Literature sex, and family history consider important risk

Literature review

Breast
cancer:

Breast
cancer is the most commonly diagnosed cancer and the top cause of cancer death among
females, approximately about 23% of the total cancer cases and 14% of the
cancer deaths (Jemal et al., 2011). At 2012 a study performed in cancer
incidence and mortality shows that  breast
cancer is the second most common cancer overall (1.7 million cases, 11.9%) but
ranks 5th as cause of death (522,000, 6.4%) because of the relatively
favourable prognosis (Ferlay et al., 2014). In the year 2012, there were 1008
cases of breast cancer in Jordan, of total breast cases 994 occurred in females
and 14 cases in males (Al-Sayaideh et al., 2012).

The
causative agents that induce breast cancer are not clear but there are risk
factors that may influence or increase the possibility of getting it. Such as Micro-RNA
miR-21 which controls the expression of several genes that regulate tumor
progression, including RAB6A, a member of the RAS oncogene family, TGFb-induced
protein, TGFb receptor II and Bcl2. Furthermore, recently it has been shown
that miRNA hsa-MiR-21 (miR-21) is up regulated in breast cancer and
significantly associated with advanced stage at presentation and the increased
levels of miR-21 are correlated with poor survival (Huang et al., 2009).

Epigenetic
factors and genes mutation also play role in breast cancer progression: silencing
of the estrogen receptor and inactivation of BRCA1 and BRCA2 can occur through
methylation. Gene mutation may involve dominant gene mutations such as
mutations in BRCA1 and BRCA2 genes or SNPs (single nucleotide pleomorphism)
which is change in a single nucleotide that result in increasing (MMP-1 and
MMP-9 correlate well with advanced tumor stage) or decreasing (MMP-8 lead to
enhanced breast cancer metastasis) genes expression (Tao et al., 2014). Risk of
developing breast cancer is increased by early menarche, late menopause, and nulliparity;
whereas, risk is reduced by higher parity and lactation. Furthermore, using
combined hormone therapy after menopause increases breast cancer risk; the higher
risk appears to apply only to recent use. In addition, age, sex, and family
history consider important risk factors (Anderson, Schwab and Martinez, 2014).

 

 

 

 

 

 

 

 

 

 

 

 

 Molecular classification of breast cancer
first proposed by Perou and Sorlie, in there study they divided breast cancer
according to gene expression as follow: Luminal which differentiated in two or
three subgroups it reflect estrogen receptor (ER), ER regulatory genes and the
expression of genes expressed in normal luminal epithelial cells. Human
epidermal growth factor receptor (HER-2) posi­tive it reflect HER-2 amplification
and overexpression. Basal breast cancer which reflect ER, progesterone (PR),
and HER-2 negative and the expression of genes expressed in normal breast basal
cells. A normal-like subgroup also been described (Perou et al., 2000). There
are special types of tumor that does not fit Perou classification and it
comprise about 15-25% of breast cancer, according to ER expression they divided
into two main groups: ER-positive group that consist of classic invasive
lobular carcinoma, tubular, micropapillary, mucinous and neuroendocrine carcinomas.
The ER-negative group includes apocrine, pleomorphic invasive lobular
carcinoma, adenoid cystic, metaplastic and medullary carcinomas (Vuong et al.,
2014).

 

Table
1: Histologic and molecular properties of specific type breast
cancer (Eliyatkin et al., 2015)

Molecular
subtype

Common
histologic types

HG

ER
status by IHC

HER2
status by ISH/IHC

Ki67
by IHC

Specific
IHC/molecular properties

Luminal
A

Classical,
lobular, tubular, cribriform

1
or 2

     +

        –

Low

Luminal
CK +
E-cadherin
+/-
 

Luminal
B

Micropapillary

2
or 3

     +/-

       -/+

High

Luminal
CK +, p53 mutations

Basal-like

Medullary,
metaplastic,
adenoid
cystic, secretory

3

      –

        –

High

Basal
CK+, p53 mDNA repair loss, EGFR+/- mutations

Molecular
apocrine

Apocrine,
plemorphic lobular

2
or 3

      –

       +/-

High

Androgene
receptor+

Claudin
low

Metaplastic

3

      –

        –

High

Cancer
like stem cell, EMT like, low E-cadherin level

 

HG: histological
grade, tumors are graded as 1, 2, 3, or 4, depending on the amount of
abnormality.

