ZEB1 serves as an oncogene in acute myeloid leukaemia via regulating the PTEN/PI3K/AKT signalling pathway by combining with P53

Abstract Acute myeloid leukaemia is a complex, highly aggressive hematopoietic disorder. Currently, in spite of great advances in radiotherapy and chemotherapy, the prognosis for AML patients with initial treatment failure is still poor. Therefore, the need for novel and efficient therapies to improve AML treatment outcome has become desperately urgent. In this study, we identified the expression of ZEB1 (a transcription factor) and focused on its possible role and mechanisms in the progression of AML. According to the data provided by the Gene Expression Profiling Interactive Analysis (GEPIA), high expression of ZEB1 closely correlates with poor prognosis in AML patients. Additionally, the overexpression of ZEB1 was observed in both AML patients and cell lines. Further functional experiments showed that ZEB1 depletion can induce AML differentiation and inhibit AML proliferation in vitro and in vivo. Moreover, ZEB1 expression was negatively correlated with tumour suppressor P53 expression and ZEB1 can directly bind to P53. Our results also revealed that ZEB1 can regulate PTEN/PI3K/AKT signalling pathway. The inhibitory effect of ZEB1 silencing on PTEN/PI3K/AKT signalling pathway could be significantly reversed by P53 small interfering RNA treatment. Overall, the present data indicated that ZEB1 may be a promising therapeutic target for AML treatment or a potential biomarker for diagnosis and prognosis.

Epithelial-mesenchymal transition (EMT) is considered to be a critical process in normal embryonic development, which has also been revealed to be involved in the tumorigenesis, metastasis and drug resistance. 8 Zinc finger E-box-binding homeobox 1 (ZEB1), a member of the zinc finger transcription factor family, regulates the expression of epithelial/mesenchymal markers (such as E-cadherin, N-cadherin and vimentin) to modulate the EMT progression. 9 Upregulation of ZEB1 has been found in a variety of tumours and positively correlated with the degree of malignancy and poor prognosis. 10 Previous studies have shown that ZEB1 acts with other transcriptional regulators and regulates tumour metastasis via EMT in lung cancer, 11 hepatoma, 12 breast cancer 12,13 and colorectal cancer. 14,15 In addition, ZEB1 is implicated in the regulation of proliferation and differentiation of various cells, such as neuronal progenitors, 16 vascular smooth muscle cells 17 and osteoblasts. 18 Wafaa Ghoneim Shousha al 19 reports that ZEB1 has potential as diagnostic and prognostic marker for AML. However, the specific molecular mechanism has not been clarified.
P53 serves as a well-known tumour suppressor that is impli-  20 The PTEN/PI3K/AKT signalling pathway plays a vital role in the initiation and progression of multiple tumours by modulating cell proliferation, differentiation, invasion and metastasis. 21 In a recent study, miR-205 suppresses the activation of AKT/mTOR signalling pathway by down-regulation of ZEB1, reverses EMT and thereby inhibits glioblastoma cells migration and invasion. 22 In addition, PTP4A1 promotes proliferation and invasion in intrahepatic cholangiocarcinoma via up-regulating ZEB1, accompanied by the activation of PI3K/AKT pathway. 23 In addition, P53 can regulate the PTEN/ PI3K/AKT signalling pathway. 24 Based on the above observations, the present study was conducted to explore whether ZEB1 regulated the PTEN/PI3K/AKT signalling pathway by associating with P53 in AML.

| Cell culture
Normal monocyte cell line SC, acute myeloid leukaemia cell lines NB4, K562 and THP-1 were all purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences and cultured in our laboratory under standard conditions. The medium was changed every 24 hours, and the subculture cells were changed every 48 hours.
Scrambled siRNA, ZEB1 siRNA and P53 siRNA were synthesized by GeneChem (Shanghai, China). GV141-ZEB1 and empty GV141control were also designed by GeneChem. Transfection of cells was conducted using Lipofectamine™ 2000 (Invitrogen, USA) according to the manufacturer's protocol. The medium was exchanged 6 hours after transfection. All experiments were repeated at least three times.

