Exploring the role of Fusobacterium nucleatum in colorectal cancer: implications for tumor proliferation and chemoresistance

Abstract

Fusobacterium nucleatum (Fn) has been extensively studied for its connection to colorectal cancer (CRC) and its potential role in chemotherapy resistance. Studies indicate that Fn is commonly found in CRC tissues and is associated with unfavorable prognosis and treatment failure. It has been shown that Fn promotes chemoresistance by affecting autophagy, a cellular process that helps cells survive under stressful conditions. Additionally, Fn targets specific signaling pathways that activate particular microRNAs and modulate the response to chemotherapy. Understanding the current molecular mechanisms and investigating the importance of Fn-inducing chemoresistance could provide valuable insights for developing novel therapies. This review surveys the role of Fn in tumor proliferation, metastasis, and chemoresistance in CRC, focusing on its effects on the tumor microenvironment, gene expression, and resistance to conventional chemotherapy drugs. It also discusses the therapeutic implications of targeting Fn in CRC treatment and highlights the need for further research.

Introduction

Fusobacterium nucleatum (Fn), a gram-negative anaerobe, has been reported as a potential pathogen in colorectal cancer (CRC) [1, 2]. Evidence suggests that enrichment of Fn affects CRC’s development, progression, and poor prognosis [3, 4]. CRC is the third most common cancer and the fourth leading cause of cancer-related death globally. By 2030, it is expected to increase to 60%, with about 2.2 million new cases and 1.1 million cancer-related deaths [5]. Historically, the majority of CRC cases occur in industrialized nations; however, its incidence is rising in countries with relatively low human development indices, such as Asia, South America, and Eastern Europe [6]. Surgery is a standard therapeutic option for CRC, especially for early-stage (stages 0- II). In addition to surgery, adjuvant treatments such as chemotherapy and targeted therapy are necessary for other stages [7, 8]. Over the past few decades, new chemotherapy regimens have improved the overall survival rate for individuals with advanced colon cancer. Despite advances in chemotherapy treatments, a major ongoing challenge is the development of chemoresistance. While nearly half of CRC patients initially respond to chemotherapy, resistance eventually develops in almost all cases, resulting in treatment failure and reduced survival rates [7, 9, 10].

Chemoresistance in CRC is a complicated process that involves various mechanisms like genetic variations, epigenetic modifications, and changes in the tumor microenvironment [11–13]. Recent studies have indicated that Fn has a major effect on the development of chemoresistance in CRC by altering the tumor microenvironment and modifying the expression of key genes involved in chemoresistance [14, 15]. Therefore, this review aims to offer a comprehensive overview of the current understanding of the involvement of Fn in the development of chemoresistance in CRC. We review how Fn contributes to chemoresistance, with a particular focus on its impact on the tumor microenvironment, gene expression, and resistance to conventional chemotherapy drugs. Additionally, we will discuss potential therapeutic approaches targeting Fn to improve treatment effectiveness in CRC patients.

Association between Fn and CRC progression

Interaction and carcinogenesis

Tumor growth and metastasis are significantly influenced by the dysbiosis and composition of the oral microbiome. A recent study suggested that there is a complex and reciprocal interaction between the oral microbial community and the tumor microenvironment [16].

Certain oral bacteria and their metabolic byproducts seem to have intrinsic tumor-promoting capabilities that may contribute to the advancement of malignant growth at both the primary and distant metastatic sites [17, 18]. In contrast, the unique conditions within the tumor environment promote the colonization and growth of specific opportunistic oral bacteria, such as Fn and Porphyromonas gingivalis [19, 20]. Fn is one of the most prevalent gram-negative bacterial species in the human oral cavity and is considered a pathobiont due to its tendency to proliferate during the dysbiotic phase, which precedes periodontal disease development [21, 22]. It contributes to the development of periodontitis by interacting with keystone species such as P. gingivalis, thereby disrupting the balance between the host and the oral microbiota [23]. The bacterium can be detected on the tongue and within biofilms consisting of multiple species at the gingival margins of teeth, where it is speculated to play a key role in the formation of subgingival dental plaque [24, 25]. Fn has various adhesion mechanisms that allow it to bind to many oral bacterial species, a phenomenon known as co-aggregation or co-adherence [26]. By forming a bridge between anaerobic secondary colonizers such as Treponema denticola, P. gingivalis, and Aggregatibacter actinomycetemcomitans and early oral colonizers such as Streptococcus species, Fn plays a crucial role in oral biofilms. This connection helps form and maintain the polymicrobial community, making it more resistant to eradication efforts [26–28]. Additionally, oral bacteria can resist human defensive mechanisms, including phagocytosis and antiseptics, by forming biofilms. P. gingivalis, in particular, promotes the growth of Fn by releasing a molecule distinct from AI-2, which is believed to facilitate interspecies communication within mixed-species communities and contribute to biofilm formation [29]. These potentially pathogenic oral microorganisms have been consistently linked to the development of various of tumors, highlighting their ability to affect carcinogenesis and disease progression through various mechanisms [16]. Similar to its effects in gum tissue, Fn can induce infection and inflammation throughout the body. It has also been demonstrated that several malignancies are linked to Fn mislocalization [30]. A recent study revealed that the risk of CRC escalated with higher levels of Porphyromonas (32.1% vs. 16.2% in cases vs. control participants) and Fn (34.3% vs. 28.1%), as measured by quantitative polymerase chain reaction (qPCR). The presence of Fn was significantly greater in patients with CRC compared to controls (31.9% vs. 11.7%), correlating with an increased risk of CRC [31]. Fn has been detected in most CRC tissues, primarily in the proximal colon. Although the direct spread of Fn from the oral cavity to the colon seems plausible, some evidence indicate that Fn migrates via a hematogenous route to distant regions. This raises the interesting question of how a bacterium found in the mouth may be associated with various illnesses and malignancies, both within and outside the mouth [32–34]. In response to this question, several mechanisms have been proposed, including the resistance of oral bacteria to the highly acidic environment of the stomach, which may cause them to survive [35]. Furthermore, persistent exposure to proton pump inhibitors may increase intestine colonization of oral bacteria, changing the stomach pH to a less acidic value [36]. In addition, the ingestion of dead bacterial components can enhance the expression of virulence genes in bacteria and trigger a cytotoxic response, which is one probable factor, known as “necrotrophy-necrovirulence” [37]. According to these possible mechanisms, the oral and fecal microbiomes exhibit significant similarities, with over 45% of the bacterial taxa being comparable, as indicated by the Human Microbiome Project. Therefore, oral microbial translocation may be a mediating factor in intestine colonization [38]. Fn survival may also be facilitated by tumor-induced conditions, such as increased blood flow, blood vessel leakage, hypoxia, and an immunosuppressive microenvironment. Nevertheless, these conditions are insufficient to prevent other oral anaerobic bacteria from colonizing and causing cancer [39]. Fn colonization may require certain methods and conditions. Current data suggest that tumor localization by Fn could be controlled by glycan-lectin interactions. The tumor ligand for fusobacterial attachment is hypothesized to be the disaccharide D-galactose-β (1-3)-N-acetyl-D-galactosamine (Gal-GalNAc) or a similar sugar molecule [40]. GalNAc and Gal-GalNAc are O-GalNAc glycans that serve as post-translational modifications to proteins. They are formed by covalently linking N-acetylgalactosamine (GalNAc) to specific Ser/Thr residues in the acceptor protein, resulting in the Tn-antigen. These glycans are usually capped with sialic acid in healthy cells, preventing exposure [41]. However, in numerous carcinomas, particularly CRC, shortened O-GalNAc glycans are generated, and sialic acid is not added. This leads to an increased presence of unmasked Tn and T-antigens, which is associated with tumor invasion and metastasis [42]. Galactose and galNAc can prevent Fn from binding and coaggregation with various gram-negative species in dental plaques. These findings suggest that Fn expresses a lectin that binds to these molecules found on the receptor of these coaggregation-partner bacteria [43, 44]. The outer-membrane Fap2 protein has been identified as the protein responsible for this lectin activity [45]. Fap2 can facilitate Fn-induced apoptosis in lymphocytes, which may help bacteria avoid detection by the immune system [46, 47]. As experimental evidence has indicated, Fap2 may also aid in binding Fn to these cancer cells, given the overexpression of Gal-GalNAc in CRC [39, 48]. Furthermore, this bacterium, like other Gram-negative bacteria, can produce outer membrane vesicles (OMVs) both in vitro and in vivo. The proteins in Fn Antigenic components of OMVs, such as FomA, FadA, FadD, Fad-I, NapA, ClpB, GroEL, TraT, and YadA, can activate Toll-like receptors (TLRs) on epithelial or immunological cells. TLR stimulation activates the NF-κB pathway, producing proinflammatory cytokines [49, 50].

