The presentation illustrates the role of epigenetic mechanisms in HCC development .De in therapy scription of the role of DNA methylation ,histone modifications,and miRNA in etiopathogenesis of the disease is provided,together with the possible use in therapy .

1. Epigenetic Mechanisms in
Hepatocellular Carcinoma
Dr.Ekbal Mohamed Abo-Hashem(MD)
Professor of Clinical Pathology
Faculty of Medicine-Mansoura University-
Egypt

2. Item 5
Epigenetics: Definition-history
Epigenetic alterations in HCC
a. Global DNA hypomethylation & cancer – linked gene specific DNA hypomethylation.
b. Cancer-linked gene specific DNA hypermethylation.
c. Altered histone modification patterns.
d. Aberrant expression of microRNAs
Hepatocellular Carinoma: A genetic and
epigenetic disease
Take-home message.
Further readings
Item 1
Item 2
Item 3
Item 4
Agenda

3. The development and progression of HCC is a
multistep and long-term process characterized by the
progressive sequential evolution of morphologically
distinct preneoplastic lesions (formed as a result of
chronic liver injury, necro-inflamation and
regeneration, small cell dysplasia, low-grade and
highgrade dysplastic nodules) that culminates in the
formation of HCC.
1.Hepatocellular Carinoma: A
genetic and epigenetic disease

4. Traditionally, the development of HCC in humans
has been viewed as a progressive multistep process
of transforming of normal cells into malignant
driven primarily by the stepwise accumulation of
genetic alterations in tumor-suppressor genes and
oncogenes, with mutations in b-catenin and P53
genes being the major genetic alterations.

5. However, over the past decade there has been a
surge in data indicating the importance of
epigenetic processes, which has largely changed
the view of HCC as a genetic disease only.
Presently, HCC is recognized as both a genetic
and epigenetic disease, and genetic and epigenetic
components cooperate at all stages of liver
carcinogenesis. While the sequential accumulation
of various genetic changes in
hepatocarcinogenesis has been extensively
studied, the contribution of epigenetic alterations
to HCC development and progression has
remained relatively unexplored until recently.

7. 2- Epigenetics: Definition-
history
The term “epigenetics” refers to all stable changes of
phenotype traits that are not coded in the DNA
sequence itself. Epigenetic mechanisms can be viewed
as an interface between the genome and risk factor/ life
style/ enviromental influence.
Epigenetic inheritance is essential for the development
of critical cellular processes such as gene transcription,
differentiation and protection against viral genomes.
Epigenetic and genetic mechanisms work together to
establish key cellular genes and destabilize the
genome, leading to oncogenic tarnsformation and the
observed complexity and heterogeneity in human
cancers, including HCC.

8. The term epigenetics was first set by Conrad
Wassington in 1940, based on the Greek prifix “epi”
meaning over or above.
In the 1970s: DNA methylation was identified as a
regulatory factor.
In the 1980s and 1990s: pioneering research on the
maintenance and regulation of DNA methylation
patterns in mammals.
In the mid 1990s: histone modification was discovered
as an epigenetic determinant.
In the 2000s, non-coding micro RNAs and their
functions were identified.
The first whole epigenome analysis was completed for
yeast in 2005.

9. a. Global DNA hypomethylation & cancer – linked
gene specific DNA hypomethylation.
* DNA methylation.
* Global DNA hypomethylation.
*Cancer- linked, gene specific hypomethylation.
3-Epigenetic alterations in HCC

10. * DNA methylation
The methylation of DNA is catalyzed by DNA
methyltransferases (DNAMT1, DNAMT3a and
DNAMT3b) and is a reversible physiological process
in the eukaryotic genome.
This biochemical event involves the donation of a
methyl group from S- adenosyl- methionine (SAM)
to the 5-position of a cytosine nucleotide linked to a
guanine nucleotide (CpG dinucleotides) by a
phosphodiester bond.
Regions with a high CpG dinucleotide content form
CpG islands, which are located in the regulatory
regions of many genes, including promotors and
enhancers. Thus, changes in methylation status can
either facilitate (hypomethylation) or inhibit
(hypermethylation) the expression of a gene.

11. Alterations of DNA methyltransferases in human HCC
Several reports distinctly established the major role of altered gene
expression of DNA methyltransferase DNMT1, a main enzyme
involved in the maintenance of genomic methylation patterns, the de
novo DNA methyltransferases DNMT3A and DNMT3B, and methyl-
binding proteins in the development and progression of HCC. This is
evidenced by a progressive marked up-regulation of DNMT1,
DNMT3A, and DNMT3B in premalignant noncancerous liver tissues
and in full-fledged HCC and by the fact that over-expression of these
DNMTs significantly correlated with CpG-island hypermethylation of
tumor-related genes.

