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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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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Cathepsin D protects human neuroblastoma cells from doxorubicin-induced cell death

Vitalia Sagulenko, Daniel Muth, Evgeny Sagulenko, Tobias Paffhausen, Manfred Schwab and Frank Westermann*

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


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
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


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 
Neuroblastoma, a tumor arising from undifferentiated neural crest-derived cells, is one of the most common solid tumors in early childhood. Amplification of MYCN oncogene in neuroblastoma is associated with advanced disease and is a strong prognostic factor of poor clinical outcome (1,2). Chemotherapy resistance of neuroblastomas with amplified MYCN is a devastating problem often emerging during clinical treatments. Overexpression of MRP1 drug efflux protein and downregulation of components of the apoptotic machinery have been shown to contribute to the development of drug resistance in neuroblastoma (3,4). However, the molecular network integrating MYCN and the response of neuroblastoma cells to chemotherapy is still poorly understood. MYCN is a member of the MYC family transcriptional factors regulating cell growth and apoptosis (5). Enforced expression of MYCN accelerates cell cycle progression (6) and promotes malignant behavior of neuroblastoma cells in vitro and in vivo (7,8). Ectopic expression of MYCN from a tetracycline-regulated promoter has been shown to sensitize neuroblastoma SH-EP cells to apoptosis induced by cytokines and cytotoxic drugs (9–11).

Doxorubicin is an antibiotic of the anthracycline group that has been used for the treatment of solid tumors, including neuroblastomas, for >30 years. Despite its proven antitumor efficacy, doxorubicin administration is frequently restrained by intrinsic and acquired resistance in tumor cells. As it is currently believed, the cytotoxic effect of doxorubicin mainly depends on its ability to inhibit topoisomerase II and to intercalate into the DNA structure, inducing formation of single-strand/double-strand DNA breaks (12). Involvement of additional mechanisms independent of DNA damage that disrupt mitochondrial function and thus contributing to high toxicity of anthracyclines have been also discussed (13). It has been suggested that in neuroblastoma doxorubicin induces apoptosis via activation of the CD95/caspase-8 pathway (9). However, growing evidence suggests that different neuroblastoma cell types may execute doxorubicin-induced apoptosis independent of the death receptor pathway or even undergo caspase-independent cell death (14–16). To expand our knowledge about genes and pathways involved in the MYCN-mediated development of drug resistance, we studied the doxorubicin-induced response in the human neuroblastoma cell line Tet21N, a subclone of SH-EP cells, harboring ectopic MYCN under a tetracycline-regulated promoter.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 Supplementary material
 Funding
 References
 
Cell cultures and transfections
The human neuroblastoma cell lines Tet21N, a subclone of SH-EP cells expressing MYCN from a tetracycline-regulated promoter, and Tet21N-EBNA-1–3, a stable subclone of Tet21N cells expressing EBNA-1, were described previously (6,17). MYCN expression was switched on by removal of tetracycline (Sigma Munich, Germany) from the growth medium. Doxorubicin (Sigma) was used at a final concentration of 0.1 µg/ml. Doxorubicin concentration was selected as a minimal dose that effectively induced apoptosis in Tet21N cells expressing MYCN (MYCN ON) but not in ‘MYCN OFF’ cells in which MYCN expression is switched off (9).

Individual plasmids (7 µg) were transfected into 6 x 106 Tet21N cells, and the episomal library (42 µg) was transfected into 4 x 107 Tet21N-EBNA-1–3 cells, using the Effectene reagent (Qiagen, Hilden, Germany). Stably transfected cells were selected with appropriate antibiotics at the following final concentrations: zeocin (Invitrogen, Karlsruhe, Germany) 250 µg/ml and puromycin (BD Clontech, Heidelberg, Germany) 2.5 µg/ml.

Construction of a subtracted episomal complementary DNA expression library
A subtracted complementary DNA (cDNA) library of Tet21N cells was constructed and cloned into the vector pTAdvance (BD Clontech) (18). Briefly, a tester cDNA fraction was generated from RNA harvested at several time points upon induction of apoptosis by doxorubicin in Tet21N ‘MYCN ON’ cells. RNA for the driver fraction was isolated in parallel from control MYCN OFF cells that were not exposed to doxorubicin. The cDNA inserts from the resulting subtracted library were subcloned into the episomal expression vector pTKO-CZ as described previously (17).

Selection, rescue and analysis of episomes
Tet21N-EBNA-1–3 cells transfected with the episomal library were treated with doxorubicin for 21 days in complete growth medium. Cells that survived the selection were cultivated for 3 weeks in doxorubicin-free complete medium supplemented with zeocin. Preparation and sequencing analysis of episomes from the cells that survived the selection were performed as described previously (17).

Apoptosis and viability assays
Cells were treated with doxorubicin or cultured without the drug for the indicated time, and apoptosis was determined by the extent of DNA fragmentation according to the FACS analysis of propidium iodide (Sigma) -stained nuclei (9). Viability of cells was evaluated by their ability to exclude Trypan blue.

Caspase activity assays
Caspase-3/CPP32 and caspase-9/Mch6 activities were measured in cellular lysates obtained from at least three experiments using fluorometric assay kits (BioVision, Mountain View, CA) according to the manufacturer’s recommendations. Fluorescence of the cleaved substrate was measured in fluoroscan Ascent FL using 400 nm excitation and 515 nm emission filters.

