International Journal of Surgery 53 (2018) 178–183

Contents lists available at ScienceDirect

International Journal of Surgery

journal homepage: www.elsevier.com/locate/ijsu

Review

DNA methylation as an epigenetic regulator of gallbladder cancer: An T

overview
Jibran Sualeh Muhammada,b,∗, Muhammad Rizwan Khanc, Kulsoom Ghiasb
a
Department of Basic Medical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates
b
Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi, Pakistan
c
Department of Surgery, The Aga Khan University, Karachi, Pakistan

A R T I C LE I N FO A B S T R A C T

Keywords: Gallbladder cancer (GBC) is a lethal health issue affecting mostly the women in their middle-age. High incidence
Gallbladder of GBC has been reported across the world specifically in Asian countries, India and Pakistan. The exact etiology
Gallbladder cancer remains unknown, although several risk factors and genetic aberrations involving mutations or epigenetic
Epigenetic regulation changes may be involved in gallbladder carcinogenesis. This article presents a review of the published literature
DNA methylation
mainly from the year 2003 onwards. The topic of main concerns was epigenetic regulation of GBC. All relevant
studies identified were included and are described according to the aforementioned subheadings. In this review,
we have discussed the role of DNA methylation in GBC, clinical implication and future prospects of biomarker
development for early diagnosis and therapeutic interventions.

1. Introduction
Gallbladder cancer (GBC) is a rare, but often fatal malignancy with
ethnic, geographic, and socioeconomic variation in its incidence.
Recent statistics reveals greater than 10,000 new cases and over 3000
deaths per year due to GBC in the United States alone [1]. High in-
cidence of GBC has also been reported in South American and South
Asian countries, including India and Pakistan [2–4]. More than 70% of
GBC patients are female, making it one of the most common malig-
nancies of the gastrointestinal tract in women from these regions [2–6].
Some of the cases of GBC are found incidentally in patients undergoing
cholecystectomy for the treatment of cholecystitis or cholelithiasis, but
most of the patients usually presents late with subtle nonspecific
symptoms. By then, the cancer has usually reached at an advanced
stage and possesses a high risk of metastases. Complete removal of the
gallbladder with adjacent liver parenchyma and the draining lymph
nodes by a specialized surgeon is the only curative treatment option,
which may not be possible in advanced cases. Research on GBC has
therefore been focused on identifying biomarkers for early diagnosis
and molecular targets for therapeutic interventions.
One particular area of focus is epigenetics, which may play a role in
disease onset and progression. Epigenetic mechanisms, such as DNA
methylation, histone modification, and regulation of gene expression by
microRNA (miRNA), are defined as involving changes to the phenotype
without any change in DNA sequence itself [7]. Of these key epigenetic
mechanisms, DNA methylation has been most extensively studied with
regards to regulation of carcinogenesis [8]. DNA methylation results in
reversible changes and therefore are an attractive therapeutic target for
cancer treatment. From the year 2003 onwards, several studies on the
role of DNA methylation in GBC were reported for global populations,
including from United States, Chile, and several Asian countries. Most
of these studies were performed on Chilean population, reporting hy-
permethylation in genes related to cell adhesion, cell cycle, and p53
pathways. On the other hand, studies from Indian population showed a
significant role of increased promoter methylation of tumor suppressor
genes with respect to advance stages of GBC [9,10]. Hence, high in-
cidence and poor prognosis of GBC reported in these Asian and Amer-
ican countries was directly related to gene regulation via promoter
hypermethylation. In this systematic review, we present the highlights
of relevant studies reporting promoter methylation of specific genes
involved in epigenetic regulation of GBC. Based on the available lit-
erature, we also provide an insight into clinical implications, ther-
apeutic approaches and future directions in the field.
2. Methods
We used the MeSH terms “gallbladder” and “gallbladder cancer”
with “epigenetics,” or “DNA methylation,” to search the scientific da-
tabases, such as SCOPUS, Ovid, PubMed and Web of science. To identify
additional studies, we hand-searched the reference lists of the studies

∗
Corresponding author. Department of Basic Medical Sciences, College of Medicine, University of Sharjah, Sharjah, United Arab Emirates.
E-mail address: dr.jibran@live.com (J.S. Muhammad).

