Amplification and Deletion of Topoisomerase IIα Associate with ErbB-2 Amplification and Affect Sensitivity to Topoisomerase II Inhibitor Doxorubicin in Breast Cancer
2000; Elsevier BV; Volume: 156; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64952-8
ISSN1525-2191
AutoresTero A.H. Järvinen, Minna Tanner, Virpi Rantanen, Maarit Bärlund, Åke Borg, Seija Grénman, Jorma Isola,
Tópico(s)Lung Cancer Treatments and Mutations
ResumoTopoisomerase IIα (topoIIα) is a key enzyme in DNA replication and a molecular target for many anti-cancer drugs called topoII inhibitors. The topoIIα gene is located at chromosome band 17q12-q21, close to the ErbB-2 oncogene (HER-2/neu), which is the most commonly amplified oncogene in breast cancer. Because of the physical proximity to ErbB-2, copy number aberrations may also occur in the topoIIα gene. These topoIIα gene copy number aberrations may be related to the altered chemosensitivity to topoII inhibitors that breast cancers with ErbB-2 amplification are known to have. We used fluorescence in situ hybridization to study copy number aberrations of both topoIIα and ErbB-2 in nine breast cancer cell lines and in 97 clinical breast tumors, which were selected for the study according to their ErbB-2 status by Southern blotting. TopoIIα-protein expression was studied with Western blot and sensitivity to doxorubicin (a topoII inhibitor) with a 96-well clonogenic in vitro assay. Two of the five cell lines with ErbB-2 gene amplification (SK-BR-3 and UACC-812) showed amplification of topoIIα. In MDA-361 cells, ErbB-2 amplification (14 copies/cell) was associated with a physical deletion of topoIIα (four copies of chromosome 17 centromere and two copies of topoIIα). The topoIIα amplification in UACC-812 cells was associated with 5.9-fold-increased topoIIα protein expression and 2.5-fold-increased sensitivity to the topoII inhibitor, doxorubicin, whereas the deletion in MDA-361 leads to decreased protein expression (45% of control) and a 2.4-fold-increased chemoresistance in vitro. Of 57 ErbB-2-amplified primary breast carcinomas, 25 (44%) showed ErbB-2-topoIIα coamplification and 24 (42%) showed a physical deletion of the topoIIα gene. No topoIIα copy number aberrations were found in 40 primary tumors without ErbB-2 amplification. TopoIIα gene amplification and deletion are common in ErbB-2-amplified breast cancer and are associated with increased or decreased sensitivity to topoII inhibitors in vitro, respectively. These findings may explain the altered chemosensitivity to topoII inhibitors reported in ErbB-2-amplified breast cancers. Topoisomerase IIα (topoIIα) is a key enzyme in DNA replication and a molecular target for many anti-cancer drugs called topoII inhibitors. The topoIIα gene is located at chromosome band 17q12-q21, close to the ErbB-2 oncogene (HER-2/neu), which is the most commonly amplified oncogene in breast cancer. Because of the physical proximity to ErbB-2, copy number aberrations may also occur in the topoIIα gene. These topoIIα gene copy number aberrations may be related to the altered chemosensitivity to topoII inhibitors that breast cancers with ErbB-2 amplification are known to have. We used fluorescence in situ hybridization to study copy number aberrations of both topoIIα and ErbB-2 in nine breast cancer cell lines and in 97 clinical breast tumors, which were selected for the study according to their ErbB-2 status by Southern blotting. TopoIIα-protein expression was studied with Western blot and sensitivity to doxorubicin (a topoII inhibitor) with a 96-well clonogenic in vitro assay. Two of the five cell lines with ErbB-2 gene amplification (SK-BR-3 and UACC-812) showed amplification of topoIIα. In MDA-361 cells, ErbB-2 amplification (14 copies/cell) was associated with a physical deletion of topoIIα (four copies of chromosome 17 centromere and two copies of topoIIα). The topoIIα amplification in UACC-812 cells was associated with 5.9-fold-increased topoIIα protein expression and 2.5-fold-increased sensitivity to the topoII inhibitor, doxorubicin, whereas the deletion in MDA-361 leads to decreased protein expression (45% of control) and a 2.4-fold-increased chemoresistance in vitro. Of 57 ErbB-2-amplified primary breast carcinomas, 25 (44%) showed ErbB-2-topoIIα coamplification and 24 (42%) showed a physical deletion of the topoIIα gene. No topoIIα copy number aberrations were found in 40 primary tumors without ErbB-2 