The role of the complement system in cancer etiology and management

Summary of key facts

The complement system is a key component of the innate immune system, responsible for facilitating immune defense mechanisms, regulating inflammation, and maintaining tissue homeostasis.
The complement system is a proteolytic cascade, comprising more than 50 highly regulated soluble proteins and membrane-bound receptors. The cascade is initiated by recognition of pathogen-associated patterns (PAMPs), damage-associated molecular patterns (DAMPs), and binding to immune complexes and proceeds via three distinct pathways: the classical, the lectin, and the alternative pathways.
Complement activation results in production of important immune mediators, including C3a, C3b, C5a, and C5b-9, which mediate innate immune responses against foreign invaders, attraction of immune effector cells, phagocytosis, and formation of the membrane attack complex (MAC) resulting in cell lysis. This process is tightly controlled by a range of soluble and membrane-bound regulatory proteins.
Though essential to immune function, dysregulated complement activation contributes to a diverse range of disease pathologies, including atypical hemolytic uremic syndrome, C3 glomerulopathies, age-related macular degeneration, rheumatoid arthritis, sepsis, atherosclerosis, ischemia-reperfusion injury, and cancer.
Complement-targeting drugs have been FDA-approved for mostly orphan conditions such as paroxysmal nocturnal hemoglobinuria, yet several more are in clinical trials or under development.
Complement is activated in response to cancer cells, and complement activation products are deposited within tumor tissue. Bioinformatics analysis indicates a negative correlation between complement signaling pathways and prognosis in many cancer types.
Complement mediators including C3a, C5b, and sublytic C5b-9 have been implicated for roles in promoting tumor growth and metastasis.
Research in preclinical models suggests the therapeutic potential of complement-targeting drugs, either alone or in combination with current cancer therapeutics.

Introduction

The complement system is critical to proper immune function, but inappropriate or excessive complement activation contributes to many pathologic inflammatory conditions including cancer. Traditionally regarded as contributing to the antitumor response through complement-dependent cytotoxicity (CDC), there is growing evidence that complement activation products including C3a, C5a, and C5b-9 can also promote tumor growth. Indeed, complement proteins have been shown to regulate primary tumor growth and metastasis, indirectly via the antitumor immune response, angiogenesis, and formation of the premetastatic niche and directly by promoting tumor cell proliferation and migration. The availability of complement-targeting drugs, with more in the developmental pipeline, suggests the potential for novel immunotherapeutic strategies to target both innate and adaptive immunity and boost the antitumor response.

Complement activation pathways

A key component of innate immunity, the complement system forms the first line of defense, aiding in the elimination of pathogens and damaged cells. The complement system is a proteolytic cascade, comprising more than 50 highly regulated soluble proteins and membrane-bound receptors. Complement activation elicits a range of proinflammatory effects, including increased vascular permeability, modulation of cytokine release, recruitment of innate immune cells such as neutrophils and macrophages to damaged tissues, enhanced phagocytosis, and lysis of pathogens and damaged cells. ^, Although it has been traditionally regarded as a mediator of innate immune activities, the complement system also contributes to efficient adaptive immune responses.

The complement cascade is activated in response to danger signals (damage- or pathogen-associated molecular patterns; DAMPs and PAMPs) via three pathways ( Fig. 3.1 ), depending on the stimuli. The classical pathway is activated via interaction of antigen–antibody complexes with the multimeric collectin C1q leading to conformational changes in the C1q molecule and complex formation with serine proteases C1r and C1s. The lectin pathway is initiated by the binding of mannan-binding lectin (MBL), ficolins, and other pattern recognition molecules that recognize aberrant carbohydrates on the surfaces of pathogens and damaged or necrotic cells, allowing for the recruitment of MBL-associated serine proteases (MASPs)-1 and -2. The alternative pathway is triggered by interaction with foreign antigens on pathogen surfaces. The activation of classical and lectin pathways, leading to formation of the C1qrs complex and activation of MASP-2, respectively, results in proteolytic cleavage of C4 into C4a and C4b and then C2 into C2a and C2b. C4b then binds to C2b, generating the classical/lectin C3 convertase (C4b2b complex). ^, The alternative pathway, however, is in a constant state of low-level activation (tickover), in which spontaneous hydrolysis of a labile thioester bond converts C3 to a bioactive form C3(H ₂ O) in the fluid phase. This pathway proceeds directly through C3 cleavage to generate an alternative pathway C3 convertase (C3bBb), thus allowing an immediate response to microbial challenge.

