograft model, JDCaP, from skin metastatic tissue of Japanese castration resistant PCa patient which was revealed to have androgen sensitivity (Kimura T, et al. Prostate. 2009;69:1660). In the present study, we established castration resistant sublines, JDCaP-HR, from JDCaP and analyzed the sequence and expression profile of molecules including full length AR and AR splice variants using whole transcriptome sequencing. METHODS JDCaP was transplanted to female or castrated male nude mice and the stable castration resistant sublines, JDCaP-HR 1, 2, 3 and 4, were established by serial passages. Whole transcriptome analysis by next-generation sequencing was performed to analyze the sequence and expression profile of molecules including full length AR and AR splice variants in these cells. RESULTS Administration of bicalutamide did not show any anti-tumor effect for all of the established JDCaP-HRs and their castration resistance was confirmed. Whole transcriptome analysis revealed that 49 genes were up-regulated and 14 genes were down-regulated in JDCaP-HR comparing to JDCaP. The expression of wild type-AR was significantly higher in JDCaP-HRs than in JDCaP (Figure). In addition, three of JDCaP-HRs expressed AR-V7 and the other subline expressed another form of truncated AR splice variant, ARV567es which was also deleted the ligand-binding domain. The parental JDCaP did not express any AR splice variants. CONCLUSIONS All castration resistant sublines expressed the truncated forms of AR splice variants. The results suggest that AR splice variants may have an important role in development of castration resistance in PCa. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1090 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takahiro Kimura More articles by this author Mariko Honda More articles by this author Kojiro Tashiro More articles by this author Shigehiro Bando More articles by this author Hiroshi Sasaki More articles by this author Yuko Kamata More articles by this author Jun Miki More articles by this author Shin Egawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP84-05 THE OESTROGEN RECEPTOR IS SIGNIFICANTLY DYSREGULATED IN A MODEL OF DOCETAXEL RESISTANT PROSTATE CANCER AND TAMOXIFEN REVERSES APOPTOTIC RESISTANCE TO DOCETAXEL IN VITRO.

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-05 THE OESTROGEN RECEPTOR IS SIGNIFICANTLY DYSREGULATED IN A MODEL OF DOCETAXEL RESISTANT PROSTATE CANCER AND TAMOXIFEN REVERSES APOPTOTIC RESISTANCE TO DOCETAXEL IN VITRO. Dara Lundon, Maria Prencipe, Amanda O'Neill, Sinead Ahearne, Stephen Madden, Padraig Doolan, and William Watson Dara LundonDara Lundon More articles by this author , Maria PrencipeMaria Prencipe More articles by this author , Amanda O'NeillAmanda O'Neill More articles by this author , Sinead AhearneSinead Ahearne More articles by this author , Stephen MaddenStephen Madden More articles by this author , Padraig DoolanPadraig Doolan More articles by this author , and William WatsonWilliam Watson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2229AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The armamentarium of agents for the treatment of advanced prostate cancer has been significantly augmented in recent years. However the optimal treatment pathway for each man is still poorly defined. Docetaxel is an important chemotherapeutic drug in the treatment of prostate cancer, being amongst the most effective chemotherapeutic agents for the treatment of metastatic castrate-resistant prostate cancer. Docetaxel-resistance is a major obstacle in such patients. Defining the mechanisms underlying resistance so as to inform treatment selection remains a challenge. Previous experiments and publications in our laboratory have shown complex changes in pro and anti-apoptotic proteins in the development of docetaxel resistance. When targeted individually these proteins do not significantly impact upon the resistant phenotype. Our hypothesis was that targeting the central signalling pathways and transcription factors (TF) which control these could represent a more appropriate mechanism for identifying potential therapeutic targets and prognostic markers. Our aims were to identify potential targets/markers and then to undertake a validation of their functional role in a model of docetaxel resistance. METHODS We have developed a number of doxetaxel resistant sublines in PC-3 cells, and undertaken a transciptomic analysis of these cells by DNA microarray analysis using the Affymetrix Human Gene 1.0 ST Array. Using novel bioinformatic techniques, including correspondence, between-group and co-inertia analyses; transcriptional targets suitable for discerning patients with a phenotype of Docetaxel-resistance were identified. Manipulation with tamoxifen and estradiol, was used to determine functional effects on the apoptotic pathway by propidium iodide DNA staining and flow cytometry. RESULTS The TF ESR1, also known as the oestrogen receptor is amongst those predicted to be responsible for the differential gene expression observed in docetaxel-resistant prostate cancer. We have mapped the cell-processes of this TF and can display how it is intimately implicated in regulating docetaxel resistance in prostate cancer cell lines. We have demonstrated the close interplay of ESR1 on apoptosis, response to stress, chemotaxis and chemosensitivity amongst a myriad of inveigling functions. Manipulation of ESR1 in the PC3 docetaxel-resistant cells resulted in altered apoptotic resistance to docetaxel; whereby tamoxifen in combination with docetaxel resulted in a significant increase in apoptosis (p<0.01), and estradiol attenuated resistance (p<0.05) in this cell line model of advanced prostate cancer. CONCLUSIONS Due to the complexity of changes associated with the development of resistance to docetaxel, manipulating the upstream transcription factors and their networks may represent a more comprehensive therapeutic targeting approach for this advanced form of prostate cancer. We have successfully identified ESR1 as being dysregulated in a model of advanced prostate cancer and commenced validation of its functional role in docetaxel resistance in-vitro. The identification of TFs such as ESR1 could be utilised to help stratify men with prostate cancer, as potential therapeutic targets, prognostic aides and predictors of response to docetaxel or combined therapy in men with significant prostate cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1091 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Dara Lundon More articles by this author Maria Prencipe More articles by this author Amanda O'Neill More articles by this author Sinead Ahearne More articles by this author Stephen Madden More articles by this author Padraig Doolan More articles by this author William Watson More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Glucose regulated protein 78 (GRP78) inhibits apoptosis and attentinutes chemosensitivity of gemcitabine in breast cancer cell via AKT/mitochondrial apoptotic pathway

