TIMI 2136 No. of Pages 14
Trends in
Microbiology
Review
Fusobacterium nucleatum, a key pathogenic
factor and microbial biomarker for
colorectal cancer
Ni Wang1,2,3,4,5 and Jing-Yuan Fang
1,2,3,4,5,
*
Colorectal cancer (CRC), one of the most prevalent cancers, has complex etiology.
The dysbiosis of intestinal bacteria has been highlighted as an important contributor
to CRC. Fusobacterium nucleatum, an oral anaerobic opportunistic pathogen,
is enriched in both stools and tumor tissues of patients with CRC. Therefore,
F. nucleatum is considered to be a risk factor for CRC. This review summarizes
the biological characteristics and the mechanisms underlying the regulatory
behavior of F. nucleatum in the tumorigenesis and progression of CRC.
F. nucleatum as a marker for the early warning and prognostic prediction of
CRC, and as a target for prevention and treatment, is also described.
Highlights
Fusobacterium nucleatum has been related to genetic and epigenetic lesions,
such as microsatellite instability (MSI),
CpG island methylator phenotype
(CIMP), and genome mutation in colorectal cancer (CRC) tissues.
F. nucleatum could promote the proliferation and metabolism, remodel the immune microenvironment, and facilitate
metastasis and chemoresistance in the
tumorigenesis and development of CRC.
CRC and gut microbiota
F. nucleatum could function as a biomarker for screening the high-risk population for CRC.
CRC has become the third most commonly diagnosed cancer and the third leading cause
of cancer death worldwide [1,2]. The initiation and progression of CRC is caused by genetic
factors and environmental factors, among which the gut microbiota is a special environmental
risk factor [3].
The human microbiome is estimated to comprise 100 trillion cells, ten times as many as human
cells, which encode 100 times more unique genes than the human genome [4]. Most microorganisms exist in the gut and play vital roles in host immunity and nutrition [5]. Hence, gut microbial
dysbiosis (see Glossary) might change the physiological function of the host and induce
diseases [6]. With the development of next-generation sequencing [7], a new perspective
has been gained on the pathogenesis of CRC. In 2012, two genomic analyses of the CRC
microbiome simultaneously reported that F. nucleatum, a common oral anaerobic bacillus, was
evidently enriched in carcinoma tissues [8,9]. F. nucleatum is an opportunistic pathogen with
the potential to function as a scaffold that binds to other oral colonizers (Box 1). Previous studies
revealed that F. nucleatum is involved in the initiation, progression, and chemoresistance of
human CRC [10,11].
The origin, transport, and colonization of F. nucleatum
The microbial communities in the mouth could anatomically connect with those in the colon via
saliva; however, in healthy adults, the microbiomes in the oral cavity and distal gut are highly
distinct [12]. F. nucleatum, as a common oral colonizer, could be detected in CRC tissues
[13]. This aroused the interest of researchers to investigate the source of F. nucleatum in
CRC tissues.
Similar coabundance networks were detected in both oral and gut microbiota datasets of individuals
with CRC [14]. The same F. nucleatum could be isolated from the colorectal and saliva samples
of the same patients with CRC, suggesting that F. nucleatum in the colorectum was derived from
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Although further in-depth studies are warranted, several virulence- or phage-based
therapeutics specific to F. nucleatum may
be novel promising methods in CRC
treatment.
1
Division of Gastroenterology and
Hepatology, Shanghai Jiao Tong
University, Shanghai, China
2
Shanghai Institute of Digestive Disease,
Shanghai Jiao Tong University, Shanghai,
China
3
NHC Key Laboratory of Digestive
Diseases, Shanghai Jiao Tong University,
Shanghai, China
4
State Key Laboratory for Oncogenes
and Related Genes, Shanghai Jiao Tong
University, Shanghai, China
5
Renji Hospital, School of Medicine,
Shanghai Jiao Tong University, Shanghai,
China
*Correspondence:
jingyuanfang@sjtu.edu.cn (J.-Y. Fang).
https://doi.org/10.1016/j.tim.2022.08.010
© 2022 Elsevier Ltd. All rights reserved.
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Box 1. Biological characteristics of F. nucleatum as an opportunistic pathogen
Fusobacteriota, an understudied phylum of bacteria, comprises two families: Leptotrichiaceae and Fusobacteriaceae. The
latter includes the genus Fusobacterium, which are Gram-negative, non-spore-forming, spindle-shaped obligate
anaerobes [25]. Among these species, Fusobacterium nucleatum resides mainly on the human oral mucosa in both
healthy and diseased individuals [112].