https://doi.org/10.1016/j.ijsu.2018.03.053
Received 14 August 2017; Received in revised form 20 March 2018; Accepted 21 March 2018
Available online 27 March 2018
1743-9191/ © 2018 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.
J.S. Muhammad et al.
Fig. 1. Flowchart outlining the search strategy employed to identify the relevant studies.
identified using MeSH terms. The flowchart in Fig. 1 depicts the search
strategy employed to identify relevant studies.
3. Epigenetic regulation of carcinogenesis
Epigenetic alterations associated with cancer development includes
silencing of tumor suppressor genes via methylation, inactivation of
DNA repair genes by histone acetylation and miRNA regulation of cell
proliferation, differentiation, and apoptosis [11]. Aberrant epigenetic
changes in the DNA causing alteration and deregulation of the normal
cell cycle processes leads to abnormal cell survival and initiation and
progression of carcinogenesis. DNA methylation involves the conver-
sion of the nucleotide cytosine, specifically found before guanine in
CpG islands, into methylcytosine [12]. The promoter region of nu-
merous tumor suppressor genes and DNA repair genes contains CpG
islands and are, therefore, highly prone to methylation. Hypermethy-
lation of the promoter region of a gene directly blocks the binding of
transcription factors essential for gene expression, thus rendering loss of
expression or gene silencing [13]. Studies have reported higher me-
thylation levels of various tumor suppressor genes, such as APC,
BRCA1, and Long interspersed nuclear elements (LINE-1), which are
associated with the development of breast cancer [14]. Hypermethy-
lation of other genes besides tumor suppressor genes has also been
strongly linked with cancer initiation and progression. A recent study
showed that hypermethylation-induced silencing of an autophagy-re-
lated gene was directly associated with carcinogenic transformation in
179
International Journal of Surgery 53 (2018) 178–183
gastric epithelial cells [15]. With regards to GBC, the role of DNA
methylation has been extensively studied as compared to histone
modification. Only a few studies have reported differential expression
of miRNAs in GBC [16–18]. However, solid evidence is still lacking with
respect to any link between methylation of miRNA or histone mod-
ifications in the development of gallbladder carcinogenesis. Therefore,
the main focus of this review is the role of DNA methylation in gall-
bladder carcinogenesis.
4. DNA methylation as an epigenetic regulator of gallbladder
cancer
Chronic inflammation of the gallbladder as a consequence of gall
stones or bacterial infections is a well-documented risk factor for GBC.
Chronic cellular irritation might be responsible for activation of in-
tracellular enzymes involved in hypermethylation of the promoter re-
gion of tumor suppressor or cell cycle regulating genes. These epige-
netic changes, in turn, contribute to early-stage tumorigenesis in GBC
and are also seen in cholelithiasis. The methylation profile of previously
reported genes and the functional significance of the epigenetic changes
in GBC are summarized in Table 1. A perspective figure explaining the
association of Gallbladder stones, and chronic inflammation leading to
DNA methylation of specific genes is shown (Fig. 2).
J.S. Muhammad et al. International Journal of Surgery 53 (2018) 178–183

Table 1
Description of the genes frequently found methylated in gallbladder cancer.
Gene Gene function (http://www.genecards.org/) Reference

APC Encodes a tumor suppressor protein and involved in cell migration, cell adhesion, transcriptional activation, and apoptosis [9, 10, 19–22]
SHP1 Regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation [10]
3-OST-2 Gene knockdown increases cell proliferation, invasion and migration [10]
CDH1 & CDH13 Loss of function of these genes is thought to contribute to cancer progression by increasing proliferation, invasion, and/or [10, 19–21, 27]
metastasis
PTEN Functions as a tumor suppressor by negatively regulating AKT/PKB signaling pathway and regulation of cell cycle and cell cycle [31, 32]
progression
CDKN2A/p16 Cell cycle regulation by slowing the progress of dividing cells from G1 to S phase [10, 19–22, 34, 35, 37, 38]
WIF-1 This gene functions as a tumor suppressor gene, and has been found to be epigenetically silenced in various cancers [40, 41]