Furthermore, CRC is a heterogeneous disease, and subtype identification is directly related to clinical treatment. Guinney et al. classified CRC into four consensus molecular subtypes (CMS) based on clinicopathological and molecular characteristics: CMS1 (microsatellite instability immune, 14%), CMS2 (canonical, 37%), CMS3 (metabolic, 13%), and CMS4 (mesenchymal,23%) [51]. Increasing evidence suggests a relationship between gut microbial dysbiosis and CRC development. Previous studies have shown that some bacterial species, such as Fusobacterium hwasookii and P. gingivalis, are more abundant in the CMS1 subtype [52]. Additionally, the Fn is more abundant in CRC tissues than adjacent normal tissues [53].

According to a recent study, several molecular characteristics, such as the CpG island methylator phenotype, microsatellite instability, and a decreased density of CD3 + T-cells, are associated with the abundance of Fn in CRC tissues. These findings indicate that bacteria may play a role in determining the immunological response of various CRC subtypes [54]. Mima et al. investigated the enrichment of Fn in several bowel subsites in CRC and reported that Fn levels gradually increased in CRC. Furthermore, CRC subtypes can be treated more effectively if they are classified based on the clinical, pathological, and molecular characteristics of Fn [55]. In 2016, a high concentration of Fn was linked to shorter survival times in more than 1000 CRC patients [56]. In 2019, Oh et al. reported the predictive effects of Fn in patients receiving adjuvant chemotherapy. These findings suggest the need for a thorough investigation into the relationships between Fn and different subtypes of CRC [57].

Invasion and metastasis

An essential aspect of cancer development is Epithelial-mesenchymal transition (EMT), which generally promotes local invasion and distant metastasis. The activation of EMT programs during cancer development confers improved migratory and invasive capabilities on epithelial-derived tumor cells, enabling them to spread from the primary site and form secondary tumors at distant locations [58]. Recent studies have shown a particular molecular pathway that connects the Fn to the enhancement of EMT in CRC. It has been demonstrated that in CRC cells, Fn infection causes the cytochrome P450 enzyme CYP2J2 to be upregulated. This increase is mediated by the activation of the TLR4/AKT/Keap1/NRF2 signaling pathway. Following an elevation in CYP2J2 expression, 12,13-epoxyoctadecenoic acid (12,13-EpOME), a carcinogenic metabolite of the enzyme, is produced at a higher rate. The overexpression of CYP2J2 or 12,13-EpOME significantly promotes CRC cell invasion and migration, indicating that the Fn-induced CYP2J2/12,13-EpOME axis is a crucial driver of EMT-mediated tumor cell dissemination [59].

Infection with Fn in human CRC-derived HCT116 cells increases migration, secretes IL-8 and CXCL1, increases autophagy, and activates NF-κB. These factors are linked to higher metastatic potential and a poor outcome [30]. Furthermore, Ou et al. found that matrix metalloproteinase 7 (MMP7) is upregulated by Fn in CRC cells in a dose-dependent manner compared to normal cells and could be associated with Fn-promoted metastasis [60]. Fn-infected cells release exosomes containing metastasis-related microRNA-126 (miR-1246), miR-92b-3p, miR-27a-3p, and CXCL16/IL-8, which greatly promote metastasis. Internalizing these exosomes leads to overexpression of cyclin D1, β-catenin, cellular MYC proto-oncogene, and mesenchymal markers in CRC cells, indicating a cancer-promoting function [61]. Fn can also secrete outer membrane vesicles (OMVs) containing lipoproteins, phospholipids, proteins, and lipopolysaccharides (LPS), which serve as a vehicle for virulence factors [62, 63]. In addition to the direct effects on tumor cells, Fn has been found to promote angiogenesis, a critical process for solid tumors’ sustained growth and metastasis. Fn can activate an autocrine function in endothelial cells, resulting in increased production of vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGFR1), and VEGFR2, all of which promote proliferation and metastasis (Fig. 1A) [64, 65].