RNA interference
The shRNAs targeting CTSD were designed using the siRNA target finder and design software available at Ambion’s web page (http://www.ambion.com/techlib/misc/siRNA_finder.htm). Two 60mer complementary oligonucleotides encoding the 19mer hairpin RNA specific to the selected target sequence were synthesized, annealed and cloned into pSilencer 3.1-Puro (Ambion, Austin, TX).

The sequences of the synthetic oligonucleotides were as follows: CTSD-1, 5'-AAGTACAACAGCGACAAGTCC-3'; CTSD-2, 5'-AAGCTGGTGGACCAGAACATC-3' and CTSD-3, 5'-AAAGGCTACAAGCTGTCCCCA-3'.

pSilencer 3.1-Puro–scramble (shRNA scr) was used as negative control. The resulting plasmids were transfected into Tet21N cells and stably transfected cells were selected with puromycin. The efficiency of CTSD silencing was evaluated by immunoblotting.

Ectopic expression of cathepsin D
Cathepsin D cDNA was isolated from pSG1-cathepsin D (a kind gift of Dr M.Garcia, University of Montpellier, France) and inserted into EcoRI-digested pcDNA4 vector (Invitrogen). Resulting plasmid, pcDNA4-CathD, was used for transfection into Tet21N cells. Ten independent zeocin-resistant cell clones were picked, and expression of cathepsin D was evaluated by immunoblotting.

Antibodies and immunoblotting
The following antibodies were used: mouse monoclonal anti-cathepsin D (clone 49), anti-DAP3 (clone 10) and rabbit polyclonal anti-phospho-Akt S-473 (BD Bioscience, Heidelberg, Germany); anti-β-Actin (clone AC-15) (Sigma); anti-MYCN (clone B8.4.B), anti-{alpha}-Tubulin (clone B-7) and anti-P53 (clone DO-1) (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit polyclonal anti-phospho-Bcl-2 S-70 (Cell Signaling, Danvers, MA) and anti-p21/WAF1/Cip1, pooled mouse monoclonal IgGs from clones CP36 and CP74 (Upstate, Millipore, Schwalbach, Germany).

Protein extracts from cells were prepared as described elsewhere (18). Isolated proteins (20 µg) were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 12 or 10–20% gradient PAA gels and blotted onto polyvinylidene difluoride membranes (Millipore). Immunodetection was performed using horseradish peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany) and BM Chemiluminescence Blotting Substrate (Roche, Mannheim, Germany).

cDNA microarray analysis
Construction of microarrays of total 42 578 cDNA clones, hybridization, image analysis and normalization were performed as have been described (19,20). The cohort available for cDNA microarray analysis consisted of tumor samples obtained from 49 patients prior to chemotherapy (19). Total RNA was isolated from 30–60 mg of snap-frozen primary neuroblastoma tumor tissue. To analyze gene expression profiles in Tet21N cells, total RNA was collected from 2 x 107 Tet21N cells after induction of MYCN expression at different time points in normal growth condition (complete medium, no drugs). The raw data of both tumor samples and cell line experiments were normalized using the variance normalization method (20). The microarray data are available online (accession numbers E-CVDE-2 and E-CVDE-3) (20) (F.Westermann, D.Muth, A.Benner, T.Bauer, K.O.Henrich, A.Oberthür, B.Brors, T.Beissbarth, F.Pattyn, J.Vandesompele, F.Speleman, R.König, M.Fischer and M.Schwab, submitted for publication).

Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed as described in F.Westermann, D.Muth, A.Benner, T.Bauer, K.O.Henrich, A.Oberthür, B.Brors, T.Beissbarth, F.Pattyn, J.Vandesompele, F.Speleman, R.König, M.Fischer and M.Schwab (submitted for publication). Briefly, ~5 x 107 cells were cross-linked and lysed. Isolated DNA was sonicated with a Diagenode Bioruptor at high power level for six 5 min pulses (30 s on and 20 s off). Immunoprecipitation was performed using 10 µg of anti-MYCN or normal mouse IgG (Santa Cruz Biotechnology) and Dynabeads Protein G (Invitrogen). Purified chromatin immunoprecipitation DNA was used as a template for 35 cycles of polymerase chain reaction. The primer sequences were CTDS_fw4: 5'-GCGTCATCCCGGCTATAAG-3' and CTDS_bw4: 5'-GAGCGCGAAAGTCACCAC-3'.

Luciferase promoter–reporter assays
The 712 bp fragment of the CTSD core promoter (bp –752/–41 from the translation initiation site +1) flanked by the restriction sites for HindIII and NheI was polymerase chain reaction amplified from cosmid G248P87136C10 (BACPAC Resources, Children’s Hospital Oakland) using the following primers: PCTSDfor: 5'-GCTAGCGAATACTTTGCCTGCCTTCG-3' and PCTSDrev: 5'-AAGCTTCGCTTATAGCCGGGATGAC-3'. HindIII/NheI-digested polymerase chain reaction fragment was subcloned into the pGL3-Basic vector (Promega, Madison, WI) and the resulting plasmid was cotransfected with plasmid pRL-SV40 (Promega) into Tet21N MYCN ON or MYCN OFF cells. pGL3-Basic or pGL3-PTMA (a gift of Dr W.Lutz, University of Marburg) was used as controls for promoter activity. Activity of the promoter constructs was assayed 24 h after transfection using Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s manual. Measurements were done in FLUOstar OPTIMA luminometer (BMG Labtech, Offenburg, Germany).

