Repurposing a plant alkaloid homoharringtonine targets insulinoma associated-1 in N-Myc-activated neuroblastoma

Abstract

High-risk neuroblastoma (NB) is a heterogeneous and malignant childhood cancer that is frequently characterized by MYCN proto-oncogene amplification or elevated N-Myc protein (N-Myc) expression. An N-Myc downstream target gene, insulinoma associated-1 (INSM1) has emerged as a biomarker that plays a critical role in facilitating NB tumor cell growth and transformation. N-Myc activates endogenous INSM1 gene expression through binding to the E2-box of the INSM1 proximal promoter in NB. We identified a plant alkaloid, homoharringtonine (HHT), from a chemical library screening showing potent inhibition of INSM1 promoter activity. This positive-hit plant alkaloid exemplifies an effective screening approach for repurposed compound targeting INSM1 expression in NB cancer therapy. The elevated N-Myc and INSM1 expression in NB constitutes a positive-loop through INSM1 activation that promotes N-Myc stability. In the present study, the biological effects and anti-tumor properties of HHT against NB were examined. HHT either down regulates and/or interferes with the binding of N-Myc to the E2-box of the INSM1 promoter and the inhibition of PI3K/AKT-mediated N-Myc stability could lead to the NB cell apoptosis. HHT inhibition of NB cell proliferation is consistent with the INSM1 expression as higher level of INSM1 exhibits a more sensitive IC₅₀ value. The combination treatment of HHT and A674563 provides a better option of increasing potency and reducing cellular cytotoxicity than HHT or A674563 treatment alone. Taken together, the suppression of the INSM1-associated signaling pathway axis promotes the inhibition of NB tumor cell growth. This study developed a feasible approach for repurposing an effective anti-NB drug.

Keywords: Alkaloid; Homoharringtonine; INSM1; MYCN proto-oncogene; Neuroblastoma.

Figure 1.. INSM1 and N-Myc expression in NB tumors.

(A) INSM1 and N-Myc expression levels were derived from a range of neurological cancers (generated from the Broad Cancer Cell Line Encyclopedia: https://depmap.org/portal). The statistical analysis of INSM1 and MYCN gene expression and the graph of 111 neurological cancer cell lines subgroup were analyzed using R Studio software. (B) Correlation between INSM1 and N-Myc expression in 31 NB cell lines was performed by R Studio software and the dataset was provided by CCLE. (C, D) A Kaplan-Meier plot shows the OS time and survival probability in NB patients. NB patients derived from Asgharzadeh and Rajbhandari cohorts were generated from R2 Genomics Analysis and Visualization Platform (r2.amc.nl). NB patients were divided into high- and low-INSM1 expression based on the scan cut-off model. A red line indicates high INSM1 expression and poor prognosis. AA, astrocytoma; GBM, Glioblastoma; GM, glioma; MB, medulloblastoma; NB, neuroblastoma; OG, oligodendroglioma.

Figure 2.. HHT inhibits INSM1 promoter activity.

(A) HHT was subjected to a short-term (8 h) treatment assay normalized by cell number in stably transfected BE2-M17 or IMR-32 cells. The cell number was determined by a viability OD₄₉₀ reading with R² (inset). (B) HHT dose responses of INSM1 promoter activity. BE2-M17 and IMR-32 cells were treated with various HHT doses for 24 h. IMR-32 cells showed extreme sensitivity to INSM1 promoter activity corresponding to the high INSM1 expression level (inset). (C) NB cells treated with 1 or 10 μM HHT for a cell viability assay using two INSM1/N-Myc-positive (BE2-M17, IMR-32) and two INSM1/N-Myc-negative cells (SKnSH, SHSY-5Y).

Figure 3.. Effect of HHT on the cell viability of three NB cell lines: BE2-M17, TH-N-MYC#1 and IMR-32.

(A) HHT chemical structure. (B) Cells were treated with different doses of HHT from 0.5 nM to 10 μM for 48 h. Following a 48-h treatment, cell viability was examined by MTS assay. Values are presented as mean ± SD. The IC₅₀ values of BE2-M17, TH-N-MYC#1 and IMR-32 were 32.6 nM, 32.8 nM and 12.2 nM. (C) Eight human NB cell lines were treated with HHT (IC₅₀ determined using a 0.5 nM-10 μM range) 48 h for a cell viability assay. (D) These cell lines were subjected to western blot analysis for INSM1 and N-Myc with β-actin as a loading control. Relative quantitative expression level against β-actin as control were performed in at least three separated experiments.

Figure 4.. Biological effects of HHT on NB cells.

