DNA Sharma et al.,2009 Cytosine residues are methylated

DNA METHYLATION

DNA
methylation is an enzyme obsessed chemical alteration to the DNA sequence that usually occurs at CpG dinucleotide
in creatures Jovanovica et al.,2010
or DNA methylation is the covalent
change taking place at the 5′ end of the CpG dinucleotide of the cytosine ring
{C5 positions of cytosine (5-methylcytosine, 5-mC) li et al.,2016 Virani et al.,2012}
and with S-adenosyl-methionine as its methyl supporter Chen et al.,2013. A
CpG dinucleotide consists on a cytosine that precedes a guanine,29 and CpG
islands are successions of 500 up to 2,000 base pairs in length of CpG
dinucleotides mainly located in the proximal promoter region of genesBasse  and Arock ,2015 . This response is
catalyzed by the de novo methyltransferase
comprising DNMT1, DNMT3A and DNMT3B. This response is catalyzed by the DNMT
family, comprising DNMT1, DNMT3A and DNMT3B. Throughout the course of embryo
development, DNMT3A and DNMT3B are required for DNA methylation from scratch
and performance free of duplication and express equal importance for both
unmethylated and hemimethylated DNA, while DNMT1 is measured to be the
methyltransferase retaining the methylation position and methylating
hemimethylated DNA Sharma et al.,2009 Chen et al.,2013. DNA methylation provides a platform for several
methyl-binding proteins. These include MBD1, MBD2, MBD3, and MeCP2. These in
turn function to convert histone-modifying enzymes to manage the
chromatin-template processes Dawson et
al.,2012 Virani et al.,2012. CpG
sites are not casually scattered in the genome, but are intense in short
CpG-rich DNA fragments or DNA fragments in the lengthy repeat so-called ‘CpG
islands’. Sharma et al.,2009 Cytosine
residues are methylated on opposite DNA strands in the palindromic sequence
CpG. The methyl groups have no effect on base combination but can effect
protein-DNA interactions by protruding into the major groove Jones et al.,1999. DNA hypo methylation can
be related with gene reactivation and chromosomal variability and might lead to
the up regulation or overexpression of proto-oncogenes, improved recombination
and alteration degrees, twisted or damage of X-chromosomal inactivation and
loss of imprinting Sharma et al.,2009.
Hypomethylation of DNA has systematic effects. Primarily, it can lead to gene
initiation.  It has been found newly that
many CpG islands are routinely methylated in somatic tissues. These methylated
islands can develop hypomethylated in cancer and nearby genes become activated
Feinberg et al.,2004. Even if
arrangements of DNA methylation are generally passed authentically to daughter
cells, these patterns can also be reconstructed or wipe out Benjamin Tycko 2000.Even
though mutations in DNA methyltransferase and MBD proteins have long been known
to add to progressive aberrations Dawson et
al., 2012.

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The
figure summarizes the effect of methylation on DNA resulting in inhibition of
transcription process.  Taby et al.,2003

 

COVALENT HISTONE MODIFICATION

The
condensation of DNA in chromosome involves coiling of DNA around a protein
molecule called histone protein Taby et
al.,2003. The structure of histone protein is octane, with two each of
histones H2A, H2B, H3, and H4 Dawson et
al.,2012. In the mid?1960s 
pioneering work of Allfrey on histone modificationAllis et al.,2006. Histone methylation occurs
at both arginine and lysine residues on the tails of histone proteins H3 and
H4. Lysine methylation is enhanced by histone-lysine- N-methyltransferases,
also recognized as K-methyltransferases, and includes the transfer of methyl
groups from the cofactor S-adenosyl methionine. A vital protein involved in
control of stem cell upkeep and variation, EZH2 (enhancer of Zeste 2) is a
K-methyltransferase which catalyzes the trimethylation of H3K27. EZH2 is a
member of the polycomb repressive complex 2, a protein complex that involves
both a K-methyltransferase which identifies H3K27me3. The 3K27me3 mark is
normally involved in silencing genes connected to development and stem cell
differentiation, including the Hox gene cluster. EZH2 shows hyper expression
both at the transcriptional and protein stages, especially in prostate cancer
increase in EZH2 protein in the cell nucleus was noticed with a progression
from benign to metastatic disease. Overexpression of EZH2 is also seen in
breast cancer (BC), lymphomas, and glioblastomas etc.Virani et al.,2012.

