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986 Detection of Drug-target Proteins on Tumor-derived Exosomes by ELISA Using Anti-CD81 Antibodies
S238
european journal of cancer 48, suppl. 5 (2012) S25–S288
samples and 2 archival cytological smears from 42 lung adenocarcinoma patients were evaluated. EGFR-activating mutations, that is, L858R (exon 21) mutation, exon 19 deletion, and EGFR-TKI-resistant T790M (exon 20) mutation, were analyzed by mutation-based polymerase chain reaction (PCR) performed using a quenching probe (MBP-QP) and Tm analysis using the i-densy system (ARKRAY Inc.). The cytological samples were analyzed in sediments of cells without DNA extraction. Paraffin-embedded biopsy and archival cytological samples were analyzed in extracted DNA. The results for the MBP-QP and Tm method were compared with those for the PCR clamp and DNA direct sequencing methods. Results: The L858R mutation, exon 19 deletion, and T790M mutation were detected in 6, 6, and 2 cases, respectively. Of the patients with L858R mutation or exon 19 deletion, 8 were treated with EGFR-TKIs. Six patients were responsive to this treatment. However, 2 patients were resistant to EGFRTKI treatment, and the T790M mutation was detected in addition to L858R mutation or exon 19 deletion in these patients. These EGFR mutations were also detected by the PCR clamp method and DNA direct sequencing. In the MBP-QP and Tm method, EGFR gene mutations in 3 exons were detectable in 1 tube after 1.5 hours. The results − with or without mutations − were easy to assess by using a visual chart. In particular, a direct assay could be performed from sediments in the lung cytological samples (PE and BALF samples) that were confirmed to contain carcinoma cells. Conclusions: The MBP-QP and Tm method used in the current study is a rapid and simple method for simultaneously detecting EGFR-activating and gatekeeper mutations. It is expected to be useful for companion diagnosis for determining EGFR-TKI treatment in lung adenocarcinomas. 986 Detection of Drug-target Proteins on Tumor-derived Exosomes by ELISA Using Anti-CD81 Antibodies H. Kamada1 , T. Yamashita1 , S. Kanasaki1 , Y. Maeda1 , M. Inoue1 , K. Nagano1 , Y. Abe1 , Y. Tsutsumi2 , S. Tsunoda1 . 1 National Institute of Biomedical Innovation, Laboratory of Biopharmaceutical Research, Ibaraki, Japan, 2 Osaka University, Laboratory of Toxicology and Safety Science, Suita, Japan Introduction: Detection and measurement of drug-target or biomarker proteins in tumor tissues are important processes for personalized cancer chemotherapy. However, current immuno-histochemical or cytogenetic analyses of biopsy specimens are highly invasive for pre-therapeutic diagnosis and are often difficult to apply to lung cancer. In this study, we established a simple method to detect membrane proteins such as epidermal growth factor receptor (EGFR) on exosomes secreted by tumor cells and evaluated its usefulness as a minimally invasive diagnosis. Material and Method: Cell culture: Human lung cancer cell lines (HARA, HARA-B and A549) were maintained in RPMI-1640 with 10% fetal bovine serum and cultured for 72 h in the serum-free culture medium to purify exosomes. Exosome preparation: The culture medium were sequentially centrifuged at 300 g for 5 min and 16,000 g for 20 min to eliminate cells and debris, followed by filtration through a 0.22 mm pore nitrocellulose membrane. Then, exosomes were precipitated by ultracentrifugation at 140,000 g for 70 min. Exosome detection by sandwich ELISA: Mouse anti-human CD81 antibody was immobilized on assay plate to capture exosomes specifically. For target protein detection, goat anti-EGFR antibody and anti-goat IgG were used. Blood plasma: As a model of patients’ plasma, peripheral blood of BALB/c nu/nu mice inoculated with HARA-B cells was collected and EGFR on exosomes was measured with the ELISA. Results and Discussion: Anti-CD81 antibody was used for capturing the exosomes because CD81 presented universally in each exosome membrane of normal and lung cancer cells. An ELISA system that captures exosomes using an anti-CD81 antibody, successfully detected exosomal EGFR, a typical target of molecular targeting drugs, from EGFR-positive tumor cell exosomes and blood plasma of tumor-bearing mice. Results of this study suggest that this novel method for exosomal protein measurement could be useful for in vitro diagnosis, especially for minimally invasive companion diagnosis in personalized cancer chemotherapy. Conclusion: The amount of EGFR expressing in plasma exosomes was quantified in order to apply to the diagnosis for lung cancer. The quantification of EGFR in plasma should be utilized for the ‘companion diagnostics’, which associated with the effectiveness of anticancer drugs such as Cetuximab to detect the EGFR level in plasma derived from lung cancer patients in the near future.
