- Email: [email protected]
p53 mutations induced by the MDM2 inhibitor nutlin-3 in p53 wild-type neuroblastoma cells
S72
Poster abstracts
Poster Session – Drug Resistance and Modifiers, Wednesday 29 November 2016
the platinum drug-based therapy of ovarian carcinoma is often limited by the occurrence of drug resistance. Because the Receptor Tyrosine kinase (RTK) Axl can be deregulated in ovarian carcinoma and can play a role in maintaining tumor cell survival, the aim of this study was to examine the phenotype of ovarian carcinoma cells over-expressing Axl undergoing molecular targeting of Axl. Preclinical pharmacology approaches were employed including growth inhibition assays, western blot analyses, antibody arrays, gene knockdown by siRNA transfection, quantitative Real-time PCR, invasion and migration assays. The drug interaction was analysed using the Chou and Talalay method. We examined the effect of molecular targeting of Axl in different ovarian carcinoma cell lines including the cisplatin-resistant variant IGROV-1/Pt1, which over-expressed Axl and exhibited enhanced invasive potential as compared to parental cells. When the IGROV-1/Pt1 cells were transfected with Axl-tailored siRNAs, a marked and persistent reduction of mRNA/protein levels was obtained. Axl-silenced cells displayed reduced growth and invasive/migratory capabilities compared to control siRNAtransfected cells, in the absence of changes in cisplatin sensitivity. Axl silencing resulted in increased activation of ErbB3 in IGROV-1/Pt1 cells. Such cells displayed reduced sensitivity to AZD8931, a small molecule which inhibits ErbB3, besides EGF receptor and ErbB2, as compared to the parental IGROV-1 cell line. In keeping with this behavior, increased phosphorylation of ErbB3 at Tyr1289 was detected in IGROV-1/Pt1 cells. When exploring the possible advantage of the combination of cisplatin and AZD8931 in IGROV-1/Pt1 cells, a favourable drug interaction was observed in cells pretreated with AZD8931 before exposure to cisplatin. An analysis of the synergistic interaction after Axl silencing showed a reduced efficacy of the drug combination. Our findings indicate that compensatory survival pathways involving ErbB3 can be hyperactivated upon Axl silencing in ovarian carcinoma cells. The activation of this RTK, associated with drug resistance of ovarian carcinoma cells, can be counteracted by treatment with a specific inhibitor which displays a synergistic effect in combination with cisplatin. The evidence of increased activation of ErbB3 in platinum-resistant cell endowed with reduced sensitivity to other target-specific agents suggests the need to simultaneously target multiple survival factors to overcome drug resistance. No conflict of interest. 207 Poster (Board P033) Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status M. Michaelis1 , F. Rothweiler2 , N. Loeschmann2 , M. Sharifi3 , T. Ghafourian4 , J. Cinatl2 . 1 University of Kent, Centre for Molecular Processing and School of Biosciences, Canterbury, United Kingdom; 2 Goethe-Universitaet, Institut fuer Medizinische Virologie, Frankfurt am Main, Germany; 3 Universities of Kent and Greenwich, Medway School of Pharmacy, Chatham, United Kingdom; 4 University of Sussex, School of Life Sciences, Brighton, United Kingdom Background: Enzastaurin is a PKCb inhibitor that has been tested in clinical trials. Here, enzastaurin was tested in neuroblastoma and rhabdomyosarcoma cell lines. Material and Methods: Cell viability in response to enzastaurin was tested in the project cell lines, their vincristine-resistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2-transduced, and p53depleted cells. Pathway activation was determined through detection of protein phosphorylation. ABC transporter function was studied using cytotoxic and fluorescent substrates, specific inhibitors, determination of ABC transporter ATPase activity. The interaction of enzastaurin and ABCB1 was studied by in silico docking studies. Results: Enzastaurin IC50s ranged from 3.3 to 9.5mM in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125mM interfered with ABCB1-mediated drug transport. PKCa and PKCb may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations 1.25mM. The investigated cell lines did not express PKCb. PKCa depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Computational docking experiments detected a direct interaction of enzastaurin and ABCB1. These data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. Conclusions: The interaction of enzastaurin with ABC transporters needs to be considered for the further development of therapies combining enzastaurin with ABC transporter substrates. No conflict of interest.
