Histone deacetylase inhibitor chidamide regulates the Wnt/β-catenin pathway by MYCN/DKK3 in B-ALL

Summary

Our previous studies revealed that MYCN downregulates the expression of DKK3, activates the Wnt/β-catenin signalling pathway at the transcriptional level, and thereby promotes the development of B cell acute lymphocytic leukaemia (B-ALL) but does not affect the methylation of the DKK3 promoter. Some studies have shown that MYCN is associated with histone acetylation. We speculate that histone deacetylase inhibitors (HDACis) can inhibit the Wnt/β-catenin signalling pathway by inhibiting MYCN and increasing the expression of DKK3. Based on previous experiments, we tested this hypothesis by analysing the changes in MYCN, DKK3 and the Wnt/β-catenin signalling pathways in B-ALL cells after treatment with the selective HDACi chidamide. The in vitro and in vivo experiments confirmed that chidamide inhibited the expression of MYCN and increased the expression of DKK3 by inhibiting the activity of histone deacetylase, and these effects resulted in inhibition of the Wnt/β-catenin signalling pathway and the proliferation of B-ALL cells. These findings indicate that chidamide might be used alone or in combination with other chemotherapy regimens for patients with B-ALL and thus provide a new approach to the treatment of B-ALL.

Keywords : B cell acute lymphocytic leukaemia . Chidamide . MYCN . DKK3 . Wnt/β-catenin

Introduction

B cell acute lymphocytic leukaemia (B-ALL) is a disease in which B lymphocytes are blocked at the immature stage, and due to the uncontrolled proliferation and accumulation of leukaemic cells in the bone marrow, these progenitor cells or precursor B leukaemic cells migrate from the bone marrow to the peripheral circulation and then infiltrate into the liver, spleen, lymph nodes, thymus, and central nervous system, which results in a high mortality rate [1]. At present, the main treatments for B-ALL include combined chemotherapy, im- munotherapy and haematopoietic stem cell transplantation, but some patients still experience drug resistance, relapse and a low long-term survival rate [2–5]. Therefore, the search for new cytogenetic and molecular biological mechanisms and the development of new targeted drugs are of great sig- nificance for the treatment of B-ALL.
The classical Wnt/β-catenin signalling pathway is related to the pathogenesis of B-ALL, and abnormal activation of the Wnt pathway can lead to the proliferation of B-ALL cells [6, 7]. The Dickkopf (DKK) family is a group of secretory glyco- proteins composed of DKK1, 2, 3, and 4 and soggy, which regulate the Wnt/β-catenin signalling pathway. DKK3, which is a negative regulator of Wnt/β-catenin, can inhibit the Wnt/ β-catenin signalling pathway and promote cell apoptosis [8]. MYCN, a member of the MYC proto-oncogene family, en- codes phosphorylated proteins of nuclear transcription activators/inhibitors that can directly upregulate or downreg- ulate genes by binding to promoters. MYCN can control cell proliferation, apoptosis and differentiation through indirect pathways and is also widely involved in tumorigenesis [9–11].

Our previous studies have found that MYCN downregulates the expression of DKK3 and activates the Wnt/β-catenin signalling pathway at the transcriptional level, which results in promotion of the development of B-ALL [12]. Treatment with the demethylation drug 5-Adc increases the expression of DKK3 and inhibits the Wnt/β-catenin sig- nalling pathway, which leads to inhibition of the proliferation of B-ALL cells. In MYCN-overexpressing Nalm6 and BALL- 1 cells, the levels of MYCN mRNA and protein remain high after treatment with 5-Adc, which indicates that MYCN does not affect the methylation of the DKK3 promoter [12]. Some studies have shown that the HDACi vorinostat can downreg- ulate the MYCN mRNA and protein levels in neuroblastomas and exerts a significant inhibitory effect on cell lines overex- pressing MYCN [13], which suggests that MYCN is associated with histone acetylation. We speculate that HDACis can in- hibit the Wnt/β-catenin signalling pathway by inhibiting MYCN and increasing the expression of DKK3.

