Runt-related transcription factor 1 promotes apoptosis and inhibits neuroblastoma progression in vitro and in vivo

Abstract

Background

Runt-related transcription factor 1 (RUNX1) is a heterodimeric transcription factor that binds to the core element of many enhancers and promoters and can accelerate apoptosis in various tumors. However, the regulatory mechanisms underlying RUNX1 expression in neuroblastoma (NB), a highly malignant tumor in childhood, remain largely unclear. In this study, we aimed to assess the role of RUNX1 in NB and to reveal the underlying mechanisms that may contribute to finding a potential therapeutics strategy against NB.

Methods

Growth, invasion, metastasis and angiogenesis were assessed using Cell Counting Kit-8 (CCK-8) immunocytochemistry, and studies involving soft agar, cell invasion, tube formation and whole animals. The levels of expression were measured using real-time quantitative PCR for RNA, Western blot and immunostaining analyses for proteins. Luciferase reporter and chromatin immunoprecipitation assays indicated that RUNX1 directly binds within the BIRC5, CSF2RB and NFKBIA promoter regions to facilitate transcription. The level of apoptosis was assessed by determining mitochondrial membrane potential and flow cytometry.

Results

RUNX1 was highly expressed in ganglioneuroma (GN) and well-differentiated (WD) tissues relative to the poorly differentiated (PD) and undifferentiated (UD) ones. Moreover, RUNX1 effectively reduced cell viability, invasion, metastasis, angiogenesis, and promoted apoptosis in vitro and in vivo. RUNX1 reduced BIRC5 transcription and increased CSF2RB and NFKBIA transcription by directly binding BIRC5, CSF2RB and NFKBIA promoters. In addition, cytotoxic drugs, especially cisplatin, significantly increased RUNX1 expression in NB cells and promoted apoptosis.

Conclusions

These data show that RUNX1 is an independent surrogate marker for the progression of NB and it can be used for monitoring NB prognosis during therapy.

Background

Neuroblastoma (NB) is the most common extracranial solid tumor in children. NB develops from neural crest-derived cells and accounts for 10 to 15% of all cancer deaths within the first 15 years of life [1]. Clinical presentation of NB is remarkably heterogeneous ranging from spontaneous regression to rapid progression and death [2, 3]. Moreover, a member of MYC transcription factor gene family, MYCN, is amplified in neuroblastoma cell lines and primary neuroblastomas and the progression of neuroblastoma disease tightly and adversely correlates with MYCN gene amplification, in that higher the amplification level poorer the clinical outcome [4]. The long-term survival rate of high-risk NB remains poor even with intensive treatments including multimodal therapy, surgery, high-dose myeloablative therapy with autologous hematopoietic stem cell rescue and GD2-directed immunotherapy [5]. Therefore, it is important to develop new and effective preventative or therapeutic approaches that are relatively nontoxic to the host yet effective against cancer cell growth.

Programmed cell death, or apoptosis, plays critical roles in the homeostasis of multicellular organisms and the development of cancer [6]. Runt-related transcription factor 1 (RUNX1) is a core transcription factor involved in apoptosis according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Recent studies have shown that RUNX1 is expressed at low levels or is mutated in numerous solid and blood malignancies and associated with enhanced growth, adhesion, invasion, metastasis and apoptosis of cancer. Published data of human cell lines and mouse models suggest that RUNX1 suppresses cancer aggression and that high RUNX1 levels are associated with better overall survival [7,8,9].

It has been reported that RUNX1 expression is reduced in metastasis-prone solid tumors [8] and that excess RUNX1 inhibits NB cell growth and cell cycle [10]. However, the regulatory mechanisms of RUNX1 in NB remain unclear. Here, we used multiple experimental methods to elucidate whether RUNX1 can effectively reduce NB invasion, metastasis and promote NB-apoptosis by activating baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), colony stimulating factor 2 receptor beta (CSF2RB) and NFKBIA. We also use animal models of cell line derived NB-tumors to assess the mechanistic role of RUNX1 in NB-progression.

Materials and methods

Primary neuroblastoma tumors

Twenty diagnostic NB tumor samples were acquired from the Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was signed by the guardians of the pediatric patients. Research protocols were approved by the Research Ethics Committees of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. The degree of tumor differentiation was determined by the department of pathology, Union Hospital, Tongji Medical College.

Immunostaining and H&E staining

Generally, the tumor tissues were fixed in 10% paraformaldehyde and embedded in paraffin. The sections were dewaxed using xylene and rehydrated in graded alcohols. After antigen retrieval and blocking with goat serum, the sections were incubated overnight with the primary antibodies RUNX1 (A0400, ABclonal 1:100 dilution) and CD31 (ab28364, Abcam, 1:100 dilution) at 4 °C. Two pathologists assessed the tumor clinicopathological features.