4.1. Adenomatous polyposis coli (APC) (5q22.2)
Tekchamen et al. reported hypermethylation and subsequent down-
regulation of the adenomatous polyposis coli (APC) gene promoter in
96% of GBC patient samples studied in the Indian population.
Methylation-specific (MS)-PCR was used to analyze the methylation of
APC gene. The expression of APC gene was studied by semi-quantitative
PCR, real-time PCR and immunohistochemistry. Although methylation
was also observed in 80% of the gallbladder tissue samples from pa-
tients with gallstone disease, functional loss of APC was seen more in
GBC suggesting that methylation silencing of APC may play a role in the
early stages of gallbladder carcinogenesis [9]. Studies from Chile also
reported methylation of the APC gene promoter region [19–22], with a
progressive increase in the level of the methylation status of several
genes from chronic cholecystitis to advanced carcinoma. Functional
loss of APC is known to activate Wnt signaling pathway and an increase
in the levels of the transcription factor β-catenin inside the nucleus,
resulting in cellular proliferation [24]. The functional significance of
methylation of APC has been studied extensively in colorectal carci-
noma, and loss of APC secondary to chronic inflammation due to
gallstones may be similarly responsible for GBC carcinogenesis.
4.2. SHP1 (12p13.31)
This protein encoded by PTPN6 gene is known to modulate sig-
naling by tyrosine phosphorylated cell surface receptors such as KIT
and the EGFR. Takahashi et al. evaluated 24 tumor suppressor genes for
aberrant promoter methylation in gallbladder tissue samples from pa-
tients with chronic cholecystitis without cancer and tissue samples from
GBC patients. The MS-PCR and combined restriction analysis methods
were used to detect methylation. The study revealed significantly
higher methylation frequencies in several genes in the cancer samples
as compared to cholecystitis samples. Of genes tested, SHP1 methyla-
tion was highest in GBC in 80% of tumor samples [10]. Interestingly,
SHP1 was also methylated in 88% of chronic cholecystitis samples.
SHP1 is a tyrosine phosphatase known to regulate cell growth and
apoptosis [25]. While abnormal SHP1 expression has been reported in
various cancers [26], its significance has not been explicitly studied in
GBC. Hypothetically, hypermethylation and silencing of SHP1 in GBC
could lead to increased cell proliferation. The same study also reported
higher methylation frequency of 3-O-sulfotransferase 2 (3-OST-2) gene
in GBC samples as compared with cholecystitis samples [26]. Previous
studies in breast cancer have suggested a role for 3-OST-2 in the pro-
gression to malignant cells in early stages of the diseases [27], with
gene knockdown strongly linked to increased cell proliferation, inva-
sion, and migration. Similar to SHP1, a decrease in expression of 3-OST-
2 is hypothesized to be involved in gallbladder carcinogenesis.
4.3. CDH1 (16q22.1)
This gene is responsible for encoding the Cadherin family of pro-
teins essential for regulating cell-cell adhesions by interacting with
themselves in a homophilic manner, and are frequently hypermethy-
lated in many types of cancers. CDH1, which encodes for a protein

Fig. 2. A perspective figure showing association of Gallbladder stones, and chronic inflammation leading to DNA methylation of specific genes.

