micro environment of breast cancer

Introduction to Laboratory medicine Review article by Farooq A et al, May, 2014

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)

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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)

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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)

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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.

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FIG; Structure of IL-1 beta, CCL5, CCL2


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

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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

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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 a reason why overweight entities have

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Figure 4: 3-D Structure of IL-6


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 growth factor for breast cancer cells and

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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 relatively wide range of cells including

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Figure 5 3-D Structure of IL-8


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

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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.

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Figure 1 3-D Structure of TNF-Alpha


As implied by its name, TNF-α may have 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 (p55and 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 aclassic 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 tumour endothelium of 66% of cases does indicate that TNF- α may be an element in

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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 tothis 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 factorsregulating 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 malignantbreast 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-γInterferon 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 supernatants were shown to inhibit growth of vesicular stomatitis virus. This interferon

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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 MechanismsCTLs 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 antibodies has been used to improve the therapeutic effectiveness of T cells against chronic viral infections and tumors.

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Blocking the action of IFN-γ in cancer patients undergoing T cell–based immunotherapy could improve their therapeutic outcome.

Transforming Growth Factor-βTransforming 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 becomes the mature TGF-β molecule following its release from the pro-region by proteolytic cleavage. The mature TGF-β

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Figure 3 3-D Structure of TGF-Beta


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 MechanismsCancer 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.

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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

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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 consisting of cytokines and growth factors (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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SANSONE, P., STORCI, G., TAVOLARI, S., GUARNIERI, T., GIOVANNINI, C., TAFFURELLI, M., CECCARELLI, C., SANTINI, D., PATERINI, P., MARCU, K. B., CHIECO, P. & BONAFE, M. 2007. IL-6 triggers malignant features in mammospheres from human ductal

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micro environment of breast cancer

Health & Medicine

cytokines of il

family il

family of cytokines

receptors il

interleukin il

members il

receptor antagonist

roles of cytokines