CD47/SIRPα pathways: Functional diversity and molecular mechanisms

Abstract

Cellular communication is required for the normal function and maintenance of homeostasis. The extracellular communications are mediated by cell surface receptors, which transmit signals for various cell functions. Cell defense also relies on distinguishing between self and non-self. The integrins belong to the transmembrane receptors family and serve a crucial function in cell-extracellular adhesion and cell–cell signaling. The cell surface integrin-associated protein, or CD47, is an important integrin regulator and performs an array of functions. The CD47 interacts with the signal regulatory protein α (SIRPα) and regulates adhesion, apoptosis, phagocytosis, proliferation, metabolism, activation, hematopoietic stem cell migration, and malignancies. The CD47 expression is increased in tumor cells to escape immune response. The anti-CD47 antibodies have shown promising therapeutic strategies in various clinical trials in the treatment of hematologic and solid tumors. In the current review, we discussed multifunctional roles, molecular mechanisms, and therapeutic strategies of tumors by utilizing CD47/SIRPα interactions.

Key Words: CD47; Signal regulatory protein α; Erythrocyte; Cancer; Immunity

Core Tip: In the current review, we discussed multifunctional roles (apoptosis, phagocytosis, metabolism, activation, hematopoietic stem cell migration, and malignancies) and molecular mechanisms of CD47/signal regulatory protein α receptors in various cells.

INTRODUCTION

CD47, or integrin-associated protein, is a 50 kDa heavily glycosylated plasma membrane transmembrane protein ubiquitously expressed in various organs and tissues[1]. It is expressed on erythrocytes and lymphocytes and a variety of cells having β3 integrin expression[1,2]. It contains an extracellular immunoglobulin domain, five transmembrane domains, and a short intracellular tail (Figure 1). It was initially recognised as a protein associated with α3β3 integrin in the placenta and neutrophils and later shown to modulate integrin function and leukocyte responsiveness to RGD-containing extracellular matrix protein[3]. Various isoforms of CD47 proteins with different functional profiles have been recognized[4]. These isoforms are generated either by post-translational changes, like glycosylation, pyroglutamate and glycosaminoglycan modification, and proteolytic cleavage, or by a pre-translational alternative cleavage, and polyadenylation[5-7]. The altered CD47 proteoform performs different functions. CD47 interacts with the signaling regulatory protein (SIRP) family and thrombospondin-1 (TSP1) (Figure 1).

Figure 1 CD47SIRPα interaction in tumour cells and macrophage has been shown. ITIM: Immunoreceptor tyrosine inhibitory motif; SIRP: Signal regulatory protein.

SIRPα is expressed on various cells, most abundantly on neurons and myeloid-lineage hematopoietic cells, polymorphonuclear leukocytes, monocytes, fibroblasts, and endothelial cells[8]. SIRPα, also known as Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) (BIT/CD172a), belongs to the Ig superfamily[9]. It has three extracellular Ig-like domains (one distal IgV-like domain and two membrane-proximal IgC-like domains) and four tyrosine residues in the cytoplasm[9]. The SIRPα cytoplasmic tyrosine residues phosphorylate and form an immunoreceptor tyrosine inhibitory motif (ITIM)[10]. ITIMs serve as binding sites for tyrosine phosphatases with dual Src-homology 2 domains, as well as inositol phosphatase and other receptors that increase tyrosine kinase activity (Figure 1). The SHP1/2 activation mediates Syk activation and phosphorylation of ITAM, which regulates phagocytosis[11]. The SIRP family also includes two other members, SIRPβ and SIRPγ proteins[10]. CD47 interacts only with the SIRPα and SIRPγ[12]. CD47's IgV domain interacts with integrins αvβ3 and α2β1, as well as its ligands TSP and SIRPα[4].

CD47/SIRPα signaling performs a wide range of functions. CD47-mediated signaling modulates metabolism and mitochondrial homeostasis in various cells[13]. CD47/SIRPα signaling performs a crucial role in immune defense, lymphocyte homeostasis, bone remodeling, hematopoietic stem cell (HSCs) engraftment, atherosclerosis, and tumor immune surveillance by activation of macrophage phagocytic response and cells of the immune system[14]. The interaction is species-specific, and responses depend on the bidirectional signalling of both the receptors. The detailed functions governed by the CD47/SIRPα interaction are described in the next sections (Table 1).

Table 1 Major cellular functions performed by CD47/SIRPα signaling.

