Regulatory mechanism and functional analysis of S100A9 in acute promyelocytic leukemia cells

Abstract

S100A9, a calcium-binding protein, participates in the inflammatory process and development of various tumors, thus attracting much attention in the field of cancer biology. This study aimed to investigate the regulatory mechanism of S100A9 and its function involvement in APL. We used real-time quantitative PCR to determine whether PML/RARα affects the expression of S100A9 in NB4 and PR9 cells upon ATRA treatment. ChIP-based PCR and dual-luciferase reporter assay system were used to detect how PML/RARα and PU.1 regulate S100A9 promoter activity. CCK-8 assay and flow cytometry were employed to observe the viability and apoptosis of NB4 cells when S100A9 was overexpressed. Results showed that S100A9 was an ATRA-responsive gene, and PML/RARα was necessary for the ATRA-induced expression of S100A9 in APL cells. In addition, PU.1 could bind to the promoter of S100A9, especially when treated with ATRA in NB4 cells, and promote its activity. More importantly, overexpression of S100A9 induced the apoptosis of NB4 cells and inhibited cell growth. Collectively, our data indicated that PML/RARα and PU.1 were necessary for the ATRA-induced expression of S100A9 in APL cells. Furthermore, S100A9 promoted apoptosis in APL cells and affected cell growth.

Keywords

S100A9 / PU.1 / PML/RARα / ATRA / APL

Introduction

Acute promyelocytic leukemia (APL) is pathologically characterized by a typical translocation between chromosomes 15 and 17. This translocation results in the formation of a promyelocytic leukemia-retinoic acid receptor (PML/RARa) fusion protein, which plays an important role in the leukemogenesis of APL. The protein functions in blocking cell differentiation at the promyelocytic stage [ 1]. The fusion protein harbors the protein–protein interaction domain of PML and the DNA binding domain of RARa, which is a strong transcriptional repressor. In the presence of pharmacological concentrations of all-trans retinoic acid (ATRA), the dissociation of corepressor molecules from PML/RARa promotes APL blasts to terminally differentiate by restoring the expression of genes that are essential for myeloid differentiation [ 2]. Our previous study identified a critical mechanism of differentiation blockage in APL, by which PML/RARa disrupts the function of the hematopoietic transcription factor PU.1 and subsequently blocks the regulation of a number of genes that are essential for myeloid differentiation [ 3]. PU.1 is an important transcription factor for myeloid differentiation. It regulates numerous myeloid genes, including granulocyte colony-stimulating factor receptor, CD18, CD11b, and IRF1 [ 4, 5]. PU.1 disruption has been shown to block the differentiation or maturation of myelomonocytes, which results in leukemia genesis in animal models, whereas overexpressed PU.1 improves AML blast differentiation [ 3]. In APL cells, ATRA restores PU.1 expression. Therefore, increased PU.1 targets molecules at the gene expression level, including PSMBs, HCK, and HOTAIRM1 [ 4].

The S100 protein family is a class of low-molecular-weight acidic, calcium-binding proteins, which play important roles in the biological activities of the body by interacting with calcium ions and participating in cell differentiation, cell migration, gene transcription, and cell cycle operation [ 6]. The S100 protein is closely related to the development of various tumors. S100A9 is an important member of the S100 calcium-binding family of proteins, which increases in level during partial inflammation and autoimmunity, especially in myeloid cells [ 7]. This protein is expressed during myeloid cell differentiation. S100A9 increases in abundance in granulocytes and monocytes by forming a heterocomplex with S100A8. However, the protein has a distinct function and regulatory mechanism [ 8]. Interestingly, S100A9 can exhibit pro- and anti-inflammatory roles depending on the tissue and cell type [ 9]. Increased expression of S100A9 has been observed in a number of tumors, such as colon, ovarian, breast, and thyroid cancers [ 10]. However, S100A9 is downregulated in several common human cancers, including head and neck squamous cell carcinoma, esophageal squamous cell carcinoma, cervical cancer, nasopharyngeal cancer, and oropharyngeal cancer [ 11]. Previous studies showed that S100A9 can inhibit cell growth and induce cell apoptosis through the classical mitochondrial pathway with its partner, S100A8, in various cell types [ 12]. Studies have also found that S100A9 is differentially expressed in adult acute myeloid leukemia and non-hematologic malignancies and increases in AML remission. These conditions indicated that an intimate connection exists between S100A9 and the pathogenesis and prognosis of leukemia [ 13].

