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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2017, Vol. 11 Issue (1) : 87-96     DOI: 10.1007/s11684-016-0469-4
Regulatory mechanism and functional analysis of S100A9 in acute promyelocytic leukemia cells
Yonglan Zhu1,Fang Zhang1,Shanzhen Zhang2,Wanglong Deng1,Huiyong Fan1,Haiwei Wang1,2,Ji Zhang1,2()
1. State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2. Medical Institute of Health Sciences, Chinese Academy of Sciences, Shanghai 200025, China
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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     
Corresponding Authors: Ji Zhang   
Just Accepted Date: 14 December 2016   Online First Date: 05 January 2017    Issue Date: 20 March 2017
URL:     OR
Fig.1  ATRA-induced expression of S100A9 is dependent on PML-RARa in APL. (A) S100A9 expression in APL patients was significantly lower than that in other AML patients. The expression values are the absolute intensities after log transformation. Data were extracted from GSE10358. (B) The expression level of S100A9 was significantly lower in NB4 cells than in U937 and HL-60 cells. (C) RNA and protein expression of S100A9 gradually increased in NB4 cells when treated with ATRA at a series of time points. (D) The relative expression of S100A9 gradually increased after PML/RARa induction of ZnSO4-treated PR9 cells in the presence of ATRA at the indicated time points. However, the expression did not change in PR9 cells without ZnSO4 treatment even in the presence of ATRA. The data represent the mean of three replicates±S.D. **P<0.01, ***P<0.001.
Fig.2  PML/RARa binds to the promoter of S100A9 in NB4 cells. (A) Schematic representation of the promoter regions of S100A9. PU.1 binding sites (rhombus) and RARE half-sites (rectangle) were defined using TRANSFAC with core and matrix similarity. The S100A9-1 region spanned PU.1 and RARE sites. The S100A9-2 region spanned PU.1 sites. (B) Schematic shows the binding of PML/RARa to the promoter regions of S100A9. ChIP assays of the NB4 cells were conducted using anti-PML and anti-RARa antibodies. The peaks represented the PML/RARa-enriched ChIP regions. S100A9 was located on the Watson (+) strand of chromosome 1. (C) ChIP was conducted on NB4 cells using anti-RARa, anti-PML, or nonspecific (normal immunoglobulin G (IgG)) antibodies. ChIP-qPCR was conducted using S100A9-1 and negative (BLNK) or positive (PLCB2) primers. Results were presented as fold enrichment of chromatin, normalized to nonspecific IgG. (D) Promoter activities of S100A9 were repressed by PML/RARa in a dose-dependent manner. Luciferase reporter assays were conducted on 293T cells. The data represent the mean of three replicates±S.D.
Fig.3  PU.1 regulates the basal expression of S100A9 by targeting the promoter regions of S100A9. (A) A positive correlation was found between S100A9 and PU.1 mRNA levels in 415 AML (M1–M6) patients from GSE14468. (B) Knockdown of PU.1 reduced ATRA-mediated activation of S100A9 at the mRNA expression level. The mRNA levels of PU.1 and S100A9 were detected in NB4 cells by RT-PCR at 48 h post-transfection with siRNA (3 µg) and in the absence or presence of ATRA for 24 h. (C) PU.1 bound to the promoter of S100A9 in APL cells. The pattern on the left is a schematic that shows PU.1 binding to the promoter regions of S100A9. ChIP assays of the NB4 cells were conducted using anti-PU.1 antibody. The peaks represent the PU.1-enriched ChIP regions. The image on the right shows the ChIP results of the assays for the NB4 cells that were conducted using anti-PU.1 or IgG antibodies. ChIP-qPCR was undertaken with S100A9-1, S100A9-2, BLNK, and PLCB2 primers. (D) PU.1 increased the promoter activities of S100A9 in a dose-dependent manner. Luciferase reporter assays were conducted on 293T cells. The data represent the mean of three replicates±S.D. *P<0.05.