12. * Global DNA hypomethylation.
Occurs due to loss of methylation at normally heavily
methylated areas of genome.
* decrease in the number of methylated cytosine bases.
Active loss of methylated cystosines is due to dysfunction
of DNA repair machinary.
Passive loss of methylated cytosines may be due to:
- Limited availability of the universal methyl donor S-
adenosyl-L- Methionine (SAM).
- Compromised integrity of DNA- Altered expression
and/or activity of DNA methyltransferases (DNMTS)

13. The loss of DNA methylation was the
first epigenetic abnormality and one of
the most common molecular alterations
identified in human cancers, including
HCC. Global DNA demethylation in
HCC primarily affects stable, methylated
areas of the genome composed
predominantly of repetitive DNA
sequences.

14. There are several molecular consequences of global
DNA demethylation that may contribute to the
progression of liver carcinogenesis via multiple
mechanisms. Specifically, genomic hypomethylation
may cause a significant elevation in mutation rates,
aberrant activation of ‘‘normally’’ silenced tumor-
promoting genes, loss of imprinting, and activation and
transposition of repetitive DNA elements leading to
chromosomal and genomic instability.

15. * Cancer-linked, gene-specific hypomethylation
Hypomethylation of ‘‘normally’’ methylated
genes is significant in the pathogenesis of HCC.
Currently, a number of hypomethylated tumor-
promoting genes, including uPA, HPA , SNCG ,
TFF3 , MAT2A , HKII , CD147, and VIM have
been identified in primary human HCC.
Gene-specific promoter DNA hypomethylation
changes are related to biological processes
critical for tumor progression, including cell
growth, cell communication, adhesion and
mobility, signal transduction, and drug
resistance.

16. B) Cancer-linked gene-specific DNA
hypermethylation in human HCC
DNA hypermethylation is the state where the methylation
of ‘‘normally’’ undermethylated DNA domains, those that
predominantly consist of CpG islands.
CpG islands (cytosine-phosphate- guanine) are present in
the promotor and transcription start sites of the genome.
They comprise only 1-2% of the genome and are normally
unmethylated. In- appropriate methylation of cytosine
residues of CpG islands interfers with promotor function of
the genes& transcriptional silencing of genes critical to the
normal anti-neoplastic process (blocking tumor suppressor
gene function).

17. Normal demethylation of CPG islands is achieved by
proteins containing (Zn) finger binding domains and
by various histone demethylases.
Gene-specific DNA hypermethylation has gathered
most attention as a critical event in liver
carcinogenesis. Several epigenetically inactivated
genes, as evidenced by association between
diminished mRNA levels with highly methylated
promoters, have been identified in HCC.
These genes are involved in the regulation of vital
biological processes, including cell-cycle control,
apoptosis, cell proliferation, and xenobiotic
metabolism.

18. The role of cirrhosis as it relates to DNA methylation. Cirrhosis plays an important
role in the development of hepatocellular carcinoma (HCC). This can result from
epigenetic changes and can also cause epigenetic and genetic changes that
ultimately lead to HCC, HCV, hepatitis C virus; HBV, hepatitis B virus

19. Proposed pathway relating epidemiological exposures to the development of
hepatocellular carcinoma (HCC). Acquired disease results in chronic hepatic insults,
which accumulate and result in the development of HCC. HCV, hepatitis C virus;
HBV, hepatitis B virus

20. The aberrant gene-specific hypermethylation of the
aforementioned genes occurs not only in HCC, but also in
premalignant pathological conditions, including chronic
viral hepatitis B and C and liver cirrhosis, suggests the
importance of gene-specific hypermethylation event in
pathogenesis and progression of HCC.
The existence of two opposing hyper- and hypomethylation
events in the same functional pathways complement or
enhance each other in the disruption of cellular
homeostasis favoring progression of HCC. For instance,
hypermethylation and transcriptional inactivation of the E-
cadherin (CDH1) gene and hypomethylation- induced up-
regulation of the Vimentin (VIM) gene in HCC may
exaggerate invasion and escalate further progression of
HCC.

21. C- Altered histone modification patterns:
Histones are basic proteins that facilitate the
packaging of DNA in the nucleus and regulation
of gene expression in cells. The histone proteins
are H1, H5, H2A, H2B H3 and H4.
Two copies of the H2A, H2B, H3 and H4
histones assemble to form an octameric histone
protein core. DNA is wrapped around the core
twice in a left handed super-helical turn to make a
structural unit called the nucleosome. Histones H1
and H5 are linker histones.