Fig. 1.

The potential mechanisms of Fn are associated with CRC progression and chemoresistance. A: Fn effect on endothelial cells, could result in increased production of VEGF, VEGFR1, and VEGFR2, leading to proliferation and metastasis. B: Fn triggers the release of inflammatory cytokines, creating a pro-inflammatory microenvironment that could trigger cancer progression. C: Fn invasion upregulates miR-21, and activates the TLR4, MyD88, and NF-kB pathway, reducing the expression of RASA1 and increasing inflammatory factors that promote CRC cell proliferation. D: Fn activates the TLR4/NF-κB pathway in CRC cells and increases the expression of BIRC3, which may reduce sensitivity to 5-Fu and induce chemoresistance

Signaling pathway

Mechanisms of the main virulence proteins

Fn’s pathogenic cascade begins with its ability to adhere to and enter human epithelial and endothelial cells. The bacterial proteins FadA and Fap2 play essential functions in this process [66]. FadA binds to host cell-junction molecules, particularly E-cadherin and VE-cadherin, found in epithelial and endothelial cells, respectively. It exists in both membrane-anchored and secreted forms [67, 68]. Through this interaction, Fn internalization and the formation of an active FadA complex are facilitated. This leads to a series of host responses that are associated with cancer development and progression. These responses include the β-catenin signaling activation and the upregulation of inflammatory or oncogenic genes [67]. Moreover, FadA’s binding to VE-cadherin on endothelial cells disrupts cell-cell junctions, increasing endothelial permeability and permitting Fn to spread to distant locations via hematogenous pathways [69]. The other Fn protein, Fap2, acts as an autotransporter, mediating bacterial enrichment through interaction with host cells. It recognizes overexpressed Gal-GalNAc and binds to it, facilitating Fn attachment to tumor epithelial cells, while inhibiting immune cell cytotoxicity and activity [39, 70]. The loss of epithelial E-cadherin, which regulates cell-cell adhesion and limits cell motility, is one of the characteristics of the EMT, a critical step in CRC metastasis. Fn has been shown to induce epithelial cells to take on a mesenchymal-like phenotype, increasing their ability to invasion [71, 72]. The dysregulation of the E-cadherin/β-catenin complex occurs in tandem with this process, leading to either the total downregulation of E-cadherin or its mislocalization from the cell membrane to the cytoplasm. Clinical observations have revealed that patients with high levels of Fn are more likely to have lymph node metastases, highlighting Fn’s role as a significant risk factor for CRC metastasis [73]. Even when overall levels of E-cadherin protein remain constant, Fn appears to trigger the endocytosis of membrane-bound E-cadherin, which disrupts calcium-dependent cell-cell adhesion. The process of CRC metastasis to distant locations may be facilitated by Fn’s intracellular position within vesicular structures enriched in E-cadherin. This suggests a potential mechanism by which Fn mediates the mislocalization of E-cadherin [67, 74].

Molecular alterations and DNA damage in the tumor microenvironment

DNA damage is a known factor in the development and spread of tumors. Interestingly, studies have shown that Fn can directly increase the activity of DNA methyltransferases, leading to the hypermethylation of tumor suppressor genes (TSGs) and high microsatellite instability (MSI-H) phenotype and the development of a CpG island methylator phenotype (CIMP) in CRC [56]. Fn may cause these epigenetic changes by controlling the production of inflammatory cytokines and reactive oxygen species (ROS), which can impact DNA methylation patterns. Furthermore, it has been demonstrated that Fn dysregulates the Chk2 signaling pathway, leading to impaired cell cycle regulation and a weakened response to DNA damage [73]. Impaired DNA repair mechanisms may indicate DNA damage caused by Fn. Furthermore, it has been reported that Fn inhibits the production of the DNA glycosylase NEIL2, which typically aids in reducing DNA damage from inflammation. This suppression contributes to the development and spread of CRC by causing an accumulation of DNA double-strand breaks (DSBs) [75]. Malignant transformation may arise from inadequate repair of these DSBs, mediated by mechanisms including non-homologous end joining (NHEJ). It’s interesting to consider how complicated the connection is between Fn and microsatellite instability in CRC [76]. Even though Fn-high CRCs with MSI-H status are strangely connected to poor clinical outcomes, MSI-H CRCs are often associated with a positive prognosis. This could be explained by Fn’s ability to create inflammation and the translocation of the mismatch repair protein MSH3, leading to enhanced microsatellite instability at selected tetranucleotide repeats (EMAST) or low-level MSI (MSI-L) [77].These findings suggest that Fn may impact the genetic landscape of CRC in various ways, potentially influencing the progression and prognosis of the disease [73]. In a study of 109 patients with stage II/III right-sided CRC, 67 patients with MSI-H and more activated cytotoxic lymphocytes were found to have better clinical outcomes [78, 79]. Nevertheless, a recent study discovered that CRCs with higher levels of Fn infection, while associated with MSI-H, have a poorer prognosis due to inflammatory agents released by the tumor [30]. However, another study found that CRCs with higher levels of Fn infection are associated with MSI-H and have a poorer prognosis. This could be related to the tumor secretes inflammatory factors in response to Fn invasion, leading to MSI-L/EMAST, a type of microsatellite instability [80]. BRAF mutation or a high-level CIMP may be the cause of MSI. CIMP can also silence the mismatch repair gene MLH1. Studies have revealed a connection between Fn and CIMP-positive CRCs. However, more investigation is needed to determine the connection between Fn, CIMP, and MSI status [69].

Induction of a pro-inflammatory tumor microenvironment

Inflammation, a key player in cancer initiation, and the NF-κB signaling pathway has a crucial role in triggering the transcription of numerous inflammatory genes. The mechanisms of how Fn contributes to the inflammatory tumor microenvironment and subsequent cancer progression are multifaceted. Fn-derived FadAc complex consists of mature FadA and pre-FadA. The FadAc protein can induce the phosphorylation and internalization of E-cadherin, leading to the aggregation of β-catenin and the activation of β-catenin-regulated transcription (CRT). Additionally, Fn LPS can activate the TLR4/PAK1/β-catenin S675 cascade, leading to further stimulating CRT [67, 81].