The underlying mechanism of gemcitabine resistance during breast cancer treatment remains unclear. Glucose regulated protein 78 (GRP78) frequently triggered by anticancer agents, was substantially elevated in gemcitabine resistant sublines. Ectopic expression of GRP78 changes gemcitabine chemosensitivity and apoptosis levels in breast cancer cells. Further experiments showed an involvement of caspase 9, not caspase 8, in gemcitabine resistance and GRP78-mediated chemosensitivity, suggesting that mitochondria apoptotic pathway was activated by GRP78. This finding was further supported by the observations of AKT activation, Bcl-2 increase, Bax and Bim decrease. Conclusively, GRP78 plays a vital role in gemcitabine resistance and clinical strategy to improve gemcitabine efficacy in breast cancer by manipulating GRP78 should be explored.

Design, synthesis, cytotoxic activity and molecular docking studies of new 20(S)-sulfonylamidine camptothecin derivatives

In an ongoing investigation of 20-sulfonylamidine derivatives (9, YQL-9a) of camptothecin (1) as potential anticancer agents directly and selectively inhibiting topoisomerase (Topo) I, the sulfonylamidine pharmacophore was held constant, and a camptothecin derivatives with various substitution patterns were synthesized. The new compounds were evaluated for antiproliferative activity against three human tumor cell lines, A-549, KB, and multidrug resistant (MDR) KB subline (KBvin). Several analogs showed comparable or superior antiproliferative activity compared to the clinically prescribed 1 and irinotecan (3). Significantly, the 20-sulfonylamidine derivatives exhibited comparable cytotoxicity against KBvin, while 1 and 3 were less active against this cell line. Among them, compound 15c displayed much better cytotoxic activity than the controls 1, 3, and 9. Novel key structural features related to the antiproliferative activities were identified by structure–activity relationship (SAR) analysis. In a molecular docking model, compounds 9 and 15c interacted with Topo I-DNA through a different binding mode from 1 and 3. The sulfonylamidine side chains of 9 and 15c could likely form direct hydrogen bonds with Topo I, while hydrophobic interaction with Topo I and π–π stacking with double strand DNA were also confirmed as binding driving forces. The results from docking models were consistent with the SAR conclusions. The introduction of bulky substituents at the 20-position contributed to the altered binding mode of the compound by allowing them to form new interactions with Topo I residues. The information obtained in this study will be helpful for the design of new derivatives of 1 with most promising anticancer activity.