amplification. TopoIIα gene amplification and deletion are common in ErbB-2-amplified breast cancer and are associated with increased or decreased sensitivity to topoII inhibitors in vitro, respectively. These findings may explain the altered chemosensitivity to topoII inhibitors reported in ErbB-2-amplified breast cancers. The ErbB-2 oncogene (also termed HER-2/neu) is the most frequently amplified oncogene in breast cancer. A number of studies have shown that the ErbB-2 oncogene is amplified in 20 to 35% of breast and ovarian cancers, and the amplification is known to be associated with shortened disease-free and overall survival.1Pauletti G Godolphin W Press MF Slamon DJ Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.Oncogene. 1996; 13: 63-72PubMed Google Scholar, 2Press MF Bernstein L Thomas PA Meisner LF Zhou J-Y Ma Y Hung G Robinson RA Harris C El-Naggar A Slamon DJ Phillips RN Ross JS Wolman SR Flom KJ HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas.J Clin Oncol. 1997; 15: 2894-2904Crossref PubMed Scopus (733) Google Scholar, 3Clark GM Should selection of adjuvant chemotherapy for patients with breast cancer be based on erbB-2 status?.J Natl Cancer Inst. 1998; 90: 1320-1321Crossref PubMed Scopus (59) Google Scholar ErbB-2 amplification has also been linked with altered sensitivity to cytotoxic drugs, especially to those targeting topoisomerase IIα (topoII inhibitors), in many clinical trials.4Wright C Cairns J Cantwell BJ Cattan AR Hall AG Harris AL Horne CHW Response to mitoxantrone in advanced breast cancer: correlation with the expression of c-erbB-2 protein and glutathione S-transferases.Br J Cancer. 1992; 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Experiments with mouse and human cells have indicated that the in vitro-induced ErbB-2 protein overexpression does not alter the chemosensitivity of cancer cells to the topoII inhibitor.16Pegram MD Finn RS Arzoo K Beryt M Pietras RJ Slamon DJ The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells.Oncogene. 1997; 15: 537-547Crossref PubMed Scopus (308) Google Scholar, 17Pegram MD Pauletti G Slamon DJ HER-2/neu as a predictive marker of response to breast cancer therapy.Breast Cancer Res Treat. 1998; 52: 65-67Crossref PubMed Scopus (235) Google Scholar Neither is the ErbB-2 protein as a transmembrane growth factor receptor known to interact physically with topoII inhibitors. Although the ErbB-2 oncogene is considered to be the target gene for 17q12-q21 amplification, the amplicon harbors other closely located genes, such as v-erbA/thyroid hormone receptor α (THRA)18van de Vijver MJ van de Bersselaar R Devilee P Cornelisse C Peterse J Nusse R Amplification of the neu (c-erbB-2) oncogene in human mammary tumors is relative frequent and is often accompanied by amplification of the linked c-erbA oncogene.Mol Cell Biol. 1987; 7: 2019-2023Crossref PubMed Scopus (289) Google Scholar; retinoic acid receptor α19Smith K Houlbrook S Greenall M Carmichael J Harris AL Topoisomerase IIα co-amplification with erbB2 in human primary breast cancer, and breast cancer cell lines: relationship to m-AMSA and mitoxantrone sensitivity.Oncogene. 1993; 8: 933-938PubMed Google Scholar; MLNs 50, 51, 62, and 6420Tomasetto C Regnier C Moog-Lutz C Mattei MG Chenard MP Lidereau S Identification of four novel human genes amplified and overexpressed in breast carcinoma and located to the q11–q21.3 region of chromosome 17.Genomics. 1995; 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74: 502-507Crossref PubMed Scopus (65) Google Scholar, 32Withoff S de Vries EGE Keith WN Nienhuis EF van der Graaf WTA Uges DRA Mulder NH Differential expression of DNA topoisomerase IIα and -β in P-gn and MRP-negative VM26, mAMSA and mitoxantrone-resistant sublines of the human SCLC cell line GLC.Br J Cancer. 1996; 74: 1869-1876Crossref PubMed Scopus (53) Google Scholar, 33Zhou Z Zwelling LA Kawakami Y An T Kobayashi K Herzog C Kleinerman ES Adenovirus-mediated human topoisomerase IIα gene transfer increases the sensitivity of etoposide-resistant human breast cancer cells.Cancer Res. 1999; 59: 4618-4624PubMed Google Scholar Studies with only a few primary breast carcinomas indicate that ErbB-2 and topoIIα can both be amplified simultaneously in breast cancer.19Smith K Houlbrook S Greenall M Carmichael J Harris AL Topoisomerase IIα co-amplification with erbB2 in human primary breast cancer, and breast cancer cell lines: relationship to m-AMSA and mitoxantrone sensitivity.Oncogene. 