The RNA-binding protein dyskerin, encoded by the DKC1 gene, functions as a core component of the telomerase holoenzyme as well as ribonuclear protein complexes involved in RNA processing and ribosome biogenesis. The diverse roles of dyskerin across many facets of RNA biology implicate its potential contribution to malignancy. In this study, we examined the expression and function of dyskerin in neuroblastoma. We show that DKC1 mRNA levels were elevated relative to normal cells across a panel of 15 neuroblastoma cell lines, where both N-Myc and c-Myc directly targeted the DKC1 promoter. Upregulation of MYCN was shown to dramatically increase DKC1 expression. In two independent neuroblastoma patient cohorts, high DKC1 expression correlated strongly with poor event-free and overall survival (P < 0.0001), independently of established prognostic factors. RNAi-mediated depletion of dyskerin inhibited neuroblastoma cell proliferation, including cells immortalized via the telomerase-independent ALT mechanism. Furthermore, dyskerin attenuation impaired anchorage-independent proliferation and tumor growth. Overexpression of the telomerase RNA component, hTR, demonstrated that this proliferative impairment was not a consequence of telomerase suppression. Instead, ribosomal stress, evidenced by depletion of small nucleolar RNAs and nuclear dispersal of ribosomal proteins, was the likely cause of the proliferative impairment in dyskerin-depleted cells. Accordingly, dyskerin suppression caused p53-dependent G1 cell-cycle arrest in p53 wild-type cells, and a p53-independent pathway impaired proliferation in cells with p53 dysfunction. Together, our findings highlight dyskerin as a new therapeutic target in neuroblastoma with crucial telomerase-independent functions and broader implications for the spectrum of malignancies driven by MYC family oncogenes. Cancer Res; 76(12); 3604–17. ©2016 AACR.

The human gene encoding dyskerin, DKC1, was first identified through its involvement in dyskeratosis congenita, a heterogeneous disease characterized by defects in highly regenerative tissues (1). Subsequent studies demonstrated high DKC1 expression in various adult cancers, with the DKC1 promoter shown to be a target of c-Myc in breast cancer (2–4). Gene expression array and proteomic studies identified DKC1 among the suite of genes upregulated in neuroblastoma in association with MYCN amplification (5–7). Despite these findings, the significance of dyskerin expression in cancer has been debated, with some studies suggesting that dyskerin may have tumor suppressor functions (8, 9).

Amplification of the MYCN oncogene is a powerful prognostic indicator used in risk stratification of neuroblastoma, in which elevated MYCN expression is known to contribute to the development of aggressive disease (10). Neuroblastoma presents an ideal malignancy for studying Myc-driven cancer, as cell lines that do not have high N-Myc generally express elevated c-Myc (11). Neuroblastoma is a malignancy of the developing sympathetic nervous system and the most common extracranial solid tumor of childhood (12). It presents as advanced disease in approximately 50% of all patients and accounts for a disproportionate 15% of pediatric cancer–related deaths from only 8% of pediatric cancer diagnoses, with children in the high-risk category having less than 50% chance of long-term survival (12). The major challenges in the treatment of high-risk cases are dose-limiting toxicities of chemotherapeutics and drug resistance. Identification and validation of new therapeutic targets will provide opportunity for the development of new drugs to improve outcomes for children with aggressive and drug-resistant disease.

Dyskerin is a highly conserved RNA-binding and modifying protein that is expressed in a broad range of normal tissues (1, 13). It functions in ribonuclear protein (RNP) complexes by directly binding and thereby stabilizing small noncoding RNAs, including the RNA component of telomerase (hTR), H/ACA-box small nucleolar RNAs (snoRNA), and Cajal body RNAs (scaRNA; refs. 14, 15). In complexes with hTR, dyskerin is essential to the biogenesis and function of the active telomerase enzyme complex, which plays a well-characterized role in immortalization and the development of 85% to 90% of all cancers (16). Telomerase activity has been reported to be highest in advanced stages of neuroblastoma (stages III and IV) and is an independent prognostic indicator of poor outcome (17, 18).

RNP complexes that include dyskerin and H/ACA snoRNAs play essential roles in ribosome biogenesis, functioning to modify rRNA and in the processing of rRNA precursors (15). Accordingly, mice featuring Dkc1 mutations harbor defects in rRNA processing and protein translation, as well as low telomerase activity (19, 20). In complexes with H/ACA-box scaRNAs, dyskerin is also involved in the processing of precursor mRNAs. Studies showing that some snoRNAs encode miRNA precursors allude to additional means by which dyskerin may influence mRNA processing and the abundance of specific proteins in cancer cells (21, 22). The diverse functions of dyskerin highlight multiple mechanisms through which it may contribute to malignancy, and further add to the rationale for investigating the regulation, function, and potential for therapeutic exploitation of dyskerin.

This study shows that the DKC1 gene promoter is targeted by both c-Myc and N-Myc, and that high DKC1 expression is an independent prognostic indicator for adverse clinical outcome in neuroblastoma. Furthermore, we show for the first time that downregulation of dyskerin arrests the replication of neuroblastoma cells, impairs anchorage-independent growth, and suppresses tumor formation. The inhibitory effect of dyskerin depletion on neuroblastoma cell proliferation was accompanied by a reduction in H/ACA snoRNAs and hallmarks of ribosomal stress, and was found to be independent of telomerase suppression.

Cell culture

IMR32, HEK293T cells, Phoenix-A packaging cells, and MRC-5 fetal lung fibroblasts were purchased from ATCC, and SK-N-AS, SK-N-BE(2), SK-N-DZ, SK-N-FI, SK-N-SH, CHP-134, Kelly, NB69 from European Collection of Cell Cultures. BE(2)-C, LAN-1, SH-EP, and SH-SY5Y were provided by Dr. June Biedler, Memorial Sloan-Kettering Cancer Center, (New York, NY) and NBL-S and NBL-W by Dr. Susan Cohn, University of Chicago (Chicago, IL). These cell lines were authenticated by short tandem repeat profiling (CellBank Australia) in May 2012 and were cultured for no more than 6 weeks. Neuroblastoma cell lines were cultured in DMEM or RPMI1640. HEK293T, Phoenix-A, and HeLa cells were cultured in DMEM and MRC-5 in α-minimal essential medium (all media from Life Technologies). Culture medium was supplemented with 10% FBS (ThermoTrace), 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Cells were cultured at 37°C with 5% CO2.

Targeted gene silencing

Cells were transfected with 50 to 100 nmol/L siRNA (Supplementary Table S1) using Lipofectamine RNAiMAX reagent (Life Technologies). Vectors and gene transfer methods are described in detail in the Supplementary Materials and Methods. Transduced cells were selected by FACS for GFP expression (LMS-based retroviral vectors) or by growth in either 0.5 mg/mL genetecin (MND retroviral vectors) or 0.5 μg/mL puromycin (BABEpuro-shRNA retroviral vectors). Expression of shRNA from Fh1t-UTG–based lentiviral vectors was induced by addition of 1 μg/mL doxycycline-hyclate (Sigma-Aldrich) to the growth medium every 3 days.

Gene and protein expression, telomerase activity, and telomere length analyses

Gene and protein expression were assayed by immunoblot analysis and qRT-PCR using standard procedures. Antibodies and primers are detailed in the Supplementary Methods and Methods and Supplementary Table S2, respectively. Telomerase enzyme activity and telomere length were measured using the quantitative telomeric amplification protocol (qTRAP) and Southern blot–based telomeric restriction fragment analysis, respectively, as described previously (23). For the qTRAP assay, negative controls were MRC-5 cells, heat-treated SK-N-SH lysate (Heat-Tx), and lysis buffer without sample (LB).

