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Article https://doi.org/10.1038/s41467-024-45572-w
Fusobacterium nucleatum promotes tumor
progression in KRAS p.G12D-mutant
colorectal cancer by binding to DHX15
Huiyuan Zhu
1,6,7
,ManLi
1,6
,DexiBi
1
, Huiqiong Yang
1
, Yaohui Gao
1
,
Feifei Song
1
, Jiayi Zheng
1
, Ruting Xie
1
, Youhua Zhang
1
,HuLiu
1
,XuebingYan
2
,
Cheng Kong
3
, Yefei Zhu
4
,QianXu
4
,QingWei
1,7
& Huanlong Qin
4,5,7
Fusobacterium nucleatum (F. nucleatum) promotes intestinal tumor growth
and its relative abundance varies greatly among patients with CRC, suggesting
the presence of unknown, individual-specific effectors in F. nucleatum-
dependent carcinogenesis. Here, we identify that F. nucleatum is enriched
preferentially in KRAS p.G12D mutant CRC tumor tissues and contributes to
colorectal tumorigenesis in Villin-Cre/KrasG12D+/- mice. Additionally, Para-
bacteroides distasonis (P. distasonis)competeswithF. nucleatum in the G12D
mouse model and human CRC tissues with the KRAS mutation. Orally gavaged
P. distasonis in mice alleviates the F. nucleatum-dependent CRC progression.
F. nucleatum invades intestinal epithelial cells and binds to DHX15, a protein of
RNA helicase family expressed on CRC tumor cells, mechanistically involving
ERK/STAT3 signaling. Knock out of Dhx15 in Villin-Cre/KrasG12D+/- mice attenu-
ates the CRC phenotype. These findings reveal that the oncogenic effect of
F. nucleatum depends on somatic genetics and gut microbial ecology and
indicate that personalized modulation of the gut microbiota may provide a
more targeted strategy for CRC treatment.
Colorectal cancer is the second most common malignancy worldwide
and is characterized by specific somatic mutations including lesions in
oncogenes, tumor suppressor genes, and DNA repair-related genes1,2.
According to The Cancer Genome Atlas (TCGA) database, APC,TP53,
KRAS,andSMAD4 are the four most frequently mutated genes in
patients with CRC, and their alterations often exhibit prognostic
relevance3. For example, KRAS mutations are found in about half of the
human CRC cases and recognized as critical determinants of ther-
apeutic response4.InKRAS, the codon 12 of exon 2 is the most pre-
valent site of mutation in CRC (mainly p.G12D). As a clinical interest,
there are currently no drugs that can effectively treat the KRAS p.G12D-
expressing tissues5.
In addition to genetic mutations, other local factors, most notably
gut microbiota, also influence CRC occurrence and progression6.
Recent studies have shown that the genotoxic pathogenicity island in
Escherichia coli causes a distinct mutational signature in clonal
organoids7and that a gut microbiota-generated metabolite, gallic acid,
switches mutant p53 from tumor-suppressive to oncogenic8.These
observations suggest that in the pathogenesis of CRC, there are cross-
talks between gut microbiota and local somatic genotypes.
Received: 24 July 2023
Accepted: 26 January 2024
Check for updates
1
Department of Pathology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China.
2
Department of Oncology,
Yangzhou University Medical College Affiliated Hospital, Yangzhou 225000, China.
3
Department of Colorectal Surgery, Fudan University Shanghai Cancer
Center, Shanghai 200032, China.
4
Research Institute of Intestinal Diseases, Tongji University School of Medicine, Shanghai 200072, China.
5
Department of
Gastrointestinal Surgery, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai 200072, China.
6
These authors contributed
equally: Huiyuan Zhu, Man Li.
7
These authors jointly supervised this work: Huiyuan Zhu, Qing Wei, Huanlong Qin. e-mail: 1801022@tongji.edu.cn;
weiqing1971@tongji.edu.cn;qinhuanlong@tongji.edu.cn
Nature Communications | (2024) 15:1688 1
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Cross-cohort meta-analyses have captured CRC-associated gut
bacteria that were consistently identified in the cancer population
spanning different continents9,10, and they include Fusobacterium
nucleatum,Parvimonas micra,Gemella morbillorum,andPeptos-
treptococcus stomatis. Although the etiologic connection remains
elusive for many of the convergent microbial signatures, consider-
able knowledge has been gained on the versatile roles of F. nucle-
atum. Studies have shown that F. nucleatum aggravates CRC
development and chemoresistance via multiple routes including
attachment to epithelial cells, modulation of immune micro-
environment, promotion of cell cycle, activation of TLR4 signaling,
and regulation of autophagy11–14. Whereas some gut microbes
potentiate CRC tumor growth, others play a beneficial role. A recent
mouse study involving a collection of 11 bacterial strains isolated
from healthy human donors showed that the microbial intervention
significantly improved the efficacy of anti-PD-1 treatment15. In addi-
tion, anti-CRC effect was also observed after introduction of single
gut microbes, such as Lactobacillus acidophilus16 and Parabacteroides
distasonis (P. distasonis)17.P. distasonis is defined as one of the 18
core members in the gut microbiota of human18 and thought to have
important physiological functions in host19. Although these results
provide the lead of substantial clinical interest, greater mechanistic
insights are needed to facilitate the translation of gut microbiota-
based findings.
Here, we attempt to elucidate the host and gut microbial factors
crucial for F. nucleatum-dependent CRC progression. Our findings
indicate that the oncogenic role of F. nucleatum is influenced by the
genotype of somatic tissue, a putative host RNA helicase, and a F.
nucleatum-competing gut bacterium. The results illustrate the intri-
cacy of local effectors in modulating F. nucleatum-dependent CRC
development, which may inform a more effective, personalized inter-
vention for the disease.
Results
F. nucleatum is enriched preferentially in KRAS p.G12D-mutant
CRC tissues
To investigate the correlation between F. nucleatum abundance and
CRC somatic mutations, we performed quantitative polymerase chain
reaction (qPCR) and exon sequencing on 24 pairs of tumor and mat-
ched paratumor tissues. The tumor samples were divided into wild-
type (WT) tissues and mutation-bearing tissues based on exon
sequencing results. Subsequent analysis revealed that F. nucleatum
abundance in the KRAS mutant tissues was higher than that in the KRAS
WT tissues and paratumor tissues (Fig. 1a), which was validated in an
independent cohort of 239 CRC patients (CRC patients information are
shown in Supplementary Table 1) ( Fig. 1b). In comparison, F. nucleatum
abundance exhibited noassociation with mutation in four testedgenes
namely APC,TP53,MSH2,andSMAD4.
Fig. 1 | F. nucleatum is enriched preferentially in KRAS p.G12D-mutant CRC
tumor tissues. a Exon sequencing and qPCR detection of the mutation status of
CRC tissues and F. nucleatum abundance in the indicated group. The relative
abundance of F. nucleatum was confirmed using abundance of F. nucleatum
/quantity of pgt gene. Significant differences are indicated: one-way ANOVA with
Sidak’s multiple comparison test, data are presented as the mean ± SEM.
bCorrelation of KRAS mutation status and F. nucleatum abundance in CRC tumor
tissues. Significant differences areindicated: Chi-squaretest, two-sided,n=239.cF.
nucleatum (Fn) positive ratesunder different KRAS mutation types in tumor tissues
of CRC (n=239).dRelative abundance of F. nucleatum in the CRC patients of the
indicated groups. Significant differences are indicated: one-way ANOVA with
Bonferroni’s multiple comparison test, data are presented as the mean ± SEM.
Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-45572-w
Nature Communications | (2024) 15:1688 2
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Among patients with CRC, mutations in KRAS concentrate at
codons 12, 13, and 61 of exon 2 and vary greatly in prevalence20.To
examine whether F. nucleatum level correlated with particular genetic
variants, we analyzed the KRAS mutation profile and F. nucleatum
abundance in cohort 2 by qPCR. We found that F. nucleatum was
detected in 42/83 (50.6%)tumors with a KRAS mutation in exon 2, 7/19
(36.8%) tumors with a KRAS mutation in exon 3 and 43/137 (31.4%) in
KRAS WT tumors (Fig. 1c). Interestingly, the tumors with the p.G12D
mutation, but not those with the adjacent p.G13D mutation, accom-
modated significantly higher F. nucleatum abundance than WT tumors
(Fig. 1d). Collectively, our data bridge a local connection between F.
nucleatum abundance and KRAS genotype.