Cell culture and oligonucleotide sets used for construct vectors

Human MYCN-amplified (IMR-32, TIN-2) and non-MYCN-amplified (SK-N-AS, SH-SY5Y, and SK-N-SH) NB cell lines were obtained from Dr. Muxiang Zhou’s laboratory, Children’s Oncology/Hematology, Emory University, and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and cultured at 37 °C in a humidified atmosphere with 5% CO2. Cisplatin and cytarabine was obtained from Selleck (Wuhan, China), both were dissolved in dimethyl sulfoxide (DMSO). The oligo sets for RUNX1 was inserted into CV186 (GeneChem Co., Ltd., Shanghai, China) and shRNAs were inserted into GV298 (Additional file 2: Table S3). Stable cells were screened by neomycin or puromycin (Invitrogen).

Cell viability assay

Cell viability under different concentrations was determined using the Cell Counting Kit-8 (Dojindo, Tokyo, Japan) according to manufacturer’s guideline. After treatment, the cells were cultured in 96-well plates. At 12, 24, 36, 48, 60, 72, 84 and 96 h, CCK8 solution (DOJINDO, Japan) was added to the 96-well plate, and cells were further incubated at 37 °C for 2 h. Absorbance was measured at 450 nm. The oligo sets primer sequences see Additional file 2: Table S1.

Cellular apoptosis assay

Apoptosis was measured by using an FITC Annexin V/propidium iodide (PI) using Apoptosis Detection Kit (BD Pharmingen, USA). Tumor cells were collected, washed twice with cold phosphate-buffered saline (PBS), resuspended with 200 μl of binding buffer at 2–5 × 10⁵ cells/ml density and stained with 2.5 μL of Annexin V-FITC and 5 μL of PI incubated at room temperature for 15 min in the dark. Apoptosis was analyzed by a flow cytometer (Becton Dickson Co.) at a wavelength of 488 nm.

Real-time qRT-PCR

Total RNA was isolated with TRIzol reagent (Invitrogen, USA), and cDNA was synthesized using a PrimeScriptTM RT Reagent Kit (Perfect Real Time, TaKaRa, Japan). Real-time PCR was performed with SYBR Green PCR Master Mix (SYBR 1 Premix Ex TaqTM II, TaKaRa, Japan) and the primer indicated in the Supplementary map. After normalization to the β-actin gene, the transcript levels of each target gene were determined according to the 2^−ΔΔCt method. The PCR and ChIP primer sequences see Additional file 2: Table S2.

Western blot analysis

Tissue or cellular protein was extracted with 1 × cell radio immunoprecipitation assay (RIPA) lysis buffer (Promega) following the provided protocol. Equal amounts of total protein were loaded onto a 12% SDS-PAGE gel, transferred onto polyvinylidene difluoride membranes (Millipore), blocked with 5% fat-free milk for 2 h at room temperature, and incubated with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Specific proteins were visualized with enhanced chemiluminescence detection reagent according to the manufacturer’s instructions (Pierce Biotechnology).

Soft agar assay

Cells (6× 10³) were mixed with 0.3% Nobel agar (Fisher Scientific, Pittsburgh, PA) and layered onto 0.6% solidified agar in DMEM/F12 containing 10% FBS in 6-well plates. After incubation 3 weeks, cells were fixed with 100% methanol and stained with 0.5% crystal violet dye.

Cell invasion assay

Matrigel invasion assays were performed with polycarbonate filter membranes with a diameter of 6.5 mm and pore size of 8 μm (Invitrogen) using membranes coated with Matrigel matrix (BDScience, Sparks, MD). Tumor cell suspensions (1 × 10⁵ cells/well) in serum-free culture medium were added to the upper chambers and incubated for 24 h at 37 °C. The filters were fixed with methanol for 30 min and stained with 0.1% crystal violet for 20 min at room temperature.

Tube formation assay

A total of 150 μl of Matrigel (BD Biosciences) solution per well was added to a 48-well plate and a gel was formed at 37 °C for 1 h. Human umbilical vein endothelial cells (HUVECs) were serum-starved by culturing in serum-free culture medium for 24 h, suspended at a density of 9 × 10³ cells/well and added to the Matrigel-coated wells.

