The signal regulatory protein (SIRP) α1, also termed CD172a or Src homology 2 domain-containing phosphatase substrate-1 (SHPS-1), is a cell surface receptor expressed predominantly in monocytes, granulocytes, dendritic cells, as well as hematopoietic stem cells [1, 11, 25]. SIRPα1 is the most characterized member of the human SIRP family. Its overexpression leads to a reduced responsiveness to tyrosine kinase ligands, such as epidermal growth factor, platelet-derived growth factor and insulin . These suggest that, SIRPα1 is considered as a negative regulator of signaling. In addition to this, SIRPα1 expression is significantly reduced in the majority of acute and chronic myeloid leukemias . Therefore, the reduced expression is considered to play a role in the pathogenesis of these diseases through aberrant signaling, such as SHP-1 and its downstream signaling KIT pathway .
It was reported that, CD47, a ligand for SIRPα1, was identified as an important “don’t eat me” signal expressed on tumor cells [9, 16]. CD47 is constitutively upregulated on human myeloid leukemias, and over-expression of CD47 on a myeloid leukemia line increases its pathogenicity by allowing it to evade phagocytosis . Therefore, the blockade of the CD47: SIRPα1 axis between tumor cells and immune cells has been attracted attention [12, 3]. However, the precise biological mechanisms of the downstream effects of SIRPα1 suppression in leukemia cells have not been clarified.
My group previously established SIRPα1-knockdown chronic myeloid leukemia K562 (K562SIRPα1KD) cells expressing reduced levels of SIRPα1 by stably transfecting SIRPα1 siRNAs, and found that expression of β-catenin is increased in these cells . Phosphorylation of Ser 9 of glycogen synthase kinase (GSK)-3β, results in the activation of this protein, leading to the up-regulation of β-catenin . We revealed that downregulation of SIRPα1 results in the aberrant phosphorylation of several signaling pathway components, such as extracellular regulated kinase (ERK), Akt and GSK-3β, which may lead to the induction of β-catenin.
In this study, I examined whether the serum deprivation enhances the activity of downstream signaling pathways, affect to the cell survival. As a result, I revealed that in serum starvation conditions, K562SIRPα1KD cells showed aberrant cell proliferation.
K562 SIRPa1KD cells and their control cells were previously established in our laboratory  and employed in this study. Exponential phase cells were collected and counted, washed twice with phosphate buffered saline (PBS) and seeded 5000 cells/well at 96 well plate in indicated serum concentrations. At indicated times, cell growth was determined by means of dye reduction assay involving a tetrazolium salt WST-8 (Dojindo, Tokyo, Japan).
Cell cycle profiles were determined by analyzing the DNA contents using propidium iodide (Dojindo) staining and flow cytometry as described . Briefly, 48 hours after the indicated proportions of the serum in the culture, the cells were washed, fixed, and stored at –20°C until analysis. These cells were collected, suspended in 300 µl of RNase/PBS solution and incubated at 37°C for 30 min. Then, the cells were resuspended in 300 µl of propidium iodide/PBS solution, and incubated in the dark at room temperature for 15 min. After filtration, all samples were applied to an Epics XL (Beckman Coulter, Nyon, Switzerland). To analyze the data, ModFitLT (Verity Software, Topsham, ME, USA) were employed.
BrdU incorporation assay was carried out as per the BrdU Staining Kit for Flow Cytometry FITC (eBioscience, San Diego, CA, USA). To examine cell proliferation in low serum condition, exponentially grown cells are incubated for 48 hours in 0.3% serum in culture. For serum stimulation assay, exponentially grown cells were cultured and collected and counted, washed twice with PBS, cultured 24 hours in serum free RPMI. Then FBS was added to a final 10% volume into the medium and incubated for 16–20 hours. These cells were washed twice with PBS and 10 µM of BrdU was added to the cell medium for 45 minutes and then the cells were washed with flow cytometry staining buffer. Cells were fixed and stained for BrdU using a FITC conjugate anti-BrdU FITC antibody (clone BU20A). Samples were analyzed using MACSQuant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany). Negative control samples without BrdU were used to establish levels of background autofluorescence and to define the BrdU positive gate.
