 of novel stress response genes exclusively modulated under MYCN amplified conditions. These include genes controlling lipid metabolism, DNA damage, and oncogenic signaling. Most of these genes correlate with significantly worse overall survival in neuroblastoma patient cohorts. Together these data are consistent with a model where MYCN acts as a co-factor with p53, altering both MYCN and p53 transcriptional responses independent of MYCN/MAX mediated DNA binding. We further discuss the biological and clinical implications of these findings.
To evaluate non-canonical MYCN functions we first tested the hypothesis that MYCN and p53 closely associate when p53 is activated in the presence of high levels of MYCN. We used a MYCN inducible system (Tet-ON) in p53 wild-type SHEP neuroblastoma cells to model these conditions. To avoid interference from fusion tags, we used a conditional construct expressing wild type MYCN with no 5’ or 3’ tags [18]. These cells stabilize and activate p53-mediated responses in response to Nutlin-3a (an MDM2 inhibitor) as shown in Figure 1A. Next, we used proximity ligation assays (PLA) to evaluate interactions of endogenous MYCN with p53. As shown, focal nuclear fluorescence signals of MYCN and p53 proximity interactions significantly increased only when MYCN and p53 were both present at high levels (Figure 1B). Of note, little to no PLA signals were detected in controls or when p53 is activated under non-MYCN conditions (Figure 1B).
Figure 1: MYCN and p53 co-localize and bind to each other. (A) The MYCN3 cell line was generated by transfecting a Tet-On plasmid containing full-length MYCN cDNA. MYCN3 cells were treated with Doxycycline to induce MYCN levels and with Nutlin-3a to induce p53 levels. Western blot showing MYCN and p53 protein levels under different treatment conditions. (B) Proximity ligation assays (PLA) for MYCN and p53 binding were performed using MYCN3 cells in the presence or absence of doxycycline and Nutlin-3a treatments. Additional controls were performed to determine antibody specificity. Representative images of control and combined doxycycline and Nutlin-3a treated cells are shown. A Scatter-plot and the mean ± SEM of the number of PLA spots per cell are shown in the bottom panel. The p-values of the difference between combination treatments and all other groups was <0.05 (Mann-Whitney test). Arrows indicating prominent PLA spots. *** p<0.001, ns= non significant.
As the PLA data suggested a close interaction of MYCN and p53 in our conditional system, we sought to validate these results by co-immunoprecipitation assays using both endogenous and recombinant GST and FLAG tagged proteins (Figure 2). We demonstrate robust association of p53 with both MYCN (Figure 2A) and C-Myc (Figure 2B). Importantly, we found that MAX, the primary transcriptional cofactor for MYC(N), was not associated with either MYCN or C-Myc protein pulled down with p53 (Figure 2A, 2B). Additionally, GST-C-Myc is also co-immunoprecipitated with p53, when incubated with cell free extract of HEK-293 cells overexpressing p53 (Figure 2C). We further verified direct protein/protein interaction using an in vitro cell free system using purified recombinant MYCN-6×HIS and GST-p53 proteins. (Figure 2D). To evaluate recombinant protein interactions in a distinct and p53 null background (p53-/-), we used the non-small cell lung cancer cell line H1299 with transient transfection of p53-GFP and MYCN-3xFlag recombinant protein vectors. We again demonstrated robust interactions of MYCN with p53 by pulldown and Western blotting (Figure 2E). These data strongly suggest that both MYCN and C-Myc can directly bind to p53 when present at high levels and that MAX is not a component of this complex.