Enzyme-linked immunosorbent assay detection of cathepsin D
Tet21N MYCN ON and MYCN OFF cells were seeded onto 12-well dishes at initial density of 1 x 106 in 1 ml complete medium. Twenty-four hours after seeding, cells were treated with doxorubicin for 48 h or left untreated for control. Equal aliquots of the conditioning media were collected, and detached cells were removed by centrifugation at 700g for 10 min. Amount of cathepsin D in the resulting cell-free conditioning media was assayed by Rapid Format Cathepsin D ELISA kit (Calbiochem, EMD Chemicals, Gibbstown, NJ), according to the protocol provided by the manufacturer. Dual-wavelength absorbance was measured in FLUOstar OPTIMA multiplate reader using A450 and A600 filters. Obtained values were normalized by number of cells. Each experiment was performed in triplicates. Bars represent mean values of triplicates ± SD.

Statistics
To test the difference between mean values, obtained from at least three measurements, the Student’s t-test for unpaired observations was applied. The differences were considered significant if the P value was <0.05. To test the correlation between candidate gene expressions and established prognostic factors, the Wilcoxon exact test was used. Statistical analysis was performed using R-software (http://www.r-project.org).


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 Supplementary material
 Funding
 References
 
Function-based selection of transcripts involved in doxorubicin-induced cell death in Tet21N cells
To identify genes that contribute to the MYCN-mediated regulation of doxorubicin sensitivity in Tet21N neuroblastoma cells, we used a function-based gene cloning approach based on the ‘technical knockout’ (TKO) strategy. The TKO strategy allows identification of death-associated genes due to their inactivation by antisense RNAs expressed from an episomal cDNA library (21). To increase the efficiency of the TKO screening, we used two modifications (17): (i) a subtractive hybridization of doxorubicin-treated MYCN ON Tet21N cells versus MYCN OFF cells not exposed to doxorubicin was performed to enrich the library for transcripts that are preferentially expressed in the cells committed to doxorubicin-induced death and (ii) the subtracted cDNA fragments were subcloned into the modified vector pTKO-CZ (17) disregarding the sequence orientation to add the features of sense-oriented cDNA fragments or genetic suppressor elements (22) to the standard TKO strategy. The resulting episomal expression library consisted of >50 000 clones. DNA sequencing of 63 randomly picked clones showed that 86% of the inserts were represented by unique transcripts (54 of 63). The remaining sequences either did not show homology to any known transcripts (7 of 63) or represented the vector sequence (2 of 63). As expected, the resulting episomal expression library was enriched for MYC-responsive transcripts (19 of 54 unique transcripts) (supplementary Table, available at Carcinogenesis Online).

The constructed library was introduced into the MYCN-expressing Tet21N-EBNA-1–3 cells, a subclone of Tet21N cells that is highly permissive for transfection with EBV-derived plasmids, such as pTKO-CZ (16). For comparison, Tet21N-EBNA-1–3 cells were transfected with the empty pTKO-CZ vector. Transfected cells were selected for the attenuated response to doxorubicin, and cells that survived doxorubicin treatment were allowed to form colonies of visible size. Tet21N-EBNA-1–3 cells transfected with the library formed about seven times more colonies than control cells (36 versus 5). Episomal DNA from 36 doxorubicin-resistant colonies was isolated, and DNA sequencing of the inserts revealed 20 different sequences. The episomes representing different unique sequence were pooled and used for the second round of selection. Tet21N-EBNA-1–3 cells transfected with the secondary pool of episomes and treated with doxorubicin for 3 weeks formed 16 times more colonies compared with control cells (150 versus 9 colonies). The screening procedure is depicted in Figure 1A. Sixty-five plasmids sequenced after the second round of doxorubicin selection contained six different inserts (Table I). Two prevalent inserts were detected in 50 of 65 analyzed plasmids. The first insert was a 220 bp antisense cDNA fragment of COX8A gene, encoding for the subunit VIII of the cytochrome c oxidase. The second insert was derived from the cDNA of CTSD gene, encoding for a lysosomal protease cathepsin D, and it was cloned in sense orientation. Two of the genes, identified in the present study, DAP3A and CTSD have been described previously as positive mediators of interferon-induced apoptosis in HeLa cells (23,24). However, the cDNA fragments of DAP3A and CTSD identified in our screening were different from those published previously. These data show that using the modified TKO strategy, we targeted genes involved in the regulation of cell death.


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  		Table I. cDNA fragments isolated from doxorubicin-resistant Tet21N colonies after the second round of selection

 