With a long rod shape, F. nucleatum has the capacity to adhere to many other oral microorganisms in the oral cavity. When
exposed to a microenvironment with Streptococcus sanguinis, a single F. nucleatum cell can interact with up to ten cells of
S. sanguinis in a corncob formation [113]. F. nucleatum has been isolated from both supragingival and subgingival
polymicrobial biofilms, and it participates in the development of dental plaque communities, which has been implicated
in the etiology of periodontitis [114]. As an opportunistic pathogen, outgrowing F. nucleatum stimulates Porphyromonas
gingivalis, an oral low-abundance keystone pathogen with a community-wide impact, to disrupt host homeostasis and
induce periodontitis [115]. In a dental plaque biofilm, F. nucleatum serves as a supportive bridge between primary colonizers (such as Streptococcus species) and anaerobic secondary colonizers (such as P. gingivalis), and as a physiological
bridge that provides anaerobic microenvironments for strict anaerobe coaggregation [116,117]. In addition to periodontal
diseases, F. nucleatum has also been isolated from patients with adverse pregnancy, IBD, appendicitis, rheumatoid
arthritis, cardiovascular disease, Lemierre's syndrome, respiratory tract infection, Alzheimer's disease, and, especially,
CRC [118]. Regarded as opportunistic and tumor-associated pathogens, F. nucleatum has been at the forefront of
scientific attention.
the oral cavity [15]. Within individual CRC tumors, the co-occurrence of Fusobacterium and its
associated – typically oral Gram-negative anaerobes (such as Prevotella, Bacteroides,
Leptotrichia, Selenomonas, and Campylobacter species) – also demonstrated that
Fusobacterium isolated from CRC tissues has an oral origin [16].
Recently, most literature demonstrated that enteral transmission is the predominant route for
CRC-tissue colonization of oral F. nucleatum. F. nucleatum, and the fusobacterial virulence
gene Fusobacterium adhesin A (FadA), are reported to have significantly increased abundance
in stool samples of patients with CRC [17,18]. In situations where the oral–intestinal barrier is
damaged, such as low stomach acid, the oral flora can be transferred to the intestine, and vice
versa. The distinct fusobacterial community diversity in matching saliva and gastrointestinal
aspirates from patients with inflammatory bowel disease (IBD) suggests selective Fusobacterium
translocation through the gastrointestinal tract [19].
Furthermore, the localization of intravenously injected F. nucleatum to CRC tissues verified the
transmission of fusobacteria to colon adenocarcinomas through the circulation [20,21]. However,
F. nucleatum is not an original inhabitant of mice, and infrequent administration of F. nucleatum
through oral gavage without prior antibiotic use makes it difficult for F. nucleatum to easily colonize mice while competing with resident bacteria. Moreover, previous studies have shown that
the establishment of a CRC mouse model by using high-frequency oral gavage of F. nucleatum
is faster and more stable than tail-vein injection. Therefore, the role of circulatory translocation
might need to be further verified in humans.
Fusobacterial Fap2, a galactose adhesion hemagglutinin, has been verified to mediate
F. nucleatum colonization and invasion of CRC cells by binding to the tumor-overexpressed
host factor galactose-N-acetyl-D-galactosamine (Gal-GalNAc) [20,22]. In addition, purified
recombinant protein of fusobacterial virulence factor FadA has been verified to bind and invade
host cells, thus stimulating CRC growth [10,23]. Intriguingly, under stress and disease conditions,
amyloid-like FadA could confer acid tolerance and further assist the gastrointestinal translocation
and colonization of F. nucleatum [24]. Meng et al. also showed that Fap2 was required for
amyloid-like FadA production, suggesting that the phenomenon caused by a fap2 mutant
might be partly caused by lack of amyloid FadA [24].
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Glossary
Coaggregation: the specific adhesive
interactions between diverse bacterial
species that contribute to facilitate the
existence of dental multispecies biofilms.
CpG island methylator phenotype
(CIMP): a cancer-specific CpG island
with high degrees of methylation in a
subset of tumors with epigenetic
instability.
Dysbiosis: an imbalance of the
microbiome structure, which is related to
the function of the microbial community
and multifactorial human diseases.
Fecal microbiota transplantation
(FMT): restoration of eubiosis of the
patient’s digestive tract by transplanting
a healthy microbiome, thus alleviating
various digestive disorders and even
other nongastrointestinal diseases.
Lipopolysaccharide (LPS): the major
component of the outer membrane of
Gram-negative bacteria, which could
activate the host innate immune
responses.
Microsatellite instability (MSI): the
spontaneous gain or loss of nucleotides
from repetitive DNA microsatellite tracts,
the spontaneous gain or loss of
nucleotides from repetitive DNA
microsatellite tracts, that is, the
molecular fingerprint of a deficient
mismatch repair system.
Myeloid-derived suppressor cells
(MDSCs): immature myeloid cells that
are activated by tumor-derived
cytokines and have shown potent
immune suppressive functions.
Next-generation sequencing:
technologies that allow for thousands to
billions of DNA fragments to be
massively parallel sequenced or deep
sequenced.
Virulence factor: the component of a
pathogen that impairs the physiological
function of the host.
Trends in Microbiology
F. nucleatum in CRC pathogenesis
Increasing evidence shows that tumor-enriched F. nucleatum is involved in multiple stages of
CRC progression [10,20,25,26]. Further research proposed various models, such as the
alpha-bug hypothesis and the driver-passenger model, to illustrate the mechanism by which
F. nucleatum promotes CRC [27,28] (Box 2). In this section, we review the current understanding
of the role of F. nucleatum in the occurrence and development of CRC.