(A) IMR-32 and BE2-M17 (5,000 cells) were plated in 0.5% top soft agar with or without 0.5 μM HHT for two weeks. (B) BE2-M17 and IMR-32 cells were treated with two concentrations of HHT (0, 25 or 100 nM) for 5 days. Cell numbers were counted using the trypan blue dye exclusion method. (C) BE2-M17 cells were treated with HHT (100 nM) for 48 h. Cell lysates were subjected to western blot analysis using the indicated antibodies. Following HHT treatment, the expression level (ratio) was shown by normalization with control β-actin or pAKT/AKT ratio.

Figure 5.. N-Myc binds to an E2-box of the INSM1 proximal promoter and activates endogenous INSM1 expression in NB cells.

(A) INSM1 promoter activity was measured by transfecting N-Myc cDNA expression plasmid (0-200 ng) in SKnSH (N-Myc negative) cells. All transfections were normalized to TK-Renilla. The graph represents three experiments. (B) A ChIP assay was performed using cross-linked chromatin from BE2-M17 cells. The endogenous N-Myc bounds to the E2-box region of human INSM1 proximal promoter. BE2-M17 chromatin, either treated or not treated with HHT was incubated with rabbit IgG, or anti-N-Myc (B.8.4) Ab. The bound DNA was compared with 2% of input DNA using PCR amplification with primers spanning the human INSM1 promoter E2-box region (170 bps). (C) SH-SY-5Y cells were infected with Ad-N-Myc or Ad-LacZ for 3 days to induce endogenous INSM1 expression. Relative quantitative expression level versus that of β-actin are shown.

Figure 6.. Overexpression of INSM1 or N-Myc rescues HTT treated NB cell viability.

(A) BE2-M17 cells were infected with Lenti-Cherry, Lenti-INSM1, or Lenti-N-Myc for 24 h and then treated with HTT (50 nM) for 48 h before subjected to cell viability assay. Lenti-Cherry was used as a negative control. Values are presented as mean ± SD, n=8. ***p<0.001 vs vehicle group. INSM1 and N-Myc expression were shown in Western blot analysis. (B) BE2-M17 cells were treated with Ad-INSM1-siRNA versus Ad-sc-siRNA for 48 h to knockdown the INSM1 expression before subjected to cell viability assay. INSM1 expression was shown in western blot analysis. Relative quantitative expression level versus β-actin is shown.

Figure 7.. PI3K/AKT signaling regulates INSM1 expression and apoptosis pathway.

(A) INSM1 promoter activity was measured after 24h of LY20 or A563 inhibitor treatment, whereas (B) cell viability was measured after 48h of inhibitor treatment. Data are presented as the mean ± SD vs. PBS control. (C) AKT inhibitor (A563; 0.5 μM) was used to treat BE2-M17 cells for 48h, and western blot analysis was then performed with the indicated antibodies, including GSK3β, N-Myc, INSM1, PARP (F+C) and p53. β-Actin was used as the loading control. Relative quantitative expression control versus A563 or β-actin are shown.

Figure 8.. HHT facilitates N-Myc protein ubiquitination and degradation.

(A) HHT (100 nM) inhibits INSM1 and N-Myc RNA expression using real-time PCR. (B) HHT inhibits N-Myc protein expression, whereas proteosome inhibitor MG132 blocks N-Myc degradation. (C) Co-IP assay of anti-N-Myc and anti-ubiquitin showed dramatic increase in ubiquitination level of N-Myc upon HHT treatment. (D) BE2-M17 cells were either pre-treated or un-treated with 100 nM HHT for six hours before addition of CHX (10 μg/ml) from 0-5 h. The N-Myc protein half-life using CHX chase assay revealed HHT facilitates N-Myc protein degradation.

Figure 9.. Combining HHT and A563 for viability assay using TH-N-MYC#1, BE2-M17, and IMR-32 cells.

(A) TH-N-MYC#1, BE2-M17, and IMR-32 cells were treated with HHT and/or A563 alone or in combination with varying concentrations for 48 h in a cell viability assay. The red circle indicates enhanced combination effect. The green line represents 70% inhibition of cell viability. (B, C) BE2-M17 and IMR-32 cells were treated with HHT (25 nM), A563 (0.25 μM) or both for 5 days. Cell numbers were counted using the trypan blue dye exclusion method. (D, E) Western blot analysis of NB cells treated with HHT (25 nM), A563 (0.25 μM) or both for 2 days. The effects of treatment with HHT or A563 alone or in combination on the expression of INSM1, PARP (F+C) or p53 shown. Relative quantitative expression level in control versus A563, HHT, A563+HHT or β-actin are shown.