Figure 1
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  		Fig. 1. TKO selection of cDNA fragments that protect neuroblastoma Tet21N cells from doxorubicin-induced cell death. (A) The TKO strategy. Viability (B) and colony formation (C) of Tet21N stable polyclonal populations transfected with pTKO-sCTSD or pTKO-asCOX8A. Cells transfected with the empty vector pTKO-CZ were used as control. (B) The stably transfected cells were treated with doxorubicin for 48 h. Floating and adherent cells were pooled, stained with Trypan blue and counted. Trypan blue-negative cells were considered viable. For each sample, at least 1000 cells were counted. (C) The same stable cell populations as in (B) were treated with doxorubicin for 2 weeks. After the treatment, cells were reseeded into complete medium supplemented with zeocin and cultivated for 3 weeks. Resulting cell colonies were fixed with methanol and stained with 1:50 diluted Giemsa reagent. (D) Protein levels of cathepsin D in Tet21N stable polyclonal population transfected with pTKO-sCTSD. Lanes 1, 3 and 5—control Tet21N-pTKO-CZ cells and lanes 2, 4 and 6—Tet21N-pTKO-sCTSD. Stable polyclonal populations of Tet21N cells transfected with pTKO-asDAP3 were used as control to monitor reduction of protein levels of endogenous DAP3 due to expression of antisense-oriented DAP3 cDNA fragment. Lanes 7, 9 and 11—Tet21N-pTKO-CZ cells and lanes 8, 10 and 12—Tet21N-pTKO-asDAP3. Cells were cultured in the presence of tetracycline to keep MYCN expression off or in tetracycline-free medium to keep MYCN expression on. Doxorubicin was added to the cells at final concentration of 0.1 µg/ml (DOXO+), and untreated cells (DOXO–) were used as control. After 48 h of MYCN induction or doxorubicin treatment, cells were harvested, and levels of cathepsin D or DAP3 were evaluated in the total protein samples by immunoblotting with the specific antibodies; {alpha}-tubulin served as loading control. (E) Sequence analyses of the insert in pTKO-sCTSD. Upper panel—nucleotide sequence; the ORF sequence is printed in bold and sequence derived from the CTSD cDNA is underlined. Cloning restriction sites NotI (destroyed) and BamHI are boxed. Lower panel—alignment of the deduced amino acid sequence of the TKO-cathD peptide (query) and cathepsin D precursor (Ref. N NP_001900 [GenBank] ) (subject). Alignment preformed by BlastP2; score = 126 bits (316), expect = 4e-30 and identities = 59/60 (99%).

 
To confirm the death-protective effect of the most abundant episomes, selected by two rounds of doxorubicin treatment: pTKO-sCTSD and pTKO-asCOX8A, we transfected them into the MYCN ON Tet21N cells. Both episomes increased the viability of Tet21N cells after short-time doxorubicin treatment (24 h, P = 0.036) (Figure 1B) and enhanced their clonogenicity upon long-time (3 weeks) doxorubicin exposure (Figure 1C). Cathepsin D can act as a proapoptotic or as a prosurvival protein depending on cell type and experimental conditions (25). Therefore, we further focused on an analysis of the role of cathepsin D in doxorubicin-induced cell death in human neuroblastoma. Expression of pTKO-sCTSD did not affect the protein level of cathepsin D in Tet21N cells, whereas expression of the antisense-oriented DAP3 cDNA fragment reduced the level of DAP3 protein (Figure 1D). This result indicates that expression of the sense-oriented CTSD fragment did not interfere with the cathepsin D translation. Within the sequence of the insert in pTKO-sCTSD, we detected an ORF for a 60 amino acid peptide spanning the 326–384 amino acid region of procathepsin D (Figure 1E). These data suggest that pTKO-sCTSD episome protects Tet21N cells from doxorubicin because of the expression of the peptide fragment of cathepsin D. Such a peptide could act in a dominant-negative way, similarly to the previously published genetic suppressor elements (22). However, we also considered the possibility that the peptide encoded by pTKO-sCTSD may retain some of the cathepsin D activities similarl to the activation peptide derived from preprocathepsin D (25). Therefore, we further clarified the role of cathepsin D in doxorubicin-induced cell death in Tet21N cells.

CTSD downregulation sensitizes Tet21N cells to doxorubicin
To analyze the effects of cathepsin D inhibition on the sensitivity of Tet21N MYCN ON cells to doxorubicin, we downregulated cathepsin D by two different approaches: (i) silencing of CTSD expression by RNA interference and (ii) inhibition of its enzymatic activity using pepstatin A. For siRNA-mediated silencing, three plasmids expressing different shRNAs targeting the CTSD messenger RNA (mRNA) at positions +497 (construct #1), +797 (#2) and +1166 (#3) were tested. Construct #2 most efficiently reduced the expression of cathepsin D (Figure 2A), and thus it was selected for further analysis. Doxorubicin-induced apoptosis was assayed in a stable Tet21N subclone with silenced CTSD (Tet21N-shCTSD) by the level of nucleosomal DNA degradation after 24 h of treatment. Tet21N-shCTSD cells were more sensitive to doxorubicin-induced apoptosis compared with control (P = 0.023) (Figure 2B). In agreement with this, pretreatment of Tet21N cells with pepstatin A increased the number of dead cells upon doxorubicin treatment (P = 0.002) (Figure 2D). These results indicate that cathepsin D downregulation sensitizes neuroblastoma cells to doxorubicin-induced apoptosis.