The activated CRT upregulates the Wnt signaling genes expression, NF-κB, pro-inflammatory cytokines, and oncogenes, creating a pro-inflammatory environment that stimulates cancer progression [67, 82]. Using advanced microRNA sequencing technology, the role of miRNAs in promoting CRC progression was investigated. They discovered that miR-21, upregulated by Fn invasion, activates TLR4 and binds to myeloid differentiation factor 88 (MyD88). The miR-21 activation subsequently triggers the NF-kB pathway and reduces the level of RAS GTPase Ras p21 protein activator 1 (RASA1), resulting in an elevation in various inflammatory factors that remarkably promote CRC cell proliferation (Fig. 1C). Moreover, knockdown/knockout of miR-21 can reduce the oncogenic role of Fn in colorectal carcinogenesis. Therefore, both the DNA of Fn and miR-21 could serve as biomarkers for poor outcomes [83, 84]. Fn also triggers the release of the inflammatory cytokines, specifically interleukin-8 (IL-8), IL-10, and Tumor Necrosis Factor-alpha (TNF-α), and facilitates the formation of inflammasomes, creating a pro-inflammatory microenvironment that stimulates cancer progression (Fig. 1B) [85]. Analysis using qPCR showed a significant elevation in IL-8 and IL-6 mRNAs in CRC cells penetrated by Fn [67, 84]. The upregulation of TNF-α, IL-8, monocyte chemotactic protein 1 (MCP-1), and IL-6 mRNAs is known to favor tumor proliferation. Inflammasomes are multiprotein complexes consisting of apoptosis-associated speck-like protein, caspase activation and recruitment domain (CARD), procaspase-1, and a sensor protein, either NOD-like or AIM2-like, which can be activated by inflammation [86]. Additionally, both immunohistochemistry and qPCR analysis of clinical CRC samples showed an elevated expression level of nuclear phosphorylated NF-κB p65 [80]. Bullman et al. proposed that the constitutive NF-κB signaling triggered by Fn contributed to the CRC progression, and the elimination of Fn could be a promising option as an adjuvant treatment for CRC patients [87].

Constructing an immunosuppressive tumor microenvironment

The ability of cancer to evade the body’s immune response is a key factor in its progression. The Fn aids immune evasion by inhibiting T-cells and natural killer (NK) cells, essential components of the antitumor immune response [88]. The innate immune system’s NK cells can eliminate various targets, including bacteria, viruses, cancer cells, and parasites [88]. Their activity is regulated by both inhibitory and activating receptors. Fn has been demonstrated to utilize the T-cell immunoglobulin and T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) inhibitory receptor, which is found on the surface of T-cells, NK cells, and tumor-infiltrating lymphocytes (TILs) [89]. Moreover, CD226, the co-stimulatory molecule in immune cells, competes with TIGIT for CD155 binding. TIGIT inhibits CD226 homodimer formation, thereby diminishing CD226’s activity in anti-viral T cell response and the antitumor. TIGIT activation mediates inhibitory signals in NK cells via ITIM and reduces T cell activation and proliferation [90–92]. Furthermore, TIGIT’s interaction with dendritic cells (DCs) enhances the IL-10 release while reducing the IL-12 synthesis, shifting immune responses toward a tolerogenic phenotype [93].TIGIT is also strongly expressed in regulatory T cells (Tregs), where it increases immunosuppressive activity and IL-10 production, contributing to a tumor-suppressive milieu [94].This interaction between TIGIT + Tregs and DCs, together with the production of immunosuppressive molecules such as fibrinogen-like protein 2 (Fgl2), inhibits Th1 and Th17 development, resulting in an environment that promotes tumor persistence by undermining effective T cell responses [92, 95]. In line with this, a study was conducted in 2022 that showed that removing CD155 in CRC cells increases the activity of CD8 + T cells, and blocking the CD155-TIGIT pathway reduces tumor growth in mouse models. Furthermore, TIGIT + cells in CRC are associated with more advanced disease, earlier recurrence, and decreased survival rates [96, 97]. The direct interaction between the Fn-derived Fap2 protein and TIGIT results in the inhibition of NK cell cytotoxicity and the induction of cytotoxic T-cell death [70]. Additionally, Fn has been shown to bind to and activate carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), another inhibitory receptor expressed on T-cells and NK cells, which is believed to be involved in mediating T-cell exhaustion. This means that tumors can evade immune cell attack by inhibiting the activities of both T-cells and NK cells [98, 99]. Consequently, it allows tumors to effectively evade immune detection through reduced levels of IFN-γ and CD107a, impairing the function of T cells and NK cells, resulting in expression of the cell adhesion molecule CEACAM1, significantly inhibiting cell invasion and migration [99, 100]. Furthermore, a study showed that the wild-type F. nucleatum strain FN726 dramatically reduced IFN-γ and CD107a degranulation compared to the Fap2-deficient mutant FNK50. The FN726 is virulent through TIGIT and CEACAM1, but FNK50 is only virulent through CEACAM1. This shows that Fn reduces antitumor immunity by activating the TIGIT receptor and CEACAM1, generating an immunosuppressive milieu for tumor cell avoidance [99, 101].

Furthermore, Fn causes lymphocyte cell death and tumor progression by inhibiting G1 phase cell MDSCs and inhibiting TIGIT receptors [102, 103].

A study on 366 cancer patients found that high tumor-infiltrating T-cell density leads to better outcomes, regardless of clinical, pathological, or molecular characteristics. Fap2 is bound to human inhibitory receptor TIGIT and can cause lymphocyte cell death, leading to immune escape and poor prognosis in CRC patients [70, 104]. In addition to the direct suppression of NK cells and T-cells, Fn has been shown to selectively attract myeloid-derived suppressor cells (MDSCs).