Chromatin immunoprecipitation assays

Chromatin immunoprecipitation (ChIP) assays were performed as described previously (24) using antibodies and primers listed in Supplementary Table S3. Relative enrichment of specific promoter regions was determined by normalization to input DNA.

Cell-cycle analysis

Cells were washed in cold PBS, then fixed in cold 70% ethanol before permeabilization with 0.5% Triton-X/0.1% BSA and staining with 25 μg/mL propidium iodide (Sigma-Aldrich)/100 μg/mL DNAse-free RNAse (Roche) in 0.1% BSA/PBS. Stained cells were analyzed by flow cytometry (FACSCalibur, BD Biosciences) using ModFit LT v3.0 software (Verity Software House).

Anchorage-independent growth and tumorigenicity

Anchorage-independent growth was assayed by colony formation in soft agarose as described previously (25). Tumorigenicity was assessed with approval of the Animal Care and Ethics Committee of the University of New South Wales (Sydney, New South Wales, Australia; ACEC #11/5B). Female 6- to 8-week old BALB/c nu/nu (nude) mice (Animal Resource Centre) were injected with 2 × 106 cells suspended in 200 μL DMEM into the hind flank. Mice with palpable tumors were switched to food laced with 600 mg/kg doxycycline (Specialty Feeds) to induce transgene expression. Mice were sacrificed before tumor volume reached 1.5 cm3, calculated as: volume (mm3) = [length (mm) × width (mm) × height (mm)]/2.

Detection of ribosomal proteins by immunofluorescence

Cells cultured on glass coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X, and blocked for 50 minutes with 10% FBS before staining with anti-rpl11 (Abcam; ab86863) or anti-rpl5 (Abcam; ab79352) for 50 minutes and then secondary antibody (Life Technologies; A-11012) for 50 minutes. Coverslips were mounted with Prolong-Gold (Life Technologies; P36930). Images were captured using a Zeiss Axiovert 200M inverted microscope with a LCI Plan-Neolfluar 63x/1.3 objective and an AxioCam HRm camera (Carl Zeiss Inc.).

The DKC1 gene is upregulated in neuroblastoma cells and is a direct transcriptional target of N-Myc and c-Myc

Quantification of DKC1 mRNA in 15 neuroblastoma cell lines (8 with MYCN amplification and 7 lacking MYCN amplification) revealed that DKC1 mRNA was elevated in the neuroblastoma cells relative to normal human myofibroblasts (MRC-5 and WI-38) and bone marrow endothelial cells (BMEC; Fig. 1A, top). On average, DKC1 expression was 4-fold higher in neuroblastoma cells than in normal cells (P < 0.01), and was higher in cells with MYCN amplification compared with those without amplification (P < 0.05). Immunoblot analysis showed that dyskerin protein levels were highest in cells with MYCN amplification, and was also elevated in cells with high c-Myc expression (Fig. 1B). There was a significant correlation between dyskerin protein and DKC1 mRNA levels (Supplementary Fig. S1; r = 0.639, P < 0.05).

Figure 1.

Expression of DKC1, TERT, and telomerase enzyme activity in neuroblastoma cell lines. A, DKC1 and TERT gene expression in neuroblastoma cell lines determined by quantitative real-time PCR (qRT-PCR) analysis. Values are mean ± SEM from three to five independent assays, with duplicates in each assay. **, P < 0.01; *, P < 0.05; n.s., not significantly different in Mann–Whitney test. B, representative immunoblot analysis showing dyskerin, N-Myc, and c-Myc protein, with GAPDH as a loading control. The vertical white line indicates rearrangement of the image for presentation purposes. C, telomerase enzyme activity measured using the telomeric repeat amplification protocol (qTRAP). Heat-Tx, heat-treated SK-N-SH lysate; LB, lysis buffer with no sample. Values are mean ± SEM from three independent assays, with duplicates in each assay. The graph at the right shows mean telomerase activity in subgroups of the cells. D, telomeric restriction fragment length analysis of telomere length in neuroblastoma cell lines.

Figure 1.

Expression of DKC1, TERT, and telomerase enzyme activity in neuroblastoma cell lines. A, DKC1 and TERT gene expression in neuroblastoma cell lines determined by quantitative real-time PCR (qRT-PCR) analysis. Values are mean ± SEM from three to five independent assays, with duplicates in each assay. **, P < 0.01; *, P < 0.05; n.s., not significantly different in Mann–Whitney test. B, representative immunoblot analysis showing dyskerin, N-Myc, and c-Myc protein, with GAPDH as a loading control. The vertical white line indicates rearrangement of the image for presentation purposes. C, telomerase enzyme activity measured using the telomeric repeat amplification protocol (qTRAP). Heat-Tx, heat-treated SK-N-SH lysate; LB, lysis buffer with no sample. Values are mean ± SEM from three independent assays, with duplicates in each assay. The graph at the right shows mean telomerase activity in subgroups of the cells. D, telomeric restriction fragment length analysis of telomere length in neuroblastoma cell lines.

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Neuroblastoma cell lines were also characterized for expression of the catalytic component of telomerase, TERT, as well as telomerase enzyme activity and telomere length. TERT mRNA expression was detected in all but one of the neuroblastoma cell lines (SK-N-FI; Fig. 1A, bottom graph). Telomerase activity varied across the panel of cell lines (Fig. 1C), correlating more closely with expression of DKC1 (r = 0.575, P < 0.05) than TERT (r = 0.375, P = 0.169; Supplementary Fig. S2A and S2B, respectively). There was also heterogeneity in telomere length among the neuroblastoma cell lines (Fig. 1D). The SK-N-FI cell line, which had no detectable TERT expression and negligible telomerase activity, exhibited very long and heterogeneous telomeres, which is typical of cells immortalized by the telomerase-independent alternate telomere lengthening mechanism (ALT; ref. 26).

Having shown that elevated DKC1 expression was associated with high MYC and MYCN expression, ChIP assays were performed to determine whether N-Myc and c-Myc proteins directly interact with the DKC1 gene. Using BE(2)-C, a MYCN-amplified neuroblastoma cell line, and SH-SY5Y, which has high c-Myc expression and lacks MYCN amplification (Fig. 1B), we found that both Myc proteins (together with their obligate binding partner, Max), bound DKC1 at sites proximal to the DKC1 promoter (Fig. 2A). We identified a noncanonical E-box within a CpG island downstream of the DKC1 transcription start site (Fig. 2A, Amplicon B) and two canonical E-boxes within the first intron (Fig. 2A, Amplicon C) as sites of N-Myc and c-Myc binding in BE(2)-C and SH-SY5Y cells, respectively. N-Myc and c-Myc also bound at canonical E-boxes proximal to the transcriptional start site of the TERT gene (Fig. 2B). No c-Myc or N-Myc binding was observed at control amplicons upstream of the DKC1 and TERT promoters (Amplicon A in Fig. 2A and B).

Figure 2.

The DKC1 gene is a target of MYC-family oncoproteins in neuroblastoma cells. ChIP was performed to assess binding of N-Myc and c-Myc proteins to the promoters of DKC1 (A) and TERT (B) in BE(2)-C (left schemes) and SH-SY5Y (right schemes). Schemes beneath each graph represent human DKC1 and TERT promoters showing the canonical and noncanonical E-box sites, and positions of the PCR amplicons (A–C). Values represent mean ± SEM from three independent experiments. C, qRT-PCR analysis of MYCN, DKC1, and TERT expression in SHEP-TET21/N cells previously transfected with a MYCN vector induced by tetracycline withdrawal.