MicroRNA expression profiles of drug-resistance breast cancer cells and their exosomes

Exosomes have been shown to transmit drug resistance through delivering miRNAs. We aimed to explore their roles in breast cancer. Three resistant sublines were established by exposing parental MDA-MB-231 cell line to docetaxel, epirubicin and vinorelbine, respectively. Preneoadjuvant chemotherapy biopsies and paired surgically-resected specimens embedded in paraffin from 23 breast cancer patients were collected. MiRNA expression profiles of the cell lines and their exosomes were evaluated using microarray. The result showed that most miRNAs in exosomes had a lower expression level than that in cells, however, some miRNAs expressed higher in exosomes than in cells, suggesting a number of miRNAs is concentrated in exosomes. Among the dysregulated miRNAs, 22 miRNAs were consistently up-regulated in exosomes and their cells of origin. We further found that 12 of the 22 miRNAs were significantly up-regulated after preneoadjuvant chemotherapy. Further study of the role of these 12 miRNAs in acquisition of drug resistance is needed to clarify their contribution to chemoresistance.

Transcriptomic Determinants of Scrapie Prion Propagation in Cultured Ovine Microglia

Susceptibility to infection by prions is highly dependent on the amino acid sequence and host expression of the cellular prion protein (PrPC); however, cellular expression of a genetically susceptible PrPC is insufficient. As an example, it has been shown in cultured cells that permissive and resistant sublines derived from the same parental population often have similar expression levels of PrPC. Thus, additional cellular factors must influence susceptibility to prion infection. The aim of this study was to elucidate the factors associated with relative permissiveness and resistance to scrapie prions in cultured cells derived from a naturally affected species. Two closely related ovine microglia clones with different prion susceptibility, but no detectable differences in PrPC expression levels, were inoculated with either scrapie-positive or scrapie-negative sheep brainstem homogenates. Five passages post-inoculation, the transcriptional profiles of mock and infected clones were sequenced using Illumina technology. Comparative transcriptional analyses identified twenty-two differentially transcribed genes, most of which were upregulated in poorly permissive microglia. This included genes encoding for selenoprotein P, endolysosomal proteases, and proteins involved in extracellular matrix remodeling. Furthermore, in highly permissive microglia, transforming growth factor β–induced, retinoic acid receptor response 1, and phosphoserine aminotranspherase 1 gene transcripts were upregulated. Gene Set Enrichment Analysis identified proteolysis, translation, and mitosis as the most affected pathways and supported the upregulation trend of several genes encoding for intracellular proteases and ribosomal proteins in poorly permissive microglia. This study identifies new genes potentially involved in scrapie prion propagation, corroborates results from other studies, and extends those results into another cell culture model.

Molecular characterization of irinotecan (SN-38) resistant human breast cancer cell lines.

BackgroundStudies in taxane and/or anthracycline refractory metastatic breast cancer (mBC) patients have shown approximately 30 % response rates to irinotecan. Hence, a significant number of patients will experience irinotecan-induced side effects without obtaining any benefit. The aim of this study was to lay the groundwork for development of predictive biomarkers for irinotecan treatment in BC.MethodsWe established BC cell lines with acquired or de novo resistance to SN-38, by exposing the human BC cell lines MCF-7 and MDA-MB-231 to either stepwise increasing concentrations over 6 months or an initial high dose of SN-38 (the active metabolite of irinotecan), respectively. The resistant cell lines were analyzed for cross-resistance to other anti-cancer drugs, global gene expression, growth rates, TOP1 and TOP2A gene copy numbers and protein expression, and inhibition of the breast cancer resistance protein (ABCG2/BCRP) drug efflux pump.ResultsWe found that the resistant cell lines showed 7–100 fold increased resistance to SN-38 but remained sensitive to docetaxel and the non-camptothecin Top1 inhibitor LMP400. The resistant cell lines were characterized by Top1 down-regulation, changed isoelectric points of Top1 and reduced growth rates. The gene and protein expression of ABCG2/BCRP was up-regulated in the resistant sub-lines and functional assays revealed BCRP as a key mediator of SN-38 resistance.ConclusionsBased on our preclinical results, we suggest analyzing the predictive value of the BCRP in breast cancer patients scheduled for irinotecan treatment. Moreover, LMP400 should be tested in a clinical setting in breast cancer patients with resistance to irinotecan.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2071-1) contains supplementary material, which is available to authorized users.