Mitochondrial membrane potential assay

The mitochondrial membrane potential was determined using tetraethylbenzimidazolylcarbocyanine iodide (JC-1) dye (KeyGEN BioTECH, China). Tumor cells were cultured in a confocal dish, and then, JC-1 was stained with 1 × incubation buffer for 20 min at 37 °C in the dark. Next, cells were washed twice with PBS, and 1 × incubation buffer was added. Fluorescence emission was observed under a confocal microscope; green fluorescence was observed using excitation and emission wavelengths of 498 and 553 nm, respectively, and red fluorescence was observed using excitation and emission wavelengths of 570 and 600 nm, respectively.

Luciferase reporter assay

Human BIRC5, CSF2RB and NFKBIA promoters and their truncated forms were amplified from genomic DNA by PCR and subcloned into pGL3-Basic (Promega). We used a luminometer to measure luciferase activity.

ChIP assay

A ChIP assay was performed according to the manufacturer’s instructions for an EZ-ChIP kit (Beyotime, China). Real-time qPCR was performed as previously described.

Animal study

Female BALB/c nude mice were purchased Beijing Vital River Laboratory Animal Technology (Beijing, China), and experimental protocols were approved by the Animal Care Committee of Tongji Medical. For in vivo tumor growth, 1-month-old male nude mice (n = 5 per group) were injected subcutaneously into the right upper back with 1 × 10⁷ tumor cells stably transfected with the indicated vectors. Thirty days later, the mice were sacrificed and examined as described above. The experimental metastasis studies were performed by tail vein injection of tumor cells (2 × 10⁶ per mouse, n = 5 per group) into nude mice as previously described. Forty-five-day-old mice were sacrificed and examined as described above.

Statistical analysis

All experiments were performed at least three times, and the data are indicated as the mean ± SEM or median. The cutoff values were calculated by the average expression of the gene. Unpaired t-test were used to compare two groups, and multiple group comparisons were analyzed by variance (*P < 0.05, **P < 0.01, and ***P < 0.001 were regarded as significant differences).

Results

Identification of RUNX1 as an independent prognostic marker for NB apoptosis and progression

To determine the crucial role of transcription factors in apoptosis, we analyzed the mining of public microarray datasets of KEGG [11] and ChIP-X [12] (Fig. 1a). Based on the number of target genes, we found HNF4A and RUNX1 as a potential transcription factors that might regulate apoptosis. In order to explore the significance of HNF4A and RUNX1 in NB, Kaplan–Meier curves of 498 patients (GSE49710) showed that high HNF4A and RUNX1 levels were correlated with favorable overall survival in NB patients (p = 0.053 and 0.019, respectively, Fig. 1b). Based on the significant of survival, we chose RUNX1 for the further examination. In addition, mining of public datasets (GSE49710) showed that RUNX1 levels were negatively correlated with MYCN amplification (P < 0.0001), progression (P < 0.0001), and advanced stage (P = 0.0014, Fig. 1c) by International Neuroblastoma Staging System (INSS). Notably, in public datasets (GSE49710), there was no correlation between RUNX1 and MYCN (R = − 0.071, P = 0.13, Supplementary Fig. S1a), which indicate RUNX1, not HNF4A, might serve as an independent prognostic factor for favorable outcome of NB. Moreover, we measured the level of RUNX1 in 5 ganglioneuroma (GN, as control) tissues, 5 well differentiated (WD) NB tissues, 5 poor differentiated (PD) NB tissues, and 5 undifferentiated (UD) NB tissues. As shown in Fig. 1d and Fig. 1e, western blot analyses and immunohistochemical staining revealed that RUNX1 was highly expressed in GN tumor and WD tumor tissues relative to the PD and UD NB tissues. Furthermore, RUNX1 expression was observed in NB cell lines, SH-SY5Y, SK-N-SH, SK-N-AS, IMR-32 and TN-2 (Fig. 1f). Collectively, these data demonstrate that RUNX1 is an independent prognostic marker for NB apoptosis and progression.

Fig. 1

Identification of RUNX1 as an apoptosis transcription factor and an independent prognostic factor in NB tissue and cell lines. a Cluster analysis of apoptosis genes in the KEGG and ChIP-X databases. We selected the top ten transcription factors (TFs), as shown. b Kaplan-Meier curves indicating the survival of 498 (GES49710, cutoff value = 292.04) NB patients with high or low HNF4A and RUNX1 expression, respectively (Log-rank test, p = 0.053, p = 0019, respectively). c Mining of public microarray datasets (GSE49710) revealing the RUNX1 expression levels in tumors with various MYCN amplification levels, progression and INSS stages. (unpaired t-test, p < 0.0001, p < 0.0001, P = 0.0014.) d and e Western blot and immunohistochemical staining images indicated protein levels of RUNX1 in g ganglioneuroma (GN, control), well differentiated (WD) NB, poor differentiated (PD) NB, and undifferentiated NB tissues (n = 5 per group). Scale bar: 100 μm. ***P < 0.001 versus GN. f Western blot assay indicating the levels of RUNX1 in NB cell lines (SH-SY5Y, SK-N-SH, SK-N-AS, IMR-32, TN-2). Data are shown as mean ± SEM and representative of three independent experiments in panels (d)–(f). Exact P values are specified in Additional file 2: Table S3