Based on previous experiments, we tested this hypothesis by analysing the changes in MYCN, DKK3 and the Wnt/β- catenin signalling pathway in B-ALL cells after treatment with chidamide. Chidamide, a novel synthetic benzamide HDACi that was independently developed in China, can se- lectively inhibit the activity of HDAC1, 2, 3, and 10 after its oral administration and was approved by the China Food and Drug Administration (CFDA) for the treatment of refractory or relapsed peripheral T cell lymphoma (PTCL) in December 2014 [14]. In vitro and in vivo experiments confirmed that chidamide inhibited the expression of MYCN and increased the expression of DKK3 by inhibiting the activity of HDAC, and these effects resulted in inhibition of both the Wnt/β- catenin signalling pathway and the proliferation of B-ALL cells. These findings indicate that chidamide can be used alone or in combination with other chemotherapy regimens for the treatment of patients with B-ALL and thus provide a new approach for the treatment of B-ALL.

Materials and methods

Reagents

Chidamide was provided by Shenzhen CHIPSCREEN BIOSCIENCES Co., Ltd.were cultured at 37 °C in an incubator with 5% CO2 (HF-90, Shanghai, China).

Cell proliferation assay

Nalm6, BALL-1 and SupB15 cells were cultured to a density of approximately 90% and inoculated in 96-well culture plates. After overnight culture, the cells were treated with chidamide (2 μM/L) [15] and cultured at 37 °C in an incubator with 5% CO2 for 24, 48 and 72 h. At each time point, 10 μL of CCK-8 was added, and after 2 h, the OD value was measured with a microplate reader at 450 nm.

Cell cycle and apoptosis analysis

For cell cycle analysis, Nalm6, BALL-1 and SupB15 cells were exposed to chidamide for 48 h, collected, washed twice with phosphate-buffered saline (PBS), resuspended in 70% ethanol and fixed for 12 h at 4 °C. The cells were then washed twice with PBS and incubated with 100 μL of RNase and 500 μL of propidium iodide staining solution in the dark for 30 min at 4 °C. Apoptotic cells were stained with Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI). The cell cycle and apoptosis were then detected by flow cytometry.

Quantitative real-time PCR

Total RNA was extracted using TRIpure (RP1001, BioTake, Beijing, China). The concentration of RNA in each sample was determined using an ultraviolet spectrophotometer (NANO 2000). The obtained RNA samples were reverse tran- scribed to obtain corresponding cDNAs. The PCR amplifica- tion system included the cDNA as the template, forward and reverse primers, SYBR Green, Taq PCR Master Mix and dis- tilled water. The fluorescence quantitative analysis was per- formed using a fluorescence quantitative PCR instrument (Exicycler 96, BIONEER, Korea). The experiments were per- formed at least three times, and the data were analysed using the 2-△△CT method. The primer sequences for the target genes are indicated in Table 1.

Western blot

Protein was extracted from the cells, and the protein concentration was calculated according to the standard curve. The total protein was separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes, and the PVDF membranes were blocked with 5% skimmed milk powder for 1 h. The PVDF membranes were incubated with the diluted primary antibodies over- night at 4 °C and then incubated with the secondary antibody for 1 h at room temperature. The substrate was emitted by ECL, and the optical density of the target strip was analysed using a gel image processing system (Gel-Pro-Analyser software). Primary antibodies for MYCN, DKK3, Ace-H3K18 and Ace-H4K8 were purchased from ABclonal Biotechnology Co., Ltd. (Wuhan, China), and cyclin D1, Bcl-2, Bax, β-catenin, p-GSK3β, GSK3β and GAPDH were purchased from Wanlei Biotechnology Co., Ltd. (Shenyang, China). GAPDH was used as the internal reference.

