**2. Inflammatory mediators as regulator of breast cancer development and metastasis**

The link between inflammation and cancer has been observed over 150 years ago when Rudolf Virchow noted that cancers tend to occur at sites of chronic inflammation. Indeed, epidemiological studies indicate that inflammatory and infectious diseases are often associated with an increased risk of cancer (Coussens & Werb, 2002). The microenvironment of tumors mimics that of tissues during the height of an inflammatory response to injury (Joyce & Pollard, 2009). However, unlike the organized morphology of normal tissue, and the ultimate resolution of the inflammation that occurs during healing, tumors exist in a state of chronic inflammation characterized by the presence of cancer cells, immune cells, aberrant vascular cells, and the persistence of inflammatory mediators, such as cytokines and chemokines.

each other and with the cancer cells playing important roles in tumorigenesis (Soto &

It is clear now that metastatic tumors represent the greatest threat to cancer patient mortality. Indeed, when breast cancer is diagnosed early and metastases are not present, 5 year survival is >88%; however, if metastases are also present, long-term survival is significantly diminished (~10%) (Jemal, et al., 2011). Thereby, the major cause of mortality of breast cancer and different types of cancer is due to metastasis to distant organs, such as lung, bone, liver and brain (Lu & Kang, 2007). A notable feature of this process is the variation in metastatic organ tropism displayed by different types of cancer (Chambers, et al., 2002, Fidler, 2002). A classic view has proposed that purely mechanical factors regulate the fate of blood-borne metastasis tumor cells (MacDonald, et al., 2002); however, this does not fully explain the non-random distribution and distinct pattern of metastasis in each tumor type (Lu & Kang, 2007). However, tumor microenvironment has also shown an important role in the regulation of this process (Valdivia-Silva, et al., 2009). A number of different molecules present in the microenvironment have been associated to the metastasis of breast cancer, among them, chemokines, which have been associated with regulation of cell migration and invasion of tumor cells into specific organs (Muller, et al., 2001, Zlotnik, 2006). Chemokines are a superfamily of chemotactic cytokines characterized by their ability to induce directed migration of leukocytes, during haematopoiesis, lymphoid organ development, and in disease (Sallusto, et al., 2000); their expression may be inducible, primarily by pro-inflammatory cytokines such as TNF-a and IL-1-b (Ben-Baruch, 2003). Chemokine receptor expression in many cancer cells have shown to be a non-random process (Shields, et al., 2007, Zlotnik, 2006) and to have a role in organ-specific metastasis: for example, CXCR4 expression and metastasis to lung, bone and lymph nodes (Muller, et al., 2001), CCR7 to lymph nodes (Shields, et al., 2007), CX3CR1 to brain (Mourad, et al., 2005), CCR9 to liver and small bowel (Amersi, et al., 2008, Letsch, et al., 2004), and CCR5 and CXCR2 to lung, liver, vessel endothelial cells and bone (Gross & Meier, 2009, Keeley, et

Here, we will discuss the ability of the chemokines to affect tumor cell–microenvironment interactions, increasing the invasive behaviour and metastasis, confirming the importance of the host inflammatory response that may differ between tumor types, disease stages, and/or many other host factors; and the role of stromal contribution of the inflammatory

**2. Inflammatory mediators as regulator of breast cancer development and** 

The link between inflammation and cancer has been observed over 150 years ago when Rudolf Virchow noted that cancers tend to occur at sites of chronic inflammation. Indeed, epidemiological studies indicate that inflammatory and infectious diseases are often associated with an increased risk of cancer (Coussens & Werb, 2002). The microenvironment of tumors mimics that of tissues during the height of an inflammatory response to injury (Joyce & Pollard, 2009). However, unlike the organized morphology of normal tissue, and the ultimate resolution of the inflammation that occurs during healing, tumors exist in a state of chronic inflammation characterized by the presence of cancer cells, immune cells, aberrant vascular cells, and the persistence of inflammatory mediators, such as cytokines

Sonnenschein, 2004; Egeblad et al., 2010).

al., 2010, Miller, et al., 1998).

**metastasis** 

and chemokines.

microenvironment to cancer progression and metastasis.