180
J.S. Muhammad et al.
called E-cadherin, was found to be methylated to varying degrees in
GBC in different populations, specifically in 41% of Japanese samples
[28] and in more than 65% of Chilean patients [19–21,27]. Another
gene related to cell adhesion, CDH13, was found to be hypermethylated
in up to 80% of gallbladder cancer patients [11,20]. Epigenetic silen-
cing, detected using MS-PCR, or loss of function of both these genes
contributes to cancer progression by increasing proliferation, migra-
tion, invasion, and metastasis.
4.4. PTEN (10q23.31)
Another tumor suppressor gene, PTEN, is often found to be silenced
by methylation in several cancers, including breast cancer [29]. PTEN
negatively regulates intracellular levels of phosphatidylinositol-3,4,5-
trisphosphate in cells and functions as a tumor suppressor by inhibiting
the AKT/PKB signaling pathway [30]. Ali et al. recruited 141 con-
secutive GBC patients in India, of which 48% showed hypermethylation
of the promoter region of PTEN, using MS-PCR. The methylation status
was not associated with the level of metaplasia and was similar in all
stages of GBC. Immunohistochemical analysis revealed decreased PTEN
expression in almost all methylated samples [31]. In another study from
India, Tekcham et al. [32] showed PTEN promoter methylation in 30%
of GBC patients and in 22% of patients with gallstones. RT-PCR analysis
of PTEN in advanced GBC cases revealed significantly lower expression,
especially in grade III cancer samples compared to those of other genes,
suggesting that PTEN promoter methylation could be a useful bio-
marker for diagnosis or prognosis of GBC.
4.5. Cyclin dependent kinase inhibitor 2A (CDKN2A)/p16 (9q21.3)
CDKN2A, commonly known as p16, is a tumor suppressor gene
frequently mutated or deleted in a wide variety of cancers. p16 plays a
key role in regulation of the cell cycle, specifically by interacting with
CDK4 and CDK6, inhibiting their ability to interact with Cyclin D, hence
slowing the progression of rapidly dividing cells from G1 to S phase.
Genetic or epigenetic reactivation of p16, therefore, is a potential ap-
proach for cancer therapy [33]. Of the 51 cancerous gallbladder spe-
cimens collected and analyzed in the Japanese population, Tadokoro
et al. reported methylation and loss of expression of p16 in 73% and
62% of cases, respectively [34]. Decreased expression was attributed
either to loss of allele heterozygosity or promoter hypermethylation, as
no p16 mutations were detected. While the sample size was limited,
another study also showed p16 gene promoter methylation in 22% of
GBC patients [35]. Several studies from Chile [11,19–22,36], Germany
[37] and China [38] have investigated the role of p16 and reported
varying frequency of gene hypermethylation, ranging from 20% to 60%
in GBC patients. All studies have used MS-PCR to analyze methylation
levels. Overall, methylation-induced loss of p16 gene expression is
significantly associated with various premalignant and malignant le-
sions in GBC and could be an early event in cancer progression [23].
4.6. WIF1 (12q14.3)
The Wnt inhibitory factor 1 gene is an inhibitor of the Wnt pathway.
Abnormal activation of the Wnt pathway is known to increase cell
proliferation and differentiation, leading to tumor development [39]. A
study in a gallbladder cancer cell line showed a low expression of WIF-
1, which was associated with promoter methylation of the WIF1 gene
[40]. Treatment of the GB-SD cells with a demethylating agent restored
expression of WIF1. Additionally, transfection with WIF1 expression
plasmid inhibited cell proliferation, invasion, and metastasis accom-
panied by increased expression of matrix metalloproteinase. Another
study has shown that the level of WIF1 protein expression is low in WIF-
1 hypermethylated specimens and multivariate analysis revealed that
WIF-1 methylation was an independent prognostic factor for 5-year
overall survival [41].
181
International Journal of Surgery 53 (2018) 178–183
4.7. Others
FHIT, DUTT1, BLU, and SEMA3B, genes present on the short arm of
chromosome 3 and involved in cell cycle regulation and DNA repair
regulation, have been reported to be associated with several epithelial
cancers. Riquelme et al. [42] hypothesized that aberrant methylation of
