This article is mentioned in:

Introduction

The complement system comprises several soluble and membrane-bound proteins that, when activated, provide a congenital defense against microbial infection. In addition to its defensive role, complement activation elicits numerous biological effects, including leukocyte recruitment, smooth muscle contraction and increased vascular permeability (1–4). Although the activation of the complement system is strictly regulated by various regulators and inhibitors in the body fluids and plasma (5,6), inappropriate or uncontrolled activation of the complement system can lead to local or systemic inflammation, tissue damage and disease (7,8). A number of studies have provided novel insights into the structure and function of complement proteins, which have improved our understanding of how different components of the complement system participate in the destruction or restoration of homeostasis in the tumor microenvironment (TME). This increased knowledge has inspired novel approaches for complement-targeted therapy (9,10).

Colorectal cancer (CRC) is one of the most frequently occurring cancers worldwide, ranking third among all cancers in incidence and second in terms of mortality (11). Despite certain improvements that have been made to treatment regimens, including surgery, radiotherapy and chemotherapy, the mortality rate of CRC remains high (12). Notably, therapies targeting the immune system have achieved significant advances in CRC treatment, suggesting that the immune system plays a critical role in the regulation of tumor progression. The tumor immune microenvironment (TIME), which contains various immune components, has received increasing attention due to its crucial role in influencing the immune response in the TME. Indeed, the balance of immunosuppressive cells, for example, myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), with antitumor immune cells, for example, cytotoxic T cells and natural killer (NK) cells, has been shown to determine the state of tumor progression (13). Currently, tumor immunotherapy is primarily focused on promoting the antitumor effects of cytotoxic T lymphocytes and modulating inflammatory responses (14). The complement system, which is the main effector of innate immunity, is widely present in the TIME, and plays a central role in regulating immune responses in the TME. Complement activation was traditionally considered to promote the killing of tumor cells via complement-mediated cytotoxicity, with blocking of the complement cascade recognized as a risk factor for tumor progression. For example, inhibiting the activity of C3 and C5 convertases, which serve as key enforcers of the complement cascade by upregulating the expression of the complement regulatory protein (CRP) CD55, has been found to be associated with a poor prognosis in patients with CRC (15). However, complement activation may fulfill complex roles in cancer progression. For example, complement components from the TME, such as C1q, C5a and C7, have been shown to facilitate tumor progression through the modulation of angiogenesis, antitumor immunity and tumor growth (16–18). Similarly, complement activation products inside tumors, such as C3a, have been shown to exhibit pro-tumor properties by inhibiting antitumor immunity (19).

In the present review, current knowledge of the role of the complement system in the pathogenesis of CRC is summarized, and the contribution, mechanism and functional modes of complement components in CRC are also discussed. In particular, the review highlights unconventional modes of complement activation in extracellular and intracellular environments.

Complement system and its roles in CRC

It is widely acknowledged that the complement system is a systemic, serum-based effector of innate immunity, with activation typically comprising a cascade of enzymatic reactions confined to the extracellular space, known as cascade-dependent activation. However, it has also been shown that the complement system can be activated by enzymes inside cells in a cascade-independent manner (20). Furthermore, several complement components, such as C1q, have been shown to function without activation (21–23). Recent experiments have established that complement components themselves and their activation products, derived from either cascade-dependent or -independent activation, are involved in regulating the progression of CRC.

Complement cascade-dependent activation pathway in CRC

Complement cascade-dependent activation pathways

The complement system can be activated by three different pathways, namely, the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP). These three pathways are activated by different activators: The CP is activated by antigen-antibody complexes, whereas the LP, also known as the mannose-binding lectin (MBL) pathway, is triggered by the direct recognition of sugar structures on the surface of pathogens by MBLs, which are mainly present in the blood. The AP, also known as the bypass pathway, is an antibody-independent pathway in which microorganisms and xenobiotics initiate a cascade of enzymatic reactions with the help of factors B, D and P (24). Activation of the CP and LP leads to the cleavage of C4 and C2 and the formation of C3 convertase (C4b2b). By contrast, the AP is activated by the spontaneous hydrolysis of C3, leading to the formation of the alternative C3 convertase (C3bBb). C3 convertase cleaves C3 into an anaphylatoxin (C3a) and a larger fragment (C3b). The deposition of C3b leads to the formation of a new C3 convertase through a bypass pathway, creating a positive feedback loop that results in the further deposition of C3b (25).

The activation of C3 also leads to the formation of C5 convertase (C4b2b3b or C3bBb3b). All three activation pathways share a common terminal pathway: C5 convertase cleaves C5 into C5a and C5b. Subsequently, C5b stably binds to C6 to form C5b6, which spontaneously binds to C7, forming C5b67. The C7 component of the complex is initially inserted into the lipid bilayer of the target cell membrane, after which C8 binds with high affinity to C5b67 inserted into the membrane, thereby forming a stable C5b678 complex that is deeply embedded in the cell membrane. This complex can bind to multiple C9 molecules to form C5b6789n (C5b-9), which is termed the ‘membrane attack complex’ (MAC) (26–28).