F. nucleatum promotes the proliferation and metabolism of CRC
In a mouse model with ApcMin/+, a mutation predisposing the mouse to spontaneous CRC [29],
oral instillation of F. nucleatum could potently facilitate adenocarcinomas, adenomas, and small
intestinal aberrant crypt foci [30]. Further research showed that F. nucleatum could promote
tumorigenesis by stimulating proliferation and metabolism in CRC cells (Figure 1) [10,31].
The fusobacterial virulence factor, FadA, was identified to bind to E-cadherin for host–epithelial
cell attachment, which is followed by triggering the Wnt/β-catenin pathway, thus provoking
oncogenic and inflammatory responses [10]. The FadA-deletion mutant was defective in inducing
colonic tumors in ApcMin/+ mice, demonstrating the driver role of FadA in tumorigenesis [23].
Annexin A1 is the Wnt/β-catenin signaling modulator that is involved in activation of Cyclin D1.
Annexin A1 was confirmed as an important component for FadA to exert its stimulatory effect,
which was demonstrated in ApcMin/+ mice [23]. Moreover, another study demonstrated that
recombinant FadA stimulated the proliferation of the CRC cell line SW480 in a time- and dosedependent manner [32]. Lipopolysaccharide (LPS) of F. nucleatum could trigger β-catenin
through the Toll-like receptor 4 (TLR4)/phosphor-p21-activated kinase 1 (PAK1) cascade in
CRC cells [33]. LPS also activates TLR4 signaling to myeloid differentiation primary response
88 (MYD88), resulting in the activation of nuclear factor kappa B (NF-κB) and increasing expression
of microRNA (miRNA) miR-21. Additionally, miR-21, an oncogene involved in colitis-associated CRC
[34], reduces the expression of RASA1 (encoding the RAS GTPase RAS P21protein activator 1) and
provokes the RAS-mitogen-activated protein kinase (MAPK) pathway, thus inducing S-phase
accumulation and enhancing the proliferation of CRC cells [26].
Most cancer cells rely on aerobic glycolysis to supply energy for tumor growth, rather than oxidative phosphorylation, which is a recognized hallmark termed the Warburg effect. F. nucleatum
Box 2. The bacterial hypothesis models for CRC
Based on their work on the capacity of enterotoxigenic Bacteroides fragilis (ETBF) to induce CRC in multiple intestinal
neoplasia (Min) mutation mice, Sears et al. provided a hypothesis named the ‘alpha-bug hypothesis’ [27]. In this
hypothesis, alpha-bugs are not merely directly carcinogenic alone, but also remodel the gut microbiome composition to aid
its induction of intestinal epithelial cell (IEC) mutations and intestinal immune responses, permitting oncogenic transformation.
In addition, alpha-bugs and their helpers might predispose the host to carcinogenesis via selectively ‘crowding out’ the
cancer-protective intestinal microbiota [27]. Subsequently, with developments in next-generation sequencing, Tjalsma et al.
proposed a bacterial ‘driver–passenger’ model which has been incorporated into the genetic paradigm of the CRC process
[28]. Bacterial drivers are certain members of the indigenous colonic microbiome that provoke IEC DNA damage and CRC
initiation. After that, colorectal carcinogenesis alters the intestinal milieu, thus facilitating the colonization and proliferation of
bacterial passengers, such as Fusobacterium spp. [28]. Notably, in contrast to sustainable cancerous mutations in genomes,
the driver bacteria initiating colorectal tumorigenesis would be gradually outcompeted by passenger bacteria that further
regulate tumorigenesis [28].
The driver bacteria portion of the driver–passenger model is similar to, but different from, the alpha-bug hypothesis [119].
The driver–passenger model states that CRC-associated bacterial drivers (which could be considered as alpha-bugs and
their helpers) would not always colonize and are eventually replaced by the passenger bacteria because of changes in the
tumor growth microenvironment, while the alpha-bug hypothesis assumes that the bacterial drivers will continue to localize to
growing tumors. Therefore, the researchers supporting the driver–passenger model suggested that driver and passenger
bacteria have a significant temporal correlation with CRC tissues and might play roles in the pathogenesis of CRC.
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Figure 1. Possible molecular mechanisms of Fusobacterium nucleatum in colorectal cancer (CRC)
carcinogenesis. Fusobacterial lipopolysaccharide (LPS) could trigger β-catenin and NF-κB expression through TLR4.
FadA also activates the Wnt/β-catenin pathway via binding to E-cadherin. Then, Fap2 could mediate F. nucleatum
colonization in CRC tissues through binding to the host factor Gal-GalNAc. These pathogen–host interactions are involved
in the carcinogenesis of CRC. Moreover, F. nucleatum could induce genetic and epigenetic lesions in CRC. F. nucleatum
promotes CIMP and induces DNA damage in CRC tissues. F. nucleatum also upregulates H3K27ac-targeting genes
ENO1 and ANGPTL4. These lesions facilitate the metabolism and proliferation of F. nucleatum-infected CRC cells.
Abbreviations: CIMP, CpG island methylator phenotype; DNMT, DNA methyltransferase; ERK, extracellular signalregulated kinase; FadA, Fusobacterium adhesin A; IECs, intestinal epithelial cells; TSGs, tumor suppressor genes.
infection is reported to activate glycolysis and carcinogenesis by upregulating H3K27ac-targeting
of the genes ENO1 (encoding enolase 1) and ANGPTL4 (encoding angiopoietin like 4) in CRC
cells [31,35].
