Annexin A2-S100A10 heterotetramer is upregulated by PML/RARα fusion protein and promotes plasminogen-dependent fibrinolysis and matrix invasion in acute promyelocytic leukemia

doi:10.1007/s11684-017-0527-6

Front. Med.

2017, Vol. 11

Issue (3) : 410-422 https://doi.org/10.1007/s11684-017-0527-6

RESEARCH ARTICLE

Annexin A2-S100A10 heterotetramer is upregulated by PML/RARα fusion protein and promotes plasminogen-dependent fibrinolysis and matrix invasion in acute promyelocytic leukemia

Dan Huang¹, Yan Yang¹, Jian Sun¹, Xiaorong Dong¹, Jiao Wang¹, Hongchen Liu¹, Chengquan Lu², Xueyu Chen¹, Jing Shao²(

), Jinsong Yan¹(

)

¹. Dalian Key Laboratory of Hematology, Liaoning Hematopoeitic Stem Cell Transplantation Medical Center, Department of Hematology of the Second Hospital of Dalian Medical University, Dalian 116027, China
². Dalian Key Laboratory of Hematology, Liaoning Hematopoeitic Stem Cell Transplantation Medical Center, Department of Environmental Health and Toxicology, School of Public Health, Dalian Medical University, Dalian 116044, China

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Abstract

Aberrant expression of annexin A2-S100A10 heterotetramer (AIIt) associated with PML/RARα fusion protein causes lethal hyperfibrinolysis in acute promyelocytic leukemia (APL), but the mechanism is unclear. To facilitate the investigation of regulatory association between ANXA2 and promyelocytic leukemia/retinoic acid receptor a (PML/RARα) fusion protein, this work was performed to determine the transcription start site of ANXA2 promoter with rapid amplification of 5′-cDNA ends analysis. Zinc-induced U937/PR9 cells expressed PML/RARα fusion protein, and resultant increases in ANXA2 transcripts and translational expressions of both ANXA2 and S100A10, while S100A10 transcripts remained constitutive. The transactivation of ANXA2 promoter by PML/RARα fusion protein was 3.29±0.13 fold higher than that by control pSG5 vector or wild-type RARα. The overexpression of ANXA2 in U937 transfected with full-length ANXA2 cDNA was associated with increased S100A10 subunit, although S100A10 transcripts remained constitutive. The tPA-dependent initial rate of plasmin generation (IRPG) in zinc-treated U937/PR9 increased by 2.13-fold, and cell invasiveness increased by 27.6%. Antibodies against ANXA2, S100A10, or combination of both all remarkably inhibited the IRPG and invasiveness in U937/PR9 and NB4. Treatment of zinc-induced U937/PR9 or circulating APL blasts with all-trans retinoic acid (ATRA) significantly reduced cell surface ANXA2 and S100A10 and associated reductions in IRPG and invasiveness. Thus, PML/RARα fusion protein transactivated the ANXA2 promoter to upregulate ANXA2 and accumulate S100A10. Increased AIIt promoted IRPG and invasiveness, both of which were partly abolished by antibodies against ANXA2 and S100A10 or by ATRA.

Keywords annexin A2-S100A10 heterotetramer PML/RARα fusion protein plasmin cell invasion acute promyelocytic leukemia

Corresponding Author(s): Jing Shao,Jinsong Yan

Just Accepted Date: 02 June 2017 Online First Date: 10 July 2017 Issue Date: 29 August 2017

Cite this article:

Dan Huang,Yan Yang,Jian Sun, et al. Annexin A2-S100A10 heterotetramer is upregulated by PML/RARα fusion protein and promotes plasminogen-dependent fibrinolysis and matrix invasion in acute promyelocytic leukemia[J]. Front. Med., 2017, 11(3): 410-422.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0527-6
https://academic.hep.com.cn/fmd/EN/Y2017/V11/I3/410