Figure 2
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  		Fig. 2. Silencing of CTSD sensitizes Tet21N cells to doxorubicin. (A) Reduction of cathepsin D protein levels in Tet21N cells by siRNA. Tet21N cells were stably transfected with different shRNA expression plasmids targeting CTSD mRNA. Total protein (20 µg per lane) from transfected cells was resolved by 10–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto membranes. Immunodetection was performed with cathepsin D-specific antibody; {alpha}-tubulin served as loading control. (B) Doxorubicin-induced apoptosis in Tet21N cells with silenced CTSD. Tet21N cells stably expressing shRNA-CTSD construct #2 (Tet21N-shRNA-CTSD) were treated with doxorubicin for 24 h and apoptosis was determined by FACS analysis of propidium iodide-stained nuclei. Indicated values are the mean values of three independent experiments ± SD. (C) Inhibition of cathepsin D enzymatic activity sensitizes Tet21N cells to doxorubicin. Pepstatin A, an enzymatic inhibitor of cathepsin D, was added to the MYCN-expressing Tet21N cells 18 h prior to doxorubicin administration. After 48 h of cotreatment with doxorubicin and pepstatin A, adherent and floating cells were pooled and their viability was evaluated by Trypan blue exclusion. For each sample, at least 1000 cells were counted. The presented data are mean values of three independent experiments ± SD.

 
Cathepsin D overexpression activates antiapoptotic signaling in Tet21N cells
The obtained results suggest the antiapoptotic activity of cathepsin D in Tet21N cells. Therefore, we further analyzed the effect of ectopically overexpressed catalytically active cathepsin D in these cells. Stable overexpression of cathepsin D resulted in significant accumulation of both the intracellular precursor protein (~50 kD) and the mature enzyme, which was detected as a 30 kD heavy chain (Figure 3A, lanes 1 and 6) in Tet21N-CTSD cells. In control cells, transfected with empty pcDNA4 vector only, low levels of the mature cathepsin D was detected (Figure 3A, lanes 2–5). Tet21N-CTSD cells were less prone to doxorubicin-induced nucleosomal DNA degradation (P = 0.01) (Figure 3B) and had lower levels of caspase-3 (P = 0.001) and caspase-9 (P = 0.05) activation (Figure 3C). These data show that cathepsin D protects Tet21N cells from doxorubicin-induced apoptosis.


Figure 3
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  		Fig. 3. Cathepsin D overexpression reduces sensitivity of Tet21N cells to doxorubicin. (A) Selection of Tet21N cells stably overexpressing cathepsin D. Tet21N cells were transfected with cathepsin D expression plasmid pcDNA4-CathD or with an empty vector pcDNA4. Stable clones were selected with zeocin. A total protein was isolated from independent zeocin-resistant cell clones, resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (20 µg per lane) and blotted onto membranes. Immunodetection was performed with cathepsin D-specific antibody; {alpha}-tubulin served as loading control. Lanes 1, 4, 5 and 6—Tet21N-pcDNA4-CathD and lanes 2 and 3—control (Tet21N-pcDNA4). (B) Doxorubicin-induced apoptosis in Tet21N-CTSD cells. Tet21N-CTSD and control Tet21N-pcDNA cells were treated with doxorubicin for 24 h and apoptosis was determined by FACS analysis of propidium iodide-stained nuclei. Presented data are mean values of three independent experiments ± SD. (C) The same cell lines as in (B) were treated with doxorubicin for 18 h and activities of caspase-3 and caspase-9 were evaluated by the fluorometric assay. Caspases activation was determined as the ratio of the fluorescent signals in treated to untreated cells. Data are the mean of three independent experiments ± SD. (D) Cathepsin D overexpression in Tet21N cells promotes Akt phosphorylation. Tet21N-CTSD (lanes 2 and 4) and control cells (lanes 1 and 3) were cultured in the complete medium (DOXO–) or treated with doxorubicin for 24 h (DOXO+). Total protein was isolated and resolved in 10–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (40 µg per lane). Immunodetection was performed with antibodies specific to phosphorylated Akt at Ser-473 or Bcl-2 at Ser-70 and total Akt, p53 and p21/Cip/WAF1. Cathepsin D overexpression was confirmed with cathepsin D-specific antibody. β-Actin was used as loading control. (E) PI3–Akt inhibition abrogates cathepsin D antiapoptotic effect on Tet21N cells. Tet21N-CTSD and control Tet21N-pcDNA cells were treated with LY294002 (at a final concentration of 20 µM), doxorubicin (0.1 µg/ml) or with combination of both reagents for 48 h. For comparison, cells were treated with dimethyl sulfoxide (DMSO) in the same concentration that was used to dissolve LY294002. Apoptosis was determined by FACS analysis of propidium iodide-stained nuclei. Each experiment was performed in triplicates. Bars are the mean values of triplicates ± SD.

 
To gain insight into molecular mechanisms of cathepsin D-mediated doxorubicin resistance in neuroblastoma cells, we analyzed cellular key pro- and antiapoptotic signaling pathways in Tet21N-CTSD cells prior and upon doxorubicin treatment. Cathepsin D overexpression was associated with phosphorylation of protein kinase B (Akt) at Ser-473 and Bcl-2 at Ser-70 (Figure 3E). Inhibition of PI3–Akt signaling by LY294002 restored the sensitivity of Tet21N-CTSD cells to doxorubicin, supporting the suggestion that role of PI3–Akt signaling has an essential role in prosurvival effect of cathepsin D (Figure 3E). Two bands of Bcl-2-phospho-Ser-70 were detected in the total protein extracts obtained from Tet21N-CTSD and control cells cultured in the complete medium without doxorubicin. Previously, multiple phosphorylation of Bcl-2 has been reported (26). Therefore, we suggested that two phospho-Bcl-2-Ser-70 bands represented mono (lower band) and multiple (upper band) phosphorylated Bcl-2. In control cells, doxorubicin-induced apoptosis was accompanied by significant reduction of monophosphorylated Bcl-2-Ser-70, whereas in doxorubicin-resistant Tet21N-CTSD cells monophosphorylated Bcl-2 was detectable. Tet21N-CTSD cells were less sensitive to doxorubicin-induced apoptosis even at higher p53 levels in these cells compared with control cells (Figure 3E). Tet21N-CTSD cells responded to doxorubicin-induced DNA damage likewise the control cells via stabilization of p53 protein and induction of its direct target p21/Cip/WAF1. These data suggest that in neuroblastoma cells, cathepsin D promotes antiapoptotic signaling via the PI3–Akt pathway and contributes to doxorubicin resistance independent of p53 signaling.

