role of cytokines in breast cancer

1. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
Page | 1
Cytokines in the habitat of breast cancer; Some of which promote
cancer while remaining cytokines help to alleviate cancer hence
helping the immune-system.
Farooq A, Ayub G, Ali H, Jamim K and Siddique R.
National university of Sciences and Technology H-12 Islamabad.
Abstract
Breast cancer like other cancers is proliferative and tumor invasive as it is made obvious
when the roles of cytokines were studied and understood. There are some cytokines which
are proliferative and tumor invasive such as interleukin-1 alpha and interleukin-1 beta
along with IL-6, IL-11, TGFβ . Simultaneously there are other cytokines which played their
role in alleviation of cancerous states such as IL-12, IL-18, IFNs which help in inhibition of
invasion of cancer/tumors. So up till now IL-2, IFNα, IFNβ and sometimes IFNγ, IL-6, IL-
12 h are now employed in the treatment against breast cancer. However the inhibitor of the
receptor of the IL-1 alpha and IL-1 beta also show the prohibition of invasiveness and
hence it is therapy given to cancerous patients.
Key words
Breast cancer, cytokines, proliferation, invasion, tumor, metastasis.
Introduction
Cytokines are low molecular weight
glycoproteins which are synthesized and
rapidly secreted by the healthy as well as the
diseased cells when they get stimulated.
They can act on many target cells and their
mode of action may be pleotropic,
synergistic and antagonistic manner. In
living organisms which are multi cellular,
cytokines serve as intercellular mediators
that help the regulation of growth, survival
and differentiation(Heinrich et al., 1998). It
is worth to mention that cytokines are not
only produced in the health states or by the
healthy cells but it should not be surprising
that the cancerous cells also produce the
cytokines and function as tumor growth
promoting or inhibiting factors. Specific or
non-specific anti-tumor growth responses
are produced by these intercellular
mediators (Kreiss et al., 1994).
The interleukin (IL) -1 family of cytokines
(IL-lα, IL-1β), the IL-1 receptor antagonist
(IL-1Ra) and receptors (IL-1RI and IL-1RII)
have been found to be frequently expressed
in breast cancer cell lines, in human breast
cancer tissue, and within the tumor
microenvironment(Miller et al., 2000,
Pantschenko et al., 2003, Singer et al., 2003)

2. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
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Immunological functions of the
family IL-1
The IL-1 family of the cytokines is
responsible to generate the immune response
relating to the innate immunity. This was
basically discovered and when it became
obvious through the experimentation that the
cytoplasmic domains of the receptors of the
IL-1 highly resemble those of the
cytoplasmic domains of the toll like
receptors. So it is intriguing to know that
what are the roles of the toll like receptors
and cytokines of IL-1 family. All the basic
inflammatory responses along with the
enlarged expression of the adhesion
molecules became the roles of TLR ligands
and IL-1 family. The IL-1 family of the
cytokines functions also in non-specific
detection of the antigen and increased
function of the lymphocytes (Dinarello,
2009) the member IL1-beta is the most
studied cytokine of the IL-1 family because
of its roles found in the auto inflammatory
diseases.
Pathways of action of IL-1 family
The IL-1 family of the cytokines comprise
of total 11 proteins which are from IL1F-1to
IL1F-11 which are duly encoded by
different 11 genes both in the species of
human and in the mice. As mentioned
previously the IL-1 family workouts the
innate immune responses. Their role in the
auto-inflammatory diseases was made
clearer when it was noticed that the
members IL-1alpha and IL-1 beta were
blocked by the antagonist of the receptor of
the IL-1 family named as IL1-RA. IL-1
alpha and beta heavily increases the
expression of then a lot of genes present
there at that time in different cell types.
When a ligand binds to the receptor then a
series of events including the
phosphorylation and a lot of ubiquitous
processes occurs which result in the
activation of the different pathways such as
the nuclear Kappa B signaling JNK and the
activated p-38 nitrogen protein kinase
reactions which co-operatively induces the
expression of different genes of the IL-1
family such as MKP-1, IL-1 beta, IL-1
alpha, I kappa-B alpha, IL-8, MCP-1 and IL-
6 by the methods of transcription and post
transcriptions. This is also very important to
mention that there are some other intra-
cellular components that respond to the
cytokines such as IL-18 and IL-33 (Weber et
al., 2010)
So it obvious now that the interleukin-1
family of the cytokines which include IL-1
alpha, IL-1 beta are playing their roles in the
inflammatory process up regulation by the
increased expression of many effector
proteins such as nitric oxide synthase and
matix-metallo proteinases along with the
expression of many cytokines and the
chemokine(Dinarello, 2002)

3. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
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Figure model showing that effect of IL-1 on tumor growth and metastasis (Lewis et al., 2006)
The hypoxia state when reached, leads to the
secretion of the following chemical
compounds including the cytokines such as
IL-1, IL-6 and vascular endothelial growth
factor (VEGF) (Balkwill and Mantovani,
2001)
Two closely related proteins named as IL-1
alpha and the other named as IL-1 beta serve
as agonists to one another and they bring
about the following roles such as
inflammation, hematopoiesis and innate
immunity. So the places where there is
tumor progressing the cytokine IL1 will be
more abundant over there and effect the
processes of carcinogenesis, tumor growth
and invasiveness proliferation of the growth
of more and more cancerous cells. But if
their individual effects are seen then it has
been proposed that the anti-tumor activity is
achieved when the IL-1 alpha is exposed to
the tumor but these were membrane
associated and were exposed to the
malignant cells but IL-1 beta derived from
the malignant cells and is in the secretable
form will activate the inflammation process
and will lead to tumor mediated suppression.
Tumor invasiveness and alleviation of the
tumor mediated suppression is achieved by
the use of the receptor of the IL-1 alpha
named as IL-1 RA hence it is an important
achievement in the cancer therapy(Apte et
al., 2006)

4. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
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Interleukin-1 beta, CCL2, CCL5
IL-1 beta is a cytokine protein; its
other name is catabolin. It is encoded by
IL1B gene in humans .Its precursor cut by
caspase 1 to form mature IL-1 beta .Igal
Gery discovered it in 1972 and referred it
lymphocyte activating factor( LAF).In 1985
it was found to consist of two different
proteins ; IL-1 alpha and IL-1 beta. It is the
member of the cytokines family interleukin-
1.Its production takes place by activated
macrophages. In inflammatory responses it
has very importance.IL-1 beta also involves
in differentiation, apoptosis and
proliferation. This cytokine induces the
cyclooxygenase-2 in the central nervous
system which in turn involves in the
inflammatory pain hypersensitivity. Its gene
present on chromosome 2.Its increased
production causes large number of
autoinflammatory syndromes(Soria et al.,
2011).
Together with interleukins we cannot ignore
the importance of inflammatory cytokines in
the breast cancer. CCL5 (chemokine ligand
5) is a protein in humans encoded by CCL5
gene. Its other name is RANTES(Donlon et
al., 1990). It is an 8kDa protein and chemo
tactic factor for Tcells, eosniphills, and
basophills.Its most important role in
recruiting leukocytes towards the
inflammatory site. The chemokine gene is
present on chromosome 17.
CCL2 is another chemokine and also
referred as monocyte chemotactic protein
1(MCP1). Basically it is a cytokines belong
to the family CC chemokines family.CCL2
recruits dendritic cells, memory T cells,
monocytes towards the inflammation site. It
is a 13 kDa protein.
FIG; Structure of IL-1 beta, CCL5, CCL2

5. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
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Role of interleukin -1 beta in cancer
and its correlation with CCL2 and
CCL5
Breast cancer is a common example of
inflammation linked malignant disease.
Breast cancer has large quantity of
inflammatory constituents’ also included
soluble factors and cells that are polarized to
tumor promoting phenotype. Many studies
indicate that huge number of inflammatory
cytokines present in the tumor
microenvironment. These are actively
involved in the progression and
development of the breast cancer. Recently
much attention also given to the IL-
1beta.IL-1 beta involves in breast cancer and
metastasis. It was determined by
immunohistochemistry in patients diagnosed
with
 Ductal Carcinoma In Situ (DCIS)
 IDC-with-relapse
 Invasive Ducal Carcinoma without
relapse
 Benign breast disorders
In normal breast epithelial cells, It expressed
at very low incidence, and it significantly
increases in tumor cells. The relation of
CCL2, CCL5 and IL-1 beta is very
significant in tumor cells. In breast cancer
chemokines and cytokines have equal
importance and they go side by side. There
is a close relationship among IL-1 beta
CCL2 and CCL5. All are expressed in breast
cancer and perform different activities
which enhance the tumor growth and
metastasis.
CCL2 and CCL5 are classified as
inflammatory chemokines and their
production is stimulated by IL-1 beta and
TNF- alpha. These are not expressed in
normal cells. Different types of association
FIG; Effects of IL-1 beta(Auron et al., 1984)

6. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
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exist between IL-1 beta and CCL2, CCL5.
Their association is different in different
stages of the tumor. While IL-1 beta is most
important in the progression of the disease.
It creates such environment and promotes
the conditions which increases the
metastasis. There is overlap among the
tumor promoting activities of CCL2, CCL5
and IL-1 beta(Soria et al., 2011)
Both chemokines CCL2 and CCL5 involves
in various types of cross talks among the
cancerous cells and cells of the tumor
microenvironment by
 Changing the balance at the tumor
site between various leukocytes by
enhancing the presence of various
deleterious tumor associated
macrophages (TAM)(Carr et al.,
1994).
 Hindering the potential anti-tumor
activities of the T cells.
 Of the two chemokines CCL2 also
involves in the angiogenesis.
 CCL5 and CCL2 expressed by the
cells of the tumor microenvironment
mesenchymal stem cells and
osteoblasts involves in the process of
breast cancer malignancy and
metastasis.
 Both chemokines act directly on the
tumor cells to enhancing the pro-
maligncy phenotype by promoting
their invasive related and migratory
properties.
Current information and different researches
indicates that CCL2 and CCL5 are
inflammatory mediators with pro-
malignancy activities in breast cancer. They
also considered as potential therapeutic
targets against the breast cancer(Auron et
al., 1984).
Fig.; Action of CCL2 and CCL5

7. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
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Interleukin 6
Interleukin-6 HGF BSF-2 or formerly
known as Interferon β-2 is a cytokine that is
a member of proteins involved in maturation
of B cells and inflammation. The interleukin
is chiefly formed at sites of acute and
chronic inflammations, from where it is
secreted in serum and induces a
transcriptional inflammatory response
through IL-6 r-α. The function of the gene is
occupied in a disease state, that includ
susceptibility to diabetes mellitus and
systemic juvenile rheumatoid arthritis.
Interleukin 6 acts as both a pro-
inflammatory cytokine and an anti-
inflammatory myo-kine. In human beings, it
is encrypted by the IL-6 gene. The gene for
IL-6 is located on chromosome 7 (7p21) and
is transcribed as and when necessary.
Interleukin-6 is secreted by macrophages
and T cells to stimulate immune response in
emergency conditions. For instance, during
infection, after traumatization and other
tissue damage leading to inflammations. IL-
6 also plays a role in combatting infections,
as in Balb-c mice, it is required for
resistance against Streptococcus
pneumoniae. Smooth muscle cells in tunica
media of many blood vessels also form IL-6
as a pro- inflammatory cytokine. In addition,
osteoblasts release IL-6 to enhance
formation of osteoclasts. Role of IL-6 as an
anti-inflammatory cytokine is arbitrated
through its inhibitory effects on TNF-alpha,
Interleukin-1, and activation of Interleukin-1
receptor-α and Interleukin-10.
Functions
IL-6 is an essential arbitrator of temperature
and of the acute phase responses. It is
accomplished of overpassing the blood-brain
barrier (BBB) and introducing PGE2
synthesis in hypothalamus, thus altering
temperature set-point of body. In fatty and
muscle tissues, IL-6 kindles energy
utilization that pointers to elevated body
heat. IL-6 can also be secreted by
macrophages in reaction to Pathogen-
Associated Molecular Patterns (PAMPs).
PAMPs fix to Pattern Recognition Receptors
(PRRs), which include Toll-like receptors
(TLRs). The receptors are present on the cell
surfaces and intracellular compartments and
induce intracellular signaling cataracts that
results in production of inflammatory
cytokines.
IL-6 is essential for growth of hybridoma
and is observed in many additional cloning
media such as briclone. IL-6 is also
fashioned by adipocytes and is thought to be
Figure 4: 3-D Structure of IL-6

8. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
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a reason why overweight entities have
advanced levels of endogeneous C-Reactive
Proteins(Smirnova et al., 2002). Blockers of
IL-6 (including estrogen) are used to treat
post-menopausal osteoporosis. Intranasaly
administered IL-6 has been shown to
improve sleep associated link of
memories(Wahl et al., 2000).
IL-6 is stimulates synthesis of acute phase
proteins and neutrophil production in the
bone marrow region. It is antagonistic to
regulatory T cells and supports the growth
of B cells. IL-6 is also reflected as a
myokine (a cytokine produced from
muscles), and is raised in reaction to
contraction of muscles. It is meaningfully
enhanced with exercise, and leads to the
appearance of other cytokines in the
circulation(Smirnova et al., 2002). During
workout, it is thought to act in a hormone-
like manner, to boost up substrate transfer.
IL-6 has wide-ranging anti-inflammatory
functions in its part as a myokine. (IL-6 was
the first myokine that was found to be
secreted into the blood stream in response to
muscle contractions).
Mechanism of Action
IL-6 signals through a cell-surface type-1
cytokine receptor complex, consisting of the
signal-transducing component gp130 (also
referred to as CD-130) and a ligand-binding
IL-6Rα chain (CD-126). CD-130 is the
mutual signal transducer for several
cytokines including leukemia inhibitory
factor (LIF), cardiotrophin-1, ciliary
neurotropic factor and is almost universally
expressed in most tissues. Comparatively,
the expression of CD-126 is restricted to
certain tissues. As IL-6 interacts with its
receptor, it triggers the gp-130 and IL-6
Receptor proteins to form a complex, thus
activating the receptor. These centers bring
together the intracellular regions of gp-130
to recruit a signal transduction cascade
through some transcription factors, Janus
kinases (JAKs) and Signal Transducers and
Activators of Transcription (STATs)
Role in Breast Malignancy
Breast malignancy is second leading cancer
in women. Chronic and low-level
inflammations play a key role in the
pathogenesis of many tumor forms including
breast cancer. IL-6 is a key player in the
systemic inflammation; regulating both the
tissue metabolism and inflammatory
response during acute stimulations.
Interleukin-6) is a pleiotropic cytokine with
tumor-promoting and tumor-inhibitory
properties. It has been observed that direct
application of IL-6 on breast cancer cells
inhibits proliferation in estrogen receptor
positive cells, while high circulating IL-6
levels are correlated with a poor prognosis
in breast cancer patients. This makes IL-6 a
good biomarker for tumor burden, impaired
metabolism. Local intra-tumoral IL-6
signaling is important for monitoring breast
cancer cell growth, self-renewal and
metastasis of cancer stem cells.
Breast lump Interleukin-6 (IL-6) expression
surges with the lump grade and raised levels
of serum IL-6 are associated with lowly
breast cancer patient survival. IL-6 mediates
hematopoiesis and lymphocyte. Recent
studies show that IL-6 alone is a powerful

