TAS4464

SOX4-induced upregulation of ARHGAP9 promotes the progression of acute myeloid leukemia

Xin He1 |Haizhu Zou1|Fengyu Wang2

Abstract

Acute myeloid leukemia (AML) is the most common acute leukemia. Rho GTPase activating protein 9 (ARHGAP9) has been reported to be positively correlated with overall survival of AML patients, but the specific molecular function remains unclear. This study aims to further explore the functional role and the molecular mechanism of ARHGAP9 in AML cells. The expression level of ARHGAP9 in AML cells was measured using quantitative real-time PCR (qRT-PCR) and western blot. Cell transfection was performed to interfere ARHGAP9. CCK-8, flow cytometry and TUNEL assays were conducted to detect cell viability, cell cycle distribution and apoptosis, respectively. The binding relationship between SOX4 and ARHGAP9 promoter was verified using luciferase reporter assay and chromatin immunoprecipitation. The results showed that ARHGAP9 was upregulated in AML cells. Interference of ARHGAP9 greatly reduced cell viability and induced cell cycle arrest in G1 phase, accompanied with the reduction of Ki67, PCNA, cyclin D1, cyclin E1, CDK4 and CDK6. In addition, Interference of ARHGAP9 greatly promoted cell apoptosis, accompanied with the decreased protein expression of Bcl-2 and the increased protein expression of Bax, cleaved caspase 3 and cleaved caspase 9. Furthermore, SOX4 directly bound to ARHGAP9 promoter and regulated ARHGAP9 expression. In conclusion, this study suggested that ARHGAP9 interference exerted an antitumor effect through inhibiting cell proliferation, blocking cell cycle progression, and promoting cell apoptosis in AML cells. ARHGAP9 may serve as a novel therapeutic target for AML.

KEYWORDS
acute myeloid leukemia, apoptosis, ARHGAP9, proliferation, SOX4

1 | INTRODUCTION

Acute myeloid leukemia (AML) is a heterogeneous group of clonal hematopoietic malignant disorder, which results from genetic alternation and/or epigenic changes in blood cell precursors-induced overproduction of neoplastic clonal myeloid stem cells (Pelcovits & Niroula, 2020). Currently, the standard treatment for AML is chemotherapy with hematopoietic stem cell transplantation. However, due to the chemotherapy resistance in leukemia cells and high relapse rates in AML patients, the prognosis of patients with AML is unsatisfactory (Chiu et al., 2016; Ofran & Rowe, 2014; Xu et al., 2015). Thus, a more in-depth knowledge of the biology of AML is urgently needed for more rational and efficient therapies.
Rho GTPase activating protein 9 (ARHGAP9), a member of Rho family of small guanosine triphosphatases (RhoGTPases) activating protein (RhoGAP), encodes four functional protein domains, including RhoGAP domain, src-homology 3 (SH3) domain, WW domain, and pleckstrin homology (PH) domain(Ang et al., 2007; Furukawa et al., 2001). RhoGAP proteins are key regulators of the actin cytoskeleton, playing a crucial role in cell adhesion, migration and invasion. As these cell functions commonly contribute to cancer cell metastasis and overall survival of cancer patients, RhoGAPs have been emerging as the novel cancer-related biomarkers in the recent years(Gujral et al., 2014; Kandpal, 2006; Kucia-Tran et al., 2016). Consistently, ARHGAP9 has been investigated in diverse cancers in recent years. Zhang H et al reported that a lower expression of ARHGAP9 was found in hepatocellular carcinoma tissues than in normal tissues, which was also closely correlated with a shorter overall survival time of cancer patients, and ARHGAP9 overexpression could suppress the migration and invasion of hepatocellular carcinoma cells(Zhang et al., 2018). In contrast, Wang and Ha (2018) revealed a high expression of ARHGAP9 in breast cancer tissues and cell lines, and knockdown of ARHGAP9 reduced cell proliferation, migration and invasion abilities in breast cancer cells. These observations indicate that ARHGAP9 may play a dual role in different types of cancers. To our surprise, a study concerning on the role of ARHGAP9 in clinical AML was just published. This study concluded that ARHGAP9 was highly expressed in AML patients associated with poor overall survival and could be used as a prognostic biomarker (Han et al., 2021). However, more biological functions of ARHGAP9 in the occurrence and development of AML remain unclear, which need an in-depth research.
In the present study, we further investigated the role of ARHGAP9 in AML, and explored its functions in regulating AML cellular biological behaviors, raising the possibility of novel targeted biomarkers for AML treatment.