F. nucleatum exacerbates colorectal tumorigenesis in Villin-Cre/
KrasG12D+/−mice
To investigate the mechanistic underpinning of the observed F.
nucleatum-KRAS (p.G12D) link, we successfully generated Villin-Cre/
KrasG12D+/−mice (Supplementary Fig. 1a, b). We hypothesized that F.
nucleatum colonization would be increased in the colon of Villin-Cre/
KrasG12D+/−mice, thus aggravating tumorigenesis. To test this, we
developed an azoxymethane/dextran sulfate sodium (AOM/DSS)-
induced CRC mouse model (the mice were intraperitoneally injected
with 10 mg/kg AOM, followed by 5-day oral administration of 2.5% DSS
starting 5 days later) in both Villin-Cre/KrasG12D+/−and Villin-Cre/
KrasG12D−/−(WT) backgrounds and gavaged the mice with F. nucleatum
(1 × 109CFU) or phosphate-buffered saline (PBS) as shown in Fig. 2a.
qPCR and fluorescence in situ hybridization (FISH) assays revealed that
F. nucleatum was enriched in Villin-Cre/KrasG12D+/−colonic tissues
compared with that in the WT tissues (Fig. 2b–d) but undetectable in
the tissues of PBS-treated rodents, regardless of genetic backgrounds
(Supplementary Fig. 1c). Moreover, we showed that F. nucleatum-
treated Villin-Cre/KrasG12D+/−mice exhibited a pronounced augmenta-
tion in tumor formation compared with F. nucleatum-treated WT lit-
termates and PBS-treated animals (Fig. 2e). Hematoxylin and eosin
(H&E) staining revealed that F. nucleatum-treated Villin-Cre/KrasG12D+/−
mice exhibited higher grades of dysplasia (Fig. 2f), tumor multiplicities
and tumor loads than F. nucleatum-treated WT littermates (Fig. 2g–i).
Additionally, to evaluate the interaction of F. nucleatum with the
immune system in KRAS mutant mice and patients, immuno-
fluorescence (IF) and qPCR were performed to evaluate the infiltrated
immune cells and inflammatory cytokine production in colonic tissues
of PBS and F. nucleatum-treated Villin-Cre/KrasG12D+/−mice. We found
that F. nucleatum led to less CD3+Tcellsinfiltrating which is consisting
with the study that F. nucleatum was inversely associated with tumor
stromal CD3+lymphocytes (Supplementary Fig. 1d, e)21. However, there
were no significant differences in the number of infiltrated CD11c+
dendritic cells in colonic tissues between PBS and F. nucleatum-treated
Villin-Cre/KrasG12D+/−mice (Supplementary Fig. 1d, e). The expression of
Il-17a,Il-6 were also elevated after F. nucleatum treatment in Villin-Cre/
KrasG12D+/−mice (Supplementary Fig. 1f). And we confirmed the results
of infiltrated immune cells in tumor tissues from KRAS G12D-mutant
patients by IF (Supplementary Fig. 1g, h). Together, our results suggest
that F. nucleatum-mediated-colorectal tumorigenesis is aggravated in
mice harboring KRAS p.G12D mutant via its increased colonization.
P. distasonis competes with F. nucleatum in a Villin-Cre/
KrasG12D+/−mouse model and human KRAS mutant CRC tissues
Given that gut microbiota is a complex ecosystem, we were curious
about whether the connection between F. nucleatum and KRAS
(p.G12D) involved other bacteria. We first surveyed the microbiota
compositions of the colon tissues from WT and Villin-Cre/KrasG12D+/−
mice and discovered that Clostridium XIVa and Ralstonia were deple-
ted in the G12D animals (Supplementary Fig. 2a), although, no differ-
ences in the appearance, colon length and histological presentation of
the colon were observed between the two groups (Supplementary
Fig. 2b–d). These findings suggested thatthe p.G12D mutation affected
the gut microbiota, which could have ramifications in F. nucleatum-
related interactions. We therefore treated the mice with F. nucleatum
(1 × 109CFU) every two days for four weeks (Supplementary Fig. 2e)
and subsequently detected substantial compositional differences, all
depleted in Villin-Cre/KrasG12D+/−mice, between the two groups, com-
prising Parabacteroides,Roseburia,Akkermansia,Bacteroides,Blautia,
Clostridium XI,Anaerostipes,Allobaculum,Alistipes,andLactobacilus
(Fig. 3a). Next, we used the AOM/DSS mouse model to induce CRC
tumorigenesis in the two backgrounds, followed by F. nucleatum
exposure (Supplementary Fig. 2f). We found that Bacteroides,Para-
bacteroides,Alistipes, Clostridium XI,andIntestinimonas were aug-
mented in WT littermates compared to Villin-Cre/KrasG12D+/−mice
(Fig. 3b). Among the four genera (Parabecteroides,Bacteroides,Clos-
tridium XI,andAlistipes) exhibiting concordant alterations in both
mouse experiments, Parabecteroides manifested F. nucleatum-depen-
dent p.G12D depletion with the highest statistical significance, sug-
gesting prominent intermicrobial antagonism.
Parabacteroides species are diverse and P. distasonis,Para-
bacteroides merdae (P. merdae), and Parabacteroides goldsteinii (P.
goldsteinii) have been recognized as the main probiotics conferring
protection against CRC and metabolic disorders17,19,22,23.Toidentify
which species interacts with F. nucleatum and correlates with KRAS
mutation in CRC development, we examined the abundance of P.
distasonis,P. merdae and P. goldsteinii in the colon of AOM/DSS-
induced rodents gavaged with F. nucleatum. qPCR analysis showed
that P. distasonis, but not the other two Parabacteroides species,
registered greater enrichment in WT littermates when compared to
Villin-Cre/KrasG12D+/−mice (Fig. 3c). Next, we showed that the inverse
association between F. nucleatum andP. distasonis was found in human
tissues with KRAS mutations, but absent in KRAS WT clinical samples
(Fig. 3d,e).ByusingalogisticregressionmodelhavingP. distasonis as
an outcome with KRAS and F. nucleatum, and their interaction term
(KRAS xF. nucleatum) as exposures, we discovered that F. nucleatum
could be regarded as a protective factor for P. distasonis (OR = 0.548),
and the abundance of P. distasonis is negatively correlated with F.
nucleatum (p<0.05). However, no significant difference was observed
between the abundance of P. distasonis and KRAS mutation (Supple-
mentary Table 2). Overall, our data indicate that P. distasonis may
antagonize F. nucleatum during colorectal tumorigenesis in both mice
and humans.
P. distasonis alleviates F. nucleatum-mediated CRC progression
The above findings suggested a possibility that P. distasonis could
compete with F. nucleatum to mitigate CRC development. To verify this
hypothesis, we administered AOM/DSS-induced Villin-Cre/KrasG12D+/−
mice and WT littermates with F. nucleatum and/or P. distasonis (Sup-
plementary Fig. 3a). To fully assess the treatment effect of P. diastonis,
we administrated DSS for three cycles to ensure successful tumor
development. Examination of the colon tissues showed that P. dis-
tasonis gavage alleviated F. nucleatum-promoted CRC progression in
Villin-Cre/KrasG12D+/−mice (Fig. 4a, b). P. distasonis-treated mice devel-
oped dramatically fewer and smaller tumors than the F. nucleatum-or
PBS-treated counterparts; moreover, concurrent exposure to F. nucle-
atum and P. distasonis led to diminished size and number of tumors
compared to that derived from F. nucleatum-treated counterparts
(Fig. 4c–e). The biotherapeutic role of P. distasonis was corroborated
when we treated the clinically derived KRAS p.G12D and KRAS WT
colonic organoids with F. nucleatum and/or P. distasonis (Fig. 4f, g). In
addition, expression of Ki67 in the organoids was significantly
decreased in both P. distasonis-treated and P. distasonis/F. nucleatum-
co-treated groups, indicating that excessive multiplication of colonic
epithelial cells induced by F. nucleatum was suppressed in the presence
Article https://doi.org/10.1038/s41467-024-45572-w
Nature Communications | (2024) 15:1688 3
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of P. distasonis (Fig. 4h and i). Collectively, our data reveal that P. dis-
tasonis could alleviate F. nucleatum-mediated progression of CRC
tumors bearing the p.G12D mutation.
