- Email: [email protected]
EBP-β-LIP turnover decides cancer chemotherapy outcome
Poster Session – Drug Resistance and Modifiers, Wednesday 29 November 2016 least in part by reducing NCOA3 expression, the critical ER coactivator in endocrine resistant breast cancer cells. No conflict of interest. 202 Poster (Board P028) Mitochondria-targeted doxorubicin: A new therapeutic strategy against drug-resistant osteosarcoma I. Buondonno1 , E. Gazzano1 , J. Sea Rin2 , V. Audrito3 , J. Kopecka1 , M. Fanelli4 , I.C. Salaroglio1 , C. Costamagna1 , I. Roato3 , E. Mungo1 , C. Hattinger4 , S. Deaglio3 , S. Kelley2 , M. Serra4 , C. Riganti1 . 1 University of Torino, Department of Oncology, Torino, Italy; 2 University of Toronto, Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, Toronto, Canada; 3 University of Torino, Department of Medical Sciences, Torino, Italy; 4 Orthopaedic Rizzoli Institute, Laboratory of Experimental Oncology, Pharmacogenomics and Pharmacogenetics Research Unit, Bologna, Italy Background: Doxorubicin (Dox) is one of the leader drugs for osteosarcoma standard chemotherapy. 40−45% of osteosarcoma patients are unresponsive to Dox, due to the overexpression of the drug efflux transporter ABCB1/P-glycoprotein (Pgp). Mitochondria metabolism is critical for the survival of drug resistant cells. Materials and Methods: We used a chemically modified Dox containing the anthracycline scaffold conjugated with a mitochondria-targeted peptide (mtDox), against human Dox-sensitive U-2OS and Saos-2 osteosarcoma cells and the corresponding variants (DX30, DX100, DX580) with progressively higher Dox-resistance and expression of ABCB1/Pgp. Results: Differently from Dox, which had a nuclear accumulation, mtDox was selectively delivered into mitochondria where its retention increased with the increase of resistance. While DOX was not cytotoxic in resistant variants, mtDOX still induced either cell necrosis or immunogenic death. Interestingly, Dox resistance increase was paralleled by the up-regulation of 111 genes controlling mitobiogenesis and mitochondria energy metabolism. mtDOX, but not Dox, down-regulated more than 2-fold 59 of these genes, decreased mitochondria biogenesis, mitochondrial transport of proteins and metabolites, mitochondrial energy metabolic pathways and ATP synthesis, while it increased ROS, mitochondrial depolarization and caspase 9/3 activation in resistant cells. mtDOX, but not DOX, reduced the growth of Dox-resistant/Pgp overexpressing osteosarcomas implanted in syngenic BALB/c mice, by killing tumor cells, increasing intratumor apoptosis and raisinig a proper anti-tumor response by the host immune system. Differently from Dox, mtDOX did not display signs of systemic toxicity and was not toxic for not-transformed osteoblasts. Furthermore, the indexes of cardiotoxicity did not differ between untreated animals and mtDox-treated animals. Conclusions: We propose a new and effective chemotherapeutic strategy for Dox-resistant osteosarcomas, by using a mitochondria-targeted Dox that exploits the metabolic signature typical of resistant cells − i.e. the hyperactive mitochondrial functions − and hits the energy pathways crucial for Dox-resistant osteosarcoma. mtDOX was effective also in Pgpoverexpressing tumors and was not cardiotoxic, overcoming the main limitations of Dox in osteosarcoma therapy. Our work may pave the way to the potential use of mtDOX in clinical settings, in particular for patients with Pgp-positive osteosarcomas or as a possible second-line treatment for relapsed patients. No conflict of interest. 203 Poster (Board P029) C/EBP-b-LIP turnover decides cancer chemotherapy outcome J. Kopecka1 , E. Panda1 , I.C. Salaroglio1 , I. Buondonno1 , S. Barak2 , M. Rubinstein2 , C. Riganti1 . 1 University of Turin, Department of Oncology, Turin, Italy; 2 The Weizmann Institute of Science, Department of Molecular Genetics, Rehovot, Israel Background: Chemotherapy often induces endoplasmic reticulum (ER) stress. ER stress activates molecular networks which first promote cell survival; if this attempt fails, cell death is induced. CAAT/enhancer binding protein-b (C/EBP-b) plays a key role in determining cell fate under ER stress. It has two isoforms: the pro-survival C/EBP b LAP and the pro-apoptotic C/EBP b LIP. LAP/LIP ratio is correlated with chemotherapy response because it regulates the expression of multidrug resistant protein 1/P-glycoprotein (Pgp), which causes chemoresistance. The regulation of LAP/LAP turnover in cancer cells and its role in chemotherapy response is not known. Material and Methods: We compared basal, chemotherapy- and ERstress-induced LAP/LIP ratio in ER stress-sensitive/chemosensitive and ER stress-resistant/chemoresistant cancer cells. We investigated the mechanism of LAP/LIP turnover, which was dictated by the rate of the reciprocal degradation, and the impact of altering LAP/LIP degradation on chemotherapy response.