1993; 8: 933-938PubMed Google Scholar, 22Keith WN Douglas F Wishart GC McCallum HM George WD Kaye SB Brown R Co-amplification of erbB2, topoisomerase IIα and retinoid acid receptor genes in breast cancer and allelic loss at topoisomerase I on chromosome 20.Eur J Cancer. 1993; 29A: 1469-1475Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 23Murphy DS McHardy P Coutts J Mallon EA George WD Kaye SB Brown R Keith WN Interphase cytogenetic analysis of erbB2 and topoIIα co-amplification in invasive breast cancer and polysomy of chromosome 17 in ductal carcinoma in situ.Int J Cancer. 1996; 64: 18-26Crossref Scopus (53) Google Scholar In accordance with coamplification, immunohistochemically detectable topoIIα expression is significantly correlated with ErbB-2 overexpression in breast cancer.34Järvinen TAH Kononen J Pelto-Huikko M Isola J Expression of topoisomerase IIα is associated with rapid cell proliferation, aneuploidy, and c-erbB-2 overexpression in breast cancer.Am J Pathol. 1996; 148: 2073-2082PubMed Google Scholar We studied ErbB-2 and topoIIα gene copy number aberrations in breast cancer cell lines and primary breast carcinomas and determined the association between topoIIα copy number aberrations, protein expression, and sensitivity to doxorubicin, a widely used topoII inhibitor. Breast cancer cell lines BT-474, DU-4475, MCF-7, MDA-157, MDA-361, SK-BR-3, UACC-812, UACC-893, and ZR-75-1 were obtained from the American Type Culture collection (ATCC, Rockville, MD) and were cultured in recommended conditions. The confluent cultures were harvested to obtain interphase nuclei from the cells that were predominantly in the G1 phase of the cell cycle. The cells were fixed in Carnoy's fluid (75% methanol, 25% acetic acid) and dropped on microscope slides.35Tanner MM Tirkkonen M Kallioniemi A Isola J Kuukasjärvi T Collins C Kowbel D Guan X-Y Trent J Gray JW Meltzer P Kallioniemi O-P Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in breast cancer.Cancer Res. 1996; 56: 3441-3445PubMed Google Scholar Primary breast tumors (97) were derived from the tumor bank of the University of Lund, Sweden. The primary tumors were selected from the set of tumors that had been studied previously for ErbB-2 amplification by Southern blotting.36Borg Å Baldetorp B Fernö M Killander D Olsson H Sigurdsson H ERBB2 amplification in breast cancer with a high rate of proliferation.Oncogene. 1991; 6: 137-143PubMed Google Scholar The primary tumors were freshly frozen and stored at −70°C. Imprint touch preparations were prepared for FISH by lightly pressing a semithawed frozen tumor piece onto Superfrost Plus microscope slides (Menzel, Germany).37Tanner MM Karhu RA Nupponen NN Borg Å Baldetorp B Pejovic T Fernö M Killander D Isola JJ Genetic aberrations in hypodiploid breast cancer: frequent loss of chromosome 4 and amplification of cyclin D1 oncogene.Am J Pathol. 1998; 153: 191-199Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar A PAC clone for ErbB-2 (RMC17P077) was obtained from Resource for Molecular Genetics (Berkeley, CA), and a p1 probe for topoIIα was obtained by a polymerase chain reaction (PCR)-based screening of a p1 library (Genome Systems Inc., St. Louis, MO).38Bärlund M Tirkkonen M Forozan F Tanner MM Kallioniemi O Kallioniemi A Increased copy number at 17q22–q24 by CGH in breast cancer is due to high-level amplification of two separate regions.Genes Chromosomes Cancer. 1997; 20: 372-376Crossref PubMed Scopus (84) Google Scholar, 39Bärlund M Nupponen NN Karhu R Tanner MM Paavola P Kallioniemi O Kallioniemi A Molecular cytogenetic mapping of 24 CEPH YACs and 24 gene-specific large insert probes to chromosome 17.Cytogenet Cell Genet. 1998; 82: 189-191Crossref PubMed Scopus (9) Google Scholar A chromosome 17 pericentromeric probe (p17H8) was used as a reference probe to determine the overall copy number of chromosome 17.38Bärlund M Tirkkonen M Forozan F Tanner MM Kallioniemi O Kallioniemi A Increased copy number at 17q22–q24 by CGH in breast cancer is due to high-level amplification of two separate regions.Genes Chromosomes Cancer. 1997; 20: 372-376Crossref PubMed Scopus (84) Google Scholar, 39Bärlund M Nupponen NN Karhu R Tanner MM Paavola P Kallioniemi O Kallioniemi A Molecular cytogenetic mapping of 24 CEPH YACs and 24 gene-specific large insert probes to chromosome 17.Cytogenet Cell Genet. 