Alterations in Genomic 5-Hydroxymethylcytosine Level in
Hepatocellular Cancer
Thesis
Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in
the Graduate School of The Ohio State University
By
Mufaddal Mustafa, B.S.
Graduate Program in Pathology
The Ohio State University
2013
Thesis Committee:
Dr. Kalpana Ghoshal, Advisor
Dr. Samson T. Jacob
Copyright by
Mufaddal Mustafa
2013
Abstract
Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death
worldwide. There are a limited number of therapeutic options currently available to delay
the advancement of HCC, and the chance of survival decreases as the disease progresses.
DNA methylation of certain tumor suppressor genes and hypomethylation of oncogenes
have been shown to initiate HCC. 5-hydroxymethylcytosine (5-hmC), recently identified
sixth base of the genome, can cause demethylation by its conversion to cytosine through
reactions catalyzed by the TET enzymes. Research on the role of this novel modification
in the liver and HCC is needed before it can be therapeutically targeted. To this end, we
planned to perform systematic analysis of the role of 5-hmC in liver biology and its
aberrations in HCC. In the present study, DNA from primary human HCC and matching
benign livers were used to compare alterations in 5-hmC level, if any, in tumors. Mouse
models of HCC were also used to further validate the results obtained from human
specimens. We observed remarkable decrease in global 5-hmC content in HCCs of both
human and rodent origin using multiple techniques that include dot blot,
immunohistochemistry, and LC/MS analysis. Furthermore, specific regions of EGFR,
H19, and 7SL loci also exhibited reduced 5-hmC levels in the tumor samples. Since
aberrations in methylation can cause cancer including HCC, it would be important to
ii
identify differentially hydroxymethylated genes in the liver and the consequence of their
differential hydroxymethylation in HCC.
iii
Dedication
Dedicated to my family
iv
Acknowledgments
I thank my advisor, Dr. Kalpana Ghoshal, for providing experimental ideas and feedback
over the last two years.
I thank my committee member, Dr. Samson Jacob, for his invaluable guidance and
motivation.
I thank Dr. Tasneem Motiwala for initially teaching me all of the lab techniques and her
continued help with everything throughout my graduate studies.
I thank the researchers at the OSU core facilities that helped generate and analyze results.
Finally, I thank all the members of my lab for their advice, generosity, and daily support.
v
Vita
June 2007 .......................................................Westerville Central High
March 2011 ....................................................B.S. Biology, The Ohio State University
Fields of Study
Major Field: Pathology
vi
Table of Contents
Abstract ............................................................................................................................... ii
Dedication .......................................................................................................................... iv
Acknowledgments ...............................................................................................................v
Vita..................................................................................................................................... vi
List of Tables ..................................................................................................................... ix
List of Figures ......................................................................................................................x
Introduction..........................................................................................................................1
Hepatocellular Carcinoma .......................................................................................1
DNA Methylation in HCC .......................................................................................3
Hydroxymethylation in HCC...................................................................................5
Project Aims ........................................................................................................................8
Methods ...............................................................................................................................9
DNA Isolation..........................................................................................................9
Immuno-Dot Blot Analysis......................................................................................9
Liquid Chromatography/ Mass Spectrometry Analysis.........................................10
Immunohistochemistry ..........................................................................................11
Quantitative Reverse Transcriptase PCR Analysis................................................11
vii
Pulldown of 5-hmC DNA ......................................................................................11
CDAA and CSAA diet model................................................................................12
Results................................................................................................................................14
Conclusions........................................................................................................................18
Discussion ..........................................................................................................................19
References..........................................................................................................................22
Appendix A: Tables and Figures .......................................................................................25
viii
List of Tables
Table 1: Primer Sequences ................................................................................................26
ix
List of Figures
Figure 1: Passive and Active DNA Demethylation ...........................................................27
Figure 2: TET1/2/3 Facilitated Demethylation..................................................................28
Figure 3: 5-hmC Content in Different Tissue Types .........................................................29
Figure 4: 5-hmC DNA Pulldown.......................................................................................30
Figure 5: Immuno-Dot Blot Analysis ................................................................................31
Figure 6: Immunohistochemical Analysis .........................................................................32
Figure 7: Liquid chromatography and mass spectrometric analysis..................................33
Figure 8: 5-hmC level in EGFR, H19, and 7SL loci..........................................................34
Figure 9: Immuno-Dot Blot analysis in Cirrhotic samples................................................35
Figure 10: TET1/2/3 mRNA expression in human HCC and benign livers ......................36
Figure 11: 5-hmC content in the livers of mice fed CDAA or CSAA diet........................38
x
Introduction
Hepatocellular Carcinoma
Hepatocellular carcinoma is a type of primary liver cancer and is the third most
common cause of cancer-related death worldwide, behind only lung and stomach cancers
[1, 2]. It has a 5-year overall survival rate of 12% due to the late diagnosis of the disease
in most patients [3]. The largest risk factors of HCC are hepatitis B and C viral infections,
cirrhosis, steatosis, alcoholic liver disease, and family history [4]. HCC occurs most
commonly in Asia and Sub-Saharan Africa where the average incidence rate is greater
than 98 per 100,000 persons [5, 6]. United States has a relatively low incidence rate with
the average rate of 8.5 per 100,000 persons, however it has been increasing every year in
both the U.S. and other developed western countries [7]. HBV is responsible for most
cases of HCC in Asia and Africa, while HCV causes the majority of cases in the United
States and Western Europe [8, 9]. Men have a higher death rate than women, and the
prevalence of the disease increases with age among both genders [10, 11].