9. Introduction to Laboratory medicine Review article by Farooq A et al,
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growth factor for breast cancer cells and
carcinoma-associated epithelial-
mesenchymal transitions (EMT). These
EMT phenotypes associate with diminished
breast cancer patient survival. Ectopic IL-6
expressing MCF-7 cells (MCF-7IL-6)
displayed a gene expression profile and
phenotype consistent with EMT promote
breast Cancer.
Jointly, the above body of research
highlights the probable impact of IL-6 on
breast cancer progression. Although it
strengthens the conception that IL-6
indorses carcinoma progression and
challenge which considers IL-6 a sheer
consequence of cancer-associated
inflammation, future clinical studies will be
needed to corroborate with these inferences.
Interleukin 8
Interleukin 8, also known as CXCL-8 and
neutrophil chemotactic factor is a adherent
of the CXC chemokine family produced by
macrophages and other cells like airway
smooth muscle cells, epithelial cells, and
endothelial cells. Endothelial cells keep IL-8
in their storage vesicles known as, the
Weibel-Palade bodies. In humans, the
interleukin-8 protein is encoded by the IL-8
gene, located on chromosome 4q(Baggiolini
and Clarklewis, 1992).
There are many receptors on the surface
membranes of cells capable of binding IL-8;
most recurrently studied receptors are the G
protein-coupled serpentine receptors CXCR2
and CXCR1. Affinity and expression for IL-
8 varies between the two receptors (CXCR1
> CXCR2). TLRs are the receptors of the
innate immune system. These receptors
identify antigen patterns through a series of
biochemical reactions chain, IL-8 is secreted
out. IL-8 induces chemo taxis in target cells,
chiefly neutrophils but also other
granulocytes, triggering them to rove
towards the spot of septicity. IL-8 also
brings phagocytosis and promotes
angiogenesis. In target cells, IL-8 brings a
series of physiological responses required
for phagocytosis and migration, such as rises
in intracellular Ca2+
, exocytosis and a
respiratory burst.
IL-8 can be secreted by any cells with TLRs
that are involved in the innate immune
response. The macrophages recognize an
antigen first, consequently are the first cells
to release IL-8 to recruit other cells. Both
monomers and homodimers of IL-8 have
been reported to be persuasive inducers of
the chemokine receptors CXCR1 and
CXCR2. The homodimer is more powerful,
but methylation of Leu25 can block the
activity of the homodimers.
Functions
Though neutrophil granulocytes are the
prime objective cells of IL-8, there is a
Figure 5 3-D Structure of IL-8

10. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
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relatively wide range of cells including
macrophages, endothelial cells and
keratinocytes that respond to iL-8.
Interleukin-8 is always associated with pro
inflammatory cytokine in psoriasis and
gingivitis. Interleukin-8 secretion is
amplified by ROS, which thereby cause the
recruitment of inflammatory cells and brings
an extra surge in oxidant stress mediators,
making it a vital stricture in localized
swelling. IL-8 is also shown to be connected
with stoutness.
If a heavy with child mother has high levels
of iL-8, there is an enlarged risk of
schizophrenia in her offsprings. High IL-8
intensities have been shown to lessen the
probability of positive responses to
antipsychotic prescription in schizophrenia.
IL-8 expression is regulated by NF-κB. NF-
κB regulation represents a novel anti-IL-8
treatment for use in inflammatory diseases
such as cystic fibrosis. Expression of IL-8 is
negatively regulated by a number of
mechanisms. MiRNA-146a/b-5p suppresses
IL-8 expression by stopping the expression
of IRAK1.Additionally, the 3'UTR of IL-8
contains a A/U-rich elements that make it
exceedingly unbalanced under certain
conditions.
The IL-8 protein contains 4 cysteine remains
partaking in disulfide bridges (Cys-50/ Cys-
9; Cys-34/ Cys-7 ).
The IL-8 receptor is a member of a G-
protein-coupled receptor proteins family.
There are at least two different IL-8 receptor
types. The type 1 receptor specifically binds
with IL8 and type 2 receptor binds to the IL-
8-related factors MGSA (Melanoma growth
stimulatory activity), MIP-2, GRO and NAP-
2 (neutrophil-activating proteins-2).
Biological Role
The actions of IL-8 are not specie-specific.
Human IL-8 is also active in rabbitt and
mice cells. The biological actions of IL-8
look like those of a related protein, NAP-2.
IL-8 differs from all other cytokines in its
ability to precisely trigger neutrophil
granulocytes. In neutrophils, IL-8 causes a
momentary upsurge in cytosolic Ca2+
levels
and the release of enzymes from granules.
IL-8 also increases the digestion of ROS and
escalates Chemotaxis.. IL-8 actually hinders
histamine release from basophil cell of
humans and is involved also in mediating
pain.IL-8 prevents the linkage of leukocytes
to galvanized endothelial cells and therefore
retains anti-inflammatory activities. The 72
amino acid form of IL-8 is approximately
ten-fold more powerful in impeding
adhesion of neutrophils than the 77 aa
variant. IL-8 is a mitogen for epidermal
cells. Macrophage-derived IL-8 supports
angiogenesis and may play a part in
disorders such as tumor growth, rheumatoid
arthritis and wound healing that unfavorably
depend on angiogenesis.
Binding of IL-8 to its receptor and
bioactivities of IL-8 are repressed by an
artificial peptide, anti-leukinate.
Role on breast Cancer
The idea of stem-like cells in tumor has been
achieving fame over the last score. Cancer
stem-like cells (CSCs), are liable for driving
tumor development and cause disease