Fig. 3.1, The complement cascade is activated via three pathways, depending on the stimulus.

Once generated, C3 convertases cleave C3 to produce the important effector molecules anaphylatoxin C3a and opsonin C3b. Formation of C3b also enables binding of the protease factor B (FB). The resulting proconvertase (C3bB) is quickly transformed by factor D (CFD) into an active C3 convertase (C3bBb) that by itself can cleave more C3 into C3b, thereby creating an amplification loop for C3b deposition. C3 convertases from the classical or alternative pathways also bind to C3b to generate C5 convertases (C4b2b3b and C3bBb3b, respectively). These in turn cleave the downstream component C5 to generate anaphylatoxin C5a and C5b, which initiates formation of the membrane attack complex. These products of complement pathway activation (i.e., C3a, C3b, C5a, C5b) are responsible for mediating many of the effects of the complement system (described in Complement effector molecules ).

Finally, although its role in human physiology and pathology has yet to be clearly demonstrated, complement activation can be triggered directly by proteolytic enzymes that cleave C3 and C5 to form C3a and C5a, respectively. This fourth extrinsic pathway can be initiated by enzymes of the coagulation cascade such as factors IX, X, XI/XIa, plasmin, and thrombin, as well as other enzymes, including cathepsin D, granzyme B, and β-tryptase, which are secreted by damaged cells or leukocytes. ^, Notably, there is also evidence for intracellular generation and function of complement activation fragments.

To maintain the balance between efficient destruction of pathogens and prevention of unwanted damage to host tissue, complement activation is tightly controlled by soluble and membrane-bound regulatory proteins. These include soluble factors such as carboxypeptidases; complement factors (CF)H, CFB, CFD, and CFI; C4b-binding protein (C4BP) and C1 inhibitor (C1inh); and membrane complement regulatory proteins (mCRPs) such as CD35 (complement receptor type-1; CR1), CD46 (membrane cofactor protein; MCP), and CD55 (decay-accelerating factor; DAF; Fig. 3.2 ). These CRPs protect both normal and neoplastic cells from damage by accelerating decay of convertases or cleaving activation fragments to inactive forms. Another membrane-bound protein, CD59 (protectin), binds to C8 and C9 to prevent assembly of the membrane attack complex (MAC). ^,

Fig. 3.2, Regulators of the complement pathway.

Complement effector molecules

Activation of the complement cascade leads to generation of potent effector molecules, including C3a, C3b, C5a, and C5b.

Opsonin c3b

C3b not only amplifies the complement response via convertase formation (see Complement activation pathways ) but also acts as a mediator of innate and adaptive immunity. Cleavage of C3 induces a conformational change in the C3b fragment, allowing it to bind to proteins or carbohydrates present on cell membranes and foreign structures in a process called opsonization . C3b binding to CR1 on immune cells enables opsonized cells to be shuttled to the spleen and liver where C3b binds to the complement receptor of the immunoglobulin family (CRIg) expressed on tissue-resident macrophages such as Kupffer cells and induces phagocytosis. In addition to its role as an opsonin, C3b mediates adaptive immune functions, improving the contact between effector and target cells and potentiating antibody-dependent cell-mediated cytotoxicity (ADCC) and CDC. Additionally, C3b deposited on antigen-presenting cells interacts with CR1/CR2 expressed on antigen-specific T cells to promote their proliferation.