1998; 82: 189-191Crossref PubMed Scopus (9) Google Scholar The specificity of the large-insert-size genomic DNA probes was confirmed by PCR with primers amplifying the sequences for ErbB-2 and topoIIα and with a probe DNA as a template. The PCR conditions were optimized for each primer pair, using PTC-100 thermocycler (MJ Research Inc., Watertown, MA). Approximately 100 ng of each template probe and 25 pmol/L of corresponding primers were used in a 25-μl reaction volume in a standard reaction mixture recommended with Dynazyme II thermostable DNA polymerase (Finnzymes Oy, Espoo, Finland). The PCR analysis showed that the ErbB-2 probe did not recognize sequences from topoIIα and vice versa (data not shown). Two-color FISH was done as previously described.34Järvinen TAH Kononen J Pelto-Huikko M Isola J Expression of topoisomerase IIα is associated with rapid cell proliferation, aneuploidy, and c-erbB-2 overexpression in breast cancer.Am J Pathol. 1996; 148: 2073-2082PubMed Google Scholar, 36Borg Å Baldetorp B Fernö M Killander D Olsson H Sigurdsson H ERBB2 amplification in breast cancer with a high rate of proliferation.Oncogene. 1991; 6: 137-143PubMed Google Scholar, 37Tanner MM Karhu RA Nupponen NN Borg Å Baldetorp B Pejovic T Fernö M Killander D Isola JJ Genetic aberrations in hypodiploid breast cancer: frequent loss of chromosome 4 and amplification of cyclin D1 oncogene.Am J Pathol. 1998; 153: 191-199Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Before hybridization, imprint touch specimens were fixed at room temperature with 50%, 70%, and 100% Carnoy's fluid (10 minutes each). The probes were labeled with biotin-14-deoxyadenosine triphosphate and digoxigenin-11-deoxyuridine triphosphate by standard nick translation. The hybridization was carried out overnight at 42°C in a hybridization mixture35Tanner MM Tirkkonen M Kallioniemi A Isola J Kuukasjärvi T Collins C Kowbel D Guan X-Y Trent J Gray JW Meltzer P Kallioniemi O-P Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in breast cancer.Cancer Res. 1996; 56: 3441-3445PubMed Google Scholar, 37Tanner MM Karhu RA Nupponen NN Borg Å Baldetorp B Pejovic T Fernö M Killander D Isola JJ Genetic aberrations in hypodiploid breast cancer: frequent loss of chromosome 4 and amplification of cyclin D1 oncogene.Am J Pathol. 1998; 153: 191-199Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar containing 5 ng of pericentromeric probe, 20 ng of gene-specific probe, and 10 μg of human placental DNA. After hybridization, the slides were washed with 0.4× standard saline citrate (2 minutes at 74°C) and 2× standard saline citrate (1 minute at room temperature). Hybridization was detected immunohistochemically with avidin-fluorescein isothiocyanate (for biotin-labeled probe) and anti-digoxigenin rhodamine. The slides were counterstained with 0.2 mm 4,6-diamidino-2-phenylindole (DAPI) in an antifade solution (Vectashield, Vector Laboratories, Burlingame, CA).35Tanner MM Tirkkonen M Kallioniemi A Isola J Kuukasjärvi T Collins C Kowbel D Guan X-Y Trent J Gray JW Meltzer P Kallioniemi O-P Independent amplification and frequent co-amplification of three nonsyntenic regions on the long arm of chromosome 20 in breast cancer.Cancer Res. 1996; 56: 3441-3445PubMed Google Scholar, 37Tanner MM Karhu RA Nupponen NN Borg Å Baldetorp B Pejovic T Fernö M Killander D Isola JJ Genetic aberrations in hypodiploid breast cancer: frequent loss of chromosome 4 and amplification of cyclin D1 oncogene.Am J Pathol. 1998; 153: 191-199Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Hybridizations were evaluated using an Olympus BX50 epifluorescence microscope equipped with a 63× oil-immersion objective (numeric aperture 1.4). A dual band-pass fluorescence filter (Chromotechnology, Brattleboro, VT) was used to visualize the fluorescein isothiocyanate and rhodamine signals simultaneously. At least 80 nonoverlapping nuclei with intact morphology (based on DAPI counterstaining) were scored to determine the number of hybridization signals for each topoIIα, ErbB-2, and 17 centromere probe. Control hybridizations to normal lymphocytes were done to ascertain that the probes recognized a single-copy target and that the hybridization efficiencies were sufficient. Both absolute copy numbers and the relative copy number ratio (ratio between mean number of ErbB-2 or topoIIα and mean number of chromosome 17 centromere signals) were determined. The amplification of ErbB-2 and to
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