Poster abstracts
S71
Results: Differently from ER stress-sensitive/chemosensitive cells, ER stress-resistant/chemoresistant cells had high LAP/LIP ratio and did not express LIP in response to ER stress or chemotherapy. This was due to the constitutive LIP ubiquitination followed by lysosomal/proteosomal degradation in resistant cells. The inducible intratumor over-expression of LIP, as well as the prevention of LIP degradation by lysosomes and proteasome inhibitors, improved chemotherapy outcome, by reducing Pgp levels and restoring chemotherapy- and ER stress-mediated cell death. Constitutive LIP degradation was detected in primary solid and hematological tumors unresponsive to chemotherapy. Conclusions: Our work demonstrates that the constitutive ubiquitination and degradation of LIP induces resistance to ER stress- and chemotherapy-induced cell death. LIP ubiquitination level may be considered a predictive biomarker of chemotherapy response. Lowering LAP/LIP ratio by preventing LIP ubiquitination and degradation represents a new approach to treat chemoresistant tumors. No conflict of interest. 205 Poster (Board P031) Pharmacokinetics and metabolite identification study of flavonoid dimer FD18: A potent P-glycoprotein modulator in reversing cancer drug resistance J.W.Y. Kan1 , C.S.W. Yan1 , I.L.K. Wong1 , K.F. Chan1 , T.H. Chan2 , L.M.C. Chow1 . 1 The Hong Kong Polytechnic University, The Department of Applied Biology and Chemical Technology, Kowloon, Hong Kong; 2 McGill University, Department of Chemistry, Montreal, Quebec, Canada Background: Overexpression of P-glycoprotein (P-gp) is one of the leading causes of multidrug resistance in chemotherapy. P-gp reduces the intracellular drug concentration below their respective therapeutic level. To reverse P-gp-mediated drug resistance, we recently reported that synthetic flavonoid dimer is a new class of potent P-gp modulator. One of the flavonoid dimers, FD18, has an effective concentration (EC50) of around 140 nM in reversing paclitaxel (PTX) resistance. FD18 can also reverse P-gp-mediated PTX resistance in human breast cancer xenograft model in vivo. Here, we report the PK profile and metabolite identification of FD18; and subsequently in vitro and in vivo P-gp modulating evaluation of FD18 metabolites. Methods: PK study of FD18 was conducted in SD rat. Metabolism of FD18 was evaluated in rat and human liver microsome assay in vitro and SD rat in vivo. Metabolite identification of FD18 was done by UPLC-MSMS QTOF and authenticated using pure, synthetic compounds. P-gp modulating activities of the metabolites were evaluated with various anticancer drugs on LCC6MDR cells in vitro and subsequently in breast cancer xenograft model in vivo. Results: IV administration of FD18 resulted in a first order kinetics and a non-linear plasma PK profile. IP administration of 45 mg/kg FD18 resulted in a mean residence time (MRT) of approximately 600 minutes. Three major metabolites of FD18 (M1, M2 and M3) were identified in vitro and in vivo. Metabolites identities were authenticated using pure, synthetic compounds. The P-gp modulating activities of M1, M2 and M3 (in reversing PTX resistance) was determined to be 305±35 nM, 70±26 nM and no activity, respectively. Surprisingly, M2 is also a potent P-gp modulator towards vinblastine, vincristine, doxorubicin, mitoxantrone and daunorubicin with EC50 of 61±13 nM, 83±11 nM, 153±39 nM, 64±27 nM and 88±52nM, respectively. Hydrochloride salt of synthetic M2 demonstrated in vivo efficacy in reversing PTX resistance in breast cancer xenograft model. P-gp-overexpressing xenograft was treated with 12 injections of M2 (28 mg/kg, IP) and PTX (12 mg/kg, IV) every other day (q1d ×12). On day 30, tumor size of animals treated with M2 and PTX was 773±114 mm3 (n = 8) while animals in the solvent control group was 1759±455 mm3 (n = 6). Tumor size of animals treated with PTX (12 mg/kg, IV) alone was 1208±66 mm3 (n = 6). Conclusion: Flavonoid dimer is a new class of safe and potent P-gp modulators. The proposed metabolism pathway of FD18 is via N-dealkylation and oxidative deamination. M2 is a metabolite of FD18 with potent in vitro and in vivo P-gp modulating activity. Conflict of interest: Other Substantive Relationships: Part of the project involves a technology licensed to Athenex Ltd. 206 Poster (Board P032) Targeting ErbB3 activation in drug-resistant ovarian carcinoma cells over-expressing the receptor tyrosine kinase Axl C. Corno1 , L. Gatti1 , N. Carenini1 , N. Zaffaroni1 , C. Lanzi1 , P. Perego1 . 1 Fondazione IRCCS Istituto Nazionale dei Tumori, Experimental Oncology and Molecular Medicine, Milan, Italy Ovarian carcinoma is the most common gynecological cancer and a major cause of cancer-related death in women. The high lethality of this cancer is mainly due to late diagnosis and treatment failure. In fact, the efficacy of