Regulation of CTSD expression by MYCN
To investigate the impact of cathepsin D on neuroblastoma in vivo, we examined the expression of CTSD in a cohort of 49 primary neuroblastoma tumors of different international neuroblastoma staging system stages, patient’s age at diagnosis and MYCN amplification status. CTSD expression was significantly higher in the MYCN-amplified tumors than in MYCN single-copy tumors (P = 0.009) (Figure 4A). These data suggested that deregulated MYCN is involved in the regulation of CTSD expression. To test this possibility, we analyzed changes in CTSD mRNA level in Tet21N cells after switching MYCN expression on. Amount of CTSD mRNA was highest at 2 h after induction of MYCN by removing tetracycline from the medium. Amount of CTSD mRNA sharply declined at 4 h after induction followed by reaccumulation at later time points. Similar mRNA accumulation profiles were previously found for a subgroup of ‘low-affinity’ MYCN/c-MYC target genes (F.Westermann, D.Muth, A.Benner, T.Bauer, K.O.Henrich, A.Oberthür, B.Brors, T.Beissbarth, F.Pattyn, J.Vandesompele, F.Speleman, R.König, M.Fischer and M.Schwab, submitted for publication). Early CTSD elevation most probably was induced by new growth factors added with fresh medium, whereas reaccumulation of CTSD mRNA in parallel with increasing MYCN levels at the later time points suggests induction of CTSD expression by MYCN. The promoter sequence of CTSD contains two canonical E-boxes: at position –718 and –115, the proximal one is in the context of CpG island, suggesting direct involvement of MYCN in the induction of CTSD transcription. Using chromatin immunoprecipitation assays, we found that endogenous MYCN protein in MYCN-amplified neuroblastoma cell line IMR5/75 and ectopic MYCN in Tet21N directly interacted with the CTSD promoter. In Tet21N cells, cathepsin D promoter fragments were precipitated from MYCN ON and MYCN OFF cells most probably due to the leaky MYCN expression. To determine whether MYCN binding to the CTSD promoter activates transcription of the CTSD gene, we performed luciferase promoter–reporter assays. The 817 bp fragment of the core CTSD promoter harboring two E-boxes (bp –877/–60) was fused to firefly luciferase gene and introduced into neuroblastoma cell lines Tet21N, MYCN-amplified IMR5/75, MYCN single-copy SH-EP and SY5Y. We found that MYCN expression resulted in a consistent and statistically significant activation of the CTSD promoter construct (P = 0.04 for Tet21N MYCN ON versus MYCN OFF and P < 0.001 for MYCN-amplified versus non-amplified cells) (Figure 4D). Together, these data indicate that MYCN positively regulates transcription of CTSD.


Figure 4
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  		Fig. 4. Regulation of cathepsin D expression by MYCN. (A) Expression of CTSD in neuroblastoma tumors as determined by cDNA microarray analysis. Differential expression was estimated by Wilcoxon exact rank-sum test. Statistical comparisons were made for MYCN-amplified versus all MYCN single-copy tumors and MYCN single-copy tumors obtained from patients >1.5 years of age at diagnosis versus MYCN single-copy tumor obtained from patients <1.5 years of age at diagnosis. Data are presented as box plots: horizontal boundaries of the box represent the 25th and 75th percentile, the median is denoted by a horizontal line in the box and whiskers above and below extend to the most extreme data points which are no more than 1.5 times the interquartile range from the box. (B) Quantification of relative changes in the amount of CTSD mRNA in Tet21N cells after induction of MYCN expression. To switch on MYCN expression, Tet21N growing in the presence of tetracycline were detached from the substratum with versene, washed several times with fresh growth medium without tetracycline and reseeded in the growth medium without tetracycline. Upper panel: mRNA was isolated at indicated time points and CTSD expression profile was analyzed using microarrays. Lower panel: total protein samples were prepared in parallel with mRNA isolation at indicated time points and levels of MYCN were assayed by immunoblotting. (C) In vivo binding of MYCN to the CTSD promoter. Immunoprecipitation of cross-linked chromatin (chromatin immunoprecipitation) with control (IgG lane) or MYCN-specific antibodies was performed in Tet21N cells with regulated ectopic MYCN and in IMR5/75 cells with endogenous amplified MYCN. Precipitated DNA fragments were used for polymerase chain reaction with primers specific for the CTSD promoter. (D) Regulation of CTSD promoter activity by MYCN. A fragment of the CTSD promoter including two E-boxes was cloned into pGL3-Basic vector and transfected into Tet21N MYCN ON, Tet21N MYCN OFF, MYCN-amplified IMR5/75, MYCN single-copy SH-EP and SY5Y cells. pGL3-Basic was used a negative control and pGL3-PTMA was used as positive control. A dual-luciferase reporter assay was used to normalize for transfection efficiency. Columns represent average promoter activity normalized to activity of pGL3-Basic (RLU—relative luminescence unit). SDs were <1% for all the samples. (E) MYCN expression destabilizes intracellular cathepsin D in Tet21N cells. Exponentially growing Tet21N MYCN ON and MYCN OFF cells were treated with doxorubicin (DOXO+) for 24 h or left untreated (DOXO–). Total cellular protein was resolved by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunodetection was performed with antibodies specific to MYCN and cathepsin D; {alpha}-tubulin served as loading control. (F) Intracellular cathepsin D negatively correlates with MYCN expression in different neuroblastoma cell lines. Neuroblastoma cells were seeded into six-well plates; 24 h after seeding, total protein was isolated, resolved by 10–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis and immunoblotted with anti-MYCN and anti-cathepsin D; β-actin served as loading control.