Notably, each step of the cascade-dependent complement activation process is strictly regulated by a series of CRPs. For example, C1 inhibitor (C1INH) is known to inactivate active enzymes C1r, C1s of CP, and mannan-binding lectin serine peptidase (MASP) of LP through by covalently binding to them (29). Factor I is a serine protease that degrades C3b and C4b in the presence of cofactors (30). Factor H is a cofactor for C3b degradation, whereas C4 binding protein is the primary cofactor for C4b degradation. Other factors, such as complement receptor 1, membrane cofactor protein and decay accelerating factor, limit the formation and stability of C3/C5 convertases. Additionally, clusterin, vitronectin and CD59 prevent the formation and membrane insertion of the C5b-9 complex (31,32) (Fig. 1).

Figure 1. Activation and regulation of complement cascade-dependent processes. The complement system is activated through three pathways: CP, LP and AP. The CP is initiated when C1 (comprising C1q, C1r and C1s) binds to an antibody; the LP is activated by the MBL-MASP complex on pathogens; and the AP is triggered by spontaneous C3 hydrolysis or C3 tickover, which is amplified by regulators detecting unprotected surfaces. All pathways lead to the formation of C3 convertases (C3bBb or C4b2a), which cleave C3 into C3a and C3b. C3b is deposited on target cells, where it promotes signaling via complement receptors and forms a feedback loop in the AP, which promotes C3b deposition and its conversion into C5 convertases. The C5 convertases cleave C5 into C5a and C5b. The latter interacts with C6, C7, C8 and C9 molecules to form the MAC. Regulation is achieved by soluble factors, namely C1-INH, Factor I, C4BP, Factor H, clusterin and vitronectin, and membrane proteins, namely CD35/CR1, CD46/MCP, CD55/DAF and CD59. These regulators control various activation stages: C1-INH influences C1r, C1s and MASP, whereas C4BP, Factor H, Factor I, CD55 and CD35 regulate C3/C5 convertase formation and stability. Clusterin and vitronectin inhibit the assembly or membrane insertion of the MAC by binding to the C5b-7 complex, and CD59 inhibits the binding of C9 to C5b-8 by competing for binding to a nascent epitope on C8, thereby disrupting the association of C9 with C5b-8. CP, classical pathway; LP, lectin pathway; AP, alternative pathway; MBL, mannose-binding lectin; MASP, mannan-binding lectin serine protease; MAC, membrane attack complex; C1-INH, C1 inhibitor; C4BP, C4 binding protein; CR1, complement receptor 1; MCP, membrane cofactor protein; DAF, decay accelerating factor.

Upstream complement components in CRC

Chronic inflammation serves as a significant driver of CRC progression (33). In the TIME, several inflammatory factors or chemokines produced during chronic inflammation participate in the occurrence and development of CRC (34,35). There is evidence to suggest that upstream complement products from the cascade-dependent activation pathway, such as C3a and C5a, are involved in influencing the TIME (36,37). C3a levels have been found to be significantly increased in the serum of patients with CRC, and C3a upregulation has therefore been recommended as an important biomarker for early diagnosis (36), suggesting that C3a may be an important contributor to CRC progression. Mehrabani et al (38) also found that C3a is present at high levels in the serum of patients with CRC, and clearly diminished after the patients received treatment, implying that C3a plays a role in tumorigenesis. However, Krieg et al (39) demonstrated that C3a receptor (C3aR) deficiency promoted CRC development by the induction of inflammatory responses characterized by pro-tumor effects, suggesting that C3a/C3aR exerts antitumor effects on CRC progression. Therefore, the role of C3a in CRC is not clear and further research is necessary to elucidate it. Furthermore, the complement cascade-dependent generation of C5a in the TIME has been shown to promote CRC progression through multiple mechanisms: Firstly, C5a induces the infiltration of immunosuppressive cells into the TIME, which inhibits their antitumor activities. For example, C5a recruits C5aR+ MDSCs into the colonic TIME to suppress the antitumor activity of CD8+T cells (40). Secondly, C5a promotes the pro-tumor activities of tumor-associated macrophages (TAMs). For example, C5a promotes colon cancer metastasis by activating NF-κB-associated signaling pathways in C5aR+ TAMs and stimulating macrophage differentiation toward an M2 phenotype with tumor-promoting activity (41). In addition, Piao et al (42) used C5aR−/−mice to demonstrate that the metastasis of CRC to the liver depends on monocyte chemoattractant protein-1 (MCP-1) produced by macrophages, with high levels of MCP-1 secreted by C5a, leading to activation of the PI3K-AKT signaling pathway in macrophages. Thirdly, C5a prompts metastasis in CRC by regulating CRC cell activities. For example, Xu et al (43) demonstrated that, in human CRC cell lines, the upregulation of C5a receptor 1 (C5aR1), accelerates epithelial-mesenchymal transition (EMT) and activates the Wnt/β-catenin signaling pathway, effects that are association with CRC progression. Also, C5a has been identified as a potential clinical biomarker for the early detection and prognosis of CRC, based on the observation of elevated C5a levels in the plasma of patients (44,45). Therefore, in addition to being vital components of pro-tumor mechanisms in CRC, C3a and C5a are also potentially valuable clinical biomarkers for this disease.

Overall, the anaphylatoxins C3a and C5a play important roles during the progression of CRC by modulating the inflammatory microenvironment within the TIME, or by directly affecting tumor processes such as growth and metastasis. However, their effects on CRC development are not consistent. Specifically, C3a and its receptor C3aR display dual effects, while C5a and its receptor C5aR display only pro-tumor effects. These differences may be due to the involvement of distinct complement components and different mechanisms. In addition, while signaling pathways such as the NF-κB, PI3K-AKT and Wnt/β-catenin signaling pathways are being considered as candidate mechanisms, further studies are necessary to elucidate the molecular mechanisms underlying the regulation of inflammatory cell function and CRC cell growth behaviors in greater detail.