F. nucleatum invades tumor cells and binds to DHX15
Given the extensive involvement of F. nucleatum in CRC
development13,14,weassessedwhetherF. nucleatum can invade tumor
cells. We first successfully constructed two mutant cell lines expressing
KRAS p.G12D and p.G13D variants namely KRAS p.G12D and p.G13D
cells. Western blot analysis confirmed that the KRAS p.G12D cells were
successfully mutated (Supplementary Fig. 4a). Confocal fluorescence
microscopy showed that F. nucleatum invaded more in the KRAS
p.G12D cell line (on an average of 6.8/cell) than in KRAS p.G13D (on an
average of 2.94/cell) or KRAS WT cell line (on an average of 0.9/cell)
Fig. 2 | F. nucleatum exacerbates colorectal tumorigenesis in Villin-Cre/
KrasG12D+/−mice. a Schematic diagram of the experimental design and timeline of
mouse models. bqPCR analysis of F. nucleatum abundance in colonic tissues
derived from Villin-Cre/KrasG12D+/−(KrasG12D) mice and WT littermates (WT) treated
with AOM/DSS and F. nucleatum.Significant differences are indicated: two-tailed
Student’st-test, n= 5 (WT) and n=4 (KrasG12D) respectively, data are presented as
the mean ± SEM. cFISH detection of F. nucleatum in colonic tissues derived from
Villin-Cre/KrasG12D+/−mice and WT littermates using a Cy3-conjugated F. nucleatum
specific probe, the red arrows indicate F. nucleatum,n= 5, scale bar: 50 μm.
dStatistical analysis of the results in (c). Significant differences are indicated: two-
tailed Student’st-test, n=5 per group, data are presented as the mean ± SEM.
e,fRepresentative images and H&E stainings of the colons of WT littermates and
Villin-Cre/KrasG12D+/−mice treated with AOM/DSS and PBS or F. nucleatum, the red
arrows indicate tumors, n= 5 per group, scale bar: 50 μm. g–iTumor numbers,
tumor loads, and size of Villin-Cre/KrasG12D+/−mice and WT littermates treated with
AOM/DSS and PBS or F. nucleatum.Significant differences are indicated: one-way
ANOVA with Bonferroni’s multiple comparison test, n=5 per group, data are pre-
sented as the mean ± SEM. Data are representative of three independent experi-
ments. Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-45572-w
Nature Communications | (2024) 15:1688 4
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(Fig. 5a, b), suggesting that the observed F. nucleatum-p.G12D link
involved a difference in bacterial capacity to penetrate the epithelial
cells. Using cryo-focused ion beam milling and cryo-electron tomo-
graphy, we visualized invasion of F. nucleatum inside the cytoplasm of
infected G12D cells and confirmed that F. nucleatum contacted the
nuclear membrane (Fig. 5c). Meanwhile, transmission electron micro-
scope (TEM) experiments were performed on organoids derived from
KRAS p.G12D-mutant CRC patients and found F. nucleatum invaded
inside the cytoplasm of the infected organoids (Supplemen-
tary Fig. 4b).
The KRAS p.G12D peptide exhibits aberrant activation, which
accompanies tumorigenesis24. To clarify whether the activation of
KRAS p.G12D is linked to increased enrichment of F. nucleatum, clini-
cally derived KRAS p.G12D or WT colonic organoids were infected with
F. nucleatum, with or without incubation of KRAS p.G12D antibody.
FISH assay revealed that F. nucleatum display elevated invasiveness in
the KRAS p.G12D-mutant colonic organoids compared to that in the
WT organoids, which was attenuated after KRAS p.G12D-specificanti-
body incubation (Fig. 5d, e). To investigate whether the surface pro-
teins on KRAS p.G12D mutation that facilitated entry of F. nucleatum,
we performed western blotto evaluate the expression of tight junction
proteins. The reduction of tight junction proteins results in intestinal
epithelial barrier dysfunction and bacterial translocation25.Results
showed that KRAS p.G12D mutation cells expressed less ZO-1 and
Claudin-1 proteins when compared with KRAS p.G13D mutation and
KRAS WT cells(Supplementary Fig.4c) which may facilitate the entry of
F. nucleatum. These data suggested that the p.G12D mutation aug-
ments the bacterium-peptide affinity, leading to increased F. nucle-
atum invasion into the tumor cells.
Next, we aimed to identify the cellular component that F. nucle-
atum interacts with. We incubated F. nucleatum lysates with biotiny-
lated proteins from KRAS p.G12D, KRAS p.G13D, and KRAS WT cells
respectively. Subsequent western blot analysis showed that three
candidate proteins from F. nucleatum might interact with KRAS p.G12D
cells (Fig. 5f). The bands of interest were then submitted for mass
spectrometry (MS) protein identification, which reported three pro-
teins, FN0488 (glutamate dehydrogenase), FN1277 (aminoacyl-histi-
dine dipeptidase), and FN1859. Among them, FN1859 was the only
putative F. nucleatum surface protein according to the Uniprot data-
base (https://www.uniprot.org).
FN1859 from F. nucleatum might interact with a cognate receptor
on KRAS p.G12D cell. To capture the unknown effectors, we purified
recombinant His-tagged FN1859 proteins from Escherichia coli and
performed His-tag pull-down assays with WT, KRAS p.G13D, and KRAS
p.G12D cell lysates. Mass spectrometry revealed the level of DHX15 was
dramatically augmented in the KRAS p.G12D cell line compared with that
in the WT and KRAS p.G13D cell lines (Supplementary Table 3). DHX15 is
an important member of the DEAH-box RNA helicase family that is
expressed in the nucleus and contributes to carcinogenesis26.Totestthe
possible interaction between FN1859andDHX15,pull-downassaywas
conducted in the three cell lines of G12D, G13D, and WT. Western blot
analysis demonstrated that DHX15 exhibited specificcouplingtoFN1859
and that the protein-protein interaction was augmented in KRAS p.G12D
cells than that in the other two cell lines (Fig. 5g). The similar results were
observed in CRC samples from WT, KRAS p.G13D and KRAS p.G12D-
mutant patients, shown in Supplementary Fig. 4e. To clarify whether P.
distasonis involved in the interaction between F. nucleatum and DHX15,
TEM was performed to assess the invasive ability of P. distasonis to
Fig. 3 | P. distasonis competes with F. nucleatum in a Villin-Cre/KrasG12D+/−mouse
model and KRAS mutant CRC tissues. a Heat map of differentially abundant
genera between Villin-Cre/KrasG12D+/−mice (n= 3) and WT littermates (n= 6) treated
with F. nucleatum (1 × 109CFU) every two days for four weeks. bHeat map of top
differentially abundant genera between Villin-Cre/KrasG12D+/−(n=7)miceand WT
littermates (n= 6) which treated by AOM/DSS and F. nucleatum.cqPCR analysis of
P. distasonis and P. goldsteinii/merdae abundance in colonic tissues derived from
Villin-Cre/KrasG12D+/−miceand WT littermatestreated by AOM/DSSand F. nucleatum.
Significant differences are indicated: two-tailed Student’st-test, n=4(WTP. dis-
tasonis), n=3(KrasG12D P. distasonis), and n=6(P. goldsteinii/merdae) respectively,
data are presented asthe mean ± SEM. d,eCorrelation analysis ofF. nucleatum and
P. distasonis abundance in KRAS mutant (n=102)andKRAS WT (n=137) CRC
patients. Significant differences are indicated: Chi-square test, two-sided. Source
data are provided as a Source Data file.
