Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death.
High incidence of chemotherapy resistance is the primary cause of treatment failure in a subset of neuroblastomas with amplified MYCN. We have reported previously that ectopic MYCN expression promotes proliferation of neuroblastoma Tet21N cells and simultaneously sensitizes them to the drug-induced apoptosis. In search for genes that are involved in MYCN-dependent regulation of drug resistance, we used a function-based gene cloning approach and identified CTSD encoding for a lysosomal aspartyl protease cathepsin D. Downregulation of cathepsin D expression by RNA interference or inhibition of its enzymatic activity increased sensitivity of MYCN-expressing Tet21N cells to doxorubicin. Overexpression of cathepsin D in Tet21N cells attenuated doxorubicin-induced apoptosis. It was accompanied by activation of protein kinase B (Akt) and persistent antiapoptotic activity of Bcl-2. In primary neuroblastomas, high CTSD messenger RNA (mRNA) levels were associated with amplified MYCN, a strong predictive marker of adverse outcome. Chromatin immunoprecipitation and luciferase promoter assays revealed that MYCN protein binds to the CTSD promoter and activates its transcription, suggesting a direct link between deregulated MYCN and CTSD mRNA expression. We further show that neuroblastoma cells can secrete mitogenic procathepsin D and that MYCN expression and especially doxorubicin treatment promote procathepsin D secretion. Extracellular exogenous cathepsin D induces Akt-1 phosphorylation and doxorubicin resistance in sensitive cells. These results demonstrate an important role of cathepsin D in antiapoptotic signaling in neuroblastoma cells and suggest a novel mechanism for the development of chemotherapy resistance in neuroblastoma.
Department of Tumor Genetics B030, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Vitalia
V
Sagulenko
Daniel
D
Muth
Evgeny
E
Sagulenko
Tobias
T
Paffhausen
Manfred
M
Schwab
Frank
F
Westermann
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000502:physiology; D002402:Cathepsin D / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics; D015513:Oncogene Proteins / Q000235:genetics; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000502:physiology; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins / Q000502:physiology; D015398:Signal Transduction
D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; D002402:Cathepsin D; D004317:Doxorubicin; D004792:Enzyme Precursors; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C097041:DAP3 protein, human; C459801:MYCN protein, human; C058345:procathepsin D