22. Histone proteins, as well as DNA are important
components of chromation representing the
physiological from of the genome. Nucleosome
is the repaeting unit of chromatin in which
genomic DNA is wrapped around a core
octamer consisting of dimers of four histone
proteins (H2A, H2B,H3, and H4 and/or their
variant isoforms). Another histone protein H1
severs as a linker to further connect individual
nucleosomes into larger chromatin fiber.

23. Histone (chromation) modifications comprise
covalent post-translational modifications of
histone proteins. The N-terminal tails of
nucleosomal histones are subjected to different
modifications, including acetylation,
melhylation, phosphorylation and
ubiquitination which apear to work together
with other epigenetic mechanisms in
establishing and maintaining gene activity
states, thus regulating a wide range of cellular
processes.

26. Changes in histone modifications in HCC occur genome-
wide and on genespecific scales. At least eight different
classes of post-translational modifications, including
methylation, acetylation, phosphorylation, ubiquitynation,
sumoylation, biotinylation, and ADP-ribosylation have been
identified on the core histones H2A, H2B, H3, H4, and the
H1 family of linker histones.
Typically, histone acetylation is associated with an active
transcription, whereas methylation may be associated with
either active or repressive states, depending on the modified
site.
Histone modifying enzymes display multifaceted roles in
co-ordinating the interaction of intracellular signaling
pathways through chromatin remodeling.

28. Roles of histone modifications in the regulation of gene
transcription.

29. D- Aberrant expression of microRNAs:
The term microRNA was first reported by Ambros in
2001. There are currently over 1000 human micro RNAs
listed in the miRNA data base (h
ttp://microrna.sanger.ac.uk/sequences/), accounting for
about 7% of the human transcriptome, although it is
predicted that the true figure is likely to be more than one
thousand.

30. miRNAs are small non-coding-single-
stranded RNAs of 16-29 nucleotides in
length that negatively regulate the
expression of many target genes at the
post-transcriptional and/or translational
levels and play a critical role in the
initiation and progression of HCC. They
regulate expression of various oncogenes
and tumor suppressor genes, thereby
contribute to proliferation, apoptosis,
epithelial to mesenchymal transition and
metastasis.

31. RNA induced silencing complex (RISC)

32. Biosynthesis
• Pri-micro RNAs are cleaved within the nucleus
by a microprocessor complex consisting of
Dorsha (an RNase III-type nuclease) and a
protein cofactor, DGCR8.
• The resulting 60-70 nucleotide Hairpin
sturcture ( pre micro RNA) encodes for a single
micro RNA sequence that is exported from the
nucleus to the cytoplasm by Exportin 5.

33. • Cytoplasmic pre-micro RNAs are further cleaved by
Dicer (another RNAase III- nuclease) in concert with
cofactors, to remove the loop sequence forming a short-
lived asymmetric duplex intermediate. (microRNA:
microRNA*) which is then leaded into the microRISC
complex.
• The micro RNA-RISC complex is then delivered to
3UTR of mRNA for degradation. This leads to
decreased synthesis of proteins which are either
inhibitors or activators of metabolic pathways
depending on cell types, tissues and developmental
stage.
• MicroRNAase can play a direct role in oncogenesis as
they can function both as oncogenes and tumor
suppressor molecules.

34. Biogenesis of microRNA and its role in the regulation of gene
expression.

35. Hepatic miRNA profile may predict the
recurrence of HCC after resection and
circulatory miRNA levels may be used as a
potential biomarker for non-invasive
diagnosis of HCC.

37. 4- Take-home message:
The epigenetic events (DNA methylation,
histone modification and noncoding
RNAs), being specific to different risk
factor exposures, form an epigenetic
signature with the potential to serve as an
important biomarker for early detection and
prevention of HCC. Unlike genetic events,
epigenetic events are reversible, and thus
hold better promise for therapeutic
interventions.,

38. Stratigies to delay or reverse the slowly
progressing preneoplastic stage, during
which gene function is perturbed mainly
by potentially reversible epigenetic
mechanisms, may prevent a slow the
subsequent development of irreversible
sturctural alterations in HCC-related genes,
and therapy prevent the emergence of HCC
during a normal life span.

39. Many HCC, contain irreversible structural changes
in multiple genes involving several regulatory
pathways simultaneously. Effective molecular
therapy of such genomically complex HCCs
presents a severe challenge. By contrast, some
HCCs such as these characterized by B-caterin
mutations, may have more limited genomic
alterations and be more amenable to molecular
therapeutic intervention prospectively,
development of global methods of analysis such as
proteomics or micro assay methods will probably
increase discovery of new genes or pathways
involved in hepatocarcinogenesis

40. Epigenetics-based therapy for HCC
These approaches are directed at
modifying DNA methylation profiles and
histone modification states in liver cancer
cells.

41. Thank you..