Although the complement-cascade activation products C3a and C5a, derived from the cleavage of C3/C5 by C3/C5 convertase, are recognized as risk factors for CRC, the body expresses several CRPs, including membrane proteins such as CD55 and CD46, and soluble proteins such as factor H and Factor I, that can inhibit C3/C5 convertase activity, and thereby regulate CRC progression. As noted by Talaat et al (46), CRC cells can evade complement-dependent cytotoxicity by the overexpression of CD55, particularly under hypoxic conditions, which prevents tumors from being eradicated. Consistent with these findings, Lin et al (47) found that the absence of CD55 led to the dysregulation of complement-cascade activation, which triggered the infiltration of inflammatory cells into the intestinal mucosa, and eventually resulted in the excessive production of pro-inflammatory cytokines, including IL-10, IL-12, IL-6 and TNF-α, and aggravation of colonic inflammation. These studies suggest that CD55 serves an antitumor role. However, other studies have contradicted this by suggesting that CD55 performs a pro-tumor role. CD55 expression has been shown to be upregulated in CRC, particularly in the more advanced stages (48). Dho et al (49) developed a novel chimeric CD55 monoclonal antibody that activated the complement system, leading to CRC cell proliferation, invasion and migration. Additionally, this antibody exhibited a synergistic inhibitory effect with 5-fluorouracil (5-FU) on CRC cell growth, highlighting the potential of CD55 as a therapeutic target for CRC. In multiple clinical trials, the presence of CD55 has been considered a potential hallmark of poor prognosis in patients with CRC (15,50,51). The current perspective is that the conflicting roles of CD55 in CRC may result from, for example, inconsistent modeling conditions, modeling durations and tumor progression stages (46,52). Mechanistically, CD55 may inhibit tumor growth by reducing the levels of activation products C3a and C5a. By contrast, its pro-tumor activities may result from its blockade of the complement cascade-dependent activation pathway, leading to lower levels of MAC formation and diminished MAC-induced antitumor effects. In addition, CD46, along with CD55, has been found to be upregulated in human colon cancer cells through the STAT3/STAT6/p38 MAPK signaling pathway, and knocking out both CD46 and CD55 induced tumor cell apoptosis and reduced colon cancer growth in mice (52). This further confirms the pro-tumor function of CRPs that inhibit C3/C5 convertases.

In addition to the aforementioned membrane CRPs, soluble CRPs such as Factor I and Factor H have also been shown to participate in the regulation of CRC tumorigenesis. In one study, the expression of Factor I was demonstrated to be significantly upregulated in colon cancer, and proposed as a diagnostic biomarker for CRC (53). In addition, the knockout of Factor I inhibited the proliferation, migration and invasion of colon cancer cells, which was attributed to the suppression of glycolysis as a consequence of blocking the Wnt/β-catenin/c-Myc signaling pathway (53). In addition, Wilczek et al (54) demonstrated that the levels of Factor H and its split variant, Factor H-like protein, were highly increased in patients with colon adenocarcinoma and metastatic foci in the liver. The authors verified that the upregulation of Factor H resulted in tumor growth and metastasis through binding to C3b on the tumor cells and preventing the subsequent formation of lytic components at the cell surface. Moreover, after blocking the function of Factor H in the SW620 CRC cell line, treatment of the cells with specific antibodies against CD55 and CD59 rendered them more readily lysed (54). This demonstrates the strong potential of either targeting Factor H or performing a combined blockade of various CRPs, including CD55, CD59 and Factor H, in the treatment of CRC.

Downstream complement components in CRC

The MAC formed in the terminal stages of complement activation has been suggested to exert cytotoxic effects on tumor cells, thereby inhibiting tumor progression (55,56). It has been reported that in patients with CRC, the expression of CD59, which prevents MAC formation, significantly correlates with tumor grade, suggesting that the high expression of CD59 in tumors is a marker of poor prognosis (57). In addition, blocking CD59 with an anti-CD59 monoclonal antibody was found to prevent MAC-mediated cytolysis in HT29 CRC cells (58). These data indicate that blocking the formation of MAC by CD59 in CRC cells contributes to tumor growth. In a separate study, mice with a deficiency of C6, an important component of the MAC, developed more severe colitis symptoms than those of wild-type mice following treatment with dextran sulfate sodium (59), suggesting that blocking MAC formation may be a risk factor for colitis-associated cancer.