the CpG islands of the promoter region of these genes might also be
linked with gallbladder carcinogenesis. All four genes were found to be
hypermethylated; 92% of surgically resected GBC cases exhibited me-
thylation in the promoter region of SEMA3B, 66% of cases in FHIT, 26%
in BLU and 22% in DUTT1, suggesting that these tumor suppressor
genes are a more fragile target for aberrant promoter methylation
contributing to the pathogenesis of GBC.
Several other genes have also been reported to be methylated in
GBC samples with intermediate to low frequencies. Gracia et al. [20]
analyzed both pre-neoplastic and neoplastic gallbladder tissue samples
in the Chilean population for methylation of DLC1, DAPK1, MGMT,
TIMP3 and RARβ2 genes. Of these, only DLC1 gene methylation was
more common in invasive cancers and was associated with poor sur-
vival [20]. In a study in a Korean population, frequent methylation was
found in RASSF1 gene (22.7%) in GBC patients [43], prompting the
authors to infer a possible role in gallbladder cancer development. In
another Korean study [44], PGP9.5 promoter hypomethylation was
found to be a reliable marker for GBC development. Another novel
tumor suppressor gene, cysteine dioxygenase type 1 (CDO1) gene was
recently reported to be epigenetically regulated in GBC. These findings
suggested that CDO1 methylation was significantly associated with the
phenotypic acquisition of invasive and metastatic properties in primary
GBC. Notably, CDO1 hypermethylation showed prognostic relevance,
especially in stage II GBC, in which it is highly anticipated to work as a
predictive marker for candidates of adjuvant therapy [45].
5. Clinical implications and future directions
Gallbladder carcinogenesis is usually considered a multistep process
attributed to both genetic and environmental factors. The carcinogenic
models in the development of gallbladder cancer have been well de-
scribed in the past and include the metaplasia-dysplasia-carcinoma se-
quence or the adenoma-carcinoma sequence [46]. There is some clin-
ical and experimental data to support both the models, but severe
dysplasia has been found in most of the cases of gallbladder cancer
suggesting that the former sequence is a frequent pathway [47]. There
is also some data from Pakistan indicating that approximately 40% of
the patients with gallstones have intestinal metaplasia of the gall-
bladder epithelium, which is considered a precursor lesion for gall-
bladder cancer [6]. Previous studies suggest a long latency period for
gallbladder carcinogenesis and approximately 15 years may be required
for progression from dysplasia to advanced carcinoma [48,49]. There-
fore, efforts are needed to identify the genetic and environmental fac-
tors responsible for the development of precursor lesions many years
before the appearance of cancer. Epigenetic changes, if detected early
in the course of the disease, may help in early diagnosis of the gall-
bladder cancer resulting in improved prognoses.
Early detection of epigenetic changes in gallbladder cancer may
help improve the clinical outcomes of the patients with gallbladder
cancer in a number of ways. Overall, a number of clinical applications
of epigenetic changes have been suggested in the literature. Fragments
of genomic DNA released from the necrotic tumor cells may enter the
circulation or be released in the body fluids where they can be used as
biomarkers for the diagnosis, staging, or post-treatment monitoring of
cancer [50]. The epigenetic changes may be used as a marker of ade-
quate surgical resection according to gene methylation profile of the
tumor tissues compared to normal tissues including the tissues at the
margin of resection [51]. Moreover, epigenetic changes may also be
used as a marker of response to local as well as systemic treatment [52].
The limiting factor is that such epigenetic markers are still in the
J.S. Muhammad et al.
experimental phase for gallbladder cancer, and there is no available
data on their clinical use. One potential factor limiting the development
of such markers is the non-uniform geographic distribution of the dis-
ease itself. Variable data reviewed in this study from different popula-
tions revealed no universal epigenetic biomarker. Additional work is
required in high-risk regions to identify population-specific epigenetic
