Download Hemolytic E. coli Promotes Colonic

Published OnlineFirst March 24, 2016; DOI: 10.1158/0008-5472.CAN-15-2083
Cancer
Research
Integrated Systems and Technologies
Hemolytic E. coli Promotes Colonic Tumorigenesis
in Females
Ye Jin1, Senwei Tang1, Weilin Li1, Siew Chien Ng1, Michael W.Y. Chan2,
Joseph J.Y. Sung1, and Jun Yu1
Abstract
Bacterial infection is linked to colorectal carcinogenesis, but
the species that contribute to a protumorigenic ecology are illdefined. Here we report evidence that a-hemolysin–positive
(hlyþ) type I Escherichia coli (E. coli) drives adenomagenesis
and colorectal cancer in human females but not males. We
classified E. coli into four types using a novel typing method to
monitor fimH mutation patterns of fecal isolates from adenoma patients (n¼ 59), colorectal cancer patients (n¼ 83), and
healthy subjects (n¼ 85). hlyþ type I E. coli was found to be
relatively more prevalent in stools from females with adenoma
and colorectal cancer, correlating with poor survival in colo-
rectal cancer patients. In mechanistic studies in female mice, we
found that hlyþ type 1 E. coli activated expression of the glucose
transporter GLUT1 and repressed expression of the tumor
suppressor BIM. hly-encoded alpha hemolysin partially
accounted for these effects by elevating the levels of HIF1a.
Notably, colon tumorigenesis in mice could be promoted by
feeding hlyþ type I E. coli to female but not male subjects.
Collectively, our findings point to hemolytic type I E. coli as a
candidate causative factor of colorectal cancer in human
females, with additional potential as a biomarker of disease
susceptibility. Cancer Res; 76(10); 2891–900. 2016 AACR.
Introduction
colorectal cancer patients and promoted colorectal cancer development in mice without affecting inflammation (5).
Although the previous evidence indicates the association
between E. coli and colorectal cancer and provides some clues
on the underlying mechanisms, many questions remain unanswered. Screening and comparison of a large number of clinical
isolates from patients with colorectal cancer, adenoma, and
healthy controls are necessary to give more information. Efforts
are also desired to identify previously unidentified colorectal
cancer–associated E. coli and to uncover their oncogenic mechanisms. Moreover, it is unknown whether gender-specific differences
exist for colorectal cancer development and if bacteria have any
role in this process. Here we identify hemolytic type I E. coli as a
type of bacteria that is significantly associated with adenoma and
colorectal cancer in female patients, by analyzing a large number
of clinical E. coli isolates. Hemolytic type I E. coli activates the
expression of the glucose transporter GLUT1 and reduces the
expression of the tumor suppressor BIM at least in part by acting
on hypoxia-induced a-subunit (HIF1a), and the hly-encoded
alpha hemolysin is required for the regulation. We finally verify
the tumorigenic capacity of hemolytic type I E. coli in two mouse
models and demonstrate the requirement of alpha hemolysin of
type I E. coli for colonic tumorigenesis.
Colorectal cancer is the third most common cancer and has
become a major public health problem worldwide. Carcinogenesis of colorectal cancer has been linked to Escherichia coli (E. coli).
E. coli is a commensal of the normal gut microflora of humans and
other mammals, and is one of the most common causes of
infections by Gram-negative bacilli (1). Enteropathogenic E. coli
was reported to downregulate expression of key DNA mismatch
repair proteins MSH2 and MLH1 (2), induce cancer cell detachment via cytoskeleton rearrangement, and reduce cellular apoptosis of the detached cancer cells (3). However, no in vivo evidence
has been provided for the role of enteropathogenic E. coli for
colorectal cancer. Another type of E. coli that belongs to B2
phylogenetic group carries a pks island and produces cyclomodulins has also been linked to colorectal cancer. pksþE. coli has been
reported to cause DNA double-strand breaks and activate the DNA
damage checkpoint pathway, leading to cell-cycle arrest and cell
death (4). Recent studies showed that pksþE. coli was present in a
significantly high percentage of inflammatory bowel disease and
1
Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing
Institute of Health Sciences, The Chinese University of Hong Kong,
Hong Kong. 2Department of Life Science, National Chung Cheng
University, Min-Hsiung, Chia, Taiwan.
Note: Supplementary data for this article are available at Cancer Research
Online (http://cancerres.aacrjournals.org/).
Corresponding Authors: Ye Jin, Institute of Digestive Disease and Department
of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka
Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong
Kong. Phone: 852-3763-6119; Fax: 852-2144-5330; E-mail: [email protected];
Joseph J.Y. Sung, [email protected]; and Jun Yu, [email protected]
doi: 10.1158/0008-5472.CAN-15-2083
2016 American Association for Cancer Research.
Materials and Methods
fimH sequencing, clustering, and typing
Given the higher rate of point mutation in the mannosebinding lectin domain than in the pilin domain (6), a gene
fragment (from 145 to þ687 relative to the start codon) of
fimH covering the lectin domain-encoding region was PCR amplified using primers fimH-F and fimH-R and then sequenced. For
each stool sample, 4–6 E. coli isolates were randomized selected
for the fimH sequencing analysis. As a control, fimH of E. coli K-12
MG1655 (a laboratory reference strain) was included for
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2891
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Jin et al.
sequencing. Low-quality sequencing data were removed (i.e.
sequencing quality score < 30 and error probability > 0.1%;
or reading rate < 50%) and the qualified fimH sequences of fecal
E. coli isolates were aligned to that of the reference strain
MG1655 using BWA-MEM (7). A binary matrix was constructed
on the basis of SNP information of the fimH sequences and
cluster analysis was performed using the R pheatmap package.
