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The contribution of ABC transporters to drug resistance in a realistic mouse mammary tumor model
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Abstracts / Cancer Genetics and Cytogenetics 203 (2010) 44e65
THE CONTRIBUTION OF ABC TRANSPORTERS TO DRUG RESISTANCE IN A REALISTIC MOUSE MAMMARY TUMOR MODEL Piet Borst1, Sven Rottenberg1, Jos Jonkers1, Serge Zander1, Marina Pajic1, Janneke Jaspers1 1. The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Although ABC transporters are brilliant in causing drug resistance in cultured cells, their contribution to drug resistance in patients has remained controversial. We have tackled this problem in a mammary tumor model in mice. These Brca1 / , p53 / tumors arise ‘spontaneously’ and closely resemble human breast cancer in BRCA1+/ carriers (1). These tumors respond to a range of standard chemotherapeutic agents (1), but eventually end up being resistant (1e3) to most drugs, but not to cisplatin (1,4). Resistance to doxorubicin in this tumor model is nearly always due to P-glycoprotein upregulation at the transcriptional level (1,3). A 5-fold increase in the low basal level of P-glycoprotein RNA in these tumors is sufficient for complete resistance. These levels of P-glycoprotein are not detectable with standard immunocytochemistry (3), showing that our current tools are inadequate to detect relevant amounts of P-gp in clinical samples. Crossing in null alleles for Mdr1a/b made the tumors hypersensitive to doxorubicin and docetaxel, showing the important contribution of P-gp to resistance in this tumor model. P-gp also causes resistance to the PARP inhibitor olaparib (2). In about half of the mice treated with topotecan, resistance is associated with upregulation of Abcg2 (Bcrp) (5). Tumors in Abcg2 / mice take longer to become resistant, showing the importance of Abcg2 as a defense system (5). We have not observed upregulation of any of the other ABC-transporters associated with topotecan resistance in cultured cells, such as Abcc2 and 4. Notwithstanding the complete remissions often obtained with some drugs, we are rarely able to eradicate the tumor (1e5). Our evidence indicates that this is not due to the hypothetical unique properties of tumor stem cells. We are also studying potential markers that determine initial drug response, but detect none at the RNA level before tumor treatment. References: (1) Rottenberg S et al. Proc Natl Acad Sci U S A 2007;104:12117e22. (2) Rottenberg S et al. Proc Natl Acad Sci U S A 2008;105:17079e84. (3) Pajic M et al. Cancer Res 2009;69:6396e404. (4) Borst P, Rottenberg S, Jonkers J. Cell Cycle 2008;7:1353e9. (5) Zander S et al. Cancer Res 2010;70: 1700e10.
GOBAL DNA METHYLATION IN FETAL HUMAN GERM CELLS AND GERM CELL TUMORS: CORRELATION WITH DIFFERENTIATION AND CISPLATIN RESISTANCE Leendert Looijenga1, Hendrik Wermann1,2, Hans Stoop1, Ad Gillis1, Friedemann Honecker2, Ruud van Gurp1, Ole Ammerpohl3, Julia Richter3, Wolter Oosterhuis1, Carsten Bokemeyer2 1. Department of Pathology, Erasmus MCeErasmus University Medical Center, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands 2. Department of Oncology, Hematology, Bone Marrow Transplantation with section of Pneumology, University Medical Center HamburgEppendorf, Hamburg, Germany 3. Institute of Human Genetics, Christian-Albrechts University Kiel/ University Hospital Schleswig-Holstein Campus Kiel, Germany
Differences in the global methylation pattern, i.e., hyper- as well as hypo-methylation, are observed in cancer, including germ cell tumors (GCTs). Related to their precursor cells, the GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n 5 103) and GCTs (n 5 251) by immunohistochemical staining for 5-mCytidine. We found that the global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, while female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) were hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumors, choriocarcinomas) showed a higher degree of methylation. Embryonal carcinomas showed an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam-2 before and after demethylation using 5-azacytidine. Exposure to 5-azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell specific marker VASA, showed increased expression. Upon treatment with 5-azacytidine, TCam-2 cells were analyzed by a high-throughput methylation screen for changes in methylation sites of 14,000 genes. Among the genes revealing changes, interesting targets were identified, i.e., demethylation of KLF11, a putative tumor suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs.