Distinct characterization of two vinorelbine-resistant breast cancer cell lines developed by different strategies.

Resistance to chemotherapy is a major obstacle to the successful treatment of breast cancer patients. Recently, we successfully established two vinorelbine-resistant sublines, BC-DS and BC-TS, from the human breast cancer cell line BCap37, with different 'two-stage screening methods'. Interestingly, though BC-DS and BC-TS were developed from the same BCap37 cell line with the same drug, they showed remarkable differences. Compared with the parental BCap37 cells both BC-DS and BC-TS had resistance to vinorelbine, but the resistant characterizations are both unstable. BC-DS showed increased migration capability while BC-TS showed reduced migration capability. When investigating their multidrug resistance, we found BC-DS became more sensitive to methotrexate, which suggested that combination of MTX and vinorelbine could be a new treatment strategy. Moreover, BC-DS and BC-TS overexpressed P-glycoprotein at different levels. Our research also showed that the present clinical usage of vinorelbine is reasonable. These findings suggest that the vinorelbine-induced multiple drug resistance (MDR) sublines may be used as an in vitro model not only to further elucidate possible mechanisms of MDR involved in the human breast cancer, but also to find methods to optimize the curative effect of vinorelbine in clinic.

Generation of Cisplatin-Resistant Ovarian Cancer Cell Lines

Ovarian cancer is the most lethal gynecological cancer in which cisplatin-based treatment plays a fundamental role as the first line chemotherapy option. However, development of platinum-resistance is a critical and poorly understood problem in ovarian cancer treatment. Although in vitro generation of platinum-resistant ovarian cancer cell lines is a long established approach to uncover the molecular mechanisms underlying resistance development, the methodology of this resistance induction is poorly explained in publications. The aim of this study was to propose a method for induction of resistance in ovarian cancer cell lines. To reach this aim, A2780 human ovarian cancer cell line was continuously exposed to stepwise increasing concentrations of cisplatin (0.5–2.6µM) over a period of 6 months and three resistant sublines were collected. Cisplatin resistance was examined by clonogenic survival assay and growth curve analysis was carried out in order to evaluate the proliferation characteristics of the established sublines. The A2780 resistant sublines exhibited 5.1 to 11.7 fold resistance to cisplatin, compared to their parental cells, and although growth rate and plateau saturation density significantly decreased by cisplatin resistance enhancement, all three resistant sublines presented a typical growth curve even though they were cultured in the cisplatin containing medium. These results suggest that reliable drug resistant human ovarian cancer cell lines can be successfully established by this method.

Abstract A10: Establishment and characterization of a patient-derived non-small cell lung cancer mouse model of acquired resistance towards anti-EGFR treatment