Figure 2.

The DKC1 gene is a target of MYC-family oncoproteins in neuroblastoma cells. ChIP was performed to assess binding of N-Myc and c-Myc proteins to the promoters of DKC1 (A) and TERT (B) in BE(2)-C (left schemes) and SH-SY5Y (right schemes). Schemes beneath each graph represent human DKC1 and TERT promoters showing the canonical and noncanonical E-box sites, and positions of the PCR amplicons (A–C). Values represent mean ± SEM from three independent experiments. C, qRT-PCR analysis of MYCN, DKC1, and TERT expression in SHEP-TET21/N cells previously transfected with a MYCN vector induced by tetracycline withdrawal.

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The relationship between MYCN expression and that of DKC1 and TERT was further tested using a well-characterized tetracycline-modulated vector system that induces MYCN expression to very high levels in SH-EP neuroblastoma cells (27). These experiments showed that MYCN levels were dramatically upregulated following removal of tetracycline (Fig. 2C). Upregulation of MYCN was accompanied by a 50- to 100-fold increase in DKC1 expression, and a 10- to 20-fold increase in TERT expression. Together, these data provide the first functional evidence of N-Myc and c-Myc targeting the DKC1 promoter in neuroblastoma cells.

High DKC1 expression is an independent prognostic indicator of poor event-free and overall survival in neuroblastoma

To investigate the clinical significance of DKC1 expression levels, we combined publically available microarray data from a large cohort of neuroblastoma patients and published clinical data for a subset of these patients (n = 477, the Kocak cohort; ref. 28). Our analyses revealed that DKC1 expression was significantly higher in tumors with amplification of the MYCN gene compared with those without (Fig. 3A; P < 0.001). Moreover, DKC1 expression strongly correlated with MYCN expression in the subset of patients with MYCN amplification (Fig. 3B; r = 0.531 P < 0.001). The strength of this association was notable in light of a poor correlation between TERT and MYCN expression in this sample subset (Supplementary Fig. S3; r = 0.215, P = 0.071). When DKC1 gene expression was dichotomized around the upper quartile, high expression of DKC1 was significantly associated with poorer event-free survival (EFS, Fig. 3C) and poorer overall survival (OS, Fig. 3D), with 5-year EFS rates of 76% ± 2% and 28% ± 5% and 5-year OS of 91% ± 2% and 36% ± 5% for tumors with low and high DKC1 expression, respectively. These correlations were also highly statistically significant when DKC1 expression was dichotomized at the median or upper decile (Supplementary Fig. S4A and S4B, P < 0.0001). The clinical significance of high DKC1 expression was further validated by analysis of an independent cohort of 197 primary tumors from neuroblastoma patients enrolled in COG protocol 9047. In the COG dataset, high expression of DKC1 was again associated with poorer EFS (P < 0.0001, Fig. 3E) and poorer OS (P < 0.0001, Fig. 3F).

Figure 3.

High DKC1 expression is a marker of poor prognosis in neuroblastoma. A, DKC1 expression in neuroblastoma tumor samples with (n = 71) and without (n = 405) MYCN amplification. The graph shows log-transformed, zero-centered expression levels obtained from microarray dataset (Kocak cohort; ref. 28). Boxes indicate 25th to 75th percentiles and whiskers illustrate minimum to maximum values. P value from two-tailed Mann–Whitney tests. B, correlation between DKC1 and MYCN expression in tumors from the Kocak cohort with MYCN amplification. r is Spearman coefficient and P value from two-tailed correlation tests. C–F, Kaplan–Meier curves showing the probability of EFS and OS for the Kocak (C and D) and Children's Oncology Group (E and F) cohorts. Values were dichotomized into ‘high’ and ‘low’ DKC1 expression around the upper quartile.

Figure 3.

High DKC1 expression is a marker of poor prognosis in neuroblastoma. A, DKC1 expression in neuroblastoma tumor samples with (n = 71) and without (n = 405) MYCN amplification. The graph shows log-transformed, zero-centered expression levels obtained from microarray dataset (Kocak cohort; ref. 28). Boxes indicate 25th to 75th percentiles and whiskers illustrate minimum to maximum values. P value from two-tailed Mann–Whitney tests. B, correlation between DKC1 and MYCN expression in tumors from the Kocak cohort with MYCN amplification. r is Spearman coefficient and P value from two-tailed correlation tests. C–F, Kaplan–Meier curves showing the probability of EFS and OS for the Kocak (C and D) and Children's Oncology Group (E and F) cohorts. Values were dichotomized into ‘high’ and ‘low’ DKC1 expression around the upper quartile.

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Multivariate analysis was performed using the larger cohort (Kocak) to determine whether the prognostic significance of high DKC1 was independent of established prognostic indicators for neuroblastoma. Age at diagnosis (<18 months vs. ≥18 months), tumor stage (1, 2, or 4s vs. 3 or 4), MYCN status (amplified vs. single copy), and DKC1 gene expression (upper quartile) were tested in a Cox proportional hazards regression model (Supplementary Table S4). DKC1 expression retained strong independent prognostic significance for EFS (HR = 2.21; 95% confidence intervals, CI, 1.41–3.45; P < 0.0005) and OS (HR = 2.60; 95% CI, 1.43–4.71; P = 0.0017). Consistent with previous reports (18), high TERT expression also correlated with poor patient outcomes in univariate analysis of the Kocak cohort (Supplementary Fig. S5A and S5B); however, TERT expression was not significant in mulitvariate analyses with independent prognostic factors (Supplementary Table S4; P = 0.8066 for EFS and P = 0.5561 for OS).

Downregulation of DKC1 inhibits the replication and tumorigenic growth of neuroblastoma cells

The functional significance of high DKC1 expression was investigated using two siRNAs (D2 and D3) designed to effectively downregulate the DKC1 gene (Fig. 4A and B). In short-term cell proliferation assays, downregulation of DKC1 to levels similar to those detected in normal cells significantly inhibited the replication of neuroblastoma cells (Fig. 4C). Across 10 neuroblastoma cell lines, there was an overall correlation between the extent of dyskerin suppression and the magnitude of the proliferative impairment (Spearman r = 0.431, P < 0.05).

Figure 4.

Downregulation of dyskerin suppresses neuroblastoma cell proliferation in vitro and tumor growth in mice. A, qRT-PCR analysis of DKC1 expression in neuroblastoma cells transfected with siRNAs targeting DKC1 mRNA (D2 and D3) or a control oligonucleotide (Sc). Values are mean ± SEM from three to five independent transfections. Upper and lower horizontal dotted line indicate DKC1 expression in HeLa and normal MRC5 myofibroblasts, respectively. B, immunoblot ofdyskerin protein in a representative experiment where SK-N-AS cells were transfected with siRNA. C, proliferation of siRNA-transfected cells. Values are mean ± SEM from three to seven independent transfections, quantified as expansion. D, SK-N-BE(2) cells were transduced with retroviral vectors that constitutively express shRNA targeting dyskerin mRNA (LMS-DKC1 shRNA), nonsilencing control shRNA (LMS-NS), or GFP only (LMS-EV). qRT-PCR was performed on GFP+ cells within 15 days of transduction. Values are mean ± SEM from at least five independent assays. **, P < 0.01; *, P < 0.05 from Bonferroni multiple comparisons test. E, anchorage-independent growth in soft-agarose measured 2 weeks after transduction. Values are mean ± SEM from four independents experiments. **, P < 0.01, Holm–Sidak multiple comparisons test. F and G, SK-N-BE(2) cells were transduced with FH1t shRNA lentiviral vectors encoding shRNA under control of a doxycycline (dox)-inducible promoter. F, qRT-PCR analysis of DKC1 expression (left axis) and cell viability determined by Trypan blue (right axis) in cells cultured with or without doxycycline. Values are the average of duplicates at each time point. G, tumor growth in BALB/c nu/nu mice engrafted with FH1t-transduced SK-N-BE(2) cells (10 mice per group). P value, two-way ANOVA.