In vivo experiments

After 1 week of adaptive feeding, 5-week-old NOD/SCID nude mice weighing 17–23 g were randomly divided into two groups of three mice each. The mice in group A were subcutaneously inoculated with untreated Nalm6 cells in the right proximal axilla, and those in group B were subcutane- ously inoculated with Nalm6 cells treated with chidamide for 48 h. After 1 week, the tumours of the mice were photographed every 3 days, and the tumours were circled with a marker. The long (a) and short diameters (b) of the xenograft tumours were measured every 3 days, and the tumour volumes were calculated as ab2/2. At the end of day 22, the mice in each group were sacrificed, and the tumour tissues were stripped, weighed and photographed. The TUNEL assay was used for the detection of apoptosis. Western blotting was used to detect the expression of MYCN, DKK3, β-caten- in, GSK3β and p-GSK3β in tumour tissues.

Statistical analysis

The data are expressed as the mean values ± standard devia- tions and were statistically analysed using a matched t test. P < 0.05 was considered to indicate significance. All the data were analysed using GraphPad Prism 5.0 (GraphPad Software, USA) software.

Results

Chidamide induces cell cycle arrest, promotes apoptosis and inhibits the proliferation of Nalm6, BALL-1 and SupB15 cells

To confirm the effect of chidamide on B-ALL cell lines, we evaluated the effects of chidamide on the proliferation, apoptosis and the cell cycle of Nalm6, BALL-1 and SupB15 cells. As shown in Fig. 1A, D and G, chidamide inhibited the proliferation of Nalm6, BALL-1 and SupB15 cells. We then detected the effect of chidamide on the cell cycle and apoptosis of Nalm6, BALL-1 and SupB15 cells through flow cytometry. After treat- ment with chidamide, the proportion of Nalm6 cells at the G1 phase increased from 46.93 ± 0.93% to 58.70 ± 0.72%, and the proportion of these cells at the S phase decreased from 42.63 ± 0.60% to 30.43 ± 0.96% (Fig. 1B and J). The proportion of BALL-1 cells at the G1 phase increased from 42.07 ± 0.75% to 53.47 ± 2.01%, and the proportion of these cells at the S phase decreased from 48.53 ± 0.78% to 36.63 ± 1.86% (Fig. 1E and K). Moreover, the proportion of SupB15 cells at the G1 phase increased from 56.83 ± 1.20% to 67.10 ± 1.57% and that at the S phase decreased from 31.70 ± 1.30% to 20.03 ± 1.82% (Fig. 1H and L). These findings confirmed that chidamide can induce cell cycle arrest at the G1 phase. In addition, the treatment of Nalm6, BALL-1 and SupB15 cells with chidamide increased the per- centages of total apoptotic cells, early apoptotic cells and late apoptotic cells, which indicated that chidamide can promote ap- optosis in B-ALL cell lines (Fig. 1C, F, I, M,N and O). We then performed Western blotting to detect the expression of the cell cycle- and apoptosis-related proteins cyclin D1, Bcl-2 and Bax in Nalm6, BALL-1 and SupB15 cells. The results showed that treatment with chidamide decreased the expression of cyclin D1 and Bcl-2 proteins and increased the expression of Bax pro- tein (Fig. 3B). All the results indicate that chidamide can induce cell cycle arrest, promote apoptosis and inhibit the proliferation of Nalm6, BALL-1 and SupB15 cells.

Chidamide decreases the expression of MYCN mRNA and protein and increases the expression of DKK3 mRNA and protein in Nalm6, BALL-1 and SupB15 cells

In our previous experiments, we analysed the promoter region of DKK3 and identified several MYCN-binding sites with transcrip- tion factor activity. Double luciferase reporter gene detection and ChIP assays have confirmed that MYCN could directly bind to the promoter region of the DKK3 gene to downregulate the ex- pression of DKK3 mRNA and protein, activate the Wnt/β- catenin pathway, promote the proliferation of B-ALL cells and serve as proto-oncogenes [12]. Treatment with the demethylated drug 5-Adc increases the expression of DKK3 and inhibits the Wnt/β-catenin pathway, which results in inhibition of the prolif- eration of B-ALL cells, and MYCN is not related to the overexpressing cell lines [13], which suggests that MYCN is associated with histone acetylation. To confirm the relationship between MYCN and histone acetylation in B-ALL cell lines, we detected the expression of MYCN and DKK3 mRNA and protein in Nalm6, BALL-1 and SupB15 cells treated with chidamide by qRT-PCR and Western blotting assays. Chidamide decreased the expression of MYCN mRNA and protein and increased the ex- pression of DKK3 mRNA and protein in Nalm6, BALL-1 and SupB15 cells (Fig. 2A-F and Fig. 3A).

