TRIM16 acts as a tumour suppressor by inhibitory effects on cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells

Abstract

The family of tripartite-motif (TRIM) proteins are involved in diverse cellular processes, but are often characterized by critical protein-protein interactions necessary for their function. TRIM16 is induced in different cancer types, when the cancer cell is forced to proceed down a differentiation pathway. We have identified TRIM16 as a DNA-binding protein with histone acetylase activity, which is required for the retinoic acid receptor β(2) transcriptional response in retinoid-treated cancer cells. In this study, we show that overexpressed TRIM16 reduced neuroblastoma cell growth, enhanced retinoid-induced differentiation and reduced tumourigenicity in vivo. TRIM16 was only expressed in the differentiated ganglion cell component of primary human neuroblastoma tumour tissues. TRIM16 bound directly to cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. TRIM16 reduced cell motility and this required downregulation of vimentin. Retinoid treatment and enforced overexpression caused TRIM16 to translocate to the nucleus, and bind to and downregulate nuclear E2F1, required for cell replication. This study, for the first time, demonstrates that TRIM16 acts as a tumour suppressor, affecting neuritic differentiation, cell migration and replication through interactions with cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells.

Figure 1

TRIM16 reduces neuroblastoma cell viability, clonogenicity and tumourigenicity. (a) The cells were stained with an antibody recognizing the Myc-tag epitope to confirm the TRIM16 plasmid transfection by immunofluorescence microscopy and immunoblot examination. (b) The viable cells were measured up to day 4 by Alamar Blue assay in BE(2)-C cells stably transfected with empty vector or with TRIM16 plasmid DNA. A statistically significant difference (**P<0.01 and ***P<0.001) compared with the empty vector control. (c) In the top panel, the clonogenicity of various BE(2)-C cell transfectants was determined using soft agar. In the bottom panel, number of colonies were compared between empty vector control (EV-1) and TRIM16 plasmid transfected BE(2)-C cell. (d) A colony-forming assay showed reduced colony size in clones overexpressing TRIM16. A statistically significant difference (P<0.0001) compared with empty vector control (EV-1). (e) The tumour volume of TRIM16 or empty vector transfected BE(2)-C cells injected into nude mice for 4 weeks. Each value shown in the figure represented data from 16 mice. A statistically significant difference (P<0.01) compared with empty vector control (EV-1). (f) In the left panel, western blotting analysis by TRIM16-specific antibody at 24, 48 and 72 h of BE(2)-C cells without or with TRIM16 siRNA transfection. Actin was used as an internal control for loading. In the right panel: the viable cells were measured up to 72 h by Alamar Blue assay in BE(2)-C cells transfected with control siRNA or TRIM16 specific siRNA.

Figure 2

TRIM16 induces neuronal differentiation in neuroblastoma cells. (a, b) Up to 6 days of 10 μ all-trans-retinoic acid (aRA) treatment or ethanol control, cell differentiation was assessed by analysing neurite outgrowth under phase contrast microscopy. Cell images were captured and stored, and neurite outgrowth was quantified. Error bars indicate s.e. A statistically significant difference (**P<0.001) compared with empty vector control. (c) Immunohistochemical staining for TRIM16 expression in a primary human neuroblastoma tumour (stage I). In the tumour tissue, endogenous TRIM16 was expressed (brown staining) higher in large mature ganglion cells and less in the neuroblasts (small blue round cells). (d) The percentage of TRIM16-positive cells were compared between differentiated regions and poorly differentiated regions in the tumour tissues from 21 neuroblastoma patients. (e) A formalin-fixed tissue section from the same patient of Figure 2c was subjected to immunohistochemical assessment of class III β-tubulin protein expression (brown staining). (f) Immunohistochemical staining for expression of TRIM16 (brown staining) in MYCN mice, using an anti-TRIM16 antibody (scale bar: 100 μm).

Figure 3

TRIM16 binds to and modulates vimentin protein expression in both neuroblastoma and lung cancer cells. (a) Interaction of TRIM16 with vimentin. Lysates of BE(2)-C and SK-MES-1 cells transfected with TRIM16 plasmids were analysed by immunoblotting using anti-vimentin and anti-Myc-tag antibodies (western blots: vimentin and TRIM16 proteins), combined without (input) or with immunoprecipitation using anti-TRIM16 antibody or anti-IgG antibody control. (b) TRIM16 overexpression downregulates exogenous vimentin. Lysates of transfected BE(2)-C and SK-MES-1 cells were analysed by anti-TRIM16 (top panel) or anti-vimentin (middle panel) or anti-actin as loading control (bottom panel). (c) BE(2)-C and SK-MES-1 cells were transfected with either control siRNA or siRNA specifically targeting TRIM16 for 72 h, followed by protein extraction and western blot. (d) Immunocytochemical expression patterns vimentin (orange/red fluorescence) and TRIM16 (green fluorescence) in BE(2)-C cells colocalized in the cytoplasm (visualized as yellow fluorescence in merge panel).

