Bladder cancers arise from transformed urothelial cells that line the bladder. These
cancers are urothelial or squamous cell carcinomas or, more rarely, additional histologic
variants such as adenocarcinoma. The most important bladder cancer risk factors worldwide
are arguably smoking and urogenital schistosomiasis. The parasitic Schistosoma haematobium
worm causes urogenital schistosomiasis in approximately 112 million people, primarily
in sub-Saharan Africa and the Middle East [1]. During infection, S. haematobium worms
lay highly inflammatory eggs in the bladder wall. This inflammation is thought to
promote carcinogenesis through unclear mechanisms. People with chronic urogenital
schistosomiasis exhibit increased risk and earlier onset of bladder cancer (up to
two decades earlier), with a predominance of squamous cell carcinoma [2]. Consequently,
S. haematobium has been categorized as a Group I carcinogen (“carcinogenic to humans”)
by the International Agency on Research on Cancer of the World Health Organization
[3].
Although S. haematobium is an accepted bladder carcinogen, many patients with schistosomal
bladder cancer present at advanced stages [4]. We posit that this is due to a combination
of poor medical infrastructure in endemic areas and a lack of diagnostic and prognostic
tools for schistosomal bladder cancer and its precursor epigenetic events.
One promising strategy for improving the diagnosis and prognostication of schistosomal
bladder cancer is analysis of urothelial DNA methylation. This epigenetic modification,
in which cytosine bases are converted to methylcytosines, is becoming increasingly
appreciated as a source of carcinogenesis in multiple cancers, including leukemias,
lymphomas, and bladder, colon, and esophageal carcinomas [5,6]. Not surprisingly,
DNA methylation also has been implicated in preneoplastic changes in many of these
same tissues, e.g., in the colon and esophagus (reviewed in [7]). Furthermore, there
is evidence that DNA methylation changes may also be elicited by uropathogenic Escherichia
coli infections, a known risk factor for bladder cancer (as in the well-established
case of Helicobacter pylori in gastric carcinogenesis) [8]. One major pathway by which
DNA methylation likely contributes to carcinogenesis is reversible, promoter methylation-induced
silencing of expression of tumor suppressor genes [7]. Over time, promoter methylation-induced
silencing of expression of tumor suppressor genes may lead to accumulation of additional
procarcinogenic DNA methylation events and outright mutations.
Indeed, DNA hypermethylation of numerous genes, including the tumor suppressor genes
RASSF1A and TIMP3, has been identified in the urine of patients with urogenital schistosomiasis[9].
Other investigators have identified DNA methylation of RARbeta2 and APC as potential
urine biomarkers of bladder cancer, with schistosomiasis-associated cases having higher
rates of methylation of these genes [10]. Yet another group reported differential
methylation of CDH1, DAPK1, CDKN2A, MGMT, ICDKN2B, FHIT, APC, RASSF1, GSTP1, RARB,
and TP73 in bladder cancer specimens, with schistosomiasis-associated specimens featuring
more differentially methylated genes than those not associated with this infection
[11]. Interestingly, Saad et al. reported that levels of N7-methylguanine, a form
of methylated guanine, were more frequently elevated in cancerous versus normal bladder
tissues from patients with bladder cancer, regardless of whether it was associated
with schistosomiasis or not [12]. However, this marker was not further increased in
the subset of patients with schistosomal bladder cancer (relative to non-schistosomiasis-associated
cancers), suggesting that DNA methylation is not unique to schistosomal bladder cancer.
Regardless, hypermethylation of genes (especially tumor suppressor genes) may be a
key cause of reversible preneoplastic lesions of the bladder urothelium exposed to
S. haematobium infection.
Despite the importance of S. haematobium as a bladder carcinogen, studies of the associated
basic cancer biology (including the role of DNA methylation) have been limited because
of a historical lack of tractable animal models for urogenital schistosomiasis. To
address the need for tractable tools to study schistosomal cancer biology, we developed
the first experimentally tractable mouse model of urogenital schistosomiasis [13].