Histone
acetylation: the histone acetylation is mainly related to the transcriptional
activity Virani et al.,2012.The
acetylation of lysine residues is a major histone alteration complexity in
transcription, chromatin construction also DNA
repair. Acetylation nullifies lysine’s positive charge of histone and may so
fade the electrostatic interaction between histones and negatively charged DNA. For this reason, histone acetylation is often
linked with a more open chromatin conformation dawson et al.,2012. Histone acetylation and chromatin conformation is
controlled by HAT (histone acetyltransfarase family) also known as
K-acetyltransferase and histone deacetylases. HAT catalyzes addition of acetyl
group to lysine using acetyl coenzyme A. HAT family consists mainly three members,
the Gen5 family, p300/CBP family, MYST family. Activity of these members plays
a vital role in cancerogenesis in positive or negative manner.the Wnt signaling
pathway become inappropriate in cancer particularly with stem cell phenotype
has been shown greater value by HAT Gen5 in breast cancer. CBP (Cyclic AMP
response element-binding protein) and p300 have shown capable of acetylation of
all four core histones as well as non-histone protein including p53,Rb, E2F and
myb.  p300 and CBP are essential tumor-suppressor

genes
that may be lost due to loss of heterozygosity in different cancersVirani et al.,2012.

Histone
deacetylation: Deacetylation of histones causes an increase in the positive
charge of these proteins. This modification raises the histone desire for DNA
or for other histones. The resulting compaction of the chromatin may block
access of transcription factors to the DNA or pressure the movement of RNA
polymerase. HDAC catalyses the removal of acetyl group in transcriptional repressor
Momparler 2003.HDAC have non histone proteins which are involved  in deacetylation of number of proteins
identifies as potential substrate. The link between histone deacetylation and
DNA methylation was the result that MeCP2 physically relates with the
transcriptional co-repressor protein Sin3A, and in so doing recruits a histone
deacetylase (HDAC) to chromatin that contains methylated DNA. This non-histone protein
involves in deacetylation and plays a major role role in carcinogenesis
including p53.YY1 and STAT3. HDAC3 a class1 HDAC in cancer cell found long term
reduced by means of RNA interference led to inhibition of ?- catenin
translocation to the nucleus in addition to that disrupting Wnt signaling pathway
Virani et al., 2012. The gene for the
multifunctional actin-binding protein gelsolin, which is epigenetically
silenced in most human breast cancers, can be reactivated either by AzaC or by
treatment with the histone deacetylase inhibitor trichostatin- A Tycko 2000.

                      Fig.
histone acetylation and deacetylation

The
direct collaboration between the chromo domain of Tip60 and histone H3
trimethylated on lysine 9 (H3K9me3) at double-strand break DSBs activates the
acetyltransferase activity of Tip60. Reduction of intracellular H3K9me3 blocks
initiation of the acetyltransferase activity of Tip60, resulting in faulty ATM
activation and extensive defects in DSB repair. Indeed, previous studies show
that site-specific histone changes associate well with particular biological
functions such as gene transcription. Such as, histone H3 lysine 9 acetylation
(H3K9ac), H3 serine 10 phosphorylation (H3S10ph), and H3 lysine 4
trimethylation (H3K4me3) are reported to be related with transcriptional
activation. Equally, H3K27me3 and hypoacetylation of H3 and H4 have been shown
to be correlated with transcriptional repression. the functions of histone
modifications are exposed by the recognition of histone code by specific
cellular mechanism such as the transcription tool. The histone H3S10
phosphorylation mark is catalyzed by mitogen and stress-activated protein
kinase 1 (MSK1) and is recognized by a 14-3-3?/14-3-3? heterodimer through its
interaction with H3K4 trimethyltransferase SMYD3 and the p52 subunit of TFIIH
(Fig.). Chen et al.,2013