F. nucleatum reprograms the immune microenvironment of CRC
In 2013, the capacity of F. nucleatum to modulate a protumorigenic inflammatory milieu for intestinal tumorigenesis was first reported [30]. F. nucleatum was demonstrated to activate the NF-κB
pathway to upregulate the expression of proinflammatory cytokines (cyclooxygenase 2 (COX-2),
tumor necrosis factor (TNF), interleukin (IL)-6, IL-8, and IL-1β), and selectively recruits myeloidderived immune cells, such as myeloid-derived suppressor cells (MDSCs), tumor-associated
neutrophils (TANs), tumor-associated macrophages (TAMs and M2-like TAMs), and dendritic cells
(DCs), thereby constructing an immune microenvironment and potentiating tumor progression
[30]. The mechanisms of immune microenvironment construction by F. nucleatum in CRC were
reported in subsequent studies (Figure 2).
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(A)
(B)
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Figure 2. Fusobacterium nucleatum reconstructs the tumor milieu in colorectal cancer (CRC) cells and immune
cells. (A) In CRC cells, F. nucleatum activates the NF-κB pathway to upregulate proinflammatory cytokines and trigger M2
macrophage polarization. F. nucleatum also recruits myeloid-derived immune cells (such as MDSCs, TANs, TAMs, and DCs).
(B) In addition, F. nucleatum inhibits the functions of human immune cells through binding TIGIT (via Fap2) and CEACAM1
(via CbpF). F. nucleatum also induces lymphocytic apoptosis via Fap2 and RadD. Additionally, F. nucleatum could create a
kynurenine-enriched toxic environment inside immune cells through tryptophan metabolism. Abbreviations: DCs, dendritic
cells; MDSCs, myeloid-derived suppressor cells; NK cells, natural killer cells; TAMs, tumor-associated macrophages; TANs,
tumor-associated neutrophils; TIGIT, T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain.
F. nucleatum creates a proinflammatory tumor milieu
Accumulating evidence has shown that inflammation plays a critical role in tumor progression.
Chronic colonic inflammation, such as Crohn’s disease (CD) and ulcerative colitis (UC), has
been widely identified as a risk factor for CRC [36]. In particular, continuous host low-grade
inflammation resulting from intestinal microbiota stimulation has been verified to contribute to
the tumorigenesis and development of CRC [37].
Through the presumed interaction of fusobacterial Fap2 and host Gal-GalNAc, F. nucleatum
could invade CRC cells to induce the secretion of IL-8 and C-X-C motif chemokine ligand 1
(CXCL1). These proinflammatory cytokines recruit neighboring immune cells and further promote
them to secrete their own cytokines (CXCL2, C-C motif chemokine ligand 3 (CCL3), and TNFα) to
remodel the tumor microenvironment [20,22]. Additionally, with F. nucleatum-induced adaptive
immune responses, increased levels of IgA and IgG antibodies against F. nucleatum are detected
in the serum of patients with CRC [38,39].
In recent years, immune checkpoint therapy, which activates the antitumor immune response by
inhibiting the interaction between T cell inhibitory receptors and their ligands, has been used
successfully to treat a variety of malignant tumors [40]. The most widely used immune checkpoints
inhibitors, such as antiprogrammed cell death 1 (PD-1), play an important role in immunotherapy
research [41]. Moreover, the intestinal microbiota could influence the efficacy of PD-1/
programmed cell death 1 ligand 1 (PD-L1)-mediated antitumor immunotherapy [42]. Qin et al.
demonstrated that F. nucleatum could activate stimulator of interferon response CGAMP interactor
1 (STING) signaling to upregulate expression of PD-L1 in CRC cells, thereby enhancing the
therapeutic effect of PD-L1 blockade [11].
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F. nucleatum inhibits anticancer immune responses
Besides the creation of a proinflammatory microenvironment, F. nucleatum also remodels the
tumor milieu via immune escape [43]. Several studies validated that the enrichment level of
F. nucleatum in CRC tissues correlates inversely with the number of tumor-infiltrating lymphocytes (TILs), thus resulting in antitumoral immune suppression [43,44]. Fusobacterial persistence
post-neoadjuvant chemoradiotherapy (nCRT) correlates with increased relapse rates in patients
with locally advanced rectal cancer (LARC), potentially arising from immune cytotoxicity suppression induced by a lack of CD8+ T cells [45].
MDSCs are tumor-permissive myeloid cells with strong immunosuppressive activity [46]. F. nucleatum
recruits MDSCs to construct the tumor microenvironment [30]. In two independent cohort studies, the
inverse association of F. nucleatum and CD3+ T cell density in CRC tissues supported the above
conclusion [43,47]. Interestingly, the presence of F. nucleatum in CRC liver metastases is associated
with reduced numbers of CD8+ T cells and increased numbers of MDSCs [48,49].
