Signal Regulatory Protein α Is Associated With Tumor Polarized Macrophages Phenotype Switch And Plays A Pivotal Role In Tumor Progression

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, and the second leading cause of cancer death in China. HCC is usually present in inflamed fibrotic and/or cirrhotic liver with extensive leukocyte infiltration. Thus, the immune status at different tumor sites is tightly associated with the biological behavior of HCC.Macrophages (Mφ) are the most prominent component of the infiltrated leukocytes in tumors. These cells are derived from circulating monocytes and recruited into tumor by cytokines and chemokines, such as CSF1and MCP-1. Mφ have remarkable plasticity that they can acquire special phenotypic characteristics with diverse functions in response to environmental signals. Tumor-associated Mφ (TAMs) closely belong to M2, they could suppress antitumor immunity and promote tumor progression. Evidences from clinical and epidemiological studies have shown a strong association between TAMs density and poor prognosis in several types of cancer, including hepatocellular carcinoma. However, some publications demonstrated that Mφ in tumor stroma were activated and displayed a HLA-DRhigh phenotype. These cells can also facilitate tumor progression. Taken together, these results indicated that tumor can take advantage of either immune suppression or activation status of Mφ at distinct tumor sites to promote tumor progression. Currently, the precise mechanism of how tumor educating Mφ to accomplish specific tasks has not been fully elucidated.Signal regulatory protein a (SIRPa) is a cell-surface protein mainly expressed on myeloid cells, including Mφ and dendritic cells. The extracellular region of SIRPa is heavily glycosylated and comprises of three immunoglobin superfamily (IgSF) domains, which are similar to the TCR and BCR, suggesting that SIRPa may have a pivotal role in immune regulation. The intracellular region contains two immunoreceptor tyrosine-based inhibition motifs (ITIM) with four tyrosine residues that are phosphorylated in response to a variety of stimuli. The phosphorylation allows SHP1or SHP2recruitment to SIRPa that, in turn, dephosphorylates specific substrates involved in various physiological effects. SIRPa can bind to either widely expressed transmembrane ligand CD47or soluble ligands, such as the surfactant proteins A and D. It is suggested that SIRPa/CD47signaling axis is important in tumor therapy. Our previous work has shown that SIRPa negatively regulate TLR signaling in Mφ. However, it is still unknown whether SIRPa expression on tumor-polarized Mφ can act on tumor progression.In this article, we identify that SIRPa is down-regulated on monocytes/Mcp isolated from peritumoral areas of hepatocellular carcinoma (HCC) samples, while its level is moderately recovered in intratumor Mφ. In vitro assay demonstrates that SIRPa expression is significantly reduced on Mφ when co-cultured with hepatoma cells. This reduction is partly due to the soluble factors in tumor microenvironment. Knockdown (KD) of SIRPa prolongs activation of NF-κB and PI3K-Akt pathways as Mφ encountering tumor cells, leading to increased capacity of Mφ for migration, survival and proinflammatory cytokine production. Enhanced Stat3and impaired Statl phosphorylation are also observed in tumor-exposure SIRPa-KD Mφ. Adoptive transfer with SIRPa-KD Mφ accelerates mouse hepatoma cells growth in vivo by remolding inflammatory microenvironment and promoting angiogenesis. SIRPa accomplishes this partly through its sequestration of the signal transducer SHP2from IKKβ and PI3K regulatory subunit p85(PI3Kp85). These findings suggest that SIRPa functions as an important modulator of tumor-polarized Mφ in hepatoma, and the reduction of SIRPa is a novel strategy used by tumor cells to benefit their behavior. Therefore, SIRPa could be utilized as a potential target for HCC therapy Mast cells are important immune effector cells and play a central role in IgE-dependent allergic disease. FcεRI aggregation contributes to inflammatory symptomology by inducing the release of proinflammatory mediators and the production of various cytokines. Upon crosslinking by IgE-allergen, FcεRI β and γ subunits are phosphorylated at ITAMs by the receptor-proximal Src family protein tyrosine kinases (SFKs), such as Lyn, Fyn and Hck. Activated FcεRI induces a large number of downstream molecules activation, such as phosphatidylinositol3’-kinase (PI3K)-Akt kinase, phospholipase Cγ (PLCγ), mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB), resulting in enhanced calcium flux, prominent degranulation and increased synthesis of various cytokines. However, the precious mechanism involved in the regulation of this process is still not fully understood.Appropriate activation of mast cells is mediated by several factors, including the ability of the cells to distinguish activating or inhibitory stimuli as well as the strength and duration of a given stimulus. Several negative intracellular regulators modulate the signaling events initiated by FcεRI in mast cells, including the SH2-containing tyrosine phosphatases1(SHP1), the lipid phosphatase SH2-containing inositol5’phosphatase1(SHIP1), and the E3ubiquitin ligase c-Cbl. In addition, once coaggregated with their ligands, cell surface receptors that contain immunoreceptor tyrosine-based inhibitory motifs (ITIM) provide inhibitory signals to dampen those FcεRI-initiated events. Through their ITIM, these receptors suppress cell activation by promoting dephosphorylation of key molecules via SHP1, SHP2or the SHIP family.Signal regulatory protein (SIRPα), also known as Src homology-containing phosphotyrosine phosphatase substrate (SHPS), is a member of the immunoglobulin-like receptor superfamily proteins. SIRPα is especially abundant in innate immune cells including mast cells. The putative extracellular region of SIRPα possesses three immunoglobulin-like domains with multiple N-linked glycosylation sites. The cytoplasmic region of SIRPα contains two ITIM with four tyrosine residues which are phosphorylated in response to a variety of growth factors and integrin-mediated cell adhesion. This phosphorylation enables recruitment of Src homology-containing tyrosine phosphatase2(SHP2). SHP2, a widely expressed cytoplasmic tyrosine phosphatase with two SH2domains, has been reported to be involved in growth factor-induced cell proliferation, probably through activation of the Ras-MAPK cascade. SIRPa/SHP2complex could negatively or positively regulate intracellular signaling initiated either by tyrosine kinase-coupled receptors for growth factors or by cell adhesion to extracellular matrix proteins. Moreover, dominant-negative form of SIRPa makes cells resistant to TNF-induced apoptosis through activation of NF-κB. Previous publication has demonstrated that coaggregation of the intracellular region of SIRPa with FcεRI inhibits immunoreceptor-dependent mast cell activation. However, the functional properties of native SIRPa molecule in mast cells have not been fully examined.Here we provide evidence to show that signal regulatory protein a (SIRPa), an ITIM-containing receptor, is an endogenous regulator of IgE-Ag induced mast cell activation. SIRPα expression is promptly reduced in mast cells in response to FcεRI aggregation. Impaired expression of SIRPa in mast cells facilitates FcεRI-evoked degranulation and de novo synthesis of cytokines (IL-4, IL-13, IL-6and TNFa). Further investigations demonstrate that SIRPa knockdown in mast cells accelerates calcium mobilization and affects cytoskeletal rearrangement (F-actin disassembly and polymeric tubulin formation) after FcεRI aggregation. Mechanistic studies highlight the prolonged activation of NF-κB and MAPKs as well as PLCy after FcεRI stimulation asa consequence of the inhibition of SIRPa expression in mast cells. Immunoprecipitation analysis shows that SIRPa knockdown markedly increases IgE-induced SHP2interaction with PI3K regulatory subunit PI3Kp85or IKKβ in mast cells, indicating that SIRPa may accomplish this through its association and sequestration of SHP2. Collectively, our results strongly indicate that SIRPa is a biologically important regulator of FcεRI signaling.