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Fig. 4 | P. distasonis alleviates the F. nucleatum-mediated CRC progression.
aRepresentative images of the colons of Villin-Cre/KrasG12D+/−mice treated with
AOM/DSS and F. nucleatum or/and P. distasonis,n= 3 per group. bRepresentative
H&E stainings of the colons treated in (a), n=3 per group, scale bar: 20 μm.
c–eTumor numbers, tumor loads, and size of Villin-Cre/KrasG12D+/−mice treated in
(a). Significant differences are indicated: one-way ANOVA with Bonferroni’smulti-
ple comparison test, n= 3 per group, data are presented as the mean ± SEM. fFISH
detection of F. nucleatum (red) and P. distasonis (green) in colonic organoids
derived from KRAS WT and KRAS p.G12D CRC patients treated by F. nucleatum or/
and P. distasonis, scale bar: 50 μm. gStatistical analysis of the results in (f). Sig-
nificant differences are indicated: two-tailed Student’st-test, n= 5 per group, data
are presented as the mean ± SEM. hRepresentative immunostainings of Ki67 in
colonicorganoids derivedfrom KRAS WT and KRAS p.G12D CRC patients treatedby
F. nucleatum or/and P. distasonis,scalebar:50μm. iStatisticalanalysis of the results
in (h). Significant differences are indicated: one-way ANOVA with Bonferroni’s
multiple comparison test, n= 5 per group , data are presented as the mean ± SEM.
Data are representative of twoindependent experiments. Sourcedata are provided
as a Source Data file.
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tumor cells. We incubated P. distasonis with KRAS p.G12D-mutant cells
and data showed that no P. distasonis was observed in the G12D cells
(Supplementary Fig. 4e). Then we purified recombinant His-tagged
DHX15 proteins from Escherichia coli and performed pull-down assays
with P. distasonis lysates. There was no protein of P. distasonis was pulled
by DHX15 (Supplementary Fig. 4f). Therefore, we supposed that P. dis-
tasonis couldn’timpacttheinteractionbetweenF. nucleatum and
DHX15. Furthermore, we investigated whether the invasive level of F.
nucleatum could be inhibited by P. distasonis, we incubated G12D cells
with F. nucleatum, F. nucleatum +P. distasonis,F. nucleatum + super-
natant of P. distasonis and carried out the bacterial recovery assays. The
results showed that the invasive level of F. nucleatum could be inhibited
by P. distasonis, especially its supernatant implying that the metabolites
of P. distasonis may involved in inhibiting F. nucleatum invasion (Fig. 5h).
Finally, DHX15 expression was verified significantly increased in the
colon tissues of Villin-Cre/KrasG12D+/−mice compared to WT littermates
(Fig. 5i, j), and the pattern was recapitulated in the three cell lines
(Fig. 5k). Together, these data reveal that DHX15 is a receptor of F.
nucleatum in tumor cells and is upregulated upon the p.G12D mutation.
The ERK/STAT3 signaling mediates the expression of DHX15
The RAF/MEK/ERK and PI3K/AKT cascades are the major pathways
downstream of KRAS activation and control the processes of cell
growth and survival27. To better understand how DHX15 upregulation
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occurs in KRAS p.G12D tumors, we investigated whether these two
pathways a re involved in regulating DHX15 expression upon KRAS
activation. Western blot analysis showed that the level of phosphory-
lated ERK, but not that of AKT, was substantially upregulated in KRAS
p.G12Dcell line compared with the KRAS WT and KRAS p.G13D cell lines
(Fig. 6a). This coincided with the increased expression of DHX15 in the
cells harboring the ma jor KRAS mutation (Fig. 6a) as well as augmented
DHX15 level and p-ERK activation in the colon tissues of Villin-Cre/
KrasG12D+/−mice (Fig. 6b, c). To investigate whether F. nucleatum could
drive ERK activation and increase DHX15 expression, we performed
experiments by co-culture F. nucleatum with KRAS WT, p.G12D and
p.G13D cells and found that the level of phosphorylated ERK and
DHX15 in the F. nucleatum-treated cells was the same with untreated
cells (Supplementary Fig. 5a, b). To analyze the level of DHX15 in
patient samples, we performed IHC on tumor tissues of patients with
CRC that harbor KRAS p.G12D, p.G13D mutations, and KRAS WT allele.
We found that KRAS p.G12D-bearing CRC expressed a greater level of
DHX15 proteins compared with KRAS p.G13D-bearing and KRAS WT-
bearing patients (Fig. 6d, e), which collectively illustrated the func-
tional connections of the putative RNA helicase within the p.G12D-
bearing tumors.
ERK is located in the cytoplasm and upon activation is transferred
to the nucleus to regulate the activity of various transcription factors
such as FOS, ELK1, MYC, and STAT1/328. To predict the putative
downstream transcription factor of ERK that could directly initiate the
transcription of DHX15, the bioinformatics tool JASPAR was used to
identify STAT3. Correspondingly, the specificERKinhibitorandSTAT3
inhibitor were employed to examine the effect of this pathway on
DHX15expressionanddetectedasignificant downregulation at the
protein level (Fig. 6f). These data indicatethat the ERK-STAT3 signaling
activation is required for DHX15 upregulation.
Curation of protein database identified a potential STAT3-binding
site in the promoter of DHX15 (Fig. 6g). Chromatin immunoprecipi-
tation (ChIP) assays showed that DHX15 promoter abundance was
increasedby2.0-foldinKRAS p.G12D cells, 1.15-fold in KRAS WT cells,
and 1.54-fold in KRAS p.G13D cells (Fig. 6h). Luciferase reporter assay
verified that STAT3 bound to the promoter element of DHX15 and
affected DHX15 expression (Fig. 6i). Taken together, our data
demonstrate that the activation of ERK/STAT3 signaling in the down-
stream of KRAS is involved in DHX15 upregulation.
Knock out of Dhx15 in Villin-Cre/Kras G12D+/−mice attenuates
the CRC phenotype
To verify the regulatory effect of DHX15 in colorectal tumorigenesis, we
knocked out DHX15 in Villin-Cre/KrasG12D+/−mice by generating Villin-Cre/
KrasG12D+/−
/Dhx15fl/flmice (Supplementary Fig. 6a). Then we developed an
AOM/DSS-induced CRC mouse model and gavaged F. nucleatum in both
Villin-Cre/KrasG12D+/−and Villin-Cre/KrasG12D+/−
/Dhx15fl/flmice (Fig. 7a). We
verified that F. nucleatum-treated Villin-Cre/KrasG12D+/−
/Dhx15fl/flmice
exhibited a relieve in tumor formation compared with F. nucleatum-
treated Villin-Cre/KrasG12D+/−mice (Fig. 7b). H&E staining confirmed that F.
nucleatum-treated Villin-Cre/KrasG12D+/−
/Dhx15fl/flmice showed lower
grades of dysplasia (Fig. 7c), tumor multiplicities and tumor loads than F.
nucleatum-treated Villin-Cre/KrasG12D+/−mice (Fig. 7d–f). To investigate
whether immune factors involved in the pro-tumorigenic activities of
DHX15-F. nucleatum interaction, IF, and qPCR were performed to eval-
uate the infiltrated immune cells and inflammatory cytokine production
in colonic tissues of F. nucleatum-treated Villin-Cre/KrasG12D+/−and Villin-
Cre/KrasG12D+/−
/Dhx15fl/flmice.Wefoundthattherewerenosignificant
differences in the number of infiltrated CD3+T cells and CD11c+dendritic
cells between the two groups (Fig. 7gand7h). However, the expression
of Il-6,Il-17a were elevated after F. nucleatum treatment in Villin-Cre/
KrasG12D+/−mice when compared to Villin-Cre/KrasG12D+/−
/Dhx15fl/flmice
(Fig. 7i) suggested that the inflammatory cytokines might contribute to
the pro-tumorigenic activities of F. nucleatum. Overall, we verified that
the F. nucleatum-mediated-colorectal tumorigenesisisattenuatedin
mice activating Kras p.G12D and lacking DHX15.