Figure 4

Downregulated vimentin is required for TRIM16 effects on cell motility. (a) Lysates of BE(2)-C cells transfected with TRIM16 plasmids were analysed by immunoblotting and an immunofluorescence assay using anti-Myc antibody. (b) Relative closure of BE(2)-C wounds. The average distance moved by the empty vector control and the average (and s.e.) movement of TRIM16 plasmid DNA transfected cells in three independent wounds is shown relative to the percentage of original wound. Three locations of three independent empty vector control and TRIM16 plasmid DNA transfected wounds were photographed at 8, 24 and 48 h post wound. (c) Representative phase contrast micrographs of closure of scratch-wounded confluent cultures of empty vector or TRIM16 plasmid DNA transfected BE(2)-C cells at time point immediately after wounding and 24 and 48 h post wounding.

Figure 5

Downregulated vimentin is required for TRIM 16 reduction of cell motility and cell migration. (a) Lysates of BE(2)-C cells transfected with indicated plasmids (lane 1: pcDNA3.1 and pCMV-6 empty vectors; lane 2: vimentin; lane 3: TRIM16; and lane 4: TRIM16/vimentin) were analysed by immunoblotting using anti-His and anti-Flag antibodies. (b) The average distance migrated by the two empty vector controls (lane 1), TRIM16 (lane 2), vimentin (lane 3) and TRIM16/vimentin (lane 4) plasmid DNA-transfected cells in three independent wounds is shown relative to the percentage of original wound. (c) BE(2) cells were transfected with different plasmid DNA for 72 h, followed by incubation with 5-bromo-2-deoxyuridine (BrdU) for the last 6 h. BrdU incorporation was measured as OD units of absorbance. (d) Invasion assay of BE(2)-C cells through collagen-coated cell culture inserts. The percentage of the migrated cells divided by the total number of cells in the wells from three independent experiments is shown.

Figure 6

TRIM16 translocates to the nucleus and decreases E2F1 levels in BE(2)-C cells. (a) The nuclear protein extract from BE(2)-C cells transfected with TRIM16 plasmids was incubated with either a mouse IgG (right lane) or an anti-Myc antibody (middle lane). The purified IP complex was analysed by western blot using antibodies for TRIM16 and E2F1 proteins. (b, c) Immunoblotting analysis of expression of TRIM16 and E2F1 in BE(2)-C cells. Cytoplasmic (CP) and nuclear (NP) proteins of cells treated with 10 μ all-trans-retinoic acid (aRA) for 24 and 48 h (Figure 6b) or transfected with TRIM16 plasmid DNA for 24 and 48 h (Figure 6c) were analysed by immunoblotting using anti-TRIM16 and anti-E2F1 antibodies. Anti-actin antibody is used as loading control, anti-histone H3 is used as control for nuclear protein and anti-GAPDH antibody is used as cytoplasmic protein control. (d, e) BE(2)-C cells were transfected with control siRNA or siRNA specifically targeting E2F1 for 72 h, followed by real-time PCR and 5-bromo-2-deoxyuridine (BrdU) cell proliferation assay. A statistically significant difference (***P<0.001) compared with control siRNA. (f) BE(2) cells were transfected with different plasmid DNA for 72 h, followed by incubation with BrdU for the last 6 h. BrdU incorporation was measured as OD units of absorbance.

Figure 7

Identification of the interaction domains of TRIM16 and E2F1. (a) Interaction of individual TRIM16 domains with E2F1: Flag-tagged E2F1 and individual domains of TRIM16 were coexpressed in BE(2)-C cells. Flag- and GFP-tagged immunoprecipitates (IPs) were analysed for the presence of Flag- and GFP-tagged constructs (co-IPs) by western blots. Transfection of E2F1 together with the empty vector was used as a control. Non-specific bands are labelled by asterisks. (b) TRIM16 downregulates the protein E2F1 level by reducing the protein's half-life: BE(2)-C cells were transfected either with E2F1 or TRIM16, as well as E2F1 and TRIM16 mutants where indicated. After 16 h the transfected cells were trypsinized, pooled and then replated. After 24 h, cycloheximide was added at a final concentration of 25 μg/ml to the media. At the specified time points the cells were harvested and the protein extracted for analysis by western blots. The western blots were probed with polyclonal rabbit anti-E2F1 or anti-actin antibodies. Actin was used a protein loading control.