The early symptoms of HCC are nonspecific and could be mistaken for other
diseases or complications. This is one of the reasons why patients are diagnosed with
HCC at a very advanced stage of the disease, rendering therapy less effective and
increasing the mortality rate. The most common early symptoms of HCC can include
1
abdominal pain, early satiety, weight loss, and jaundice. Increased surveillance is
recommended for individuals displaying known risk factors of HCC to ensure early
detection [2]. Serum α-fetoprotein (AFP) is the most common biomarker tested during
screenings [12]. An evaluation of AFP levels combined with abdominal ultrasonography
was tested as a screening method during a randomized control trial. The study concluded
that by combining these two screening methods, the diagnosis of HCC in patients
occurred earlier and consequently improved survival [13].
There are multiple options for the treatment of HCC depending on the progression
of the disease. For patients with early HCC, liver transplantation, surgical resection, or
ablation is often recommended [2]. The Milan criteria are used to determine if a patient is
suitable for a liver transplant. The criteria states that the patient can only have one tumor
less than 5 cm, or three or fewer tumors less than 3 cm without any vascular invasion in
either scenario [14]. Surgical resection involves the use of a CT or MRI scan of the liver
to locate lesions and subsequently removing them without compromising normal liver
function [15]. Ablation also requires CT or MRI imaging to locate tumors and uses
radiofrequency or toxic chemicals such as ethanol to cause necrosis of the target [16].
Liver transplantation and surgical resection have the greatest five year survival and
lowest recurrence rate from any of the early stage HCC treatments [17].
In patients with highly advanced HCC involving tumor vascularization and
metastasis, systemic therapy is the only treatment option. However, with the exception of
Sorafenib, most chemotherapy or other anti-cancer drug treatments are ineffective in
improving long term survival [18]. Sorafenib causes the suppression of tumor cell
2
proliferation and angiogenesis by inhibiting both the Raf kinase and VEGFR intracellular
kinase pathways [19]. It has been approved by the FDA to treat advanced HCC and
shown to increase survival in multiple studies [2].
DNA Methylation in HCC
Epigenetic modifications are heritable post-replicative changes in DNA that can
have an impact on gene expression and function. Methylation at C-5 of cytosine is the
most prevalent epigenetic modification of DNA, which is essential for mammalian
development. Aberrations in DNA methylation are associated with multiple cancers,
including HCC [20, 21]. In somatic cells, most methylation occurs on CpG dinucleotides
where approximately 60%-90% of them always remain in a methylated state.
Unmethylated CpG sites are normally observed as clusters near gene promoters and are
called CpG islands [22]. DNA methyltransferases (DNMTs) facilitate the enzymatic
addition of the methyl group on the cytosine. They also ensure that during DNA
replication, the methylation pattern of the parent strand is correctly copied on the newly
synthesized strand [23]. Another group of important components involved in gene
regulation through DNA methylation are the methyl-binding proteins (MBPs). In the
presence of their specific methyl-binding domain (MBD), these proteins can bind
methylated CpG dinucleotides to inhibit transcription and promote gene silencing by
inducing chromatin remodeling [24]. Methylation can also directly inhibit transcription
by physically blocking transcriptional proteins on promoters and as a result prevent gene
expression [20].