However, other studies have shown that MAC may exacerbate tumor progression (55,60). As summarized in a previous review, several studies have shown that, in order to exert its pro-tumor effects, MAC must exist mainly in its sublytic form (60). The specific mechanisms that have been proposed for its pro-tumor effects are as follows: i) Inducing the transcription of oncogenes and the proliferation of tumor cells; ii) protecting tumor cells from apoptosis; iii) promoting tumor angiogenesis; and iv) regulating the expression of response gene to complement 32 (RGC-32), a gene that regulates the cell cycle via the activation of Akt and cell division cycle protein 2 kinases (60). Based on current understanding, altering the transcriptional activity of CRC cells to promote the expression of tumor-promoting molecules may be the main mechanism by which sublytic MAC regulates CRC progression. Epidermal growth factor (EGF) performs a vital role in tumor development (61). Towner et al (62) showed that sublytic MAC induces the expression of the EGF receptor in CRC cells, thereby driving CRC tumor progression. Additionally, other studies have shown that strongly upregulating RGC-32 in colon cancer cells promotes tumor growth via the regulation of cytoskeletal reorganization or chromatin assembly (63,64). Given that the activation of sub-lytic MAC significantly upregulates the level of RGC-32, and considering the key role of RGC-32 in the underlying mechanism of CRC, the role of the sub-lytic MAC/RGC-32 signaling axis in the pathogenesis of CRC merits further attention in the future. Although further studies of CRC models are required to fully determine the pro-tumor effects of sub-lytic MAC, the research that has already been completed to elucidate the underlying pro-tumorigenic mechanisms has shed great insights into the role of MAC in CRC.

These findings indicate that MAC exerts a dual role in CRC, possessing both anti-cancer and cancer-promoting effects, depending primarily on the pattern of MAC attack. In one scenario, MAC initiates lethal attacks that directly induce cancer cell death, which is the primary manifestation of its anticancer effect. In the other scenario, sublytic MAC attacks, which are not sufficiently robust to directly cause cell death, may stimulate cancer cells to release growth factors or activate survival signaling pathways, thereby promoting their survival, growth and migration. This may be the primary mechanism underlying the cancer-promoting effects of MAC. It should be emphasized, however, that while the role of MAC in cancer is recognized, further in-depth research and validation experiments are required in more cancer models, such as CRC models, to fully elucidate the complex association between MAC and cancer.

Complement cascade-independent activation pathway in CRC

The complement system is a systemic, serum effector in innate immunity, and complement activation is traditionally viewed as a cascade of enzymatic reactions confined to the extracellular space, driven by three cascade-dependent activation pathways. Notably, the latest research has shown that the complement system is also activated by enzymes inside cells in a cascade-independent manner, and that this has an important role in the pathogenesis of CRC. For example, C3 and C5, the core complement components, have been found to be activated within cells in CRC (65,66).

Intracellular C3 activation in CRC

Liszewski et al (65) initially discovered the intracellular activation of C3 in human CD4+ T cells, and confirmed that T-cell-intrinsic C3 can be cleaved into C3a and C3b by the protease cathepsin L (CTSL). The CTSL-mediated intracellular generation of C3a is essential for the survival of resting T cells, as it sustains the tonic mammalian target of rapamycin signaling by engaging C3aR expressed on the lysosomes. By contrast, T cell receptor (TCR) activation induces the translocation of intracellular C3a to the cell surface and induces T helper 1 (Th1) immunity. In addition, the intracellular C3 product, C3b, can increase CD8+ T-cell immunity by binding to the C3b receptor CD46 in an autocrine manner (67) (Fig. 2). Notably, in non-immune cells, such as Caco-2 cells, pathogen-deposited C3 can be transported into the cells during pathogen endocytosis, resulting in the activation of multiple signaling pathways, including those regulated by NF-κB and activator protein 1 (68).

Figure 2. Role of the complement system in the regulation of immune cells and its impact on non-immune cells. Intracellularly, complement primarily regulates immune cell function whilst also influencing non-immune cells. The left panel details the activation of C3 and C5 within a T-cell. CTSL cleaves C3 into C3a and C3b. Intracellular C3a stabilizes mTOR signaling via lysosomal C3aR, which is crucial for T-cell survival. Intracellular C5a binds to C5aR1, inducing the production of ROS and activating the NLRP3 pathway to promote the Th1 response. With TCR activation, intracellular C3a translocates to the cell surface, triggering Th1 immunity, while C3b binds to CD46 in an autocrine manner, which enhances T-cell immunity. C5 in T cells can also be activated by the co-stimulatory effect of TCR and CD46. The right panel show the role of complement components in non-immune cells, using epithelial cells as an example. Pathogen-deposited C3 is endocytosed, activating pathways such as the NF-κB and AP-1 pathways. In addition, CTSD cleaves C5 in lysosomes and endosomes, generating C5a. C5a binds to C5aR1, recruiting KCTD5, cullin-3 and Roc1 to form a complex that stabilizes β-catenin through K63-linked polyubiquitination. This stabilizes β-catenin in the nucleus, thereby regulating gene transcription. Elevated β-catenin levels and nuclear translocation are closely associated with poor prognosis in colorectal cancer. CTSL, cathepsin L; C3aR, C3a receptor; C5aR1, C5a receptor 1; mTOR, mammalian target of rapamycin; ROS, reactive oxygen species; NLRP3, NOD-, LRR- and pyrin domain-containing protein 3; Th1, T helper 1; TCR, T-cell receptor; AP-1, activator protein-1; CTSD, cathepsin D; KCTD5, K+ channel tetramerization domain 5; Roc1, regulator of cullins-1; COX-2, cyclooxygenase-2.

Recently, studies have indicated that intracellular C3, particularly C3a, contributes to the promotion of CRC progression. For example, Liu et al (69) showed that the proportions of M0 and M1 macrophages and quiescent mast cells were increased in CRC samples with high C3 expression, whereas the proportions of activated dendritic cells (DCs), activated mast cells and memory CD4+ T cells were decreased, implying that high levels of C3 in CRC cells may promote CRC progression by altering the TIME. In addition, the C3a produced by tumor cells has been shown to reduce the infiltration of NK cells into solid tumors, such as CRC, by promoting the interaction between C3aR and lymphocyte function-associated antigen-1 in NK cells, which stimulates tumor growth (70).