In the present study, we investigated the mechanism and function involved in the differential expression of S100A9 in APL. We discovered that PU.1 regulated the basal expression of S100A9, and PML/RARa repressed the expression of S100A9 in APL. However, PML/RARa was necessary for ATRA-induced expression of S100A9 in NB4 cells. Furthermore, ATRA significantly increased S100A9 expression. In addition, the increased expression of S100A9 contributed to the apoptosis and growth inhibition of NB4 cells. Our data highlighted the regulatory mechanism of S100A9 and important function of S100A9 in APL.

Materials and methods

Cell culture and reagents

NB4, U937-PR9, U937, and HL-60 cells were grown in RPMI 1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). Human embryonic kidney 293T cells were maintained in DMEM (Gibco) supplemented with 10% FBS. The cells were cultured in a humidified incubator with 5% CO₂ at 37 °C. ZnSO₄ (Sigma, St Louis, MO, USA) was dissolved in sterile water as a stock solution at 100 mmol/L. ATRA (Sigma) was dissolved in ethanol as a stock solution at 1 mmol/L and used in the dark during experiments. Both ZnSO₄ and ATRA were diluted by 1000-fold when used.

Plasmid constructions

The human S100A9 promoter (chr1:153329870-153330800) harboring two transcription factor motifs was amplified by genomic PCR starting from U937 and inserted into the pGL3-basic vector (Promega, Madison, WI, USA) upstream of the firefly luciferase coding region at the XhoI and HindIII sites. The renilla luciferase plasmid pRL-SV40 was used as control for transfection efficiency.

Transient transfection and luciferase assays

293T cells were transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. Transfected cells were assayed for luciferase activity using dual-luciferase reporter assay system reagents (Promega, Madison, WI, USA). The detailed information for the procedure was described previously [ 3]. Each transfection was performed three times.

Chromatin immunoprecipitation (ChIP) assay

ChIP was undertaken according to the manufacturer’s recommendation for the ChIP-IT High Sensitivity Kit (Active Motif). We used 4.5×10⁶ NB4 cells for each ChIP reaction. Chromatin was sheared to a size of 200–1000 bp. In brief, NB4 cells were incubated with the following antibodies: anti-PU.1 (T-21x), anti-PML (H-238x), and anti-RARa (C-20x) from Santa Cruz, and normal rabbit IgG (ab46540). The total input and immunoprecipitated DNA was analyzed by PCR using the following primers: S100A9-1 (forward: 5′-TAGTGGAACCTCGGATTGGGT-3′; reverse: 5′-TGATTGGTCAGAGTGTGGCAA-3′), S100A9-2 (forward: 5′-TAGTGGAACCTCGGATTGGGT-3′; reverse: 5′-TGATTGGTCAGAGTGTGGCAA-3′), PLCB2, a positive primer, (forward: 5′-GAGGGATGGCTGCTCTGGTT-3′; reverse: 5′-GCTGGGCTAAGAAGGGCGATA-3′), and BLNK, a negative primer, (forward: 5′-GGCCCTGACTGATGGAAATAC-3′; reverse: 5′-CAGCAGGTGACCATCCCTTTAG-3′). Each experiment was performed in triplicate, and the fold enrichment of the chip PCR product was calculated as previously described [ 3].

RNA extraction and quantitative real-time PCR

RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and 1 µg of the total RNA was reverse transcribed into cDNA by using SuperScript II Reverse Transcriptase kit (Invitrogen) according to the manufacturer’s protocol. Quantitative real-time PCR was performed in the ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the SYBR Green PCR master mix (Toyobo, Osaka, Japan). GAPDH was used as a reference gene for internal control. The relative gene expression where calculated as follows: 10⁴ × 2^−∆Ct, where ∆Ct= Ct (TARGET)−Ct (GAPDH). Primer sequences of S100A9, PU.1, and GAPDH were as follows: S100A9 (forward: 5′-GGTCATAGAACACATCATGGAGG-3′; reverse: 5′-GGCCTGGCTTATGGTGGTG-3′); PU.1 (forward: 5′-GTGCCCTATGACACGGATCTA-3′; reverse: 5′-AGTCCCAGTAATGGTCGCTAT-3′); and GAPDH (forward: 5′-GAAGGTGAAGGTCGGAGTC-3′; reverse: 5′-GAAGATGGTGATGGGATTTC-3′).