3
DNA methylation is essential for early development and differentiation, and
responsible for the maintenance of normal gene expression later in life. Cancer can
develop when the methylation status of oncogenes and tumor suppressor genes is altered,
either as a result of hypomethylation or hypermethylation, respectively. Through
hypomethylation, oncogenes can become activated, while hypermethylation can induce
the suppression of tumor suppressor genes [20, 25]. In HCC, many tumor suppressor
genes and other cancer related genes that function in cell-cycle regulation, apoptosis,
DNA repair, cellular homeostasis, and cell adhesion and invasion, are commonly
hypermethylated and thus inactivated [20]. Hypomethylation is also observed in multiple
genes whose activation correlated significantly with increased tumor size, tumor number,
AFP levels, and overall poor survival [26].
Drugs that can cause demethylation have been explored as options to treat HCC
and other cancers. 5-Azacytidine and its congener, decitabine, are two of the first DNA
methylation inhibitors currently being used in therapy [20]. Zebularine is also a drug that
can inhibit methylation and has been shown to reduce proliferation and increase
apoptosis, resulting in enhanced survival in mice [27]. Other demethylating drugs have
been successful in reducing cell viability and inducing apoptosis in multiple cancer cell
lines. Currently, however, the lack of specificity of these drugs prevents them from being
approved as treatment options for human patients. Global hypomethylation could also
cause the activation of oncogenes and potentially result in a more aggressive form of
cancer [20]. Even if modifications in methylation status cannot be presently used for
therapy, the ability to detect changes in methylation can still be helpful as a biomarker.
4
Genes that are abnormally hypomethylated or hypermethylated serve as biomarkers for
early detection of cancer, tumor classification, and response to treatments [20].
Hydroxymethylation in HCC
The hydroxylated form of 5-methylcytosine (5-mC) is 5-hydroxymethylcytosine
(5-hmC). It was first discovered in the DNA of bacteriophages, and during the early
1970s was shown to account for modification observed on 15% of all cytosines in the
DNA extracted from the brains of mice, rats, and frogs [28, 29]. However, only recently
5-hmC has begun to be considered an important epigenetic modification. The formation
of 5-hmC has been proposed to lead to demethylation both through a passive and an
active process. Passive demethylation can occur since DMNT1, the maintenance
methyltransferase, does not recognize 5-hmC, resulting in unmethylated cytosines on the
DNA of the newly synthesized cells. The active method suggests that 5-hmC is
recognized by DNA repair proteins and removed, leading to demethylation (Fig.1). The
ability of 5-hmC to cause demethylation can have significant consequences in the
regulation of transcription and gene activation [30].
The oxidation of 5-mC to 5-hmC is catalyzed by the family of Fe(II)- and αketoglutarate dependent dioxygenases, the Ten-Eleven- Translocation (TET) family of
proteins, called TET1, TET2, and TET3 [31]. TET1 is highly expressed in ES cells and is
very important in regulating methylation and gene expression. The knockdown of TET1
has been shown to increase 5-mC and decrease the 5-hmC levels [30, 31]. TET2 is
mutated in patients with multiple myeloid malignancies, including acute myeloid
5
leukemia (AML), which results in a loss of hydroxymethylation in those patients. This
triggers aberrant DNA methylation and the silencing of important genes, further causing
the progression of AML [33]. These findings suggest that TET2 may have a tumor
suppressor function [30]. The overexpression of TET1 and TET2, but not TET3,
promotes a global decrease in 5-mC [34]. However, TET3 is the only Tet family member
that is expressed in mouse oocytes and zygotes, and is responsible for the
hydroxymethylation of DNA during the advanced pronuclear-stage of zygotes. It has also
been shown to regulate essential genes for eye and neural development in frogs [35]. TET
enzymes can also further oxidize 5-hmC to 5-formylcytosine (5fC) and 5carboxylcytosine (5caC); eventually leading to demethylation once the carboxyl group is
removed [32] (Fig. 2).
Multiple studies have discovered a large amount of variation in the 5-hmC level
between human tissues. The brain has the highest amount, followed by breast and liver
tissues (Fig.3). Similarly the 5-mC levels are the greatest in the brain; however the
disparity among different tissues types is not as prevalent when comparing 5-mC content.
There is also no significant correlation between global 5-mC and 5-hmC levels among
the tissue types [36]. When breast, liver, lung, pancreas, and prostate cancer tissues are
compared with their normal counterpart, a substantial decrease in 5-hmC is observed in
the diseased samples. The mRNA expression of TET1/2/3 is downregulated in multiple
cancers as well, highlighting some correlation between their expression and global 5hmC content [37]. Since the 5-hmC level and Tet expression are reduced in many
6
different cancers, they can be used as biomarkers for diagnosis and possibly become
therapeutic targets in the future.
7
Project Aims
Aim 1: Determine alterations in
(a) 5-hmC levels in tumors compared to benign livers of human livers in diseased state
such as cirrhosis and HCC and,
(b) enzymes (TET1, TET2, TET3) that catalyze hydroxymethylation.