Strikingly, M2-like TAMs exhibit protumoral functions, including control of the inflammatory
response and adaptive immunity [50]. F. nucleatum triggers M2 macrophage polarization and
stimulates CRC growth in a TLR4-dependent manner involving activation of the IL-6/p-signal
transducer and activator of transcription 3 (STAT3)/c-MYC pathway and the NF-κB/ S100
calcium-binding protein A9 (S100A9) pathway [51,52]. F. nucleatum also promotes M2 macrophage
polarization and infiltration to enhance CRC metastasis by activating the NF-κB/miR-1322/CCL20
cascade [53].
F. nucleatum could dampen the tumor-killing function of natural killer (NK) cells via the binding of
fusobacterial Fap2 to the human T-cell immunoglobulin and immunoreceptor tyrosine-based
inhibitory motif domain (TIGIT) [54]. TIGIT, an inhibitory receptor expressed on many immune cells,
could dampen the cytotoxic function, thus protecting F. nucleatum and adjacent CRC cells from immune cell attack [55]. Recent studies showed that, in the immune evasion mechanism, F. nucleatum
utilizes surface trimeric autotransporter adhesin CbpF to bind and activate another human inhibitory
receptor, CEA cell adhesion molecule 1 (CEACAM1), thus inhibiting the functions of T cells and NK
cells [56,57]. F. nucleatum also induced lymphocytic apoptosis via Fap2, thereby increasing tumor
growth [58,59]. Besides Fap2, RadD is another F. nucleatum outer-membrane protein that could induce cell death of human lymphocytes [59]. Additionally, intracellular F. nucleatum triggers tryptophan metabolism to create a kynurenine-enriched toxic environment inside macrophages,
thus escaping attack by cytotoxic T lymphocytes (CTLs) [60].
F. nucleatum induces genetic and epigenetic lesions in CRC
In addition to the common specific genetic mutations in TP53 (encoding tumor protein P53), KRAS
(encoding Kirsten rat sarcoma viral oncogene homolog), and Apc (encoding adenomatous
polyposis coli protein) genes, epigenetic alterations, including promoter DNA and RNA methylation,
and histone methylation and acetylation, are also common causes of CRC [61]. Accumulating
evidence indicates that there are numerous links between F. nucleatum and CRC genetics and
epigenetics, covering the whole range of CRC occurrence and development (Figures 1–3).
Epidemiological associations suggested that increased levels of F. nucleatum promote
microsatellite instability (MSI), CpG island methylator phenotype (CIMP), and genome
mutations in CRC tissues [15,62]. In contrast to low-level CIMP (CIMP-low, CIMP2), typical
high-level CIMP (CIMP-high, CIMP1) CRCs are associated with MSI through epigenetic silencing
of a mismatch repair gene, MLH1 (encoding MutL homolog 1), which could affect DNA damage
[63,64]. Indeed, studies suggested that F. nucleatum could induce DNA damage, thus leading to
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(B)
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Figure 3. The potential mechanism underlying the promotion of Fusobacterium nucleatum in colorectal cancer (CRC) metastasis and
chemoresistance. (A) The Fap2-Gal-GalNAc- and LPS-TLR4-interactions could activate YAP/METTL3 and the NF-κB signaling pathway, followed by upregulation of
KIF26B, KRT7, and 12, 13-EPOME, as well as inhibition of the phosphorylation of ERK, thus stimulating CRC metastasis. Moreover, various miRNAs (including miR4474, miR-4717, and miR-21) are involved in CRC metastasis. (B) Fusobacterial LPS could interplay with TLR4, thus activating the NF-κB signaling pathway and
inhibiting downstream miR-18a* and miR-4802, resulting in upregulation of ULK-1 and ATG7, respectively. Consequently, the apoptosis of CRC cells is inhibited,
which leads to CRC chemoresistance. Moreover, ANO1 in F. nucleatum-infected cells could inhibit apoptosis. Abbreviations: CREBBP, CREB-binding protein; 12,13EPOME, the 12,13-cis epoxide form of linoleic acid; ERK, extracellular signal-regulated kinase; YAP, yes-associated protein.
tumor growth and inflammation in CRC [65,66]. Furthermore, the correlations between increased
F. nucleatum levels and the tumor localization in the proximal colorectum, CIMP, and MSI, were
corroborated in a subsequent investigation using 16S rRNA sequencing of mucosal or fecal
F. nucleatum in patients with CRC [67]. F. nucleatum was confirmed to increase DNMT (encoding
DNA methyltransferase) expression and mediate promoter hypermethylation of tumor suppressor genes (TSGs), thereby turning off their expression [68]. In accordance with the above results,
intratumoral F. nucleatum enrichment was reported to correlate with promoter CpG island
hypermethylation of CDKN2A (encoding cyclin-dependent kinase inhibitor 2A) in MSI-high
CRCs [69]. Recently, N6-methyladenosine (m6A) methylation in RNA has been identified to be
related to tumor progression [70]. F. nucleatum was revealed to induce the downregulation of
methyltransferase-like 3 (METTL3) via the yes-associated protein (YAP)/forkhead box D3
(FOXD3) cascade, resulting in the overexpression of KIF26B (encoding kinesin family member 26B)
and enhanced metastasis of CRC cells [71].