RNA interference experiments

Two specific sequences of siRNA against PU.1 were purchased from GenePharma. Their sequences were 5′-GCUUCGCCGAGAACAACUUTT-3′ and 5′-GAGGUCAAGAAGGUGAAGATT-3′ named as siPU.1-1 and siPU.1-2, respectively. The sequence of the negative control was 5′-UUCUCCGAACGUUGUCACGUTT-3′. NB4 cells (2×10⁶) were suspended in 100 µl of Amaxa Nucleofector Solution V and then mixed with 3 µg of siRNA transfected by electroporation using the Amaxa Nucleofector II device and program X-001 (Lonza). After transfection for 24 h, the cells were added to ATRA for another 24 h.

Retroviral constructs and transfection

Full-length human S100A9 cDNA was amplified by RT-PCR using the U937 cells and cloned into an MSCV2.2-derived vector, MigR1, at the NotI and BamHI sites. The entire cDNA sequence that was inserted into the plasmids was verified through sequencing. MigR1-S100A9 was transfected into 293T packaging cells by using Lipofectamine 3000. The cell culture supernatants containing the virus were then collected at 48 h after transfection. NB4 cells were infected with the viral supernatant and 8 µg/ml of polybrene (Sigma) and then centrifuged at 800 ×g for 90 min at 32 °C. After 6 h, the medium was changed.

Cell apoptosis and proliferation analysis

Annexin V-APC and PI were used (BD Biosciences) to detect apoptotic cells in GFP-positive cells via flow cytometry following the manufacturer’s recommendation. We used CCK-8 assay (Dojindo Laboratories, Japan) to detect cell viability. Cells were seeded into 96-well plates at a density of 2×10³ cells/well. After incubation for 1, 2, 3, 4, and 5 days, 10 µl of CCK-8 solution was added to each well. The optical density was measured at 450 nm, and cell proliferation was determined.

Western blot

Total protein extraction and Western blot analysis were performed as previously described [ 11]. Rabbit anti-S100A9 monoclonal antibody, mouse anti-β actin monoclonal antibody, and mouse anti-Bcl-2 monoclonal antibody were purchased from Abcam, Cambridge (UK). Rabbit cleaved caspase-3 was purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Gene expression analysis

GSE10358 and GSE14468 were obtained from the Gene Expression Omnibus.

Statistical analysis

Statistical analysis was conducted using GraphPad Prism software (GraphPad). For two-group comparisons, significance was determined using two-tailed t-tests. P-value<0.05 was considered statistically significant.

Results

S100A9 is an ATRA-responsive gene, and PML/RARa is necessary for ATRA-induced expression of S100A9 in NB4 cells

S100A9 is abundant in granulocytes and monocytes, and it is upregulated during the differentiation of hematopoietic cells. These properties potentially indicated that S100A9 played an important role in myeloid differentiation. First, we analyzed the expression level of S100A9 using the published expression profiles of 43 APL patients and 236 other AML specimens from GSE10358 [ 14]. As indicated in Fig. 1A, S100A9 expression was significantly lower in APL samples than in other AML patients. All data from patients were seen in supplemental data Table S1. Real-time RT-PCR was conducted to compare S100A9 expression among several hematopoietic cells. Fig. 1B shows that S100A9 expression was significantly lower in NB4 cells (PML/RARa-positive) than in U937 and HL-60 cells (PML/RARa-negative). This observation indicated that S100A9 expression was inversely correlated with PML/RARa expression. Given that APL is characterized by a unique clinical response to a differentiation inducing agent, ATRA, S100A9 expression was examined in APL cells after ATRA treatment. As shown in Fig. 1C, RNA and protein expression levels of S100A9 gradually increased after the NB4 cells were treated with ATRA in a time series. The same conclusion could be made on Fig. 1D showing PR9 cells treated with ZnSO₄ and ATRA. PR9 is a PML/RARa-inducible model [ 15] constructed from U937 cells treated with 100 µmol/L ZnSO₄ for 4 h. PR9 can eventually induce many features that are similar to those of APL. However, S100A9 RNA expression did not change when the cells were treated with ATRA without the induction of PML/RARa, as shown in Fig. 1D. The above results indicated that S100A9 was responsive to ATRA, and its expression was closely related to PML/RARa.

Considering that arsenic trioxide (As₂O₃) combined with ATRA can effectively cure APL patients, we also conducted an experiment to determine whether As₂O₃ affects S100A9 expression. We observed that As₂O₃ had minimal effect on the expression of PU.1 on NB4 cells, but it increased S100A9 expression. However, S100A9 expression induced by As₂O₃ at different time points was significantly lower than that induced by ATRA (Fig. S1).

