The role of the complement system in cancer (original) (raw)
The surge of interest in cancer immunotherapy is mainly focused on manipulating function or number of cytotoxic T cells. However, two important reasons justify studying the role of complement activation in cancer progression and the effect of complement manipulation in cancer therapy. First, the complement system is an important component of the inflammatory response, and inflammation is involved in various stages of tumorigenesis and cancer progression (35). Second, complement activation regulates adaptive immune response (15) and might have a role in regulating T cell response to tumors.
Complement system in inflammation and tumorigenesis. Tumor-promoting inflammation has an important role in carcinogenesis and cancer progression (36–38). A series of elegant experiments established that activation of the complement system is an important component of tumor-promoting inflammation. Bonavita et al. showed that C3-deficient mice were protected against chemical carcinogenesis in mesenchymal and epithelial tissues (39), mainly because of reduced inflammation. Authors identified a humoral component of innate immunity, the long pentraxin PTX3, as an important negative regulator of inflammation and complement activation. PTX3-deficient mice were susceptible to chemical carcinogenesis, displaying an increased number of tumor-associated macrophages with M2 phenotype and increased concentration of CCL2 chemokine inside tumors. The tumor-promoting inflammation induced by PTX3 deficiency was complement-dependent and completely reversed after removal of C3, as manifested by a reduction in the susceptibility of Ptx3–/– C3–/– mice to chemical carcinogenesis. Similarly, treatment with the C5aR antagonist PMX-53 reversed the susceptible phenotype of Ptx3–/– mice without affecting the rate of tumorigenesis in Ptx3+/+ mice.
Activation and regulation of complement pathways in tumors. Expression of complement and CRPs is increased in malignant tumors and cancer cell lines (summarized in Table 1). Complement proteins, C3 degradation products, and complement activation products (i.e., C5a, C3a, and C5b-9) are easily detectable in various types of cancer, consistent with complement activation inside these tumors.
Complement proteins in cancer
The main pathway involved in activation of complement inside tumors is unclear, and evidence supports activation of each complement pathway in malignant tumors (40). To make matters more complicated, cancer cell membrane-bound serine proteases can also cleave C5 and generate C5a without complement activation (41). Additionally, complement proteins expressed in tumors might also play a role in cancer progression independent of complement activation, as was shown for C1q in a syngeneic murine model of melanoma, where C1q expression affected angiogenesis, tumor progression, and metastasis (42). In this murine model, C1q was expressed in endothelial cells, spindle-shaped fibroblasts, and tumor-infiltrating myeloid cells independently of C4. Lack of C4 coexpression in C1q-expressing tumors hints at a role for C1q in tumor progression independent of the classical pathway.
Expression of CRPs, including both membrane proteins (CD55, CD59, MCP, or CD46) and soluble proteins (factor H and factor H–like proteins), is increased in cancer cells (43), although the overexpression of CRPs is heterogeneous among different cancer types and even between different tumor specimens of the same type of cancer (44).
One interpretation of the presence of both complement activation products and CRPs in tumors is that complement activation is a host defense mechanism against cancer, and cancer cells resist complement attack by overexpressing CRPs. However, as discussed later in this Review, several recent studies do not support this interpretation and suggest another scenario in which local complement activation inside tumors enhances tumor growth.
Complement activation: antitumor or protumor? Evidence for the effects of complement on malignant transformation of epithelial cells and progression of cancer has evolved based on several recent studies showing complex and sometimes contradictory findings. This complexity is similar to the complex role of inflammation in cancer (45). Although inflammatory cells and cytokines are important in immune surveillance, exemplified by the benefit of bacillus Calmette-Guérin therapy in early stages of bladder cancer, chronic inflammation promotes carcinogenesis and tumor growth. Even immune cells, such as macrophages, can have both pro- and antitumor phenotypes. Despite this multifaceted picture, most evidence points toward a protumor effect of chronic inflammation (45).
The long-held view of complement activation as an antitumor defense mechanism is based on two main concepts: first, the complement system’s participation in immune surveillance against malignant cells, and second, complement-dependent cytotoxicity of therapeutic monoclonal antibodies. I will discuss these concepts below, and summarize new information pointing toward a protumor effect of complement activation inside tumors.
Complement and immune surveillance. The complement system’s ability to distinguish self from non-self makes it an important part of the innate immune response to invading pathogens (46). Expression of non-self antigens and lack of CRPs on microbes make them optimal targets for complement detection and, later on, complement-mediated elimination. Similarly, expression of danger signals and neoantigens by apoptotic cells and cellular debris optimizes their detection and removal by the complement system. Cancer cells, on the other hand, mostly express the same proteins as their normal epithelial cell counterparts, albeit occasionally with a different density. Furthermore, overexpression of CRPs by cancer cells limits immune surveillance by the complement system (3, 43, 46, 47). Putting these findings together, one can conclude that cell-mediated immunity plays a more important role than humoral immunity in immune surveillance against cancer cells (48, 49), and effectiveness of complement in early detection and elimination of cancer cells is uncertain (50).