Aim 2: Identify genes that are hydroxymethylated in normal human liver and compare
their 5-hmC levels between tumors and matching livers in human liver cancer patients.
The primary objective is to examine alterations in genomic 5-hmC level in
various liver diseases including cancer and to identify genomic wide localization of 5hmC in the liver and tumor. Moreover, it would be of interest to examine the biological
consequence of differential hydroxymethylation of the genome in HCC, which may
potentially lead to the development of novel therapeutics.
8
Methods
DNA Isolation: for use in dot blot and 5-hmC pulldown experiments.
Approximately 100µg of powdered tissue was suspended in 5mL DNA lysis
buffer containing 150mM EDTA (pH 8), 1% SDS, 10mM Tris-HCl (pH 8), and 60µg/mL
proteinase K. The mixture was incubated while shaking slowly overnight in a 50°C water
bath. Next morning, the DNA was purified using Phenol-Chloroform Isoamyl Alcohol
(PCI) and dialyzed against 10mM Tris-HCl (pH 8) and 1mM EDTA (pH 8) buffer for
one hour. The samples were dialyzed once more in fresh buffer overnight at 4°C. On the
following day, 1.6µg/mL of RNaseA was added for every 5mL sample volume and
incubated in 37°C for 2 hours. The DNA was precipitated by using two volumes 100%
ethanol, washed with 70% ethanol and dissolved in TE.
Immuno-Dot Blot analysis: to estimate 5-mC and 5-hmC levels in human and mouse
genomic DNA.
Purified DNAs were fragmented to approximately 200bp in size by sonicating for
15 pulses, followed by measuring concentration by Nanodrop. DNAs were denatured by
adding 0.4M NaOH and 10mM EDTA, followed by immediate incubation at 95°C for 10
minutes. Samples were placed on ice and neutralized by adding equal volume of ice cold
2M ammonium acetate pH 7. Mouse DNA samples (1µg and 500ng of each) and human
9
DNA samples (500ng and 250ng of each) were blotted onto a Zeta probe membrane
(BioRad, Hercules, CA) using dot blot apparatus. The DNA was fixed to the membrane
by UV cross linking using Stratalinker, and following blocking (10% milk in 0.1% TBE
for 5-hmC and 5% milk in 0.05% TBE for 5-mC) for 30 minutes it was incubated
overnight with primary antibodies specific to 5-mC or 5-hmC (Active Motif, Carlsbad,
CA). The next day, the blot was washed (0.1% TBE for 5-hmC and 0.05% TBE for 5mC) two times for 10 minutes followed by incubation with HRP-tagged secondary
antibody (anti-rabbit for 5-hmC and anti-mouse for 5-mC) for 1 hour. Next, the blot was
washed with the respective TBE solution followed by development of signal with the
Enhanced Chemiluminescence reagent (ThermoFisher, Waltham, MA). The blot was then
probed with [32P]-labeled 18S rDNA probe to determine the signal of total DNA on the
blot, which was used as the normalizer. The 5-mC and 5-hmC signals were normalized to
the 32P signal by quantifying the spot intensity through ImageJ image processing software
(NIH). To ensure that the antibodies did not cross-react, synthetic DNA containing 5hmC, 5-mC, or only cytosine were blotted on each membrane.
Liquid Chromatography / Mass Spectrometry Analysis: to detect 5-mC and 5-hmC
content in human DNA.
5µg of each DNA sample was sent for LC/MS-MS analysis at the
Pharmacoanalytical Shared Resource at The Ohio State University Comprehensive
Cancer Center. The results revealed the mass of 5-mC and 5-hmC in ng/µg of total DNA.
10
Immunohistochemistry: to observe 5-hmC content in the nucleus of human hepatocytes.
Normal liver and HCC liver samples on a tissue microarray slide (developed by
Pathology core at OSUCCC) were stained with an antibody (Active Motif) specific for 5hmC at 1:500 dilution. The stain intensity of the samples was visualized under a
microscope and the normal and HCC samples were compared in each pair.
Quantitative Reverse Transcriptase PCR analysis: to quantify Tet1, Tet2 and Tet3
expression in human and mice.
RNA was extracted from human and mouse tissue using TRIzol reagent
(Invitrogen, Carlsbad, CA) by following the manufacturer’s protocol. Following
treatment with DNase I, cDNA was generated from 2µg RNA using a Reverse
Transcriptase kit (Applied Biosystems, Grand Island, NY), and subsequently subjected to
real-time RT-PCR (qRT-PCR) analysis in Stratagene Mx3000p using Power SYBR
Green master mix (Applied Biosystems) and primers (Table 1) specific to Tet1/2/3 and
GAPDH. Relative expressions of Tet1-3 were determined using ΔCT method.