The presence and significance of leukocyte infiltrates in developing neoplasms is now undisputed (Allen, et al., 2007, Moser & Loetscher, 2001, Moser & Willimann, 2004). It has been demonstrated that leukocyte infiltration in developing tumors is one of the host´s main immune mechanisms to eradicate malignant cells. However, while some leukocytes certainly have this potential, i.e., cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells (Luster, 1998), other leukocyte cell types, most notably innate immune cells, i.e., mast cells (MCs), immature myeloid cells, granulocytes, and macrophages, instead potentiate tumor progression (Baggiolini, et al., 1997, Chen, et al., 2006, Joyce & Pollard, 2009), and enhance neoplastic cell survival. Upon entry into the neoplastic microenvironment, infiltrating leukocytes become alternatively activated and manifest a pro-tumor phenotype as defined by activation of cellular programs involved in immune tolerance and tissue remodelling (Mishra, et al., 2011, Strieter, et al., 2006). During premalignant progression, a consequence of alternative activation of leukocytes is promotion and elaboration of a microenvironment rich in extracellular matrix (ECM) remodelling proteases, and increased presence of pro-survival, pro-growth and pro-angiogenic factors that further enhance proliferative and invasive capacities of neoplastic cells (Li, et al., 2007, Orimo, et al., 2005). Such pro-tumor inflammatory microenvironments promote not only malignant conversion and development of solid tumors, but also dissemination of neoplastic cells into blood vasculature by driving invasive capacity of malignant cells, expansion of angiogenic vasculature, and neoplastic cell entry into blood vessels (and lymphatics) (Keeley, et al., 2010).

Breast carcinomas are highly infiltrated by different types of host leukocytes, including primarily T cells, and monocytes that differentiate into tumor-associated macrophages (TAM) at the tumor site (Ben-Baruch, 2003, Crowther, et al., 2001). The presence of the cellular infiltrate in breast tumors was initially regarded as evidence for the potential activity of immune mechanisms against the growing neoplasm. As explained above, several studies suggest that T-cell antitumor responses are impaired in advanced stages of breast carcinoma, and there is no definite conclusion regarding the efficacy of T-cell-dependent immune mechanisms, or regarding the correlation between the type of T-cell infiltration and tumor progression in most subtypes of breast carcinoma (Hsiao, et al., 2010). The only exception is the relatively infrequent type of medullary carcinoma, in which favourable prognosis was correlated with intensive lymphoid infiltration (Hadden, 1999). In contrast to T lymphocytes, large evidence suggests that high levels of TAM are correlated with poor prognosis in breast carcinoma. Many studies have shown a positive relationship between high levels of TAM and lymph node metastases, and suggested that the density of TAM is associated with clinical aggressiveness (Crowther, et al., 2001, O'Sullivan & Lewis, 1994). Again, the potential contribution of TAM to tumor elimination, in view of several potential antimalignant activities that may be exerted by these cells, such as antigen presentation, cytotoxicity, or/and phagocytosis, was contradictory with the promalignant activities of TAM in breast carcinoma. These promalignant activities of TAM are the result of their ability to express numerous tumor-promoting mediators, such as growth factors for breast tumor cells, angiogenic molecules, ECM degrading enzymes, inflammatory cytokines, and chemokines (Balkwill & Mantovani, 2001, Colotta, et al., 2009). In addition, TAM might contribute to tumor progression by the release of reactive oxygen intermediates, which may induce mutagenic changes that could result in increased DNA damage and generation of new subtypes of cancer cells within the tumor (Colotta, et al., 2009). A major TAM-derived

Novel Insights Into the Role of Inflammation in Promoting Breast Cancer Development 133