2 | MATERIALS AND METHODS

2.1 | Cell culture

The human normal bone marrow cell line HS-5 and AML cell lines, including Kasumi-1, ME-1, HL60 and KG-1a, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). MOLM-14 cell line was obtained from Yaji Biological (Shanghai, China). All cells were cultured in RPMI-1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco, Rockville, MD) and 1% penicillin– streptomycin mixture in a humidified atmosphere with 5% CO2 at 37C.

2.2 | Quantitative real-time PCR

Total RNA was extracted from cells by using Trizol agent (Invitrogen), and then reversely transcribed into complementary DNA (cDNA) in accordance with the instructions of Takara PrimeScript RT Master Mix (Takara, Dalian, China). Quantitative real-time PCR (qRT-PCR) was carried out using the SYBR Premix Ex Taq (Perfect Real Time, Takara). The expression of β-actin was used for normalization, and the fold differences were analyzed using 2ΔΔCt method.

2.3 | Cell transfection

Cells were transfected in accordance with the instructions of Lipofectamine 3000 (Invitrogen, Carlsbad, CA). Cells were transfected with short hairpin RNA targeting ARHGAP9 (shRNA-ARHGAP9) and the negative control (shRNA-NC), which were generated from GenePharma (Shanghai, China). 48 h post transfection, cells were harvested for transfection efficacy detection using qRT-PCR.

2.4 | Cell counting kit-8 assay

Cell viability was detected using cell counting kit-8 (CCK-8; Dojindo, Kumamoto, Japan). In brief, cells were incubated in 96-plates for 24, 48, and 72 h, respectively. Then, 10 μm of CCK-8 agent was added to each well for a further incubation at 37C for 1–2 h. The absorbance at 450 nm was recorded using a microplate reader.

2.5 | Flow cytometry assay

Cell cycle was analyzed using flow cytometry assay. In brief, after transfection for 48 h, cells were harvested, washed with PBS and fixed with 70% cold ethanol overnight. Then, cells were incubated with propidium iodide (PI) for 25 min in the dark. The samples were analyzed using a FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences).

2.6 | Western blot

The total protein was extracted from cells using RIPA lysis buffer. After the protein concentration was determined using BCA protein assay kit (CoWin Biotechnology, Beijing, China), the equal amount of proteins were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene difluoride membranes (PVDF; Millipore, MA). After blocking with skimmed milk for 2 h at room temperature, the membranes were incubated with the primary antibodies at overnight at 4C, followed with further incubation with the appropriate horseradish peroxidase (HRP)conjugated secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 h at room temperature. Eventually, the immunoreactive bands were visualized by enhanced chemiluminescence (Thermo Fisher Scientific, Bremen, Germany).

2.7 | TUNEL staining

The apoptosis of HL-60 cells was detected by TdT-mediated dUTP nick-end labeling (TUNEL) staining. The experiment was carried out strictly according to the instructions of the TUNEL kit (Elabscience®). The images were selected at 200 magnification, and five fields were captured randomly.

2.8 | Luciferase reporter assay

The full length of ARHGAP9 promoter was cloned into pGL3 vector (Promega, Madison, WI). Cells were co-transfected with Renilla luciferase expression vector (pRL-RSV), pGL3-ARHGAP9 promoter and pcDNA3.1-SOX4 or pcDNA3.1-NC. 48 h after transfection, the luciferase activity was measured using the Dual Luciferase Kit (Promega). The Renilla luciferase acted as a normalization control.

2.9 | Chromatin immunoprecipitation

ChIP was carried out to examine the binding of SOX4 to the promoter of ARHGAP9 using the Magna ChIP™ A/G Kit (Millipore) following the guide of the manufacturer’s instructions. An SOX4 antibody was applied for immunoprecipitation. The IgG was used as a negative control. The same amount of DNA without antibody precipitation acted as the input control. The ratio of DNA precipitated by the SOX4 antibody over the input control was obtained to indicate the enrichment of transcription factors bound to the promoter of ARHGAP9.