11. Introduction to Laboratory medicine Review article by Farooq A et al,
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development. In breast malignancy, there is
good sign that CSCs are basically resilient to
orthodox radio-, endocrine and chemo-
therapies. To increase cure, there is a need to
develop CSC-targeted therapies. IL-8 is up
regulated in breast cancer and linked with
poor analytical factors. IL-8, through its
receptors CXCR2/1, is a significant regulator
of breast CSC activity. Impeding CXCR2/1
signalling has proved effective in pre-
clinical replicas of breast cancer providing a
good rationale for aiming CXCR1/2
clinically. Recent studies have associated
angiogenesis and inflammatory
developments with tumor distortion. There
is a converse association between IL-8
expression and local recurrence and
metastasis. Female patient that received
post-surgery chemotherapy and radiotherapy
was found to have a local recurrence and
lower IL-8 expression Multivariate logistic
regression shows progesterone positivity,
estrogen receptor negativity and metastasis.
The role of IL-8 in tumor micro-
environment is very different than macro-
environment and supports its classification
as a possible prognostic marker,
Though, more studies are necessary for its
inclusion in clinical practice.
Tumor Necrosis Factor Alpha and
Thymidine phosphorylase
Tumor necrosis factor (TNF), cachexin, or
cachectin, and formerly known as tumor
necrosis factor alpha or TNFα is an
adipokine involved in systemic
inflammation and is a member of a group of
cytokines that stimulate the acute phase
reaction. It is produced chiefly by activated
macrophages (M1), although it can be
produced by many other cell types such as
CD4+ lymphocytes, NK cells and neurons.
The primary role of TNF is in the regulation
of immune cells. TNF, being an endogenous
pyrogen, is able to induce fever, apoptotic
cell death, cachexia, inflammation and to
inhibit tumorigenesis and viral replication
and respond to sepsis via IL1 & IL6
producing cells. Dysregulation of TNF
production has been implicated in a variety
of human diseases including Alzheimer's
disease, cancer, major depression and
inflammatory bowel disease (IBD). While
still controversial, studies of depression and
IBD are currently being linked by TNF
levels. Recombinant TNF is used as an
immunostimulant under the INN
tasonermin. TNFα can be produced
ectopically in the setting of malignancy and
parallels parathyroid hormone both in
causing secondary hypercalcemia and in the
cancers with which excessive production is
associated.
As implied by its name, TNF-α may have
Figure 1 3-D Structure of TNF-Alpha

12. Introduction to Laboratory medicine Review article by Farooq A et al,
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cytotoxic and apoptotic activities when
administered to breast tumor cell lines.
However, these effects may depend on
multiple factors, such as treatment by
estrogen and the expression of members of
the epidermal growth factor receptor family.
The fact that TNF-α activities vary under
different physiological conditions and in a
cell-type-dependent manner contributes to a
sense of ambiguity regarding its antitumor
effects. Indeed, recent investigations
strongly suggest that the chronic expression
of TNF-α in breast tumors actually supports
tumor growth. The number of cells
expressing TNF-α in inflammatory breast
carcinoma was found to be correlated with
increasing tumor grade and node
involvement, and TAM-derived TNF-α
expression was suggested to play a role in
the metastatic behavior of breast
carcinomas. Furthermore, patients with more
progressed tumor phenotypes were shown to
have significantly higher TNF-α serum
concentration.
Mechanism of Action
Angiogenesis, the development of new
blood vessels from an existing vascular
network, is an essential requirement for
tumour growth, and progression, and is
regulated by a complex network of
cytokines, enzymes and adhesion molecules
(Hemler et al., 1990). Recent studies have
shown that macrophages, as well as
malignant cells, are an important source of
such angiogenic factors in solid
tumours(1994) This is supported by recent
findings that high levels of tumour
infiltration by macrophages is associated
with increased tumour angiogenesis and
reduced survival in ductal invasive
carcinoma of the breast (Leek et al, 1996).
Others have demonstrated recently that the
cytokine tumour necrosis factor alpha (TNF-
α) and the intracellular enzyme thymidine
phosphorylase (TP) are two key angiogenic
molecules produced by focal areas of
tumour-associated macrophages (TAMs). In
the case of TNF- α, various techniques have
been used to visualize the production of
TNF-α mRNA intracellular TNF- α protein
(Miles et al, 1994; Pusztai et al, 1994)and
secreted TNF-α by TAMs in breast
carcinoma (Lewis and McGee, 1996). TP,
on the other hand, is produced not only by
TAMs, but also by malignant epithelial cells
and endothelial cells in such malignant
breast tissue (Fox et al, 1996; Relf et al,
1997). In experimental systems, TNF- α can
both inhibit and stimulate angiogenesis in a
dose-dependent manner, with high doses in
the 1 to 5-ug range being inhibitory, whereas
low doses in the 0.01- to 1-ng range are
stimulatory (Fajardo et al, 1992; Leek et al,
1994). Moreover, it has been shown that
both forms of TNF- α receptor (p55
and p75) are expressed by endothelial cells
in such tissues, with the smaller (p55) form
of the TNF- α receptor also expressed by
neoplastic cells and macrophage-like
stromal cells, and the larger (p75) variant by
infiltrating stromal cells (Miles et al, 1994;
Pusztai et al, 1994). As it is unlikely that
TNF- α could reach the high levels in the
tumour microenvironment needed to be
antiangiogenic, and, as tumour angiogenesis
and growth proceeds in the presence of
TNF- α in most tumours, it is likely that the
net effect of this cytokine on tumour