Cell	Physiological function
Erythrocytes	Serves as a marker of self. Maintain membrane integrity and erythrophagocytosis
Macrophages	Regulation of phagocytosis
Dendritic cells	Regulation of phagocytosis. Activation. Mediates immune-inhibitory signals. Transendothelial migration
Neutrophils	Adhesion. Phagocytosis. Transendothelial migration. Inflammatory response. Apoptosis
T-lymphocyte	T-cell survival and differentiation. Activates T-cell receptor-mediated pathways
Monocytes	Transendothelial migration
Endothelial cells	Insulin growth factor signaling
Platelets	Integrins α2β1 and αIIbβ3 expression
Cancer cell	Migration. Invasion. Immune resistance
Natural killer cells	NK cell homeostasis. Graft rejection
Osteoclast cells	Proliferation
Hematopoietic stem cells	Migration

The major functions of CD47/SIRPα signaling on various cells have been shown.

MAJOR CELLULAR FUNCTIONS PERFORMED BY CD47/SIRPα SIGNALLING

CD47 receptor functions on erythrocytes

Although mature erythrocytes lack integrin expression, CD47 is extensively expressed on their surfaces. It is coupled with the erythrocyte cytoskeleton and the non-cytoskeleton portion and performs different functions[15]. CD47 regulates the erythrocyte membrane integrity by creating an important connection between the band 3 complex and the Rh complex[16]. It serves as a marker of self on erythrocytes[17]. CD47-lacking erythrocytes have reduced survival in circulation. Spleen- or bone marrow-derived macrophages phagocytose CD47-deficient erythrocytes at a much faster rate than control[17]. CD47/SIRPα interaction sends a negative signal to macrophages to prevent erythrophagocytosis[17,18] (Figure 2). The negative signalling through the CD47-SIRPα interaction is mediated by the SHP-1, which forms a complex with the SIRPα[19]. CD47/SHPS-1 interaction causes the phosphorylation of SHPS-1 tyrosine and prevents the Fc-R from breaking down the SHPS-1-SHP-1 complex and inhibits the erythrophagocytosis[20]. CD47-SIRPα signalling activates CDC42, a GTP-binding protein that causes cytoskeleton movement[21]. This also leads to the induction of myosin IIA and induces a phagocytic response.

Figure 2 CD47SIRPα interaction in erythrocyte and macrophage has been shown. SIRP: Signal regulatory protein.

The loss of the CD47/SHPS-1 interaction facilitates erythrocyte removal, as seen in the pathogenesis of autoimmune hemolytic anemia or immune thrombocytopenia[22]. CD47 expression decreases with the maturation of reticulocytes, erythrocyte aging, and in vitro-stored erythrocytes[23,24]. The downregulation of CD47 expression on erythrocytes in sickle cell disease, Gaucher disease, smokers, and obese patients has also been shown[25-28]. The concentrated ambient PM_2.5 also suppresses the CD47 expression of erythrocyte membranes[28]. CD47 also plays a crucial role in hematoma clearance after intracerebral hemorrhage[29]. The paraquat and carbon nanotube administration induces the CD47 expression on erythrocytes as a defensive mechanism[30,31]. Experimental aging changes the confirmation of the CD47 receptor from an inhibitory into an activating signal, and it also acts as an “eat me signal” in a subset of the aged erythrocyte population[32]. Recently it has been seen that CD47 monomers are distributed randomly on the erythrocyte membrane, and 14% of them are found attached to the cytoskeleton[33].

Role of CD47 expression in immune response

The discrimination between self and non-self-elicits the immune responses. CD47 regulates innate and adaptive immune responses. It performs two important functions in the immune system. It interacts with the SIRPα on macrophages and dendritic cells (DC) and regulates the phagocytic responses[19]. The CD47–SIRPα interaction also regulates the neutrophil adhesion, phagocytosis, transendothelial migration, and inflammatory response[34].

CD47 expression is upregulated during infection, and anti-CD47 antibody increases DC activation and CD8⁺ T cell activation to chronic viral infection[35]. This interaction is also important in the development of CD11⁺ DC and secondary lymphoid organs. It is a ligand for SIRPα, a protein found in dendritic and macrophage cells, and a self-marker on various cells. CD47–SIRPα pathways mediate immune-inhibitory signals to DCs and reduce the cytokine interleukin-12 production[36]. It regulates integrin function and leukocyte response to RGD-containing extracellular matrix protein. CD47/TSP1 interactions regulate T-cell survival and differentiation and T-cell receptor-mediated pathways and antigen presentation by DC[37]. CD47 regulates the fate of activated T cells. Activated CD47 triggers CD3, CD4, CD45, or p56 Lck and Fas or TNF receptor signaling independent rapid T cell apoptosis[38].