Complement-dependent cytotoxicity. Complement activation was considered detrimental to cancer cells via complement-dependent cytotoxicity, which causes cancer cell lysis via MAC accumulation or phagocytosis of opsonized cancer cells by macrophages and neutrophils. Complement-dependent cytotoxicity is considered to be the main mechanism for the effectiveness of antitumor monoclonal antibodies. Rituximab, an anti-CD20 antibody against malignant B cells, is among the oldest and most widely used therapeutic monoclonal antibodies. Although in vitro and in vivo studies show that rituximab activates the classical complement pathway (51, 52), the notion that its therapeutic benefits are mainly mediated by induction of complement attack on malignant B cells is questionable. In fact, the antitumor effect of rituximab was inhibited by deposited complement proteins on B cells (53), and was enhanced in complement-deficient mice (54). Therefore, the extent to which complement-dependent cytotoxicity contributes to other immunologic effects of rituximab, i.e., antibody-dependent cellular cytotoxicity and antibody-dependent phagocytosis, is unknown. Other studies on the therapeutic mechanism of rituximab also showed a complement-independent, proapoptotic effect mediated by cross-linking of CD20 (55), as well as antiproliferative and antisurvival effects that were mediated by inhibition of B cell receptors (56). Furthermore, many in vitro antitumor effects of complement-fixing antibodies on cancer cell lines were not reproduced in vivo (57).
Complement activation promotes tumor growth. Considering that complement is not efficient in immune surveillance against cancer cells and that the main antitumor effect of monoclonal antibodies might not arise from complement activation, the data supporting an antitumor role for complement activation are scant. The question remains: If complement does not attack cancer cells, how does local complement activation and deposition of complement proteins affect tumors? To understand the consequence of complement activation inside tumors, it is helpful to reexamine the biological functions of complement activation products. C3b and its degradation products binding to CR1, CR2, and CR3 provide ligands and receptors for cell-cell and stroma-cell interactions in many physiologic and pathologic conditions. Complement activation generates C3a and C5a and MAC. The anaphylatoxin receptors C3aR and C5aR are G protein–coupled receptors present on many cell types, including lymphocytes, monocytes/macrophages, myeloid cells, hematopoietic stem cells, mesenchymal cells, and epithelial cells, including cancer cells. Anaphylatoxin receptor signaling has been studied extensively (58). Activation of C5aR promotes a range of responses depending on the cell type. Relevant to its role in cancer, C5aR activation generates prosurvival and antiapoptotic responses. For example, C5a binding to C5aR decreases apoptosis in neutrophils (59) and T cells (22), and increases cell proliferation in endothelial (60) and colon cancer cell lines (61). Activation of C3aR plays an important role in guiding collective cell migration (26) and epithelial-mesenchymal transition (62, 63), both important mechanisms in metastasis. In a sublytic density, MAC accumulation on the cell membrane promotes cell proliferation (64) and differentiation, inhibits apoptosis (10, 65), and protects cells against complement-mediated lysis (66).
Markiewski et al. showed that the activation of the classical complement pathway inside implanted orthotopic tumors in mice enhanced tumor growth (67). Complement’s progrowth effect on tumors was C5a-dependent and was eliminated in C5aR-deficient mice and in WT mice treated with a C5aR antagonist. C5a modulates the immune response to tumors by acting as a chemotactic factor, increasing infiltration of myeloid-derived suppressor cells (MDSCs) and reducing the number of CD8+ cytotoxic T cells inside tumors. MDSCs are immature myeloid cells that increase in blood, bone marrow, and spleen of tumor-bearing mice and cancer patients (68, 69) and assist tumor cells in evading the antitumor immune response. MDSCs reduce proliferation and increase apoptosis in CD8+ T cells by generating ROS and reactive nitrogen species (70). Depletion of CD8+ T cells in mice eliminated the protective effect of complement deficiency against tumor growth. In summary, this study showed that the immunomodulatory effect of activated classical complement pathway inside tumors enhances tumor growth. The origin of complement proteins was the host, but activation of complement occurred inside the tumor microenvironment, and the final effect on the tumor was an indirect immunomodulatory effect mediated by MDSCs (Figure 2).