208 Poster (Board P034) p53 mutations induced by the MDM2 inhibitor nutlin-3 in p53 wild-type neuroblastoma cells M. Michaelis1 , M. Wass1 , J. Cinatl2 . 1 University of Kent, Centre for Molecular Processing and School of Biosciences, Canterbury, United Kingdom; 2 Goethe-Universitaet, Institut fuer Medizinische Virologie, Frankfurt am Main, Germany Background: We and others showed that adaptation of wild-type p53 cell lines from different cancer entities to the MDM2 inhibitor nutlin-3 results in p53 mutations in the majority of the resulting nutlin-3-resistant sublines. In contrast, adaptation of wild-type p53 cancer cell lines to RITA (inhibits the p53-MDM2 interaction by binding to p53) did not result in lossof-p53 function. Using single p53 wild-type cell-derived clonal cell lines we demonstrated that nutlin-3 induces de novo p53 mutations. However, the origin of the nutlin-3-induced p53 mutations remains unclear. Material and Methods: In the meantime, we have established 94 nutlin-3resistant cancer cell lines by continuous exposure to step-wise increasing nutlin-3 concentrations. The p53 mutational status of these cell lines was determined by sequencing. Results: 72 out of these 94 nutlin-3-adapted cell lines (77%) harboured various p53 mutations while only 2 out of 28 sub-lines of the same cell lines adapted to various cytotoxic drugs harboured p53-mutations. The panel of nutlin-3-resistant cell lines includes 67 cell lines that were established from seven single p53 wild-type cell-derived sub-clones of the neuroblastoma cell line UKF-NB-3. Individual sub-clones differed in their p53 mutation profiles in response to adaptation to nutlin-3. Conclusions: Our data suggest that cell-specific features that differ between clonal sub-lines of the same parental cell line influence the nature and frequency of nutlin-3-induced p53 mutations. No conflict of interest. 209 Poster (Board P035) Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport M. Michaelis1 , F. Rothweiler2 , M. Wurglics3 , N. Aniceto4 , M. Dittrich3 , H. Zettl3 , M. Wass1 , T. Ghafourian5 , M. Schubert-Zsilavecz3 , J. Cinatl2 . 1 University of Kent, Centre for Molecular Processing and School of Biosciences, Canterbury, United Kingdom; 2 Goethe-Universitaet, Institut fuer Medizinische Virologie, Frankfurt am Main, Germany; 3 Goethe-Universitaet, Institut fuer Pharmazeutische Chemie, Frankfurt am Main, Germany; 4 Universities of Kent and Greenwich in Medway, Medway School of Pharmacy, Chatham, United Kingdom; 5 University of Sussex, School of Life Sciences, Brighton, United Kingdom Background: Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARa, PPARg, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARa, and PPARg represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drugresistant neuroblastoma cells. Material and Methods: Effects of pirinixic acid derivatives on cell viability was studied in a cell line panel including ABCB1-expressing cell lines. ABCB1 function was studied using fluorescent and cytotoxic substrates, specific inhibitors, and the determination of ABCB1 ATPase activity. Moreover, in silico docking studies were performed to investigate substrate interaction with ABCB1. Results: Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARa, or PPARg. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2mM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calceinAM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Conclusions: Pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport. No conflict of interest.