 
In contrast to the induction of CTSD mRNA by MYCN, the protein levels of cathepsin D in proliferating Tet21N MYCN ON cells were much lower than in MYCN OFF cells (Figure 4E). In line with this in different neuroblastoma cell lines, intracellular cathepsin D levels were also negatively correlated with MYCN protein expression (Figure 4F). These results suggest that MYCN negatively regulates cathepsin D protein expression via posttranscriptional destabilization of the intracellular cathepsin D.

These results suggest that MYCN can negatively affect cathepsin D expression via posttranscriptional destabilization of the intracellular cathepsin D.

Doxorubicin treatment promotes secretion of procathepsin D by Tet21N cells
The presented data show that cathepsin D is involved in prosurvival signaling in neuroblastoma cells. The prosurvival role of cathepsin D is well documented for breast cancer cells, where secreted procathepsin D acts as an autocrine regulator, promoting growth of cancer and endothelial cells (27,28). Therefore, we assayed secretion of procathepsin D by Tet21N cells in response to doxorubicin administration and induction of MYCN expression. Tet21N-CTSD had more extracellular cathepsin D than control cells (P < 0.001) (Figure 5A). Induction of MYCN expression resulted in a slight increase of cathepsin D secretion (P = 0.05 for control cells and P = 0.06 for Tet21N-CTSD). Doxorubicin treatment resulted in significant increase in secreted cathepsin D in both cells lines (P = 0.005 for control cells and P = 0.007 for Tet21N-CTSD cells).


Figure 5
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  		Fig. 5. Cathepsin D is an autocrine prosurvival factor for Tet21N cells. (A) Doxorubicin promotes secretion of cathepsin D by Tet21N cells. About 1 x 105 of Tet21N MYCN ON and MYCN OFF cells were seeded onto 12-well cell culture dishes. Cells were treated with doxorubicin for 48 h (DOXO+) or left untreated (DOXO–). Each experiment was performed in triplicates. Relative amount of cathepsin D in the conditioning media of Tet21N-CTSD (white bars) and control Tet21N-pcDNA4 cells (black bars) was estimated spectrophotometrically using Rapid Format Cathepsin D ELISA kit (Calbiochem). Obtained absorbance values were normalized by number of cells in the respective sample (RAU—relative absorbance unit). Bars represent mean values of triplicates ± SD. Similar results were obtained in two independent experiments. (B) Upper panel: conditioning media obtained from MYCN ON or MYCN OFF Tet21N-CTSD (gray bars) and control Tet21N cells (black bars) were mixed with complete growth medium in the ratio 1:3 and added to Tet21N cells. Cells were treated with doxorubicin and apoptosis was determined by FACS analysis of propidium iodide-stained nuclei. Presented data are mean values of three independent experiments ± SD. Lower panel: total protein was isolated from the cells treated as described for the upper panel, resolved in 10–20% gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (40 µg per lane) and immunoblotted using antibodies specific to phospho-Akt Ser-473; β-actin was used as loading control.

 
To test whether the extracellular cathepsin D may reduce sensitivity of neuroblastoma cells to doxorubicin, Tet21N MYCN ON cells were treated with doxorubicin for 48 h in the growth medium supplemented with conditioning medium from Tet21N-CTSD or control Tet21N-pcDNA4 cell. Tet21N cells supplemented with the conditioning medium containing cathepsin D were more resistant to doxorubicin (P = 0.046) (Figure 5B) and had more phosphorylated Akt-1 (Figure 5C).


   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
Supplementary material
 Funding
 References
 
In the present study, we investigated the genetic control of doxorubicin-induced cell death in neuroblastoma cells with regulated expression of MYCN. To target new genes involved in neuroblastoma response to cytotoxic therapy, a modification of TKO function-based gene cloning was used. In addition to the classical TKO strategy (21), our approach utilized a subtractive hybridization, ensuring enrichment of the library for transcripts of MYCN target genes and genes that are expressed in cells undergoing doxorubicin-induced cell death (16). We isolated an episome containing a cDNA fragment of cathepsin D that protected Tet21N cells from doxorubicin-induced cell death. The CTSD fragment in the episome was cloned in sense orientation and encoded for a 60 amino acid peptide. On the one hand, previously published studies that utilized different peptide-coding genetic suppressor elements reported dominant-negative action (22). On the other hand, a small peptide derived from the N-terminus of procathepsin D has been shown to retain mitogenic activity of the whole protein (24). Therefore, it was not clear whether the sense-oriented CTSD cDNA fragment acted as a dominant-negative mutant or whether it retained some of the cathepsin D activities. Besides its fundamental function in intracellular catabolism, cathepsin D plays an essential role in tumor progression stimulating proliferation and inhibiting apoptosis in vivo and in vitro (25,27,28). In contrast to this, cathepsin D has been shown to translocate into the cytosol upon oxidative damage of lysosomal membranes, where it may act as a mediator of apoptosis (29). In summary, cathepsin D may either inhibit or promote apoptosis via different mechanisms dependent on cell type and environmental conditions.