Discussion
Colorectal cancer is characterized by gut microbiota abnormalities
and specific somatic mutations. Recent studies revealed that high F.
nucleatum abundance in the tumor tissues wassignificantly associated
with BRAF mutations and CpG island methylator phenotype (CIMP)-
positive CRC patients in univariate analyses and the amount of F.
nucleatum was also associated with microsatellite instable (MSI)-high
tumors independent of CIMP and BRAF mutation status, supporting
the notion that gut microbiota has links to the intratumor genetics and
epigenetics of CRC29–31. Our study presents a greater complexity of
niche elements converged in the F. nucleatum-dependent CRC
tumorigenesis, as the functional links comprise a somatic mutation, a
putative host RNA helicase, a signaling pathway crucial for multiple
malignancies, and a F. nucleatum-antagonizing bacterium. The findings
indicate that a comprehensive approach is needed to disentangle the
connection between the gut microbiota shift and CRC development.
Considerable effort has been devoted to understand and manage
KRAS mutant-mediated therapeutic resistance. The biophysical dif-
ferences between individual KRAS mutant alleles including the p.G12D,
p.G12V and p.G13D variants are sufficient to generate a range of sen-
sitivities to EGFR inhibitor32. Previous evidence has shown that in both
early-onset and later-onset tumors, KRAS mutation prevalence was
higher in the cecum compared with that in the other subsites33.Among
CRCs in all colorectal sites, cecal cancers show the highest KRAS
mutation frequency34. Therefore, cecal cancers appeared to represent
a unique subtype, characterized by a high frequency of KRAS
mutation35. Interestingly, F. nucleatum is most abundant in cecal
cancers36 which support our findings in this study that patients har-
boring KRAS p.G12D mutation are correlated with an elevated F.
nucleatum presence. In fact, the relationship between F. nucleatumn
Fig. 5 | F. nucleatum invades tumor cells and binds to DHX15. a Representative
confocal images of F.nucleatum (MOI = 100)invading to indicated tumor cells. The
F. nucleatum expressed mcherry are in red and tumor cells expressed GFP are in
green. Scale bar: 20 μm. bStatistical analysis of the results in (a). Significant dif-
ferences are indicated: one-way ANOVA with Bonferroni’s multiple comparison
test, n= 5 p er group, data are presented a s the mean ± S EM. cF. nucleatum was
visible inside the G12D cells and contacted the nuclear membrane by cryo-focused
ion beam milling and cryo-electron tomography, data are representative of three
independe nt experiments. dFISH detection of F. nucleatum in colonic organoids
derived from indicated patients treated by F. nucleatum or KRAS p.G12D antibody,
scale bar:50 μm. eStatisticalanalysis of the results in (d). Significant differencesare
indicated: one-way ANOVA with Sidak’s multiple comparison test, n=3pergroup,
data are presented as the mean ± SEM. fWestern blot analysis of F. nucleatum
proteins incubated by biotinylatedproteins fromindicated cellsand detectedusing
avidin-conjugated horseradish peroxidase. The experiment was performed in two
biological replicates. gPull-down assays were performed and validation of the
FN1859-DHX15 interaction in KRAS WT, KRAS p.G12D, and KRAS p.G13D cells by
western blot. The experiment was performed in three biological replicates. hThe
invasive level of F. nucleatum when KRAS p.G12D cells were incubated with F.
nucleatum, F. nucleatum +P. distasonis,F. nucleatum + supernatant of P. distasonis.
Significant differences are indicated: one-way ANOVA with Bonferroni’smultiple
comparison test, n= 5 per group, data are presented as the mean ± SEM. iWestern
blot analysis of DHX15 expression in Villin-Cre/Kra sG12D+/−mice and WT littermates.
Lanes representative of separate mice. jStatistical analysis of the results in (i).
Significant differences are indicated: two-tailed Student’st-test, n=4 per group,
data are presented as the mean± SEM. kWestern b lot analysis of DHX15 expression
in KRAS WT, KRAS p.G12D, and KRAS p.G13D tumor cells. The experiment was
performed in three biological replicates. Source data are provided as a Source
Data file.
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Nature Communications | (2024) 15:1688 8
Content courtesy of Springer Nature, terms of use apply. Rights reserved
and KRAS mutations is not conclusive based on the previous studies.
Kosuk Mima et al. have observed 1069 CRC samples and found that the
amount of F. nucleatum was not associated with KRAS mutation30.Ivan
Borozan et al. investigated 1,994 CRC cases and concluded that
mutation status of APC,PIK3CA,KRAS,BRAF,ERBB2,andSMAD4 were
not associated with F. nucleatum prevalence31.Inthetwostudies
above, the genomic DNA of F nucleatum was extracted from FFPE
tissue sections of CRC. However, in another research, KRAS mutation
was found to be more frequently observed in CRC samples infected
with F. nucleatum and the genomic DNA of F nucleatum was extracted
from 43 fresh-frozen CRC tissue samples and the matched adjacent
normal tissues37. Consistent with this, we evaluated 24 fresh-frozen
CRC tissue samples and the matched adjacent normal tissues and 239
fresh-frozen CRC tumor tissues and verified that F. nucleatum abun-
dance was associated with KRAS mutation. The potential differences in
different researches maybe due to the tissue status (fresh frozen vs.
FFPE). What’smore,hereweidentified that KRAS genotype affects the
activation of downstream ERK signaling pathway and KRAS p.G12D
mutation-induced activation of ERK signaling is stronger than KRAS
p.G13D and KRAS WT which is similar with a previous report that BRAF
Article https://doi.org/10.1038/s41467-024-45572-w
Nature Communications | (2024) 15:1688 9
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V600E could induce stronger ERK activity than KRAS p.G12V
mutation38.
The activation of ERK signaling leads to expression of DHX15
which is a member of the DEAH-box RNA helicase family and is
required for virus-induced innate immune gene express ion39,40.Studies
have reported that DHX15 contributes to carcinogenesis in several
malignancies including prostate cancer, non-small cell lung cancer,
and hepatocellular carcinoma26,41,42. However, a functional tie with
disease-related gut microbes remains poorly characterized, althoughit
has recently been reported that DHX15 deficient mice are susceptible
to infection by enteric bacteria Citrobacter rodentium and that the
protein plays an important role in the antimicrobial response in
colitis43. Here we show that the elevated expression of DHX15 is per-
tinent to KRAS p.G12D mutation and DHX15 as a specificreceptorinthe
nuclei of tumor cells interact with F. nucleatum to mediated-colorectal
tumorigenesis.
P. distasonis negatively correlates with several adverse health
conditions including inflammatory bowel disease, multiple sclerosis,
and obesity19,44, which coincides with its protective role against
intestinal epithelial da mage and colonic tumorigenesis in mice45.Inthis
study, we show a competition between P. distasonis and F. nucleatum
that is more pronounced in p.G12D-bearing mice and patients than
their counterparts with the WT allele. These findings collectively raise
the possibility of developing the bacterium as probiotics or auxiliary
therapeutics for colorectal cancer, although more in-depth explora-
tion is needed. Recently, combination of KRAS inhibitor AMG510 and
anti-PD-1 monotherapy delayed tumor growth, with complete regres-
sion in nine of ten tumors and generated most attention46. Whether the
combination of microbiota such as P. distasonis and anti-PD-1 mono-
therapy could represent a potentially trans-formative therapy for
patients for whom effective treatments are lacking also deserves fur-
ther investigation.
Taken together, we propose a pathogenic model of CRC depen-
dent on somatic genotype. In KRAS p.G12D mutation CRC tissues, F.
nucleatum invades more and the ERK signaling is activated and sub-
sequently induces DHX15 expression. The interaction of FN1859-
DHX15 potentiates colorectal tumorigenesis and could be alleviated by
P. distasonis.Ourfindings may provide a basis for personalized therapy
for the subset of treatment-recalcitrantCRC patients carryingthe KRAS
p.G12D mutation.
Methods
Mice
Villin-Cre/KrasG12D+/−mice and Villin-Cre/KrasG12D−/−littermates con-
structed in C57BL/6J background were purchased from The Shanghai
Model Organisms Center. Mice were bred and maintained under spe-
cific pathogen-free (SPF) conditions. Age- and sex-matched mice at 6-8
weeks of age and mixed mice were randomly used for all experiments.