RUNX1 overexpression inhibits the proliferation, migration, invasion and angiogenesis of NB

To explore the function of RUNX1 in NB, we further investigated the effects of overexpression or knockdown of RUNX1 on cell proliferation, migration, invasion and tumorigenesis in NB cell lines (SH-SY5Y and SK-N-SH). Stable transfection of RUNX1 led to its overexpression in SH-SY5Y and SK-N-SH, while two independent short hairpin RNAs (shRNAs), sh-RUNX1#1 and sh-RUNX1#2, were used to deplete RUNX1 in the SH-SY5Y and SK-N-SH cell lines (Fig. 2a). The subsequent finding from CCK-8 (Fig. 2b), soft agar (Fig. 2c) and matrigel invasion (Fig. 2d) revealed that SH-SY5Y and SK-N-SH cells transfected with RUNX1 showed a decreased in cell growth, viability, invasion and migration. However, silencing of RUNX1 had opposite results with the aforementioned factors. Next, tube formation assays indicated that overexpression or silencing of RUNX1 respectively decreased and facilitated tube formation of endothelial cells, than those transfected by mock or scramble shRNA. (Fig. 2e). Taken together, these data show that RUNX1 plays a major role in regulating cell growth, proliferation, aggressiveness and tumorigenesis in NB cells.

Fig. 2

RUNX1 suppresses the growth, migration, invasion and angiogenesis of NB cells in vitro. a Western blot assays showing the expression of RUNX1 in SH-SY5Y and SK-N-SH cells stably transfected with empty vector (mock), RUNX1, scramble (sh-Scb), sh-RUNX1#1 or RUNX1#2 shRNA. b CCK8 assays depicting the change in cell viability of NB cells stably transfected with RUNX1, sh-RUNX1#1, sh-RUNX1#2, or sh-RUNX1#2 after culture for 96 h. c Representative images (left panel) and quantification (right panel) of soft agar plates indicating anchorage-independent growth of NB cells stably transfected as indicated. d Transwell Matrigel invasion assays of representative images (left panel) and quantification (right panel) for 48 h indicating the invasion capability of NB cells stably transfected as indicated. e Representative images (left panel) and quantification (right panel) of the tube formation of endothelial HUVECs treated with medium preconditioned (for 6 h) with NB cells stably transfected as indicated . *P < 0.05 vs. mock or sh-Scb, **P < 0.01 vs. mock or sh-Scb, ***P < 0.001 vs. mock or sh-Scb. Data are shown as mean ± SEM and representative of three independent experiments in panels (a)–(e). Exact P values are specified in Additional file 2: Table S3

RUNX1 overexpression promoted apoptosis and knockdown of RUNX1 suppressed apoptosis in NB cells

To test the potential predictive role of RUNX1 in NB therapy, we first examined the direct effect of RUNX1 on NB cell apoptosis. Flow cytometric assays (Fig. 3a) and JC1 staining (Fig. 3b) were used to determine the apoptosis of NB cells that were stably transfected with RUNX1, sh-RUNX1#1, or sh-RUNX1#2. The number of apoptotic cells was significantly increased in NB cells with stable overexpression of RUNX1 compared with cells mock transfected; conversely, RUNX1 knockdown decreased apoptosis compared with sh-Scb transfected. We further examined apoptosis-related cell signaling events by real-time quantitative (Fig. 3c) and Western blot analyses (Fig. 3e). The results indicated that the overexpression of RUNX1 significantly decreased the levels of Bcl-2 but increased the transcription and expression levels of Bax and cleaved caspase-3 in both SH-SY5Y and SK-N-SH cells. The ratio of the Bcl-2/Bax was decreased and increased by expression or knockdown of RUNX1 in NB cells. Analysis of the ratio of the Bcl-2/Bax show that RUNX1 induced proapoptotic potential (Fig. 3d and Fig. 3f), and efficiently activated the downstream caspase-3 and apoptosis. Thus, these results revealed that the overexpression of RUNX1 promoted apoptosis in SH-SY5Y and SK-N-SH cells.