| Western blot analysis
Total lysates from cells or tissue samples were extracted using RIPA lysis buffer (Beyotime) containing protease inhibitor cocktail. After being mixed with 5× loading buffer, samples were boiled at 100°C for 10 minutes and then subjected to SDS/PAGE on a 10% gel.

| Differentiation marker analysis
After the respective treatment, cells were harvested and washed twice with pre-cooling PBS, followed by incubation with 1 µL

| Cell cycle analysis
After the respective treatment, cells were harvested and washed twice with pre-cooling PBS and then fixed in 75% cold ethanol at −20℃ overnight. After centrifugation at 1000 × g for 10 minutes, the cells were resuspended in PBS containing RNAse A (20 μg /mL) for 30 minutes at 37°C water bath, and then, 400 µl PI was added and incubated on ice for 30 minutes. Stained cells were evaluated by a flow cytometry (Becton Dickinson) to determine cell cycle distribution. Data were analysed using ModFit software (Verity Software House).

| Double-immunofluorescent staining
Double-immunofluorescent staining was performed to assess colocalization of ZEB1 and P53. Cells were plated in six-well plates and fixed with 4% paraformaldehyde for 10 minutes, blocked with 10% BSA for 10 minutes and incubated with primary antibodies and secondary antibody, respectively. Afterwards, DAPI was used to stain the nucleus and cells were observed under a laser scanning confocal microscope. 6-week-old male NCG nude mice were purchased from the Nanjing

| Samples
Bone human bone marrow specimens from newly diagnosed AML

| Co-immunoprecipitation (Co-IP) assay
The co-immunoprecipitation assay (Co-IP) assay was conducted using the Co-IP Pull-Down Kit (Ribobio Guangzhou) according to the manufacturer's protocol. NB4 and THP-1 cells were lysed in prechilled lysis buffer containing phosphatase inhibitors and protease inhibitors. Lysates were incubated with protein A/G beads and anti-ZEB1 antibodies with gentle shaking overnight at 4°C. Beads were then washed extensively, eluted with the buffer and analysed by an immunoblot analysis as previously described.

| Statistical analysis
SPSS software (version 19.0) was used for statistical analysis of data, and all values are represented by mean ± standard deviation (SD). Students' t test was used for comparison among groups, and single ANOVA was used for comparison among multiple groups, P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01).

| ZEB1 was up-regulated in AML and suggests poor prognosis
To investigate potential significance of ZEB1 in AML, we first analysed the expression levels of ZEB1 in AML using the available data sets from the GEPIA database (http://gepia.cance r-pku.cn/detail.php). Relatively high level of ZEB1 was detected in AML patients (left bar) compared with normal cases (right bar) ( Figure 1A). Importantly, Kaplan-Meier analysis demonstrated that AML patients with overexpression of ZEB1 exhibited worse overall survival ( Figure 1B). These results indicate a possible link between ZEB1 and AML progression. To further confirm our findings, the Western blot analyses were conducted to evaluated ZEB1 expression in AML patients and the healthy controls.
We found AML group have higher ZEB1 levels than the healthy group ( Figure 1C). In addition, compared with normal human monocytes, multiple leukaemia cell lines, such as NB4, THP-1 and K562, exhibited a significantly elevated protein level of ZEB1 ( Figure 1D). From this, we could infer that ZEB1 is closely related to AML progression.