PML/RARa regulates S100A9 activity

To determine whether PML/RARa can directly bind to the S100A9 promoter, we conducted ChIP assays on the DNA samples of NB4 cells that were either treated or untreated with ATRA using PML and RARa-specific antibodies, respectively. Fig. 2A shows the PU.1 and PML/RARa binding sites at the S100A9 promoter region that were determined by TRANSFAC software. Two sets of primers were designed based on the binding sites. One covered the -450 and -300 bp PU.1 and PML/RARa sites (S100A9-1). The other set covered the 300 and 450 bp PU.1 sites (S100A9-2). As illustrated by the genomic occupancy profiles in Fig. 2B, both PML and RARa could bind the promoter of S100A9 with or without the presence of ATRA. However, a higher peak of enrichment on the S100A9 promoter was observed in ATRA-treated cells than in untreated NB4 cells. To verify this finding, we conducted chromatin immunoprecipitation quantitative real-time PCR (ChIP-qPCR) assays in NB4 cells by using the primers that contained the peak region and used anti-PML, anti-RARa, and nonspecific (IgG) antibodies. As expected, we obtained the same results (Fig. 2C), which further indicated that PML/RARa was necessary for ATRA-induced expression of S100A9. To further determine whether S100A9 transcriptional expression is repressed by PML/RARa, luciferase reporter assays of 293T cells were conducted. We observed that luciferase activity of the S100A9 promoter declined in a dose-dependent manner after transfection with PML/RARa (Fig. 2D). This behavior explains the low expression of S100A9 in APL.

PU.1 regulates the basal expression of S100A9 by targeting the promoter regions of S100A9

PU.1, which is highly expressed in granulocytic cells, regulates genes that are essential for myeloid differentiation. As shown in Fig. 2A, PU.1 binding sites were found in the S100A9 promoter region. Simultaneously, a positive correlation was noted between S100A9 and PU.1 mRNA levels in a sample of 415 AML patients, including M1–M6 (the expression data downloaded from GSE14468; Fig. 3A). We conducted the following experiments to verify this phenomenon. First, siRNA against the PU.1 experiment was ordered from GenePharma. The data in Fig. 3B showed the interference efficiency of siRNA. Knockdown of PU.1 reduced the ATRA-mediated activation of S100A9 at the mRNA expression level. Subsequently, we scanned the genomic occupancy profiles (Fig. 3C, left) on the S100A9 promoter. A high peak of enrichment was observed with ATRA treatment. Our ChIP-qPCR of NB4 cells demonstrated that PU.1 was enriched in the promoter region of S100A9 and exhibited a slightly higher enrichment fold with ATRA treatment (Fig. 3C, right). This result explains why ATRA could induce an increase in S100A9 expression. To verify our hypothesis that the reductive expression of S100A9 in APL is possibly mediated by PU.1, we conducted luciferase reporter assays on 293T cells (a non-hematopoietic cell line). As illustrated in Fig. 3D, S100A9 promoter activity increased with increasing amounts of PU.1 expression plasmid. Collectively, the data indicated that PU.1 targeted S100A9 and transactivated the promoters of S100A9.

Overexpression of S100A9 induces apoptosis and growth suppression in NB4 cells

A previous study showed that S100A9 induces cellular apoptosis and growth inhibition in various tumor cells [ 12]. To verify whether overexpression of S100A9 is involved in the apoptosis and proliferation of our model cells, we first treated NB4 cells by retroviral transduction to induce high S100A9 expression (Fig. 4A). We discovered that overexpressed S100A9 inhibited NB4 cell growth (Fig. 4B). In addition, S100A9 induced apoptosis. As shown in Fig. 4C, forced expression of S100A9 increased the early and late total apoptosis rates from 5.64%±0.86% to 18.88%±1.32% (P<0.01) in NB4 cells. In these experiments, cells with GFP were sorted for analysis to avoid a non-transfected negative background. Simultaneously, overexpressed S100A9 increased protein expression of cleaved caspase-3 and decreased Bcl-2 protein expression in NB4 cells (Fig. 4D).

Discussion

Extensive studies have been carried out to understand the function and regulatory mechanisms of S100A9 in cancer. In addition, many studies have revealed that S100A9 can be used as a biomarker for acute appendicitis, Alzheimer’s disease, systemic sclerosis, muscle invasive bladder cancer, and so on [ 16– 19]. However, reports on S100A9 in relation to leukemia are still relatively few. A previous study showed that S100A9 is one of the most downregulated genes in human and murine leukemia samples. S100A9 is enriched in extracellular space in a GO term and involved in the response to stimulus and defense response [ 20]. In this study, we observed that S100A9 expression was not only significantly lower in APL samples than in non-APL samples, but it was also lower in NB4 cells than in U937 and HL-60 cells (Fig. 1). However, the distinct difference between APL and other AML was the existence of PML/RARa, which indicated that PML/RARa restrained S100A9 activity in APL.