The high levels of inducible nitric oxide synthase and arginase-1 expression occur in immature myeloid cells, which inhibit T-cell proliferation and lead to the induction of T-cell apoptosis [105]. Along with other infiltrating cells like tumor-associated macrophages (TAMs), M2-like TAMs, neutrophils, conventional myeloid dendritic cells (DCs), and regulatory DCs, MDSCs are attracted to cancer-associated Fn. These cells significantly influence tumor-promoting inflammation, facilitating angiogenesis, invasion, and metastasis [30]. Fn promotes the expansion of myeloid-derived immune cells in CRC. Chemokines such as MCP-1, IL-8, human granulocyte chemotactic protein-2 (huGCP-2/CXCL6), and macrophage inflammatory protein-1α (MIP-1α/CCL3) are produced by CRC and other tumors, acting as activators for neutrophils and potent chemoattractants [106–108]. Furthermore, a recent study has shown that Fn increases the expression of CXCL8 and reduces the expression of CXCL10 in the HT29 cell line. As a result, this alternation could be an Fn tool to modify tumor proliferation, invasion, and chemoresistance [109]. TGF-β secreted by Fn stimulates tumor-associated neutrophils to differentiate into pro-tumor N2 phenotypes, promoting tumorigenesis. Additionally, increased reactive oxygen species contribute to DNA impairment and tumorigenesis [110–112]. Contrary to previous beliefs, neutrophils, initially considered protective against tumor cells, promote tumor growth, angiogenesis, invasion, and metastasis in CRC and other cancers [80, 113]. In vitro co-culture assays have shown that Fn can stimulate macrophage activation [80]. In Apc^Min/+ mice, Fn triggers M2 polarization of macrophages in the microenvironment through TLR4-dependent signaling, thereby promoting tumorigenesis [114, 115].TAMs, which exhibit similar characteristics to M2 macrophages, play essential roles in controlling inflammation responses, adaptive immunity, tissue remodeling, and promoting neovascularity [116]. The presence of TAMs in the cancer microenvironment is associated with increased cancer proliferation and metastasis probability, indicating a poor prognosis [80]. Moreover, CD4⁺ T helper cell-mediated immune responses are suppressed by myeloid-derived cells, such as MDSCs. When comparing Fn-enriched tumors to normal tissues, the density of CD4⁺ T-cells is much lower, indicating that Fn has an immunosuppressive effect [117]. Fn has evolved through interactions with both human cells and tissues, as well as oral bacteria. Its long rod structure allows it to bind with a variety of microbial cells, including Streptococcus sanguinis [118]. Furthermore. a single Fn can bond with up to 10 S. sanguinis cells. Fn’s biological activity facilitates biofilm formation and interactions with host cells [87, 119]. Additionally, Fn commonly co-occurs with Campylobacter spp., a key gastrointestinal pathogen, in cancer tissues [119].

CRC chemotherapy

CRC treatments include surgery, radiotherapy, and chemotherapy, which can be selected based on the specific characteristics and progression of the disease [120]. Chemotherapy is a therapeutic strategy that uses several drugs or combinations to inhibit the development of cancer cells [121]. Chemotherapeutic drugs disrupt cell division and reproduction, inhibiting growth factors, inducing cell death, preventing new blood vessel formation, cutting off nutrients, and inhibiting DNA or RNA formation by imitating nucleotides known as antimetabolites, which are most effective during the S phase of the cell cycle [121, 122]. The approved medications for treating CRC include irinotecan, fluoropyrimidines, oxaliplatin, trifluridine/tipiracil, 5-fluorouracil (5-FU), and capecitabine, which are often used as chemotherapeutic agents [123]. Irinotecan prevents topoisomerase I, which impairs DNA replication and transcription, resulting in cell death [124]. Oxaliplatin, a platinum-based compound, inhibits DNA replication and transcription by creating intrastrand connections between neighboring guanine residues or guanine and adenine [125]. Trifluridine/tipiracil, commonly known as TAS-102, is an orally active medication containing a thymidine-based nucleoside analog (trifluridine) and a thymidine phosphorylase inhibitor (tipiracil hydrochloride) [126]. Trifluridine, a thymidine derivative, acts as the cytotoxic component of trifluridine/tipiracil, primarily exerting its antitumor activity attributed to its integration into DNA. Thymidine kinase phosphorylates it to form trifluridine triphosphate, which inhibits cell proliferation and tumor growth. Tipiracil is included in the formulation for its capacity to impede thymidine phosphorylase [126]. Fluoropyrimidines include 5-FU and capecitabine. Capecitabine, an oral prodrug, converts to 5-FU in cancer cells via thymidine phosphorylase, which inhibits thymidylate synthase and incorporates it into RNA and DNA [127]. 5-FU primarily exerts its anticancer effects by inhibiting thymidylate synthase, disrupting the intracellular deoxynucleotide pools necessary for DNA replication. It also disrupts RNA synthesis during anabolism and gets incorporated into DNA, which causes fragmentation [128].

CRC patients were frequently treated with 5-FU and capecitabine in combination with platinum-based chemotherapy, which proved to be initially effective. However, many patients eventually develop resistance to these medications and unfortunately succumb due to a lack of effective treatment [14, 129, 130]. The complex interaction between the environment and genetic control contributed to CRC chemoresistance. Recent studies have demonstrated that the gut microbiota can influence local immune responses, affecting both immunotherapy and chemotherapy [14, 131–134]. The hypothesis that specific components of the microbiota could inhibit the antitumor activity of chemotherapeutic medicines is intriguing, considering that earlier animal studies have revealed a favorable function of the gut microbiota in enhancing cancer treatment [135, 136]. For instance, a recent study found a substantial reduction in the anticancer impact of oxaliplatin in germ-free or antibiotic-treated animals [131]. Certain bacteria, such as Lactobacillus johnsonii, Lactobacillus murinus, and Bacteroidetes, have been found to improve chemotherapy effectiveness [133, 134]. However, some bacteria, like Fn, seem to induce strong chemoresistance [137, 138].

Fusobacterium nucleatum and chemoresistance

Despite significant developments in cancer treatment, resistance to therapy remains a major challenge [139]. Chemoresistance to CRC is a multifaceted process influenced by intrinsic and extrinsic variables such as gene regulation, hypoxia, epigenetic modification, and gut microbiota [128, 137, 140]. The role of Fn in the development and progression of CRC has been studied [141]. In particular, Fn is known to cause chemoresistance, affecting how patients respond to therapy for the disease [79]. Fn enhances chemoresistance in CRC by regulating several cellular mechanisms. It also reduces CRC cell chemosensitivity to 5-FU by increasing baculoviral IAP repeat containing 3 (BIRC3). Inhibitor of apoptosis proteins (IAPs) are a family of proteins defined by the presence of baculoviral IAP repeat (BIR) domains. These BIR domains are crucial because they enable IAPs to bind and inhibit caspases [142–144]. They can increase tumor cell survival and induce chemotherapy resistance [15].