In addition to clustering, NMF package in R (8) was used to
determine how many types that fimH should be grouped into.
Factorization rank r, which defines the number of basis effects
used to approximate the target matrix, was decided on by trying
different values. 100 NMF runs were performed for a range of
factorization rank r from 2 to 10. To choose the optimal rank r, we
employed approaches including cophenetic correlation efficient
and an approach based on the variation of the residual sum of
squares (RSS) between the target matrix and its estimate. For the
cophenetic correlation efficient approach, we chose the smallest
value of r for which this coefficient started decreasing. For the
RSS approach, we chose the value of r for which the plot of the
RSS showed an infection point. We employed WebLogo (9, 10)
to show the SNP composition of different types of fimH.
Genetic engineering of E. coli isolates
Gene deletion from the chromosome of E. coli isolates was
achieved by using the l-Red recombination and verified by colony
PCR (11).
Primer design for detection of virulence genes
For detection of type I fimH, primers CRC-F and CRC-R3
were designed. Annealing temperature was adjusted to 62 C,
at which the PCR had 100% sensitivity and 90% specificity.
For detection of the pks island, primers published previously
(4) were used in this study. For detection of hly and other
virulence genes, conserved sequence regions were first identified across different E. coli strains by evaluation of multiple
sequence alignments. Then, primers were designed to be specific to the conserved regions. All the primers are listed in
Supplementary Table S4.
PCR quantification of hlyþ and pksþ type I E. coli in stools
We determined percentage of hlyþ or pksþE. coli relative to
total E. coli by absolute quantification real-time PCR. Genomic
DNA was isolated from stools using QIAamp Fast DNA Stool
Mini Kit (Qiagen), and was isolated from colonic mucosa using
TRIzol reagent (Life Technologies). Total E. coli were quantified
by determining lacZþ bacteria, given that most E. coli strains are
positive with lacZ (12). As one E. coli cell harbors one copy of
the lacZ gene, we determined the number of E. coli by quantifying the lacZ gene. This is also the case for hlyþ or pksþtype I
E. coli. We performed absolute quantification real-time PCR for
lacZ, hly, pks, and type I fimH, described in Supplementary
Materials and Methods.
Mouse models for colorectal tumorigenesis
Six to 8-week-old female BALB/c mice were used in this
study. Studies were approved by the Animal Experimentation
Ethics Committee. In some experiments, mice were not treated
with any carcinogen. In some experiments, mice were pretreated with azoxymethane (AOM), a colonic genotoxic carcinogen. In this study, AOM treatment was used to increase the
basal level of colorectal tumorigenesis but not for direct induc-
2892 Cancer Res; 76(10) May 15, 2016
tion of colorectal tumor formation, so that oncogenic effects of
E. coli could be detected. On day 0, mice were intraperitoneally
injected with AOM (10 mg/kg), according to a published
protocol (13). On day 5, mice were treated with neomycin
(1 g/L), vancomycin (0.5 g/L), ampicillin (1 g/L), and metronidazole (1 g/L) in drinking water to eliminate commensal
flora (14) and facilitate the colonization of fed bacteria.
Dextran sulfate sodium (DSS) is typically used together with
AOM to induce colorectal cancer in mice (13). However, the
oncogenic action of the combination of AOM and DSS are
strong and may overshadow the oncogenic effects of fed bacteria. To avoid this, DSS was not used in our experiments. After
the antibiotic treatment, mice were fed by oral gavage with PBS
or E. coli (1 108 bacteria per mouse) every other day. During
the experiments, mice were weighed weekly. At the end of the
experiments, mice were anesthetized with sodium pentobarbital intraperitoneally (60 mg/kg) and the large intestine was
excised. The large intestine was cut open longitudinally along
the main axis, and a piece (approximately 2 mm in length) of
tissue free of tumors was immediately stored in RNAlater
(Qiagen). In some experiments, the large intestine was rolled
up and fixed in 10% buffered formalin for at least 24 hours.
Paraffin-embedded sections of the Swiss roll of the large intestine were then made by routine procedures. We did not rely on
macroscopic evaluation of colonic tumor growth, as it did not
discriminate colonic tumors from normal lymphoid aggregates.
Instead, we quantified colonic tumor growth microscopically.
Each colonic Swiss roll was cut into nine cross sections, which
were prepared at an equal distance apart. Distance between two
adjacent sections was calculated so that the entire colonic Swiss
roll was sectioned. The sections of each Swiss roll were subjected to hematoxylin and eosin staining and then microscopically examined for colonic tumors arising from the epithelial
tissues. Colonic inflammation in the mouse model was evaluated histomorphologically and scored using a previously
published criterion (15).
Results
E. coli is classified into four types based on fimH mutation
patterns
The fimH gene of E. coli is prone to mutation and the mutation patterns help predict pathogenic potential (6). To see
whether E. coli with certain fimH mutation patterns is associated
with colonic adenoma and colorectal cancer, we isolated fecal
E. coli from 59 adenoma patients, 83 colorectal cancer patients,
and 85 healthy subjects. fimH was successfully sequenced
from 711 E. coli isolates (Supplementary Table S1), and displayed single nucleotide polymorphisms (SNP) at numerous
sites. Cluster analysis of the SNPs (Supplementary Fig. S1A)
and nonnegative matrix factorization (Supplementary Fig. S2A
and S2B) showed that fimH could be classified into four types.