Studies have also shown that the intracellular activation of C3, independent of the complement cascade, plays a critical role in maintaining T-cell homeostasis and supporting T-cell differentiation (65,71). This pair of studies were in general agreement that, intracellularly, C3a inhibits the differentiation of CD4+ T cells into Tregs, leading to a reduction in the level of the immunosuppressive cytokine IL-10, thereby inhibiting antitumor activity (72). However, no studies to date have explored whether C3a in T cells performs a similar role in CRC progression. Given that T cells have an important role as immune factors in the pathogenesis of CRC, investigation of the impact of intracellular C3 on CRC progression may emerge as an important area of future research.

Intracellular C5 activation in CRC

Within the lysosomes of colonic epithelial cells, C5 is cleaved by cathepsin D (CTSD) into C5a and C5b. Subsequently, C5a binds to C5aR1, recruiting K+ channel tetramerization domain 5, cullin-3 and regulator of cullins-1 to form a complex that promotes the K63-linked polyubiquitination of β-catenin, thereby stabilizing its expression in the nucleus. As β-catenin is a pivotal component of the Wnt signaling pathway, it stabilization regulates the transcription of downstream genes, thereby influencing cell proliferation and differentiation (66). As already mentioned, in immune cells such as T cells, C3 can be activated by CTSL (65), and the intracellular C3 products subsequently strengthen T-cell-mediated immune responses. In addition, the C3 products can translocate into non-immune cells and trigger multiple immune responses. Similarly to the activation of intracellular C3 catalyzed by protease CTSL in human CD4+ T cells, C5 can also undergo intracellular activation upon TCR and CD46 co-stimulation in CD4+ T cells (71). The intracellularly generated C5a binds to C5aR1 and induces reactive oxygen species production, which activates the NOD-, LRR-, and pyrin domain-containing protein 3 signaling pathway, thereby promoting Th1 responses (71) (Fig. 2).

A recent study reported that C5 inside colonic cancer cells can be cleaved by CTSD to produce C5a, and the subsequent C5a/C5aR1 signaling potentiates β-catenin stability and promotes colorectal tumorigenesis (66). Therefore, C5a/C5aR1 signaling in CRC cells has the potential to directly promote tumor growth. In addition, the intracellular activation of urokinase-type plasminogen activator-positive macrophages results in the release of C5a, which has been demonstrated to regulate the tumorigenic properties of C5aR1+ mast cells and macrophages, as well as to inhibit the cytotoxicity of CD8+ T cells, thereby promoting tumor growth (73). Furthermore, intracellular complement activation products such as C5a that are generated via the cascade-independent activation pathway have been shown to have tumor-promoting effects (66).

Advances in complement system research have deepened understanding of complement activation, expanding it from extracellular cascade-dependent activation, as an extracellular danger-sensing mechanism, to also include intracellular activation as a cascade-independent intracellular effector system. Moreover, this intracellular activation of complement proteins, such as C3a and C5a, appears to have a key pro-tumor role in CRC progression.

In summary, C3a and C5a, generated by complement cascade activation and intracellular activation, function as inflammatory mediators, capable of activating downstream signaling pathways and triggering a series of biological effects. However, these two activation pathways exhibit distinct differences in their generative mechanisms, modes of action and resulting effects. Complement cascade activation follows strict regulatory mechanisms, producing C3a and C5a as immunomodulatory factors. Upon excessive complement activation, these may induce inflammatory responses and regulate CRC cell behavior, thereby promoting or inhibiting tumor growth. By contrast, the generation of C3a and C5a by intracellular activation may involve non-canonical complement activation or direct cleavage via intracellular proteases. This process allows them to directly influence the function of immune cells, for example, T cells, DCs and MDSCs, as well as CRC cells. Also, the extracellular release of C3a and C5a enables them to exert complex effects on the TIME.

C1q protein in CRC beyond the borders of activation

C1q consists of three distinct polypeptides, designated C1qa, C1qb and C1qc, and functions as an initiating component of the classical complement cascade (74). It is a type-II transmembrane protein that is anchored to the membrane via the C1qa chain, prior to being cleaved into its soluble form (74,75). A number of studies have suggested that C1q functions beyond the borders of complement activation; that is, C1q directly participates in tumor progression in either its secreted or membrane-anchored form (21–23,76,77). Distinct from the majority of complement proteins, which are derived from the liver, C1q is mainly secreted by macrophages, and intestinal macrophages have been identified as the primary source of C1q (78). C1q+ macrophages have been observed in both healthy and tumor tissues, such as CRC and lung and liver cancers (79). And C1q produced by TAMs is considered to facilitate tumor progression by inducing immunosuppression, promoting T-cell exhaustion or facilitating neoangiogenesis through interactions with endothelial cells (76,77,80). Similarly, C1q+ macrophages have been detected in both healthy and tumor tissues in the colon, and single-cell analyses have suggested a pro-inflammatory function of C1qc+ TAMs, which preferentially express phagocytosis- and antigen presentation-associated genes (21). Another study suggested that in patients with colon cancer, a high expression level of C1qc in the cancerous tissue is associated with a worse prognosis (22), suggesting that C1q may serve as a promoting factor of CRC progression. However, a subsequent study revealed that, in addition to its action in the soluble form, the membrane-anchored form of C1q plays a protective role in inflammatory bowel diseases by enhancing the phagocytic capability of macrophages against bacteria and inflammatory cells in the colon (23). This study confirmed the protective role of the monoterpenoid glycoside paeoniflorin, which is mediated by the targeting of C1qa to increase the population of membrane-anchored C1q, while reducing C1q secretion. Therefore, the function of C1q depends on whether it is secretory or membrane-bound. Secretory C1q exhibits a pathogenic role in colon inflammation and cancer, even without activation of the complement cascade, whereas membrane-anchored C1q has a protective role in chronic colitis.