Fig. 3

Overexpression of RUNX1 promoted NB cell apoptosis, and knockdown of RUNX1 led to suppression of NB apoptosis. a Representative images (left panel) and quantification (right panel) of flow cytometry showing the apoptosis of NB cells (stably transfected with mock, RUNX1, sh-Scb, sh-RUNX1, or sh-RUNX1#2, n = 3) were double stained with an FITC-conjugated anti-Annexin V antibody and PI. The cells in the upper right and lower right quadrants depict late and early apoptosis. b Representative images (left panel) of the mitochondrial membrane potential was evaluated with JC-1 (2 μg/ml) dye. The right panel indicating the ratio of red/green fluorescence was calculated as an indicator of mitochondrial membrane potential. Red fluorescence (Ex/Em 550 nm/600 nm) and green fluorescence (Ex/Em 485 nm/535 nm) were measured using a fluorescence microscope. Scale bar, 100 μm. c-f Real-time RT-PCR and Western blot assays showing the transcript and protein levels of RUNX1, Bax, Bcl-2, and cleaved caspase-3 after stable transfection of NB cells with mock, RUNX1, sh-Scb, sh-RUNX1#1, or sh-RUNX2 and the ratio of Bcl-2/Bax. *P < 0.05 vs. mock or sh-Scb, **P < 0.01 vs. mock or sh-Scb, ***P < 0.001 vs. mock or sh-Scb. Data are shown as mean ± SEM and representative of three independent experiments in panels (a)–(f). Exact P values are specified in Additional file 2: Table S3

RUNX1 promoted apoptosis by regulating the transcription and protein expression levels of BIRC5, CSF2RB, and NFKBIA

To determine how RUNX1 transcription factor regulates apoptosis, we first analyzed the publicly available datasets of 498 NB cases (GSE49710) and 649 NB cases (GSE45547) derived from the Gene Expression omnibus (GEO) which correlated with RUNX1 and the apoptosis gene of RUNX1 derived from the KEGG [13] datasets and ChIP-X (Fig. 4a). Based on the three overlapping analyses, we focused on 7 genes (BIRC5, TUBA1A, FOS, NFKBIA, TNFSF10, CSF2RB, PARP4) regulated by RUNX1 that were associated with apoptosis and analyzed the expression of these 7 genes in 498 patients (GSE49710, Fig. 4b). We found that the correlation between RUNX1 and TUBA1A was inconsistent in the two databases, so it was removed from further analyses (Supplementary Fig. S2a-g). Next, we examined the mRNA and protein levels of BIRC5, CSF2RB, NFKBIA, FOS, PARP4 and TNFSF10 following stable transfection with RUNX1, sh-RUNX1#1 or sh-RUNX1#2 in NB cells (Fig. 4 c and d). The results indicated that stable overexpression of RUNX1, increased the expression of CSF2RB and NFKBIA, decreased the expression of BIRC5. However, stable knockdown of RUNX1 had opposite results with the aforementioned factors. Meanwhile, the expression of FOS, PARP4 and TNFSF10 was not affected by ectopic overexpression or knockdown of RUNX1. These findings indicated that transcription factor RUNX1 facilitated the apoptosis in NB through regulating the expression of CSF2RB, NFKBIA and BIRC5.

Fig. 4

RUNX1 promoted apoptosis in NB cells and altered the transcription and protein levels of BIRC5, CSF2RB, and NFKBIA. a Venn diagram heatmap showing the genes correlated with RUNX1 in NB cases public datasets (GSE45547 and GSE49710, p < 0.05) and RUNX1 target genes of apoptosis in KEGG datasets. b Heatmap derived from public datasets (GSE49710) revealing the expression of the RUNX1 target gene (BIRC5, TUBA1A, FOS, NFKBIA, TNFSF10, CSF2RB, and PARP4). c and d Real-time quantitative RT-PCR and western blot indicating the mRNA and protein level of the RUNX1 target gene in NB cells stably transfected with mock, RUNX1, sh-Scb, sh-RUNX1#1 and sh-RUNX1#2. *P < 0.05 vs. mock or sh-Scb, **P < 0.01 vs. mock or sh-Scb, ***P < 0.001 vs. mock or sh-Scb. Data are shown as mean ± SEM and representative of three independent experiments in panels (c)–(d). Exact P values are specified in Additional file 2: Table S3