Micro RNA

Epigenetic
changes are not limited to CpG methylation and post-translational modifications
of histones, but include also regulation by microRNAs .miRNA are short
endogenous nonprotein-coding single stranded RNA molecules which act as
negative gene regulators through their post-transcriptional ability to regulate
the translation of mRNA on binding. the expression of the target mRNA is then
repressed by two mechanisms: the target mRNA is degraded by ribonucleases from
the multiprotein RNA induced silencing complex (RISC) in the cytosol or by the
translation is just still without degradation of the target mRNA. In both cases
the target gene is not translated. miRNAs play a key role in many cellular
processes such as cellular growth, differentiation and apoptosis. Many tumor
suppressor miRNAs that target growth-promoting genes are repressed in cancer.
Aberrant expression of FmiRNAs is seen in BCSharma et al., 2009  Basse and Arock 2015 .The let-7 family of miRNAs
is aberrantly down-regulated in breast and lung tumors, leading to RAS pathway
oncogenic activation Taby and Issa 2010.

 

EPIGENETICS INFLUENCE ON BREAST
CANCER

Breast
cancer is the most frequently diagnosed cancer and the second leading cause of
cancer death in women. Epigenetic changes are dominant in breast cancers,
giving much interest in their clinical importance and whether these can be
altered. Aberrant HDAC activity has been reported in many tumors. HDAC 1
expression is related to an advanced stage and aggressiveness in certain
cancers .However, in breast cancer HDAC 1 expression is associated with
estrogen receptor (ER) and progesterone receptor (PR) expression, earlier stage
of disease at diagnosis, and improved disease-free survival. HDAC 6 messenger
RNA (mRNA) is more repeatedly expressed in BC patients with small (?2 cm), low
grade, ER and PR-positive tumors. However, the analysis failed to prove its independent
reason for BC. DNA methylation in hormone receptor positive and negative breast
cancer: Approximately 70% of breast tumors are positive for ER expression at
the time of diagnosis. The methylation of ER CpG islands was specifically
confirmed in breast tumor tissues Lipidus et al.,1998. Considering aberrant
DNA methylation in breast cancer, global DNA hypomethylation is far more
widespread in breast cancer specimens (up to 50 %) compared to that observed in
other tumor types. DNA hypomethylation can also affect individual breast cancer
genes. Breast cancer-related genes are hypermethylated and thus silenced
compared to non-cancerous tissue. Methylated genes in breast cancer include
those important for growth, illusion of apoptosis, invasion and metastasis as
well as cell differentiation. Methylation of promoter CpG islands of genes in
breast cancer: CpG island hypermethylation and breast cancer progression:
Promoter CpG islands hypermethylation have been associated with breast cancer
progression. Recent study assessed methylation levels of tumor suppressor
genes, RAR?2 and RASSF1A, MINT17, and MINT13 during key steps of breast cancer
development. The study identified a significant increase in the expression
levels of these genes during the breast cancer development. Hypermethylation of
promoter CpGs of RAR?2 and RASSF1A have been confirmed to play a role in breast
cancer. the report found RAR?2 and RASSF1A methylation within lesions from both
in situ lobular (LCIS) and ductal carcinoma (DCIS) Jovanovic et al., 2010.
Another study screened 57 promoter CpG loci in 20 invasive ductal carcinomas
(IDC) and their paired normal breast tissues. The study demonstrated that
methylation of 15 genes (DLEC1, GRIN2B, HOXA1, MT1G, SFRP4, TMEFF2, APC, GSTP1,
HOXA10, IGF2, RAR?, RASSF1A, RUNX3, HIN-1 and SFRP1) increased stepwise from
normal to atypical ductal hyperplasia (ADH)/ flat epithelial atypical (FEA) to
ductal carcinoma (DCIS) Park et al.,2010.even the methylation of APC, CDH1
and CTNNB1 or of the 14-3-3 Sigma gene (a tumor suppressor gene involved in the
cell cycle, DNA repair and apoptosis) has been linked to BC developmentBasse  and Arock
,2015 . AR, BARD1, BRIP1, ATM, CHEK2, DIRAS3, NBN, ERBB2, PALB2, RAD50,
and RAD51, ATM, p53, CHEK2, PTEN, CDH1are some of gene which even involved in
formation of breast cancerBehera 2017.   Promoter
hypermethylation of tumor suppressor genes in breast cancer: Epigenetic
inactivation of the tumor suppressor genes, such as BRCA1 have been implicated
as important events in sporadic breast cancer. Early reports showed that one of
the key mechanisms of BRCA1 expression loss was epigenetic silencing DiNardo
et al.,2001. DNA methylation and histone modification in breast cancer: DNA
(5-cytosine)- methyltransferases (DNMTs) are enzymes that methylate the
cytosine residue of CpGs. Four major types of DNMTs have been identified
(DNMT1, 2, 3a and 3b), among which DNMT1, DNMT3a and DNMT3b are primarily
active Sharma et al.,2009. Although breast tumors are also frequently hypomethylated
on genome-wide scale, the number of genes reported as hypomethylated in breast
cancer is relative small Wu et al.,2015