Sunday 8 − Tuesday 10 July 2012
987 Molecular Mechanisms of Sorafenib-induced Apoptosis in Cancer Cells C. Kuznia1 , B. Klinger2 , J. Keil3 , B. Seliger4 , C. Falk3 , R. Schafer ¨ 1, 1 N. Bluthgen ¨ , C. Sers1 . 1 Charite´ Berlin, Institute of Pathology, Berlin, Germany, 2 Humboldt University of Berlin, Institute for Theoretical Biology, Berlin, Germany, 3 Hannover Medical School, Integrated Research and Treatment Center Transplantation, Hannover, Germany, 4 Martin Luther University, Institute for Medical Immunology, Halle, Germany Introduction: The RAS/MEK/ERK pathway is a key oncogenic pathway involved in human cancer. Point mutations in RAS genes resulting in constitutive activation of RAS proteins occur in ~30% of human cancers. RAS activation results in an overactivation of signaling pathways thereby inducing cell proliferation, invasion and apoptosis inhibition. We investigated the Raf kinase inhibitor Sorafenib and the MEK inhibitor U0126 in human embryonal kidney cells transformed by oncogenic HRAS. Both substances target the same pathway; yet, treatment with Sorafenib induces apoptosis while U0126 does not. A time-resolved analysis of this effect by protein and mRNA arrays, revealed an involvement of DDIT4 (also known as Redd1) and mTOR signaling in the differential action of Sorafenib and U0126. The present study aims to examine whether that observation also applies to human cancer cells and to get a better understanding of the molecular mechanisms behind it. Materials and Methods: We treated six different renal carcinoma cell lines with Sorafenib or U0126 and tested apoptosis by cleaved caspase-3 measurement via flow cytometry. In addition, expression of DDIT4 was analyzed using Real-Time-PCR. To further investigate the link between DDIT4 and apoptosis we performed a siRNA-mediated knock-down of DDIT4 as well. The activation of mTOR and associated components of that signaling network were examined using a Bio-Plex assay. The activating transcription factor ATF4 described in literature as a regulator of DDIT4 was also analyzed using RealTime-PCR. Results and Discussion: Four cell lines induced caspase-3 cleavage and a significant increase of DDIT4 after Sorafenib treatment only. One cell line could not induce DDIT4, one cell line did induce DDIT4, but no cleaveage of caspase-3. siRNA-mediated knock-down of DDIT4 inhibited caspase-3 cleavage. A closer examination of the mTOR pathway using the Bio-Plex assay revealed no differences in mTOR inhibition between cells with and without DDIT4-mediated apoptosis. A significant upregulation of ATF4 in combination with an increased RNA level of DDIT4 could only be confirmed in one out of four selected cell lines. Conclusion: These results encourage the hypothesis that the induction of DDIT4 plays an important role for apoptosis induction by Sorafenib in human renal cancer. However, successful induction of apoptosis might be hampered by currently unknown mechnisms interfering with DDIT4-upregulation or caspase-cleavage. 988 Pharmacological Approach of the Role of Transcriptional Activation and Degradation in Acute Promyelocytic Leukemia M. Leiva Arjona1 , F. Jollivet1 , H. de The´ 1 . 1 Institute Universitaire ˆ d’Hematologie. Hopital Saint Louis, CNRS/INSERM/University Paris 7 UMR 7212-U944, Paris, France Background: Acute promyelocytic leukemia (APL) is caused by a variety of chromosomal translocations into the retinoic acid receptor-alpha (RARA) gene. The X-RARA fusions tightly associate with corepressor complexes under physiologic concentrations of retinoic acid (RA), leading to transcriptional repression and differentiation block. Only very high RA concentrations allow APL differentiation and clinical remission. It has been proposed that while transcriptional activation likely controls differentiation, may not be the primary basis for the