Westernblot was performed as described previously [15, 8]. Briefly, total cellular extracts were prepared from the cells cultured with 0.3% FBS for indicated period. To examine the signaling, anti-phospho-p44/42 mitogen-activated protein kinase (MAPK) (Thr202/Tyr204), anti-phospho-p38 (T180/Y182), anti-p-Akt (S473), anti-Akt1 rabbit polyclonal antibodies and p44/42 MAPK mouse monoclonal antibody were purchased from Cell Signaling Technology (Beverly, USA). Anti-38 α/stress-activated protein kinase (SAPK) 2α mouse monoclonal antibody was obtained from BD Biosciences (San Jose, CA, USA) and employed for this study.
I first examined the cell proliferation characteristics of K562SIRPα1KD cells in various concentrations of serum. As my group reported previously, there are sufficient expression of SIRPα1 mRNA and protein in K562 cells and these were obviously suppressed by the stable transfection of SIRPα1 siRNA into K562 cells . By employing these cells, I observed that in low serum culture from 0.1% to 0.3%, there is a marked increment of the cell proliferation in both (1–2, 2–6) K562SIRPα1KD cells, compared to their controls (vec 5, vec 6)(Figure 1). The cell growth was increased until day 5 in K562SIRPα1KD cells, whereas control cells did not proliferate beyond day 2 under the same serum conditions (Figure 1). This phenomenon was not observed in more than 1% serum in the medium. Consistently, cell cycle analyses after low serum (0.1% to 0.3%) culture for 48 hours revealed that the proportions of S-phase cells are significantly increased in K562SIRPα1KD cells, but not in their controls (Figure 2A, 2B). This was also not observed in more than 1% serum. I next examined the percentages of BrdU incorporated cells to examine the cell proliferation. After 48h incubation with low serum culture (0.3% serum), the percentages of BrdU incorporated cells were slightly increased in K562SIRPα1KD cells (Figure 3A). Moreover, serum stimulation experiment was also performed, and found that Brd-U positive cells were significantly increased in K562SIRPα1 KD cells (Figure 3B, 3C). Collectively, these suggest that in the low serum condition, downregulation of SIRPα1 result in the increase of the viable cells.
I next examined the signaling status in these conditions, because there are difference of viability and cell growth between K562SIRPα1 KD cells and their controls. Leukemia cell growth and survival responses to environmental triggers are controlled by the activation of multiple intracellular signaling pathways, in which ERK, Akt and p38 mitogen-activated protein kinases (p38MAPK). The function of the p38 kinases is required for the generation of various activities, including regulation of apoptosis, cell-clycle arrest and differentiation . Among these kinase pathways examined, as shown in Figure 4, I found that in this low serum culture (0.3% serum), ERK is potently phosphorylated in K562SIRPα1KD cells. I also observed obvious phosphorylation of Akt in K562SIRPα1KD cells. In addition, there are some increase of p38 phosphorylation in 1–2 at 2 and 5 days, and slight increase in 2–6 at 5 days. These suggest that suppression of SIRPα1 may have marginal effect on p38 phosphorylation. This issue will be discussed in the next section.
