Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma

Abstract

The chromatin-modifying enzyme lysine-specific demethylase 1, KDM1A/LSD1 is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells and its overexpression correlated with aggressive disease, poor differentiation and infaust outcome. Here, we show that LSD1 physically binds MYCN both in vitro and in vivo and that such an interaction requires the MYCN BoxIII. We found that LSD1 co-localizes with MYCN on promoter regions of CDKN1A/p21 and Clusterin (CLU) suppressor genes and cooperates with MYCN to repress the expression of these genes. KDM1A needs to engage with MYCN in order to associate with the CDKN1A and CLU promoters. The expression of CLU and CDKN1A can be restored in MYCN-amplified cells by pharmacological inhibition of LSD1 activity or knockdown of its expression. Combined pharmacological inhibition of MYCN and LSD1 through the use of small molecule inhibitors synergistically reduces MYCN-amplified Neuroblastoma cell viability in vitro. These findings demonstrate that LSD1 is a critical co-factor of the MYCN repressive function, and suggest that combination of LSD1 and MYCN inhibitors may have strong therapeutic relevance to counteract MYCN-driven oncogenesis.

Keywords: LSD1; MYCN; neuroblastoma; transcription.

Figure 1. MYCN physically interacts with LSD1

A., co-immunoprecipitation interaction between endogenous LSD1 and MYCN in Tet-21/N cells. Cell lysates from Tet-21/N cells Tetracycline-treated (6days) (MYCN-OFF) and untreated (MYCN-ON) were immune-precipitated with a MYCN antibody and a No-Ab sample was used as negative control. Western blot analysis was performed on immuno-purified extracts with MYCN, LSD1 and MAX antibodies as indicated; * indicates IgG. B., schematic representation of MYCN deletion mutants d1, d2 and d3 used in the CoIP assay described in panel C and of GST-MYCN constructs used in GST-pull down described in panel D. The MYCN segments cloned in the GST expression vector are in black, and numbers indicate amino acid positions. C. MYCN-LSD1 interaction. 293T were cells co-transfected with an LSD1 expression vector together with different MYCN deletion expression vectors indicated in panel B. Extract from transfected cells were Immuno-precipitated with a MYCN antibody and analyzed by western blotting. D. Immobilized GST-MYCN polypeptides were incubated with equal amounts of extract prepared from HEK 293T cells transfected with the recombinant vector 3xFLAG-LSD1protein, separated by SDS-PAGE, and probed with an anti-LSD1 antibody.

Figure 2

A. Relative expression levels of MYCN, LSD1, p21, and p53 proteins were determined by Western blot analysis with the indicated antibodies at 12 and 24 hrs after TCP treatment (lanes 3, 4, 7, 8) in MYCN-ON (lanes 1-4) and MYCN-OFF (lanes 5-8) Tet-21/N cells. MYCN-ON cells were treated with control siRNA (lane 9) or with two concentrations (20nM lane 10 and 100nM lane 11) of specific LSD1 silencing by siRNA. Actinin was used for loading normalization. B. MYCN-ON cells, lane 1, were treated for 6 days with tetracycline and these cells are referred as MYCN-OFF, lane 2. MYCN-OFF cells were depleted of tetracycline and treated with TCP. Cells cultivated for 12 and 24hrs lane 3 and 4, are collected for protein and mRNA analysis. C. TCP relieves p21 protein expression. MYCN-OFF cells were depleted of tetracycline for 12 and 24 hrs in absence, lane 3,4 and presence of TCP, lane 5, 6. D., p21 mRNA expression. As in C., MYCN-OFF cells (0) were depleted of tetracycline for 12 and 24 hrs in absence and presence of TCP.

Figure 3

A, B. LSD1 and MYCN bind and repress p21. Chromatin immunoprecipitation assays. MYCN, LSD1, antibodies were used in IPs. Immunoprecipitated samples were analyzed by qPCR using specific primers for CDKN1A promoter Transcriptional Start Site (TSS) and two upstream regions (−3.3 and −2,2 KB. MYCN-OFF (black bars), MYCN-ON (light gray bars), MYCN-ON TCP treated (slanting bars), MYCN-ON shLSD1 (dark gray bars) MYCN-ON sh-control (dotted bars). LSD1 silencing in Tet-21/N cells transduced with shLSD1 and with sh-control was assayed by western blot shown in upper right. C., D. and E., Histone modifications at p21 promoter. H3Ac, H3K27me3 and H3K4me2 antibodies were used in IPs. Immunoprecipitated samples were analyzed by qPCR using specific primers for CDKN1A promoter Transcriptional Start Site (TSS). Data from three independent Chromatin-IP assays were used to make % of input graphs presented along with standard deviations, n = 3.

Figure 4. LSD1 and MYCN cooperatively repress CLU expression

CLU gene expression was analyzed by qRT-PCR, A. or by western blot, B. using samples prepared from MYCN-OFF cells and MYCN-ON cells untreated and treated with TCP or siLSD1 as indicated. C. D. MYCN and LSD1 binding to CLU chromatin. Cell treatments are indicated at the bottom of the figure and described in the legend of Figure 3. qPCR was performed with primers for CLU TSS, −1kb and +1kb. E. F. and G.. Histone modifications at CLU gene; ChIPs were carried out using the indicated antibodies and analyzed with primers encompassing the TSS region. Data from three independent CoIP assays and presented along with standard deviations, n = 3.

Figure 5

A. MTT assays of Tet-21/N and SK-N-BE cells treated with 1mM TCP, 75 μM 10058-F4, alone and in combination for 24 and 48 hours. Data from two independent experiments were used. B. Percentage of cell-cycle distribution of Tet-21/N and SK-N-BE (2) cells, treated with MYCN and LSD1 inhibitors as indicated, was measured by Flow cytometry analysis. Cells were treated with TCP and 10058-F4 for 24 and 48 hours and stained with Propidium Iodide for cell cycle profile; the average values from three independent experiments are reported in the tables; all standard deviations are <15%. C. LSD1 and MYCN inhibitors co-treatment increases apoptosis in NB cells. Western blotting of protein extract from Tet-21/N and SK-N-BE cells, treated with TCP, 10058-F4 or both for 48 hrs, using PARP (detecting both full length protein and cleaved fragment) p21 and MYCN antibodies. Actin has been probed as loading control.