therapeutic efficacy of retinoic acid in clearing APL. Indeed, only PML-RARA degradation ensures efficient APL eradication. The renewed importance of PML/RARA degradation led us to search molecules that could trigger RARA. Material and Method: To identify new compounds with modulate RARA degradation we have constructed an human GFP-RARA cell line to carry out a cell-based fluorescent assay using libraries of FDA-approved compounds. These we tested for their ability to oppose RA-induced RARA degradation. Results: The screen blindly identified expected positive controls such as RA itself, rexinoids or proteasome inhibitors. We also identified Etretinate, a synthetic retinoid used in skin disease, as a compound that antagonizes RARA degradation. Unexpectedly, Etretinate, and its active metabolite Acitretin, both induced differentiation in several APL models ex vivo or in vivo, but did not exert significant anti-leukemic effects. Ex vivo, Etretinate or Acitretin treatment did not result in quantitative differences in clonogenic ability of pre-leukemic APL cells. In vivo, Etretinate differentiates APLs without significant loss of clonogenic activity. Importantly Etretinate or Acitretin, in sharp contrast to RA, did not trigger PML/RARA or RARA degradation. Conclusion: Our results uncouple retinoic-induced degradation and transactivation of RARA. They establish the role of PML/RARA degradation in retinoic acid response. They pave the way to screens where these two effects of
european journal of cancer 48, suppl. 5 (2012) S25–S288
samples and 2 archival cytological smears from 42 lung adenocarcinoma patients were evaluated. EGFR-activating mutations, that is, L858R (exon 21) mutation, exon 19 deletion, and EGFR-TKI-resistant T790M (exon 20) mutation, were analyzed by mutation-based polymerase chain reaction (PCR) performed using a quenching probe (MBP-QP) and Tm analysis using the i-densy system (ARKRAY Inc.). The cytological samples were analyzed in sediments of cells without DNA extraction. Paraffin-embedded biopsy and archival cytological samples were analyzed in extracted DNA. The results for the MBP-QP and Tm method were compared with those for the PCR clamp and DNA direct sequencing methods. Results: The L858R mutation, exon 19 deletion, and T790M mutation were detected in 6, 6, and 2 cases, respectively. Of the patients with L858R mutation or exon 19 deletion, 8 were treated with EGFR-TKIs. Six patients were responsive to this treatment. However, 2 patients were resistant to EGFRTKI treatment, and the T790M mutation was detected in addition to L858R mutation or exon 19 deletion in these patients. These EGFR mutations were also detected by the PCR clamp method and DNA direct sequencing. In the MBP-QP and Tm method, EGFR gene mutations in 3 exons were detectable in 1 tube after 1.5 hours. The results − with or without mutations − were easy to assess by using a visual chart. In particular, a direct assay could be performed from sediments in the lung cytological samples (PE and BALF samples) that were confirmed to contain carcinoma cells. Conclusions: The MBP-QP and Tm method used in the current study is a rapid and simple method for simultaneously detecting EGFR-activating and gatekeeper mutations. It is expected to be useful for companion diagnosis for determining EGFR-TKI treatment in lung adenocarcinomas. 986 Detection of Drug-target Proteins on Tumor-derived Exosomes by ELISA Using Anti-CD81 Antibodies H. Kamada1 , T. Yamashita1 , S. Kanasaki1 , Y. Maeda1 , M. Inoue1 , K. Nagano1 , Y. Abe1 , Y. Tsutsumi2 , S. Tsunoda1 . 