Mitui et al. , previously reported that MEK-ERK signaling pathway is required for serum-dependent survival of liver cancer HepG2 cells. They also revealed that transient expression of active MEK1 prevented apoptosis in serum-deprived condition . I speculate that activation of ERK (Figure 4), observed in low serum culture, may leads to the escape from the cell death by serum withdrawal. Although there were no growth differences between K562SIRPα1KD cells and their controls in the full culture condition (Figure 1, 10%), I observed obvious difference for the growth in low serum culture (Figure 1, 0.1–0.3%). Serum is a complex mix of albumins, growth factors and inhibitors . I speculate that in the full culture condition, redundant signals mask the important SIRPα1 related signal, which is necessary for cell growth and survival, such as ERK signaling. This may be explained by the fact that there was very modest difference for ERK phosphorylation between K562 SIRPα1KD cells and their controls in normal culture conditions , whereas very strong difference for phosphorylated ERK in low serum culture (Figure 4) in the present study. In addition, I observed marginal phosphorylation of p38 MAPK in K562 SIRPα1KD cells in low serum culture (Figure 4). In general, the activation of the p38 MAPK pathway tends to promote apoptosis whereas the activation of the ERK pathway tends to be anti-apoptotic . Birkenkamp et al. , have previously demonstrated by using TF-1 human erythroleukemia cells, that the effects of the ERK signaling pathway can overcome the pro-apoptotic effects of the p38 signaling pathway. They revealed that although the activation of the p38 pathway may be required for growth factor withdrawal-induced apoptosis, in the presence of high enough levels of ERK activation, p38 activation may not be sufficient in itself for apoptosis to occur . Based on my findings (Figures 1 and 4) and a literature , these suggest that survival effect of SIRPα1KD (Figure 1) is mainly regulated by ERK activation.
The Ras-ERK-MAPK pathways are frequently activated in hematological malignancies [17, 20]. Lunghi et al.,  previously reported that among 20 AML patients, 14 cases of AML patients had high ERK1/2 activity whereas only four cases of AML had low ERK1/2 activity. It is possible to speculate that the down-regulation of SIRPα1 is contributing to the activation of ERK1/2 in primary AML cells. Cortez et al.,  demonstrated that BCR-ABL expression activates Ras, ERK and Jnk pathways as a primary consequence of expression, induces G1-to-S phase transition, DNA synthesis, and activation of cyclin dependent kinases in cells that were arrested in G0 by growth factor deprivation . The baseline activation of ERK by BCR-ABL in K562 cells may not enough to confer serum starvation, but additional aberration, such as down-regulation of SIRPα1, result in further activation of ERK and several other pathways, may be required to obtain resistance to serum deprivation. Although alterations affecting the functions of transcription factors that regulate myeloid maturation play important roles in leukemogenesis [22, 29], inappropriate MAPK may also play a role in leukemic transformation [17, 20]. It has been shown that the interruption of this pathway profoundly affects the growth of AML cell lines and primary AML samples with constitutive MAPK activation [17, 24]. As down-regulation of SIRPα1, as well as inappropriate MAPK activation, is frequently observed in hematological malignancies, our study may shed light on the importance of SIRPα1 expression, which is relatively easy to examine by flow cytometry, in predicting the efficacy to employ molecularly targeted therapies including MAPK inhibitors.
Although SIRPα1 expression is widely expressed, the down regulation of SIRPα1 is also reported in various cancers. In prostate cancer, SIRPα1 expression is downregulated in prostate cancer tissues and cell lines, and over-expression of SIRPα1 resulted in the reduction of the number of live prostate cancer tissues, whereas SIRPα1 silencing increased prostate cancer proliferation . Consistently, it was demonstrated that SIRPα1 receptors interact with SHP-2 to inhibit wild-type epidermal growth factor receptor (EGFR) mediated tumor migration, survival and cell transformation . Same group also demonstrated that SIRPα1 receptors interfere with EGFR to inhibit glioblastoma cell transformation and migration . The expression of SIRPα1 was down-regulated in hepatocellular carcinoma tissues and cells, and over-expression of SIRPα1 in these cells results in a decrease in the growth of hepatocellular carcinoma cells . Oshima et al.  demonstrated SHPS-1 (SIRPα) is down-regulated in breast cancer tissues compared with the matched normal tissues. In v-src-transformed BALB/c3T3 cells, over-expression of SHPS-1 resulted in the suppression of anchorage independent cell growth in soft agar, as well as peritoneal dissemination in nude mice . This is quite consistent with the fact that the inhibition was observed for human neutrophil migration in the presence of soluble, recombinant CD47 consisting SIRPα binding loop . As SIRPα signal is inhibition of cell growth, the addition of CD47 ligand expected to inhibit the growth of serum starved cells. Seiffert et al. , reported that SIRPα1 expression is reduced in myeloid malignancies. Concretely, no expression in 4 out of 4 chronic myeloid leukemia patients and 26 out of 59 acute myeloid leukemia patients analyzed, while there is sufficient expression in normal myelo-monocytic cells, as well as with bone marrow hematopoietic stem/progenitor cells . However, the biological significance of this down-regulation of SIRPα1 in myeloid leukemias has not been clarified until now.