Hypoxia is also an important cellular stressor that triggers a survival program by which cells attempt to adapt to the new environment. This primarily involves adaptation of metabolism and/or stimulation of oxygen delivery. These cell-rescuing mechanisms can be conducted rapidly by a transcription factor that reacts to hypoxic conditions, the hypoxiainducible factor-1 (HIF-1a) (Semenza & Wang, 1992). HIF-1a stimulates processes such as angiogenesis, glycolysis, and erythropoiesis (Jiang, et al., 1996) by activating genes that are responsible for these processes. Cancer cells are able to survive and proliferate in extreme microenvironmental conditions and show changes in oncogenes and tumor suppressor genes. Hypoxia and HIF-1a have been implicated in carcinogenesis and in clinical behaviour of tumors. Upregulation of HIF-1a was noted during breast carcinogenesis (Bos, et al., 2001) especially in the poorly differentiated pathway. Hypoxia is related to poor response to therapy in various cancer types. In invasive breast cancer, high HIF-1a concentrations were associated with poor survival in lymph node-negative patients (Bos, et al., 2003). As prognosis in breast cancer is closely related to proliferation rate (van Diest & Baak, 1991) and poorly differentiated tumors usually exhibit high proliferation and HIF-1a overexpression, the prognostic value of HIF-1a might well be explained by a close association between HIF-1a and proliferation. Additionally, HIF-1a has shown to be a master regulator for surviving hypoxia interacting with cell cycle-related proteins. High concentrations of HIF-1a are associated with overexpression of p53 and markers of

proliferation during the late SG2 phase of the cell cycle (Bos, et al., 2004).

Barraza, et al., 2010, Valdivia-Silva, et al., 2009).

**metastasis** 

**3. Role of chemokines and their receptors in breast cancer progression and** 

While most evidence presented above suggests that proinflammatory cytokines and enzymes play an important role in mediating tumorigenesis, and tumor progression, the molecular mechanisms of metastasis and its relationship with the organotropism of cancer cell remain unclear. However, recent studies focused on the chemokines and their receptors, and the different interactions with inflammatory cytokines in the tumor microenvironment have provided additional information that might better explain the non-random patterns of organotropism during metastasis, including atypical metastasis to rare organs (Franco-

Chemokine activities in different malignancy including breast cancer are mediated primarily by their ability to induce chemotaxis of leukocytes, endothelial cells, and/or the tumor cells. Chemokines induce migration of leukocyte subpopulations to tumor sites that may promote antitumor activities (such as Th1 cells or natural killer cells), while other chemokines are responsible for large quantities of deleterious tumor-associated macrophages (TAM) at tumor sites (Allavena, et al., 2008, Ben-Baruch, 2008, Soria & Ben-Baruch, 2008) as discussed above. Moreover, specific chemokines upregulate endothelial cell migration and proliferation, and promoting angiogenesis, whereas other chemokines have powerful angiostatic properties (Strieter, et al., 2006, Struyf, et al., 2011). Another very important activity of chemokines is induction of tumor cell invasion and migration, thereby playing key roles in dictating site-directed metastasis formation (Ben-Baruch, 2008, Zlotnik, 2006). Chemokines and their receptors can execute such multifaceted roles in malignancy because cells of the tumor microenvironment, and in many cases also by the tumor cells themselves express them. As such, they can affect through autocrine pathways the ability of

inflammatory cytokine shown to be highly expressed in breast carcinomas is tumor necrosis factor alpha (TNF-a) (Leek, et al., 1998), which is a multifactorial cytokine. Tumor necrosis factor alpha was first isolated as an anti-cancer cytokines more than two decades ago (Aggarwal, 2003). However, these effects may depend on multiple factors, such as estrogen therapy and the expression of members of the epidermal growth factor receptor family. The fact that TNF-a activities vary under different physiological conditions and in a cell-typedependent manner contributes to a sense of ambiguity regarding its antitumor effects (Kanoh, et al., 2001, Offersen, et al., 2002). A number of reports indicate that TNF-a induces cellular transformation, proliferation, and tumor promotion (Balkwill & Mantovani, 2001, Li, et al., 2007). A interesting study reported that human TNF-a is more effective than the chemical tumor promoters okadaic acid and 12-O-tetradecanoylphorbol-13-acetato in inducing cancer (Komori, et al., 1993).

The number of cells expressing TNF-a in inflammatory breast carcinoma has been correlated with increasing tumor grade and node involvement (Ben-Baruch, 2003, Leek, et al., 1998). Furthermore, patients with more progressed tumor phenotypes were shown to have significantly higher TNF-a and IL-2 serum concentration (Tesarová, et al., 2000). The tumorpromoting functions of TNF-a may be mediated by its ability to induce pro-angiogenic 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 (Garg & Aggarwal, 2002).