2.10 | Statistical analysis

The data here represent mean ± SD from at least three independent experiments. The statistical analysis was performed using GraphPad Prism 7 (GraphPad Software Inc., La Jolla). The difference was assessed by one-way analysis of variance followed by Tukey’s post hoc test. For overall survival analysis, Mantel–Cox test was used. Differences with p values of <.05 were regarded as statistically significant. 3 | RESULTS 3.1 | ARHGAP9 is upregulated in AML FIGURE 1 ARHGAP9 is upregulated in AML. (a) ARHGAP9 expression in tumor samples and normal samples in AML patients was analyzed using GEPIA database. The red box indicated the tumor samples, and the gray box indicated the normal samples. *p < .05 (b) Effect of ARHGAP9 on overall survival of AML patients was analyzed using GEPIA database. The expression level of ARHGAP9 in the human normal bone marrow cell line HS-5 and AML cell lines, including Kasumi-1, ME-1, HL60, MOLM14 and KG-1a, was detected using qRT-PCR (c) and western blot (d). ***p < 0.001 versus HS-5 Using the GEPIA database ((http://gepia.cancer-pku.cn), we found that the transcriptional level of ARHGAP9 in tumor samples of AML patients was substantially higher compared to that in the normal samples (Figure 1a). In addition, the high expression of ARHGAP9 was closely correlated with a low overall survival of AML patients (Figure 1b). Then, the expression level of ARHGAP9 was further detected in the human normal bone marrow cell line HS-5 and AML cell lines, including Kasumi-1, ME-1, HL60, MOLM-14 and KG-1a.  As shown from Figure 1c and d, compared to HS-5, both the mRNA level and the protein expression of ARHGAP9 were greatly high in AML cell lines, further indicating that ARHGAP9 was overexpressed in AML. Among them, the expression of ARHGAP9 in HL-60 was highest, for which the experiments on HL-60 cells were conducted in the subsequent experiments. 3.2 | Interference of ARHGAP9 inhibits proliferation and cycle progression in AML cells To explore the biological functions of ARHGAP9 in AML cells, HL-60 cells were transfected with shRNA-ARHGAP9#1/2 or shRNA-NC. As shown in Figure 2a, the expression level of ARHGAP9 was significantly decreased after transfection with shRNA-ARHGAP9#1/2, and shRNAARHGAP9-2 was further applied in the following experiments for its higher transfection efficacy. According to the results obtained from CCK-8 assay, the cell viability was significantly reduced after transfection with shRNA-ARHGAP9 (Figure 2b). The reduced expression of Ki67 and proliferation cell nuclear antigen (PCNA), classical markers of cell proliferation, in shRNA-ARHGAP9 group, demonstrated that ARHGAP9 interference lowered the proliferation level of HL-60 cells (Figure 2c). Subsequently, flow cytometry assay exhibited that the cell proportion in G1 phase was greatly elevated, and the cell proportion in S phase was greatly reduced following shRNA-ARHGAP9 transfection, indicating that interference of ARHGAP9 induced HL-60 cell arrest in G1 phase (Figure 2d). Furthermore, interference of ARHGAP9 obviously reduced the protein expressions of cyclin D1, cell cycle dependent kinase 4 (CDK4), CDK6 and cyclin E1 (Figure 2e). The results above suggested that ARHGAP9 interference suppressed proliferation ability and cell cycle progression in HL-60 cells. 3.3 | Interference of ARHGAP9 promotes apoptosis in AML cells Next, we also explored the regulatory effect of ARHGAP9 on cell apoptosis in HL-60 cells. The images obtained from TUNEL assay revealed that the apoptotic cells were increased after ARHGAP9 interference (Figure 3a). Moreover, Bcl-2, an anti-apoptosis-related protein, was greatly downregulated, and Bax, cleaved caspase 3 and cleaved caspase 9, which were pro-apoptosis-related proteins, were greatly upregulated after transfection with shRNA-ARHGAP9 (Figure 3b). The results above suggested that ARHGAP9 interference promoted cell apoptosis in HL-60 cells. 3.4 | SOX4 directly targets and regulates ARHGAP9 in AML cells As illustrated in Figure 4a, SOX4 bound to the ARHGAP9 promoter region at 262 to 371 (S2) and 448 to 457 (S1). To further verify their binding relationship, HL-60 cells were transfected with shRNA-SOX4#1/2 to interfere SOX4 and were transfected with pcDNA3.1-SOX4 to overexpress SOX4. The results in Figure 4b exhibited a successful transfection, and shRNA-SOX4#2 was used for the following experiments. As shown from Figure 4c, the expression level of ARHGAP9 was downregulated when SOX4 was interfered, and was upregulated when SOX4 was overexpressed, suggesting that SOX4 positively regulated the expression of ARHGAP9. Next, luciferase assays revealed a lower change in luciferase activity when the S1 region of ARHGAP9 promoter was mutated than that of S2 region (Figure 4d–e), indicating that S2 region of ARHGAP9 promoter was mainly responsible for binding relationship with SOX4. Furthermore, ChIP assay exhibited that the SOX4 was enriched at S2 region of ARHGAP9 promoter (Figure 4f). The results above suggested that SOX4 could bind to the promoter of ARHGAP9 and positively regulate the expression of ARHGAP9. 