inactive PI3K/AKT pathway‖ state to an active ―PI3K/AKT pathway – inactive JUN/MAPK pathway‖ state.
Triple-negative (TN) breast cancers represent about 20% of all breast cancers, and follows an aggressive course. Basal/TN cancers have elevated Jag 1 levels, and BRCA-1 mutant breast cancers, which are typically of the basal/TN subtype, show elevated Jag1 expression compared with their BRCA2 (predominantly luminal) counterparts (Al-Hussaini et al., 2011). Resection specimens from TN breast cancers show a statistically significant association between elevated expression of Notch ligands/receptors and the basal/TN subtype (Reedijk et al., 2008).
Tumour necrosis factor alpha is produced by activated macrophages in response to microbes, especially the lipopolysaccharide (LPS) of Gram negative bacteria. It is an important mediator of acute inflammation. It mediates the recruitment of neutrophils and macrophages to sites of infection by stimulating endothelial cells to produce adhesion molecules and by producing chemokines which are chemotactic cytokines. TNF- α also acts on the hypothalamus to produce fever and it promotes the production of acute phase proteins (Mayer, 2010). Its central role in inflammation has led to the development of TNF-alpha antagonists as effective therapies for rheumatoid arthritis and inflammatory bowel disease (Szlosarek and Balkwill, 2003).
Tumor necrosis factor (TNF); formerly known as (TNF-alpha) and lymphotoxin (LT) alpha, originally characterized by their ability to induce tumor cell apoptosis and cachexia, are now considered as central mediators of a broad range of biological activities (Pfeffer, 2003). These activities encompass beneficial effects for the host in inflammation and in protective immune responses against a variety of infectious pathogens.
In response to inflammation and infection, the pro-inflammatory cytokine TNF-α is produced by macrophages, lymphocytes, fibroblasts and keratinocytes. TNF-α elicits its pro-inflammatory signals by initially binding to receptors, TNFR1 (p55) and TNFR2 (p75), on the cell surface (Englaro et al., 1999). The TNFR1 and TNFR2 elicit cellular response to TNF-α via three distinct signaling pathways leading to the activation of caspases and the activation of AP-1 and NFkB transcription factors. Activation by TNF-α result in the trimerization of the TNFR1 receptor and association of death domains located on the cytoplasmic region of the TNFR1 protein (Englaro et al., 1999)
These diverse signaling cascades lead to a range of cellular responses, which include cell death, survival, differentiation, proliferation and migration. Vascular endothelial cells respond to TNF by undergoing a number of pro-inflammatory changes, which increase leukocyte adhesion, transendothelial migration and vascular leak and promote thrombosis.
The central role of TNF in inflammation has been demonstrated by the ability of agents that block the action of TNF to treat a range of inflammatory conditions, including rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease and psoriasis (Bradley, 2008).
The increased incidence of infection in patients receiving anti-TNF treatment has highlighted the physiological role of TNF in infectious diseases.
Interleukin 1 (IL-1) is another inflammatory cytokine produced by activated macrophages.
Its effects are similar to that of TNF-α and it also helps to activate T cells (Guille et al.,
2013). Interleukin-1 (IL-1) is the prototypic pro-inflammatory cytokine. There are two forms of IL-1, IL-1alpha and IL-1beta and in most studies; their biological activities are indistinguishable (Dinarello, 1997; Dinarello, 2009). IL-1 affects nearly every cell type, often in concert with another pro-inflammatory cytokine, tumor necrosis factor (Englaro et al., 1999).
Interleukin (IL)-1 cytokines (IL-1α, IL-1β and IL-1Ra) play an important role in immune regulation and inflammatory processes by inducing expression of many effector proteins, e.g. cytokines/chemokines, nitric oxide synthetase and matrix metalloproteinases (MMPs) (Barksby et al., 2007). Excessive and/or dysregulated activity of these mediators is associated with tissue destruction and therefore the synthesis, secretion and biological activity of IL-1 cytokines have been identified as therapeutic targets for common inflammatory disorders such as rheumatoid arthritis (RA) and periodontitis.
Although IL-1 can upregulate host defenses and function as an immunoadjuvant, IL-1 is a highly inflammatory cytokine. The synthesis, processing, secretion and activity of IL-1, particularly IL-1beta, are tightly regulated. A unique aspect of cytokine biology is the naturally occurring IL-1 receptor antagonist (IL-1Ra) (Dinarello, 1997).