Further experiments revealed that PML/RARa could directly bind to the S100A9 promoter and repress S100A9 transcriptional expression (Fig. 2). Similarly, the repression that was induced by PML/RARa could be restored by pharmacological concentrations of ATRA (Fig. 1C). Earlier reports revealed that both IPA and VD3 upregulated S100A9 in the early stages of differentiation in HL-60 cells [ 21, 22]. These findings implied that S100A9 was essential for myeloid differentiation. Interestingly, in our study, ATRA alone could not upregulate the expression of S100A9 in U937-PR9 (Fig. 1D). In addition, a higher fold enrichment of PML/RARa was observed in the S100A9 promoter of NB4 cells treated with ATRA than in the untreated samples (Fig. 2C). Therefore, PML/RARa was necessary for the ATRA-induced expression of S100A9. This condition seems incomprehensible considering that PML/RARa inhibited S100A9 expression in NB4 cells. We then speculated that PML/RARa may be a ligand that recruits more transcription factors to increase S100A9 expression during the ATRA-induced differentiation of APL cells. A study has already reported that PML-RARa mediates the differentiation response of APL to ATRA treatment [ 23]. In addition, inhibiting the degradation of PML-RARa does not prevent ATRA-induced differentiation in APL [ 24]. Induction of PML-RARa expression can enhance the degree of cell differentiation that is induced by ATRA in PR9 cells [ 25]. Our data suggested that PML-RARa was involved in the regulation of ATRA-responsive genes in APL cells.

In addition, previous studies have demonstrated that S100A9 expression can be upregulated by IL-6, IL-8, IL-1a, and P53 [ 26– 28]. However, the vital transcription factors in AML, which are indispensable for S100A9 basal expression, were not found. A large number of key transcriptional factors, including AML1, CBFb, C/EBPa, Myb, and PU.1, are involved in granulocytic differentiation of hematopoietic stem cells. PU.1 controls numerous genes that are important for APL differentiation in the ATRA-induced maturation of APL blasts [ 4, 5]. In this study, we showed that the hematopoietic transcription factor PU.1 regulates the basal expression of S100A9 in APL cells. PU.1 is specifically expressed in hematopoietic cells. Disrupted PU.1 expression can cause terminal differentiation defect in myeloid cells [ 29]. In this research, we demonstrated that PU.1 targeted S100A9 and transactivated the promoters of S100A9 (Fig. 3). We proposed a model to better understand how PML/RARa, PU.1, and ATRA affect the regulatory transcription of S100A9. As illustrated in Fig. 5, PML/RARa interacted with PU.1 and inhibited S100A9 transcription in APL cells. However, when APL cells were treated with ATRA, PML/RARa acted as a ligand and recruited PU.1 and other unidentified transcription factors to activate S100A9 transcription. Furthermore, in accordance with previous reports stating that S100A9 promoted apoptosis and inhibited proliferation of cancer cells [ 12], we discovered that overexpression of S100A9 induced NB4 cell apoptosis and suppressed proliferation. These results suggested that S100A9 may serve as a potential molecular therapeutic target of cancers associated with repressed expression of S100A9.

The combined treatment of ATRA and As₂O₃ has been widely used for APL patients in clinical settings. Results have achieved very high complete remission rates and long-term outcomes [ 30]. As₂O₃ is considered the first line of treatment for elderly patients [ 31]. Previous studies have indicated that the combination of the molecules is better than treatment with ATRA or As₂O₃ alone [ 32]. Our data demonstrated that ATRA substantially increased the expression of S100A9, whereas As₂O₃ slightly affected S100A9 expression. These results indicated the importance of S100A9 in APL treatment. Our findings further supported that combination therapy, rather than As₂O₃ alone, is a wise and desirable method for most APL patients.

In conclusion, the present study investigated the transcriptional regulatory mechanism of S100A9 in APL cells. This study provides a better understanding of how PML/RARa blocks differentiation at the promyelocytic stage. Moreover, we found that PML/RARa and PU.1 regulated S100A9 transcription during neutrophil differentiation, and overexpressed S100A9 induced APL cell apoptosis and growth inhibition. Such results indicated that S100A9 may be a crucial tumor suppressor in APL and a potential therapeutic target in other cancer types with underexpressed S100A9. This work adds to our understanding of the basal regulatory mechanism of the S100 family and the role of S100A9 in the development of carcinomas.

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