Sequencing experiments showed that F. nucleatum infection was related to histone modification
genes, and remodeling of chromatin states in human intestinal epithelial cells (IECs) [72]. Besides
methylation, histone acetylation also plays important roles in the development of CRC with
bacterial exposure. F. nucleatum activates the long noncoding RNA (lncRNA) ENO1-IT1-lysine
acetyltransferase 7 (KAT7, a histone acetyltransferase) axis to stimulate acetylation of histone
H3 lysine 27 and ENO1 expression, subsequently inducing glycolysis in CRC [31]. Moreover,
H3K27ac-induced ANGPTL4 overexpression was observed in CRC cells with F. nucleatum
infection [35]. Additionally, Y. Yang et al. proposed that miR-21 is regulated by the TLR4/MYD88/
NF-κB axis to promote proliferation and invasion in CRC cells with F. nucleatum infection [26].
Other miRNAs, such as miR-1322, miR-4474, miR-4717, and exosomal miR-1246/92b-3p/
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27a-3p, also play essential roles in the tumorigenesis and development of CRC via posttranscriptional regulation [34,73].
F. nucleatum promotes the metastasis and chemoresistance of CRC
The presence of invasive Fusobacterium has been detected in hepatic and lymphatic metastases
arising from primary human CRC [8,9,20,74]. The overabundance of Fusobacterium in CRC has
a persistently positive association with regional lymph node metastasis and distant metastases
(Figure 3A) [9,74,75]. F. nucleatum stimulates epithelial–mesenchymal transition (EMT) and
metastasis in CRC via activation of a cytochrome P450 (CYP2J2)/12,13-EPOME (the 12,13-cis
epoxide form of linoleic acid) axis through TLR4/Kelch like ECH associated protein 1 (KEAP1)/
nuclear factor, erythroid 2 like 2 (NRF2) pathway [76]. In addition, infection with F. nucleatum
could activate the NF-κB pathway, thus upregulating lncKRT7-AS/keratin 7 (KRT7) to promote
CRC metastasis [77]. F. nucleatum-induced inhibition of m6A modifications could contribute to
CRC aggressiveness via the YAP/FOXD3/METTL3/KIF26B axis [71]. Through upregulation of
CARD-containing IL-1β ICE-kinase (CARD3), F. nucleatum infection could also activate autophagy
to mediate CRC metastasis [78]. Moreover, Fap2-dependent F. nucleatum colonization and
invasion of CRC cells could stimulate the secretion of IL-8 and CXCL1, which could function as
a metastatic signal to accelerate CRC cell migration [20,22]. Various miRNAs have also been confirmed to be involved in CRC metastases. F. nucleatum could accelerate the invasion of CRC cells
through the miR-1322/CCL20, TLR4/MYD88/miR21 axis, miR-4474 and miR-4717/CREB-binding protein (CREBBP) axis [26,53,79]. F. nucleatum-infected cells might deliver exosomes that selectively carry miR-1246/92b-3p/27a-3p and CXCL16/RhoA/IL-8 to noninfected cells to enhance
prometastatic behavior [80].
Currently, the chemotherapeutic drugs 5-fluorouracil (5-FU) and oxaliplatin are widely used to
treat CRC. However, chemotherapy resistance has been a major contributor to CRC recurrence
and poor patient outcome. The enrichment of F. nucleatum is detected in patients with recrudescent CRC after chemotherapy, which suggests the potential promotion by F. nucleatum of CRC
chemoresistance (Figure 3B) [11]. F. nucleatum might function in CRC via the TLR4/MYD88
innate immune response and selectively downregulate miR-18a* and miR-4802 levels, thus
activating the autophagy pathway and inducing chemotherapy failure [11]. F. nucleatum infection
could upregulate baculoviral IAP repeat containing 3 (BIRC3) via TLR4/NF-κB signaling, consequently mediating chemosensitivity to 5-FU in CRC. Interestingly, Annexin A1 expression has
also been reported to be associated with 5-FU resistance in CRC, which could be upregulated
via F. nucleatum infection [23,81]. In addition, the abundance of F. nucleatum correlates with
drug resistance in patients with advanced CRC treated with standard 5-Fu-based adjuvant
chemotherapy after radical surgery [82]. Moreover, upregulated anoctamin 1 (ANO1) in
F. nucleatum-infected cells could prevent apoptosis induced by oxaliplatin or 5-FU [83].
The potential clinical value of F. nucleatum in CRC
F. nucleatum is a marker for early warning, early diagnosis and prognosis prediction of CRC
Effective screening biomarkers for the early detection of premalignant lesions or cancers would
significantly reduce CRC-related mortality [84]. Microbiome based biomarkers may be employed
as screening and prognostic methods for CRC. High levels of F. nucleatum are observed to exist
in preneoplastic and neoplastic tissues [30,85,86], and F. nucleatum levels are higher in CRCs
than in premalignant lesions and correlate with CRC pathological stage [75,87]. Ito et al. revealed
that F. nucleatum increased according to histological grade of colorectal neoplasia and was more
frequently associated with CIMP-high premalignant lesions, suggesting the contribution of
F. nucleatum to the progression of colorectal neoplasia [86]. Moreover, F. nucleatum is also
significantly more prevalent within proximal hyperplastic polyps (HPs) and sessile serrated
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adenomas (SSAs) compared with that within proximal and distal traditional adenomas (TAs)
[86,88]. In SSAs, F. nucleatum positivity gradually increases from the sigmoid colon to the
cecum [86]. Collectively, these data indicated the potential of F. nucleatum as a risk factor in
screening high-risk populations and as a pathogenic bacterium in the early warning of CRC.