Abstract Non-small cell lung cancer (NSCLC) is the largest subgroup of lung cancer, occurring at a frequency of over 80% of lung cancer cases. In up to 30% of NSCLC patients the oncogenic driver of tumor growth is a constitutively activated EGF receptor (EGFR), which plays a critical role in regulating multiple cellular processes, including proliferation, survival and apoptosis. Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors (TKI, e.g. erlotinib or gefitinib), development of resistance is inevitable. To model the emergence of drug resistance, an EGFR driven, gefitinib sensitive, patient-derived xenograft (PDX) NSCLC model was treated continuously with gefitinib in immunocompromised mice. The dose of daily treatment was adjusted according to tumor growth over a period of up to 91 days. In a first phase, dosing was high (40-50 mg/kg) to eradicate EGFR TKI sensitive cells. At the time point of maximal antitumoral activity dosing was reduced to 20-30 mg/kg ( = low dose) to preserve selection pressure. Between 69 and 91 days after dosing was initiated, drug-resistant tumors emerged in 4 out of 10 mice under high dose treatment. Resistant tumor fragments, which were re-implanted into a new cohort of mice and continuously treated with gefitinib, kept resistance also under high dose treatment. A comprehensive analysis using Western blot (WB), qPCR and sequencing was performed to identify the reason for resistance. In WB analysis we could show that signalling through EGFR was completely abrogated in all four resistant tumor sublines. Neither secondary mutations in EGFR (ex19-21) or KRAS (ex 1+2) could be detected, nor was the expression of cMET, AXL, HGF, PTEN or HER3 significantly increased in resistant tumors as shown by sequencing and qPCR respectively. However a more comprehensive WB analysis revealed several genes being activated in resistant compared to primary tumors. Depending on the subline, phospho-(p)-cMET, p-AKT, AXL &amp; p-AXL, p-cRAF, p-MEK, p-ERK as well as HER3, IGF-R and ALK were up-regulated in resistant tumors. Based on these data we determined that signalling pathways such as the (RAS)-cRAF-MEK-ERK signalling cascade or enhanced cMET/AKT signalling were activated in drug-resistant tumors. These signalling pathways are known to be alternative pathways for EGFR signalling also in patients with acquired resistance indicating the clinical relevance of these models. To shed more light into the mechanism of resistance, whole exome sequencing analyses of the four resistant sublines as well as the original tumor model are underway. In summary, we have developed four NSCLC tumor sublines each harbouring a different mechanism of resistance to EGFR TKI treatment, modelling the emergence of drug-resistant NSCLC in patients. Herewith a strong preclinical tool for the development of innovative compounds targeting acquired EGFR resistance is now available. Citation Format: Cordula Tschuch, Kerstin Klingner, Dorothee Lehnhard, Anne Löhr, Yana Raeva, Anne-Lise Peille, Eva Oswald, Julia B. Schüler. Establishment and characterization of a patient-derived non-small cell lung cancer mouse model of acquired resistance towards anti-EGFR treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A10.

Abstract B71: DEN-50R - establishment of a novel and unique cell line based drug screening platform for cancer treatment

Abstract Today, chemotherapy and molecularly targeted therapies are the main therapeutic options in cancer treatment, but their effectiveness is limited by drug resistance, a major problem facing current cancer research. Since its establishment in the late 1980s, the NCI60 cell line panel has generated tremendous value, economic as well as societal, by contributing to the identification of mechanisms of drug action. This information, in turn, has been used to create new anti-cancer drugs for the benefit of patients. However, the cell lines comprised in the NCI60 panel are mostly derived from treatment of chemo-naïve patients (no medical treatment). Since early clinical trials of anti-cancer drugs most often recruit patients who have failed several different prior treatments, there is a need to develop a platform that builds on the concept underlying the NCI60 cell line panel and extend it to allow testing of potential new drugs in the setting of drug resistance. Moreover, it is also well-known that molecular drug resistance mechanisms might first appear following drug exposure and access to panels of matched drug-sensitive and drug-resistant isogenic cell lines, is thus mandatory for studying the underlying mechanisms of acquired drug resistance. We have initiated the establishment of DEN-50R, a novel and unique cell line-based drug screening platform within cancer treatment. The platform will, when fully developed, consist of human cancer cell lines representing the five most common cancer forms (colorectal, prostate, lung, breast cancer and malignant melanoma) and matched drug-resistant sublines for each of the five cancer forms. In a second phase additional cancer forms will be included. Resistance is developed against drugs and drug combinations currently used in the clinic, and with the introduction of novel drugs and regimens, new corresponding resistant sublines will be generated. All pairs of sensitive and resistant cell lines are thoroughly characterized with regard to cross-resistance to other drugs as well as to genomics, epigenomics, transcriptomics, and proteomics profiling. All data generated with the DEN-50R platform will be placed in the new National Danish Life Science Supercomputer with secure, regulated access providing a unique and innovative integrative analysis resource for academic, clinical and industrial researchers involved in anti-cancer drug development and cancer diagnostics at both pre-clinical and clinical levels. We envision that the DEN-50R be used to: • Test novel chemical entities for anti-cancer effects, including potential lack of cross-resistance thereby facilitating selection of analogs for further preclinical and clinical testing. • Test already known drugs for potential effects in drug resistant cell lines facilitating “repurposing” of drugs. • Reveal novel molecular drug resistant mechanisms allowing for targeted development of new drug entities that can circumvent drug resistance • identify novel predictive biomarkers for the most common drug entities further facilitating a personalized treatment approach. Citation Format: Jan Stenvang, Jose Moreira, Niels Frank Jensen, Signe Lykke Nielsen, Torben Orntoft, Ulrik Lassen, Cathrine Nordgaard, Stine Ninel Hansen, Haatisha Jandu, Anna Vind, Maria Andreasen, Camilla Cederbye, Julie Noer, Thiago Lima, Birgitte Martine Viuff, Soeren Brunak, Thomas Skoet Jensen, Peter Buhl Jensen, Joergen Drejer, Henrik Ditzel, Nils Brünner. DEN-50R - establishment of a novel and unique cell line based drug screening platform for cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B71.