Fig.4  Overexpression of S100A9 induces cell apoptosis and growth inhibition in NB4 cells. (A) Relative expression of S100A9 in NB4 cells that were harvested 72 h after MigR1 (Ctrl) or MigR1-S100A9 infection. (B) S100A9 overexpression inhibited the proliferation of NB4 cells. NB4 cells were infected with MigR1 (Ctrl) or MigR1-S100A9 retroviral component, and cell proliferation was detected by CCK-8 assay. Results showed that the proliferation rate of NB4 cells infected with MigR1-S100A9 was reduced significantly at days 4 and 5 when compared with the MigR1 (Ctrl) group and blank group. *P<0.05. (C) Overexpression of S100A9 induced apoptosis of NB4 cells. GFP-positive cells were analyzed by flow cytometry. The left panel depicts the apoptosis of MigR1-infected NB4 cells, and the right panel shows the result for MigR1-S100A9-infected NB4 cells. The values represent the three replicates±S.D. (D) Western blot analysis of cleaved caspase-3 and Bcl-2 expression in total cell lysates.
Fig.5  Possible model for the transcription regulation of S100A9 is proposed in APL.
1 de Thé H, Chen Z. Acute promyelocytic leukaemia: novel insights into the mechanisms of cure. Nat Rev Cancer 2010; 10(11): 775–783
doi: 10.1038/nrc2943 pmid: 20966922
2 Hu J, Liu YF, Wu CF, Xu F, Shen ZX, Zhu YM, Li JM, Tang W, Zhao WL, Wu W, Sun HP, Chen QS, Chen B, Zhou GB, Zelent A, Waxman S, Wang ZY, Chen SJ, Chen Z. Long-term efficacy and safety of all-trans retinoic acid/arsenic trioxide-based therapy in newly diagnosed acute promyelocytic leukemia. Proc Natl Acad Sci USA 2009; 106(9): 3342–3347
doi: 10.1073/pnas.0813280106 pmid: 19225113
3 Wang K, Wang P, Shi J, Zhu X, He M, Jia X, Yang X, Qiu F, Jin W, Qian M, Fang H, Mi J, Yang X, Xiao H, Minden M, Du Y, Chen Z, Zhang J. PML/RARα targets promoter regions containing PU.1 consensus and RARE half sites in acute promyelocytic leukemia. Cancer Cell 2010; 17(2): 186–197
doi: 10.1016/j.ccr.2009.12.045 pmid: 20159610
4 Wei S, Zhao M, Wang X, Li Y, Wang K. PU.1 controls the expression of long noncoding RNA HOTAIRM1 during granulocytic differentiation. J Hematol Oncol 2016; 9(1): 44
doi: 10.1186/s13045-016-0274-1 pmid: 27146823
5 Qian M, Jin W, Zhu X, Jia X, Yang X, Du Y, Wang K, Zhang J. Structurally differentiated cis-elements that interact with PU.1 are functionally distinguishable in acute promyelocytic leukemia. J Hematol Oncol 2013; 6(1): 25
doi: 10.1186/1756-8722-6-25 pmid: 23547873
6 Donato R. S100: a multigenic family of calcium-modulated proteins of the EF-hand type with intracellular and extracellular functional roles. Int J Biochem Cell Biol 2001; 33(7): 637–668
doi: 10.1016/S1357-2725(01)00046-2 pmid: 11390274
7 Nacken W, Roth J, Sorg C, Kerkhoff C. S100A9/S100A8: myeloid representatives of the S100 protein family as prominent players in innate immunity. Microsc Res Tech 2003; 60(6): 569–580
doi: 10.1002/jemt.10299 pmid: 12645005
8 Hunter MJ, Chazin WJ. High level expression and dimer characterization of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and 14. J Biol Chem 1998; 273(20): 12427–12435
doi: 10.1074/jbc.273.20.12427 pmid: 9575199
9 Srikrishna G. S100A8 and S100A9: new insights into their roles in malignancy. J Innate Immun 2012; 4(1): 31–40
doi: 10.1159/000330095 pmid: 21912088
10 Salama I, Malone PS, Mihaimeed F, Jones JL. A review of the S100 proteins in cancer. Eur J Surg Oncol 2008; 34(4): 357–364