1 National Institute of Biomedical Innovation, Laboratory of Biopharmaceutical Research, Ibaraki, Japan, 2 Osaka University, Laboratory of Toxicology and Safety Science, Suita, Japan Introduction: Detection and measurement of drug-target or biomarker proteins in tumor tissues are important processes for personalized cancer chemotherapy. However, current immuno-histochemical or cytogenetic analyses of biopsy specimens are highly invasive for pre-therapeutic diagnosis and are often difficult to apply to lung cancer. In this study, we established a simple method to detect membrane proteins such as epidermal growth factor receptor (EGFR) on exosomes secreted by tumor cells and evaluated its usefulness as a minimally invasive diagnosis. Material and Method: Cell culture: Human lung cancer cell lines (HARA, HARA-B and A549) were maintained in RPMI-1640 with 10% fetal bovine serum and cultured for 72 h in the serum-free culture medium to purify exosomes. Exosome preparation: The culture medium were sequentially centrifuged at 300 g for 5 min and 16,000 g for 20 min to eliminate cells and debris, followed by filtration through a 0.22 mm pore nitrocellulose membrane. Then, exosomes were precipitated by ultracentrifugation at 140,000 g for 70 min. Exosome detection by sandwich ELISA: Mouse anti-human CD81 antibody was immobilized on assay plate to capture exosomes specifically. For target protein detection, goat anti-EGFR antibody and anti-goat IgG were used. Blood plasma: As a model of patients’ plasma, peripheral blood of BALB/c nu/nu mice inoculated with HARA-B cells was collected and EGFR on exosomes was measured with the ELISA. Results and Discussion: Anti-CD81 antibody was used for capturing the exosomes because CD81 presented universally in each exosome membrane of normal and lung cancer cells. An ELISA system that captures exosomes using an anti-CD81 antibody, successfully detected exosomal EGFR, a typical target of molecular targeting drugs, from EGFR-positive tumor cell exosomes and blood plasma of tumor-bearing mice. Results of this study suggest that this novel method for exosomal protein measurement could be useful for in vitro diagnosis, especially for minimally invasive companion diagnosis in personalized cancer chemotherapy. Conclusion: The amount of EGFR expressing in plasma exosomes was quantified in order to apply to the diagnosis for lung cancer. The quantification of EGFR in plasma should be utilized for the ‘companion diagnostics’, which associated with the effectiveness of anticancer drugs such as Cetuximab to detect the EGFR level in plasma derived from lung cancer patients in the near future.
Sunday 8 − Tuesday 10 July 2012
987 Molecular Mechanisms of Sorafenib-induced Apoptosis in Cancer Cells C. Kuznia1 , B. Klinger2 , J. Keil3 , B. Seliger4 , C. Falk3 , R. Schafer ¨ 1, 1 N. Bluthgen ¨ , C. Sers1 . 1 Charite´ Berlin, Institute of Pathology, Berlin, Germany, 2 Humboldt University of Berlin, Institute for Theoretical Biology, Berlin, Germany, 3 Hannover Medical School, Integrated Research and Treatment Center Transplantation, Hannover, Germany, 4 Martin Luther University, Institute for Medical Immunology, Halle, Germany Introduction: The RAS/MEK/ERK pathway is a key oncogenic pathway involved in human cancer. Point mutations in RAS genes resulting in constitutive activation of RAS proteins occur in ~30% of human cancers. RAS activation results in an overactivation of signaling pathways thereby inducing cell proliferation, invasion and apoptosis inhibition. We investigated the Raf kinase inhibitor Sorafenib and the MEK inhibitor U0126 in human embryonal kidney cells transformed by oncogenic HRAS. Both substances target the same pathway; yet, treatment with Sorafenib induces apoptosis while U0126 does not. A time-resolved analysis of this effect by protein and mRNA arrays, revealed an involvement of DDIT4 (also known as Redd1) and mTOR signaling in the differential action of Sorafenib and U0126. The present study aims to examine whether that observation also applies to human cancer cells and to get a better understanding of the molecular mechanisms behind