Poster abstracts
Poster Session – Drug Resistance and Modifiers, Wednesday 29 November 2016
the platinum drug-based therapy of ovarian carcinoma is often limited by the occurrence of drug resistance. Because the Receptor Tyrosine kinase (RTK) Axl can be deregulated in ovarian carcinoma and can play a role in maintaining tumor cell survival, the aim of this study was to examine the phenotype of ovarian carcinoma cells over-expressing Axl undergoing molecular targeting of Axl. Preclinical pharmacology approaches were employed including growth inhibition assays, western blot analyses, antibody arrays, gene knockdown by siRNA transfection, quantitative Real-time PCR, invasion and migration assays. The drug interaction was analysed using the Chou and Talalay method. We examined the effect of molecular targeting of Axl in different ovarian carcinoma cell lines including the cisplatin-resistant variant IGROV-1/Pt1, which over-expressed Axl and exhibited enhanced invasive potential as compared to parental cells. When the IGROV-1/Pt1 cells were transfected with Axl-tailored siRNAs, a marked and persistent reduction of mRNA/protein levels was obtained. Axl-silenced cells displayed reduced growth and invasive/migratory capabilities compared to control siRNAtransfected cells, in the absence of changes in cisplatin sensitivity. Axl silencing resulted in increased activation of ErbB3 in IGROV-1/Pt1 cells. Such cells displayed reduced sensitivity to AZD8931, a small molecule which inhibits ErbB3, besides EGF receptor and ErbB2, as compared to the parental IGROV-1 cell line. In keeping with this behavior, increased phosphorylation of ErbB3 at Tyr1289 was detected in IGROV-1/Pt1 cells. When exploring the possible advantage of the combination of cisplatin and AZD8931 in IGROV-1/Pt1 cells, a favourable drug interaction was observed in cells pretreated with AZD8931 before exposure to cisplatin. An analysis of the synergistic interaction after Axl silencing showed a reduced efficacy of the drug combination. Our findings indicate that compensatory survival pathways involving ErbB3 can be hyperactivated upon Axl silencing in ovarian carcinoma cells. The activation of this RTK, associated with drug resistance of ovarian carcinoma cells, can be counteracted by treatment with a specific inhibitor which displays a synergistic effect in combination with cisplatin. The evidence of increased activation of ErbB3 in platinum-resistant cell endowed with reduced sensitivity to other target-specific agents suggests the need to simultaneously target multiple survival factors to overcome drug resistance. No conflict of interest. 207 Poster (Board P033) Enzastaurin inhibits ABCB1-mediated drug efflux independently of effects on protein kinase C signalling and the cellular p53 status M. Michaelis1 , F. Rothweiler2 , N. Loeschmann2 , M. Sharifi3 , T. Ghafourian4 , J. Cinatl2 . 1 University of Kent, Centre for Molecular Processing and School of Biosciences, Canterbury, United Kingdom; 2 Goethe-Universitaet, Institut fuer Medizinische Virologie, Frankfurt am Main, Germany; 3 Universities of Kent and Greenwich, Medway School of Pharmacy, Chatham, United Kingdom; 4 University of Sussex, School of Life Sciences, Brighton, United Kingdom Background: Enzastaurin is a PKCb inhibitor that has been tested in clinical trials. Here, enzastaurin was tested in neuroblastoma and rhabdomyosarcoma cell lines. Material and Methods: Cell viability in response to enzastaurin was tested in the project cell lines, their vincristine-resistant sub-lines, primary neuroblastoma cells, ABCB1-transduced, ABCG2-transduced, and p53depleted cells. Pathway activation was determined through detection of protein phosphorylation. ABC transporter function was studied using cytotoxic and fluorescent substrates, specific inhibitors, determination of ABC transporter ATPase activity. The interaction of enzastaurin and ABCB1 was studied by in silico docking studies. Results: Enzastaurin IC50s ranged from 3.3 to 9.5mM in cell lines and primary cells independently of the ABCB1, ABCG2, or p53 status. Enzastaurin 0.3125mM interfered with ABCB1-mediated drug transport. PKCa and PKCb may phosphorylate and activate ABCB1 under the control of p53. However, enzastaurin exerted similar effects on ABCB1 in the presence or absence of functional p53. Also, enzastaurin inhibited PKC signalling only in concentrations 1.25mM. The investigated cell lines did not express PKCb. PKCa depletion reduced PKC signalling but did not affect ABCB1 activity. Intracellular levels of the fluorescent ABCB1 substrate rhodamine 123 rapidly decreased after wash-out of extracellular enzastaurin, and enzastaurin induced ABCB1 ATPase activity resembling the ABCB1 substrate verapamil. Computational docking experiments detected a direct interaction of enzastaurin and ABCB1. These data suggest that enzastaurin directly interferes with ABCB1 function. Enzastaurin further inhibited ABCG2-mediated drug transport but by a different mechanism since it reduced ABCG2 ATPase activity. Conclusions: The interaction of enzastaurin with ABC transporters needs to be considered for the further development of therapies combining enzastaurin with ABC transporter substrates. No conflict of interest.