My current study revealed the contribution of the down-regulation of SIRPα1 to the constitutive activation of these aberrant signaling, as well as abnormal growth, in leukemia cells. To clarify the mechanisms the down-regulation of SIRPα1 in myeloid malignancies, as well as solid cancer, and to develop the restoration of SIRPα1 expression in these tumors, may open a door to new therapeutic strategies.
Initial experiments in this study were performed at Hematology laboratory in Kitasato University School of Allied Health Science, which is the author’s previous affiliation, and author thanks to my ex-laboratory technician Ms. Hiroko Nakano, and undergraduate student Ms. Sari Nakamura for obtaining most of the data including BrdU assay, cell cycle analysis and Western blot. The author also thanks to my former postgraduate student in the current lab., Mr Yutaro Takabuchi for discussions and confirming the data. This work was supported in part by a Grant-in-Aid for Scientific Research (No. 17K09019, 21K07346) from the Ministry of Education, Science and Culture, Japan, Daiichi-Sankyo Research Support (Daiichi-Sankyo Inc.).
The author has no competing interests to declare.
Barclay, AN and Brown, MH. The SIRP family of receptors and immune regulation. Nat Rev Immunol. 2006; 6: 457–64. DOI: https://doi.org/10.1038/nri1859
Birkenkamp, KU, Dokter, WH, Esselink, MT, Jonk, LJ, Kruijer, W and Vellenga, E. A dual function for p38 MAP kinase in hematopoietic cells: involvement in apoptosis and cell activation. Leukemia. 1999; 13: 1037–45. DOI: https://doi.org/10.1038/sj.leu.2401447
Chen, J, Zhong, MC, Guo, H, Davidson, D, Mishel, S, Lu, Y, Rhee, I, Perez-Quintero, LA, Zhang, S, Cruz-Munoz, ME, Wu, N, Vinh, DC, Sinha, M, Calderon, V, Lowell, CA, Danska, JS and Veillette, A. SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin. Nature. 2017; 544: 493–497. DOI: https://doi.org/10.1038/nature22076
Cortez, D, Reuther, G and Pendergast, AM. The Bcr-Abl tyrosine kinase activates mitogenic signaling pathways and stimulates G1-to-S phase transition in hematopoietic cells. Oncogene. 1997; 15: 2333–42. DOI: https://doi.org/10.1038/sj.onc.1201400
Ding, Q, Xia, W, Liu, JC, Yang, JY, Lee, DF, Xia, J, Bartholomeusz, G, Li, Y, Pan, Y, Li, Z, Bargou, RC, Qin, J, Lai, CC, Tsai, FJ, Tsai, CH and Hung, MC. Erk associates with and primes GSK-3beta for its inactivation resulting in upregulation of beta-catenin. Mol Cell. 2005; 19: 159–70. DOI: https://doi.org/10.1016/j.molcel.2005.06.009
Franklin, RA and Mccubrey, JA. Kinases: positive and negative regulators of apoptosis. Leukemia. 2000; 14: 2019–34. DOI: https://doi.org/10.1038/sj.leu.2401967
Hirako, N, Nakano, H and Takahashi, S. A PU.1 suppressive target gene, metallothionein 1G, inhibits retinoic acid-induced NB4 cell differentiation. PLoS One. 2014; 9: e103282. DOI: https://doi.org/10.1371/journal.pone.0103282
Hirosawa, M, Nakahara, M, Otosaka, R, Imoto, A, Okazaki, T and Takahashi, S. The p38 pathway inhibitor SB202190 activates MEK/MAPK to stimulate the growth of leukemia cells. Leuk Res. 2009; 33: 693–9. DOI: https://doi.org/10.1016/j.leukres.2008.09.028