As noted above, previous studies have suggested the potential of F. nucleatum as a biomarker in
CRC; therefore, the next step is to discuss methods to effectively measure F. nucleatum. As an
alternative to traditional invasive colonoscopy, noninvasive cancer biomarker identification can
significantly reduce patient discomfort. Fecal occult blood testing (FOBT), a noninvasive screening test, is used widely to screen for CRC [89]. Considering that fecal occult blood might be a
precursor of many other diseases in addition to CRC, FOBT has relatively low sensitivity and
specificity. Significantly high fecal F. nucleatum DNA levels are consistently found in patients
with CRC and premalignant lesions [30,90,91]. A recent review and meta-analysis described
the detection of enriched F. nucleatum in stools from colorectal adenoma and carcinoma patients
via quantitative PCR [92]. The specificity and sensitivity of FOBT can be improved by combining it
with fecal quantification of F. nucleatum to diagnose CRC [93]. Moreover, an altered ratio of
F. nucleatum to probiotics Faecalibacterium prausnitzii and Bifidobacterium was found in CRC
fecal samples, which might also be valuable for screening early CRC [90].
The fecal immunochemical test (FIT) is another recommended noninvasive CRC screening
method; however, it has the limitation of low sensitivity to detect advanced neoplasia. Nevertheless,
FIT is reported to be more predictive for detecting colonic lesions in combination with a microbiotabased model [94]. The stable fecal microbial load is higher in FIT samples of CRC and high-grade
dysplasia, suggesting the potential importance of microbiota assessment in FIT screening [95].
Combining FIT with F. nucleatum showed significantly superior sensitivity and specificity than FIT
alone to detect CRC, especially for advanced neoplasia, which detected advanced lesions missed
by FIT alone [96].
In addition of quantitative PCR detection of F. nucleatum DNA levels, novel antibody-based
detection methods might be more conducive to CRC screening. Immune assays based on
serum detection, such as IgA or IgG antibodies against F. nucleatum, might be used as CRC
screening tools [38,97]. The levels of F. nucleatum IgA and IgG antibodies from patients with
cancer were higher than those in matched controls. Combined with carcinoembryonic antigen
(CEA) and carbohydrate antigen 19-9 (CA19-9), anti-F. nucleatum-IgA in sera could have the
potential to detect early CRC [38]. However, considering the confounding factors, such as prior
history of other diversified fusobacterial infections, the diagnostic capabilities of these serum
antibodies should be verified by more clinical studies.
Enriched fecal F. nucleatum is an independent risk factor for metachronous adenomas after
endoscopic polypectomy [91]. Accumulating research has illustrated the significant positive
relationship between F. nucleatum abundance and poor overall survival of patients with CRC in
several independent cohorts [62,98]. After CRC surgery, high levels of F. nucleatum in patients
with CRC are similarly associated with a worse prognosis [99]. The fusobacterial presence
post-nCRT is also related to a high recurrence rate of LARC [45]. The above results imply that
F. nucleatum is a candidate prognostic biomarker for CRC.
F. nucleatum as a therapeutic target of CRC
Based on the vital role of F. nucleatum in CRC, F. nucleatum-targeted treatment has been
validated as a new therapeutic strategy for CRC (Figure 4). Several antibiotics, such as metronidazole and β-lactams, could be effective approaches to eradicate F. nucleatum [100,101]. In
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Trends in Microbiology
Figure 4. Colorectal cancer (CRC) bacteriotherapy through targeting Fusobacterium nucleatum. Several antibiotics (such as metronidazole and β-lactams)
could eradicate F. nucleatum effectively. Besides antibiotics, nonsteroidal anti-inflammatory drugs (NSAIDs, such as aspirin) could also reduce the F. nucleatum
abundance in CRC tissues. The phage-based biotic/abiotic hybrid system and virulence-based therapeutics targeting F. nucleatum might be used to achieve precise
F. nucleatum eradication. Through remodeling intestinal eubiosis by transplanting a healthy microbiome, fecal microbiota transplantation (FMT) might represent a
promising approach to treat CRC. In addition, dietary intervention, probiotics, prebiotics, and postbiotics have also shown promise in CRC treatment by reconstructing
the gut bacteria community.
particular, metronidazole treatment has been verified to eliminate the Fusobacterium burden to
suppress tumor volumes in mice bearing patient-derived CRC xenografts, indicating the
effectiveness of antibiotics in treating Fusobacterium-colonized CRC [74]. Intriguingly, besides
antibiotics, aspirin, a nonsteroidal anti-inflammatory drug (NSAID) that modulates COX-2 to
repress prostaglandin biosynthesis, has also been shown to prevent and manage CRC, not
only via its chemopreventive effects but also by reducing the F. nucleatum abundance in CRC
tissues [102]. Nevertheless, in addition to the issue of antibiotic resistance, interference with
antitumor therapy caused by the attenuating effect of these interventions on other bacteria also
needs to be considered. Hence, novel narrow-spectrum antibiotics specific to F. nucleatum
might help to avoid these problems.