Pulldown of 5-hmC DNA: to identify 5-hydroxymethylated loci and monitor 5-hmC level
in individual genes.
In order to pulldown DNA containing only 5-hmC, the hydroxyl group is
modified by adding a UDP-Azide-Glucose moiety catalyzed by the β-Glucosyltransferase
enzyme. After the conversion, the glucose is biotinylated which allows specifically the 5hmC DNA to attach to magnetic streptavidin beads. The DNA bound to the beads is
11
captured by a magnet, while the unattached DNA is washed. The 5-hmC DNA is isolated
by eluting from the beads and purified using PCR purification columns (Fig.4).
DNA samples were fragmented to approximately 200bp in size by sonicating for
15 pulses, and the concentrations were measured by nanodrop. 2.5µg starting DNA of
each sample was used for pulldown using the Hydroxymethyl Collector kit (Active
Motif). To ensure the specificity of the kit, negative control reactions lacking UDPAzide-Glucose were also performed for each sample. The 5-hmC DNA precipitation was
executed following the manufacturer’s protocol. After purification, the 5-hmC DNA was
eluted in 50µl buffer and 5µl was used for each PCR reaction with specific primers for
candidate genes.
For sequencing of 5-hmC enriched loci in the liver genome, DNA was initially
fragmented to ~300bp in size using a Covaris sonicator. DNAs from multiple human liver
samples were combined following precipitation of 5-hmC DNA as described above, to
acquire the total 200ng needed for sequencing. A library was generated using the
Illumina TruSeq kit and following the manufacturer’s procedure. The 5-hmC DNA was
sequenced using the Illumina HiSeq 2000 at the Nucleic Acid Shared Resource at The
Ohio State University Comprehensive Cancer Center.
Choline Deficient Amino Acid Defined (CDAA) and Choline Sufficient Amino Acid
Defined (CSAA) diet model: to develop a HCC model in mice.
C57BL6 were maintained in a sterile room at 25°C with 12h light-dark cycle and
provided food and water ad libitum. 9 male, 6 weeks old mice for each diet group were
12
fed either the CDAA or CSAA (Dyets Inc, Philadelphia, PA) diet for 84 weeks. Most
CDAA fed mice developed tumors, and CSAA fed mice exhibited steatosis and only
minor liver damage. DNA was isolated and 5-hmC level was detected using immuno-dot
blot analysis. This animal experiment was carried out under protocols approved by the
Ohio State University Institutional Laboratory Animal Care and Use Committee.
13
Results
Global 5-hmC level are reduced in HCCs compared to matching normal liver tissues
To observe the difference in 5-hmC and 5-mC levels between HCC and normal
liver, a dot blot assay was performed using 26 human DNA samples, 13 from HCC tumor
tissues and 13 from matching normal liver tissues. After immobilizing the DNA on the
membrane, it was probed with anti-5hmC and anti-5mC antibodies, and normalized with
18S rDNA. The results showed on average a 32.9% decrease of 5-hmC (P=2.27E-04)
(Fig.5B) and a 9.9% decrease of 5-mC levels (P=0.0028) (Fig.5C) in the HCC samples.
Note that two pairs in the 5-mC blot failed to display a signal, and therefore were
removed from the calculations. To ensure that the antibodies did not cross-react, synthetic
DNA containing 5-hmC, 5-mC, or only cytosine were blotted on each membrane. As
shown in figure 5A, the antibodies were specific to the respective modification profile of
DNA.
We also probed a tissue microarray containing 31 normal and HCC tissue pairs in
duplicate with anti-5hmC antibody. However, only 23 of the pairs were properly stained
and considered informative. Higher staining intensity was observed in 85% of the benign
liver tissues (Fig.6A, 6B), confirming that 5-hmC is indeed reduced in HCC tumors. The
remaining 15% of the tissues showed decreased 5-hmC staining in the benign liver
tissues.
14
Next, liquid chromatography and mass spectrometry analysis were used to
quantify the 5-hmC and 5-mC levels in 7 pairs of HCC and benign liver tissues. On
average, the normal liver DNA contained 0.45ng 5-hmC per µg of total DNA, while
DNA from HCC only contained 0.10ng 5-hmC. This shows that 5-hmC accounts for
0.25% of the cytosines in the benign liver and 0.057% in HCC, revealing a dramatic
77.5% decrease in 5-hmC level in tumors (P=2.62E-04) (Fig.7A). The HCC specimens
analyzed exhibited a small (24.6%) but significant (P=0.021) decrease in 5-mC level,
with 5-mC accounting for 2.9% of the cytosines in normal liver and 2.2% in tumor
(Fig.7B).