In this model, microinjection of S. haematobium parasite eggs into the bladder walls
of mice leads to rapid urothelial hyperplasia [13] and squamous metaplasia. Indeed,
urothelial hyperplasia persists for at least 3 months after egg exposure [13]. Thus,
our approach recapitulates key preneoplastic changes in the bladder associated with
urogenital schistosomiasis.
To identify what bladder urothelial DNA methylation events occur in the preneoplastic
period in our mouse model of urogenital schistosomiasis, we microinjected S. haematobium
eggs into the bladder walls of mice and 2 weeks later microdissected the urothelium
of each bladder from the remaining bladder tissue. The granuloma and the detrusor
tissue were discarded (S1 Fig). Granuloma formation was noted in all S. haematobium-injected
bladders. We then used reduced representation bisulfite sequencing (RRBS) [14,15]
to profile DNA methylation across the urothelial genome. RRBS focuses on methylation
of cytosines within CpG dinucleotide “islands,” which are genomic regions enriched
for potential DNA methylation sites. In brief, DNA was extracted from each specimen
and the restriction enzyme Msp1 used to fragment DNA at CpG islands. After bisulfite
treatment samples were fragmented to a length of 175–225 bp, they were then amplified
by PCR. Multiplexed sequencing was performed using the Illumina Hi-Seq platform. The
output was aligned to the Mouse Genome Reference Consortium Mus musculus genome (GRCm38/mm10)
using Bismark software. Methylation analysis was performed with methylKit and the
Integrative Genomics Viewer (IGV).
Using thresholds of >10x coverage, >25% difference in methylation, and p < 0.05, we
found that short-term exposure of the mammalian bladder to S. haematobium infection
led to massive changes in DNA methylation of the urothelium. S. haematobium egg-injected
mice featured major alterations in their methylome (versus control mice) 2 weeks post-egg
exposure (Fig 1). 13,333 cytosines were hypermethylated, and 6,244 were hypomethylated.
These data were processed using a promoter identification algorithm and subsequently
fed into Database for Annotation, Visualization and Integrated Discovery (DAVID) pathways
analysis. Of these differentially methylated cytosines, 1,019 were found to be within
1,000 base pairs of a transcription start site for a known gene (i.e., putative promoter
regions). Six of these genes are part of the Wnt canonical pathway (Fig 2), which
is related to cell proliferation. A CpG in the promoter of the Wnt inhibitory factor-1
(Wif1) gene, a gene silenced by hypermethylation in bladder tumors and other cancers
[16,17], was methylated 54% of the time in egg-injected mice, 34% in nitrosamine-fed
mice, and 7% in control mice. Thus, even short-term exposure of the mammalian bladder
to S. haematobium eggs results in profound alterations in DNA methylation, including
in the promoters of known tumor suppressor genes such as Wif1. Indeed, through qPCR
we discovered that WIF-1 expression was decreased in mouse urothelia after subepithelial
bladder injections with eggs versus control vehicle (0.58-fold expression in six egg-injected
mice compared to two control-injected mice, 95% CI 0.475–0.713). Primary sequencing
data can be found at http://www.ncbi.nlm.nih.gov/bioproject/PRJNA278470.
10.1371/journal.pntd.0003696.g001
Fig 1
Exposure of the bladder to S. haematobium eggs induces massive shifts in the urothelial
methylome.
Genome map demonstrating that multiple loci are hypermethylated (magenta dots) and
hypomethylated (green dots) in the mouse urothelium exposed to S. haematobium eggs
versus vehicle controls. Representative map from one of two experimental replicates
shown.
10.1371/journal.pntd.0003696.g002
Fig 2
S. haematobium eggs induce differential methylation of multiple members of the Wnt
signaling pathway, including the bladder cancer-associated tumor suppressor gene Wif1.
Differentially methylated gene members of the Wnt signaling pathway are circled in
red. Note that Wif1, a tumor suppressor gene implicated in bladder carcinogenesis,
is differentially methylated and sits far upstream along the Wnt pathway. Figure generated
using DAVID (http://david.abcc.ncifcrf.gov/).