Figure 2: (A) Endogenous MYCN and p53 co-IP. Nuclear extracts from the neuroblastoma cell line IMR-32 treated with Nutlin-3a were co-immunoprecipitated using anti-p53 (Ab-7) antibody or IgG (negative control). Western blots of immunoprecipitated proteins were performed using anti-p53 (DO-1), anti- MYCN (B8.4.B), or anti-Max (C-17) antibodies. (B) Endogenous MYC and p53 co-IP. HeLa cells treated with Nutlin-3a were co-immunoprecipitated using anti-p53 antibody or negative control IgG. Immunoprecipitated proteins were analyzed by Western blotting, using with anti-p53 (DO-1), anti- MYC (N262), and anti-MAX (C-17) antibodies. (C) in vitro GST-C-MYC pull-down. Crude nuclear protein extract from transient p53 over-expressing HEK-293T cells was incubated overnight with full-length GST-MYC or GST control proteins immobilized on glutathione-agarose beads. Pull-down samples were immunoblotted with the anti-p53 antibody. Membrane Ponceau S staining is shown as a loading control. (D) MYCN and p53 in vitro pull-down. Purified recombinant MYCN-6×His, GST-p53 (full length), and GST-control proteins were loaded as input samples. Recombinant MYCN- 6×His protein was incubated with GST-p53 or GST-control proteins immobilized on glutathione-agarose beads. GST proteins were pulled down and associated MYCN was detected by Western Blotting. Stain-Free total protein staining was used as a loading control. (E) Recombinant p53 and MYCN co-immunoprecipitation. The p53-null, non-small cell lung carcinoma cell line H-1299 was transiently transfected with plasmids overexpressing p53-GFP and MYCN-3×Flag. Crude nuclear protein extract collected from cells cultured under different transfection conditions were immunoprecipitated (IP) with either anti-p53 (Ab-7) or anti-FLAG (M2) antibody, and Western blots were performed using either anti-FLAG (M2) or anti-p53 (DO-1) antibody. (F) MYCN interacts with tetrameric form of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE2-(C) cells were incubated with GST alone and a series GST-p53 purified proteins: p53-WT (dimeric-tetrameric), p53-L344A (dimeric only) and p53-L344P (monomeric only). Input and pull-down samples were immunoblotted using anti-MYCN antibody and Ponceau staining was used as loading control.
As p53 can be present in the cytoplasm and nucleus as monomers, dimers and tetramers, but only the tetrameric form binds to chromatin [19]. To determine which p53 state most actively bound to MYCN, we used two well-characterized mutants of p53: p53 L344A that forms dimer, but not tetramers, and p53 L344P, which is exclusively monomeric [20, 21]. The GST pulldown and MYCN Western blotting showed that MYCN bound to wild type tetrameric p53 with high affinity, to monomeric p53 with low affinity, and did not bind to dimeric p53 (Figure 2F).
We next generated a series of p53- and MYCN- GST truncation mutants to map the respective interacting protein domains (Figure 3A). Nuclear extracts from MYCN-amplified SK-N-BE2-(C) neuroblastoma cells were incubated with different p53 truncations or GST alone. Interestingly, we find that the C-terminal 20 amino acids of p53 (aa 373-393) is required for MYCN interaction and pull down (Figure 3B). Next, crude extracts of HEK-293T transiently expressing full length p53 were incubated with different GST-MYCN truncation proteins. The results here were less specific likely due to the inherent disordered nature of MCYN protein, however, they suggest an interaction of p53 with the MycBOX II / IIIa domains, which are conserved between MYCN and C-Myc (Figure 3C). Taken together, these data clearly demonstrate a strong, context specific protein-protein interaction of MYCN or C-Myc with tetrameric form of p53 at c-terminal domain.
Figure 3: (A) Graphical representations of p53 and MYCN proteins. p53 (upper panel) and MYCN (lower panel) protein domains and truncation constructs. p53 protein domains: Trans Activation Domain (TAD), SRC Homology 3 domain (SH3), DNA binding domain, Nuclear Localization Signal (NLS), Tetramerization domain (TET), Regulatory domain (REG). MYCN protein domains: MYC boxes (MB), the basic region helix loop helix (BR-HLH), and the leucine zipper. The GST protein fragments are indicated with bars, and numbers refer to amino-acid positions. p53 and MYCN protein fragments were cloned in frame with the N-terminal GST in a pGEX-2T vector. GST-p53 and GST-MYCN fragments were cloned, expressed in BL-21 E.Coli strain and purified using gluthatione-agarose beads. (B) MYCN interacts with the C-terminus of p53. Crude nuclear protein extracts from MYCN-amplified SK-N-BE-(2)-c cells were incubated with the different p53 truncations or GST alone (negative control) immobilized