Carcinogenesis Advance Access originally published online on June 19, 2008
Carcinogenesis 2008 29(10):1869-1877; doi:10.1093/carcin/bgn147
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Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death
Department of Tumor Genetics B030, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
* To whom correspondence should be addressed. Tel: +49 6221 423275; Fax: +49 6221 423277; Email: f.westermann{at}dkfz.de
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.
Abbreviations: Akt, protein kinase B; cDNA, complementary DNA; CTSD, cathepsin D; EBV, Eppstein-Barr virus; FACS, Fluorescence-Activated Cell Sorting; ORF, open reading frame; PAA, polyacrylamide; PI3, phosphatidylinositol 3-phosphate; mRNA, messenger RNA; shRNA, small hairpin RNA; siRNA, small interfering RNA; TKO, technical knockout
Received January 17, 2008; revised May 16, 2008; accepted June 13, 2008.