RUNX1 regulates BIRC5, NFKBIA and CSF2RB by directly binding its promoter

To investigate the direct effects of RUNX1 on BIRC5, NFKBIA and CSF2RB transcription and expression in NB cell lines, we used JASPAR (http://jaspar.genereg.net) to first identify the binding profile of the transcription factor RUNX1 [14, 15] in vertebrates (Fig. 5a). Subsequently, we used performed chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) assays for promoter-binding as well as a dual-luciferase assay for promoter-activity assays. ChIP and qPCR assays indicated that overexpression of RUNX1 facilitated the binding of BIRC5, NFKBIA and CSF2RB promoter regions in NB cells; conversely, stable knockdown of RUNX1 decreased the binding of BIRC5, NFKBIA and CSF2RB promoter regions (Fig. 5b). Dual-luciferase assays revealed that the activity of BIRC5 was significantly decreased in NB cells with stable overexpression and increased by silencing of RUNX1, whereas NFKBIA and CSF2RB showed opposite results (Fig. 5c). To further verify the above results, we analyzed the survival of BIRC5, NFKBIA and CSF2RB expressing patients. Kaplan-Meier curves of 498 (GSE49710) NB patients revealed a difference in patient survival between the high and low BIRC5, NFKBIA and CSF2RB expression groups (Fig. 5d). Patients with low BIRC5, high NFKBIA and CSF2RB expression had higher survival probability. Collectively, these results indicated that RUNX1 directly interacted with the binding site within the BIRC5, NFKBIA and CSF2RB promoters to alter their transcription and protein levels in NB cells.

Fig. 5

RUNX1 altered the transcription and protein levels of BIRC5, CSF2RB, and NFKBIA through direct binding of its promoter. a Overview of the RUNX1 motifs found at RUNX1 binding sites. b ChIP and qPCR assays indicating enrichment of RUNX1 on target gene promoters in SH-SY5Y and SK-N-SH cells transfected with mock, RUNX1, sh-Scb, sh-RUNX1#1, and sh-RUNX1#2. c Dual-luciferase assay showing the relative activity (36 h post transfection) of BIRC5, CSF2RB, and NFKBIA promoters in cells stably transfected as indicated. d Kaplan-Meier curves showing the survival of 498 patients (GSE49710) with high or low BIRC5, CSF2RB, and NFKBIA expression (cutoff values = 13,969.57, 151.17 and 19,483.97, respectively). *P < 0.05 vs. mock or sh-Scb, **P < 0.01 vs. mock or sh-Scb, ***P < 0.001 vs. mock or sh-Scb. Data are shown as mean ± SEM and representative of three independent experiments in panels (b)–(c). Exact P values are specified in Additional file 2: Table S3

Ectopic expression of RUNX1 suppresses NB cell growth, metastasis, and angiogenesis and promotes apoptosis in vivo

We next investigated the effects of RUNX1 overexpression on tumor growth, metastasis, and angiogenesis and apoptosis in vivo. Consistent with previous findings, the growth rate as judged by the in vivo fluorescence of the fluorescent SH-SY5Y xenografted tumors as well as their final tumor-weights were significantly decreased when stably overexpressing RUNX1. Consistently suppression of RUNX1 expression by stable transfection of sh-RUNX1#1 resulted in a significant increase in the growth and weight of xenograft tumors (Fig. 6 a and b). Stable transfection of RUNX1 or sh-RUNX1#1 into SH-SY5Y cells resulted in a significant decrease or increase in Ki-67 proliferation index and CD31-positive microvessels levels of subcutaneous.

Fig. 6

Ectopic expression of RUNX1 suppresses NB cell growth, metastasis, and angiogenesis and promotes apoptosis in vivo. a Timeline (left panel), after tumor cells injection 30 days, athymic nude mice (n = 5) from each group were sacrificed. In vivo growth curves (middle and left panel) of xenografts in athymic nude mice formed by subcutaneous injection of SH-SY5Y cells stably transfected with mock, RUNX1, sh-Scb, or sh-RUNX1#1 into the dorsal flanks (n = 5 per group). b Representative in vivo fluorescence images (left panel) of athymic nude mice bearing xenografts; the weight of xenografts at the end points (right panel) formed by subcutaneous injection of SH-SY5Y cells stably transfected as indicated in nude mice (n = 5 per group). c Representative images (left panel) and quantification (right panel) of immunohistochemical staining showing the expression of Ki-67 and CD31 within xenograft tumors formed by hypodermic injection of SH-SY5Y cells stably transfected as indicated (n = 5 per group). Scale bars: 100 μm. d Western blot assays (normalized to β-actin) showing the expression of RUNX1 and its target genes and apoptosis genes in xenograft tumors formed by subcutaneous injection of SH-SY5Y cells stably transfected as indicated in nude mice into dorsal flanks of athymic nude mice (n = 5 per group). e Timeline (left panel), after tumor cells injection 45 days, athymic nude mice (n = 5) from each group were sacrificed. in vivo imaging of lungs (middle upper panel), metastatic counts of lungs (middle lower panel) and H&E staining images (right panel) of nude mice (n = 5 per group) treated with tail vein injection of SH-SY5Y cells stably transfected as indicated . Scale bar: 100 μm. *P < 0.05 vs. mock or sh-Scb, **P < 0.01 vs. mock or sh-Scb, ***P < 0.001 vs. mock or sh-Scb. Data information: Data are presented as mean ± SEM. Exact P values are specified in Additional file 2: Table S3