Figure 4.

Downregulation of dyskerin suppresses neuroblastoma cell proliferation in vitro and tumor growth in mice. A, qRT-PCR analysis of DKC1 expression in neuroblastoma cells transfected with siRNAs targeting DKC1 mRNA (D2 and D3) or a control oligonucleotide (Sc). Values are mean ± SEM from three to five independent transfections. Upper and lower horizontal dotted line indicate DKC1 expression in HeLa and normal MRC5 myofibroblasts, respectively. B, immunoblot ofdyskerin protein in a representative experiment where SK-N-AS cells were transfected with siRNA. C, proliferation of siRNA-transfected cells. Values are mean ± SEM from three to seven independent transfections, quantified as expansion. D, SK-N-BE(2) cells were transduced with retroviral vectors that constitutively express shRNA targeting dyskerin mRNA (LMS-DKC1 shRNA), nonsilencing control shRNA (LMS-NS), or GFP only (LMS-EV). qRT-PCR was performed on GFP+ cells within 15 days of transduction. Values are mean ± SEM from at least five independent assays. **, P < 0.01; *, P < 0.05 from Bonferroni multiple comparisons test. E, anchorage-independent growth in soft-agarose measured 2 weeks after transduction. Values are mean ± SEM from four independents experiments. **, P < 0.01, Holm–Sidak multiple comparisons test. F and G, SK-N-BE(2) cells were transduced with FH1t shRNA lentiviral vectors encoding shRNA under control of a doxycycline (dox)-inducible promoter. F, qRT-PCR analysis of DKC1 expression (left axis) and cell viability determined by Trypan blue (right axis) in cells cultured with or without doxycycline. Values are the average of duplicates at each time point. G, tumor growth in BALB/c nu/nu mice engrafted with FH1t-transduced SK-N-BE(2) cells (10 mice per group). P value, two-way ANOVA.

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The impact of dyskerin expression levels on the malignant properties of neuroblastoma cells was further investigated by transducing SK-N-BE(2) cells with either an LMS-based retroviral vector encoding a miR30-based shRNA targeting DKC1, a control vector encoding nonsilencing shRNA (NS) or the empty vector (EV). DKC1 mRNA was reduced in cells transduced with LMS-DKC1 shRNA to 35% to 40% of that in control-vector transduced cells (Fig. 4D). When plated in semi-solid media, the LMS-DKC1 shRNA–transduced cells generated significantly fewer colonies than control vector–transduced cells (Fig. 4E; P < 0.01), demonstrating that high levels of dyskerin are required for anchorage-independent growth.

To test the impact of dyskerin suppression on tumor growth, DKC1 shRNA (shD3) and nonsilencing shRNAs (NS) were cloned into a doxycycline-inducible expression cassette within the Fh1t-UTG lentiviral vector. Upon addition of doxycycline to culture media, DKC1 mRNA was initially reduced by approximately 90% in shD3-transduced cells; however, there was a gradual recovery of DKC1 mRNA until expression eventually plateaued at 40% to 50% of basal levels (Fig. 4F). Tumorigenicity was then tested by subcutaneous engraftment of BALB/c nu/nu mice with transduced SK-N-BE(2). Upon establishment of a palpable tumor, mice received dietary doxycycline to suppress DKC1 expression. Over a 4-week period, tumor growth was markedly suppressed in mice implanted with shD3 cells compared with those implanted with control vector–transduced cells (Fig. 4G; P < 0.05). Indeed tumor growth did not progress after initiation of doxycycline in 2 of 8 mice transplanted with shD3 cells. In contrast, all tumors grew at a steady and rapid rate in mice transplanted with control cells.

Downregulation of dyskerin halts cell replication independently of telomerase suppression

Consistent with previous studies showing that dyskerin insufficiency destabilizes hTR and dampens telomerase activity (14), NB69 and SK-N-BE(2) cells transfected with DKC1 siRNAs had lower hTR levels and telomerase activity than control cells (Fig. 5A and B). In contrast, TERT mRNA levels were not impacted by depletion of DKC1 mRNA in either cell line (Fig. 5C). To test whether suppression of hTR and telomerase activity was necessary for the growth arrest induced by dyskerin depletion, NB69 cells were transduced with a retroviral vector expressing hTR (MND-hTR). siRNA-mediated repression of DKC1 reduced both hTR and telomerase activity in control vector (MND) and hTR-overexpressing cells (Fig. 5D); however, telomerase activity in hTR-overexpressing cells transfected with dyskerin siRNA remained substantially above that detected in control vector–transduced cells (Fig. 5D, iii). Strikingly, despite the maintenance of high telomerase activity, MND-hTR cells still underwent proliferative arrest upon siRNA-mediated repression of dyskerin (Fig. 5E). These results demonstrate that neuroblastoma cells are addicted to a telomerase-independent function of dyskerin.

Figure 5.

Proliferative arrest following repression of dyskerin does not require depletion of hTR or telomerase. siRNA-transfected NB69 and SK-N-BE(2) cells assayed for hTR by qRT-PCR (A), telomerase activity by qTRAP (B), and TERT mRNA by qRT-PCR 48 and 96 hours posttransfection (C). D and E, NB69 transduced with MND-hTR or an empty vector (MND), selected in Geneticin, and then transfected with siRNA. D, DKC1 mRNA (i) and hTR levels (ii) quantified by qRT-PCR. Telomerase activity measured by qTRAP (iii). E, proliferation expressed relative to cell number at the time of transfection. Values are mean ± SEM from three to five experiments. ****, P < 0.0001; **, P < 0.01; *, P < 0.05; two-way ANOVA with Bonferroni multiple comparison of D2 and D3 with Sc.

Figure 5.

Proliferative arrest following repression of dyskerin does not require depletion of hTR or telomerase. siRNA-transfected NB69 and SK-N-BE(2) cells assayed for hTR by qRT-PCR (A), telomerase activity by qTRAP (B), and TERT mRNA by qRT-PCR 48 and 96 hours posttransfection (C). D and E, NB69 transduced with MND-hTR or an empty vector (MND), selected in Geneticin, and then transfected with siRNA. D, DKC1 mRNA (i) and hTR levels (ii) quantified by qRT-PCR. Telomerase activity measured by qTRAP (iii). E, proliferation expressed relative to cell number at the time of transfection. Values are mean ± SEM from three to five experiments. ****, P < 0.0001; **, P < 0.01; *, P < 0.05; two-way ANOVA with Bonferroni multiple comparison of D2 and D3 with Sc.

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Downregulation of dyskerin depletes H/ACA snoRNAs and causes ribosomal stress

Aside from its interaction with hTR, dyskerin binds and stabilizes H/ACA snoRNAs that guide rRNA modification and function in the processing of rRNA precursors (15). We therefore postulated that the proliferative arrest induced by dyskerin depletion resulted from reduced availability of snoRNAs, and consequently ribosomal stress. We therefore quantified the abundance of three H/ACA snoRNAs, SNORA16, SNORA42, and SNORA75 (U23), which are involved in rRNA processing in human cancer cells, in siRNA-transfected NB69 cells (29, 30). qRT-PCR analyses confirmed that the level of each of these snoRNAs was substantially less in dyskerin-depleted cells than in control siRNA–transfected cells (Fig. 6A).