Fig.1 PML/RARa fusion protein upregulated AIIt expression. (A) Using house-keeping gene β actin as the internal control, ANXA2 transcripts increased from constitutive level up to 106.3±0.37 fold at 72 h in U937/PR9 cells with induction of 100 µmol/L zinc at varied time points. (B) S100A10 transcripts maintained no more than 1.74±0.98 fold with the internal control gene of β actin at varied time points. (C) U937/PR9 cell nuclear extracts expressed PML/RARa fusion protein with induction of 100 µmol/L zinc, and U937/PR9 cell lysates with anti-ANXA2 and anti-S100A10 antibodies showed that the expression of ANXA2 and S100A10 increased 2 h after zinc induction. (D) By flow cytometry analysis, U937/PR9 cell-surface ANXA2 and S100A10 increased by 26.9% and 18.9%, respectively, after a 24 h exposure to zinc. *P<0.05.

Fig.2 Determination of transcription start site of ANXA2 promoter by 5′-RACE. The 3′-primers (outer primer, 5′-CTTGTAGACTCTGTTAATTTCCTG-3′; inner primer, 5′-TACTGAGCAGGTGTCTTCAATAGG-3′) specific for ANXA2CDS and designated primers in SMARTer RACE 5′/3′Kit (634859; Takara Bio, Japan) were used to amplify ANXA2 fragments by PCR, followed by a nested-PCR reaction. The amplified ANXA2 fragments were resolved on a 1% agarose gel and purified with TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (9762; Takara, Japan). The extracted ANXA2 fragments were sequenced with 3′-end inner primer of ANXA2, and nucleotide base G, which is located at 60397986 of the genomic sequence NC_000015.10 on chromosome 15, was determined as the TSS of ANXA2 promoter.

Fig.3 PML/RARa fusion protein transactivated ANXA2 promoter to upregulate ANXA2, which led to the increases of S100A10 subunit. (A) U937 cells transfected with ANXA2 cDNA overexpressed ANXA2 and S100A10 with Western blotting; however, U937 transfected with S100A10 cDNA showed an overexpression of S100A10 but no effect on ANXA2 subunit. (B) U937 cells transfected with S100A10 cDNA or ANXA2 cDNA showed an elevated expression of S100A10 or ANXA2 transcripts, respectively. (C) Eukaryocytic expression plasmids, including pSG5-PML/RARa, pSG5-RARa, or pSG5 vector, and pRL-SV40 vector, along with pGL3-A2 promoter reporter or pGL3-S100A10 promoter reporter, were co-transfected into COS-7 cells; luciferase activity showed that pSG5-PML/RARa plasmid led to a markedly elevated luciferase activity, which was abolished by 24-h exposure of 10⁻⁶ mol/L ATRA. (D) No transactivated luciferase activity occurred on S100A10 promoter by pSG5-PML/RARa, pSG5-RARa plasmid.

Fig.4 AIIt promoted initial rate of plasmin generation (IRPG) and invasiveness in zinc-induced U937/PR9 and NB4 cells, which were abrogated by monoclonal antibodies against ANXA2 and/or S100A10. (A) U937/PR9 cells showed an increase in IRPG from 1.75±0.84 RFU/min² to 3.73±1.67 RFU/min² in the presence of zinc at 24 h, when addition of monoclonal antibodies against ANXA2 or S100A10, or a combination of both, markedly inhibited the increase of the IRPG in zinc-induced U937/PR9 cells compared with that in U937/PR9 cells (*P<0.05). (B) Cell invasion in zinc-treated U937/PR9 cells increased to 87.13%±1.11% from 60.29%±0.71% in U937/PR9 cells without zinc induction. Blockage with antibodies against ANXA2 or S100A10, or a combination of both, markedly inhibited the invasion of zinc-treated U937/PR9 cells (*P<0.05). (C) IRPG in NB4 cells was 3.49±0.77 RFU/min² and was inhibited in the presence of monoclonal antibodies against ANXA2 or S100A10, or a combination of both (*P<0.05, **P<0.01). (D) Cell invasion was 93.62%±7.62% in NB4 cells and was inhibited in the presence of monoclonal antibodies against ANXA2 or S100A10, or a combination of both (*P<0.05, **P<0.01).