markers with enhanced clinical outcomes for patients.
Gallbladder cancer remains a fatal disease as it is usually diagnosed
at very late stages and there are no specific epigenetic biomarkers in
consideration. Probably combination of the several genes discussed in
this review may prove to be an effective biomarker. However, a novel
and population-specific biomarker facilitating early diagnosis, and later
enabling prognostic monitoring are urgently required. Epigenetic
modifications of the DNA are hopeful for the development of bio-
markers for specific cancers.
Here we have summarized the role of several genes that are fre-
quently methylated in GBC and described the relationship of methyla-
tion of various gene promoters to disease development in specific po-
pulations. We regard the utility of immunohistochemistry in the
expression of specific proteins in diagnosing and predicting prognosis of
GBC, however, promoter methylation levels could be detected from
genomic DNA obtained from the blood plasma of GBC patients em-
phasizing its use as less invasive diagnostic and prognostic applications.
Identification of population-specific epigenetic biomarkers in genomic
DNA and prospective correlation with clinical outcomes has the po-
tential to further aid in earlier diagnosis and management of gall-
bladder cancer.
Ethical approval
Not applicable.
Sources of funding
Not applicable.
Author contribution
JSM, MRK and KG conceived the idea and planned the study. JSM
performed literature search, designed study methodology and drafted
the initial manuscript including the table and figure. MRK and KG
critically analyzed and revised the manuscript. Final manuscript was
approved by all authors.
Conflicts of interest
None.
Research registration Unique Identifying Number (UIN)
Not applicable.
Guarantor
Dr. Jibran Sualeh Muhammad MBBS; Ph.D.
Assistant Professor.
Department of Basic Medical Sciences, College of Medicine,
University of Sharjah, Sharjah, United Arab Emirates.
Email: dr.jibran@live.com.
Disclosure
None.
182
International Journal of Surgery 53 (2018) 178–183
References
[1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015, CA A Cancer J. Clin. 65
(1) (2015 Jan-Feb) 5–29, http://dx.doi.org/10.3322/caac.21254.
[2] L.M. Stinton, E.A. Shaffer, Epidemiology of gallbladder disease: cholelithiasis and
cancer, Gut Liver 6 (2) (2012 Apr) 172–187.
[3] C. Are, H. Ahmad, A. Ravipati, D. Croo, D. Clarey, L. Smith, R.R. Price, J.M. Butte,
S. Gupta, A. Chaturvedi, S. Chowdhury, Global epidemiological trends and varia-
tions in the burden of gallbladder cancer, J. Surg. Oncol. 115 (5) (2017) 580–590.
[4] Y. Bhurgri, A. Bhugri, S.H. Hassan, Cancer incidents in karachi Pakistan: first results
from karachi cancer registry, Int. J. Canc. 85 (2000) 325–329.
[5] A.R. Alvi, N.A. Siddiqui, H. Zafar, Risk factors of gallbladder cancer in Karachi-a
case-control study, World J. Surg. Oncol. 9 (2011 Dec 9) 164.
[6] M.R. Khan, S.A. Raza, Z. Ahmad, S. Naeem, S. Pervez, A.A. Siddiqui, M. Ahmed,
R. Azami, Gallbladder intestinal metaplasia in Pakistani patients with gallstones,
Int. J. Surg. 9 (6) (2011) 482–485.
[7] C. Dupont, D.R. Armant, C.A. Brenner, Epigenetics: definition, mechanisms and
clinical perspective, Semin. Reprod. Med. 27 (5) (2009 Sep) 351–357.
[8] Q.W. Chen, X.Y. Zhu, Y.Y. Li, Z.Q. Meng, Epigenetic regulation and cancer (review),
Oncol. Rep. 31 (2) (2014 Feb) 523–532.
[9] D.S. Tekcham, S.S. Poojary, S. Bhunia, M.A. Barbhuiya, S. Gupta, B.R. Shrivastav,
P.K. Tiwari, Adenomatous polylosis coli (APC) gallbladder: epigenetic regulation of
APC in the molecular pathogenesis of gallbladder cancer, Indian J. Med. Res. 143
(S1) (2016) 82–90.
[10] T. Takahashi, N. Shivapurkar, E. Riquelme, H. Shigematsu, J. Reddy, M. Suzuki,
K. Miyajima, X. Zhou, B.N. Bekele, A.F. Gazdar, Wistuba II, Aberrant promoter
hypermethylation of multiple genes in gallbladder carcinoma and chronic chole-
cystitis, Clin. Canc. Res. 10 (2004 Sep 15) 6126–6133 (18 Pt 1).
[11] Y. Tian, Q. Xie, J. He, X. Luo, T. Zhou, Y. Liu, Z. Huang, Y. Tian, D. Sun, K. Yao,
Radioactive (125)I seeds inhibit cell growth and epithelial-mesenchymal transition
in human glioblastoma multiforme via a ROS-mediated signaling pathway, BMC
Canc. 15 (2015 Feb 19) 1.
[12] M. Esteller, Epigenetics in cancer, N. Engl. J. Med. 358 (11) (2008 Mar 13)