4 | DISCUSSION AML is a common aggressive malignancy characterized by a clonal proliferation of immature myeloid precursor cells, which can spread into other organs such as skin and central nervous system(Arber et al., 2016; Cancer Genome Atlas Research et al., 2013). Even though great progression in the molecular understanding of AML has been achieved in the recent years, the 5-year overall survival of patients is still only 20–25% (Kell, 2016). Thus, exploration of novel biomarkers and molecular mechanism underlying the development of AML is required to improve the clinical outcomes of patients with AML. In the present study, we disclosed a novel gene involved in the occurrence and progression of AML. ARHGAP9 expression was greatly elevated in AML. Analysis with cell lines revealed that interference of ARHGAP9 could suppress cell proliferation, hinder cell cycle progression and promote cell apoptosis in HL-60 cells. In addition, further analysis illustrated a binding relationship between SOX4 and ARHGAP9 promoter, and the expression of ARHGAP9 could be regulated by SOX4 in HL-60. Together, we revealed a novel molecular mechanism involving the anti-tumor activity of SOX4-mediated ARHGAP9 in AML cells, which might provide new clues for the development of targeted therapy for AML. It is widely acknowledged that the uncontrolled proliferation and blocked apoptosis in immature myeloid progenitors are classical features of AML (Ferrara & Schiffer, 2013). Assessing the degree of proliferation and apoptosis in cells provides important clues into the efficacy of potential anti-cancer therapies and seeking for novel targeted genes for cancer therapies. For example, depletion of Ars2 inhibited cell proliferation and culminated in cell cycle arrest at the G1 phase in AML cells, playing a crucial role in the regulation of leukemogenesis and providing a critical therapeutic target for AML treatment(Hu et al., 2019); IARS2 knockdown significantly suppressed the proliferation, induced cycle arrest at the G1 phase and promoted apoptosis in HL-60 cells, which was helpful for exploring potential therapeutic strategies for AML therapy(Li et al., 2019). Apoptosis, a form of programmed cell death, is a key mechanism that nascent neoplastic cells manipulate to transform into malignant cells. Interestingly, apoptosis can be induced by cell cycle arrest(Orren et al., 1997). In the present study, we also found a decreased cell proliferation ability and an elevated cell apoptosis ability upon ARHGAP9 interference, as well as the blockage of cell cycle progression at G1 phase. Meanwhile, the anti-tumor activity of ARHGAP9 interference was further verified by the detection of proliferation-, cycle- and apoptosis-related protein detection. Taken together, our findings indicate that ARHGAP9 interference inhibited cell proliferation by inducing cell cycle arrest in G1 phase and promoting cell apoptosis. We also investigated the molecular mechanism how ARHGAP9 interference suppressed the tumorigenesis and progression of AML. SOX4, a member of Sry-related high mobility group (SOX4) transcriptional factor family, can directly bind to the minor groove of DNA within a conserved region, and is an important regulator of mammalian development(Omidvar et al., 2013; van de Wetering & Clevers, 1992). Interestingly, SOX4 was identified as a transcriptional activator in lymphocytes and played an essential role in B-cell development(Schilham et al., 1996; van de Wetering et al., 1993). What is more, a recent study has reported that SOX4 is a crucial tumorigenic target for C/EBPα mutant AML (Zhang et al., 2013). In addition, AML patients with low SOX4 expression had higher remission rates and longer overall survival than AML patients with high SOX4 expression, therefore the expression of SOX4 could act as a biomarker for the clinical prognosis of AML patients(Lu et al., 2017; Sun et al., 2018). In the present study, we found that a binding relationship between SOX4 and ARHGAP9 promoter, and the expression level of ARHGAP9 could be regulated by SOX4. Taking the oncogenic role of SOX4 in AML into consideration, the high expression of SOX4 in AML may be partly responsible for the elevated ARHGAP9, and the functional activity of ARHGAP9 on AML progression may be partly mediated by SOX4. 5 | CONCLUSION In conclusion, our data demonstrated that ARHGAP9 was higher expressed in AML cells. The interference of ARHGAP9 inhibited cell proliferation, blocked cell cycle progression by TAS4464 inducing cell arrest in G1 phase, and promoted cell apoptosis. SOX4 is a transcription factor to bind to the ARHGAP9 promoter, and to regulate the expression of ARHGAP9, which is a possible reason for this abnormal expression of ARHGAP9 in AML cells and its regulatory function. This study provides a promising molecular target for AML therapy.

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