Figure 6.

Depletion of dyskerin reduces availability of H/ACA snoRNAs and induces ribosomal stress. A, relative abundance of DKC1 mRNA and H/ACA snoRNAs (SNORA16A, SNORA42, and SNORA75) in siRNA-transfected cells determined by qRT-PCR. Values are mean ± SEM from three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; Holm-Sidak multiple comparison test of D2 and D3 with Sc. B, representative images from immunofluorescence staining of rpl11 in NB69 cells 96 hours after transfection with siRNA. Treatment with 5 nmol/L actinomycin D (ActD) for 24 hours was used as a positive control (bottom). Overlay images show rpl11 in red and nuclei in blue (DAPI stained). The size bar represents 10 μm.

Figure 6.

Depletion of dyskerin reduces availability of H/ACA snoRNAs and induces ribosomal stress. A, relative abundance of DKC1 mRNA and H/ACA snoRNAs (SNORA16A, SNORA42, and SNORA75) in siRNA-transfected cells determined by qRT-PCR. Values are mean ± SEM from three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; Holm-Sidak multiple comparison test of D2 and D3 with Sc. B, representative images from immunofluorescence staining of rpl11 in NB69 cells 96 hours after transfection with siRNA. Treatment with 5 nmol/L actinomycin D (ActD) for 24 hours was used as a positive control (bottom). Overlay images show rpl11 in red and nuclei in blue (DAPI stained). The size bar represents 10 μm.

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A hallmark of disrupted ribosome biogenesis is dispersal of ribosomal proteins from nucleoli into the nucleoplasm (31). Accordingly, immunofluorescence staining showed diffuse nucleoplasmic staining of ribosomal protein rpl11 (Fig. 6B) in D2- and D3-transfected cells. This contrasted with Sc-transfected cells, in which there was very little rpl11 in the nucleoplasm and more obvious staining of rpl11 in nucleoli. Similar results were obtained from immunofluorescence staining of rpl5 (Supplementary Fig. S6). The effect of dyskerin depletion on ribosomal proteins mirrored the impact of low-dose treatment with actinomycin D (5 nmol/L), which disrupts ribosome biogenesis by inhibiting rRNA synthesis (Fig. 6B and Supplementary Fig. S6). Thus, together these results provide compelling evidence that downregulation of dyskerin-induced disrupted ribosome biogenesis in NB69 neuroblastoma cells, thus implicating ribosomal stress as a cause of proliferative arrest in dyskerin-depleted cells.

Dyskerin depletion activates p53 and G1 cell-cycle arrest in NB69 cells

During ribosomal stress, ribosomal proteins within the nucleoplasm recruit hdm2, resulting in p53 stabilization, upregulation of p21cip1, and G1 cell-cycle arrest (32). Consistent with this mechanism, we detected an accumulation of p53 (Fig. 7A, left) in NB69 cells transfected with DKC1 siRNA, coinciding with upregulation of p21cip1 mRNA (Supplementary Fig. S7A) and protein (Fig. 7A, left). In contrast with NB69 cells, there was no induction of p21cip1 protein or mRNA when dyskerin was repressed in SK-N-BE(2) cells, which express a mutant p53 protein defective in transactivation properties (Fig. 7A, right and Supplementary Fig. S7B; ref. 33). Propidium iodide staining showed there was a significant increase in the proportion of NB69 cells with sub-G1 DNA content following dyskerin repression (Fig. 7B, left graph and Supplementary Fig. S8A), consistent with a loss of viability evidenced by Trypan blue staining (Supplementary Fig. S8B, P < 0.05). Dyskerin-depleted NB69 cells accumulated in G1 phase, with a corresponding decrease in S and G2–M phase (Fig. 7C, left graph and Supplementary Fig. S8C). In contrast with NB69 cells, the viability and cell-cycle kinetics of SK-N-BE(2) cells were unaffected by dyskerin repression (Fig. 7B and C, right graphs and Supplementary Fig. S8D–S8F), despite a marked effect on cell proliferation (Fig. 4C).

Figure 7.

p53 is activated, but is not required for the proliferative impairment induced by dyskerin depletion. A, immunoblot analysis of dyskerin, p53, and p21cip1 in siRNA-transfected NB69 (left) and SK-N-BE(2) (right). M, mock-transfected cells. GAPDH was used as a loading control. The white vertical line indicates rearrangement of the image for presentation purposes. B and C, flow cytometric analysis of siRNA-transfected NB69 (left graphs) and SK-N-BE(2) (right graphs) stained with propidium iodide (B) sub-G1/G0 (dead and dying cells) and viable G1, S and G2–M phase cells (C). Bar graphs show mean ± SEM from four independent experiments. ***, P < 0.001; *, P < 0.05; Holm-Sidak multiple comparisons. D and E, NB69 transduced with retroviral vectors encoding p53 shRNA or GFP shRNA (control), then subjected to siRNA transfection. D, immunoblot analysis of transduced NB69 cells following transfection with siRNA. E, proliferation assessed using Trypan blue. Values are mean ± SEM from four independent experiments; ***, P < 0.001; **, P < 0.01, Bonferroni multiple comparisons test of D2 and D3 against Sc.

Figure 7.

p53 is activated, but is not required for the proliferative impairment induced by dyskerin depletion. A, immunoblot analysis of dyskerin, p53, and p21cip1 in siRNA-transfected NB69 (left) and SK-N-BE(2) (right). M, mock-transfected cells. GAPDH was used as a loading control. The white vertical line indicates rearrangement of the image for presentation purposes. B and C, flow cytometric analysis of siRNA-transfected NB69 (left graphs) and SK-N-BE(2) (right graphs) stained with propidium iodide (B) sub-G1/G0 (dead and dying cells) and viable G1, S and G2–M phase cells (C). Bar graphs show mean ± SEM from four independent experiments. ***, P < 0.001; *, P < 0.05; Holm-Sidak multiple comparisons. D and E, NB69 transduced with retroviral vectors encoding p53 shRNA or GFP shRNA (control), then subjected to siRNA transfection. D, immunoblot analysis of transduced NB69 cells following transfection with siRNA. E, proliferation assessed using Trypan blue. Values are mean ± SEM from four independent experiments; ***, P < 0.001; **, P < 0.01, Bonferroni multiple comparisons test of D2 and D3 against Sc.

Close modal

In light of the above results with SK-N-BE(2) cells, suggesting that p53 function was not necessary for dyskerin repression to impact on cell proliferation, we further investigated the role of p53 in the response of dyskerin-depleted NB69 cells. For these investigations, NB69 cells were transduced with BABEpuro-based retroviral vectors encoding p53 shRNA or control shRNA (GFPshRNA). Suppression of p53 and p21cip1 in the p53 shRNA-transduced cells was confirmed by immunoblotting (Fig. 7D) and cell counts showed that the p53 shRNA and control vector–transduced cells proliferated with identical kinetics. Proliferation was impaired by D2 and D3 irrespective of p53 and p21cip1 expression status (Fig. 7E). Therefore, although dyskerin depletion induced ribosomal stress and a robust upregulation of p53 and p21cip1 in p53-competent cells (Figs. 6B and 7A), p53 function was not essential for impairment of proliferation.

