ZNF281 inhibits neuronal differentiation and is a prognostic marker for neuroblastoma

Abstract

Derangement of cellular differentiation because of mutation or inappropriate expression of specific genes is a common feature in tumors. Here, we show that the expression of ZNF281, a zinc finger factor involved in several cellular processes, decreases during terminal differentiation of murine cortical neurons and in retinoic acid-induced differentiation of neuroblastoma (NB) cells. The ectopic expression of ZNF281 inhibits the neuronal differentiation of murine cortical neurons and NB cells, whereas its silencing causes the opposite effect. Furthermore, TAp73 inhibits the expression of ZNF281 through miR34a. Conversely, MYCN promotes the expression of ZNF281 at least in part by inhibiting miR34a. These findings imply a functional network that includes p73, MYCN, and ZNF281 in NB cells, where ZNF281 acts by negatively affecting neuronal differentiation. Array analysis of NB cells silenced for ZNF281 expression identified GDNF and NRP2 as two transcriptional targets inhibited by ZNF281. Binding of ZNF281 to the promoters of these genes suggests a direct mechanism of repression. Bioinformatic analysis of NB datasets indicates that ZNF281 expression is higher in aggressive, undifferentiated stage 4 than in localized stage 1 tumors supporting a central role of ZNF281 in affecting the differentiation of NB. Furthermore, patients with NB with high expression of ZNF281 have a poor clinical outcome compared with low-expressors. These observations suggest that ZNF281 is a controller of neuronal differentiation that should be evaluated as a prognostic marker in NB.

Keywords: MYCN; ZNF281; miR34a; neuroblastoma; p73.

Fig. 1.

ZNF281 expression during neuronal differentiation and in NB cell lines. (A) Immature murine cortical neurons were cultured in vitro, as described in Materials and Methods. Western blot (WB) analysis was carried out with antibodies to ZNF281 and Synapsin II (Syn II). Equal loading was checked by GAPDH detection. DIV, days in vitro. Numbers below the corresponding blot represent densitometric analysis normalized to the housekeeping gene and relative to DIV1. (B) Heat map (Upper) and box plot (Lower) analyses of ZNF281 expression during normal brain development. mos, months; pcw, postcoitum weeks; yrs, years. Data were retrieved from the R2 dataset (r2.amc.nl) Normal Brain Development (524 samples)–BrainSpan atlas of the developing human brain. Statistical significance of the difference between ZNF281 was calculated by unpaired two-tailed t test. (C and D) Reverse transcriptase real-time PCR (qPCR) (C) and Western blot (D) analyses of ZNF281 expression in human NB cell lines. qPCR carried out was repeated twice in triplicate. Western blot analysis was carried out with antibodies to ZNF281, c-Myc, and MYCN. β-actin was used for normalization. (E) WB analyses of ZNF281 expression during neuronal differentiation of human NB cell lines induced by ATRA 10 μM for the indicated times. Antibodies used were as in D. Blots are representative of two to four biological replicates. Numbers below the corresponding blot in D and E represent densitometric analysis normalized to the housekeeping gene. (F) ZNF281 negatively affects dendritic outgrowth of cortical neurons. DIV 1 murine cortical neurons were transfected with expression vectors encoding ZNF281 and a GFP-Spectrin fusion protein (pGFP-Spectrin) at a 15:1 ratio. After 48 h, neurons were analyzed by fluorescence microscopy. Total neurite length and quantification of branch number after transfection of ZNF281 or empty vector (EV) were performed as described in Methods. In each experiment, 10–11 cells were analyzed. Data represent mean ± SD of two different experiments (*P < 0.05, unpaired two-tailed Student’s t test). (Right) Representative image. (Scale bar, 25 μm.) (G) Quantification of branch number in DIV 3 murine cortical neurons was carried out the day after transfection. In each experiment, 15–18 cells were analyzed. Data represent mean ± SD of two different experiments (*P < 0.05, unpaired two-tailed Student’s t test). (Right) Representative image. (Scale bar: 25 μm.) (H) Neurite length of human NB cell lines transfected with a pool of siRNA against ZNF281 (siZNF281) or with scr was calculated by counting at least 60 cells. (I) WB analysis of ZNF281 and NEFM expression in the same cell lines transfected with siZNF281 or with scr. Numbers below the corresponding blot represent densitometric analysis performed by taking the expression of scr-transfected cells or controls as 1. (J) Neurite length of LAN-5 cells infected with the lentiviral vector pLVTHM-sh-scrambled, pLVTHM-sh281-I, and pLVTHM-sh281-II. Two independent clones infected with sh-ZNF281 (sh-281-I and sh-281-II) and a mixed population infected with sh-scrambled (sh-ctrl) were analyzed. Neurite counting was carried out as in H. (K) WB analysis of ZNF281 and NEFM expression in parental, sh-ctrl, sh-281-I and sh-281-II LAN-5 cells. Numbers below the corresponding blot represent densitometric analysis normalized to the housekeeping gene and relative to sh-ctrl cells. (L) Neurite length of LAN-5 cells treated with ATRA at the indicated concentrations and transfected with an expression vector for human ZNF281 was calculated by counting as in H. Differences in neurite length were evaluated for statistical significance taking the neurite length of the untreated cells as 1. Asterisks in H, J, and L indicate statistical significance (*P < 0.05; **P < 0.01) of the difference between scr and siZNF281 calculated with unpaired two-tailed Student’s t test. (M) WB analysis of LAN-5 cells treated with ATRA and transfected with an expression vector for ZNF281. Numbers below the corresponding blot represent densitometric analysis normalized to the housekeeping gene and relative to EV ctrl cells. Blots are representative of two to four biological replicates.