1148–1159.
[13] P.A. Jones, D. Takai, The role of DNA methylation in mammalian epigenetics,
Science 293 (5532) (2001 Aug 10) 1068–1070.
[14] R. Radpour, Z. Barekati, C. Kohler, Q. Lv, N. Bürki, C. Diesch, J. Bitzer, H. Zheng,
S. Schmid, X.Y. Zhong, Hypermethylation of tumor suppressor genes involved in
critical regulatory pathways for developing a blood-based test in breast cancer,
PLoS One 6 (1) (2011 Jan 24) e16080.
[15] J.S. Muhammad, S. Nanjo, T. Ando, S. Yamashita, T. Maekita, T. Ushijima,
Y. Tabuchi, T. Sugiyama, Autophagy impairment by Helicobacter pylori-induced
methylation silencing of MAP1LC3Av1 promotes gastric carcinogenesis, Int. J.
Canc. 140 (10) (2017) 2272–2283.
[16] G. Li, Y. Pu, MicroRNA signatures in total peripheral blood of gallbladder cancer
patients, Tumor Biol. 36 (2015) 6985–6990.
[17] B. Yang, B. Liu, P. Bi, T. Wu, Q. Wang, J. Zhang, An integrated analysis of differ-
ential miRNA and mRNA expressions in human gallstones, Mol. Biosyst. 11 (2015)
1004–1011.
[18] P. Letelier, P. Garcia, P. Leal, et al., miR-1 and miR-145 act as tumor suppressor
microRNAs in gallbladder cancer, Int. J. Clin. Exp. Pathol. 7 (2014) 1849–1867.
[19] J.C. Roa, L. Anabalón, I. Roa, A. Melo, J.C. Araya, O. Tapia, X. de Aretxabala,
S. Muñoz, B. Schneider, Promoter methylation profile in gallbladder cancer, J.
Gastroenterol. 41 (3) (2006 Mar) 269–275.
[20] P. García, C. Manterola, J.C. Araya, M. Villaseca, P. Guzmán, A. Sanhueza,
M. Thomas, H. Alvarez, J.C. Roa, Promoter methylation profile in preneoplastic and
neoplastic gallbladder lesions, Mol. Carcinog. 48 (1) (2009 Jan) 79–89.
[21] S.J.C. Roa, M.P. García, A.A. Melo, E.O. Tapia, H.M. Villaseca, O.J.C. Araya,
G.P. Guzmán, Gene methylation patterns in digestive tumors, Rev. Med. Chile 136
(4) (2008 Apr) 451–458.
[22] L.T. Kagohara, J.L. Schussel, T. Subbannayya, N. Sahasrabuddhe, C. Lebron,
M. Brait, L. Maldonado, B.L. Valle, F. Pirini, M. Jahuira, J. Lopez, P. Letelier,
P. Brebi-Mieville, C. Ili, A. Pandey, A. Chatterjee, D. Sidransky, R. Guerrero-Preston,
Global and gene-specific DNA methylation pattern discriminates cholecystitis from
gallbladder cancer patients in Chile, Future Oncol. 11 (2) (2015) 233–249.
[23] M.G. House, Wistuba II, P. Argani, M. Guo, R.D. Schulick, R.H. Hruban,
J.G. Herman, A. Maitra, Progression of gene hypermethylation in gallstone disease
leading to gallbladder cancer, Ann. Surg Oncol. 10 (8) (2003 Oct) 882–889.
[24] A.B. Sparks, P.J. Morin, B. Vogelstein, K.W. Kinzler, Mutational analysis of the APC/
beta-catenin/Tcf pathway in colorectal cancer, Canc. Res. 58 (6) (1998 Mar 15)
1130–1134.
[25] M.D. Christensen, C. Geisler, Recruitment of SHP-1 protein tyrosine phosphatase
and signalling by a chimeric T-cell receptor-killer inhibitory receptor, Scand. J.
Immunol. 51 (6) (2000 Jun) 557–564.
[26] C. Wu, M. Sun, L. Liu, G.W. Zhou, The function of the protein tyrosine phosphatase
SHP-1 in cancer, Gene 306 (2003 Mar 13) 1–12.
[27] D. Dietrich, R. Lesche, R. Tetzner, M. Krispin, J. Dietrich, W. Haedicke, M. Schuster,
G. Kristiansen, Analysis of DNA methylation of multiple genes in microdissected
cells from formalin-fixed and paraffin-embedded tissues, J. Histochem. Cytochem.
57 (5) (2009 May) 477–489.
[28] Y. Koga, Y. Kitajima, A. Miyoshi, K. Sato, K. Kitahara, H. Soejima, K. Miyazaki,
Tumor progression through epigenetic gene silencing of O(6)-methylguanine-DNA
methyltransferase in human biliary tract cancers, Ann. Surg Oncol. 12 (5) (2005)
354–363.
[29] S. Siddiqui, N. Akhter, S.V. Deo, N.K. Shukla, S.A. Husain, A study on promoter
J.S. Muhammad et al.
methylation of PTEN in sporadic breast cancer patients from North India, Breast
Canc. 23 (6) (2016 Nov) 922–931.
[30] G. Tamura, Alterations of tumor suppressor and tumor-related genes in the devel-
opment and progression of gastric cancer, World J. Gastroenterol. 12 (2) (2006 Jan
14) 192–198.
[31] A. Ali, P.K. Mishra, S. Sharma, A. Arora, S.S. Saluja, Effects of PTEN gene alteration
in patients with gallbladder cancer, Cancer Genet. 208 (12) (2015 Dec) 587–594.
[32] D.S. Tekcham, S. Gupta, B.R. Shrivastav, P.K. Tiwari, Epigenetic downregulation of
PTEN in gallbladder cancer, J. Gastrointest. Canc. 48 (1) (2017 Mar) 110–116.
[33] R. Zhao, B.Y. Choi, M.H. Lee, A.M. Bode, Z. Dong, Implications of genetic and
