CD47 is involved in a range of cellular processes, includingapoptosis,proliferation,adhesion, andmigration. Furthermore, it plays a role in insulin secretion, as well asimmune andangiogenic responses. CD47 is ubiquitously expressed in human cells and has been found to be overexpressed in many different tumor cells.[6][8] Expression in equine cutaneous tumors has been reported as well.[9]
Form 2 is the most widely expressed form that is found in all circulating and immune cells. The second most abundant isoform is form 4, which is predominantly expressed in the brain and in the peripheral nervous system. Onlykeratinocytes expressed significant amounts of form 1. Little is known about the functional significance of this alternative splicing. However, these isoforms are highly conserved between mouse and man, suggesting an important role for the cytoplasmic domains in CD47 function.[6][10][11]
CD47 is a high affinity receptor forthrombospondin-1 (TSP-1), a secreted glycoprotein that plays a role in vascular development and angiogenesis, and in this later capacity the TSP1-CD47 interaction inhibits nitric oxide signaling at multiple levels in vascular cells.[12] Binding of TSP-1 to CD47 influences several fundamental cellular functions including cell migration and adhesion, cell proliferation or apoptosis, and plays a role in the regulation of angiogenesis and inflammation.[6]
CD47 interacts with several membrane integrins, most commonlyintegrin αVβ3. These interactions result in CD47/integrin complexes that affect a range of cell functions including adhesion, spreading and migration.[6][14]
Due to the ubiquitous expression of CD47, signaling differs according to cell type. It is likely that intracellular and membrane-associated partners are crucial in determining the cellular response of CD47 signaling.
The role of CD47 in promoting cell proliferation is heavily dependent on cell type as both activation and loss of CD47 can result in enhanced proliferation.
Activation of CD47 with TSP-1 increases proliferation of humanU-87 MG and U373astrocytoma cells but not normalastrocytes. Additionally, CD47 blocking antibodies inhibit proliferation of unstimulated astrocytoma cells but not normal astrocytes. Though the exact mechanism is unclear, it is likely that CD47 promotes proliferation via thePI3K/Akt pathway in cancerous cells but not normal cells.[15]
Loss of CD47 allows sustained proliferation of primary murineendothelial cells and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. Expression of severalstem cell markers, includingc-Myc, is elevated in CD47-null endothelial cells and a human T cell line lacking CD47. Activation of CD47 with TSP-1 in wild-type cells inhibits proliferation and reduces expression of stem cell transcription factors.[16]
CD47ligation leads to cell death in many normal and tumor cell lines via apoptosis orautophagy.The activation of CD47 induces rapid apoptosis of T cells.Jurkat cells and peripheral blood mononuclear cells (PBMC) incubated with themonoclonal antibody Ad22 results in apoptosis within 3 hours. However, apoptosis was not observed following culture with other anti-CD47 antibodies. The apoptosis inducing function of CD47 appears to be dependent on activation of specificepitopes on the extracellular domain.[17]
Similarly, CD47 ligation rapidly induces apoptosis inB-cell chronic lymphocytic leukemia (CLL) cells. Treatment with a disulfide-linked antibody dimer induces apoptosis of CD47-positive primary B-CLL and leukemic cells (MOLT-4 and JOK-1). In addition, administration of the antibody prolonged survival ofSCID mice implanted with JOK-1 cells. Apoptosis induction appears to be regulated by the hypoxia inducible factor 1α (HIF-1α) pathway.[18]
The RAS-transformed cell lines MDFB6 and B6ras show near complete loss of TSP-1 expression. Activation of CD47 with TSP-1 results in loss of viability in these RAS-expressing cells. Affected cells do not exhibit hallmarks of apoptosis but rather autophagy as seen by staining withacridine orange and immunoreactivity for LC3.[19]
Cell migration appears to be universally stimulated by CD47 ligation and activation. The role of CD47 in cell migration was first demonstrated forneutrophils, where CD47 blocking antibodies inhibited transmigration of neutrophils andmonocytes through the endothelium. These effects were shown to be dependent on avb3 integrins, which interact with and are activated by CD47 at the plasma membrane.[6][14]
Loss of CD47 promotes proliferation and increases asymmetric division of primary murine endothelial cells.[16] Additionally, activation of CD47 with TSP-1 in wild-type primary mouse cerebral endothelial cells induces cytotoxicity, which is significantly decreased in cerebral endothelial cells derived from CD47 knockout mice.[23]
CD47 signaling may suppress angiogenesis as TSP-1 activation significantly inhibited endothelial cell migration and tube formation in vitro.[23] In vivo, injections of TSP-1 in mice after hindlimbischemia induces a significant decrease of blood flow recovery.[24] The mechanism of the anti-angiogenic activity of CD47 is not fully understood, but introduction of CD47 antibodies and TSP-1 have been shown to inhibitnitric oxide (NO)-stimulated responses in both endothelial and vascularsmooth muscle cells.[12] CD47 signaling influences theSDF-1 chemokine pathway, which plays a role in angiogenesis.[24]
Interactions between endothelial cell CD47 and leukocyte SIRPγ regulate T cell transendothelial migration (TEM) at sites of inflammation. CD47 knockout mice show reduced recruitment of bloodT cells as well as neutrophils and monocytes in areas of inflammation.[25] CD47 also functions as a marker of self on murine red blood cells which allows RBC to avoid phagocytosis. Red blood cells that lack CD47 are rapidly cleared from the bloodstream bymacrophages, a process that is mediated by interaction with SIRPα.[26] Mousehematopoietic stem cells (HSCs) and progenitors transiently upregulate CD47 during their migratory phase, which reduces macrophage engulfment in vivo.[27]
Tumor cells can also evade macrophage phagocytosis through the expression of CD47.[8] CD47 is highly expressed in bladder tumor initiating cells (T-ICs) compared with the rest of the tumor. Blockade of CD47 with a monoclonal antibody results in macrophage engulfment of bladder cancer cells in vitro.[28] CD47 is also upregulated in mouse and human myeloid leukemias, and overexpression of CD47 on a myeloid leukemia line allows these cells to evade phagocytosis.[27]
CD47 receptor signaling inhibits insulin release from human as well as mouse pancreatic β cells and that it can be pharmacologically blocked to boost insulin secretion in both models.[29]
CD47 was first identified as a tumorantigen on human ovarian cancer in the 1980s. Since then, CD47 has been found to be expressed on multiple human tumor types including acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, and other solid tumors.[8] CD47 is also highly expressed on pediatric and adult brain tumors.[30]
High levels of CD47 allows cancer cells to avoid phagocytosis despite having a higher level ofcalreticulin - the dominant pro-phagocytic signal.[31] This is due to engagement of the SIRP-α of macrophage by CD47. Engagement of SIRP-α leads to inhibition of phagocytosis. Thus blocking CD47 with antibody turns off “don’t eat me” signal and favors phagocytosis.
Anti-CD47 antibody–mediated phagocytosis of cancer fosters the activation of cancer-specific lymphocytes: cancer cells display mutant proteins to which the immune system can now react.[32][33] Based on significant activity in preclinical models and in synergistic combinations with other antibodies,[34] Humanized anti-CD47 antibody is being evaluated for the treatment of various cancers, e.g.diffuse large B-cell lymphoma (DLBCL),follicular lymphoma (FL),[35]Primary Effusion Lymphoma[36] and acute myeloid leukemia (AML).[37]
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