Effect of complement activation in the tumor microenvironment. Activation of the complement system inside tumors releases C5a and C3a into the tumor microenvironment and promotes tumor growth. C5a attracts myeloid cell, including MDSCs, into the tumor. MDSCs then reduce cytotoxic T cell responses to the tumor by inducing apoptosis and inhibiting CD8+ TILs via generation of ROS and reactive nitrogen species and depletion of arginine. In melanoma, secretion of C3 by CD8+ TILs and complement activation in the vicinity of these cells reduce IL-10 production by TILs and inhibit their function. Some cancer cell types secrete complement proteins into the tumor microenvironment and initiate an autocrine loop that increases cell proliferation and promotes metastasis. The effect of complement activation on MDSCs, TILs, and cancer cells is mediated by the C5a and C3a receptors (C5aR and C3aR) on these cells.
In a follow-up study, Nunez-Cruz et al. investigated complement’s role in tumorigenesis in a murine model of spontaneous ovarian cancer (71, 72). C3 or C5aR deficiency in these mice prevented the development of ovarian tumors, permitting no tumors or only small and poorly vascularized tumor formation (71). C3 deficiency was associated with a change in the immune profile of leukocytes infiltrating into the tumors, but C5aR deficiency reduced ovarian tumor size without altering the immune profile of infiltrating leukocytes. This result suggested the existence of a protumor effect of complement that was independent of its immunomodulatory effect.
We investigated the effect of complement in murine models of ovarian cancer and confirmed activation of complement in the tumor microenvironment (73). However, complement proteins detected inside ovarian tumors originated not from the host, but from tumor cells themselves. Complement activation products were present even inside tumors implanted in C3-deficient mice lacking a functional complement system. Although orthotopic ovarian cancer tumors in C3-deficient mice reached to the same size as those in WT mice, reducing C3 or C5 production in cancer cells significantly reduced the tumor growth independent of the host’s complement sufficiency status. C3 synthesis can be detected in malignant epithelial cells originating from several different organs, particularly lung and ovary. Inhibiting synthesis of complement proteins in cancer cells altered the immune profile of leukocytes infiltrating into tumors, manifested by an increase in the number of CD8+ T cells and reduction in myeloid cells. However, immunomodulatory effect of complement inhibition was not the main mechanism responsible for the observed reduction in tumor growth. Inhibiting complement protein synthesis in cancer cells implanted in CD8+ T cell–deficient mice reduced tumor growth to the same magnitude as in WT mice. We investigated the possibility of an autocrine stimulation of cancer cells as a result of complement activation. Anaphylatoxin receptors are present on ovarian cancer cells, and stimulation of these receptors by C3a or C5a agonist peptides increased proliferation and invasiveness of ovarian cancer cells in vitro. Furthermore, knockdown of these receptors on cancer cells reduced growth of orthotopic ovarian tumors in mice. Our studies showed that local complement activation inside the tumor microenvironment enhances tumor growth via a direct autocrine effect on ovarian cancer cells increasing cell proliferation (Figure 2).
In a murine model, Wang et al. reported another mechanism for the progrowth effect of complement activation in melanoma, showing that production of IL-10 by CD8+ tumor-infiltrating lymphocytes (TILs) is constitutively inhibited in an autocrine fashion by C3 originating from CD8+ TILs themselves, acting through C5aR and C3aR on the surface of these lymphocytes (74). C3aR and C5aR antagonists increased IL-10 production and activated CD8+ TILs that in turn reduce tumor growth. The IL-10–dependent antitumor activity of complement inhibitors in melanoma was independent of the PD-1/PD-L1 axis or MDSCs. This study provides evidence that local complement activation in the tumor microenvironment results in suppression of the immune response to melanoma by inhibiting CD8+ TIL function (Figure 2).
The studies above describe different mechanisms by which complement activation in the tumor microenvironment can enhance tumor growth: (a) by altering the immune profile of tumor-infiltrating leukocytes, (b) by increasing cancer cell proliferation, and (c) by directly suppressing CD8+ TIL function. It is possible that different cancer types use different mechanisms to take advantage of ectopic complement activation inside tumors. For example, ovarian cancer cells synthesize a significant amount of complement proteins and initiate an autocrine loop resulting in increased cell proliferation by a direct effect of anaphylatoxins on cancer cells. Conversely, melanoma cells do not secrete complement proteins, and complement proteins produced by CD8+ TILs reduce their IL-10 production and antitumor activity.
An important question remains whether complement activation has any role in malignant transformation of normal cells or only affects the expansion of already established malignant clones. Most available data are based on orthotopic murine models of ovarian cancer or mice genetically engineered to develop ovarian cancer by overexpression of oncogenes. These studies showed that complement promotes growth and expansion of malignant tumors. Bonavita et al. showed that complement promotes malignant transformation of cells exposed to chronic inflammation induced by chemical carcinogens (39). However, additional studies are required to dissect the effect of early versus late stages of complement activation on various stages of oncogenesis.