The CD47 expression also regulates transendothelial migration of monocytes and DC. The interaction between lymphocytes and CD47 is mediated by the signal regulatory protein (SIRP) family. The SIRP family included SIRPα, SIRPβ, and SIRPγ proteins[12]. SIRPα and SIRPβ are expressed on the majority of T cell and few B cell populations and mediate bidirectional signalling while SIRPγ is not expressed on myeloid cells and uses unidirectional signaling with CD47 only. CD47 expression decreases on apoptotic fibroblast and neutrophil cells[39,40].

CD47 receptor functions on endothelial cells

CD47 receptors intercommunicate with VEGFR2 and integrins in endothelial cells. It plays a crucial role in SHP2-dependent insulin growth factor signaling. Extracellular vesicles that originate from T-cell-mediated endothelial cell responses to vascular endothelial growth factor depend on CD47 expression[41].

CD47 receptor functions on platelets

CD47 regulates the integrins α2β1 and αIIbβ3 on platelets. Transfused CD47-deficient platelets cleared rapidly in wild-type recipients. CD47-deficient mice exhibit experimental thrombocytopenia. Platelet-derived exosomes also express CD47 receptors on their surface[42].

Role of CD47 expression in cancer

Cancer cells can be recognised and killed by the immune cells. Cancer cells exhibit immune resistance by altering various immunoregulatory pathways. CD47 expression is increased in ovarian carcinoma cell lines and various solid tumors, including non-Hodgkin lymphoma, murine myeloid leukemia, gastric and breast cancer, lung adenocarcinoma, osteosarcoma, head and neck squamous cell carcinoma, colon cancer, and leukemic HSCs[43-44]. Various transcription factors like nuclear factor Kappa-B, hypoxia-inducible factor 1, myelocytomatosis oncogene, and signal transducer and activator of transcription 3-mediated signalling regulate CD47 expression[45]. The migration and invasion of cancerous cells can also be impacted by CD47. Despite increased expression of "eat me" signals such as phosphatidylserine and calreticulin, many cancer cells upregulate CD47 expression to evade the innate immune system[46].

CD47-SIRPα-based immunotherapies have been implicated in the treatment of various cancers. Acute myeloid leukemia and solid tumor patients with increased CD47 expression have reduced survival[47]. CD47/SIRPα signalling causes cancer immune escape by triggering a ‘don’t eat me’ signal by inhibiting the phagocytosis of cancerous cells. CD47-SIRPα blockage and SHPS-1 activation trigger Syk activation, which induces macrophage-mediated phagocytosis[48]. It also increases antigen presentation and induces the CD8+ T cell response and neutrophil-mediated apoptosis of tumor cells. The neutrophil-mediated removal of tumor cells caused by IgA or IgG-mediated response[49]. CD47 expression in cancer cells decreases with the increase in ROS[50].

Anti-CD47 antibodies have proven to have a significant effect in cancer treatment. The therapy targeting CD47/SIRPα has been prescribed for the treatment of various cancers. CD47 and PD1/PDL1 are involved in the T-lymphoblastic lymphoma/Leukemia (T-LBL/ALL) progression. T-LBL/ALL patients have increased CD47, PDL1, and PD1 expression levels. The targeting of CD47 and PD1/PDL1 could be a new therapeutic target for the treatment of T-LBL/ALL[51]. CD47 expression is increased in intestinal non-GCB-type diffuse large B-cell lymphoma[52].

CD47 is overexpressed on myeloma cells and is a potential therapeutic target for myeloma therapies. The anti-CD47 blocking antibodies increased phagocytosis of myeloma cells and non-Hodgkin lymphoma cells and inhibited the tumor growth[53]. Exosomes with high levels of CD47 expression may facilitate tumor translocation and aid in the spread of malignancies in the microenvironment[54].

The CD47 agonist peptides produced from the C-terminal domain of TSP1 activate phospholipase C gamma-1, which induces Ca²⁺-mediated, caspase-independent programmed cell death in chronic lymphocytic leukemia B cells[55]. The antitumor response mediated by anti-CD47 antibodies depends on the FcγRs. The antibody FCγ induces macrophage and T-cell responses and increases antitumor immunity.

CD47 receptor functions on NK cells

Anti-CD47 antibody induces NK cell death in head-and-neck squamous carcinoma cells in vitro. Increased CD47 expression on HNSCC cell lines have reduced NK cell-mediated cytotoxicity as compared with the low CD47 expression[56]. In CD47−/− NK cells, proapoptotic gene expression was linked to stress-mediated elevations in reactive oxygen species, mitochondrial proton leak, and apoptosis. CD47 regulates NK cell homeostasis and functions in both a positive and negative manner in response to a viral infection[57]. The recruitment of NK cells into the tumor microenvironment is induced by increased CD47 expression. Melanoma-bearing CD47−/− mice were shown in a study to have a decreased number of splenic NK cells. TSP1/CD47 signalling negatively regulates NK cell functions, which can improve NK immune surveillance[58]. CD47 blocking activates DC, which trigger the NK cells, which induce antitumor activity in murine hepatocellular carcinoma[59].