it. Materials and Methods: We treated six different renal carcinoma cell lines with Sorafenib or U0126 and tested apoptosis by cleaved caspase-3 measurement via flow cytometry. In addition, expression of DDIT4 was analyzed using Real-Time-PCR. To further investigate the link between DDIT4 and apoptosis we performed a siRNA-mediated knock-down of DDIT4 as well. The activation of mTOR and associated components of that signaling network were examined using a Bio-Plex assay. The activating transcription factor ATF4 described in literature as a regulator of DDIT4 was also analyzed using RealTime-PCR. Results and Discussion: Four cell lines induced caspase-3 cleavage and a significant increase of DDIT4 after Sorafenib treatment only. One cell line could not induce DDIT4, one cell line did induce DDIT4, but no cleaveage of caspase-3. siRNA-mediated knock-down of DDIT4 inhibited caspase-3 cleavage. A closer examination of the mTOR pathway using the Bio-Plex assay revealed no differences in mTOR inhibition between cells with and without DDIT4-mediated apoptosis. A significant upregulation of ATF4 in combination with an increased RNA level of DDIT4 could only be confirmed in one out of four selected cell lines. Conclusion: These results encourage the hypothesis that the induction of DDIT4 plays an important role for apoptosis induction by Sorafenib in human renal cancer. However, successful induction of apoptosis might be hampered by currently unknown mechnisms interfering with DDIT4-upregulation or caspase-cleavage. 988 Pharmacological Approach of the Role of Transcriptional Activation and Degradation in Acute Promyelocytic Leukemia M. Leiva Arjona1 , F. Jollivet1 , H. de The´ 1 . 1 Institute Universitaire ˆ d’Hematologie. Hopital Saint Louis, CNRS/INSERM/University Paris 7 UMR 7212-U944, Paris, France Background: Acute promyelocytic leukemia (APL) is caused by a variety of chromosomal translocations into the retinoic acid receptor-alpha (RARA) gene. The X-RARA fusions tightly associate with corepressor complexes under physiologic concentrations of retinoic acid (RA), leading to transcriptional repression and differentiation block. Only very high RA concentrations allow APL differentiation and clinical remission. It has been proposed that while transcriptional activation likely controls differentiation, may not be the primary basis for the therapeutic efficacy of retinoic acid in clearing APL. Indeed, only PML-RARA degradation ensures efficient APL eradication. The renewed importance of PML/RARA degradation led us to search molecules that could trigger RARA. Material and Method: To identify new compounds with modulate RARA degradation we have constructed an human GFP-RARA cell line to carry out a cell-based fluorescent assay using libraries of FDA-approved compounds. These we tested for their ability to oppose RA-induced RARA degradation. Results: The screen blindly identified expected positive controls such as RA itself, rexinoids or proteasome inhibitors. We also identified Etretinate, a synthetic retinoid used in skin disease, as a compound that antagonizes RARA degradation. Unexpectedly, Etretinate, and its active metabolite Acitretin, both induced differentiation in several APL models ex vivo or in vivo, but did not exert significant anti-leukemic effects. Ex vivo, Etretinate or Acitretin treatment did not result in quantitative differences in clonogenic ability of pre-leukemic APL cells. In vivo, Etretinate differentiates APLs without significant loss of clonogenic activity. Importantly Etretinate or Acitretin, in sharp contrast to RA, did not trigger PML/RARA or RARA degradation. Conclusion: Our results uncouple retinoic-induced degradation and transactivation of RARA. They establish the role of PML/RARA degradation in retinoic acid response. They pave the way to screens where these two effects of