 

STUDIES RELEVANT TO EPIGENETICS OF
BREAST CANCER

The
team worked on bax gene using methylation specific PCR as a tool which shows
absence of CpG island hypermethylation in promoter region of the bax gene in
T47D, MCF7 (as ER positive), MDA-MB-231 and MDA-MB-468 (as ER negative) breast
carcinoma-derived cell linesAlipour et al.,2013.

Breast
cancer tissue and adjacent normal parenchymal tissue was obtained at the time
of surgery in 24 women. Normal breast tissues from 22 patients undergoing
breast surgery without breast cancer were used as controls. The tests were
carried out for HIN-1, RIL, RASSF1A, CDH13 and RAR?2, by methylation specific-PCR.
Methylated DNA products were qualitatively scored and a positive correlation
identified between tumour tissue and ipsilateral duct lavage in the breast
cancer group (n = 24)  HIN-1 (58 and
50%), RIL (63 and 42%), RASSF1A (71 and 54%), CDH13 (42 and 33%) and RAR?2 (38
and 25%). Methylation was significantly higher in tumour and ipsilateral duct
lavage fluid when compared with adjacent normal tissue and contralateral duct
lavage. Methylation was less when scored as accumulated methylation events in
benign breast tissue and duct lavage of 22 non-cancer patients: HIN (4% and
4%), RIL (8% and 0%), RASSF1A (13 and 0%), CDH13 (13 and 4%), RAR?2 (0%). No
difference was found in plasma methylation of cancer patients versus controlsGui
et al.,2009.

As
per the report Expression of 14-3-3 ? (?) is induced in response to DNA damage,
and causes cells to arrest in G2. By serial analysis of gene expression, it
reported as a gene whose expression is 7-fold lower in breast carcinoma cells
than in normal breast epithelium. The Northern blot analysis is used as tool to
identify it. Unusually, ? mRNA was undetectable in 45 of 48 primary breast
carcinomas. Genetic alterations at ? such as loss of heterozygosity were rare
(1/20 informative cases), and no mutations were detected (0/34). On the other
hand, hypermethylation of CpG islands in the s gene was detected in 91% (75/82)
of breast tumors and was associated with lack of gene expression Ferguson et
al., 2000.