Jaiswal, S, Jamieson, CH, Pang, WW, Park, CY, Chao, MP, Majeti, R, Traver, D, Van Rooijen, N. and Weissman, IL. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell. 2009; 138: 271–85. DOI: https://doi.org/10.1016/j.cell.2009.05.046
Kapoor, GS and O’rourke, DM. SIRPalpha1 receptors interfere with the EGFRvIII signalosome to inhibit glioblastoma cell transformation and migration. Oncogene. 2010; 29: 4130–44. DOI: https://doi.org/10.1038/onc.2010.164
Kharitonenkov, A, Chen, Z, Sures, I, Wang, H, Schilling, J and Ullrich, A. A family of proteins that inhibit signalling through tyrosine kinase receptors. Nature. 1997; 386: 181–6. DOI: https://doi.org/10.1038/386181a0
Liu, J, Wang, L, Zhao, F, Tseng, S, Narayanan, C, Shura, L, Willingham, S, Howard, M, Prohaska, S, Volkmer, J, Chao, M, Weissman, IL and Majeti, R. Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential. PLoS One. 2015; 10: e0137345. DOI: https://doi.org/10.1371/journal.pone.0137345
Liu, Y, Buhring, HJ, Zen, K, Burst, SL, Schnell, FJ, Williams, IR and Parkos, CA. Signal regulatory protein (SIRPalpha), a cellular ligand for CD47, regulates neutrophil transmigration. J Biol Chem. 2002; 277: 10028–36. DOI: https://doi.org/10.1074/jbc.M109720200
Lunghi, P, Tabilio, A, Dall’aglio, PP, Ridolo, E, Carlo-Stella, C, Pelicci, PG and Bonati, A. Downmodulation of ERK activity inhibits the proliferation and induces the apoptosis of primary acute myelogenous leukemia blasts. Leukemia. 2003; 17: 1783–93. DOI: https://doi.org/10.1038/sj.leu.2403032
Maekawa, T, Imoto, A, Satoh, T, Okazaki, T and Takahashi, S. Induction of beta-catenin by the suppression of signal regulatory protein alpha1 in K562 cells. Int J Mol Med. 2011; 27: 865–72. DOI: https://doi.org/10.3892/ijmm.2011.630
Mccracken, MN, Cha, AC and Weissman, IL. Molecular Pathways: Activating T Cells after Cancer Cell Phagocytosis from Blockade of CD47 “Don’t Eat Me” Signals. Clin Cancer Res. 2015; 21: 3597–601. DOI: https://doi.org/10.1158/1078-0432.CCR-14-2520
Milella, M, Kornblau, SM, Estrov, Z, Carter, BZ, Lapillonne, H, Harris, D, Konopleva, M, Zhao, S, Estey, E and Andreeff, M. Therapeutic targeting of the MEK/MAPK signal transduction module in acute myeloid leukemia. J Clin Invest. 2001; 108: 851–9. DOI: https://doi.org/10.1172/JCI12807
Mitsui, H, Takuwa, N, Maruyama, T, Maekawa, H, Hirayama, M, Sawatari, T, Hashimoto, N, Takuwa, Y and Kimura, S. The MEK1-ERK map kinase pathway and the PI 3-kinase-Akt pathway independently mediate anti-apoptotic signals in HepG2 liver cancer cells. Int J Cancer. 2001; 92: 55–62. DOI: https://doi.org/10.1002/1097-0215(200102)9999:9999<::AID-IJC1143>3.0.CO;2-O
Platanias, LC. Map kinase signaling pathways and hematologic malignancies. Blood. 2003; 101: 4667–79. DOI: https://doi.org/10.1182/blood-2002-12-3647
Qin, JM, Yan, HX, Liu, SQ, Wan, XW, Zeng, JZ, Cao, HF, Qiu, XH, Wu, MC and Wang, HY. Negatively regulating mechanism of Sirpalpha1 in hepatocellular carcinoma: an experimental study. Hepatobiliary Pancreat Dis Int. 2006; 5: 246–51.