Among the four types, detection frequency of type I E. coli was
similar among the three male groups, but was significantly
higher in the female colorectal cancer patients than in the other
female groups (P < 0.001; Supplementary Fig. S1B and S1C).
These data suggested gender-dependent specificity of type I
E. coli to colorectal cancer. It is noteworthy that the analyses
based on isolate sequencing just roughly estimated detection
frequency of each type of E. coli, as only a few isolates were
sequenced for each stool sample. Frequency of type I E. coli
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Published OnlineFirst March 24, 2016; DOI: 10.1158/0008-5472.CAN-15-2083
Role of Hemolytic Escherichia Coli for Colonic Tumorigenesis
C
NCM460
Viability (%)
80
ns
NCM460
60
40
20
HCT116
0
NCM460
Viability (%)
B
D
Type 1
100
80
60
40
20
0
Others
SW480
Type 1
Others
MG1655
0
2
3
4
Time (hours)
Blood
agar
NCM460 Viability (%)
A
100
80
60
40
20
0
Δhly
Δhly
/pUC
Δhly
/phly
hly +
–
–
+
Hemolysis +
–
–
+
Cytotoxicity +
–
–
+
J47
Figure 1.
Cytotoxicity of type I E. coli killers. A, viability percentage (%) of NCM460 after 3 hours of coculture with type I E. coli (32 isolates) and other types of E. coli
(64 isolates). B, time-course viability percentage of NCM460 cocultured with an isolate of the type I E. coli, three randomly selected isolates of other
types of E. coli, and the reference strain MG1655. Type I, n ¼ 6; other types, n ¼ 18; MG1655, n ¼ 6. C, cytotoxicity of type I E. coli was correlated with
hemolysis. Three cell lines, including NCM460, HCT116, and SW480, were tested. In the box are the 32 isolates belonging to type I E. coli. D, effects of
þ
deleting the hly operon and restoring the hly expression on cytotoxicity and hemolysis of type I E. coli. J47, a wild-type isolate of hly type I E. coli; Dhly,
an isogenic mutant deleted for the hly operon; Dhly/pUC, the hly-null mutant carrying an empty control vector pUC; Dhly/phly, the hly-null mutant
carrying a vector pUC-hly. n ¼ 6; error bar, SEM. ns, statistically not significant; , P < 0.001.
in the female patients with adenoma was later found to be
underestimated by the sequencing-based method, as revealed
by quantitative PCR analyses of DNA isolated from stools
containing a large number of E. coli. Therefore, E. coli detection
rates were later precisely determined by quantitative PCR in
this study.
One-third of type I E. coli produce hly-encoded alpha hemolysin
We then examined the interaction between colon epithelial
cells and E. coli isolates including 32 type I isolates and 63
isolates belonging to other types. A human commensal intestinal E. coli reference strain MG1655, which was found not to
promote colorectal carcinogenesis (16), was included as an
additional control. After 3 hours of bacterium cell coculture at a
multiplicity of infection (MOI) of 100, 10 of 32 type I E. coli
killed most NCM460 cells (a normal human colon mucosal
epithelial cell line), whereas other isolates did not (Fig. 1A).
The type I killers at a MOI of 100 started to kill cells 1 hour after
the coculture (Fig. 1B). The cell death caused by the type I killers
was also observed with human colon cancer cell lines HCT116
and SW480 (Fig. 1C). The hly-encoded alpha hemolysin was
reported to kill host cells (17), leading us to speculate that the
cell death caused by the type I killers was due to alpha
hemolysin. As speculated, all type I killers induced hemolysis
but none of type I non-killers and other E. coli isolates did so
(Fig. 1C). To further verify the role of hly for the type I E. colimediated cytotoxicity, we randomly selected a hlyþ type I E. coli
isolate, which was isolated from the stool of a female colorectal
cancer patient, for mutagenesis analysis. This fecal isolate was
hereafter referred to as J47. We deleted the entire hly operon
from J47, generating a hly-null mutant named J47Dhly. Deleting
hly abolished hemolysis and cytotoxicity, and transformation
of the hly mutant with a hly-overexpressing plasmid (18)
restored them (Fig. 1D). Thus, the hly-encoded alpha hemolysin is responsible for the cytotoxic effects of type I killers on
colon epithelial cells.
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hly and pks coexist in some hlyþ type I E. coli
We then detected by PCR the presence of a set of well-known
E. coli virulence genes in 97 E. coli isolates including hlyþ type I
(16 isolates), hly type I (26 isolates), other types of E. coli
isolates (38 isolates), and the reference strain MG1655. We also
determined the phylogenetic type and assayed the ability of
each isolate to cause hemolysis (Supplementary Table S2).
Among the genes tested, only the hly operon and the pks island
were present in all hlyþ type I isolates but absent from hly
type I and other types of E. coli isolates. The hly operon was
accompanied with the pks island [a pathogenic island encoding
giant modular nonribosomal peptide and polyketide synthases
(4)] in all the 16 hlyþ type I isolates, but 3 of 19 pksþ type I
E. coli isolates did not carry hly. Phylogenetic typing showed
that 73.8% (31/42) of the type I E. coli isolates belonged to type
B2 and the other 26.2% belonged to type D. Among them, all
the hlyþ type I E. coli and pksþ type I E. coli belonged to type B2.