G1:
Well differentiated (low grade), G2: Moderately differentiated (intermediate grade). G3: Poorly
differentiated (high
grade) (National Cancer Institute, 2018)

IHC: immunohistochemistry 

+/-  : mostly
positive,           -/+  :
mostly negative  

 

 

 

 

 

Triple-negative
breast cancer (TNBC):

This
type of cancers lack ER, PR, and HER2 amplification, treatment of these type
cancers are difficult due to its heterogeneity and the absence of well-defined
molecular target (Carey et al., 2007). Using gene expression and cluster
analysis 6 TNBCs subtypes identified that show unique GE and ontologies, including
2 basal-like (BL1 and BL2), an immunomodulatory,

 a mesenchymal, a mesenchymal stem–like, and a
luminal androgen receptor (LAR) subtype. In this section, the focus will be on
the LAR because the MDA 453 belong to this subtype, this subtype is ER
negative, but gene ontologies are heavily enriched in hormonally regulated
pathways including steroid synthesis, porphyrin metabolism, and androgen/
estrogen metabolism. Furthermore there are an increase in the expression of AR
mRNA and numerous of downstream AR targets and coactivators (DHCR24, ALCAM,
FASN, FKBP5, APOD, PIP, SPDEF, and CLDN8).
LAR subtype lack basal cytokeratin expression and express high levels of
luminal cytokeratins and other luminal markers (FOXA1 and XBP1),
five cell lines matched to the LAR subtype (MDA-MB-453, SUM185PE, HCC2185,
CAL-148, and MFM-223) (Lehmann et al., 2011).

 

MDA-MB-453:

Cell
lines representative of the LAR subtype (MDA-MB-453, SUM185PE, CAL-148, and
MFM-223) express high levels of AR mRNA and protein. Furthermore they were more
sensitive to bicalutamide than basal-like cell lines, also the LAR cell lines
were more sensitive to Hsp90 (which is chaperon required for AR proper folding)
inhibitor 17-dimethylaminoethylamino-17- demethoxy-geldanamycin (17-DMAG)
compared to basal-like and mesenchymal like cell lines.

MDA-MB-453
was transfected with AR targeting siRNA to investigate AR dependence on this
cell line, the knockdown verified at the mRNA and protein level, the ability of
MDA-MB-453 to form colonies was significantly reduced after knockdown of AR expression
as compared with control samples (Lehmann et al., 2011).

MDA-MB-453
cells harbor a K-RAS mutation at codon 13 (Gly 13 Asp GGC>GAC) which result
in constitutive activation of K-RAS also it has been confirmed that ERK1/2 is
highly phosphorylated in cells and it retain high ERK activity irrespective of FBS
concentration (VRANIC, GATALICA and WANG, 2011).

Mutation
in AR gene have been identified in MDA-MB-453, the resulting AR-Q865H variant
has compromised activity in response to DHT in relation to the wild type AR but
it retains transcriptional responsiveness to DHT and it does not confer
responsiveness to non-androgenic ligands. It also possibly regulates a different
set of endogenous genes in relation to wild type AR (Moore et al., 2012).

 

 

 

 

 

 

Epithelial
mesenchymal transition (EMT):

EMT
process initially recognized during several stages of embryonic development and
it has the ability to convert the epithelial cell to motile mesenchymal cell.
It is responsible of tissue remodeling events, including mesoderm formation,
neural crest development, heart valve development and secondary palate
formation etc. (Yang and Weinberg, 2008).

EMT requires
modifications in morphology, cellular architecture, adhesion, and movement capacity.
Commonly used molecular markers for EMT include increased expression of
N-cadherin, vimentin, Fibronectin, Snail1 (Snail), Snail2 (Slug), Twist, Goosecoid,
FOXC2, Sox10, MMP-2, MMP-3, MMP-9. Nuclear localization of ?-catenin, Smad-2/3,
Snail1 (Snail), Snail2 (Slug), and Twist that inhibit E-cadherin production. Furthermore,
the cells morphology change into elongated mesenchymal shape and possess
increased migration capacity, the ability to invade, and scattering (Lee et
al., 2006).

EMT
is induced by signaling pathways mediated by transforming growth factor ?
(TGF-b) and bone morphogenetic protein (BMP), Wnt–?-catenin, Notch, Hedgehog,
and receptor tyrosine kinases (Gonzalez and Medici, 2014).