Abstracts / Cancer Genetics and Cytogenetics 203 (2010) 44e65
THE CONTRIBUTION OF ABC TRANSPORTERS TO DRUG RESISTANCE IN A REALISTIC MOUSE MAMMARY TUMOR MODEL Piet Borst1, Sven Rottenberg1, Jos Jonkers1, Serge Zander1, Marina Pajic1, Janneke Jaspers1 1. The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Although ABC transporters are brilliant in causing drug resistance in cultured cells, their contribution to drug resistance in patients has remained controversial. We have tackled this problem in a mammary tumor model in mice. These Brca1 / , p53 / tumors arise ‘spontaneously’ and closely resemble human breast cancer in BRCA1+/ carriers (1). These tumors respond to a range of standard chemotherapeutic agents (1), but eventually end up being resistant (1e3) to most drugs, but not to cisplatin (1,4). Resistance to doxorubicin in this tumor model is nearly always due to P-glycoprotein upregulation at the transcriptional level (1,3). A 5-fold increase in the low basal level of P-glycoprotein RNA in these tumors is sufficient for complete resistance. These levels of P-glycoprotein are not detectable with standard immunocytochemistry (3), showing that our current tools are inadequate to detect relevant amounts of P-gp in clinical samples. Crossing in null alleles for Mdr1a/b made the tumors hypersensitive to doxorubicin and docetaxel, showing the important contribution of P-gp to resistance in this tumor model. P-gp also causes resistance to the PARP inhibitor olaparib (2). In about half of the mice treated with topotecan, resistance is associated with upregulation of Abcg2 (Bcrp) (5). Tumors in Abcg2 / mice take longer to become resistant, showing the importance of Abcg2 as a defense system (5). We have not observed upregulation of any of the other ABC-transporters associated with topotecan resistance in cultured cells, such as Abcc2 and 4. Notwithstanding the complete remissions often obtained with some drugs, we are rarely able to eradicate the tumor (1e5). Our evidence indicates that this is not due to the hypothetical unique properties of tumor stem cells. We are also studying potential markers that determine initial drug response, but detect none at the RNA level before tumor treatment. References: (1) Rottenberg S et al. Proc Natl Acad Sci U S A 2007;104:12117e22. (2) Rottenberg S et al. Proc Natl Acad Sci U S A 2008;105:17079e84. (3) Pajic M et al. Cancer Res 2009;69:6396e404. (4) Borst P, Rottenberg S, Jonkers J. Cell Cycle 2008;7:1353e9. (5) Zander S et al. Cancer Res 2010;70: 1700e10.
GOBAL DNA METHYLATION IN FETAL HUMAN GERM CELLS AND GERM CELL TUMORS: CORRELATION WITH DIFFERENTIATION AND CISPLATIN RESISTANCE Leendert Looijenga1, Hendrik Wermann1,2, Hans Stoop1, Ad Gillis1, Friedemann Honecker2, Ruud van Gurp1, Ole Ammerpohl3, Julia Richter3, Wolter Oosterhuis1, Carsten Bokemeyer2 1. Department of Pathology, Erasmus MCeErasmus University Medical Center, Daniel den Hoed Cancer Center, Rotterdam, The Netherlands 2. Department of Oncology, Hematology, Bone Marrow Transplantation with section of Pneumology, University Medical Center HamburgEppendorf, Hamburg, Germany 3. Institute of Human Genetics, Christian-Albrechts University Kiel/ University Hospital Schleswig-Holstein Campus Kiel, Germany
Differences in the global methylation pattern, i.e., hyper- as well as hypo-methylation, are observed in cancer, including germ cell tumors (GCTs). Related to their precursor cells, the GCT methylation status differs according to histology. We investigated the methylation pattern of normal fetal, infantile, and adult germ cells (n 5 103) and GCTs (n 5 251) by immunohistochemical staining for 5-mCytidine. We found that the global methylation pattern of male germ cells changes from hypomethylation to hypermethylation, while female germ cells remain unmethylated at all stages. Undifferentiated GCTs (seminomas, intratubular germ cell neoplasia unclassified, and gonadoblastomas) were hypomethylated, whereas more differentiated GCTs (teratomas, yolk sac tumors, choriocarcinomas) showed a higher degree of methylation. Embryonal carcinomas showed an intermediate pattern. Resistance to cisplatin was assessed in the seminomatous cell line TCam-2 before and after demethylation using 5-azacytidine. Exposure to 5-azacytidine resulted in decreased resistance to cisplatin. Furthermore, after demethylation, the stem cell markers NANOG and POU5F1 (OCT3/4), as well as the germ cell specific marker VASA, showed increased expression. Upon treatment with 5-azacytidine, TCam-2 cells were analyzed by a high-throughput methylation screen for changes in methylation sites of 14,000 genes. Among the genes revealing changes, interesting targets were identified, i.e., demethylation of KLF11, a putative tumor suppressor gene, and hypermethylation of CFLAR, a gene previously described in treatment resistance in GCTs.