In contrast, most of other types of E. coli belonged to type A, B1,
or D. The data based on the 97 E. coli isolates might not provide
precise and full information on the association between pks and
hly. We therefore further determined the association by PCR
analysis of DNA isolated from at least 50 mg of stools that
contained a large number of E. coli. We found that stool
samples could be positive with both pksþ type I E. coli and
hlyþ type I E. coli, but could also be positive with only one of
them (Fig. 2A). This indicates that pks and hly do not always
coexist. pksþE. coli has previously been demonstrated to promote colorectal cancer development (4, 5), which promoted
us to ask whether hly is a colorectal cancer–associated factor
in addition to pks and if they play different roles in colorectal
cancer development.
hlyþ but not pksþ type I E. coli is associated with both adenoma
and colorectal cancer in females
Quantitative PCR analyses of stool DNA showed that detection rates of neither pksþ nor hlyþ type I E. coli were significantly
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Jin et al.
A
pks+ Type I
1
5
2
H
2 6
5
2
AD
8
2
CRC
1 1
3
0 3
H
20
0
ns
ns
40
20
0
pks+ Type I (%)
hly – Type I (%)
60
ns
60
hly + Type I (%)
hly + Type I (%)
60
C
ns
40
CRC
60
60
40
0
40
20
0
ns
ns
40
20
H
(n = 25)
AD
(n = 30)
CRC
(n = 47)
ns
40
20
0
H
(n = 60)
Males
different among male subject groups (Fig. 2B). When female
subjects were analyzed, detection rate of pksþ type I E. coli was
higher in the colorectal cancer patients than in the healthy
subjects, but did not differ between the healthy subjects and
adenoma patients. In contrast, hlyþ type I E. coli was detected in
36.1% (13/36) of the female colorectal cancer patients, 39.3%
(11/28) of the female adenoma patients, but in only 6.7%
(4/60) of the female healthy subjects (Fig. 2C). Thus, unlike
pksþ type I E. coli that is linked with colorectal cancer but not
with adenoma in females, hlyþ type I E. coli is associated with
both colorectal cancer and adenoma in females. This association is independent of colorectal cancer stage (P ¼ 0.882) or age
(P ¼ 0.445), as revealed by backward elimination analysis.
Smoking is a risk factor for many cancers (19), which led us to
ask whether colonization of hlyþ type I E. coli in the female
patients is associated with smoking. None of the female colorectal cancer patients with smoking information were smokers
or ex-smokers (Supplementary Table S1), ruling out this possibility. In contrast, the three female groups displayed similar
detection rates of hly type I E. coli, so did the three male groups
(Fig. 2C). This indicates that hly type I E. coli is not associated
with colorectal cancer or adenoma in either males or females.
hlyþ but not pksþ type I E. coli correlates with poor survival in
female colorectal cancer patients
Survival analyses showed that the presence of hlyþ type I E.
coli did not affect survival in male colorectal cancer patients
(Fig. 3A) but was associated with shorter survival in female
colorectal cancer patients (Kaplan–Meier, P ¼ 0.026; Fig. 3B).
Survival of the female colorectal cancer patients was not affect-
2894 Cancer Res; 76(10) May 15, 2016
Figure 2.
þ
þ
Detection rates of hly and pks type I
E. coli in different subject groups. A,
þ
þ
partial overlap of hly and pks type I
E. coli in stools of different subject
þ
groups. B, detection rates of pks
þ
type I E. coli, hly type I E. coli, and
hly type I E. coli in stools of male
subject groups. C, detection rates of
þ
þ
pks type I E. coli, hly type I E. coli,
and hly type I E. coli in stools of female
subject groups. H, healthy subjects;
AD, adenoma patients; CRC, colorectal
cancer patients; ns, statistically not
significant; , P < 0.001.
ns
20
ns
0
hly + Type I
Females
hly – Type I (%)
pks + Type I (%)
60
2 10 4
AD
Males
B
8
AD
(n = 29)
CRC
(n = 36)
Females
ed by age (P ¼ 0.836) or colorectal cancer stage (P ¼ 0.516) at
the point of sample collection, as revealed by stepwise regression analysis through backward elimination. Thus, hlyþ type I
E. coli influenced survival independently of these host factors.
When colorectal cancer patients carrying hly type I E. coli (i.e.
carrying type I E. coli but negative with hly) were compared
with other colorectal cancer patients, no difference was observed in survival in either male or female patients (Fig. 3C and D).
Thus, hly type I E. coli is not associated with survival in
colorectal cancer patients. Unlike hlyþ type I E. coli that specifically correlated with shorter survival in female colorectal
cancer patients, pksþ type I E. coli had no effects on survival in
either male or female colorectal cancer patients (Fig. 3E and F).