Furthermore, high levels of Fn are associated with chemoresistance in advanced CRC patients receiving 5-FU-based adjuvant chemotherapy after radical resection [15]. Fn significantly induces BIRC3 expression in CRC cell lines, possibly related to chemoresistance [15, 145]. BIRC3, part of the inhibitor of apoptosis proteins (IAPs) family, can prevent apoptosis by blocking the caspase cascade [15, 146]. Previous studies have shown that Fn activates the TLR4/NF-κB pathway in CRC cells [15, 147, 148]. NF-κB activation increases the expression of many target genes, including BIRC3 [149]. Based on the results, the TLR4/NF-κB pathway may be implicated in the Fn-induced elevation of BIRC3 (Fig. 1D) [15].

The discovery that Fn can induce autophagy in CRC cells during chemotherapy has significant implications for CRC treatment [136]. Autophagy is a cellular process that breaks down damaged components to help maintain balance within the cell. While it was once believed to be primarily associated with cell death, recent research has revealed its essential function in promoting cell survival during stressful situations such as hypoxia, nutrient deprivation, oxidative stress, DNA damage, chemotherapy, and infections by intracellular pathogens. Autophagy is crucial for recycling cellular materials, eliminating microbes, and regulating innate and adaptive immune responses [150]. Autophagy can enable cancer cells to survive chemotherapy [151, 152]. Fn increases microtubule-associated protein light chain 3 (LC3)-II expression, autophagic flux, and autophagosome formation in CRC cells, thereby promoting the expression of autophagy-related proteins, including unc-51 like autophagy activating kinase 1 (ULK1), pULK1, and Autophagy Related 7 (ATG7) [153]. ATG7 catalyzes the binding of phosphatidylethanolamine (PE) to LC3-I, transforming it into LC3-II, which is required for autophagosome membrane expansions and specific cellular component identification [154–156].

However, it appears that Fn does not affect the transcription level of ULK1 or ATG7. However, miR-18a* and miR-4802 target ULK1 and ATG7, respectively, and are preferentially lost during Fn co-culture. These miRNAs can impact CRC chemoresistance both in vitro and in vivo. Typically, these miRNAs decrease the expression of these genes by targeting their 3’-untranslated regions (3’UTR) [157]. Therefore, Fn may mediate chemoresistance by specifically targeting certain autophagy components and microRNAs [145, 153]. Fn causes the reduction of miR-18a* and miR-4802 through the TLR4 and MyD88 signaling pathways [153]. It is suggested that Fn exposure stimulates the TLR4 and MyD88 signaling pathways, downregulating the synthesis of miR-18a and miR-4802. This shift causes apoptosis to be replaced by autophagy and chemoresistance in cancer cells [26]. Autophagy inhibitors might be used with standard chemotherapy to improve therapeutic success by overcoming the resistance given by Fn [153].

Fn may have a biological role in developing CRC chemoresistance through the Anoctamin-1 (ANO1) pathway [14]. The ANO1 gene codes for the human chloride channel protein ANO1, which is often overexpressed in some human carcinoma types, such as colorectal, prostate, breast, gastrointestinal stromal tumor, and esophageal squamous cell carcinoma [14, 158, 159]. It has been found that ANO1 promotes carcinogenesis and invasion by triggering the mitogen-activated protein kinase (MAPK) signaling pathway [160]. In addition to increasing MAPK signaling, ANO1 interacts with growth factor receptors like the epidermal growth factor receptor (EGFR) and modifies apoptotic pathways, enabling cancer cells to resist apoptosis caused by therapeutic drugs [161–163]. Additionally, by enhancing the expression of matrix metalloproteinases (MMPs) and factors linked to the epithelial-mesenchymal transition (EMT), ANO1’s activation of the MAPK pathway enhances the invasive characteristics of CRC cells, enabling tumor cells to invade adjacent tissues and possibly dissemination [163–165]. ANO1 inhibits apoptosis in CRC cells subjected to chemotherapy. The presence of ANO1 enables cancer cells to survive the apoptotic signals received by these chemotherapy drugs [14].

Apoptosis is a highly regulated biological process that is essential for cell proliferation and the development of tissues [166]. Apoptosis loss can lead to tumor development, growth, and progression [14, 167]. 5-FU and oxaliplatin can trigger apoptosis in tumor cells by disrupting and inhibiting DNA replication [168]. However, this effect may be reduced when tumor cells are co-cultured with Fn [14]. Lu et al. found that Fn-induced ANO1 expression and ANO1 overexpression can counteract the apoptotic effects caused by oxaliplatin and 5-FU. This suggests that Fn inhibits apoptosis in CRC through the ANO1 pathway (Fig. 2B). The possible mechanism could involve the regulation of miRNA levels [14]. ANO1 is a target of miR-132 that plays an essential role in CRC progression, and Fn can prevent apoptosis by selective loss of miR-18a* and miR-4802 [14, 169]. Functionally, miR-132 causes CRC cells to endure apoptosis and suppresses cell division. Research has demonstrated that miR-132 functions in tumor suppression by causing growth arrest and elevated apoptosis rates when induced to express itself in CRC cell lines [170]. Consequently, the possible role of Fn in CRC chemoresistance via the ANO1 pathway seems to make traditional chemotherapy approaches such as capecitabine and oxaliplatin ineffective for patients with high Fn levels [14]. ANO1 plays a major role in the progression of CRC, so targeting it may offer a promising therapeutic approach. It may disrupt its channel activity and downstream signaling effects and act as a biomarker to identify patients at high risk for CRC who could help from additional aggressive therapy approaches [14, 165, 171].

Fig. 2.

The potential role of Fn in chemoresistance. A: Fn may enhance the expression of CD44 and CD133, which are associated with the development of cancer stem cells. B: Fn-induced ANO1 expression might counteract the apoptotic effects of oxaliplatin and 5-FU

Furthermore, ferroptosis is a form of cell death that depends on iron accumulation and lipid peroxidation in cells [172]. A recent study reported that Fn can induce oxaliplatin resistance by inhibition of ferroptosis. Fn triggers the expression of glutathione peroxidase 4 (GPX4) via the E-cadherin/β-catenin/TCF4 pathway. GPX4 plays an important role in avoiding ferroptosis by converting lipid hydroperoxides to nontoxic forms. High amounts of this enzyme can protect cells against oxidative stress, preventing ferroptotic cell death [173, 174]. This procedure is associated with oxaliplatin-induced ferroptosis, eventually resulting in chemoresistance in CRC [175].