More than any other cytokine family, the interleukin (IL)-1 family is closely linked to the innate immune response. This linkage became evident upon the discovery that the cytoplasmic domain of the IL-1 receptor type I is highly homologous to the cytoplasmic domains of all Toll-like receptors (TLRs). Thus, fundamental inflammatory responses such as the induction of cyclooxygenase type 2, increased expression of adhesion molecules, or synthesis of nitric oxide are indistinguishable responses of both IL-1 and TLR ligands. Both families nonspecifically affect antigen recognition and lymphocyte function (Dinarello, 2009).
Most cancers arise in association of chronic inflammation and contain inflammatory infiltrates (Grivennikov and Kari, 2011). Immune cell have broad impact on tumour initiation, growth, and progression and many of these effects are mediated by profile cytokines. Among these cytokines, the pro-tumourigenic functions of tumour necrosis factors, IL-6, IL-1, and IL-12 are well established (Moore et al., 1999; Suganuma et al., 1999; Suganuma et al., 2002; Scott et al., 2003; Zhaorigetu et al., 2003; Grivennikov and Kari, 2011).
During carcinoma formation, cancer cells release various cytokines and growth factors into their surroundings and recruit and reprogram many other types of cells in order to establish a tumor
microenvironment. Consequently, the tumor tissues almost always contain a large number of endothelial cells, fibroblasts, and infiltrating inflammatory cells that in turn produce a variety of cytokines. The cytokines produced by these cells have been posited as key factors in modulating immune response either against or in favor of tumorigenesis in the microenvironment. The interactions that take place between immune and cancer cells are complex, involving multiple cascades of cytokines, chemokines, and/or growth factors (Sheu et al., 2008). Some cytokines are found to be particularly relevant to tumor immunology (i.e., interleukin IL-10, transforming growth factor-beta, interferon-gamma, IL-2, IL-4, and IL-12) (Mocellin and Marincola, 2001).
The role of TNF and IL-6 as master regulators of tumour associated inflammation and tumourigenesis makes them attractive targets for adjuvant treatment in cancer (Grivennikov and Kari, 2011). Discrete roles of cytokines – IL-6, IL-1 and TNF-alpha in tumour promotion and cell transformation have been demonstrated (Suganuma et al., 2002). Prior to that, the same author in 1999 had demonstrated that TNF-alpha is the key cytokine for tumour promotion in mouse skin and possibly for carcinogenesis in human as well (Suganuma et al., 1999).
Pro-inflammatory cytokine TNF-alpha is required for de novo carcinogenesis and is important to early stages of tumour promotion (Moore et al., 1999). Therefore strategies that neutralize TNF-alpha production may be useful in cancer treatment and prevention. The tumourigenic effect of IL-12 was demonstrated (Sharma et al., 2009), further confirming that endogenous IL-12 modulates the tumour promoter stimulation of inflammatory responses. Many tumors, including breast cancer, are maintained by a subpopulation of cells that display stem cell properties, mediate metastasis, and contribute to treatment resistance (Korkaya et al., 2011). These cancer stem cells (CSCs) are regulated by complex interactions with the components of the tumor microenvironment; including mesenchymal stem cells, adipocytes, tumor associated fibroblasts, endothelial cells, and immune cells through networks of cytokines and growth factors. Since these components have a direct influence on CSC properties, they represent attractive targets for therapeutic development. Elevated levels of cytokines and growth factors produced by tumor cells enhance the proliferation and survival of CSCs, induce angiogenesis (Leek et al., 1996;
Korkaya et al., 2011) and recruit tumor-associated macrophages, neutrophils, and mast cells, which secrete additional growth factors, forming a positive feedback loop that promotes tumor cell invasion and metastasis.
Inflammatory cells and cytokines play important roles in breast cancer. Breast tumours are heavily infiltrated by different types of host leukocytes, including primarily T cells, and monocytes that differentiate into tumor-associated macrophages (TAM) at the tumor site (Leek et al., 1996; Stewart and Heppner, 1997; Lee and Margolin, 2011). Studies however, suggested that T-cell antitumor responses are impaired in advanced stages of breast carcinoma (O‘Sullivan et al., 1994; Stewart and Heppner, 1997).