doi: 10.1016/j.ejso.2007.04.009 pmid: 17566693
11 Khammanivong A, Wang C, Sorenson BS, Ross KF, Herzberg MC. S100A8/A9 (calprotectin) negatively regulates G2/M cell cycle progression and growth of squamous cell carcinoma. PLoS ONE 2013; 8(7): e69395
doi: 10.1371/journal.pone.0069395 pmid: 23874958
12 Yui S, Nakatani Y, Mikami M. Calprotectin (S100A8/S100A9), an inflammatory protein complex from neutrophils with a broad apoptosis-inducing activity. Biol Pharm Bull 2003; 26(6): 753–760
doi: 10.1248/bpb.26.753 pmid: 12808281
13 Kuwayama A, Kuruto R, Horie N, Takeishi K, Nozawa R. Appearance of nuclear factors that interact with genes for myeloid calcium binding proteins (MRP-8 and MRP-14) in differentiated HL-60 cells. Blood 1993; 81(11): 3116–3121
pmid: 8499645
14 Tomasson MH, Xiang Z, Walgren R, Zhao Y, Kasai Y, Miner T, Ries RE, Lubman O, Fremont DH, McLellan MD, Payton JE, Westervelt P, DiPersio JF, Link DC, Walter MJ, Graubert TA, Watson M, Baty J, Heath S, Shannon WD, Nagarajan R, Bloomfield CD, Mardis ER, Wilson RK, Ley TJ. Somatic mutations and germline sequence variants in the expressed tyrosine kinase genes of patients with de novo acute myeloid leukemia. Blood 2008; 111(9): 4797–4808
doi: 10.1182/blood-2007-09-113027 pmid: 18270328
15 Grignani F, Ferrucci PF, Testa U, Talamo G, Fagioli M, Alcalay M, Mencarelli A, Grignani F, Peschle C, Nicoletti I, Pelicci PG. The acute promyelocytic leukemia-specific PML-RAR a fusion protein inhibits differentiation and promotes survival of myeloid precursor cells. Cell 1993; 74(3): 423–431
doi: 10.1016/0092-8674(93)80044-F pmid: 8394219
16 Bealer JF, Colgin M. S100A8/A9: a potential new diagnostic aid for acute appendicitis. Acad Emerg Med 2010; 17(3): 333–336
doi: 10.1111/j.1553-2712.2010.00663.x pmid: 20370768
17 Horvath I, Jia X, Johansson P, Wang C, Moskalenko R, Steinau A, Forsgren L, Wågberg T, Svensson J, Zetterberg H, Morozova-Roche LA. Pro-inflammatory S100A9 protein as a robust biomarker differentiating early stages of cognitive impairment in Alzheimer’s disease. ACS Chem Neurosci 2016; 7(1): 34–39
doi: 10.1021/acschemneuro.5b00265 pmid: 26550994
18 van Bon L, Cossu M, Loof A, Gohar F, Wittkowski H, Vonk M, Roth J, van den Berg W, van Heerde W, Broen JC, Radstake TR. Proteomic analysis of plasma identifies the Toll-like receptor agonists S100A8/A9 as a novel possible marker for systemic sclerosis phenotype. Ann Rheum Dis 2014; 73(8): 1585–1589
doi: 10.1136/annrheumdis-2013-205013 pmid: 24718960
19 Kim WT, Kim J, Yan C, Jeong P, Choi SY, Lee OJ, Chae YB, Yun SJ, Lee SC, Kim WJ. S100A9 and EGFR gene signatures predict disease progression in muscle invasive bladder cancer patients after chemotherapy. Ann Oncol 2014; 25(5): 974–979
doi: 10.1093/annonc/mdu037 pmid: 24631944
20 Li Z, Luo RT, Mi S, Sun M, Chen P, Bao J, Neilly MB, Jayathilaka N, Johnson DS, Wang L, Lavau C, Zhang Y, Tseng C, Zhang X, Wang J, Yu J, Yang H, Wang SM, Rowley JD, Chen J, Thirman MJ. Consistent deregulation of gene expression between human and murine MLL rearrangement leukemias. Cancer Res 2009; 69(3): 1109–1116
doi: 10.1158/0008-5472.CAN-08-3381 pmid: 19155294
21 Ishii Y, Kasukabe T, Honma Y. Immediate up-regulation of the calcium-binding protein S100P and its involvement in the cytokinin-induced differentiation of human myeloid leukemia cells. Biochim Biophys Acta 2005; 1745(2): 156–165