Collectively, hlyþ type I E. coli but not pksþ type I E. coli
correlates with poor survival in female colorectal cancer
patients.
hlyþ type I E. coli colonizes colonic mucosa, which is
dependent on FimH
Colorectal cancer–causing/promoting bacteria have to be
capable of colonizing the colon. As all the four types of E. coli
produced the fimH-encoded pilus (FimH) that binds to D-mannose structures on the surface of host cells (20), we speculate
that the FimH might facilitate the intestinal colonization of
hlyþ type I E. coli. We examined this possibility in female
BALB/c mice. Specifically, mice were pretreated with antibiotics
to eliminate native bacteria and to facilitate colonization of
fed bacteria. Mice were then daily fed by oral gavage with J47,
its isogenic mutant J47Dhly or J47DfimH for 3 days. E. coli
feeding was then stopped and the fed bacteria in stools were
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Role of Hemolytic Escherichia Coli for Colonic Tumorigenesis
A
B
Males (P = 0.226)
hly + Type I (n = 10)
60
Others (n = 37)
40
20
Survival (%)
Survival (%)
80
Survival (%)
10
20
30
40
60
hly + Type I (n = 14)
40
Others (n = 22)
20
Males (P = 0.638)
D
100
80
hly - Type I (n = 21)
Others (n = 26)
60
40
20
0
0
E
10
20
30
40
0
50 (months)
Survival (%)
0
10
30
40 (months)
Females (P = 0.87)
100
80
hly - Type I (n = 13)
Others (n = 23)
60
40
20
0
10
F
Males (P = 0.208)
20
0
50 (months)
100
20
30
40 (months)
Females (P = 0.476)
100
80
pks + Type I (n = 10)
60
Others (n = 37)
40
20
0
0
10
20
30
40
50 (months)
Survival (%)
Survival (%)
80
0
0
C
Females (*P = 0.026)
100
100
80
pks + Type I (n = 12)
60
Others (n = 24)
40
20
0
0
10
20
30
40 (months)
Figure 3.
þ
þ
Association between hly type I E. coli and survival of colorectal cancer patients. Survival of male (A) or female (B) colorectal cancer patients carrying hly
type I E. coli and those free of this type of E. coli. Survival of male (C) or female (D) colorectal cancer patients carrying hly type I E. coli and those
þ
free of this type of E. coli. Survival of male (E) or female (F) colorectal cancer patients carrying pks type I E. coli and those free of this type of E. coli.
quantified. Compared with J47 and J47Dhly, colonization levels
of J47DfimH were lower and dropped more quickly after E. coli
feeding was stopped. In contrast, colonization levels of J47 and
J47Dhly were comparable (Supplementary Fig. S3A). In another
experiment, antibiotic-pretreated mice were fed by oral gavage
with J47, J47Dhly, or J47DfimH every other day for 4 weeks and
then left untreated. Colonization of the fed bacteria in the
rectum mucosa was tested at 10 weeks. J47 accounted for 2.86%
(SEM, 1.33%) of total mucosal E. coli and J47Dhly accounted
for 0.36% (SEM, 0.21%). There was no significant difference
between J47 and J47Dhly in colonization in the colonic mucosa
(P ¼ 0.095). In contrast, J47DfimH was barely detected in the
colonic mucosa (Supplementary Fig. S3B). These in vivo data
indicate that FimH is required for colonization of the intestinal
tract by hlyþ type I E. coli, making it possible for the bacteria to
interact with the host cells.
þ
hly type I E. coli activates multiple cancer pathways in vitro
We next performed a Cancer Pathway Finder PCR array to
evaluate the tumorigenic potential of hlyþ type I E. coli. The
NCM460 cell line was co-cultured with J47, J47Dhly or PBS for 3
hours, followed by PCR array analysis. MOI was reduced to 25
so that most cells were not killed by bacteria after the coincubation. The reference strain MG1655, which did not promote
colorectal carcinogenesis (16), was employed as an additional
control so that gene regulation caused by irrelevant E. coli could
be ruled out (Fig. 4A). Compared with PBS, MG1655 enhanced
expression of only 10 oncogenes, whereas J47 upregulated
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expression of 17 oncogenes involved in angiogenesis, antiapoptosis, cell cycle, glucose transportation, epithelial-to-mesenchymal transition, metabolism, and response to hypoxia
(Supplementary Table S3), suggesting that hlyþ type I E. coli
activates multiple carcinogenic pathways in vitro. J47 also
increased expression of 8 genes responsible for apoptosis, cell
senescence, and DNA repair, and MG1655 elevated expression
of 6 such genes, showing that induction of these genes is a
common feature of both colorectal cancer–associated and
-irrelevant E. coli. Thus, compared with the irrelevant strain
MG1655, J47 has a carcinogenic potential. Deleting hly abolished regulation of 10 oncogenes that were induced by J47
(10/17 oncogenes, 58.8%), suggesting that the hly-encoded
alpha hemolysin is a key player in the in vitro carcinogenesis
induction mediated by hlyþ type I E. coli.
hlyþ type I E. coli activates GLUT1 and represses BIM through
HIF1a in vivo
We next focused on those that were upregulated by J47 relative
to J47Dhly (>2-fold change). These included three oncogenes
CCL2, FOXC2, and SLC2A1 and one tumor suppressor gene
BCL2L11. BCL2L11 encodes a proapoptotic protein Bcl-2–like
protein 11 (BIM) involved in apoptosis of colorectal cancer cells
(21), whereas CCL2 (22, 23), FOXC2 (24, 25), and SLC2A1
(encoding GLUT1; refs. 26, 27) are overexpressed in human
colonic tumors or indicative of poor prognosis. The above in vitro
data might not reflect in vivo expression regulation. We therefore
tested if the hly-encoded alpha hemolysin was required for
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Jin et al.
A
PBS
J47 Dhly
MG1655
J47
Max (Magnitude of expression)
B
GLUT1
HIF1a
FOXC2
CCL2
Normalized
protein levels
BIM
HPRT1
1 2 3 4 5
J47
C
0.5
1
0
5
10
GLUT1
2
1
P = 0.028
Figure 4.