Erratum to: PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells

[This corrects the article DOI: 10.1186/s12935-015-0239-4.].

Synergistic cytotoxicity from combination of imatinib and platinum-based anticancer drugs specifically in Bcr-Abl positive leukemia cells

Imatinib, a multitargeted tyrosine kinase inhibitor, exhibits potent anticancer activity against leukemia harboring the Bcr-Abl oncogene and some solid tumors overexpressing c-kit and PDGFR. However, its clinical efficacy is severely compromised by the emergence of resistance primarily due to acquired mutations in the Bcr-Abl kinase domain. In this study, we showed that combination of imatinib with platinum (Pt)-based anticancer agents, including cisplatin and oxaliplatin, exhibited synergistic cytotoxic effect specifically in Bcr-Abl+ human chronic myeloid leukemia cell line K562 but not in Bcr-Abl− RPMI8226 cells. Importantly, the synergistic effect was also found to circumvent imatinib resistance in an imatinib-selected resistant subline K562 ima1.0. The combination treatment increased apoptosis and DNA damage. Mechanistic study revealed that increased inhibition of Bcr-Abl and downstream ERK phosphorylation by the drug combination may contribute to the synergistic effect.

P108: Evaluation of the complex changes associated to the YB-1protein in resistant sublines of breast cancer cells

Background multidrug resistance (MDR) remains a major problem in the fight against tumors. MDR – multidimensional phenomenon, which occurs as a consequence of activation of several systems, associated both with the release of xenobiotics from the cell by ABC-transporters, and with stimulation of pathways that enhance cell survival. One of the possible regulators of MDR is multifunctional protein YB-1. YB-1 acting as a transcription factor in cell’s nucleus, can upregulate expression of MDR1, as well as some other ABC-transporters genes. Furthermore, YB-1 is secreted by cells and binds to the protein Notch3, and that stimulates proliferation and migration of cells. Also, resistance of cells may increase upon stimulation of VEGF-VEGER systems. Our aim was to study the mechanisms of MDR at various levels in the pairs of parental and resistant breast cancer cells: changes in the expression and localization of YB-1, evaluation expression of ABC-transporters, activation of protein Notch, as well as changes in the VEGF system. Materials and methods Long-term cultivation of cells MCF-7 (breast adenocarcinoma) and HBL-100 (transformed cells) in the presence of doxorubicin to obtain resistant sublines, MTT assay,real-time PCR, immunocytochemistry, flow cytofluorometry. Results we obtained the sublines of cells MCF-7 and HBL-100 resistant to doxorubicin: MCF-7/D7 and HBL-100/D85, respectively. The level of resistance to doxorubicin in resistant MCF-7/D6 was 7 times higher than in parent MCF-7 (IC50 was 0.7 ± 0.01 mcM vs 0.1 ± 0.002 mcM, respectively), and the level of resistance HBL-100/D85 was 44.7 times higher than the HBL-100 (IC50 8.5 ± 0.17 mcM vs 0.19 ± 0.03 mcM). Also, MCF-7/D7 acquired cross-resistance to cisplatin (6.7 ± 0.1 mcM vs 48 ± 0.2 mcM) and paclitaxel (2 ± 0.03 nM vs 50 ± 1 nM), but not to vinblastine and 5-fluorouracil, and HBL-100/D85 – about 100 times to paclitaxel (13 ± 0.2 nM vs 1100 ± 19 nM) and vinblastine (2.4 ± 0.3 nM vs 250 ± 5 nM), but not to the cisplatin and 5-fluorouracil. The expression profile of ABC-transporter genes showed significant activation of the MDR1 expression in subline HBL-100/D85 (more than 1000 times), but not in MCF-7/D6. Expression of other genes of the family ABC-transporters - MRP1, BCRP, MVP - increased slightly. Expression of mRNA YB-1 was almost unchanged. YB-1 protein detected in cytoplasm of parental lines, but it detected in nuclei in resistant sublines. Also, protein Notch3 translocated from membrane into nucleus. Evaluation of VEGF expression profile revealed that in a couple of VEGFA-VEGFR2 didn’t occur any changes. However, we observed a significant upregulation of expression mRNA VEGFC and down-regulation of mRNA VEGFR3 in resistant sublines MCF-7/D6 and HBL-100/D85. Conclusion the results of our study show that the appearance of MDR associated with various changes in cells. Common changes in the resistant sublines were translocation of protein YB-1 and Notch3 in cells nuclei, as well as the dynamics of expression changes in pair VEGFCVEGFR3.The last observation is an interesting finding and requires more detailed study.