Chidamide inhibits the expression of MYCN and increases the expression of DKK3 by inhibiting the activity of HDAC, which results in inhibition of both the Wnt/β-catenin signalling pathway and the prolif- eration of B-ALL cells

HDACis can inhibit the proliferation of tumour cells by inhibiting the activity of HDAC, changing the level of histone acetylation and regulating the expression of cytokines and transcription factors. As shown in Fig. 3C, the expression of Ace-H4K8 and Ace-H3K18 proteins in B-ALL cell lines in- creased after treatment with chidamide, which indicated that chidamide upregulated the expression of Ace-H4K8 and Ace- H3K18 proteins by inhibiting the activity of HDAC and there- by inhibited the proliferation of B-ALL cell lines.

Our previous experiments confirmed that MYCN regulates the expression of the Wnt/β-catenin signalling pathway by inhibiting DKK3 in B-ALL cell lines [12], and some studies have speculated that MYCN is related to histone acetylation [13]. To study the effect of chidamide on the Wnt/β-catenin signalling pathway, we detected the expression of GSK3β, p- GSK3β and β-catenin (cytoplasmic and nuclear) proteins, which are related to the Wnt/β-catenin pathway, in Nalm6, BALL-1 and SupB15 cells by western blotting. As shown in Fig. 3D, treatment with chidamide decreased the expression of p-GSK3β protein in Nalm6 and BALL-1 cells and the expres- sion of nuclear β-catenin protein in Nalm6, BALL-1 and SupB15 cells. The results showed that the Wnt/β-catenin sig- nalling pathway was inactivated by chidamide, which indi- cates that this HDACi can inhibit the Wnt/β-catenin signalling pathway in B-ALL cell lines to inhibit cell proliferation.

Chidamide exerts antitumour effects in mice with Nalm6 cell xenografts

To evaluate the effect of chidamide on B-ALL cell lines in vivo, we examined the antitumour effect of chidamide in a mouse model. Compared with the control group, chidamide treatment significantly reduced the tumour volume (Fig. 4A and C) and weight (Fig. 4B) during the 22-day observation period. TUNEL staining of the xenograft tumour tissues showed that chidamide treatment increased the number of apoptotic cells, decreased the size of apoptotic cells, decreased nuclear and cytoplasmic pyknosis (Fig. 4D), and increased the number of TUNEL- positive cells (Fig. 4E). All the results show that chidamide can promote the apoptosis of Nalm6 cell xenografts.

To further explain the effects of chidamide on MYCN, DKK3 and the Wnt/β-catenin signalling pathways in vivo, we analysed the expression of related proteins by Western blotting. Treatment with chidamide decreased the expression of MYCN protein, increased the expression of DKK3 protein, and decreased the expression of p-GSK3β and nuclear β- catenin protein (Fig. 3E and F). These findings indicate that chidamide inhibits the expression of MYCN and increases the expression of DKK3 by inhibiting the activity of HDAC, which results in inhibition of both the Wnt/β-catenin signal pathway and the proliferation of B-ALL cells in vivo.

Discussion

The treatment of B-ALL has greatly improved since the 1970s, when the survival rate was only 10% [16]; however, the currently overall 5-year survival rate of adult B-ALL is only 30%–40%, whereas that of children is 90% [1, 17]. Therefore, the search for new cytogenetic and molecular bio- logical mechanisms and the development of new targeted drugs are vital for the treatment of adult B-ALL.