The results of the functional analysis of cathepsin D in doxorubicin-treated Tet21N cells clearly showed its antiapoptotic role. The siRNA-mediated silencing of cathepsin D expression or inhibition of its enzymatic activity by pepstatin A sensitized Tet21N cells to doxorubicin. In agreement with this, prolonged enzymatic inhibition of cathepsin D induces apoptosis in neuroblastoma cells (30). Overexpression of cathepsin D in Tet21N cells suppressed the activation of caspases and significantly reduced sensitivity of the cells to doxorubicin. The antiapoptotic effect of cathepsin D in Tet21N cells was detected despite p53 induction in response to doxorubicin-induced DNA damage. In general, p53 mutations or deletions are rare in neuroblastoma. However, the function of wild-type p53 upon DNA damage is thought to be impaired in different neuroblastoma cell lines, but the mechanisms involved are poorly understood (reviewed in ref. 31). In Tet21N cells, overexpression of cathepsin D was associated with increased phosphorylation of Bcl-2 at Ser-70. Ser-70 phosphorylation enhances Bcl-2 antiapoptotic functions via inhibition of its interaction with p53 in response to DNA damage (32). Thus, cathepsin D-mediated activation of Bcl-2 may contribute to the development of drug resistance in neuroblastoma cells harboring a functional p53.

We show here that cathepsin D exerts its prosurvival effect on Tet21N cells also via the PI3–Akt pathway, which has been recently suggested as a novel prognostic marker of aggressive neuroblastomas (33). In agreement with this finding, cathepsin D deficiency resulting in profound decrease of PI3–Akt signaling and persistent neurodegeneration has been reported for mouse in vivo models (34–36).

Activation of PI3–Akt prosurvival signaling resulting in increased resistance to doxorubicin in neuroblastoma cells has been associated with various secreted factors produced by doxorubicin-resistant cells (37,38). Neuroblastoma cells are able to secrete procathepsin D, a mitogenic form of cathepsin D that has been detected previously in breast cancer cells, and are recognized as a mitogen that promotes proliferation of cancer cells, invasive growth of fibroblasts and angiogenesis (25,28). In conclusion, we suggest that cathepsin D is one of the prosurvival factors secreted by neuroblastoma cells. The finding that doxorubicin treatment promotes secretion of mitogenic procathepsin D reveals a new mechanism that may contribute to the acquisition of drug resistance in vivo. As a side effect of doxorubicin treatment, secreted procathepsin D may promote proliferation of surrounding endothelial cells and tumor cells that had survived a chemotherapeutic insult.

In line with the prosurvival role of cathepsin D in neuroblastoma cells in vitro, increased CTSD mRNA levels were found in MYCN-amplified neuroblastoma tumors. Similarly, the correlation of elevated expression of cathepsin D with advanced disease or metastatic tumor growth has been documented previously in breast cancer and glioblastoma (39–42), but has never been reported for neuroblastoma. Immunohistochemical evaluation of intracellular cathepsin D in developing neurons and childhood neuroblastic tumors, including a limited number of neuroblastomas, had linked accumulation of intracellular cathepsin D to ganglionic cell differentiation (43,44). However, the genomic status of MYCN was not reported in both studies, which precludes a retrospective interpretation of these studies. The data presented here are pointing toward a versatile mechanism of cathepsin D regulation by MYCN. It is well defined that CTSD expression is regulated by estrogens and certain growth factors in cell type-specific manner (25). Our data now suggest that MYCN directly interacts with the CTSD promoter and activates CTSD transcription. At the same time, MYCN also contributes to the downregulation of the intracellular level of cathepsin D possibly via posttranscriptional destabilization and its extracellular secretion.

In summary, the presented results demonstrate that resistance to doxorubicin in human neuroblastoma with amplified MYCN may arise due to deregulated expression of cathepsin D. Cathepsin D in neuroblastoma cells attenuates apoptosis via the PI3–Akt pathway and promotes drug resistance independent of p53 signaling. The fact that doxorubicin administration promotes secretion of mitogenic procathepsin D by neuroblastoma cells provides a basis for designing novel antitumor therapeutic strategies combining anthracyclines with inhibitors of Bcl-2 and/or PI3–Akt prosurvival signaling.


   Supplementary material
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
Funding
 References
 
Supplementary Table can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
 Funding
References
 
Deutsche Krebshilfe; Bundesministerium für Bildung und Forschung (Nationales Genomforschungsnetz-2, grant N2KR-S19T03); European Union (FP6 EET-Pipeline, grant #037260).


   Acknowledgments
 
The authors thank Dr M.Garcia for pSG1-Cathepsin D.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Supplementary material
 Funding
 References
 

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Received January 17, 2008; revised May 16, 2008; accepted June 13, 2008.


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