xenograft tumors in nude mice (Fig. 6c). Western blot assays (Fig. 6d) indicated that stable transfection of RUNX1 into SH-SY5Y cells resulted in a significant increase in the levels of CSF2RB, NFKBIA, Bax and cleaved caspase-3 and decrease in the levels of BIRC5 and Bcl-2 of subcutaneous xenograft tumors in nude mice. However, silencing of RUNX1 had opposite results with the aforementioned factors. The ratio of the Bcl-2/Bax was decreased and increased by expression or knockdown of RUNX1 in vivo (n = 5, per group). In addition, in vivo metastasis assays involving athymic nude mice infused systemically (via tail vein) with RUNX1-overexpressing fluorescent SH-SY5Y cells resulted in a significant decreased in lung metastatic colonies, while the RUNX1-knocked down cells increased these lung metastases (Fig. 6e). Taken together, these results indicated that RUNX1 suppresses NB cell growth, metastasis, and angiogenesis and promotes apoptosis in vivo.

RUNX1 is upregulated by cytotoxic drugs and promotes apoptosis

It has been reported that RUNX1 can be upregulated in blood tumors by cytotoxic drugs [16]. We hypothesized that RUNX1 could be upregulated by cytotoxic drugs in NB. The CCK-8 assay showed that different concentrations of cytarabine and cisplatin affected the growth of SH-SY5Y cells after 24 h and IC₅₀ values 2 μM and 15 μM, respectively (Fig. 7a). Meanwhile, both cytarabine and cisplatin treatment of SH-SY5Y cells in a time-dependent manner (Fig. 7b). Treatment with cytarabine and cisplatin (2 μM and 15 μM, respectively) significantly increased the level of RUNX1 in time-dependent manner in SH-SY5Y cells. RUNX1 was obviously increased with cisplatin treatment compared with cytarabine treatment in SH-SY5Y cells (Fig. 7c). To further analyze whether cisplatin regulates the apoptosis via RUNX1, we performed the rescue experiments. Western blot (Fig. 7d), flow cytometry assays (Fig. 7e) and JC-1 (Fig. 7f) staining assays showed that the treatment of SH-SY5Y cells with cisplatin resulted in increased the ratio of Bcl-2/Bax and cleaved caspase-3, the late and early apoptosis and decreased mitochondrial depolarization, respectively compared with control group (DMSO + sh-Scb). Meanwhile, knockdown of RUNX1 abolished the change in apoptosis treated with cisplatin. Collectively, our data show that cisplatin could promote apoptosis through promoting RUNX1 expression in NB cells.

Fig. 7

Cisplatin promoted apoptosis by promoting RUNX1 expression in NB cells. a SH-SY5Y cells were treated with cytarabine (0, 2, 4, 6 and 8 μM) and cisplatin (0, 20, 40, 60 and 80 μM) for 24 h, the number of viable SH-SY5Y was analysed using CCK8 assay. b Cell counting kit-8 assay depicting the change in cell viability of SH-SY5Y cells under different time (0, 12, 24, 36, 48, 60, 72 h) of cytarabine and cisplatin treatment. c Western blot assays indicating the RUNX1 levels (normalized to β-actin, n = 5) in SH-SY5Y cells treated with DMSO, cytarabine and cisplatin at indicated time points. *p < 0.05 vs. DMSO. d Western blot assays (left panel) and the ratio of Bcl-2/Bax (right panel) showing the protein levels of RUNX1, Bax, Bcl-2, and cleaved caspase-3 in SH-SY5Y cells stably transfected with sh-Scb or sh-RUNX1#1, and those treated with cisplatin and DMSO for 48 h. e Representative images (left panel) and quantification (right panel) of flow cytometry showing the apoptosis of SH-SY5Y cells stably transfected with sh-Scb or sh-RUNX1 and treated with DMSO or cisplatin. Cells were double stained with an FITC-conjugated anti-Annexin V antibody and PI. The cells in the upper right and lower right quadrants depict late and early apoptosis. f Representative images (left panel) and quantification (right panel) of the mitochondrial membrane potential were evaluated with JC-1 (2 μg/ml) dye of SH-SY5Y cells stably transfected with sh-Scb or sh-RUNX1 and treated with DMSO or cisplatin. Scale bar, 100 μm. **P < 0.01 vs. sh-Scb + DMSO, ***P < 0.001 vs. sh-Scb + DMSO. Data are shown as mean ± SEM and representative of three independent experiments in panels (a)–(f). Data information: Data are presented as mean ± SEM. Exact P values are specified in Additional file 2: Table S3