Past studies suggest that elevated dyskerin expression levels correlate with more aggressive disease (2, 3, 6); however, before the current investigation, there was a paucity of functional evidence supporting the significance of dyskerin expression levels in any cancer type, and no previous report of dyskerin suppression inhibiting tumor growth in vivo. The current study not only identifies DKC1 expression as a powerful independent prognostic indicator of poor clinical outcome, but also shows that high DKC1 expression has functional significance and potential as a therapeutic target in the MYC-driven malignancy neuroblastoma. We have shown for the first time that suppression of dyskerin dramatically and immediately inhibits neuroblastoma cell proliferation, impairs anchorage-independent growth, and reduces tumor progression in a xenograft model. Notably, the effect of dyskerin depletion on tumor cell proliferation was mediated by a mechanism that is independent of telomerase suppression.

Although dyskerin repression effectively lowered hTR levels and suppressed telomerase enzyme activity in neuroblastoma cell lines, several lines of evidence indicate that suppression of telomerase activity and telomere shortening was not the cause of the proliferative impairment. First, impaired proliferation was evident immediately upon dyskerin repression, and there was no correlation between the proliferative response and telomere length among the different neuroblastoma cell lines. Second, downregulation of dyskerin inhibited proliferation of the SK-N-FI cell line, which does not express the catalytic component of telomerase and exhibits ALT-like telomeres (34). Finally, the proliferative defect induced by dyskerin depletion was not rescued by overexpression of hTR and elevation of telomerase activity to levels significantly exceeding those of control cells.

The alternate mechanistic explanation for the acute proliferative arrest induced by downregulation of dyskerin is destabilization of H/ACA snoRNAs and consequential disruption of ribosome biogenesis inducing a ribosomal stress response. This conclusion is supported by our results showing reduced levels of snoRNAs (SNORA16A, SNORA42 and SNORA75) that guide modification of rRNAs (18s and 28s) and dispersal of the ribosomal proteins rpl11 and rpl5. A body of literature has shown that free rpl11 and rpl5, liberated by disruption of ribosome biogenesis, bind the p53-targeting E3 ubiquitin ligase Mdm2, resulting in robust activation of p53 (31, 35, 36). Thus, the p53-mediated G1 arrest and cell death demonstrated in NB69 neuroblastoma cells are consistent with the known effects of rpl11 and rpl5 dispersal. The consequences of dyskerin ablation on ribosomal proteins, p53, and G1 cell-cycle progression also concur with the reported effects of disrupting ribosome biogenesis by pharmacologic inhibition of rRNA transcription (37).

TP53 mutation is infrequently observed at diagnosis in neuroblastoma (38); however, TP53 mutations and defects in the p53 pathway, including inactivation of tumor suppressor p14ARF, have been reported in recurrent and drug-resistant disease (38). Our results show that dyskerin repression inhibited proliferation of cell lines with known defects in TP53 (SK-N-BE(2), SK-N-FI, SK-N-AS), and genomic loss of p14ARF (SK-N-AS), as well as NB69 cells transduced with p53 shRNA (39). Thus, in addition to showing that p53 is activated in response to dyskerin depletion, our investigations provide evidence of a secondary p53-independent mechanism that inhibits proliferation of neuroblastoma cells with defective p53 pathways. It is shown that downregulation of dyskerin in NB69 cells, which express wild-type p53 and p14ARF (39, 40), caused a G1 cell-cycle arrest and cell death. In contrast, SK-N-BE cells, which express mutant p53 (33), remained viable but exhibited dramatically impaired proliferation following downregulation of dyskerin. These results concur with a study that showed p53-independent proliferative arrest, without cell death, induced by ribosomal stress following treatment of cancer cell lines with an inhibitor of rRNA synthesis (41).

Contrary to the conclusions of the current study, it was previously proposed that dyskerin functions as a tumor suppressor, a hypothesis drawn from studies that showed a hypomorphic DKC1 mutation impaired IRES-mediated translation of tumor suppressors p27kip1 and p53 (8, 9, 42). However, consistent with the current findings, an independent study demonstrated that an alternate Dkc1 mutation in mice induced a proliferative defect associated with p53 activation (43). One possible explanation for these apparently conflicting findings is that the distinct DKC1 mutations modeled in mice were functionally different. The current study, which used dyskerin knockdown rather than gene mutation, is uncomplicated by potential gain-of-function effects of DKC1 mutations, and unequivocally shows that downregulation of dyskerin halts cell replication and induces a robust p53 response. These effects would not be expected if dyskerin functioned as a tumor suppressor, but rather are consistent with addiction to a gene upregulated during the transformation process. Studies that found p53 was not induced in U2OS and MCF-7 cell lines subject to siRNA-mediated downregulation of dyskerin may be explained by the defects in p14ARF known to be harbored by those cells (9, 44–46). This possibility is supported by a recent investigation that showed p14ARF bolsters p53 activation during ribosomal stress (47). It will be of interest for future studies to further characterize the mechanism of proliferative arrest induced by ribosomal stress in cancer cells with defects in the p53/p14ARF pathway.

In the context of previous investigations of dyskerin suppression, our data suggest that neuroblastoma cells may be particularly sensitive to the depletion of dyskerin. Contrasting with the robust effect on neuroblastoma cell proliferation demonstrated herein, previous investigations have reported no apparent effect of dyskerin depletion on proliferation of UM-SCC1 squamous cell carcinoma or HeLa cervical carcinoma cells (22), a moderate effect on the proliferation of U2OS osteosarcoma and MCF-7 breast cancer cells (9, 44) and a varied response in two different prostate cancer cell lines (3). Here, we have shown that repression of dyskerin impaired the proliferation of several neuroblastoma cell lines that represent the molecular heterogeneity of the disease, including cells with or without MYCN amplification, TP53 mutation, p14ARF deletion, and telomerase enzyme activity. Among the neuroblastoma cell lines tested, the one cell line that consistently did not respond to robust dyskerin mRNA suppression was the Kelly cell line (data not shown). Kelly cells feature extremely high N-Myc expression due to extraordinary amplification of the MYCN locus (>100 copies), as well as an activating mutation in the oncogenic tyrosine kinase anaplastic lymphoma kinase (ALK; ref. 48). The molecular basis for the apparent resistance of the Kelly cell line to dyskerin depletion warrants future investigation.

The capacity for dyskerin ablation to arrest proliferation of neuroblastoma cells with defective p53, loss of p14ARF expression, or amplified MYCN highlights the potential for therapeutic effect in neuroblastoma cases that are the most difficult to treat. Toward the therapeutic exploitation of dyskerin, further studies are needed to explore the impact of dyskerin depletion on various types of normal cells. Although inactivation of the DKC1 locus is embryonic lethal in mice (49), the pattern of inheritance of DKC1 mutations observed in individuals with dyskeratosis congenita indicate that a partial reduction in dyskerin in normal cells is compatible with normal cell replication and development, at least in the short to medium term. Consistent with that notion, fibroblasts from patients with DKC1 mutations were shown to proliferate at a similar rate to normal fibroblasts, even though long-term cell replication was limited by eventual critical telomere shortening (50).

Collectively, the results from these investigations demonstrate that neuroblastoma cells are addicted to a telomerase-independent pathway that is activated by N-Myc and c-Myc–induced upregulation of dyskerin. This study reveals dyskerin as an exciting new therapeutic target that is not dependent upon p53 function or telomerase, with potential for exploitation in the broad spectrum of pediatric and adult cancers that are driven by MYC oncogenes.

No potential conflicts of interest were disclosed.

