LSD1 mediates MYCN control of epithelial-mesenchymal transition through silencing of metastatic suppressor NDRG1 gene

Abstract

Neuroblastoma (NB) with MYCN amplification is a highly aggressive and metastatic tumor in children. The high recurrence rate and resistance of NB cells to drugs urgently demands a better therapy for this disease. We have recently found that MYCN interacts with the lysine-specific demethylase 1 (LSD1), a histone modifier that participates in key aspects of gene transcription. In cancer cells, LSD1 contributes to the genetic reprogramming that underlies to Epithelial-Mesenchymal Transition (EMT) and tumor metastasis. Here, we show that LSD1 affects motility and invasiveness of NB cells by modulating the transcription of the metastasis suppressor NDRG1 (N-Myc Downstream-Regulated Gene 1). At mechanistic level, we found that LSD1 co-localizes with MYCN at the promoter region of the NDRG1 gene and inhibits its expression. Pharmacological inhibition of LSD1 relieves repression of NDRG1 by MYCN and affects motility and invasiveness of NB cells. These effects were reversed by overexpressing NDRG1. In NB tissues, high levels of LSD1 correlate with low levels of NDRG1 and reduced patients survival. Collectively, our findings elucidate a mechanism of how MYCN/LSD1 control motility and invasiveness of NB cells through transcription regulation of NDRG1 expression and suggest that pharmacological targeting of LSD1 represents a valuable approach for NB therapy.

Keywords: EMT; LSD1; MYCN; NDRG1; neuroblastoma.

Figure 1. A. Venn diagram of the DEG present in both LSD1-knockdown (LSD1-KD) and TCP treatment

B. Gene set of regulated genes by TCP treatment and LSD1-KD. C. Gene set enrichment analysis (GESA) plots show enrichment of gene sets regulated by LSD1-KD and TCP treatment. In each panel, nominal NES and false discovery rates (FDRs) are indicated. D. NDRG1 gene expression was analyzed by qRT-PCR, using samples prepared from Tet-21/N cells and treated with TCP or siRNA-LSD1 and siRNA-control as indicated. LSD1 protein level in Tet-21/N cells transfected with siRNA-LSD1 or control was determined by western blot. *, statistical significance (P < 0.01; Student t test). E. Western blotting of protein extracts from Tet-21/N cells prepared as described in D, using NDRG1 and phospho-NDRG1 (Thr 346) antibodies. F. NDRG1 silencing using sh-NDRG1 in Tet-21/N cells treated with TCP or vehicle, was assayed by western blot. G. Western blotting of protein extract from Tet-21/N treated with vehicle, TCP or 10058-F4 for 48 hrs, using NDRG1 antibody. Actinin has been probed as loading control.

Figure 2. A. Schematic representation of the NDRG1 promoter

B. and C. MYCN and LSD1 binding to NDRG1 chromatin. Cell treatments are indicated at the bottom of the figure. qPCR was performed with primers for NDRG1 TSS, and -10kb. D. and E. Histone modifications at NDRG1 chromatin; ChIPs were carried out using the indicated antibodies and analyzed with primers encompassing the TSS region and -10kb from TSS. Values from three independent ChIP assays are presented along with standard deviations, n = 3. Changes in % input are shown normalized over IgG controls and are all statistically significant (P < 0,05; Student t test).

Figure 3. A. NDRG1 gene expression was determined by qRT-PCR or by western blot in Tet-21/N cells treated with SP2509 at different concentrations, as indicated. *, statistical significance (P < 0.01; Student t test)

B. Co-Immunoprecipitation with MYCN antibody was performed in 293T cells co-transfected with LSD1 and MYCN expression vectors and treated with TCP, SP2509 or vehicle. Extract were analyzed by western blotting with MYCN, LSD1 and MAX antibodies as indicated. C. Interaction between endogenous LSD1 and MYCN in Tet-21/N cells, treated with TCP, SP2509 or vehicle, was assessed by co-Immunoprecipitation. Cell lysates were immune-precipitated with a LSD1 antibody Western blot analysis was performed on immuno-precipitated extracts with LSD1 and RCOR1 antibodies. IgG-sample was used as negative control. D. Tet-21/N cells were treated for 48 h with SP2509 or vehicle and then released into fresh medium for the indicated times. Cellular extracts were prepared and stained with anti-NDRG1 and phospho-NDRG1 (Thr 346). E. and F. Cell extracts from SK-N-BE (2) and SH-SY5Y cells treated with SP2509 at the indicated concentrations were prepared and probed with NDRG1 antibody. Actinin was probed as loading control.

Figure 4. A. Tet-21/N cells were treated with SP2509 or vehicle, fixed and processed for anti-β-catenin immunofluorescence and DAPI staining

B. Western blot assay of protein extracts of Tet-21/N and SH-SY5Y cells treated as indicated using β-catenin and Cyclin D1 antibodies. α-tubulin has been probed as loading control. *, P < 0,01.

Figure 5. LSD1 inhibition reduces migration of Neurobastoma cells

A. Wound healing of Tet-21/N (High MYCN), Tetracycline-treated Tet-21/N (Low MYCN) and B. SH-SY5Y cells treated with vehicle, TCP or SP2509. C. Wound healing was performed in Tet-21/N and SH-SY5Y cells 3XFlag-NDRG1 or mock transfected. Migration was assessed under treatment conditions at several time points using a scratch wound assay. Representative phase contrast images were shown acquired at 24hrs after scratch. Western blot shows NDRG1 protein levels in 3xFlag-NDRG1 or mock transfected Tet-21/N and SH-SY5Y cells. Actinin was used as loading control. Graphs showing the results represent the mean ± SD of three independent experiments carried out in duplicate. Statistical significance P < 0,01.

Figure 6. Trans-membrane migration assay of Tet-21/N and SH-SY5Y cells treated with vehicle, TCP or SP2509, or NDRG1-transfected

Graphs showing the results represent the mean ± SD of three independent experiments carried out in duplicate. *, P < 0,05.

Figure 7. A. SK-N-BE (2) cells were treated with RA up to 9 days. LSD1, NDRG1 and MYCN protein levels were detected in differentiated SK-N-BE (2) cells at the indicated days by western blotting

NDRG1 expression is associated with good outcome and differentiated tumors. B. and C. Low NDRG1 expression is associated with negative prognosis. The number of tumors is indicated in parentheses. Kaplan-Meier analysis is shown, with individuals grouped by median of expression of NDRG1. Log-rank P values are shown. Changes in expression for LSD1 D. and NDRG1 E. in ganglioneuroblastoma (GNB), ganglioneuroma (GN) and neuroblastoma (NB). F. Inverse correlation between the expression values of NDRG1 and LSD1 in NB tumors (Pearson's correlation coefficient is shown). G. Box plot showing differential NDRG1 expression in NB tumors without (Low) or with (High) MYCN amplification.