Methylation
levels of 13 genes were analysed by quantitative multiplex methylation specific
PCR (QM-MSP) in nipple fluid samples from breasts of healthy women, and from
the affected and contralateral breasts of breast cancer patients. Methylation
analysis of the low-volume nipple fluid samples was possible. The genes which
are checked for methylation are AKR1B1, ALX1, RASSF1A and TM6SF1 which showed
promising resultsGroot et al.,2016.

The
report shows the frequency of aberrant methylation of four candidate genes
(APC, GSTP1, Rassf1A, and RAR?2) in primary breast cancer tissues from West
African women with predominantly advanced cancers. Quantitative
methylation-specific polymerase chain reaction is used to examine plasma from
93 women with breast cancer and 76 controls for the presence of four methylated
genes. Cutoff values for gene positivity of the plasma-based assay and the gene
panel were determined by receiver operating characteristic curves in the
training data set and subsequently evaluated as a screening tool in the
validation data set Methylation of at least one gene resulted in a sensitivity
of 62% and a specificity of 87%. the assay successfully detected 33% (eight of
24) of early-stage tumors Hoque et al.,2006.

A
team worked on RbL2/p130 gene using 76 breast cancer tumors along with normal
tissues (n = 76), blood (n =76) of respective individuals and control blood (n
= 50) were examined. Rbl2/p130 expression was analyzed by quantitative real
time PCR. Promoter methylation status was studied through methylation specific
PCR of bisulfite converted genomic DNA. Data was analyzed using various
statistical tests.the results were reflecting the epigenetic regulation of
RbL2/p130 gene expression in BC Ullah et al., 2015.

DNA
methylation analysis for the ALKBH3 promoter region was carried out by pyro
sequencing in 265 primary breast tumors and 30 proximal normal breast tissue
samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein
expression were analyzed in cell lines using RT-PCR and Western blotting,
respectively. DNA alkylation damage assay was carried out in cell lines based
on immunofluorescence and confocal imaging. The ALKBH3 gene undergoes CpG
promoter methylation and transcriptional silencing in breast cancer.  In group of 265 primary breast cancers, 72
cases showing aberrantly high CpG promoter methylation over the ALKBH3
promoter.  ALKBH3 is a novel addition to
the catalogue of DNA repair genes found inactivated in breast cancer Stefansson
et al., 2017.

QM-MSP
was done on ductal lavage cells from a set of 37 ductal lavage samples from
women undergoing mastectomy (27 with cancer and 3 without). Duct histology
information was available for each lavaged duct. QM-MSP data was assessed by
measuring cumulative methylation index and by receiver operating characteristic
threshold analysis. To determine the baseline level of methylation for each
gene in this population, cells from 60 ducts of women at high risk of
developing breast cancer were analyzed. QM-MSP findings on a panel of nine
genes were correlated to duct histology and ductal lavage cytology. Cytology
detected cancer in 33% (7 of 21ducts) with a specificity of 99% (92 of 93).
QM-MSP detected cancer as calculated by cumulative methylation index with a
sensitivity of 62% (13 of 21) and specificity of 82% (62 of 76) and by receiver
operating characteristic threshold analysis with a sensitivity of 71% (15 of
21) and specificity of 83% (63 of 76). Compared with cytology, QM-MSP doubled
the sensitivity of detection of cancer Fackler et al.,2006

A
group of scientist worked on the functional effects of DNA methylation in the
BRCA1 promoter and to clarify the functional status of the BRCA1 CRE (cAMP
response element) motif using MCF-7 breast carcinoma cells. Luciferase reporter
assays confirm that an intact CRE is required for BRCA1 expression in transient
transfections. Luciferase activities were reduced in constructs where the CRE
recognition sequence was changed and when constructs were methylated in vitro. Gel mobility shift and
competition assays were used to identified a DNA-protein complex identifying
the CRE motif that which were able to super shift using CREB- specific
antibody. Furthermore this CRE is methylation sensitive, and we localized this
methylation effect to a CpG dinucleotide within the BRCA1 CRE motif DiNardo et
al.,2001

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