Discussion

Previous studies have demonstrated that the transcription factor RUNX1 regulates apoptosis in acute myeloid leukemia [16], colorectal cancer [17], gastric cancer [18] and breast cancer [19]. However, the role of RUNX1 in NB and how it modulates apoptosis in NB remains unclear. In this study, we identified the transcription factor RUNX1 as an independent prognostic marker for progression and a favorable outcome of NB. We demonstrate that apoptosis genes BIRC5, CSF2RB and NFKBIA are direct targets of RUNX1 in NB, highlighting a novel role of RUNX1 in NB. In addition, cytotoxic drugs, especially cisplatin, significantly increased RUNX1 expression in NB cells and promoted apoptosis.

RUNX1 belongs to the Runt-related transcription factor family of genes and is also known as core binding factor-α (CBFα) [20, 21], which forms heterodimeric complexes with core binding factor β (CBFB) [22]. RUNX1 regulates the transcription of target genes in vertebrates by identifying the core consensus binding sequence 5′-TGTGGT-3’ [23] within the target area via the Runt domain; however, CBFB is a non-DNA-binding regulatory subunit that allosterically increases the capacity of RUNX1 DNA-binding sequence specificity. A previous study reported that co-occupancy of RUNX1 and AP-1 factors such as FOSL2 was enriched with distant regions, which demonstrated RUNX1 promoter occupancy for chromatin looping [24]. Low expression of RUNX1 is associated with the development of multiple types of tumors. In addition, overexpression of RUNX1 in U87 cells resulted in diminished tumor growth [24]. RUNX1 has been demonstrated to be downregulated in glioma, acute myeloblastic leukemia (AML) and gastric cancer; however, overexpression of RUNX1 in glioma, AML and gastric cancer reduced malignant progression [24]. It has been established that drugs that can promote RUNX1 expression are useful as single therapeutic agents. A recent study showed that RUNX1 facilitates the apoptosis of AML cells and that this effect of RUNX1 can be further enhanced by cytotoxic drugs [16]. Our evidence presented here indicates that RUNX1 directly binds to the promoters of BIRC5, CSF2RB and NFKBIA, which regulate apoptosis of NB. Interestingly, cytotoxic drugs work as agonists promoting the expression of RUNX1 thus further promoting apoptosis of NB cells. Underlying mechanisms of how cytotoxic drugs such as cisplatin act on the transcription factor RUNX1 warrant further investigations.

BIRC5 is a member of the inhibitor of apoptosis gene family, which prevents apoptosis through encoding negative regulatory proteins [25]. BIRC5 is a multitasking protein that promotes cell proliferation and prevents apoptosis [26]. NFKBIA inhibits the activity of dimeric NF-kappa-B/REL complex resulting from masking of their nuclear localization signals. Previous studies have demonstrated that NFKBIA serves as a tumor suppressor in glioblastomas and low expression of NFKBIA is associated with poorer survival in patients [27]. CSF2RB is the common beta chain of the high affinity receptor for IL-3, IL-5 and CSF and is weakly expressed in breast cancer cells [28]. Importantly, we demonstrated that a high expression of BIRC5 is indicative of a bad prognosis of NB-therapy and indicative of lower survival of the high-risk NB patients and these high levels of BIRC5 can be suppressed by overexpression of RUNX1. We want to note however that high levels of RUNX1 also inhibit the expression of CSF2RB and NFKBIA that are not always correlated with a better prognosis. Further studies of CSF2RB and NFKBIA levels will shed more light on their prognostic role in NB-thereputic outcoms.

Conclusions

In summary, we demonstrate here for the first time that RUNX1 is downregulated in NB tumors and it aids tumor growth by suppressing apoptosis. This is because, forced expression of RUNX1 facilitates apoptosis. RUNX1 does so by directly binding with the BIRC5, CSF2RB and NFKBIA promoters. Overexpression of RUNX1 suppresses NB cell growth, invasion, angiogenesis and metastasis in vitro and in vivo. We also show that RUNX1 is an independent prognostic factor for a favorable therapeutic outcome of NB. Surprisingly moreover, the classic cytotoxic chemotherapy drug, Cisplatin, induces the expression of RUNX1 in NB tumor cells. Thus the transcription factor RUNX1 may also be a potential therapeutic target for cisplatin-resistant NB.