208 Poster (Board P034) p53 mutations induced by the MDM2 inhibitor nutlin-3 in p53 wild-type neuroblastoma cells M. Michaelis1 , M. Wass1 , J. Cinatl2 . 1 University of Kent, Centre for Molecular Processing and School of Biosciences, Canterbury, United Kingdom; 2 Goethe-Universitaet, Institut fuer Medizinische Virologie, Frankfurt am Main, Germany Background: We and others showed that adaptation of wild-type p53 cell lines from different cancer entities to the MDM2 inhibitor nutlin-3 results in p53 mutations in the majority of the resulting nutlin-3-resistant sublines. In contrast, adaptation of wild-type p53 cancer cell lines to RITA (inhibits the p53-MDM2 interaction by binding to p53) did not result in lossof-p53 function. Using single p53 wild-type cell-derived clonal cell lines we demonstrated that nutlin-3 induces de novo p53 mutations. However, the origin of the nutlin-3-induced p53 mutations remains unclear. Material and Methods: In the meantime, we have established 94 nutlin-3resistant cancer cell lines by continuous exposure to step-wise increasing nutlin-3 concentrations. The p53 mutational status of these cell lines was determined by sequencing. Results: 72 out of these 94 nutlin-3-adapted cell lines (77%) harboured various p53 mutations while only 2 out of 28 sub-lines of the same cell lines adapted to various cytotoxic drugs harboured p53-mutations. The panel of nutlin-3-resistant cell lines includes 67 cell lines that were established from seven single p53 wild-type cell-derived sub-clones of the neuroblastoma cell line UKF-NB-3. Individual sub-clones differed in their p53 mutation profiles in response to adaptation to nutlin-3. Conclusions: Our data suggest that cell-specific features that differ between clonal sub-lines of the same parental cell line influence the nature and frequency of nutlin-3-induced p53 mutations. No conflict of interest. 209 Poster (Board P035) Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport M. Michaelis1 , F. Rothweiler2 , M. Wurglics3 , N. Aniceto4 , M. Dittrich3 , H. Zettl3 , M. Wass1 , T. Ghafourian5 , M. Schubert-Zsilavecz3 , J. Cinatl2 . 1 University of Kent, Centre for Molecular Processing and School of Biosciences, Canterbury, United Kingdom; 2 Goethe-Universitaet, Institut fuer Medizinische Virologie, Frankfurt am Main, Germany; 3 Goethe-Universitaet, Institut fuer Pharmazeutische Chemie, Frankfurt am Main, Germany; 4 Universities of Kent and Greenwich in Medway, Medway School of Pharmacy, Chatham, United Kingdom; 5 University of Sussex, School of Life Sciences, Brighton, United Kingdom Background: Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARa, PPARg, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARa, and PPARg represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drugresistant neuroblastoma cells. Material and Methods: Effects of pirinixic acid derivatives on cell viability was studied in a cell line panel including ABCB1-expressing cell lines. ABCB1 function was studied using fluorescent and cytotoxic substrates, specific inhibitors, and the determination of ABCB1 ATPase activity. Moreover, in silico docking studies were performed to investigate substrate interaction with ABCB1. Results: Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARa, or PPARg. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2mM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calceinAM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Conclusions: Pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport. No conflict of interest.