5-hmC level in specific genes is also reduced in HCC compared to matching liver
To test if the global decrease of 5-hmC in HCC correlated with a similar decrease
in specific genes, regions of EGFR, H19, and 7SL previously shown to be
hydroxymethylated were tested in human liver tissues [38]. Using the pulldown kit from
Active Motif, 5-hmC DNA enriched from six normal/tumor pairs were used in the PCR
reaction for each gene. The promoter and coding regions of EGFR were amplified.
Within the promoter, 5-hmC level was not significantly altered in tumors compared to
livers among these 6 pairs (data not shown). However, 5-hmC levels in the coding region
were higher in the livers compared to tumors as they demonstrated increased level of the
amplicon in the livers (Fig.8A). There was an average of 53.3% reduction of 5-hmC in
the HCC samples (P=0.027) (Fig.8D). The promoter of H19 and an upstream region
termed genic were also tested, with both regions showing a decrease of 5-hmC in the
15
tumor samples (Fig.8B). In H19 genic, 5-hmC was reduced on average by 38.0% in HCC
samples (P=0.0017) (Fig.8E). Lastly, a region of 7SL, a signal recognition particle RNA,
followed the similar trend revealing an increased 5-hmC content in the benign liver
(Fig.8C). The average 5-hmC level in the tumor samples was 39.0% less (P=0.032)
(Fig.8F). These results suggest that 5-hmC levels at least in 3 different loci are
downregulated in HCC compared to adjacent benign livers.
5-hmC levels are minimally reduced in cirrhotic livers
Since liver cirrhosis often leads to HCC development in humans, we next
measured global 5-hmC levels in 11 cirrhotic livers by immune-dot blot assay with anti5hmC antibody. The results revealed that the 5-hmC content in cirrhotic liver DNA was
minimally reduced (1.2%) compared to normal liver, as opposed to ~33% decrease in
HCCs (Fig.9). These results demonstrate that downregulation of 5-hmC is a characteristic
of HCC.
TET expression is dysregulated in HCC
TET1/2/3 enzymes catalyze the conversion of 5-mC to 5-hmC [31]. To determine
if the expression of these enzymes correlates with 5-hmC levels in normal and HCC
samples, qRT-PCR was performed in 24 normal/HCC pairs using gene specific primers
(Table 1). The results showed that the expressions of TET1 and 3 were higher in tumor
samples in 58% and 70% of the HCC specimens compared to the matching livers,
respectively, and in 63% of the pairs, TET2 expression was greater in normal tissues
16
(Fig.10A,B,C). Among these three TET enzymes, only TET2 expression correlated with
the reduced 5-hmC levels in tumor DNA. It should be noted that the expression of all
TET enzymes varied greatly among the pairs, and when averaged together, the change in
overall expression is not significant (data not shown). These results suggest that possibly
activities of these enzymes are more critical than their expression in regulating 5-hmC
levels in HCC.
Trends observed in mouse models are similar to those as humans
Next, we evaluated changes in 5-hmC level in normal and tumor bearing mice in
a diet model. In this model, C57BL6 mice are fed either the choline-deficient and amino
acid defined (CDAA) or the choline-sufficient and amino acid defined (CSAA) diet. The
CDAA diet has been shown to promote nonalcoholic steatohepatitis (NASH)-induced
HCC when fed in mice for 84 weeks, while the CSAA diet resembles a control causing
steatosis and only minor liver damage [39].
DNA from a total of 18 mice fed either the CDAA or CSAA diet for 84 weeks
was isolated, and the dot blot assay was performed to observe 5-hmC content. In the
CDAA fed mice, which had progressed to an advanced stage of HCC, the 5-hmC level
was reduced by 17.5% (P=0.0049) compared to the CSAA fed mice (Fig.11). Resembling
the same pattern detected in human HCCs, mouse model of diet induced HCC also
showed a 5-hmC decline.
17
Conclusions
Multiple experiments which included the dot blot assay, LC/MS analysis, and
TMA immunohistochemistry, revealed that global 5-hmC content in the DNA from a
HCC tumor was significantly less than in the DNA from normal liver tissue within the
same patient. To further verify this finding, 5-hmC content was observed in a HCC
mouse model, and was once more proved to be lower in the DNA obtained from diseased
tissues. Consistent with the global trend, specific regions of EGFR, H19, and 7SL genes
were also shown to have reduced 5-hmC levels in human tumor samples. A minimal
decline in 5-hmC content in the cirrhotic DNA suggests that downregulation of 5-hmC is
a characteristic of only HCC.