Almost all human cancers are involved in epigenetic changes, which contribute to the development of cancer due to their regulation of epigenetic modifications during the pro- cess of gene transcription [18–21]. Disorder of the epigenetic structure, which leads to the early abnormal cloning and ex- pansion of stem cells/progenitor cells, is considered the most important event in cancer formation [22]. Abnormal histone acetylation is involved in the regulation of permanent changes in phenotypic gene expression in ALL. Because histone acet- ylation is reversible, the development of drugs for the treat- ment of ALL that target proteins and enzymes involved in the regulation of histone acetylation has become a new therapeu- tic strategy [23]. Studies have shown that HDACis are poten- tial options for the treatment of B-ALL, and HDAC1 and HDAC2 inhibitors might exert therapeutic effects on patients [24]. Chidamide, a novel synthetic benzamide HDACi that was independently developed by China, can selectively inhibit (cytoplasmic and nuclear) (D) in Nalm6, BALL-1 and SupB15 cells are shown. (E, F) Representative Western blots showing the effects of chidamide on the expression of MYCN, DKK3, p-GSK3β, GSK3β and β-catenin (cytoplasmic and nuclear) proteins in two groups of transplanted mouse models are shown. The results originated from three independent experiments, and GAPDH served as the internal reference chemotherapy regimens can be used for the treatment of AML [25–29], lymphoma [30–32], and T-ALL [33], but this HDACi has rarely been used for the treatment of B-ALL. In this study, we found that treatment with chidamide arrested B- ALL cell lines at the G1 phase, decreased the expression of cyclin D1 and Bcl-2 protein and increased the expression of Bax protein, which indicated that chidamide can induce cell cycle arrest at the G1 phase and promote apoptosis. Our CCK- 8 assay also showed that chidamide can inhibit the prolifera- tion of B-ALL cell lines, and further experiments confirmed that chidamide can promote the apoptosis of Nalm6 cell xe- nografts in mice.

Fig. 3 Effects of chidamide on the expression of MYCN and DKK3 protein, cell cycle- and apoptosis-related proteins, histone deacetylated proteins and Wnt/β-catenin signalling pathway-related proteins. Representative Western blots showing the effects of chidamide on the protein expression of MYCN, DKK3 (A), cyclin D1, Bcl-2, Bax (B), Ace-H4K8, Ace-H3K18 (C), p-GSK3β, GSK3β and β-catenin.

Fig. 4 Chidamide inhibited the expression of MYCN and increased the expression of DKK3 by inhibiting the activity of HDAC, which resulted in inhibition of the Wnt/β-catenin signalling pathway and the pro- liferation of B-ALL cells in vivo. The changes in the tumour vol- ume (A, C) and weight (B) in mice with Nalm6 cell xenografts before and after chidamide treat- ment were detected. (D) TUNEL assay of transplanted mouse models. Positive cells were stained brown, and the original magnification was 400X. Representative fields are shown. The arrows indicate tumour cells. Bar = 50 μm. (E) Comparison.