In summary, the complicated effects on CRC exerted by complement activation products, such as C3a and C5a, and complement proteins that function without undergoing activation, such as C1q, have deepened our understanding of the role of complement components in CRC. Firstly, the roles of activation products, including C3a, C5a, and MAC, generated by the complement cascade-dependent activation pathway are not always consistent. In detail, the roles of C5a and C3a can be summarized as follows: C5a promotes CRC progression by i) regulating antitumor immunity in the TIME by recruiting immune cells, including MDSCs and TAMs, and activating the NF-κB and PI3K-AKT signaling pathways; and/or ii) directly promoting tumor behaviors, including tumor growth, proliferation and invasion behavior, by activating the Wnt/β-catenin signaling pathway. However, the role of C3a in CRC is more complex, as the high plasma levels of C3a in patients with CRC can significantly decrease after treatment, indicating that C3a may have pro-tumor role in CRC, while C3a has also been indicated to play an antitumor role via the suppression of inflammatory responses typically associated with pro-tumor effects. Furthermore, MAC can directly form on the surface of tumor cells, where it can influence cell behavior. When MAC is formed in sufficient amounts, it exerts cytotoxic effects that inhibit tumor progression. However, when MAC is deposited at sublytic levels, it triggers inflammatory signals in cells, which induce CRC progression. It is worthy of note that the roles of C3a, C5a and MAC in CRC are also regulated by various CRPs, including Factor I, Factor H, CD55 and CD59. Secondly, some C3a and C5a is generated in cells, for example, in tumor cells and macrophages, through cascade-independent activation pathways in the TIME. These cells can directly activate intracellular complement components by cathepsins and secrete activation products such as C3a and C5a into the TIME, where they exert their pro-tumor effects. Thirdly, secretory C1q promotes tumorigenesis, whereas membrane-anchored C1q is protective against chronic colitis. This progression is beyond the borders of activation. The various associations between the complement system and colon cancer are summarized in Table I.

Table I. Associations between complement components and CRC.

Table I.

Associations between complement components and CRC.