Transforming
growth factor-? (TGF?) induces EMT through the formation of tetrameric complex
of type I and type II receptors (T?RI and T?RII) to activate SMAD2 and SMAD3,
which then bind with SMAD4. The SMAD complex translocates into the nucleus and
cooperates with transcription regulators in the repression or activation of
target genes. TGF? can also induce non-SMAD signalling pathways through the
activation of PI3K–AKT–mammalian TOR complex 1 (mTORC1) signalling. Activation
of Frizzled by Wnt ligands results in phosphorylation of low-density
lipoprotein receptor–related protein 6 (LRP6) by GSK-3? and the recruitment of
Dishevelled (Dvl) and Axin to the plasma membrane enabling ?-catenin to
translocate to the nucleus, nuclear ?-catenin binds to members of the TCF/LEF
family of transcription factors to promote EMT. Wnt-mediated induction of EMT
through Snail2 by decreasing the expression of E-cadherin and increasing the
expression of fibronectin after the accumulation of ?-catenin in the nucleus (Lamouille,
Xu and Derynck, 2014), (Gonzalez and Medici, 2014).

Figure
1: signaling pathways involved in EMT (Lamouille, Xu and Derynck, 2014)

Role
of EMT in cancer:

EMT
process during cancer progression facilitate cancer cells invasion and
metastasis to distant sites, the role of EMT in oncogenesis achieved by the
ability of EMT transcriptional regulator to enhance cancer infiltration and
metastasis, such as SNAIL1 which correlate with increased aggressiveness and
poor survival in human breast cancer (Moody et al., 2005).   

The
role of EMT is not confined to tumor invasion and metastasis only, it also play
roles in cancer progression such as; resistance to cell death and senescence,
resistance to chemotherapy and immunotherapy, evading immune system by inducing
tolerance or modifying its phenotype and immunosuppression (Thiery et al.,
2009).

 

Figure
2: Cellular cytoskeletons in epithelial?mesenchymal
transition (Zhang et al., 2017)

In
breast cancer, many transcription factors that regulate EMT have been found to
be activated in breast tumor resulting in EMT initiation and tumor progression
by forming a complex signaling network (Wang and Zhou, 2011). In breast cancer,
partial or total loss of E?cadherin expression and increased N-cadherin
expression correlates with loss of differen­tiation characteristics,
acquisition of invasiveness, increased metastatic ability (Liu et al., 2017).
Cluster of differentiation CD44 (which is cell?surface protein that modulates
cellular signaling) expression is upregulated in high?grade human breast cancers,
and is associated with the level of the mesenchymal marker N?cadherin in these cancers
(Brown et al., 2011). In normal non-invasive epithelial cells, ?-catenin
located at the cell membrane binding to the cytoplasmic portion of E-cadherin,
in EMT ?-catenin dissociate from cell membrane and located in cytoplasm then
translocate in the nucleus to promote the transcription of genes that induce
EMT (Zeisberg
and Neilson, 2009). Furthermore, there are clinical evidences to support the
upregulation of Wnt/? ?catenin signaling in invasive breast ductal carcinoma (Prasad et al., 2009).

Increased
expression of SLUG in invasive ductal carcinoma result in inducing EMT through
downregulation of E-cadherin (Lopez et al., 2009), finally further studies on
the link between EMT markers and breast cancer will contribute to the identification
of biomarkers for early breast cancer metastasis prediction and to identify new
therapeutic targets in breast cancer (Liu et al., 2016).

Androgen
Receptor (AR):

Androgen
receptor belong to nuclear steroid hormone receptor (nuclear receptor
sub-family 3, group C, gene 4) along with estrogen, progesterone,
mineralocorticoid, and glucocorticoid receptors, it plays important roles in
the development and maintenance of the reproductive, musculoskeletal,
cardiovascular, immune, neural and haemopoietic systems  (Nuclear Receptors Nomenclature Committee,
1999)( Davey et al., 2016). AR consists of
three major functional domains: N-terminal domain (NTD), DNA binding domain
(DBD) and C-terminal ligand binding domain (LBD) which connected to DBD by a
flexible hinge region. The DBD is highly conserved among all steroid hormone
nuclear receptors and it consist of two zinc fingers that recognize specific
DNA consensus sequences (Heinlein and Chang, 2002).

DBD
enable direct DNA binding of AR to the promoter and enhancer regions of
AR-regulated genes, facilitating the activation functions of the N-terminal and
ligand binding domains to induce or repress the transcription of these genes,
the highly conserved nature of DBD allow specific binding to the androgen
responsive element in genes promoter regions (Schoenmakers et al., 2000). The
ligand binding domain linked through hinge region to DBD and it mediates the
interaction between the AR and heat shock and chaperone proteins, also it
facilitate the interaction with the N-terminus of the AR to stabilize bound
androgens (Heinlein, 2002).