Regulation of cancer pathways by
þ
hly type I E. coli. A, PCR array gene
expression clustergram of NCM460
treated with PBS or E. coli strains. B,
Western blot analysis of expression of
colorectal cancer-associated genes
þ
that were regulated by hly type I
E. coli J47 via the hly-encoded
alpha hemolysin. C, correlation
between expression of GLUT1, BIM,
and HIF1a in the rectum mucosa of
mice fed for 10 weeks with J47 or
J47Dhly. , P < 0.05;
, P < 0.001.
0.8
0.4
0.4
0.8
1.2
BIM
activation of their expression in vivo. These four genes were
evaluated for their expression in the colonic tissues of mice fed
for 10 weeks with J47 or J47Dhly. No difference was detected
between the two groups of mice in terms of expression of FOXC2
(P ¼ 0.177) or CCL2 (P ¼ 0.368). In contrast to the in vitro
data, feeding J47 resulted in a reduction in the expression of
BIM, which is a tumor suppressor, as compared with its isogenic
mutant J47Dhly (P ¼ 0.022). In agreement with the in vitro PCR
array data, J47 significantly increased the in vivo expression
of GLUT1 than J47Dhly (P ¼ 0.018; Fig. 4B). GLUT1 is known
to be activated by hypoxia-induced factor-1 (HIF1), a transcription factor consisting of a constitutively expressed b-subunit
and HIF1a (28, 29). This led us to examine whether the
hly-encoded alpha hemolysin somehow upregulated HIF1a.
Western blot analysis showed that J47 significantly increased the
levels of HIF1a, compared with J47Dhly (P < 0.001; Fig. 4B).
Moreover, the GLUT1 levels of the colonic mucosa were linearly
correlated with the HIF1a levels (P ¼ 0.001; Pearson correlation
coefficient, r ¼ 0.876; Fig. 4C), indicating that the J47-mediated
upregulation of GLUT1 is dependent on increased HIF1a
levels. BIM is known to be repressed by HIF1 (30, 31). In
agreement with this, our Western blot analysis of protein expression in the colonic mucosa showed that the BIM levels were
negatively correlated with the HIF1a levels (P ¼ 0.048; Pearson
correlation coefficient, r ¼ 0.637). An inverse correlation was
also observed between GLUT1 and BIM (P ¼ 0.028; Pearson
correlation coefficient, r ¼ 0.688; Fig. 4C). Taken together,
these in vivo data show that the hly-encoded alpha hemolysin is
involved in the induction of GLUT1 expression and repression of
BIM at least partially by acting on HIF1a. The inconsistency of
in vitro and in vivo BIM regulation by hlyþ type I E. coli could be
due to the fact that the in vitro cell–E. coli interaction is greatly
2896 Cancer Res; 76(10) May 15, 2016
J47Dhly
1.2
0
0
BIM
HIF1a
P = 0.048
3
1
0
0
GLUT1
J47
HIF1a
HIF1a
2
J47Dhly
2
1
5
0
1 2 3 4 5
P = 0.001
3
3
10
BIM
Min
0
5
10
GLUT1
different from the complex interplay between host cells and E. coli
in the colon.
Alpha hemolysin confers the ability of hlyþ type I E. coli to
promote colorectal tumorigenesis in wild-type mice
Elevated glucose transport and GLUT1 expression are required for the oncogenic transformation of mammalian cells, and
are associated with poor survival of cancer patients (26, 27, 29).
BIM repression is regarded as an important step during colonic
tumorigenesis (32). As the above data showed that hlyþ type I
E. coli activates GLUT1 expression and reduces BIM expression
in a manner dependent on alpha hemolysin, we asked whether
this type of E. coli could induce colonic tumorigenesis in vivo
and if alpha hemolysin is required for this process. We
employed two mouse models for testing these possibilities. In
the first model, AOM-pretreated female BALB/c mice were fed
by oral gavage with J47, J47Dhly, or PBS alone every other
day for 10 weeks. Feeding J47 reduced body weight growth of
mice (Fig. 5A), but had no significant effects on colon length or
inflammation scores (Supplementary Fig. S4A–S4C; ref. 15).
There were polyp-like outgrowths in the colons of all the
groups, and most of them were not real tumors but lymphoid
aggregates that are normally present in the colon (Supplementary Fig. S4D). To correctly quantify colonic tumors, each
colon was made into a Swiss roll, which was then cut into
nine cross sections. Microscopic evaluation of the Swiss rolls
revealed that mice fed with J47 developed significantly more
colonic tumors than mice fed with PBS (P ¼ 0.013), demonstrating that hlyþ type I E. coli promotes colonic tumorigenesis.
Comparison of the J47 group and the J47Dhly group showed
that deleting hly reduced the ability of type I E. coli to induce
tumor growth, but the effects were marginally significant in the
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Role of Hemolytic Escherichia Coli for Colonic Tumorigenesis
Figure 5.
þ
Effects of hly type I E. coli on
colorectal tumorigenesis in female
mice pretreated with AOM. A,
reduction of body weight of mice as a
result of oral gavage with J47. B,
number of colonic tumors determined
by microscopic examination of nine
cross sections of each colonic Swiss
roll stained with hematoxylin and
eosin. C, representative cross-section
picture of colonic Swiss roll of mice fed
with J47, J47Dhly, or PBS. Magnified
are three representative colonic
tumors (red arrows) detected in the
colon of mice fed with J47. Error bar,
SEM; , P < 0.05.