A, Complement cascade-dependent pathway
Complement components	Associations with CRC	(Refs.)
C3a	C3a serves as a potential clinical biomarker for the early detection and prognosis assessment of CRC	(36)
	High expression of C3a in CRC and its significant reduction after treatment suggest C3a plays a role in tumorigenesis (pro-tumor effects)	(38)
	C3aR deficiency promotes CRC development by inducing an inflammatory response with pro-tumor effects, indicating that C3a also exerts antitumor effects on CRC (antitumor effects)	(39)
C5a	C5a recruits C5aR+ MDSCs, indirectly inhibiting CD8+ T cells and promoting a tumor-friendly environment (pro-tumor effects)	(40)
	By activating NF-κB-related signaling pathways in C5aR+ TAMs and stimulating the differentiation of TAMs towards the M2 phenotype, C5a exhibits tumor-promoting activity (pro-tumor effects)	(41)
	By promoting the secretion of macrophage-derived MCP-1 and activating the PI3K-AKT signaling pathway in macrophages, C5a promotes the liver metastasis of CRC (pro-tumor effects)	(42)
	C5a promotes CRC development by inducing EMT in CRC cells and activating Wnt/β-catenin signaling pathways (pro-tumor effects)	(43)
	C5a is a potential clinical biomarker for early detection and prognosis of CRC	(44,45)
MAC	Blocking CD59 with an anti-CD59 monoclonal antibody prevents HT29 CRC cells from undergoing MAC-mediated cytolysis, thereby contributing to tumor growth (antitumor effects)	(58)
	Sublytic MAC induces the expression of EGF receptors in CRC cells, thereby driving the progression of CRC (pro-tumor effects)	(62)
	Upregulated RGC-32 in colon cancer cells promotes tumor growth through the regulation of cytoskeletal reorganization or chromatin assembly. It is speculated that, given that sublytic MAC triggers the upregulation of RGC-32 in tumor cells, sublytic MAC/RGC-32 may mediate pro-tumor mechanisms in CRC	(63,64)
CD46	CD46 upregulation in colon cancer cells via STAT3/STAT6/p38 MAPK reduces apoptosis and induces mouse cancer growth (pro-tumor effects)	(52)
CD55	CRC tumors are protected by the overexpression of CD55, especially during hypoxia (pro-tumor effects)	(46)
	CD55 deficiency abnormally activates the complement system, causing intestinal inflammation, excess cytokine release, and worsening colitis (antitumor effects)	(47)
	Novel CD55 monoclonal antibody activates complement and inhibits CRC proliferation, invasion and migration (pro-tumor effects)	(49)
	CD55 is highly upregulated in CRC, especially in the more advanced stages	(15,48,50,51)
CD59	Expression of CD59 significantly correlates with tumor grade, and is a marker of poor prognosis in patients with CRC
	Blocking CD59 with anti-CD59 monoclonal antibody prevents CRC cells from undergoing MAC-mediated cytolysis, contributing to tumor growth (pro-tumor effects)	(58)
Factor I	Factor I is significantly increased in colon cancer cells, and considered to be a diagnostic biomarker for CRC	(53)
	Knocking out Factor I inhibits colon cancer cell proliferation, migration and invasion by blocking the Wnt/β-catenin/c-Myc pathway and suppressing glycolysis (pro-tumor effects)	(53)
Factor H	Levels of factor H and its split variant, factor H-like protein, are highly increased in patients with colon adenocarcinoma and metastatic foci in the liver	(54)
	Upregulation of factor H results in tumor growth and metastasis through binding to C3b on tumor cells and preventing subsequent lytic component formation at the cell surface (pro-tumor effects)	(54)
B, Complement cascade-independent pathway
Complement components	Associations with CRC	(Refs.)
C3a	High C3 in CRC increases M0/M1 macrophages and quiescent mast cells, but decreases activated DCs, activated mast cells and memory CD4+ T cells, suggesting C3 promotes CRC progression by altering the TIME (pro-tumor effects)	(69)
	C3a produced by tumor cells reduces the infiltration of NK cells into solid tumors, such as CRC, by promoting the interaction between C3aR and lymphocyte function associated antigen 1 in NK cells, which stimulates tumor growth (pro-tumor effects)	(70)
	C3a inhibits CD4+ T cell differentiation into Tregs and reduces IL-10 levels, suppressing antitumor activity. It is speculated that since T cells play an important role as immune factors in the pathogenesis of CRC, the impact of intracellular C3 may exert pro-tumor effects on CRC by this mechanism (pro-tumor effects)	(72)
C5a	C5a/C5aR1 signaling stabilizes β-catenin, promoting CRC cell proliferation and differentiation (pro-tumor effects)	(66)
	uPA+ macrophages release C5a, which regulates the carcinogenic properties of	(73)
	C5aR1+ mast cells and macrophages, and inhibits CD8+ T cell toxicity, thereby promoting tumor growth (pro-tumor effects)
C, Beyond the borders of activation
Complement components	Associations with CRC	(Refs.)
C1q	In the colon, C1q+ TAMs are present in both healthy and tumor tissue, and single-cell analyses suggest a pro-inflammatory function of C1qc+ TAMs (pro-tumor effects)	(21)
	Patients with colon cancer who express a high level of C1qc in the colon cancer tissue exhibit a worse prognosis (pro-tumor effects)	(22)
	C1q plays a protective role in inflammatory bowel diseases by enhancing the phagocytic capability of macrophages against bacteria and inflammatory cells in the colon (antitumor effects)	(23)

[i] CRC, colorectal cancer; C3aR, complement 3a receptor; C5aR, complement 5a receptor; MDSC, myeloid-derived suppressor cell; TAMs, tumor-associated macrophages; MCP-1, monocyte chemoattractant protein-1; EMT, epithelial-mesenchymal transition; MAC, membrane attack complex; EGF, epidermal growth factor; RGC-32, response gene to complement 32; DCs, dendritic cells; TIME, tumor immune microenvironment; NK, natural killer; Tregs, regulatory T cells; uPA, urokinase-type plasminogen activator.

Complexity of complement system in CRC progression

As discussed above, complement components exhibit complicated roles in CRC, with both pro-tumor and antitumor effects. Differences in the roles of various complement components may be responsible for this. For example, C5a produced by the complement cascade-dependent activation pathway exerts pro-tumor effects on CRC, whereas C3a produced analogously has antitumor effects. Also, the distinct complement activation pathways appear to contribute to these conflicting effects; while C3a produced by the complement cascade-dependent activation pathway plays an antitumor role in CRC, C3a produced by the complement cascade-independent activation pathway promotes tumorigenesis. In addition, the varying effects the complement system has on CRC may be attributed to different CRC subtypes. According to the Consensus Molecular Subtypes (CMS) classification system and gene expression analysis, CRC may be divided into four subtypes: CMS1 (microsatellite instability immune), which exhibits clear immune infiltration and activation resulting in poor survival after relapse; CMS2 (canonical), which mainly exhibits epithelial differentiation, although it is associated with superior survival rates following relapse; CMS3 (metabolic), which is associated with the occurrence of KRAS-activating mutations, and exhibits prominent metabolic adaptation; and CMS4 (mesenchymal), which is associated with poorer relapse-free and overall survival rates, and displays high levels of complement-mediated inflammation, EMT and activation of transforming growth factor-β signaling pathways (81,82). Different CRC subtypes may exhibit significant heterogeneity of the complement system, with distinct patterns of complement expression potentially contributing to the differences in tumor aggressiveness that have been observed among CRC subtypes. For example, CMS4, characterized by complement-mediated inflammation, has poorer relapse-free and overall survival rates compared with those of other subtypes. These heterogeneous features not only highlight the diverse effects of the complement system on CRC, but also provide crucial insights that may assist the development of personalized treatment strategies. Understanding this variability is essential to facilitate the development targeted therapies that can regulate complement activity in CRC.