Anaphylatoxins C3a and C5a

C3a and C5a are powerful immune mediators through which the complement system exerts many of its effects. Small polypeptides comprising 77 and 74 amino acids, respectively, the anaphylatoxins have ∼36% overall homology but higher homology in the C-terminal “active” regions of the molecules. C5a binds two specific receptors, C5a receptor (R)1 (CD88) and C5aR2 (C5a-like receptor 2; C5L2), and C3a binds to a single receptor, C3aR. ^, All three receptors (C5aR1, C5aR2, and C3aR) belong to the superfamily of seven transmembrane spanning G protein–coupled receptors and are expressed primarily by myeloid cells, including monocytes, macrophages, eosinophils, basophils, and neutrophils. Expression by nonmyeloid cells has also been reported, especially in lung and liver. C5a binds both C5aR1 and C5aR2 with high affinity, but is thought to exert most of its biologic activity via the former.

C5a binding to C5aR1 downregulates cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling and activates signaling pathways such as phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) to induce a range of proinflammatory responses. These include chemoattraction of macrophages, neutrophils, basophils, and mast cells ^, ; enhanced phagocytosis ; and modulation of cytokine release. C5a triggers histamine release from basophils and mast cells, which in turn stimulates vasodilation and increased vascular permeability. It also stimulates neutrophil degranulation and release of toxic mediators such as reactive oxygen species (ROS) and neutrophil extracellular trap (NET) formation after priming with interferon (IFN)-γ. Additionally, C5a has been reported to stimulate angiogenesis by promoting the migration of microvascular endothelial cells. It also links to the adaptive immune system, influencing the trafficking and migration of B-cell populations ^, and modulating T-cell responses; it provides survival signals for naïve CD4 ⁺ cells, inhibits induction and function of regulatory T cells (Tregs), and promotes T-cell activation during interaction with antigen-presenting cells (APCs) in vitro and in vivo. The alternate receptor C5aR2 lacks G protein coupling and thus was originally thought to be a “decoy” or scavenger receptor, binding excess C5a without exerting direct physiologic effects. However, there is emerging evidence to suggest that C5aR2 can independently induce and moderate biologic functions of C5a through β-arrestin and p90RSK activation.

C3a has been reported to exert effects in mast cells, macrophages/monocytes, T cells, and APCs. It induces calcium mobilization from intracellular stores, ^, activation of extracellular signal-regulated kinases (ERK)1/2, and release of extracellular adenosine triphosphate (ATP) in monocytes and macrophages. Despite the lack of evidence for C3aR expression by T cells, C3a/C3aR has been reported to activate phosphoinositide-3-kinase (PI3K)-γ and induce phosphorylation of Akt, upregulating the antiapoptotic protein Bcl-2 and downregulating the proapoptotic molecule Fas, to decrease T cell apoptosis and enhance proliferation. However, the activity of C3a is short-lived, because it is rapidly cleaved at the C-terminal arginine to form C3a des-Arg, which can no longer bind to C3aR.

Compared with C5a, C3a is a much weaker chemoattractant but has been reported to exert a range of immunomodulatory functions including degranulation of eosinophils, basophils, and mast cells. ^, C3aR is thought to negatively regulate the mobilization of hematopoietic stem and progenitor cells from the bone marrow ^, and has also been shown to prevent neutrophil egress into the circulation, thus reducing acute tissue injury after ischemia or neurotrauma. Like C5a, C3a signaling may contribute to the regulation of adaptive immunity, inhibiting natural (n)Treg function and enhancing the survival and function of effector Th1 and Th17 cells. Conversely, the absence of C3aR signaling in CD4 ⁺ T cells is reported to be associated with enhanced interleukin (IL)-10, transforming growth factor (TGF)-β expression, and Foxp3 ⁺ -induced (i)Treg-mediated immunosuppression.

Both C3aR and C5aR have been shown to regulate Toll-like receptor (TLR)-induced cytokine production, with C5aR1 synergizing with TLR-2 and TLR-4 to elicit stronger inflammatory responses and C3aR regulating TLR9 signaling. ^, Moreover, TLR-induced inflammatory cytokines such as interleukin (IL)-6 can upregulate the expression of C3aR and C5aR.

Despite their critical roles in the development of effective immune responses, excess production of C5a and C3a can contribute to pathogenic proinflammatory responses, resulting in tissue damage and, eventually, multiorgan failure. Indeed, the anaphylatoxins are implicated in a range of inflammatory diseases including arthritis, ischemia-reperfusion injury, sepsis, neurodegenerative diseases, and cancer.

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