As is, Fn may indirectly enhance CRC chemoresistance via other microenvironmental factors. Cancer-associated fibroblasts (CAF), which appear to be naturally chemo-resistant to cisplatin, produce exosomes to enhance CRC growth and chemotherapy resistance [80]. CAF is a key component in TME, helping tumor cells communicate and maintain a supportive network [176, 177]. These cells have several origins, including epithelial or endothelial cells, cancer stem cells, and adipocytes, although they are mainly produced from normal fibroblasts (NF) during tumor inflammation [177, 178]. CAF promotes cancer progression by secreting various growth factors and proinflammatory cytokines, including TGF-β, IL-6, VEGF, and CXCL-12 [177, 179, 180]. In Addition, by secreting IL-6, CRC-conditioned macrophages decreased drug-induced apoptosis and enhanced CRC chemoresistance; this effect may be countered using an anti-IL-6 antibody [80].

Previous research has also shown that they play a role in metastasis, therapeutic resistance, angiogenesis regulation, metabolism, immune cell modulation, and the expression or induction of inhibitory checkpoints like cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) [177, 181–183]. Various components of Fn may have diverse effects on fibroblast cells, and CAF cells may mediate some of the bacterium’s action in the TME [177].

Fn may promote chemoresistance in CRC by increasing the percentage of cancer stem cells [80]. cancer stem cells are a type of cancer cell that exhibits stem cell-like characteristics such as self-renewal, differentiation, and the potential to initiate and maintain tumor development. These cells are also accountable for developing chemoresistance in cancer [184]. Fn may promote CRC cells to express more stem cell markers like CD44 and CD133, which are linked to the development of cancer stem cells (Fig. 2A) [80, 145]. Furthermore, Fn may enhance the self-renewal of CRC stem cells (CCSCs) and induce non-CCSCs to develop stem cell-like characteristics [147, 185]. Fn regulates this process by manipulating cellular lipid accumulation. It decreases lipid accumulation in CCSCs by increasing fatty acid oxidation, which promotes self-renewal [185]. On the other hand, Fn promotes lipid accumulation in non-CCSCs by increasing fatty acid synthesis. The lipids accumulate as droplets [185]. The accumulation of lipid droplets in tumor cells has been related to cancer development and drug resistance [186]. Therefore, Fn abundance is associated with increased CCSC population within cancer cells. This has been associated with cancer initiation, metastasis, and resistance [185]. Fn levels in surgically removed tumor tissues might predict treatment outcomes. While chemotherapeutic medicines primarily destroy differentiated cells, they have minimal impact on CSCs, often leading to cancer recurrence [187, 188].Targeting CSCs may increase therapy effectiveness by blocking essential stemness pathways or targeting markers such as CD44 and CD133. Modifying lipid metabolism may damage CSCs’ defensive systems, making them susceptible to chemotherapy. Combining conventional chemotherapy with medicines that target CSCs or impair lipid metabolism may enhance therapeutic results for CRC patients resistant to standard therapies [189–192].

Given Fn’s critical involvement in cancer development and resistance, it is anticipated that Fn-targeted therapies will introduce a new paradigm in cancer treatment. As a result, combining chemotherapy with anti-Fn medication may be a promising approach for CRC patients with elevated levels of Fn [185, 193].

Inhibition of chemoresistance induced by Fusobacterium

It has been observed that over 30% of patients with stage II and III colon cancer who undergo surgery develop resistance to 5-FU-based chemotherapy throughout an 8-year follow-up period [137]. Approximately 50% of patients who suffer from metastatic CRC are resistant to 5-FU-based chemotherapy, with a 5-year survival rate below 15%. As a result, it is essential to understand the molecular processes behind CRC chemoresistance and create innovative and efficient ways to increase chemosensitivity [137, 194]. As mentioned, Fn abundance is linked to CRC properties like tumorigenesis, development, metastasis, and recurrence, with abnormal proliferation causing CRC chemoresistance and recurrence [27, 153].

Broad-spectrum antibiotics can harm the healthy gut microbiota. Several studies have shown that antibiotic therapy can lead to the development of dysbiotic microbiota. This imbalance reduces the generation of microbiota-derived ROS, inhibiting the tumor response to oxaliplatin therapy and resulting in less tumor cell death [195]. Furthermore, this treatment strategy significantly reduces the efficacy of immunostimulatory medicines (CpG, cyclophosphamide) and platinum-based chemotherapy [196]. As a result, it seems that targeting the tumor with bacteria is a promising technique for tumor-selective therapy; however, because of a lack of antibacterial drugs specific to Fn and the bacterium’s chemical resistance, this remains a problematic issue in practical practice [87]. Given the limited effectiveness of current tumor treatments, it is important to consider the role of Fn in biofilm production and its synergistic interactions with bacteria such as Escherichia coli and Clostridium, which are known to metabolize irinotecan [196–198]. These interactions lead to increased toxicity and reduced drug efficacy. Therefore, there is an urgent need for the development of new drug strategies that focus on reducing toxicity while enhancing drug permeability [196]. Furthermore, it is worth noting that bacteria can be both enemies and friends in chemotherapy. For instance, the non-enterotoxigenic strain of Bacteroides fragilis can improve oxaliplatin effectiveness, but an enterotoxigenic strain causes CRC [199].

Metformin has been shown to reduce Fn-induced carcinogenesis in a previous study [200]. Another study investigated the impact of metformin on Fn-induced chemoresistance. Surprisingly, metformin inhibited Fn-induced chemoresistance in xenograft mice and CRC cells [137]. They revealed that metformin and Fn influence the stemness of CRC cells. Metformin reduced the expression of stemness-related proteins NANOG and SOX2, while also inhibiting tumorsphere development in CRC cells cocultured with Fn. This suggests that metformin reduces Fn-induced chemoresistance by reducing the stemness in CRC cells. They also investigated how metformin controls Fn-induced stemness in CRC cells. Moreover, the sonic hedgehog pathway, associated with stem cell self-renewal and chemoresistance, was discovered to be a connection between metformin- and Fn-mediated stemness. Metformin suppressed Fn-induced stemness, signaling of the sonic hedgehog pathway, and chemoresistance through a mechanism that dependent on miR-361-5p. This study demonstrates that metformin inhibits the MYC/miR 361-5p/sonic hedgehog signaling pathway, altering Fn-induced stemness and CRC chemoresistance [137, 201].