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angiogenesis tends towards stimulation
rather than inhibition.
Thymidine phosphorylase, an enzyme
originally isolated from platelets and also
known as platelet-derived endothelial cell
growth factor (PDECGF), catalyses the
reversible phosphorolysis of thymidine to
deoxyribose 1-phosphate and thymine. TP
has been shown to exhibit a chemotactic and
mitogenic capacity on endothelial cells in
several angiogenic model systems, and its
expression in human breast cancer cells has
been shown to correlate with microvessel
density in some studies (Fajardo et al, 1992;
Folkman, 1996; Fox et al, 1996). Moreover,
TP expression is 260- fold higher in invasive
bladder cancer (O'Brien et al, 1995) and 27-
fold higher in invasive breast carcinoma
than normal tissue (Patterson et al, 1995). In
ovarian carcinomas, areas of increased
expression of TP have been associated with
high blood velocity as measured by colour
Doppler imaging (Reynolds et al, 1994).
In most normal organs, TP is most highly
expressed in resident tissue macrophages,
and may be part of a mechanism controlling
angiogenesis in response to injury (Fox et al,
1995a). TP is not a
classic type of pro-angiogenic factor in
tumours as it is thought to exert its
angiogenic effects via the metabolites of its
enzymatic activity (Moghaddam and
Bicknell, 1992). DNA released from dying
cells and engulfed in apoptotic nuclei may
be degraded to thymidine, which can freely
enter cells, including tumour cells and
TAMs, which then metabolize thymidine via
TP to angiogenically active metabolites such
as deoxyribose-1-phosphate. TP also
catalyses the
phosphorolytic cleavage of the
chemotherapeutic pro-drug 5'-deoxy- 5-
fluorouridine (5'-DFUR) to its
therapeutically active form 5-fluorouracil (5-
FU) (Patterson et al, 1995), and it is thought
that resistance to 5'-DFUR therapy may be
due to low TP activity in some tumours. As
TNF- α has been shown to up-regulate
markedly TP activity in tumour cell lines in
vitro (Eda et al, 1993), the purpose of one of
the studies was to investigate whether TNF-
α may be involved in the regulation of TP in
vivo. To do this, non-neutralizing antibodies
for TNF- α that recognized both unbound
and receptor-bound TNF- α to correlate the
cellular distribution of TNF-a protein (both
TNF- α expression by TAMs and TNF-a
bound to receptors on tumour and
endothelial cells) with that of TP protein
expression by tumour cells in a consecutive
series of primary invasive human breast
carcinomas were studied. The study also
tried to correlate the cellular distribution of
TNF- α with a range of important tumour
variables in breast cancer, such as
angiogenesis, receptor status, axillary lymph
node involvement, focal macrophage
infiltration and prognosis.
This present study did not, however, find
any relationship between either TAM
production of TNF- α or TNF- α bound to
endothelial cells and vascular grade (i.e.
angiogenesis). Angiogenesis is thought to be
regulated by a large network of inter-relating
factors rather than one factor alone (Leek et
al, 1994; Folkman, 1996). The presence,
however, of receptor-bound TNF-1 α on the

14. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
Page | 14
tumour endothelium of 66% of cases does
indicate that TNF- α may be an element in
the complex regulation of angiogenesis. This
is also supported by the observation that
there is an association between increased
vessel staining and larger tumour size.
Increased angiogenesis is a requirement for
tumour growth and may be particularly
important in larger tumours where diffusion
distances are greater. That increased TP
expression was seen in this study to be
associated with increased TNF- α
immunoreactivity of the malignant cell
population of breast carcinomas strongly
suggests that malignant breast epithelial
cells may be a target cell population for
TAMderived TNF- α and that they may up-
regulate TP in response to
this cytokine. This may account for the
relationship between tumour macrophage
infiltration and angiogenesis, and this
pathway may be an important component in
the overall network of factors
regulating angiogenesis in breast carcinoma.
However, it is noteworthy that there was no
significant association of TP with
angiogenesis in this study, although earlier
studies reported a positive correlation in this
disease. Nor did TP correlate with other
clinical and pathological variables. The
reason for the discrepancy is unclear but
may indicate the importance of multiple
factors in the regulation of tumour
angiogenesis, and the complexity of cellular
interactions and cytokine networks with
multiply redundant pathways that influence
tumour progression. Indeed, it has been
recently shown that the presence of at least
six vascular growth factors in malignant
breast tumours (Relf et al, 1997).
In conclusion, the relationship between
TNF- α protein expression and angiogenesis
and tumour progression is complex. It may
be able to stimulate angiogenesis directly by
its actions on endothelial cells, and
importantly it may also affect angiogenesis
indirectly by its ability to modulate
expression of other factors such as TP,
which appears to be up-regulated in breast
cancer. The involvement of TNF- α in these
processes, and its association with TP
expression in particular, also underlines the
importance of theTAM population, in breast
cancer, as the most likely source of this
cytokine, and may account in part for the
strong association of focal macrophage
infiltration with increased angiogenesis and
reduced survival described earlier. It also
reinforces the concept of TAMs as
therapeutic targets for future anti-cancer and
antiangiogenic therapies. This could be
achieved in a number of ways; for example,
it may be desirable to further up-regulate TP
using TNF- α therapy in order to render the
tumour more sensitive to 5- FU.
Alternatively, drugs such as vesnarinone
(Kambayashi et al, 1996) could be used to
inhibit TNF-ox production in the TAM
population, thus reducing the angiogenic or
pro-metastatic stimulus provided by this
cytokine.
Interferon-γ
Interferon gamma (IFN-γ) is a dimerized
soluble cytokine that is the only member of
the type II class of interferons. The existence
of this interferon was recognized when
human blood lymphocytes or mouse
peritoneal lymphocytes obtained from
tuberculin-sensitized individuals were
challenged with PPD and resulting

15. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
Page | 15
supernatants were shown to inhibit growth
of vesicular stomatitis virus. This interferon
was later called macrophage-activating
factor, a term now used to describe a larger
family of proteins to which IFN-γ belongs.
In humans, the IFN-γ protein is encoded by
the IFNG gene.
The IFN-γ monomer consists of a core of six
α-helices and an extended unfolded
sequence in the C-terminal region. The
biologically active dimer is formed by anti-
parallel inter-locking of the two monomers.
IFN-γ, or type II interferon, is a cytokine
that is critical for innate and adaptive
immunity against viral and intracellular
bacterial infections and for tumor control.
IFN-γ is an important activator of
macrophages. Aberrant IFN-γ expression is
associated with a number of auto-
inflammatory and autoimmune diseases. The
importance of IFN-γ in the immune system
stems in part from its ability to inhibit viral
replication, and most importantly from its
immune-stimulatory and immune-
modulatory effects. IFN-γ is produced
predominantly by natural killer (NK) and
natural killer T (NKT) cells as part of the
innate immune response, and by CD4 Th1
and CD8 cytotoxic T lymphocyte (CTL)
effector T cells once antigen-specific
immunity develops.
Tumor Inflammatory Mechanisms
CTLs express the CD8 co-receptor and
recognize antigen on tumor cells as
peptide/major histo-compatibility class I
(MHC-I) complexes. As a consequence of
antigen recognition, CD8 CTLs exert
antitumor function via the perforin-
granzyme cytolytic pathway or through
cytokines such as interferon gamma (IFN-γ)
and tumor necrosis factor alpha (TNF-α),
which exhibit cytostatic activity. The MHC-
I–binding peptides recognized by tumor-
reactive CD8 T lymphocytes are usually
derived from genes preferentially expressed
by transformed cells or from tissue-
differentiation antigens. The identification
of MHC-I–binding peptides that serve as
tumor-rejection CD8 T-cell epitopes has
opened the door to developing synthetic
peptide cancer vaccines.
IFN-γ has long been considered to provide
antitumor benefits through its anti-
proliferative activity and its ability to
enhance antigen processing for both MHC-I
and MHC-II pathways but it also generates
tumor resistance to MHC-I–restricted CD8
T lymphocytes. This is primarily done by
two mechanisms:
1- Induction of high levels of
Programmed Death Ligand-1 PD-L1 (B7-
H1), which inhibits the survival and function
of T lymphocytes.
2- By reducing the capacity of the
tumor cells to be recognized by CD8 T cells.
IFN-γ enhances the expression of PD-L1 in
numerous cell types, including tumors.
Because signaling of PD1 reduces the ability
of T lymphocytes to become fully activated,
proliferate, and exert effector function, PD1
blockade using anti-PD1 or anti PD-L1

16. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
Page | 16
antibodies has been used to improve the
therapeutic effectiveness of T cells against
chronic viral infections and tumors.
Blocking the action of IFN-γ in cancer
patients undergoing T cell–based
immunotherapy could improve their
therapeutic outcome.
Transforming Growth Factor-β
Transforming growth factor beta (TGF-β) is
a protein that controls proliferation, cellular
differentiation, and other functions in most
cells. It is a type of cytokine which plays a
role in immunity, cancer, bronchial asthma,
heart disease, diabetes, hereditary
hemorrhagic telangiectasia, Marfan
syndrome, Vascular Ehlers-Danlos
syndrome, Loeys–Dietz syndrome,
Parkinson's disease and AIDS.
TGF-beta is secreted by many cell types,
including macrophages, in a latent form in
which it is complexed with two other
polypeptides, latent TGF-beta binding
protein (LTBP) and latency-associated
peptide (LAP). Serum proteinases such as
plasmin catalyze the release of active TGF-
beta from the complex. This often occurs on
the surface of macrophages where the latent
TGF-beta complex is bound to CD36 via its
ligand, thrombospondin-1 (TSP-1).
Inflammatory stimuli that activate
macrophages enhance the release of active
TGF-beta by promoting the activation of
plasmin. Macrophages can also endocytose
IgG-bound latent TGF-beta complexes that
are secreted by plasma cells and then release
active TGF-beta into the extracellular fluid.
TGF-β is a secreted protein that exists in at
least three iso-forms called TGF-β1, TGF-
β2 and TGF-β3. It was also the original
name for TGF-β1, which was the founding
member of this family. The TGF-β family is
part of a super-family of proteins known as
the transforming growth factor beta super-
family, which includes inhibins, activin,
anti-müllerian hormone, bone
morphogenetic protein, decapentaplegic and
Vg-1.
Most tissues have high expression of the
genes encoding TGF-β. That contrasts with
other anti-inflammatory cytokines such as
IL-10, whose expression is minimal in
unstimulated tissues and seems to require
triggering by commensal or pathogenic
flora. TGF-beta acts as an anti-proliferative
factor in normal epithelial cells and at early
stages of onco-genesis.
Some cells that secrete TGF-β also have
receptors for TGF-β. This is known as
autocrine signaling. Cancerous cells increase
their production of TGF-β, which also acts
on surrounding cells.
The peptide structures of the TGF-β iso-
forms are highly similar. They are all
encoded as large protein precursors; TGF-β1
contains 390 amino acids and TGF-β2 and
TGF-β3 each contain 412 amino acids. They
each have an N-terminal signal peptide of
20-30 amino acids that they require for
secretion from a cell, a pro-region, and a
112-114 amino acid C-terminal region that
Figure 2 3-D Structure of IFN Gamma
Figure 3 3-D Structure of TGF-Beta

17. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
Page | 17
becomes the mature TGF-β molecule
following its release from the pro-region by
proteolytic cleavage. The mature TGF-β
protein dimerizes to produce a 25 KDa
active molecule with many conserved
structural motifs. TGF-β has nine cysteine
residues that are conserved among its
family; eight form disulfide bonds within the
molecule to create a cysteine knot structure
characteristic of the TGF-β super-family
while the ninth cysteine forms a bond with
the ninth cysteine of another TGF-β
molecule to produce the dimer. Many other
conserved residues in TGF-β are thought to
form secondary structure through
hydrophobic interactions. The region
between the fifth and sixth conserved
cysteines houses the most divergent area of
TGF-β molecules that is exposed at the
surface of the molecule and is implicated in
receptor binding and specificity of TGF-β.
Tumor Inflammatory Mechanisms
Cancer cells lose the tumor-suppressive arm
of the TGF-β pathway and accrue
tumorigenic effects that directly enhance
tumor growth and invasion. However,
regardless of how they avert the tumor-
suppressive action, cancer cells can benefit
from tumor-derived TGF-β by using it as a
shield against antitumor immunity.
Epithelial-mesenchymal transition (EMT) is
a well-coordinated process during
embryonic development and a pathological
feature in neoplasia and fibrosis. Cells
undergoing EMT lose expression of E-
cadherin and other components of epithelial
cell junctions. Instead, they produce a
mesenchymal cell cytoskeleton and acquire
motility and invasive properties. EMT is key
in gastrulation and in the genesis of the
neural crest, the somites, the heart, and
craniofacial structures. It is driven by a set
of transcription factors including the zinc-
finger proteins Snail and Slug, the bHLH
factor Twist, the zinc-finger/ homeodomain
proteins ZEB-1 and -2, and the forkhead
factor FoxC3.
The competence of epithelial precursor cells
to undergo EMT becomes manifest in
response to cues that prominently feature
TGFβ (Figure 6A). As such, TGFβ-induced
EMT is observed in transformed epithelial
progenitor cells with tumor-propagating
ability EMT-like processes contribute to
tumor invasion and dissemination owing to
the cell junction-free, motile phenotype that
they confer. Carcinoma cells with
mesenchymal traits have been observed in
the invasion front of carcinomas and may
reflect a series of interconnected features:
that carcinomas are propagated by
transformed progenitor cells, that progenitor
cells are competent to undergo EMT, that
EMT is triggered by cues at the invasion
front, and that EMT augments the
disseminative capacity of these cells. That
said, not all cells that undergo EMT are
tumor propagating cells, and not all tumor-
propagating cells are necessarily competent
to undergo EMT.

18. Introduction to Laboratory medicine Review article by Farooq A et al,
May, 2014
Page | 18
TGFβ is a potent inducer of EMT. A role of
TGFβ-induced EMT in human cancer is
suggested by the gene expression analysis of
tumor-propagating breast cancer cell
populations expressing the cell surface
markers CD44+/CD24lo. The common gene
expression pattern of these cells from
different cancer patients suggested the
presence of an active TGFβ pathway.
Furthermore, treatment with a TβR-I kinase
blocker induced these cells to adopt a more
epithelial phenotype. Thus, CD44+/CD24lo
breast cancer cells may represent a tumor
cell population that has undergone EMT. In
human carcinomas, cells with features
characteristic of EMT have been observed in
the invasion front, a location that is rich in
stromal TGFβ and other cytokines that may
cooperate in EMT induction.
TGF-β promotes EMT by a combination of
Smad-dependent transcriptional events and
Smad-independent effects on cell junction
complexes. Smad-mediated expression of
HMGA2 (high-mobility group A2) induces
expression of Snail, Slug, and Twist.
Independent of Smad activity, TβRII-
mediated phosphorylation of Par6 promotes
the dissolution of cell junction complexes.
In mouse tumors and cell lines, TGFβ-
induced EMT is Smad dependent and
enhanced by Ras signaling. TGFβ can also
enhance cell motility by cooperating with
HER2 signals, as observed in breast cancer
cells overexpressing HER2.
Table 1 The micro-environment of tumor
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(Polyak et al., 2009, Sansone et al., 2007,
Balkwill and Mantovani, 2001, Hu et al.,
2005, Farmer et al., 2009, Bhati et al., 2008,
Holash et al., 1999, Paez-Ribes et al., 2009)
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