B
30
28
J47 (n = 9)
26
J47Dhly (n = 6)
24
22
PBS (n = 6)
20
Tumor number
per mouse
4
P = 0.059
3
2
1
0
18
0
2
4
6
8
10
J47
(n = 9)
Weeks
J47Dhly
(n = 6)
PBS
(n = 6)
C
J47
J47Dhly
AOM-pretreated mice (P ¼ 0.059; Fig. 5B and C). We further
examined the tumorigenic potential of J47 in mice without
AOM pretreatment. At 22 weeks, 4 of 12 mice (33.3%) fed with
J47 developed colonic tumors, whereas mice fed with J47Dhly
did not develop any tumors (P ¼ 0.039; Fig. 6A and B). We
finally tested whether hlyþ type I E. coli had any effects on
colonic tumorigenesis in male mice. AOM-pretreated male
BALB/c mice were fed by oral gavage with J47, J47Dhly, or PBS
alone every other day for 10 weeks, and then examined for
colonic tumor formation as described above. Although feeding
J47 resulted in colonic tumor formation in some male mice
(1 of 11 male mice; 9.1%), the tumorigenic effects of J47 were
not significant in the male mice as compared with either
J47Dhly (P ¼ 0.478) or PBS (P ¼ 0.519; Fig. 7). Thus, in contrast
to what was observed in female mice in which hemolytic E. coli
A
Tumor number
per mouse
Body weight (g)
A
PBS
significantly increased colonic tumor formation as compared
with PBS buffer (P ¼ 0.013), the effects of hemolytic E. coli on
colonic tumorigenesis were not significant in male mice.
Discussion
In this study, we reveal that hemolytic type I E. coli is a
causative factor for colonic adenoma and colorectal cancer in
females but is not associated with these diseases in males.
Prevalence of hemolytic type I E. coli is dramatically increased
in female patients with colonic adenoma and colorectal cancer
compared with healthy female subjects, and correlates with
poor survival in female colorectal cancer patients (P ¼ 0.026).
In contrast, prevalence of hemolytic type I E. coli is similar
between male colorectal cancer patients, male adenoma
B
2
1 mm
1
0
J47
(n = 12)
J47Dhly
(n = 8)
J47
J47Dhly
Figure 6.
þ
Effects of hly type I E. coli on colorectal tumorigenesis in female mice without AOM pretreatment. A, number of colonic tumors developed in mice,
determined by microscopic examination of nine cross sections of each colonic Swiss roll stained with hematoxylin and eosin. Error bar, SEM. B,
representative cross-section picture of colonic Swiss roll of mice fed with J47 or J47Dhly. Magnified is a representative colonic tumor detected in the
colon of mice fed with J47. , P < 0.05.
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Jin et al.
A
B
Tumor number
per mouse
2
ns
ns
1
0
0.5 mm
J47 J47Dhly PBS
(n = 11) (n = 6) (n = 5)
J47
J47Dhly
Figure 7.
þ
Effects of hly type I E. coli on colorectal tumorigenesis in male mice pretreated with AOM. A, number of colonic tumors developed in mice, determined
by microscopic examination of nine cross sections of each colonic Swiss roll stained with hematoxylin and eosin. Error bar, SEM. B, representative
cross-section picture of colonic Swiss roll of mice fed with J47 or J47Dhly. Magnified is a representative colonic tumor detected in the colon of mice fed
with J47. ns, P > 0.05.
patients and healthy male subjects, and has no effects on the
survival in male colorectal cancer patients. Animal experiments
showed that hemolytic type I E. coli induces tumorigenesis by
activating the oncogenic protein GLUT1 expression and repressing the tumor suppressor BIM expression by elevating levels
of HIF1a, in a manner dependent on the hly-encoded alpha
hemolysin. The carcinogenic capacity of hemolytic type I E. coli
and its requirement for alpha hemolysin were verified in female
mice.
pksþE. coli have been reported to be associated with colorectal
cancer (4, 5). In this study we found that pksþE. coli sometimes
overlap with hlyþ type I E. coli, which is frequently observed in
male subjects and in female colorectal cancer patients but
relatively rare in healthy female subjects or females with adenoma. Although pksþE. coli partially overlap with hlyþ type I
E. coli, their roles for colorectal cancer and adenoma are different in at least two aspects. First, hlyþ type I E. coli correlates with
shorter survival in female patients while pksþ type I E. coli does
not; second, hlyþ type I E. coli is associated with both adenoma
and colorectal cancer, whereas pksþ type I E. coli is associated
with colorectal cancer but not with adenoma in females. The
link between hlyþ type I E. coli and adenoma suggests that these
bacteria pre-exist the onset of colorectal cancer and supports
our in vitro and in vivo evidence that they are the cause but not
the consequence of colorectal cancer. As it takes a long time for
adenoma to develop into colorectal cancer, the adenoma specificity of hlyþ type I E. coli suggest that these bacteria can be used
as potential antecedent biomarkers for colorectal cancer.