An 11-aa inhibitory peptide derived from the FadA-binding site in E-cadherin was reported to prevent F. nucleatum from binding to CRC cells in vitro, as well as colonizing xenograft CRC tumor
[10]. The anti-FadA monoclonal antibody was also shown to inhibit F. nucleatum binding to
CRC cells in vitro [24]. In addition, by targeting FomA, another outer-membrane protein of
F. nucleatum that mediates adhesion, halitosis vaccines were reported to inhibit bacterial
coaggregation and biofilm formation [103]. However, whether it would reduce the incidence
10
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Trends in Microbiology
of CRC is unknown. Therefore, further in-depth studies are warranted to validate the clinical
values of these virulence-based therapeutics in CRC.
Moreover, given the exquisite selectivity of phages, phage-based methods have been considered
as a promising novel approach [104]. The phage-guided irinotecan-loaded nanoparticles, which
target the tumor colonized Fusobacteriota, have been shown to regulate CRC growth [105].
Furthermore, specifically F. nucleatum-binding phage surface-attached silver nanoparticles
were constructed subsequently. This phage-based biotic/abiotic hybrid system was verified to
achieve precise F. nucleatum eradication and reduce MDSC amplification for tumor-immune
therapy [106].
Fecal microbiota transplantation (FMT) is another option that functions by remodeling the
intestinal microenvironment. Previous studies have shown that FMT could obtain a high cure
rate in treating Clostridium difficile infection (CDI), especially in the patients with recurrent and
refractory disease [107]. Therefore, considering its efficacy, FMT might be a promising approach
to treat CRC, although further in-depth studies are required.
In addition, several other strategies, including dietary intervention, probiotics, prebiotics, and
postbiotics, have also been shown to be promising in CRC treatments by targeting intestinal
bacteria. Dietary intervention, such as higher-fiber diets, might be considered as the most
effective and economical method to treat CRC [108]. Probiotics might aid the prevention and
treatment of CRC by repressing colonization of pathogenic bacteria, modulating colonic immunity, and enhancing gut barrier function [109]. Prebiotics are nondigestible food ingredients that
selectively stimulate beneficial bacterial species, thus improving host health [110]. Postbiotics
are microbial-derived biomolecules with antioxidant, antiproliferative, anti-inflammatory, and anticancer effects [111]. In summary, F. nucleatum might be a therapeutic target in CRC, while more
approaches targeting F. nucleatum should be investigated and applied clinically.
Outstanding questions
What is the causal relationship
between colonization of F. nucleatum
and initiation of CRC?
In addition to membrane proteins
reported so far (such as FadA and
Fap2), which other virulence factor
of F. nucleatum can mediate the
interaction between F. nucleatum
and CRC cells to promote tumor
tumorigenesis and progression?
What are the specific ways and
mechanisms by which F. nucleatum
transports and colonizes from primary
to metastatic sites of CRC?
Could F. nucleatum as well as its
virulence factors be the markers of
diagnosis and prognosis of CRC?
What are the underlying mechanisms
by which F. nucleatum adhesion leads
to a poorer prognosis of CRC?
Could these novel treatments targeting
virulence factors of F. nucleatum be
utilized to reduce the incidence of CRC
via specific control of F. nucleatum?
Concluding remarks
Studies suggest that the intestinal microbiota, once considered as a ‘forgotten organ’, plays
critical roles in the pathogenesis of many human diseases, especially CRC. F. nucleatum can
change from a harmless common oral bacterium to a pathogenic one through interacting with
other microorganisms and human cells. In particular, F. nucleatum, recruited by tumor cells,
could in turn help cancer cells to reconstruct the immune microenvironment to promote the
development of tumor. These findings indicate that F. nucleatum might function as a diagnostic
biomarker, prognostic predictor, and therapeutic target in CRC. However, the subgroup of
F. nucleatum with the most significantly increased abundance in CRC needs to be further
identified. The complex interaction between F. nucleatum and other microorganisms might
contribute to a multispecies community in CRC development. The binding of F. nucleatum
virulence proteins and corresponding CRC receptors could guide the preparation of novel and
specific treatments for CRC. There are still many challenges to be overcome in clinical application
because of the wide varieties of bacteria, cross-species transformation, tumor progression, and
individual differences in patients (see Outstanding questions). Further in-depth investigation
combined with preclinical models are urgently needed to better reveal the comprehensive biological
characteristics of F. nucleatum in humans as well as the causal relationship between F. nucleatum
and human CRC.
Acknowledgments
We thank all patients and individuals for their participation in our study. This project was supported in part by grants from the
National Key R&D Program of China (2020YFA0509200), National Natural Science Foundation of China (81830081,
Trends in Microbiology, Month 2022, Vol. xx, No. xx
11
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31970718), Shanghai Municipal Health Commission, Collaborative Innovation Cluster Project (2019CXJQ02), Clinical
Research Plan of SHDC (SHDC2020CR1034B), Shanghai Sailing Program (21YF1425600) and Innovative research team
of high-level local universities in Shanghai.
Declaration of interests
The authors declare no competing interest.
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