Two
transcriptional activators have been identified: the ligand-independent
activation factor (AF-1), positioned in the N-terminal domain and required for
maximal activity of the AR, and the ligand-dependent activation factor (AF-2),
placed in the ligand binding domain responsible for making the coregulator
binding site as well as enabling direct interactions between the N-terminal and
ligand binding domains (Wilson, 2011). Nuclear localization signal (NLS)
located between the DBD and hinge region and its responsible for AR entrance into
the nucleus, while nuclear export signal (NES) responsible for exporting AR to
the cytoplasm upon ligand removal and it located in the ligand binding domain (Tan
et al., 2014).

 

Figure
3: Functional domains of the androgen receptor (Davey et al., 2016).

In DNA
binding-dependent actions of AR (canonical AR signalling), androgens bind to
the AR resulting in detachment of chaperone proteins, conformational change and
exposure of NLS which induce nuclear localization of AR, where it dimerises and
binds to AR responsive elements within target genes promoters to enhance or
repress gene transcription in the presence of co-regulators. (van de Wijngaart
et al., 2012). DNA binding independent actions of AR (non-canonical) in which
androgen/AR complex activate second messenger pathways including ERK, Akt and
MAPK, also indirect gene transrepression can happen, by the AR binding and
sequestering transcription factors such as activator protein-1 (AP-1) (Lamont
and Tindall, 2011).

 

Role
of AR in EMT:

AR
receptor can induce EMT through several mechanism, direct inhibition of
E-cadherin through the ability of AR to bind to potent repressive element in
E-cadherin promoter after being activated by DHT, and induce morphological
changes from epithelial to mesenchymal-like appearance. Furthermore, clinical
samples from invasive breast ductal carcinoma show high levels of AR and
reduced E-cadherin expression (Liu et al., 2008).

AR
can induce EMT in prostate cancer through the activation of Snail zinc finger
transcription repressor resulting in significant migration and invasion
potential (Zhu and Kyprianou, 2010).

Zinc
finger E-box-binding protein 2 (ZEB2) which is one of the EMT transcriptional
mediator was found to be significantly upregulated after androgen stimulation,
decrease in ZEB2 expression have been observed after AR silencing in
androgen-dependent prostate cancer cell line. This finding propose AR as
positive regulator of ZEB2 expression in androgen-dependent cells (Jacob
et al., 2014). In prostate cancer activated AR can significantly increase the
expression of Slug even after 2hr from activation (Wu et al., 2012).

 Splice variants of AR contribute to
castration-resistant prostate cancer and EMT induction, AR splice variants 3
(AR3) modulates the expression of TGF-?, IGF1, and several EMT associated genes
(Khan et al., 2015). In triple negative breast cancer AR upregulate ZEB1
(which is transcription factor involved in EMT) after being activated with DHT to
promote EMT and cell migration (Graham et al., 2009).

 

SLUG:

Slug
belongs to Slug/Snail family of transcription factors, Proteins of this family
include Snail1, Slug/Snail2, Snail3/Smuc, and Scratch. They have a highly
conserved carboxy-terminal region that contain four to six C2H2-type zinc
fingers that bind to a subset of E box (CAGGTG) sites in the promoter regions
of genes that regulated by this family (Inukai et al., 1999). The N-terminal
region contains the SNAG transactivation domain, which is essential for
Snail2-mediated repression and specific 28 amino-acid sequence (amino acids 96
to 123) called the SLUG domain, both the N-terminal SNAG and the central SLUG
domains are required for efficient repression of the E-cadherin promoter. Nuclear receptor co-repressor 1 (NCoR) interacts with Snail2
through the SNAG domain, while C-Terminal Binding Protein 1 (CtBP1) is recruited
through the SLUG domain (Molina-Ortiz et al., 2012).

In
normal development SLUG was first recognized in the neural crest, developing
mesoderm and the limb of chick embryos and it shows dynamic pattern of
transcription during early development, furthermore knockdown of Slug gene using
antisense oligos result in specific developmental failure suggesting its
important role in embryo development (Nieto et al., 1994).

Slug
display increased expression during organogenesis, especially in proliferating
chondrocytes in bone of both neural crest and mesodermal origin, intestinal and
stomach walls, craniofacial mesenchyme and mesenchyme of the lung and kidney
(Oram et al., 2003).

Study
performed by (Shi et al., 2011)
recognize Snail2 as a key regulator of the signals involved in
mesodermal induction of neural crest, and its loss results in changes in the
RNA levels of a number of BMP and Wnt agonists and antagonists.

Polycomb
repressive complex 2 (PRC2) consist of Eed, Ezh2 and Suz12 core component which
expressed in neural crest cells and are required for neural crest marker
expression, during neural crest development slug and PRC2 cooperate with each
other in order to express the genes that associate with neural crest
specification and migration (Tien et al., 2015).