Rosenbauer, F, Wagner, K, Kutok, JL, Iwasaki, H, Le Beau, MM, Okuno, Y, Akashi, K, Fiering, S and Tenen, DG. Acute myeloid leukemia induced by graded reduction of a lineage-specific transcription factor, PU.1. Nat Genet. 2004; 36: 624–30. Epub 2004 May 16. DOI: https://doi.org/10.1038/ng1361
Seiffert, M, Cant, C, Chen, Z, Rappold, I, Brugger, W, Kanz, L, Brown, EJ, Ullrich, A and Buhring, HJ. Human signal-regulatory protein is expressed on normal, but not on subsets of leukemic myeloid cells and mediates cellular adhesion involving its counterreceptor CD47. Blood. 1999; 94: 3633–43. DOI: https://doi.org/10.1182/blood.V94.11.3633.423k01_3633_3643
Takahashi, S. Inhibition of the MEK/MAPK signal transduction pathway strongly impairs the growth of Flt3-ITD cells. Am J Hematol. 2006; 81: 154–5. DOI: https://doi.org/10.1002/ajh.20520
Takahashi, S. Molecular functions of SIRPalpha and its role in cancer. Biomed Rep. 2018; 9: 3–7. DOI: https://doi.org/10.3892/br.2018.1102
Valent, P, Ghannadan, M, Hauswirth, AW, Schernthaner, GH, Sperr, WR and Arock, M. Signal transduction-associated and cell activation-linked antigens expressed in human mast cells. Int J Hematol. 2002; 75: 357–62. DOI: https://doi.org/10.1007/BF02982124
Van Der Valk, J, Brunner, D, De Smet, K, Fex Svenningsen, A, Honegger, P, Knudsen, LE, Lindl, T, Noraberg, J, Price, A, Scarino, ML and Gstraunthaler, G. Optimization of chemically defined cell culture media--replacing fetal bovine serum in mammalian in vitro methods. Toxicol In Vitro. 24: 1053–63. DOI: https://doi.org/10.1016/j.tiv.2010.03.016
Wu, CJ, Chen, Z, Ullrich, A, Greene, MI and O’rourke, DM. Inhibition of EGFR-mediated phosphoinositide-3-OH kinase (PI3-K) signaling and glioblastoma phenotype by signal-regulatory proteins (SIRPs). Oncogene. 2000; 19: 3999–4010. DOI: https://doi.org/10.1038/sj.onc.1203748
Yan, M, Burel, SA, Peterson, LF, Kanbe, E, Iwasaki, H, Boyapati, A, Hines, R, Akashi, K and Zhang, DE. Deletion of an AML1-ETO C-terminal NcoR/SMRT-interacting region strongly induces leukemia development. Proc Natl Acad Sci U S A. 2004; 101: 17186–91. Epub 2004 Nov 29. DOI: https://doi.org/10.1073/pnas.0406702101
Yao, C, Li, G, Cai, M, Qian, Y, Wang, L, Xiao, L, Thaiss, F and Shi, B. Prostate cancer downregulated SIRP-alpha modulates apoptosis and proliferation through p38-MAPK/NF-kappaB/COX-2 signaling. Oncol Lett. 2017; 13: 4995–5001. DOI: https://doi.org/10.3892/ol.2017.6070