The TET1/2/3 proteins which are responsible for catalyzing the conversion of 5mC to 5-hmC, were shown to have sporadic expression levels between multiple human
normal and tumor pairs. From the three enzymes, only TET2 correlated with 5-hmC
content in the DNA by averaging reduced expression in tumors in majority of the human
pairs. This conflicts with the quantity of 5-hmC known to be present in HCC DNA and
requires the measurement of protein expression and activity of the Tet enzymes before a
direct correlation can be proved.
18
Discussion
Experimental results in both humans and mice prove that the 5-hmC content is
reduced in DNA acquired from liver tumors when compared with DNA from normal
liver tissues. A similar outcome has been observed in a publication where it is shown
through a dot blot assay that a normal liver tissue sample contained a greater amount of
5-hmC than HepG2, a HCC cell line [36]. This report however only compares between
two samples, while our experiments duplicate these results in multiple samples from both
human and mouse origin. Since 5-hmC is decreased in HCC, it suggests that certain
genes are likely to get hypermethylated and silenced in tumors. Normally, when 5-hmC is
present on the DNA, it will lead to demethylation of that region and allow for
transcription to proceed. If the hydroxylation of the methyl group is prevented, as
detected in HCC, the CpG islands remain methylated causing silencing of the nearby
promoters. Some of these affected loci can be tumor suppressor genes whose promoters
retain the methyl group and are therefore, unable to impede the onset of HCC. The next
logical step is to identify the genes whose 5-hmC status differ between normal and
diseased conditions, and possibly attempt to hypomethylate them in order to restore their
expression. Sequencing 5-hmC pulldown DNA from both normal and HCC tissues is the
best method to locate the affected genes. Our lab has sequenced 5-hmC DNA isolated
from multiple normal human liver tissues using the library generation kit from Illumina.
19
We also plan to sequence 5-hmC DNA from HCC tissues, along with examining
methylation levels in the genome. Comparison between the methylation status and the 5hmC variation in the normal and tumor DNA will provide a broader understanding of the
relationship amongst these epigenetic modifications. After proper bioinformatics analysis
and validation of the targets, connections can be recognized and therapeutic options can
be considered in preventing or delaying the progression of HCC.
There was extensive variation in the mRNA expression levels of the TET1/2/3
enzymes within the human tissues. A conclusion cannot be drawn from this data since
none of the results from the human experiments were significant. Considering that the
TET enzymes facilitate the conversion of 5-mC to 5-hmC, a decline in their expression
levels should ideally decrease the quantity of 5-hmC generated. Therefore, when 5-hmC
reduction is observed in HCC, TET expression should also follow a similar pattern. Even
though this was not an occurrence in the data being presented, another group has
succeeded in proving this hypothesis. Yang et al. presented a significant decrease of
TET1/2/3 expression in both breast and liver tumors, alongside the display of 5-hmC
decline in the DNA from the same tissues [37]. A possible explanation for our inability to
obtain a significant result could be that there was a large sample size with 24
normal/HCC pairs as opposed to only three pairs in the Yang et al. experiment. Normally,
a large amount of variation exists between human samples and increasing the sample size
raises the chance for that disparity. Comparing a smaller number of pairs in our
experiments might have shown the down-regulation of TET expression in most if not all
tumor samples. However, we cannot rule out that the activities of one or more of these
20
enzymes are reduced in HCCs thereby reducing 5-hmC levels during rapid proliferation
of the tumor cells. Protein expression levels of TET1/2/3 also needed to be compared
between normal and HCC patients. Lastly, siRNA mediated knockdown of each
individual TET protein followed by the measurement of 5-hmC can reveal the roles of
each specific enzyme. If the protein expression is also reduced in tumor extracts and the
knockdown of the enzymes lead to decreased 5-hmC levels, it can definitely be stated
that they regulate 5-hmC conversion. The failure of the TET proteins to express leads to
the retention of methylation, thus silencing potential genes that aid in the prevention of
HCC.
The understanding of the synthesis and biological function of 5hydroxymethylcytosine has greatly improved over the last few years. This surge of
curiosity has led to the discovery in how the level of 5-hmC is altered throughout the
progression of leukemia. Since global 5-hmC is reduced in tumors, it can be employed as
a molecular biomarker for early diagnosis of HCC and other carcinomas. It is important
to elucidate the exact mechanism and identify all of the components in 5-hmC mediated
gene regulation so that we can modulate its level in different diseases as a therapeutic
option. Manipulating the processes involved in 5-hmC has incredible potential, and it can
hopefully be available for cancer therapy in the near future.