Clinical significance of complement system in CRC

Several studies have investigated the possibility of targeting the complement system to block tumor progression in CRC. For example, Downs-Canner et al (83) demonstrated that cobra venom factor, which depletes C3, and Staphylococcus aureus superantigen-like protein 7, which inhibits C5, both effectively suppressed tumor growth in a CRC mouse model. These antitumor effects were mediated via the enhancement of immune cell infiltration, specifically that of CD8+ T cells, and the expression of chemokines, including C-C motif chemokine ligand 5, and C-X-C motif chemokine ligands 10 and 11. Due to the important role of the C5a/C5aR axis in immune cell infiltration, Ding et al (84) discovered that blocking this pathway, either by knocking out complement C5 or C5aR1, or by using the C5aR inhibitor PMX205, resulted in the near-complete blockade of CRC progression. This was achieved through the inhibition of MDSC infiltration and an increase in the proportion of CD8+ T cells. Notably, the study also demonstrated that the knockout of C5 and C5aR1 resulted in a smaller tumor size and fewer tumors, along with a greater reduction in the number of infiltrating MDSCs and a more pronounced increase in the proportion of CD8+ T cells compared with that achieved with C3 knockout. This suggests that targeting C5 is likely to more effective than targeting C3 in complement-based therapies for CRC. However, although no clinical treatment method has yet been devised to target the C5a/C5aR axis for the treatment of CRC, the STELLAR-001 phase I trial has explored the possibility of combining the anti-C5aR antibody IPH5401 with the programmed cell death ligand 1 inhibitor durvalumab for the treatment of advanced solid tumors (46).

Targeting CRPs has also been shown to delay the progression of CRC, in addition to targeting the C5a/C5aR axis. The expression of CD55 is upregulated in CRC cells under hypoxic conditions, which protects the tumor from lysis (46). In this regard, Dho et al (49) developed a novel CD55 chimeric monoclonal antibody that inhibits the proliferation, invasion and migration of CRC cells by activation of the complement system. In addition, a combination of the anti-CD55 antibody with 5-FU was found to provide synergistically improved therapeutic effects on CRC (49).

In summary, while clinical applications are not yet available, preclinical studies in which the complement system is targeted have revealed promising therapeutic effects on CRC. However, based on current research findings, several key areas require further investigation to optimize the targeting of the complement system for CRC treatment. For example, the complement system is an important component of innate immunity, and continuously blocking its activation may impair opsonization and bacteriolytic activity, thereby increasing the risk of infection (46). To overcome this challenge, novel drug formulations, such as a next-generation ‘recycling’ form of eculizumab, are being developed for potential use (85,86). In addition, targeting C5aR instead of directly blocking C3 or C5 enables opsonization, which should protect cancer patients from the risk of bacterial infection (46). Therefore, it is necessary to carefully consider the complexity of the function of a drug when studying the role of the complement system in tumor progression, as this will provide a solid theoretical foundation for the development of targeted therapeutic strategies. Moreover, most current research efforts have focused on in vitro experiments and animal models, which may not fully represent the complex situation in the human body (87–89). Therefore, further validation and adjustment of these strategies are required prior to clinical application.

Conclusions and prospects

CRC remains one of the most commonly occurring tumors worldwide, and the prognosis of patients with CRC is unsatisfactory following treatment with traditional methods. As a key component of the TIME, complement activation products derived from both complement cascade-dependent activation in body fluids and intracellular, cascade-independent activation play various roles in CRC. These include the regulation of inflammatory responses during tumorigenesis, the modulation of antitumor immunity in the TME, and the control of tumor growth. At times, complement proteins can affect tumor progression without being activated. However, the effects of these components of the complement system are not always consistent, and in some cases have been shown to be contradictory. Further research is necessary to explore the main factors of the complement system that contribute to CRC progression. In addition, some early studies focused on the changes of complement components in the plasma or feces rather than the tumor tissues and did not explore the underlying mechanisms. Comprehensive research elucidating the signaling pathways involved in the interactions of complement components with other immune cells or tumor cells within the TIME, particularly at various developmental stages of CRC, are lacking. A deeper exploration of these aspects may not only reveal novel therapeutic targets for CRC, but also provide a more comprehensive analysis and understanding of the conflicting roles of the complement system in CRC.

Additionally, while several studies have suggested that complement components play an antitumor role in CRC development, current research is primarily focused on the pro-tumor effects of these complement components. The inhibition of C3, C5 and CD55 has been shown to alleviate CRC development. The therapeutic effects achieved by therapies targeting the complement system indicate that the pro-tumor effects of the system play a more dominant role than the antitumor effects in CRC progression. In conclusion, given the important role of the complement system in the pathogenesis of CRC, further exploration of therapies targeting the complement system is likely to be invaluable.

Acknowledgements

Not applicable.

Funding

The present study was supported by the National Natural Science Foundation of China (grant nos., 82000525 and 81873883) and Science and Technology Support Plan for Youth Innovation of Colleges and Universities of Shandong Province of China (grant no. 2021KJ106). In addition, it was funded by Youth Innovation Team Project for Talent Introduction and Cultivation in Universities of Shandong Province and the domestic visiting project of Shandong Second Medical University.

Availability of data and materials

Not applicable.

Authors' contributions

YX, ML and SL designed and edited the manuscript. JZ, YW, JS and XF contributed to the search and analysis of literature and the design of the manuscript. Data authentication is not applicable. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References