| Down-regulation of ZEB1 inhibited proliferation and induced differentiation of AML cells in vitro
Considering high ZEB1 expression in AML, we further explored its role in the proliferation and differentiation of AML cells. First, the siRNA-ZEB1 or negative control (siRNA-NC) was transfected into NB4 and THP-1 cell lines. As shown in Figure 2A, silencing efficiency was confirmed by Western blotting. We next performed a series of experiments to explore the functional significance of ZEB1. Flow cytometric assays showed ZEB1 silencing resulted in increased G0/G1 phase arrest and decreased the population of S phase ( Figure 2B).
Consistently, the cell cycle markers (CDK4, cyclin A2, cyclin D1, p-Rb) were detected to be down-regulated in ZEB1-silenced group ( Figure 2C). Flow cytometry also showed that ZEB1 could inhibit proliferation-related protein Ki67 ( Figure 2D). To further study the effects of ZEB1 on cell differentiation, the flow cytometry ( Figure 2E) and Western blot analysis ( Figure 2F) were performed, showing that knock-down of ZEB1 promoted CD11b and CD14 (relatively classic markers of differentiation) expression in NB4 and THP-1 cells. In addition, similar results were obtained when cell morphology was evaluated using Wright-Giemsa staining ( Figure 2G). In the si-ZEB1 group, cells were characterized by matured appearances that were the smaller nucleoli, the decreased nuclear/cytoplasm ratio. Taken together, these data above indicated that silencing of ZEB1 suppressed AML proliferation and promoted differentiation.

| ZEB1 can interact with P53 and regulate its expression in AML cells
All above data suggested that ZEB1 was essential for the differentiation and proliferation of AML. We next sought to identify the involved molecular mechanisms. In order to verify the interaction between ZEB1 and P53, double-immunofluorescent staining Co-IP and were performed. The Co-IP experiments in AML cells revealed apparent binding of ZEB1 and P53 ( Figure 3A). According to the combined fluorescence images, obvious colocalization of ZEB1 and P53 was found in the cytoplasm ( Figure 3B). Finally, we examined the correlations between ZEB1 and P53 at protein levels. Western blotting showed that ZEB1 silencing promoted the expression of P53 ( Figure 3D, F), whereas ZEB1 overexpression inhibited the expression of P53 ( Figure 3C, E). Altogether, ZEB1 could bind to P53 and regulate its expression.

| The regulatory function of ZEB1 on PTEN/ PI3K/AKT signalling pathway can be reversed by knock-down of p53
PTEN/PI3K/AKT is an important intracellular signalling pathway, participating in the regulation of various cellular functions such as cell proliferation, differentiation, apoptosis and glucose transport.
In view of the above results, we explored whether ZEB1 exerted its effect via the PTEN/PI3K/AKT pathway. Western blot analysis revealed that the protein levels of PTEN, p-PI3K and p-AKT were significantly reversed in ZEB1 silenced cells ( Figure 4A). As expected, P53 knock-down also reversed the inhibition of PTEN/PI3K/AKT signalling by ZEB1 silencing in AML cell lines ( Figure 4B). Taken together, ZEB1 might mediate the PTEN/PI3K/AKT signalling by p53 in AML cells.  Cell cycle arrest is one of the indicators of proliferation inhibition and occurs due to the loss of cyclins expression and cdks activity. 28 The regulation of cell proliferation, cell cycle and differentiation is considered an effective strategy for leukaemia treatment. Having Notably, whether ZEB1 is involved in apoptosis and drug resistance in AML cells remain to be further investigated.

| D ISCUSS I ON
Previous reports have indicated that several zinc finger transcription factors, including Snail1, Slug and Twist, directly or indirectly regulate P53 function in vitro and in vivo. 29 As we all known, P53 is a classical tumour suppressor protein.
Overall, ZEB1 can bind to P53 and regulate its expression, which may be another mechanism of ZEB1 regulating AML. The silencing of p53 significantly restored the inhibition of PI3K/AKT signalling induced by ZEB1 silencing in AML cell lines ( Figure 6). In general, ZEB1 may be a new therapeutic target for AML. F I G U R E 5 Effects of ZEB1 on tumour growth in vivo. A, B, Tumour images and weights at experimental end-points in NC and shZEB1 NB4 xenografts (n = 5 for each group). C, Western blot analysis of cyclin D1, cyclin A2, P-Rb and CDK4 in tumour tissues of NC and shZEB1 groups. D, Western blot analysis of CD11b and CD14 in tumour tissues of NC and shE2F4 groups. β-actin was used as an internal control. Bar graphs (mean ± SD) and representative images are shown. *P < 0.05, **P < 0.01, compared with the NC group NC shZEB1 NC shZEB1 Conceptualization (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon request.