21
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24
Appendix A:
Tables and Figures
25
Table 1: Primer Sequences
26
Figure 1: Passive and Active DNA Demethylation. (A) 5-hmC is not recognized by
DNA methyltransferases (DNMTs), preventing maintenance methylation during DNA
replication and resulting in passive demethylation. (B) 5-hmC may be recognized by
DNA repair proteins, such as 5-hmC specific DNA glycosylase (5hmC-DG), which
converts 5-hmC to cytosine, leading to active demethylation [30].
27
Figure 2: TET1/2/3 Facilitated Demethylation. Flowchart displaying the TET1/2/3
enzyme mediated conversion of 5-mC to 5-hmC through hydroxylation, followed by
multiple oxidations forming 5-fC and 5-caC. The end product after decarboxylation is a
completely demethylated cytosine.
28
Figure 3: 5-hmC Content in Different Tissue Types. Global 5-hmC levels are the
highest in brain, followed by breast and liver tissues. All values are scaled relative to
brain [36].
29
Figure 4: 5-hmC DNA Pulldown. A flowchart outlining the glycosylation of the
hydroxyl group, followed by biotinylation, streptavidin attachment, and eventual elution
of the enriched 5-hmC DNA. The reagents and protocol were obtained from Active
Motif. [http://www.activemotif.com/catalog/775/hydroxymethyl-collector]
30
A
N=1
3
N=1
1
Figure 5: Immuno-Dot Blot Analysis. (A) Dot blot displaying 5-hmC and 5-mC
antibody specificity and the signals of a small number of human normal liver and
matching tumor samples along with their corresponding 18S rDNA signal used to
normalize. (B) Average 5-hmC content among 13 normal and tumor pairs significantly
reduced in tumor samples. (C) Average 5-mC content among 11 normal and tumor pairs
significantly reduced in tumor samples.
31
A
Figure 6: Immunohistochemical analysis with anti-5hmC antibody showed
significantly reduced 5-hmC level in tumor compared to the benign liver tissues. (A)
5-hmC antibody stained tissue section of a representative normal and tumor pair on a
tissue microarray. (B) 85% of the 23 normal and tumor pairs had greater 5-hmC staining
in the normal samples.
32
Figure 7: Quantification of 5-hmC and 5-mC levels in genomic DNA by liquid
chromatography and mass spectrometric analysis. Genomic (A) 5-hmC and (B) 5-mC
levels were significantly reduced in tumor DNAs compared to those in matching livers.
However the decrease in 5-hmC was more pronounced than 5-mC in tumors.
33
N=6 N=6 N=6 Figure 8: 5-hmC level in EGFR, H19, and 7SL loci. (A) A region of EGFR gene was
amplified in 5hmC-pulldown DNA as well as in the negative control and input. The
results of 3 normal and tumor pairs are displayed on an ethidium bromide stained agarose
gel. (B) A region of H19 was amplified and displayed on a gel. (C) A region of 7SL was
amplified and displayed on a gel. (D-F) After normalizing to input, on average among 6
normal and tumor pairs, 5-hmC levels in the EGFR, H19 and 7SL amplicons were
significantly reduced in tumors compared to those in benign livers.
34
Figure 9: Estimation of 5-hmC level by immune-dot blot analysis in cirrhotic
samples. 5-hmC levels in 11 cirrhotic samples were minimally reduced compared to
those observed in normal samples. All values were normalized to 18S rDNA.
35
Figure 10: TET1/2/3 mRNA expression in human HCC and benign livers. (A) TET1
expression significantly increased in tumor samples in 58% of the 24 pairs. (B) TET2
expression significantly increased in normal samples in 63% of the 24 pairs. (C) TET3
expression significantly increased in tumor samples in 70% of the 10 pairs. All values are
displayed in a log scale and calculated based on the fold change over respective normals.
Tet1-3 expressions were normalized to that of GAPDH. (continued)
36
Figure 10 continued
37
Figure 11: 5-hmC content in the livers of mice fed choline deficient amino acid
defined (CDAA) or choline sufficient amino acid defined (CSAA) diet. On average, 5hmC content in CDAA fed mice was significantly reduced when compared to the DNA
of CSAA fed mice. 9 mice in each diet group were compared. 5-hmC levels were
determined using dot blot analysis and normalized to 18S rDNA.
38