Fig. 2.

The expression of ZNF281 is controlled by p73 and MYCN. (A) WB analysis of NB cell lines transfected with an expression vector encoding TAp73β-HA. Numbers below the corresponding blot represent densitometric analysis normalized to the housekeeping gene and relative to EV. (B) qPCR to measure the expression of miR34a. Analysis was performed twice in triplicate. Error bars represent SD. Asterisks indicate statistical significance (**P < 0.01) of the difference between EV and TAp73β-HA-transfected cells calculated with unpaired two-tailed Student’s t test. (C) WB analysis of the indicated NB cell lines transfected with premiR34a. Numbers below the corresponding blot represent densitometric analysis normalized to the housekeeping gene. (D) WB analysis of H1299 cells transfected with a backbone-modified anti miR34a or with anti-nc control and subsequently retransfected with an expression vector encoding for TAp73β-HA or with the EV. Numbers below the corresponding blot represent densitometric analysis normalized to the housekeeping gene and relative to the EV. (E) WB analysis of the indicated human NB cell lines transfected with a pool of anti-MYCN siRNA or with scrambled siRNAs control (src) for 48 and 96 h. Numbers below the corresponding blot represent densitometric analysis normalized to the housekeeping gene. (F) qPCR to measure the expression of miR34a in the cells of E. Asterisks indicate statistical significance (*P < 0.05; **P < 0.01) of the difference between scr (at 48 h) and other samples calculated with unpaired two-tailed Student’s t test. Error bars represent SD of triplicate biological replicates.

Fig. 3.

Array analysis of genes differentially expressed in BE2(M17) cells silenced for ZNF281 expression: ZNF281 binds to the promoters of GDNF and NRP2. (A) Gene ontology grouping of differentially expressed genes. The expression of ZNF281 was analyzed by Western blot in BE2(M17) cells silenced for ZNF281 compared with scr controls before microarray analysis. FDR, false discovery rate. (B) WB representative of four biological replicates used for the microarray analysis. Numbers below the blot represent densitometric analysis normalized to the housekeeping gene. (C) Validation analysis: the expression of ZNF281, GDNF, and NRP2 was analyzed by qPCR. Expression of ZNF281 was also analyzed in BE2(M17) cells silenced for ZNF281 by WB analysis (a representative result is shown). Graphs show the mean of 4 independent experiments (*P < 0.05; **P < 0.01). (D) The expression of GDNF and NRP2 was analyzed by qPCR in BE2(M17) cells treated with 10 μM ATRA for 6 d (*P < 0.05; **P < 0.01). (E) Scheme of the promoter regions of GDNF, NRP2, and Axin2 (positive control) and chromosome 16q22 region (negative control). TSS, transcription start site; arrows indicate the position of the primers used for the ChIP analysis; solid boxes represent GC-rich regions. (F) ChIP results analyzed by semiquantitative PCR. Inp, input; IgG immuno-precipitation carried out with IgG. (G) ChIP analyzed by real-time qPCR; results are expressed as fold enrichment respect to IgG (**P < 0.01). ChIP analyses were repeated twice with similar results. Error bars represent SD of triplicate biological replicates. Statistical analysis was performed using two-tailed Student’s t test. (H) Scheme of the controls operating on ZNF281 expression. Arrow-headed and bar-headed lines indicate activation and inhibition respectively.

Fig. 4.

Expression of ZNF281 as a prognostic marker in NB datasets. (A) Expression of ZNF281 in NB patients at stage 1 versus stage 4. All data used for the analysis are from the GSE 45547 Tumor Neuroblastoma–Kocak dataset (649 patients) in R2 genomics analysis and visualization platform (r2.amc.nl), and from the GSE49710 NB dataset (260 patients). Boxes indicate the median (horizontal line); whiskers indicate distances from the highest and lowest value to each end of the box that are within 1.5× box length; outliers are represented as dots. Mean values were compared with the two-tailed unpaired t test. (B) Kaplan-Meier (KM) overall and event-free survival analysis of the total Kocak cohort. (C) KM analyses in the MYCN nonamp and in the MYCN-amp subcohorts according to ZNF281 expression. The R2 system gives the cutoff value of ZNF281 expression levels. The difference between the curves for ZNF281 high and ZNF281 low groups are compared by χ² test.