Fig.5 ATRA downregulated the AIIt expression in cells harboring PML/RARα and subsequently led to the reduction of initial rate of plasmin generation (IRPG) and cell invasion. (A) ATRA reduced the expression of ANXA2 and S100A10 in zinc-induced U937/PR9 cells after exposure to 10⁻⁶ mol/L ATRA. (B) Cell surface expression of AIIt subunits decreased following ATRA treatment. As revealed by flow cytometry, the expression of ANXA2 at cell surface was 93.9%±6.2% prior to ATRA treatment and then dropped to 33.4%±1.3% at 48 h and 17.2%±4.2% at 120 h post-treatment. The expression of S100A10 was 88.3%±5.7% prior to ATRA treatment and dropped to 43.8%±2.3% at 48 h and 14.9%±2.7% at 120 h post-treatment. **P<0.01. (C) ANXA2 transcripts decreased after ATRA treatment by TaqMan PCR with house-keeping gene β actin as the internal control. (D) S100A10 transcripts showed a decreased expression with β actin as the internal control at varied time points. (E) After exposure to ATRA, the IRPG in zinc-induced U937/PR9 cells decreased to 0.57±0.04 RFU/min² at 96 h post-ATRA from 2.18±0.47 RFU/min² prior to ATRA. *P<0.05. (F) After exposure to ATRA, the cell invasion of zinc-induced U937/PR9 cells decreased to 13.18%±8.38% at 96 h after exposure to ATRA from 82.84%±6.99% before treatment with ATRA. **P<0.01. (G) After exposure to ATRA, the IRPG in NB4 cells started to decrease to 2.38±1.08 RFU/min² at 24 h and 1.83±0.90 RFU/min² at 96 h from 3.95±0.41 RFU/min² prior to ATRA. *P<0.05. (H) The cell invasion in NB4 cells decreased from 74.55%±19.37% pre-treatment of ATRA to 8.26%±5.26% at 96 h post-ATRA treatment. **P<0.01.

Fig.6 Effects of ATRA treatment on the mRNA levels of ANXA2 and S100A10, the cell surface expression of ANXA2 and S100A10, and the IRPG and the cell invasiveness in the isolated APL blasts. The APL blasts were isolated from nine patients newly diagnosed with APL. (A) ATRA treatment decreased ANXA2 transcripts from onefold at pre-treatment to 0.35±0.19 fold at day 6 post-treatment, with β actin as the internal control. (B) ATRA treatment decreased S100A10 transcripts from onefold at pre-treatment to 0.38±0.12 fold at day 6 post-treatment, with β actin as the internal control. (C) Flow cytometry analysis on cell surface expression of ANXA2 expression. ANXA2 expression reduced from 15.80%±4.81% prior to treatment to 3.74%±1.49% at day 6 post-ATRA treatment. (D) Flow cytometry analysis on cell surface expression of S100A10 expression. S100A10 expression decreased from 27.9%±8.7% prior to treatment to 2.94%±1.41% at day 6 post-treatment. (E) ATRA treatment decreased the level of IRPG from the pre-treatment level of 1.66±0.38 fold to 1.08±0.50 fold at day 3 and 0.33±0.26 fold at day 6 post-treatment. In addition, blockage with antibodies against ANXA2 or S100A10, or a combination of both, further reduced the IRPG level of the isolated APL blasts from days 1 to 3. However, no inhibitory effect from antibodies was observed at day 6. (F) ATRA treatment decreased the level of the invasiveness of the isolated APL blasts from 27.9%±8.70% prior to treatment to 8.08%±4.28% at day 3 and 2.95%±1.41% at day 6. Antibodies against ANXA2 or S100A10, or a combination of both, further inhibited the invasiveness. However, the inhibitory effects became weaker along with ATRA treatment, particularly at day 6.

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