Conception and design: R. O'Brien, S.L. Tran, J.I. Fletcher, K.L. MacKenzie

Development of methodology: R. O'Brien, S.L. Tran, M.F. Maritz, S. Purgato, G. von Jonquieres, P.H. Gunarantne, G. Perrini

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. O'Brien, S.L. Tran, M.F. Maritz, C.F. Kong, C. Yang, J. Murray, A.J. Russell, C.L. Flemming, G. von Jonquieres, H.A. Pickett, W.B. London, M. Haber

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. O'Brien, S.L. Tran, M.F Maritz, B. Liu, S. Purgato, C. Yang, A.J. Russell, G. von Jonquieres, M. Haber, G. Perrini, J.I. Fletcher, K.L. MacKenzie

Writing, review, and/or revision of the manuscript: R. O'Brien, S.L. Tran, J. Murray, C.L. Flemming, M. Haber, M.D. Norris, J.I. Fletcher, K.L. MacKenzie

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): R. O'Brien, S.L. Tran, M.F. Maritz, B. Liu, W.B. London

Study supervision: K.L. MacKenzie

The authors thank Elysse McIlwain and Paul Young for technical assistance.

This work was supported by funds from the Cancer Council New South Wales (Project Grants RG 11-01 and RG 15-16), National Health and Medical Research Council (Career Development Fellowship 510378), Cancer Institute New South Wales (Career Development Fellowship 09CDF217), and the Italian Association for Cancer Research (AIRC).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Heiss
NS
,
Knight
SW
,
Vulliamy
TJ
,
Klauck
SM
,
Wiemann
S
,
Mason
PJ
, et al
X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions
.
Nat Genet
1998
;
19
:
32
8
.
2.
Liu
B
,
Zhang
J
,
Huang
C
,
Liu
H
. 
Dyskerin overexpression in human hepatocellular carcinoma is associated with advanced clinical stage and poor patient prognosis
.
PLoS ONE
2012
;
7
:
e43147
.
3.
Sieron
P
,
Hader
C
,
Hatina
J
,
Engers
R
,
Wlazlinski
A
,
Muller
M
, et al
DKC1 overexpression associated with prostate cancer progression
.
Br J Cancer
2009
;
101
:
1410
6
.
4.
Alawi
F
,
Lee
MN
. 
DKC1 is a direct and conserved transcriptional target of c-MYC
.
Biochem Biophys Res Commun
2007
;
362
:
893
8
.
5.
Westermann
F
,
Muth
D
,
Benner
A
,
Bauer
T
,
Henrich
KO
,
Oberthuer
A
, et al
Distinct transcriptional MYCN/c-MYC activities are associated with spontaneous regression or malignant progression in neuroblastomas
.
Genome Biol
2008
;
9
:
R150
.
6.
von Stedingk
K
,
Koster
J
,
Piqueras
M
,
Noguera
R
,
Navarro
S
,
Pahlman
S
, et al
snoRNPs regulate telomerase activity in neuroblastoma and are associated with poor prognosis
.
Transl Oncol
2013
;
6
:
447
57
.
7.
Chen
QR
,
Song
YK
,
Yu
LR
,
Wei
JS
,
Chung
JY
,
Hewitt
SM
, et al
Global genomic and proteomic analysis identifies biological pathways related to high-risk neuroblastoma
.
J Proteome Res
2010
;
9
:
373
82
.
8.
Bellodi
C
,
Krasnykh
O
,
Haynes
N
,
Theodoropoulou
M
,
Peng
G
,
Montanaro
L
, et al
Loss of function of the tumor suppressor DKC1 perturbs p27 translation control and contributes to pituitary tumorigenesis
.
Cancer Res
2010
;
70
:
6026
35
.
9.
Montanaro
L
,
Calienni
M
,
Bertoni
S
,
Rocchi
L
,
Sansone
P
,
Storci
G
, et al
Novel dyskerin-mediated mechanism of p53 inactivation through defective mRNA translation
.
Cancer Res
2010
;
70
:
4767
77
.
10.
Brodeur
GM
,
Seeger
RC
,
Schwab
M
,
Varmus
HE
,
Bishop
JM
. 
Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage
.
Science
1984
;
224
:
1121
4
.
11.
Sadee
W
,
Yu
VC
,
Richards
ML
,
Preis
PN
,
Schwab
MR
,
Brodsky
FM
, et al
Expression of neurotransmitter receptors and myc protooncogenes in subclones of a human neuroblastoma cell line
.
Cancer Res
1987
;
47
:
5207
12
.
12.
Maris
JM
. 
Recent advances in neuroblastoma
.
N Engl J Med
2010
;
362
:
2202
11
.
13.
Watkins
NJ
,
Gottschalk
A
,
Neubauer
G
,
Kastner
B
,
Fabrizio
P
,
Mann
M
, et al
Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure
.
RNA
1998
;
4
:
1549
68
.
14.
Mitchell
JR
,
Wood
E
,
Collins
K
. 
A telomerase component is defective in the human disease dyskeratosis congenita
.
Nature
1999
;
402
:
551
5
.
15.
Yu
YT
,
Meier
UT
. 
RNA-guided isomerization of uridine to pseudouridine–pseudouridylation
.
RNA Biol
2014
;
11
:
1483
94
.
16.
Hanahan
D
,
Weinberg
RA
. 
Hallmarks of cancer: the next generation
.
Cell
2011
;
144
:
646
74
.
17.
Hiyama
E
,
Hiyama
K
,
Yokoyama
T
,
Matsuura
Y
,
Piatyszek
MA
,
Shay
JW
. 
Correlating telomerase activity levels with human neuroblastoma outcomes
.
Nat Med
1995
;
1
:
249
55
.
18.
Poremba
C
,
Scheel
C
,
Hero
B
,
Christiansen
H
,
Schaefer
KL
,
Nakayama
J
, et al
Telomerase activity and telomerase subunits gene expression patterns in neuroblastoma: a molecular and immunohistochemical study establishing prognostic tools for fresh-frozen and paraffin-embedded tissues
.
J Clin Oncol
2000
;
18
:
2582
92
.
19.
Ruggero
D
,
Grisendi
S
,
Piazza
F
,
Rego
E
,
Mari
F
,
Rao
PH
, et al
Dyskeratosis congenita and cancer in mice deficient in ribosomal RNA modification
.
Science
2003
;
299
:
259
62
.
20.
Ge
J
,
Rudnick
DA
,
He
J
,
Crimmins
DL
,
Ladenson
JH
,
Bessler
M
, et al
Dyskerin ablation in mouse liver inhibits rRNA processing and cell division
.
Mol Cell Biol
2010
;
30
:
413
22
.
21.
Ender
C
,
Krek
A
,
Friedlander
MR
,
Beitzinger
M
,
Weinmann
L
,
Chen
W
, et al
A human snoRNA with microRNA-like functions
.
Mol Cell
2008
;
32
:
519
28
.
22.
Alawi
F
,
Lin
P
. 
Loss of dyskerin reduces the accumulation of a subset of H/ACA snoRNA-derived miRNA
.
Cell Cycle
2010
;
9
:
2467
9
.
23.
Taylor
LM
,
James
A
,
Schuller
CE
,
Brce
J
,
Lock
RB
,
Mackenzie
KL
. 
Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization
.
J Biol Chem
2004
;
279
:
43634
45
.
24.
Iraci
N
,
Diolaiti
D
,
Papa
A
,
Porro