P38: The role of intercellular interactions in the regulation of hormonal sensitivity of breast cancer cells

The efficiency of endocrine therapy for tumors is limited by the development of hormone resistance and progression of tumor cells to hormone-independent phenotype. Among these tumors – breast cancers, for which hormone therapy is one of the most common and effective methods of treatment, but only in cases, when the tumors retain their hormonal dependence. The mechanism of hormonal independence was found to be based on the fundamental properties of cancer cell including both downregulation of specific hormone receptors, and affecting of intracellular signalling, particularly – estrogen-independent growth signaling pathways. However, the role of the intercellular interactions in the progression of hormonal resistance is still unclear. We hypothesize, that the formation of the clone of the hormone-resistant cells in the tumor, and the subsequent common growth of the hormone-resistant and sensitive cells may lead to spread the hormonal resistance to the sensitive cells – as a result of the secretion of the specific factors acting in the paracrine manner or via the direct cell–cell contacts. Here, using the estrogen-dependent breast cancer cells MCF-7 and the resistant subline MCF-7/T developed by long-term cultivation of MCF-7 cells in the presence of antiestrogen tamoxifen, we investigated the possible changes in the hormonal sensitivity of these cells caused by the co-cultivation in vitro. For this purpose MCF-7/T cells were transfected with the plasmid containing the gene of the green fluorescent protein (GFP), and GFP-positive hormone-resistant subline MCF-7/T/GFP+ was developed. The GFP expression should allow to distinguish the resistant and parental cells during co-cultivation. To study the influence of the co-cultivation on the cell sensitivity to tamoxifen the parent MCF-7 cells (GFP-negative) were co-cultivate with the resistant MCF-7/T/GFP+/cells for 10 days, then the cells were treated with tamoxifen and the efficiency of growth inhibitory tamoxifen action was determined. We found, that the co -cultivation of the parent and resistant cells lead to increase in the resistance of the parent cell to tamoxifen, indicating the important role of the contacts, direct or indirect, between hormone sensitive and resistant cells in the development of hormone resistance. In general, we evaluate the established results as the first evidence of the possible involvement of the cell–cell underrelations in the realization of the hormonal response, and suppose that the next investigations will give a new insights in the molecular mechanisms of this effects and its role in the formation of the hormone-resistant phenotype of the tumors.

PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells.