The octapeptide Jelleine-I, extracted from honeybee royal jelly, has shown promising biological activity and minimal toxicity [202, 203]. It has been found to effectively prevent CRC growth by targeting Fn, a key player in CRC formation. The halogenated derivative Br-J I efficiently decreases Fn load, colon inflammation, and Fn-induced CRC development, indicating its potential for cancer therapy [204]. A study investigated Br-J-I’s antimicrobial mechanism and revealed that it damages the bacterial cell membrane, killing Fn by triggering cell membrane disruption, which results in the formation of protrusions and disassembling the bacterial membrane [205]. In addition, combining Br-J-I with 5-FU decreases cell viability by over 40%, indicating that Br-J-I sensitizes 5-FU’s cytotoxicity and synergizes with 5-FU to improve antitumor activity. Consequently, Br-J-I might be an effective supplementary treatment for CRC and help reduce chemoresistance [205]. Furthermore, the tumor-targeted nano assembly described by Li et al. has been shown as a potential therapeutic agent for improving CRC therapy by targeting and removing intratumoral Fn [206]. The nano assembly consists of two main components: oxaliplatin prodrug-modified polyglycidyl ether (PP) and oligomethyleneimine (OLP) [206]. The nano assembly targets explicitly tumor cells, guiding cellular uptake and effectively eliminating extracellular and intracellular Fn. Moreover, its biocompatibility and efficient antibacterial activity make it a promising candidate for CRC treatment [206].

Zheng et al. presented a gut microbiota-modulating therapeutic based on phage-guided biotic abiotic hybrid nanomaterials [207]. The researchers developed a phage strain from human saliva capable of specifically lysing Fn. Then, they enclosed irinotecan into dextran nanoparticles (DNPs), resulting in irinotecan-loaded DNPs. Finally, using a bioorthogonal process, they covalently connected IDNPs to azide-modified phages (A-phages), resulting in a phage-guided biotic-abiotic hybrid nanosystem [207, 208]. In vivo investigations were subsequently performed to show that A-phages accumulated in CRC tumors and that oral administration of the nanosystem removed intra-tumor Fn, which suggests potential therapeutic methods for malignancies with Fn abundance [207, 208].

Fap2 and TIGIT/TLR4 are potential targets due to their role in Fn enrichment, antitumor immunity, and chemoresistance. An Fn-directed vaccination targeting FomA (an outer membrane protein produced by Fn that plays a role in bacterial biofilm formation) that creates an immune response has previously been studied [208, 209]. However, data on the incidence of CRC after getting the vaccination are currently missing. Furthermore, Brennan et al. contended that, even if vaccination might trigger specific types of immune responses like T cell responses, some Fn strains can evade the immune-killing impact in the intracellular phase [27, 208, 210]. Alternatively, T cell-inducing vaccinations, such as those used to treat malaria and tuberculosis, may create a more practical approach for Fn. Microbial ecosystem replacement, which employs consortia of engineered microorganisms or tailored cocktails of human-derived isolates, might be another alternative for altering the tumoral microbiota that may harbor Fn enrichment. This method is currently being investigated in clinical trials with Clostridium difficile and may be utilized in the future to exclude Fn [208].

The interaction between Fn and Annexin A1 appears to contribute to chemotherapy resistance, metastatic progression, and poor prognostic outcomes in CRC. Consequently, Annexin A1 emerges as a promising candidate for therapeutic intervention aimed at inhibiting Wnt/β-catenin signaling. Unlike E-cadherin, which is widely expressed and causes significant challenges as a therapeutic target, Annexin A1 exhibits increased and selective expression in proliferative cancer cells. This characteristic positions it as a viable therapeutic target with minimal adverse side effects [211, 212]. Zhang S et al. reported that BIRC3 was shown to be the most upregulated gene in CRC cells via the TLR4/NF-κB signaling pathway after Fn infection. Furthermore, in vitro and in vivo studies have shown that Fn infection decreases the chemosensitivity of these cancer cells to 5-FU. Moreover, a substantial correlation exists between high Fn frequency and chemoresistance in advanced CRC patients receiving routine adjuvant treatment with 5-FU following radical resection. The study’s findings imply that targeting Fn and BIRC3 might be a viable therapeutic strategy for reducing chemoresistance to 5-FU in advanced CRC, necessitating more investigation into their roles as possible clinical targets [5, 213].

In addition, miRNAs, such as hsa-miR-515-5p, can be delivered by intestinal epithelial cells to influence Fn proliferation. Alternate miR-515-5p expression in CRC patients may disrupt the balance, potentially affecting Fn proliferation and responsiveness to chemotherapeutic treatments and prognosis [214]. Therefore, gaining a thorough understanding of the mechanisms and potential of miRNAs involved in cancer development and progression is crucial for therapeutic goals.

Conclusion

Fn contributes significantly to tumor proliferation and chemoresistance in CRC patients. It is commonly found in CRC tissues and is associated with a poor prognosis and treatment failure. Fn affects the process of autophagy, targeting the TLR4 and MyD88 innate immune signaling pathways, and it also alters the response to chemotherapy. In addition, it has been demonstrated that Fn is associated with the inflammatory factors that create an inflammatory tumor microenvironment promoting cancer spreading. Additionally, it affects endothelial cells, and the resulting increase in VEGF levels could lead to CRC metastasis. Explore the current molecular mechanisms and underscore the importance of understanding how Fn induces chemoresistance could offer valuable insights for developing targeted medications to improve treatment outcomes. Chemoresistance presents a significant challenge in cancer treatment. While the role of Fn in inducing chemoresistance has been reported, further research is needed to fully understand the molecular mechanisms involved. Developing new strategies and supplemental therapies to reduce chemoresistance by interfering with Fn-induced processes is of the utmost importance.

Author contributions

AK conceived and designed the study. AK, ZE, and LD wrote the paper. AS, SR, and AzK participated in manuscript editing. Notably, all authors have reviewed and approved the manuscript.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

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Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.