According to the guidelines (GB/T 35892-2018), the ethics committee
specified that the maximal tumor burden is no more than 10% of the
body weight of animals and the average diameter is less than 20 mm.
During the experiment, the tumor sizes of the mice complied with the
regulations. All animal protocols were reviewed and approved by the
Ethics Review Committee for Animal Experimentation at Shanghai
Tenth People’sHospital.
Human subjects
The primary tumor tissues and the adjacent normal tissues were
obtained from patients with colorectal adenocarcinoma who under-
went a surgical resection. 254 fresh tumor tissues and 24 adjacent
normal tissues were collected at the time of surgical resection and
were immediately frozen in liquid nitrogen and stored at −80 °C.
Additionally, formalin-fixed, paraffin-embedded specimens (one from
each case) were collected from Department of Pathology of Shanghai
Tenth People’s Hospital Affiliated to Tongji University. According to
the TNM staging system, tumors were classified independently by two
pathologists. The clinic pathological parameters included age, sex,
tumor size, tumor location, pathological grade, and tumor size. All
individuals provided informed consent and the ethics approval also
covered the “Establishment of patient-derived organoids”. The study
was performed in accordance with the Declaration of Helsinki Princi-
ples and approved by the Research Ethics Board of Shanghai Tenth
People’s Hospital, Tongji University School of Medicine (SHDSYY-
2019-2751).
16S rRNA sequencing analysis
Colon tissues from patients or mice were collected. The total DNA was
then extracted with the QIAamp DNA Mini kit (QIAGEN). The 16S rRNA
high-throughput sequencing was performed by Realbio Genomics
Institute (Shanghai, China) using the Illumina MiSeq. Variable regions
V3-V4 on 16S rRNA genes of bacteria were amplified with forward
primer F341 5′-ACTCCTACGGGRSGCAGCAG-3′and reverse primer
R806 5′-GGACTACVVGGGTATCTAATC-3′. The raw data were then
subjected to a quality control procedure using UPARSE. The qualified
reads were clustered to generate operational taxonomic units (OTUs)
at the 97% similarity level using Usearch. Principal components ana-
lysis (PCA), heatmap analysis, Bray-Curtis similarity cluster, and spe-
cies abundance analysis were performed.
Targeted gene sequencing
Five genes were screened for the detection of mutations in patients
with CRC by sequencing using the Illumina NovaSeq 6000. To identify
the mutations in these genes, we designed PCR primers using the
primerXL pipeline. Three hundred and eighty oligonucleotide pairs
were produced and encompassed all of the CDSs and most of the
untranslated regions of the 5 genes. The amplification reactions were
conducted using an AB 2720 Thermal Cycler (Life Technologies Cor-
poration) with the following cycling conditions: 95 °C for 2 min; 11
Fig. 6 | The ERK/STAT3 signaling mediates the expression of DHX15. a Western
blot analysis of p-ERK,p-AKT, and DHX15 expression in KRAS WT, KRASp.G12D, and
KRAS p.G13D tumor cells. The experiment was performed in three biological
replicates. bWestern blot analysis of p-ERK and DHX15 expression in Villin-Cre/
KrasG12D+/−mice and WT littermates. Lanes representative of separate mice.
cStatistical analysis of the results in (b). Significant differences are indicated: two-
tailed Student’st-test, n=4(KrasG12D)andn= 6 (WT) respectively, data are pre-
sented as the mean ± SEM. dRepresentative immunohistochemical detection of
DHX15 in human CRC tissues of KRAS WT, KRAS p.G12D, and KRAS p.G13D groups,
scale bar: 20 μm. eStatistical analysis of theresults in (d).Significant diff erences are
indicated: one-wayANOVA with Sidak’smultiple comparison test,n= 7 (WT), n=10
(G12D), and n= 9 (G13D) respectively, data are presented as the mean ± SEM.
fWestern blotting analysis of DHX15 expression in KRAS p.G12D tumor cells after
the specific inhibitor (SCH772987 for ERK and APTSTAT3-9Rfor STAT3) treatment.
The experiment was performed in two biological replicates. gThe schematic dia-
gram shows one potential binding site of STAT3 inthe putative promoter element
of DHX15. hSTAT3 was immunoprecipitated from indicated cells. Immunopreci-
pitateswere assayed for theenrichment of DHX15promoter. Significant differences
are indicated: one-way ANOVA with Bonferroni’s multiple comparison test, n=3
per group, data are presented as the mean ± SEM. iLuciferase activity in lysates of
293T cellstransfectedwith luciferasereporter plasmids of pGL3-basic emptyvector
(basic), DHX15 promoter (p-DHX15-WT) or DHX15 promoter with mutation on pre-
dicted STAT3-binding site (p-DHX15-MUT), together with STAT3 plasmid or not.
Results are presented as the ratio of firefly luciferase to renilla luciferase activity,
relative to that of 293T cells transfected with pGL3-basic empty vector. Significant
differences are indicated: one-way ANOVA with Bonferroni’s multiple comparison
test, n= 6 per group, data are presented as the mean ±SEM. Source data are pro-
vided as a Source Data file.
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Nature Communications | (2024) 15:1688 10
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cycles of 94 °C for 20 s, 63°C per cycle for 40s, 72 °C for 1 min; 24
cycles of 94 °C for 20 s, 65 °C for 30 s, 72 °C for 1 min and 72°C for
2 min. The PCR products were used generate a library for further
detection, and the DNA-adapter-ligated and -indexed fragments from
ten libraries were then pooled and hybridized. After hybridization of
the sequencing primer, base incorporation was performed using the
Illumina NovaSeq 6000 in a single lane following the manufacturer’s
standard cluster generation and sequencing protocols for 250 cycles
Fig. 7 | Knock out Dhx15 in Villin-C re/Kras G12D+/−mice attenuated the CRC phe-
notype. a Schematic diagram of the experimental design and timeline of mouse
models. b,cRepresentative images and H&E stainings of the colons of Villin-Cre/
KrasG12D+/−mice and Villin-Cre/KrasG12D+/−
/Dhx15fl/flmice treated with AOM/DSS and F.
nucleatum, the red arrows indicate tumors, n=5per group,scale bar:50μm.
d–fTumor numbers, tumor loads, and size of Villin-Cre/KrasG12D+/−mice and Villin-Cre/
KrasG12D+/−
/Dhx15fl/flmice treated with AOM/DSS and F. nucleatum.Significant differ-
ences are indicated: two-tailed Student’st-test, n= 5 per group,data are presented as
the mean ± SEM. gRepresentative immunofluorescence detection of CD3 and CD11c
positive cells in Villin-Cre/KrasG12D+/−mice and Villin-Cre/KrasG12D+/−
/Dhx15fl/flmice treated
with AOM/DSS and F. nucleatum, scale bar: 50 μm. hStatistical analysis of the results in
(g). Significant differences are indicated: two-tailed Student’st-test, n=5pergroup,
data are presented as the mean ± SEM. iqPCR analysis of Il-17a,Il-6 mRNA expression
Villin-Cre/KrasG12D+/−mice and Villin-Cre/KrasG12D+/−
/Dhx15fl/flmice treated with AOM/DSS
and F. nucleatum.Significant differences are indicated: two-tailed Student’st-test, n=4
(KrasG12D)andn=5 (KrasG12D/Dhx15−/−
) respectively, data are presented as the mean ±
SEM. Data are representative of two independent experiments. Source data are pro-
vided as a Source Data file.
Article https://doi.org/10.1038/s41467-024-45572-w
Nature Communications | (2024) 15:1688 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved
of sequencing per read to generate paired-end reads, including 250 bp
at each end and 8 bp of the index tag. The primer sequences are pro-
vided in Source Data file.