Role of CD47/SIRPα in bone homeostasis and atherosclerosis

CD47/SIRPα interaction regulates bone homeostasis by the production of osteoclast cells[60]. CD47-/- mice had decreased body weight and areal bone mineral density and have a decreased number of osteoclast cells[61]. The femurs are shorter and have lower trabecular bone[61]. CD47/SIRPα signalling induces osteoclastogenesis by the dephosphorylation of myosin protein[60]. Another study in SIRPα mutant mice suggests that SIRPα negatively regulates this function in osteoclasts[62]. The role of CD47 in bone injuries in murine fracture model was recently investigated[63]. There was reduced callus bone formation and decreased mesenchymal progenitor proliferation in CD47-/- mice[63].

CD47 and integrins α_Vβ₃ play a crucial role in osteoarthritis[64]. Osteoarthritic joints have increased CD47 expression and ligand attachment capability of integrin α_Vβ_3. The triggering of α_Vβ₃ and CD47 promotes inflammation and joint injury in osteoarthritis[64].

Role of CD47 expression in transplantation

CD47/SIRPα interaction plays a crucial role in macrophage-mediated graft transplantation. CD47 overexpression reduces NK cell response and macrophage-mediated phagocytosis in cell transplantation[65,66]. The role of CD47/CD274 expression in transplanted hepatocyte graft rejection has been described[67]. CD47/CD274 overexpression suppressed the macrophage and T cell responses in vitro as well as reduced innate and adaptive immune responses during hepatocyte transplantation[68].

Stem cells

The CD47 expression up-regulates with the maturation of HSCs[69]. It also helps in the migration of HSCs in response to inflammation[70]. The aged muscular stem cells have increased CD47 expression[71].

Therapeutic challenges and limitations

The prevention of the CD47/SIRPα interaction between tumor cells and macrophages causes tumor cells to die in several ways. The macrophages can phagocytose the tumor cells by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) or directly by apoptosis by caspase-independent mechanisms[43]. Antibodies and fusion proteins targeting CD47/SIRPα pathways have been implicated in various clinical applications[72]. Blocking of CD47 causes anemia; however, use of a low priming dose overcomes this problem. The combination therapy, including antibodies along with increased phagocytosis agents and antitumor antibodies like rituximab, cetuximab, and penitumumab, had synergistic effects and increased clinical efficacy[72]. An anti-CD47 antibody Magrolimab, in combination therapy has been used in clinical trials in hematologic and solid tumors. It also shows good therapeutic effects in human hematological malignancies, in ovarian cancer cell lines, in patient-derived xenograft models, and in pediatric brain tumor types[73]. Another anti-CD47 antibody, CC-90002, induces phagocytosis of hematologic and tumor cells and antitumor effects in the myeloma cells. AO-176 also promotes apoptosis of tumor cells in various hematologic and solid tumors. Most anti-SIRPα and CD47 antibodies engaging the Fc receptor only show promising results in the preclinical models[73].

The major therapeutic challenge with anti-CD47 therapy includes on-target toxicity, as CD47 is expressed ubiquitously on all cells. Since erythrocytes express CD47, anti-CD47 antibody induces anemia and other blood-related disorders by non-selectively phagocytizing erythrocytes by macrophages. The Fc regions of antibodies activate ADCC and CDC pathways and induce toxicity of normal cells. Another challenge in cancer therapeutics is the development of resistance in tumor cells by reducing the CD47 expression or by increasing the immune response, which will reduce the extended therapeutic effects response.

CONCLUSION

CD47/SIRPα interactions accomplish a wide range of physiological functions. This pathway-mediated downstream signaling is crucial in immune defense, lymphocyte homeostasis, bone remodeling, HSCs engraftment, atherosclerosis, and tumor immune surveillance. It activates macrophages' phagocytic response and modulates metabolism and mitochondrial homeostasis in various cells. Cancerous cells in various tumors increased CD47 expression, which helps the migration and invasion and helps to evade the innate immune system. The therapy utilizing the CD47 target offers a new approach for the treatment of cancer. The phase1 trial using antibodies have shown significant effects on tumors. The dose for phase II trials has not been measured yet. However, challenges like on-target and off-target toxicity, nonselective phagocytosis, anemia, and resistance development are the major concerns that require further research. There is also a need to identify potential biomarkers and examine biosafety, efficacy, and side effects and develop guidelines for therapeutic approaches.