epigenetic alterations of CDKN2A (p16(INK4a)) in cancer, EBioMedicine 8 (2016
Jun) 30–39.
[34] H. Tadokoro, T. Shigihara, T. Ikeda, M. Takase, M. Suyama, Two distinct pathways
of p16 gene inactivation in gallbladder cancer, World J. Gastroenterol. 13 (47)
(2007 Dec 21) 6396–6403.
[35] T. Tozawa, G. Tamura, T. Honda, S. Nawata, W. Kimura, N. Makino, S. Kawata,
T. Sugai, T. Suto, T. Motoyama, Promoter hypermethylation of DAP-kinase is as-
sociated with poor survival in primary biliary tract carcinoma patients, Canc. Sci.
95 (9) (2004 Sep) 736–740.
[36] J.C. Roa, Q. Vo, J.C. Araya, M. Villaseca, P. Guzmán, G.S. Ibacache, X. de
Aretxabala, I. Roa, Inactivation of CDKN2A gene (p16) in gallbladder carcinoma,
Rev. Med. Chile 132 (11) (2004 Nov) 1369–1376.
[37] B. Klump, C.J. Hsieh, S. Dette, K. Holzmann, R. Kiebetalich, M. Jung, U. Sinn,
M. Ortner, R. Porschen, M. Gregor, Promoter methylation of INK4a/ARF as detected
in bile-significance for the differential diagnosis in biliary disease, Clin. Canc. Res. 9
(5) (2003 May) 1773–1778.
[38] T. Ueki, A.W. Hsing, Y.T. Gao, B.S. Wang, M.C. Shen, J. Cheng, J. Deng,
J.F. Fraumeni Jr., A. Rashid, Alterations of p16 and prognosis in biliary tract can-
cers from a population-based study in China, Clin. Canc. Res. 10 (5) (2004 Mar 1)
1717–1725.
[39] J. Behrens, B. Lustig, The Wnt connection to tumorigenesis, Int. J. Dev. Biol. 48
(5–6) (2004) 477–487.
[40] Y. Huang, Q. Du, W. Wu, F. She, Y. Chen, Rescued expression of WIF-1 in gall-
bladder cancer inhibits tumor growth and induces tumor cell apoptosis with altered
expression of proteins, Mol. Med. Rep. 14 (3) (2016 Sep) 2573–2581.
[41] B. Lin, H. Hong, X. Jiang, C. Li, S. Zhu, N. Tang, X. Wang, F. She, Y. Chen, WNT
inhibitory factor 1 promoter hypermethylation is an early event during gallbladder
183
International Journal of Surgery 53 (2018) 178–183
cancer tumorigenesis that predicts poor survival, Gene 622 (2017 Jul 30) 42–49.
[42] E. Riquelme, M. Tang, S. Baez, A. Diaz, M. Pruyas, Wistuba II, A. Corvalan, Frequent
epigenetic inactivation of chromosome 3p candidate tumor suppressor genes in
gallbladder carcinoma, Canc. Lett. 250 (1) (2007 May 18) 100–106.
[43] S.K. Kee, J.Y. Lee, M.J. Kim, S.M. Lee, Y.W. Jung, Y.J. Kim, J.Y. Park, H.I. Bae,
H.S. Hong, Y.K. Yun, S.G. Kim, D.S. Kim, Hypermethylation of the Ras association
domain family 1A (RASSF1A) gene in gallbladder cancer, Mol. Cell. 24 (3) (2007
Dec 31) 364–371.
[44] Y.M. Lee, J.Y. Lee, M.J. Kim, H.I. Bae, J.Y. Park, S.G. Kim, D.S. Kim,
Hypomethylation of the protein gene product 9.5 promoter region in gallbladder
cancer and its relationship with clinicopathological features, Canc. Sci. 97 (11)
(2006 Nov) 1205–1210.
[45] K. Igarashi, K. Yamashita, H. Katoh, K. Kojima, Y. Ooizumi, N. Nishizawa,
R. Nishiyama, H. Kawamata, H.1 Tajima, T. Kaizu, Y. Kumamoto, M. Watanabe,
Prognostic significance of promoter DNA hypermethylation of the cysteine dioxy-
genase 1 (CDO1) gene in primary gallbladder cancer and gallbladder disease, PLoS
One 12 (11) (2017 Nov 21) e0188178.
[46] P. Letelier, P. Brebi, O. Tapia, J.C. Roa, DNA promoter methylation as a diagnostic
and therapeutic biomarker in gallbladder cancer, Clin. Epigenet. 4 (1) (2012 Jul
13) 11.
[47] S. Gourgiotis, H.M. Kocher, L. Solaini, A. Yarollahi, E. Tsiambas, N.S. Salemis,
Gallbladder cancer, Am. J. Surg. 196 (2008) 252–264.
[48] I. Roa, J.C. Araya, M. Villaseca, X. De Aretxabala, P. Riedemann, K. Endoh, et al.,
Preneoplastic lesions and gallbladder cancer: an estimate of the period required for
progression, Gastroenterology 111 (1996) 232e6.
[49] I. Roa, J.C. Araya, M. Villaseca, J. Roa, X. de Aretxabala, G. Ibacache, Gallbladder
cancer in high risk area: morphological features and spread patterns, Hepato-
Gastroenterology 46 (1999) 1540e6.
[50] A.R. Sepulveda, D. Jones, S. Ogino, W. Samowitz, M.L. Gulley, R. Edwards,
V. Levenson, V.M. Pratt, B. Yang, K. Nafa, L. Yan, P. Vitazka, CpG methylation
analysis–current status of clinical assays and potential applications in molecular
diagnostics: a report of the association for molecular pathology, J. Mol. Diagn. 11
(2009) 266–278.
[51] E.L. Sceusi, D.S. Loose, C.J. Wray, Clinical implications of DNA methylation in
hepatocellular carcinoma, HPB 13 (2011) 369–376.
[52] K. Ghoshal, S. Bai, DNA methyltransferases as targets for cancer therapy, Drugs
Today 43 (2007) 395–422.