doi: 10.1016/j.bbamcr.2005.01.005 pmid: 16129123
22 Roth J, Goebeler M, van den Bos C, Sorg C. Expression of calcium-binding proteins MRP8 and MRP14 is associated with distinct monocytic differentiation pathways in HL-60 cells. Biochem Biophys Res Commun 1993; 191(2): 565–570
pmid: 8499645
23 Wang JG, Barsky LW, Davicioni E, Weinberg KI, Triche TJ, Zhang XK, Wu L. Retinoic acid induces leukemia cell G1 arrest and transition into differentiation by inhibiting cyclin-dependent kinase-activating kinase binding and phosphorylation of PML/RARα. FASEB J 2006; 20(12): 2142–2144
doi: 10.1096/fj.06-5900fje pmid: 16935935
24 Nervi C, Ferrara FF, Fanelli M, Rippo MR, Tomassini B, Ferrucci PF, Ruthardt M, Gelmetti V, Gambacorti-Passerini C, Diverio D, Grignani F, Pelicci PG, Testi R. Caspases mediate retinoic acid-induced degradation of the acute promyelocytic leukemia PML/RARα fusion protein. Blood 1998; 92(7): 2244–2251
pmid: 9746761
25 Grignani F, Gelmetti V, Fanelli M, Rogaia D, De Matteis S, Ferrara FF, Bonci D, Grignani F, Nervi C, Pelicci PG. Formation of PML/RAR alpha high molecular weight nuclear complexes through the PML coiled-coil region is essential for the PML/RARα-mediated retinoic acid response. Oncogene 1999; 18(46): 6313–6321
doi: 10.1038/sj.onc.1203029 pmid: 10597230
26 Kim JH, Oh SH, Kim EJ, Park SJ, Hong SP, Cheon JH, Kim TI, Kim WH. The role of myofibroblasts in upregulation of S100A8 and S100A9 and the differentiation of myeloid cells in the colorectal cancer microenvironment. Biochem Biophys Res Commun 2012; 423(1): 60–66 PMID:22634002
doi: 10.1016/j.bbrc.2012.05.081
27 Bando M, Zou X, Hiroshima Y, Kataoka M, Ross KF, Shinohara Y, Nagata T, Herzberg MC, Kido J. Mechanism of interleukin-1a transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line. Biochim Biophys Acta 2013; 1829(9): 954–962
doi: 10.1016/j.bbagrm.2013.03.010 pmid: 23563247
28 Li C, Chen H, Ding F, Zhang Y, Luo A, Wang M, Liu Z. A novel p53 target gene, S100A9, induces p53-dependent cellular apoptosis and mediates the p53 apoptosis pathway. Biochem J 2009; 422(2): 363–372
doi: 10.1042/BJ20090465 pmid: 19534726
29 McKercher SR, Torbett BE, Anderson KL, Henkel GW, Vestal DJ, Baribault H, Klemsz M, Feeney AJ, Wu GE, Paige CJ, Maki RA. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J 1996; 15(20): 5647–5658
pmid: 8896458
30 Chau D, Ng K, Chan TSY, Cheng YY, Fong B, Tam S, Kwong YL, Tse E. Azacytidine sensitizes acute myeloid leukemia cells to arsenic trioxide by up-regulating the arsenic transporter aquaglyceroporin 9. J Hematol Oncol 2015; 8(1): 46
doi: 10.1186/s13045-015-0143-3 pmid: 25953102
31 Zhang Y, Zhang Z, Li J, Li L, Han X, Han L, Hu L, Wang S, Zhao Y, Li X, Zhang Y, Fan S, Lv C, Li Y, Su Y, Zhao H, Zhang X, Zhou J. Long-term efficacy and safety of arsenic trioxide for first-line treatment of elderly patients with newly diagnosed acute promyelocytic leukemia. Cancer 2013; 119(1): 115–125
doi: 10.1002/cncr.27650 pmid: 22930197
32 Shen ZX, Shi ZZ, Fang J, Gu BW, Li JM, Zhu YM, Shi JY, Zheng PZ, Yan H, Liu YF, Chen Y, Shen Y, Wu W, Tang W, Waxman S, De Thé H, Wang ZY, Chen SJ, Chen Z. All-trans retinoic acid/As2O3 combination yields a high quality remission and survival in newly diagnosed acute promyelocytic leukemia. Proc Natl Acad Sci USA 2004; 101(15): 5328–5335
doi: 10.1073/pnas.0400053101 pmid: 15044693
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