Given that S. haematobium eggs induced differential methylation of many urothelial
genes, we sought to reverse this process pharmacologically. We gave the DNA methylation
inhibitor 5-fluoro-2ʹ-deoxycytidine (FdCyd, 12.5 mg/kg) and tetrahydrouridine (THU,
an inhibitor of FdCyd metabolism, 25 mg/kg) on an every other day basis (14 days total)
intraperitoneally to S. haematobium egg-injected mice. This drug combination suppressed
S. haematobium egg-induced urothelial hyperplasia, a key preneoplastic change in the
bladder (Fig 3). Specifically, mice treated with FdCyd and THU had a mean urothelial
thickness of 67 μm (±19) and those treated with vehicle injections had a mean thickness
of 101 μm (±26, p = 0.043—unpaired Student’s t test).
10.1371/journal.pntd.0003696.g003
Fig 3
In vivo inhibition of DNA methylation prevents S. haematobium egg-induced urothelial
hyperplasia, a potential preneoplastic lesion of the bladder.
(A) Every other day administration of the DNA methylation inhibitor 5-fluoro-2ʹ-deoxycytidine
(FdCyd) (12.5 mg/kg) and tetrahydrouridine (THU, 25 mg/kg, an inhibitor of metabolism
of FdCyd) for 14 days prevents S. haematobium egg-induced urothelial hyperplasia (n
= 4 mice, right panel) compared to vehicle control (n = 3 mice, left panel). The yellow
and black arrows denote the thickness of the urothelium overlying the egg-induced
bladder granuloma. (B) Bar graph depicting urothelial thickness from (A) in graphical
format (“DNMTI” and “Vehicle” indicate the DNA methylation inhibitor- and vehicle-treated
groups, respectively).
To our knowledge, this is the first in vivo demonstration that pharmacologic inhibition
of DNA methylation can prevent preneoplastic changes in the bladder. Thus, manipulating
DNA methylation may be a promising approach to slow or prevent bladder carcinogenesis
in high-risk patient populations, namely those with extensive exposure to carcinogens
such as urogenital schistosomiasis. Mass drug administration campaigns, snail control,
better water hygiene, and public education regarding schistosomiasis are likely to
have the greatest impact on bladder cancer associated with S. haematobium. However,
a period of chronic bladder inflammation caused by parasite eggs may be enough to
predispose individuals to subsequent metaplastic changes, even after successful treatment
[18].
Admittedly, our suggestions are highly speculative, and much more detailed work is
required to characterize any mechanistic relationships between urogenital schistosomiasis-induced
urothelial DNA methylation and bladder oncogenesis. Nonetheless, there are indications
that chronic cystitis and squamous cell carcinoma of the bladder, both linked to urogenital
schistosomiasis, may have causal associations with bladder urothelial DNA methylation
[19,20]. If a relationship between S. haematobium infection-mediated bladder urothelial
DNA methylation and bladder oncogenesis is confirmed, then we propose that it may
be possible to identify S. haematobium-infected or former patients at high risk of
bladder carcinogenesis through urine testing for urothelial DNA methylation patterns.
Administration of DNA methylation inhibitors such as FdCyd, which are currently in
clinical trials for urothelial carcinoma, could then be evaluated for their ability
to reduce the risk of these patients developing advanced schistosomal bladder cancer.
Ethics Statement
All animal work was conducted according to relevant United States and international
guidelines. Specifically, all experimental procedures were carried out in accordance
with the Administrative Panel on Laboratory Animal Care (APLAC) protocol and the institutional
guidelines set by the Veterinary Service Center at Stanford University (Animal Welfare
Assurance A3213-01 and US Department of Agriculture [USDA] License 93-4R-00). Stanford
APLAC and institutional guidelines are in compliance with the US Public Health Service
Policy on Humane Care and Use of Laboratory Animals. The Stanford APLAC approved the
animal protocol associated with the work described in this publication.
Supporting Information
S1 Fig
Dissection of the urothelium of the mouse bladder.
(A) An S. haematobium egg-injected bladder fileted open and immobilized with pins.
The black oval denotes a subepithelial egg granuloma. (B) Hematoxylin and eosin staining
of the muscular detrusor layer of the bladder, which has been dissected away, leaving
the (C) isolated urothelium available for downstream analyses.
(TIF)
Click here for additional data file.