Alpha hemolysin binds nonspecifically to host cells and triggers
cellular reactions without the need for a receptor (33). We
speculate that hemolytic type I E. coli elevates levels of HIF1a
by indirect mechanisms. Alpha hemolysin of E. coli is a poreforming toxin, generating small cation-permeable channels in the
host cell membrane. Alpha hemolysin enables hemolytic E. coli to
induce "focal leaks" both in colonic cells and tissues, within which
hemolytic E. coli accumulates (34, 35). E. coli is a group of facultative anaerobic bacteria, making ATP by aerobic respiration in the
presence of oxygen. Thus, the hemolytic E. coli trapped in colonic
tissues would deplete oxygen, generating a hypoxic microenvironment, which is known to stabilize HIF1a and increase HIF1a
activity (36). Therefore, hemolytic type I E. coli mediates activation of HIF1a and consequently induces colonic tumorigenesis
at least in part by generating focal hypoxia. Although many
intestinal bacteria consume oxygen, most of them are incapable
of making pores in the colonic tissue and generating a hypoxic
2898 Cancer Res; 76(10) May 15, 2016
microenvironment surrounding colonic cells. Thus, the hypoxic
mechanisms could be specific to these "pore-making" hemolytic
bacteria, which are primarily hemolytic type I E. coli in the colon.
By creating channels in the host cell membrane, hemolytic E. coli
also cause ion oscillations such as elevation of intracellular
calcium (33, 37), which in turn could lead to calcium-dependent
HIF1a activation (38). Thus, it is speculated that hemolytic E. coli
regulates HIF1a levels and activity probably through multiple
pathways.
HIF1 regulates diverse oncogenic pathways (31), and accordingly hemolytic E. coli could regulate a quite number of downstream genes through HIF1. We showcased that the HIF1activated GLUT1 and HIF1-repressed BIM were induced and
repressed in vivo by hemolytic E. coli, respectively. Elevated
GLUT1 expression and glucose uptake are features of oncogenic
transformation of mammalian cells. GLUT1 is also associated
with poor survival of cancer patients (26, 27, 29). Therefore,
not only the carcinogenic capability of hemolytic E. coli but also
the poor survival in female colorectal cancer patients carrying
this type of E. coli could be partially attributed to the HIF1mediated activation of GLUT1.
Despite the requirement of alpha hemolysin for tumorigenesis mediated by this type of E. coli, alpha hemolysin is also
cytotoxic to colon epithelial cells. It is a paradox that the
hemolysin acts as a carcinogenic factor but is meanwhile cytotoxic and apoptotic to both normal and cancerous cells. Unlike
in vitro conditions, under which cultured cells are quickly killed
by alpha hemolysin, in vivo environments support continuous
repairment of colonic tissues so that alpha hemolysin is carcinogenic to colonic tissues but is not as potent as to inhibit
tumor development. These may explain why the net outcome of
hlyþ type I E. coli infection is increased colorectal cancer carcinogenesis. In addition, in vitro expression regulation may sometimes fail to mimic in vivo regulation. Indeed, our data showed
that the tumor suppressor BIM was induced by hlyþ type I E. coli
in vitro but repressed by this bacterium in vivo.
Another interesting finding is that hemolytic type I E. coli is
specifically associated with adenoma and colorectal cancer in
females. It is beyond the scope of this study but warrants further
investigation why females are more vulnerable to colorectal
cancer carcinogenesis mediated by hemolytic type I E. coli.
Gender-dependent differences have been known to exist in the
etiology and pathogenesis of a long list of diseases such as heart
failure (39), viral infections (40), and Alzheimer disease (41).
Mechanisms underlying the gender difference remain poorly
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Published OnlineFirst March 24, 2016; DOI: 10.1158/0008-5472.CAN-15-2083
Role of Hemolytic Escherichia Coli for Colonic Tumorigenesis
understood and may involve complex processes. In the case of
the gender specificity of hemolytic E. coli-mediated colonic
tumorigenesis, the genetic factor is ruled out because our in
vitro data showed that hemolytic E. coli had cytotoxic and
tumorigenic effects on cell lines that are derived from men. It
is therefore speculated that the gender specificity observed in
this study might be associated with immunological, hormonal,
or behavioral factors that have been linked to gender difference
in disease development (40–43). Our animal experiments
showed that hemolytic E. coli significantly induced colonic
tumorigenesis in female mice but failed to do so in male mice,
confirming the gender specificity of hemolytic E. coli-mediated
colonic tumorigenesis. It is, however, noteworthy that gender
difference in mice does not fully mimic that in humans, as the
latter is further complicated by social factors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): Y. Jin, W. Li, S.C. Ng, M.W.Y. Chan
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): Y. Jin, S. Tang, M. Chan
Writing, review, and/or revision of the manuscript: Y. Jin, S.C. Ng, J.J.Y. Sung,
J. Yu
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): Y. Jin, W. Li, J. Yu
Study supervision: Y. Jin, S.C. Ng, J. Yu
Grant Support
This project was supported by 863 Program China (2012AA02A506
to J. Yu), 973 Program China (2013CB531401 to J. Yu), National Natural
Science Foundation of China (NSFC) for Young Scientists (C010301
to Y. Jin), Shenzhen Technology and Innovation Project Fund
(JSGG20130412171021059 to J. Yu), Shenzhen Virtual University Park
Support Scheme to CUHK Shenzhen Research Institute (J. Yu), and 973
Program China (2014CB745200 to Y. Jin).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Authors' Contributions
Conception and design: Y. Jin, J.J.Y. Sung, J. Yu
Development of methodology: Y. Jin, J. Yu
Received July 30, 2015; revised January 20, 2016; accepted February 18, 2016;
published OnlineFirst March 24, 2016.
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Published OnlineFirst March 24, 2016; DOI: 10.1158/0008-5472.CAN-15-2083
Hemolytic E. coli Promotes Colonic Tumorigenesis in Females
Ye Jin, Senwei Tang, Weilin Li, et al.
Cancer Res 2016;76:2891-2900. Published OnlineFirst March 24, 2016.
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