In our previous experiments, we found that treatment with the demethylation drug 5-Adc can increase the expression of DKK3 and inhibit the Wnt/β-catenin signalling pathway,which results in inhibition of the proliferation of B-ALL cells. In MYCN-overexpressing Nalm6 and BALL-1 cells, the levels of MYCN mRNA and protein remain high after treatment with 5-Adc, which indicates that MYCN does not affect the methylation of the DKK3 promoter [12]. MYCN protein can recruit the histone acetyltransferase complex to maintain the acetylation of chromosomes and can thus enhance transcrip- tion, particularly that of genes involved in cell cycle progres- sion and proliferation [34]. The acetylation levels are coordi- nated by the synergy of histone acetyltransferases (HATs) and HDACs. HDACs consist of a series of enzymes that can re- verse the acetylation of nuclear and cytoplasmic proteins, which results in reductions in the expression of many differentiation-related genes and regulation of the function of acetylated nuclear and cytoplasmic proteins. Therefore,MYCN can recruit HDACs to promote histone deacetylation, DNA aggregation and the inhibition of genes, including genes that encode proapoptotic factors [14]. HDACis can inhibit the deacetylation of histones and other proteins, which results in cell cycle arrest at the G2/M phase and thereby the induction of tumour cell differentiation and apoptosis, and exhibit low toxicity to normal cells [35]. Some studies have evaluated the relationship between the effect of SAHA on the viability of neuroblastoma cells and the amplification and overexpression of MYCN in different human neuroblastoma cell lines [13]. These studies demonstrated that SAHA decreases the viability of all tested cell lines and simultaneously induces a decrease in MYCN expression [13]. Based on these findings, we speculat- ed that MYCN is related to histone acetylation in B-ALL. Our results showed that chidamide inhibits the expression of MYCN and increases the expression of DKK3 in B-ALL cell lines in vitro and in vivo, which indicates that MYCN is related to histone acetylation in B-ALL.

Evolutionarily conserved Wnt signalling pathways play an important role in the development of many organ systems [36]. An abnormal Wnt signalling pathway is one of the char- acteristics of many solid tumours, particularly colon and he- patocellular carcinoma [37, 38]. The classical Wnt/β-catenin signalling pathway is related to the pathogenesis of B-ALL, and abnormal activation of the Wnt pathway can lead to the proliferation of B-ALL cells [6, 7]. In our previous experi- ments, we found that MYCN can activate the Wnt/β-catenin signalling pathway by targeting the inhibition of DKK3 ex- pression to promote B-ALL cell proliferation and inhibit apo- ptosis [12]. In this study, we found that treatment chidamide decreases the expression of p-GSK3β protein in Nalm6 and BALL-1 cells and the expression of nuclear β-catenin protein in Nalm6, BALL-1 and SupB15 cells, which indicates that chidamide can inhibit the Wnt/β-catenin signalling pathway in B-ALL cell lines and thereby inhibits cell proliferation. Our study also showed that chidamide can inhibit the growth of Nalm6 cell xenografts in mice, promote the apoptosis of transplanted tumour cells, decrease the expression of MYCN protein, increase the expression of DKK3 protein, and de- crease the expression of p-GSK3β and nuclear β-catenin pro- tein, and these effects result in inhibition of the Wnt/β-catenin signalling pathway and inactivation of the Wnt/β-catenin sig- nalling pathway in vivo. Our previous experiments showed that MYCN siRNA can increase the expression of DKK3 mRNA and protein, decrease the expression of p-GSK3β and nuclear β-catenin protein and inhibit the activity of the Wnt/β-catenin pathway in Nalm6 and BALL-1 cells [12]. We also previously confirmed that Lv-MYCN shRNA can upregulate the expression of DKK3 mRNA and protein, inhibit the Wnt/β-catenin pathway and pro- mote the apoptosis of B-ALL cells in vivo [12]. The findings suggest that both chidamide and the silencing of MYCN can increase the expression of DKK3, inhibit the Wnt/β-catenin signalling pathway and promote the apoptosis of B-ALL cells. Therefore, we concluded that chidamide can inhibit the Wnt/β-catenin signalling path- way and cell proliferation by inhibiting MYCN and in- creasing the expression of DKK3 in B-ALL.However, our study did not involve B-ALL patients; thus, further research is needed to confirm the effect of chidamide in B-ALL patients. In the present study, we confirmed that chidamide can inhibit the proliferation of B-ALL cells by inducing cell cycle arrest and promoting apoptosis. The in vitro and in vivo experiments demonstrated that chidamide inhibits the expression of MYCN and increases the expression of DKK3 by inhibiting the activity of HDAC, which results in inhibition of the Wnt/β-catenin signalling pathway and the proliferation of B-ALL cells. The findings indicate that chidamide might be used alone or in combination with other chemotherapy regimens for patients with B-ALL and thus provide a new approach for the treatment of B-ALL.