PurposeAKT plays a pivotal role in the signal transduction of cancer cells. MK2206, an AKT inhibitor, has been shown to be an effective anti-cancer drug to a variety of cancer cell lines. However, some cancer cells acquire resistance to MK2206 and new strategies to suppress these cell lines remain to be developed.Experimental designAcquired MK-2206-resistant neuroblastoma (NB) cell sublines were induced by stepwise escalation of MK-2206 exposure (4–12 weeks). MTT assay was used to validate cell proliferation. Flow cytometry was performed for cell cycle analysis. Western blot assay was used for cell signaling study.ResultsMK2206 (5–10 µmol) significantly suppressed cell growth of MK2206 non-resistant NB cells (LAN-1, KP-N-SIFA, NB-19 and SK-N-DZ), but is less efficient in inhibiting that of resistant sublines, even after 2-week MK2206-free incubation. MK2206 acted in mTOR-S6K dependent and independent methods. MK-2206 resistant sublines (LAN-1-MK, KP-N-SIFA-MK, and SK-N-DZ-MK) showed lower IC50 of GSK2334470 (PDK1 inhibitor). The cell growth of all sublines was prohibited by AZD8805 (mTOR inhibitor), with IC50 of AZD8805 3–10 times lower than MK2206 non-resistant cells. The signaling profiles of these resistant sublines were characterized by elevated PDK1-mTOR-S6K activity, accompanying by low phosphorylation of AKT compared with non-resistant counterparts. GSK2334470 and AZD8055 effectively inhibited phosphorylation of PDK1 and mTOR, respectively, and induced higher G0–G1 ratio in LAN-1-MK than that in LAN-1 as well. PDK1 and mTOR inhibitors effected on phosphorylation of GSK3β in some of resistant sublines.ConclusionNB cells can acquire MK2206 resistance after exposure for 4–12 weeks. Resistant cells feature reliance on PDK1-mTOR-S6K pathway and are more sensitive to PDK1 and mTOR inhibitors than the non-resistant counterparts. Thus, suppression of PDK1-mTOR-S6K signaling pathway is an effective way to overcome the MK2206 resistance, and this may be a promising strategy for targeted therapy.

Proapoptotic activity and ABCC1-related multidrug resistance reduction ability of semisynthetic oleanolic acid derivatives DIOXOL and HIMOXOL in human acute promyelocytic leukemia cells

One of the main problems of present-day oncology is the ability of neoplastic cells to develop different mechanisms of resistance to chemotherapeutic agent. A natural compound oleanolic acid (OA) was found to be active against many types of neoplastic cells. This paper examines the influence of eight semisynthetic oleanolic acid derivatives on drug-sensitive human acute promyelocytic leukemia cell line HL-60 and its multidrug resistant subline ABCC1 overexpressing HL-60/AR. Viability inhibition, proapoptotic activity, as well as influence on the ABCC1 gene expression level, ability to inhibit the transport function of multidrug resistance associated protein 1 (ABCC1) and to alter its level by the tested compounds, were evaluated.The most potent compounds were DIOXOL (methyl 3,11-dioxoolean-12-en-28-oate) and HIMOXOL (methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate). DIOXOL was most efficient in inducing apoptosis of HL-60 cells. It activated both intrinsic and extrinsic pathways of apoptotic cell death. Proapoptotic properties of DIOXOL were probably related to the significant decrease of p65 NFκB level and inhibition of its translocation to the nucleus. In turn, HIMOXOL was the most potent compound against resistant HL-60/AR cells. It inhibited ABCC1 transport function (short time response) and decreased the level of ABCC1 protein (long time response) as a result of reduction of ABCC1 expression.

TGF Beta1 Expression Correlates with Survival and Tumor Aggressiveness of Prostate Cancer.

Although biopsy Gleason score and clinical stage can be used to inform treatment decisions for prostate cancer, identifying molecular markers of tumor aggressiveness could lead to a more tailored approaches to therapy. In the present study, we investigated the association of transforming growth factor (TGF)-β1 levels and various markers of tumor aggressiveness and explore some potential mechanisms underlying the associations. We used human and murine prostate cancer cell lines and their respective hormone resistance sub-lines, in vitro and in vivo to examine the changes in tumor aggressiveness, as well as the pathway responsible for these changes. Furthermore, 105 prostate cancer biopsy specimens were analyzed to correlate the level of TGF-β1 with the clinical characteristics of patients. Our data revealed that activated TGF-β1 signaling resulted in more aggressive tumor growth and augmented the epithelial-mesenchymal transition. Activated IL-6 signaling was associated with TGF-β1 levels and the aggressive tumor features noted in TGF-β1-positive prostate cancers in vitro and in vivo. Furthermore, the TGF-β1 levels significantly correlated with Tregs accumulation in vivo. The clinical data indicated that TGF-β1 immunoreactivity had a moderate positive correlation with IL-6 staining, advanced clinical stage, higher Gleason score, and pretreatment PSA in patients with prostate cancer. TGF-β1 levels are significantly associated with aggressive prostate features. In vitro and in vivo alternations of TGF-β1 expression impacts tumor invasiveness, tumor growth rate and recruitment of immunosuppressive Treg cells in the tumor microenvironment. TGF-β1 expression may represent a clinical useful biomarker to guide prostate cancer treatment decisions.