In contrast to T lymphocytes, TAM is associated with clinical aggressiveness in breast cancer (O‘Sullivan et al., 1994; Leek et al., 1996; Stewart and Happner, 1997). The tumor-promoting activities of tumor-associated macrophages (TAM) has been linked to its ability to express numerous tumor-promoting characteristics, such as growth factors for breast tumor cells, angiogenic mediators, extracellular matrix degrading enzymes and inflammatory cytokines (Crowther et al., 2001; Balkwill and Mantovani, 2001). Tumour-associated macrophages (TAM) also contribute to tumor progression by the release of reactive oxygen intermediates that may induce mutagenic changes that could result in increased DNA damage and generation of diversity within the tumor (Mantovani et al., 1992; Balkwill, 2002).
A major TAM-derived inflammatory cytokine shown to be highly expressed in breast carcinomas is tumor necrosis factor alpha (TNF-α) which is a multifactorial cytokine with cytotoxic and apoptotic activities when administered to breast tumor cell lines (Balkwill and Mantovani, 2001; Crowther et al., 2001). These effects may depend on multiple factors, such as treatment by estrogen and the expression of members of the epidermal growth factor receptor family. Furthermore, patients with more progressed tumor phenotypes have been shown to have significantly higher TNF-α serum concentration (ibid). The tumor-promoting functions of TNF-α is associated to its ability to induce proangiogenic functions, to promote the expression of matrix metalloproteinases (MMP) and endothelial adhesion molecules, and to cause DNA damage via reactive oxygen, the overall effect of which is promotion of tumor-related processes (Stewart and Happner, 1997).
The role of two other inflammatory cytokines; IL-6 and IL-1, is also of clinical importance in breast carcinoma. The IL-6 contributes to disease progression while IL-1 is used to differentiate between invasive carcinoma and ductal carcinoma in situ (Chaloob, 2014). The IL-1 levels is significantly higher in invasive carcinoma than in ductal carcinoma in situ or
in benign lesions, implying that elevated levels of IL-1 are directly correlated with a more advanced disease (Jin et al., 1997). The two cytokines (IL-6 and IL-1) and TNF-α are interrelated and may act in an additive manner, suggesting that these three cytokines form a network of related factors that may affect tumor cell progression in a cooperative manner.
Elevated levels of cytokines and growth factors produced by tumor cells enhance the proliferation and survival of cancer stem cells (CSCs), induce angiogenesis, and recruit tumor-associated macrophages, neutrophils, and mast cells, which secrete additional growth factors, forming a positive feedback loop that promotes tumor cell invasion and metastasis (Korkaya et al., 2011).
The outcome of breast cancer is usually determined by multiple factors. Serum tumor necrosis factor alpha (TNF-α) concentration has been found to be increased in the circulation of patients with malignancy. Preoperative evaluation of serum TNF-α concentration has been found to be a valuable parameter for reflecting the severity of staging for invasive breast cancer (Sheen-Chen et al., 2004). Serum level of TNF-α has also been found to correlate with response to neoadjuvant chemotherapy in locally advanced breast cancer (Berberoglu et al., 2004). These results suggest that the serum concentration of TNF-alpha can be an indicator of response and could be used in clinical decision-making for patients with locally advanced breast cancer.
The TNF- alpha and IL-1 can be estimated by use of ELISA (Koppolu et al., 2013;
Chaloob, 2014) that has be found to be sensitive, reproducible and can be applied in clinical conditions.
CHAPTER THREE MATERIALS AND METHOD 3.1 Materials
3.1.1 Area of the Study
This is a prospective study that was carried out at the University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu State and Federal Teaching Hospital Abakaliki, Ebonyi State; both in southeastern Nigeria. Enugu State is a mainland state in southeastern Nigeria with the coordinates of 6o20‘N 7030‘E, a population of 5,590, 513 and density of 780/km2 (William, 2008). The University of Nigeria Teaching Hospital (UNTH) is centrally located in Enugu State and this serves as a major referral centre for hospitals and clinics in the state and neighboring states.
Abakaliki is the capital of Ebonyi State in southeastern Nigeria, lies within the coordinates of 6020‘N 8006‘E with a population density of 141,438 according to 2006 National Census (Nigerian Data Portal, 2016). Abakaliki hosts many quarry factories, rice mill, vibrant farming population, State University and Federal Teaching Hospital that serve the town and environs.
3.1.2 Study population
Patients who visited Surgical and Oncology Clinics and Wards of the University of Nigeria Teaching Hospital Ituku-Ozala, Enugu and Federal Teaching Hospital, Abakaliki with signs and symptoms suggestive of breast tumour, between October, 2012 and February, 2015 were recruited into the study. Sex/aged-matched apparently healthy individuals were also recruited as controls.