Animal experiments
Following the single dose of AOM (10mg/kg, intraperitoneal) injec-
tion, mice were treated 2.5% DSS (molecular weight 36,000–50,000,
MP biomedicals) in drinking water for 5 successive days (Toinvestigate
whether F. nucleatum exacerbates colorectal tumorigenesis in Villin-
Cre/KrasG12D+/−mice in early time, micewere administrated DSS for one
cycle. To assess the treatment effect of P. diastonis and the role of
DHX15 respectively, mice were administrated DSS for three cycles to
ensure successful tumor development.) and then gavaged with 1× 109
CFU of F. nucleatum and/or P. distasonis every 2 days for 4 weeks. The
animals in the control group were gavaged by the same volume of PBS.
Then mice were sacrificed to harvest colon tissues for analysis.
Bacterial recovery assay
1×10
5cells were grown in a 24-well plate and co-cultured with bacteria
for 1 h (MOI =100) under anaerobic conditions. After co-culture,
Ampicillin (200 mg/ml) and Gentamicin (200 mg/ml) were used to
eliminate extracellular bacteria for 1 h, and medium was removed and
cellswerewashedwithPBSthreetimes.Tolysesthecells,100μlof
H
2
O was added for 20 min, followed b y the addition of 900 μlof
Wilkins–Chalgren anaerobe broth to homogenize the cells. The inva-
ded F. nucleatum colonies were recovered on Wilkins–Chalgren
anaerobe agar plate under anaerobic conditions; the number of colo-
nies was counted.
FISH
Frozen colon tissues were fixed in Carnoy’s solution overnight and
embedded in paraffin; 5 μm thick sections were hybridized in the
hybridization buffer (0.9 M NaCl, 20 mM Tris/HCl, pH 7.3, 0.01% SDS).
Stringency was used with the form amide concentration from 0to 30%
(v/v). Pre-warmed hybridization buffer (20 ml) was mixed with
approximately 5 pmol of the oligonucleotide probe and carefully
applied to the tissue sections. After incubation for 5 h in a dark humid
chamber at 46 °C, each of the slides were rinsed with sterile double-
distilled water, air-dried in the dark, and mounted with ProLong Gold
Antifade Mountant with DAPI (Thermo Fisher Scientific). The probe
sequences used to detect F. nucleatum are listed as followed: 5′-
CGCAATACAGAGTTGAGCCCTGC-3′47.
DNA extraction and qPCR
Mucosaltissues were digested in PBS containing an enzymatic cocktail
of mutanolysin (250 U/ml) and lysozyme (1mg/ml) (Sigma-Aldrich) at
37 °C for 1 h, total genomic DNA was extracted with the QIAamp DNA
Mini Kit (QIAGEN) according to the manufacturer’s instructions. qPCR
was performed to detect the F. nucleatum or P. distasonis level by using
40 ng genomic DNA in 20 μl universal SYBR Green PCR Master Mix
(Roche) in a ViiA 7 Real-Time PCR System (Applied Biosystems).
F. nucleatum or P. distasonis quantitation was measured relative to the
pgt gene. The primers used are listed as followed: F. nucleatum for-
ward: CAACCATTACTTTAACTCTACCATGTTCA, F. nucleatum reverse:
GTTGACTTTACAGAAGGAGATTATGTAAAAATC, P. distasonis forward:
CCACGCAGTAAACGATGA, P. distasonis reverse: 5′-CTTAACGCTTTCG
CTGTG-3′, prostaglandin transporter (pgt) forward: 5′-ATCCCCAAAG-
CACCTGGTTT-3′, pgt reverse: 5′-AGAGGCCAAGATAGTCCTGGTAA-3′.
Cell culture and stable cell lines construction
The human colon cancer cell line HT-29 was obtained from Stem Cell
Bank, Chinese Academy of Sciences, and was maintained in Dulbecco’s
modified Eagle’s medium (DMEM) (Hyclone, #SH30243.01) supple-
mented with 10% FBS (Gibco, #10270-106), 1% penicillin/streptomycin
(Beyotime, #C0222). All cells were cultured at 37 °C supplied with 5%
CO
2
.ThecelllineswithstableoverexpressionofKRAS p.G12D, p.G13D,
or control cells were generated by infection HT-29 cells with KRAS
G12D-sgRNA-Cas-EGFP, KRAS G13D-sgRNA-Cas-EGFP and negative
scramble control-EGFP lentiviral plasmids which were purchased from
GeneChem (Shanghai, China) according to the manufacturer’s
instructions.
Establishment of patient-derived organoids (PDOs)
Biopsies from CRC patients were collected in 5 ml PBS containing
penicillin/streptomycin on ice. Following washing and mincing tissues
into around 1–2mm
3, samples were digested with 10 ml of cell dis-
sociation reagent (Stem Cell, #07174) on ice on a rocking platform for
30 min. Dissociated cells were passed through 100 μm cell strainer,
and then pelleted and suspended in ice-cold PBS. Centrifuge cells for
300 g, 5 min, and then resuspend cells in growth factor reduced (GFR)
matrigel (Corning, #356231),and seed cells on48-well cell culture plate
(Corning, #3548). Following solidified in 37 °C and 5% CO
2
incubator
for 30 min, 300 μl of human IntestiCult™Organoid Growth Medium
(Stem Cell, #06010) which were additionally added 10 μMofY27632
(Stem Cell, #72304) for the primary culture and were overlaid in the
well coated with matrigel. As for the passaging of PDOs, organoids
were harvested with ice-cold PBS and pipetted with mechanical force
through 1 ml pipette (160 times per well). Dissociated PDOs were
pipetted and washed with ice-cold PBS. Resuspend the dissociated
cells in GFR matrigel and re-seeded on 48-well flat bottom cell culture
plate. PDOs were frozen in FBS containing 10% DMSO for biobanking.
The information of the patients from whom the organoids derived is
provided in Supplementary Table 1.
Far-western assay
The far-western assay was performed as the reference48. To prepare
the biotinylated cell proteins, cell proteins were extracted with PBS
containing 1% Triton X-100 (Sigma-Aldrich) for 1h at room tempera-
ture, then labeled with 1 mM EZ-Link Sulfo-NHS-LC-Biotin (Thermo
Fisher Scientific) for 2 h at 4 °C. Whole F. nucleatum proteins were
extracted with PBS containing 1% Triton X-100, separated by
SDS–polyacrylamide gel electrophoresis (PAGE) and transferred onto
a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane
was blocked with 5% BSA for 1 h and incubated with biotinylated cell
proteins overnight at 4 °C. Biotin-labeled proteins were detected using
avidin-conjugated horseradish peroxidase (HRP). The corresponding
bands in SDS-PAGE were excised for identification by mass
spectrometry.
His pull-down assay
The recombinant His-FN1859 or His-DHX15 was produced in Escher-
ichia coli strains. To perform the His pull-down assay, His-FN1859 or
His-DHX15 was incubated with Ni-NTA Magnetic Beads, followed by
adding cell or bacteria proteins indicated overnight at 4 °C. The beads
were then washed and boiled with SDS-PAGE gel loading buffer. Eluted
proteins were separated by SDS-PAGE and analyzed by mass
spectrometry.
Mass spectrometry
Scoop out the area where the protein is located in SDS-PAGE and cut it
into small pieces about 1 mm3. Add double steaming water to soak the
glue block, shake for 10 minutes, absorb the lotion. The glue block was
immersed in 50% ACN/100 mM NH
4
HCO
3
(pH 8.0) solution, shook for
10 min, and the lotion was absorbed. The process was repeated three
times. Add 100% ACN-impregnated glue block, shake for 10 min,
absorb the lotion, and then drain the glue block in a vacuum draining
machine. 10 mM DTT/50 mM NH
4
HCO
3
(pH 8.0) solution was added to
the glue block, incubated at 56 °C for 1 h for reduction reaction, and
then the leaching solution was removed. After that, 55 mM iodoace-
tamide/50 mM NH
4
HCO
3
(pH 8.0) solution was added, and the
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Nature Communications | (2024) 15:1688 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved
solution was incubated at room temperature and dark place for
30 min. Add 100% ACN, shake for 20 min, absorb the infusion, drain
the glue block. Appropriate amount of trypsin was added to the glue
blocks, and 50 mM NH
4
HCO
3
solution was added to completely cover
the glue blocks, and the glue blocks were incubated at 37 °C overnight
for enzyme digestion. Then 60% ACN/5% formic acid was added,
ultrasonic shock was performed for 10 min, and the supernatant was
absorbed into the new centrifuge tube after centrifugation. After the
extraction process is repeated twice, the extracted liquid is combined
and drained in the centrifugal concentrator. The peptide was desalted
using C18 column and frozen at −20 °C for machine detection.