A
,
Valli
E
,
Gherardi
S
, et al
A SP1/MIZ1/MYCN repression complex recruits HDAC1 at the TRKA and p75NTR promoters and affects neuroblastoma malignancy by inhibiting the cell response to NGF
.
Cancer Res
2011
;
71
:
404
12
.
25.
MacKenzie
KL
,
Franco
S
,
Naiyer
AJ
,
May
C
,
Sadelain
M
,
Rafii
S
, et al
Multiple stages of malignant transformation of human endothelial cells modelled by co-expression of telomerase reverse transcriptase, SV40 T antigen and oncogenic N-ras
.
Oncogene
2002
;
21
:
4200
11
.
26.
Bryan
TM
,
Englezou
A
,
Gupta
J
,
Bacchetti
S
,
Reddel
RR
. 
Telomere elongation in immortal human cells without detectable telomerase activity
.
EMBO J
1995
;
14
:
4240
8
.
27.
Lutz
W
,
Stohr
M
,
Schurmann
J
,
Wenzel
A
,
Lohr
A
,
Schwab
M
. 
Conditional expression of N-myc in human neuroblastoma cells increases expression of alpha-prothymosin and ornithine decarboxylase and accelerates progression into S-phase early after mitogenic stimulation of quiescent cells
.
Oncogene
1996
;
13
:
803
12
.
28.
Kocak
H
,
Ackermann
S
,
Hero
B
,
Kahlert
Y
,
Oberthuer
A
,
Juraeva
D
, et al
Hox-C9 activates the intrinsic pathway of apoptosis and is associated with spontaneous regression in neuroblastoma
.
Cell Death Dis
2013
;
4
:
e586
.
29.
Yoshihama
M
,
Nakao
A
,
Kenmochi
N
. 
snOPY: a small nucleolar RNA orthological gene database
.
BMC Res Notes
2013
;
6
:
426
.
30.
Kiss
AM
,
Jady
BE
,
Bertrand
E
,
Kiss
T
. 
Human box H/ACA pseudouridylation guide RNA machinery
.
Mol Cell Biol
2004
;
24
:
5797
807
.
31.
Golomb
L
,
Volarevic
S
,
Oren
M
. 
p53 and ribosome biogenesis stress: the essentials
.
FEBS Lett
2014
;
588
:
2571
9
.
32.
Pestov
DG
,
Strezoska
Z
,
Lau
LF
. 
Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition
.
Mol Cell Biol
2001
;
21
:
4246
55
.
33.
Goldschneider
D
,
Horvilleur
E
,
Plassa
LF
,
Guillaud-Bataille
M
,
Million
K
,
Wittmer-Dupret
E
, et al
Expression of C-terminal deleted p53 isoforms in neuroblastoma
.
Nucleic Acids Res
2006
;
34
:
5603
12
.
34.
Farooqi
AS
,
Dagg
RA
,
Choi
LM
,
Shay
JW
,
Reynolds
CP
,
Lau
LM
. 
Alternative lengthening of telomeres in neuroblastoma cell lines is associated with a lack of MYCN genomic amplification and with p53 pathway aberrations
.
J Neurooncol
2014
;
119
:
17
26
.
35.
Dai
MS
,
Lu
H
. 
Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5
.
J Biol Chem
2004
;
279
:
44475
82
.
36.
Lohrum
MA
,
Ludwig
RL
,
Kubbutat
MH
,
Hanlon
M
,
Vousden
KH
. 
Regulation of HDM2 activity by the ribosomal protein L11
.
Cancer Cell
2003
;
3
:
577
87
.
37.
Bywater
MJ
,
Poortinga
G
,
Sanij
E
,
Hein
N
,
Peck
A
,
Cullinane
C
, et al
Inhibition of RNA polymerase I as a therapeutic strategy to promote cancer-specific activation of p53
.
Cancer Cell
2012
;
22
:
51
65
.
38.
Tweddle
DA
,
Malcolm
AJ
,
Bown
N
,
Pearson
AD
,
Lunec
J
. 
Evidence for the development of p53 mutations after cytotoxic therapy in a neuroblastoma cell line
.
Cancer Res
2001
;
61
:
8
13
.
39.
Dreidax
D
,
Gogolin
S
,
Schroeder
C
,
Muth
D
,
Brueckner
LM
,
Hess
EM
, et al
Low p14ARF expression in neuroblastoma cells is associated with repressed histone mark status, and enforced expression induces growth arrest and apoptosis
.
Hum Mol Genet
2013
;
22
:
1735
45
.
40.
Carr
J
,
Bell
E
,
Pearson
AD
,
Kees
UR
,
Beris
H
,
Lunec
J
, et al
Increased frequency of aberrations in the p53/MDM2/p14(ARF) pathway in neuroblastoma cell lines established at relapse
.
Cancer Res
2006
;
66
:
2138
45
.
41.
Drygin
D
,
Lin
A
,
Bliesath
J
,
Ho
CB
,
O'Brien
SE
,
Proffitt
C
, et al
Targeting RNA polymerase I with an oral small molecule CX-5461 inhibits ribosomal RNA synthesis and solid tumor growth
.
Cancer Res
2011
;
71
:
1418
30
.
42.
Yoon
A
,
Peng
G
,
Brandenburger
Y
,
Zollo
O
,
Xu
W
,
Rego
E
, et al
Impaired control of IRES-mediated translation in X-linked dyskeratosis congenita
.
Science
2006
;
312
:
902
6
.
43.
Gu
BW
,
Bessler
M
,
Mason
PJ
. 
A pathogenic dyskerin mutation impairs proliferation and activates a DNA damage response independent of telomere length in mice
.
Proc Natl Acad Sci U S A
2008
;
105
:
10173
8
.
44.
Alawi
F
,
Lin
P
. 
Dyskerin is required for tumor cell growth through mechanisms that are independent of its role in telomerase and only partially related to its function in precursor rRNA processing
.
Mol Carcinog
2011
;
50
:
334
45
.
45.
Park
YB
,
Park
MJ
,
Kimura
K
,
Shimizu
K
,
Lee
SH
,
Yokota
J
. 
Alterations in the INK4a/ARF locus and their effects on the growth of human osteosarcoma cell lines
.
Cancer Genet Cytogenet
2002
;
133
:
105
11
.
46.
Bisogna
M
,
Calvano
JE
,
Ho
GH
,
Orlow
I
,
Cordon-Cardo
C
,
Borgen
PI
, et al
Molecular analysis of the INK4A and INK4B gene loci in human breast cancer cell lines and primary carcinomas
.
Cancer Genet Cytogenet
2001
;
125
:
131
8
.
47.
Dai
MS
,
Challagundla
KB
,
Sun
XX
,
Palam
LR
,
Zeng
SX
,
Wek
RC
, et al
Physical and functional interaction between ribosomal protein L11 and the tumor suppressor ARF
.
J Biol Chem
2012
;
287
:
17120
9
.
48.
Mosse
YP
,
Laudenslager
M
,
Longo
L
,
Cole
KA
,
Wood
A
,
Attiyeh
EF
, et al
Identification of ALK as a major familial neuroblastoma predisposition gene
.
Nature
2008
;
455
:
930
5
.
49.
He
J
,
Navarrete
S
,
Jasinski
M
,
Vulliamy
T
,
Dokal
I
,
Bessler
M
, et al
Targeted disruption of Dkc1, the gene mutated in X-linked dyskeratosis congenita, causes embryonic lethality in mice
.
Oncogene
2002
;
21
:
7740
4
.
50.
Westin
ER
,
Chavez
E
,
Lee
KM
,
Gourronc
FA
,
Riley
S
,
Lansdorp
PM
, et al
Telomere restoration and extension of proliferative lifespan in dyskeratosis congenita fibroblasts
.
Aging Cell
2007
;
6
:
383
94
.