Poster abstracts
S71
Results: Differently from ER stress-sensitive/chemosensitive cells, ER stress-resistant/chemoresistant cells had high LAP/LIP ratio and did not express LIP in response to ER stress or chemotherapy. This was due to the constitutive LIP ubiquitination followed by lysosomal/proteosomal degradation in resistant cells. The inducible intratumor over-expression of LIP, as well as the prevention of LIP degradation by lysosomes and proteasome inhibitors, improved chemotherapy outcome, by reducing Pgp levels and restoring chemotherapy- and ER stress-mediated cell death. Constitutive LIP degradation was detected in primary solid and hematological tumors unresponsive to chemotherapy. Conclusions: Our work demonstrates that the constitutive ubiquitination and degradation of LIP induces resistance to ER stress- and chemotherapy-induced cell death. LIP ubiquitination level may be considered a predictive biomarker of chemotherapy response. Lowering LAP/LIP ratio by preventing LIP ubiquitination and degradation represents a new approach to treat chemoresistant tumors. No conflict of interest. 205 Poster (Board P031) Pharmacokinetics and metabolite identification study of flavonoid dimer FD18: A potent P-glycoprotein modulator in reversing cancer drug resistance J.W.Y. Kan1 , C.S.W. Yan1 , I.L.K. Wong1 , K.F. Chan1 , T.H. Chan2 , L.M.C. Chow1 . 1 The Hong Kong Polytechnic University, The Department of Applied Biology and Chemical Technology, Kowloon, Hong Kong; 2 McGill University, Department of Chemistry, Montreal, Quebec, Canada Background: Overexpression of P-glycoprotein (P-gp) is one of the leading causes of multidrug resistance in chemotherapy. P-gp reduces the intracellular drug concentration below their respective therapeutic level. To reverse P-gp-mediated drug resistance, we recently reported that synthetic flavonoid dimer is a new class of potent P-gp modulator. One of the flavonoid dimers, FD18, has an effective concentration (EC50) of around 140 nM in reversing paclitaxel (PTX) resistance. FD18 can also reverse P-gp-mediated PTX resistance in human breast cancer xenograft model in vivo. Here, we report the PK profile and metabolite identification of FD18; and subsequently in vitro and in vivo P-gp modulating evaluation of FD18 metabolites. Methods: PK study of FD18 was conducted in SD rat. Metabolism of FD18 was evaluated in rat and human liver microsome assay in vitro and SD rat in vivo. Metabolite identification of FD18 was done by UPLC-MSMS QTOF and authenticated using pure, synthetic compounds. P-gp modulating activities of the metabolites were evaluated with various anticancer drugs on LCC6MDR cells in vitro and subsequently in breast cancer xenograft model in vivo. Results: IV administration of FD18 resulted in a first order kinetics and a non-linear plasma PK profile. IP administration of 45 mg/kg FD18 resulted in a mean residence time (MRT) of approximately 600 minutes. Three major metabolites of FD18 (M1, M2 and M3) were identified in vitro and in vivo. Metabolites identities were authenticated using pure, synthetic compounds. The P-gp modulating activities of M1, M2 and M3 (in reversing PTX resistance) was determined to be 305±35 nM, 70±26 nM and no activity, respectively. Surprisingly, M2 is also a potent P-gp modulator towards vinblastine, vincristine, doxorubicin, mitoxantrone and daunorubicin with EC50 of 61±13 nM, 83±11 nM, 153±39 nM, 64±27 nM and 88±52nM, respectively. Hydrochloride salt of synthetic M2 demonstrated in vivo efficacy in reversing PTX resistance in breast cancer xenograft model. P-gp-overexpressing xenograft was treated with 12 injections of M2 (28 mg/kg, IP) and PTX (12 mg/kg, IV) every other day (q1d ×12). On day 30, tumor size of animals treated with M2 and PTX was 773±114 mm3 (n = 8) while animals in the solvent control group was 1759±455 mm3 (n = 6). Tumor size of animals treated with PTX (12 mg/kg, IV) alone was 1208±66 mm3 (n = 6). Conclusion: Flavonoid dimer is a new class of safe and potent P-gp modulators. The proposed metabolism pathway of FD18 is via N-dealkylation and oxidative deamination. M2 is a metabolite of FD18 with potent in vitro and in vivo P-gp modulating activity. Conflict of interest: Other Substantive Relationships: Part of the project involves a technology licensed to Athenex Ltd. 206 Poster (Board P032) Targeting ErbB3 activation in drug-resistant ovarian carcinoma cells over-expressing the receptor tyrosine kinase Axl C. Corno1 , L. Gatti1 , N. Carenini1 , N. Zaffaroni1 , C. Lanzi1 , P. Perego1 . 1 Fondazione IRCCS Istituto Nazionale dei Tumori, Experimental Oncology and Molecular Medicine, Milan, Italy Ovarian carcinoma is the most common gynecological cancer and a major cause of cancer-related death in women. The high lethality of this cancer is mainly due to late diagnosis and treatment failure. In fact, the efficacy of