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) assays were performed using
the SimpleCHIP enzymatic chromatin immunoprecipitation kit (Cell
Signaling Technology, #9002) according to the manufacturer’s pro-
tocol with minor modifications. The genomic DNA recovered from the
ChIP assays was amplified with primers specifictotheSTAT3-binding
elements of the DHX15 promoter region. The specificity of the primer
set was verified by analyzing the dissociation curve of each gene-
specific PCR product and listed in Supplementary Table 4.
Luciferase reporter assays
PGL3-basic vector (Promega, #E1751) was used to clone the promoter
of DHX15. Site-specific mutant was generated by PCR. 293T cells were
provided from the cell bank of Chinese Academy of Sciences and
seeded in a 96-well plate with a density of 0.8× 105/well 1 day before
transfection. Wells was transfected with a mixture of 100 ng pGL3
luciferase vector, DHX15 WT plasmid or DHX15 mutant plasmid or/and
100 ng STAT3 plasmid and 25 ng pRL-TK renilla vector using Lipo-
fectamine 3000 Transfection Reagent (Invitrogen, #L3000-15).Twelve
hours post-transfection, luciferase activity was measured on a micro-
plate reader (Berthold, TriStar LB941) by using the Dual-Luciferase
Reporter Assay System (Promega, #2920). Theratio of fireflyluciferase
to renilla luciferase was calculated for each well.
RNA extraction, reverse transcription, and qPCR
Total RNA was extracted from colon tissues from mice using the TRIzol
reagent (Invitrogen, #15596-026), and NanoDrop spectrophotometer
(ND-1000) was used for RNA quality control. cDNA was synthesized
using PrimeScriptTM RT Master Mix (TaKaRa, #RR036A). qPCR was
carried out with the TB Green Premix Ex TaqTM II (TaKaRa, #RR820A) in
a ViiA 7 Real-Time PCR System (Applied Biosystems). The relative
expression of target genes was confirmed using quantity of target
gene/quantity of β-actin. Primer sequences are listed in Supplementary
Table 4.
Western blotting
Mouse colons or cultured cells were lysed in radio immunoprecipita-
tion assay buffer supplemented with protease and phosphatase inhi-
bitor cocktail (Thermo Scientific, #78440). Antibody used are listed in
Supplementary Table 5. The signal was detected with ECL Western
Blotting Substrate (Thermo Scientific, #34095) and Amersham Imager
600 (GE Healthcare). Images have been cropped for presentation.
Immunohistochemistry and immunofluorescence
The human or mouse colons were fixedinformalinandembeddedin
paraffin. Sections (6 μm) were stained with hematoxylin and eosin
(H&E). For immunohistochemistry, Ki67 or DHX15 expression was
evaluated in colon sections using rabbit anti-Ki67 Ab (1:100 dilution,
Cell Signaling Technology, #12202) or rabbit anti-DHX15 Ab (1:100
dilution, Proteintech, #12265-1-AP), following the manufacturer’s
instructions. For immunofluorescence, CD3 or CD11c expression was
evaluated in colon sections using rat anti-CD3 Ab (1:100 dilution,
Abcam, #ab11089) or mouse anti-CD11c Ab (1:100 dilution, Abcam,
#ab254183), following the manufacturer’s instructions. For observa-
tion F. nucleatum invasion, cells were seeded in 35 mm plates and
exposed to F. nucleatum with a MOI of 100 under anaerobic condi-
tions. Then the cells were washed for three times and imaged imme-
diately or fixed in 4% formaldehyde containing 0.1% Triton X-100 at
room temperature for 15min. DAPI was used to visualize the nuclei.
Images were obtained by Olympus BX51 microscope or Zeiss LSM900
confocal microscope.
Bacterial strains and culture conditions
F. nucleatum (25586) was purchased from ATCC and P. distasonis was
isolated from CRC patients and has a 99.72% identity with the 16S rRNA
gene sequence of Parabacteroides distasonis strain ATCC 8503 (NCBI
accession number NR_074376.1) (Supplementary Table 6). Identifica-
tion of cultured F. nucleatum and P. distosonis by Nanopore Sequen-
cing is shown in Supplementary Table7. The strainswere maintained in
bottled Thioglycollate anaerobe broth (Sanyao, #15611) or Columbia
agar in an anaerobic chamber (5% CO
2
,2%H
2
, and 93% N
2
)at37°C.
Transmission electron microscope
Cells were harvested after trypsin digestion, and fixed with 2.5% glu-
taraldehyde solution. PDOs were dissociated by TrypLETM Express
(Gibco, 12604-013), and fixed in 2.5% glutaraldehyde solution. TEM
(Hitachi) was operated at a voltage of 80 kV and equipped for mor-
phological observation after sample preparation.
Statistics and reproducibility
Each experiment was performed at least two biological replicates. The
data were analyzed with Graphpad Prism 5 and Spss 22.0. Acquired
data were presented as mean values, and error bars represent the SEM.
Student’st-test was used when two conditions were compared, and
one-way ANOVA with Bonferroni post-test or Sidakʼs post-test was
used for multiple comparisons. The correlations between F. nucle-
atum,P. distasonis abundance and KRAS mutation status were analyzed
using Chi-square. The probability values of <0.05 were considered
statistically significant. Exact p-value was provided.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
The sequencing data in thisstudy have been deposited in the GSA with
accession number CRA013275,CRA013274,CRA013276,CRA013455,
HRA006025. Dataset HRA006025 is available under restricted access
for human genetics data privacy concerns; access can be obtained by
the DAC (Data Access Committees) of the GSA-human database. The
approximate response time for accession requests is about 3 days.
Once access has been approved, the data will be available for3 months.
The user canalso contact the corresponding author directly. The mass
spectrometry proteomics data have been deposited to the Proteo-
meXchange Consortium via the PRIDE partner repository with the
dataset identifier PXD048684,PXD048686. The remaining data are
available within the Article, Supplementary Information or SourceData
file. Source data are provided as a Source Data file. Source data are
provided with this paper.
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Acknowledgements
This work was supported by grants from the National Nature Science
Foundation of China (81972221, 82072634, 81801564, 81902422);
Shanghai Sailing Program (18YF1419400); Clinical Research Plan of
SHDC (SHDC2020CR2069B); Jiangsu Natural Science Foundation
(BK20231245); Program of Jiangsu Commission of Health (No.
M2020024). We are very grateful for the kind offer of Dhx15-floxed mice
from Prof. Shu Zhu (Division of Life Sciences and Medicine, University of
Science and Technology of China, Hefei, 230027, China), and the help
for using cryo-focused ion beam milling and cryo-electron tomography
from Prof. Quan Wang and Xuebo Yang (Shanghai Institute for Advanced
Immunochemical Studies and School of Life Science and Technology,
ShanghaiTech University, Shanghai, 201210, China).
Author contributions
H. Zhu, Q. Wei, and H. Qin designed the research. H. Zhu, M. Li, and H.
Yang conducted most of the experiments. M. Li helped with organoids
culture. D. Bi and H. Liu offered help in data analysis. F. Song helped in
RNA interference. C. Kong and Y. Zhang helped with mouse model
construction. Y. Gao and Y. Zhu helped with the experimental details. R.
Xie and J. Zheng contributed to specimen preparation. X. Yan and Q. Xu
helped with data discussion. H. Zhu wrote the manuscript. All authors
approved the final version of the manuscript.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-024-45572-w.
Correspondence and requests for materials should be addressed to
Huiyuan Zhu, Qing Wei or Huanlong Qin.
Peer review information Nature Communications thanks Federica Fac-
ciotti and the other, anonymous, reviewer(s) for their contribution to the
peer review of this work. A peer review file is available.
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