In addition, several inflammatory interleukins have been linked with carcinogenesis and tumor progression. Among these, IL-6 and IL-1 have been widely studied in breast carcinoma. In different types of cancer, IL-1 promotes growth and confers chemoresistance (Arlt, et al., 2002, Woodworth, et al., 1995). Furthermore, IL-1 secretion into the tumor milieu also induces several angiogenic factors from tumor and stromal cells that promotes tumor growth through hyperneovascularization (Zhou, et al., 2011). IL-6 may act as a paracrine growth factor for multiple myeloma, non-Hodgkin's lymphoma, bladder cancer, colorectal cancer, and renal carcinoma (Angelo, et al., 2002, Landi, et al., 2003, Okamoto, et al., 1995, Voorzanger, et al., 1996). However, contradictory studies suggested that elevated levels of IL-6 might contribute to breast cancer progression (Karczewska, et al., 2000, Kurebayashi, 2000). Initial analyses regarding IL-1b indicated that its levels were significantly higher in invasive carcinoma than in ductal carcinoma *in situ* or in benign lesions, implying that elevated levels of IL-1b are directly correlated with a more advanced disease (Jin, et al., 1997). Of interest is the fact that the two cytokines (IL-6 and IL-1b) and TNF-a 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.

Cyclooxygenase (COX)-2, an inducible enzyme with expression regulated by NF-kb, mediates tumorigenesis. COX-2, the inducible isoform of prostaglandin H synthase has been implicated in the growth and progression of a variety of human cancers, and its expression can be induced by various growth factors, cytokines, oncogenes, and other tumor factors. IL-1 has been reported to upregulate COX-2 expression in human colorectal cancer cells via multiple signalling pathways (Liu, et al., 2003). COX-2 is expressed at an intermediate or high level in epithelial cells of invasive breast cancers (Chang, et al., 2005, Half, et al., 2002). Expression of COX-2 in breast cancer correlates with poor prognosis, and COX-2 enzyme inhibitors reduce breast cancer incidence in humans. COX-2 overexpression has also been found in the mammary gland of transgenic mice induced mammary cancer (Kundu & Fulton, 2002).

inflammatory cytokine shown to be highly expressed in breast carcinomas is tumor necrosis factor alpha (TNF-a) (Leek, et al., 1998), which is a multifactorial cytokine. Tumor necrosis factor alpha was first isolated as an anti-cancer cytokines more than two decades ago (Aggarwal, 2003). However, these effects may depend on multiple factors, such as estrogen therapy and the expression of members of the epidermal growth factor receptor family. The fact that TNF-a activities vary under different physiological conditions and in a cell-typedependent manner contributes to a sense of ambiguity regarding its antitumor effects (Kanoh, et al., 2001, Offersen, et al., 2002). A number of reports indicate that TNF-a induces cellular transformation, proliferation, and tumor promotion (Balkwill & Mantovani, 2001, Li, et al., 2007). A interesting study reported that human TNF-a is more effective than the chemical tumor promoters okadaic acid and 12-O-tetradecanoylphorbol-13-acetato in

The number of cells expressing TNF-a in inflammatory breast carcinoma has been correlated with increasing tumor grade and node involvement (Ben-Baruch, 2003, Leek, et al., 1998). Furthermore, patients with more progressed tumor phenotypes were shown to have significantly higher TNF-a and IL-2 serum concentration (Tesarová, et al., 2000). The tumorpromoting functions of TNF-a may be mediated by its ability to induce pro-angiogenic 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

In addition, several inflammatory interleukins have been linked with carcinogenesis and tumor progression. Among these, IL-6 and IL-1 have been widely studied in breast carcinoma. In different types of cancer, IL-1 promotes growth and confers chemoresistance (Arlt, et al., 2002, Woodworth, et al., 1995). Furthermore, IL-1 secretion into the tumor milieu also induces several angiogenic factors from tumor and stromal cells that promotes tumor growth through hyperneovascularization (Zhou, et al., 2011). IL-6 may act as a paracrine growth factor for multiple myeloma, non-Hodgkin's lymphoma, bladder cancer, colorectal cancer, and renal carcinoma (Angelo, et al., 2002, Landi, et al., 2003, Okamoto, et al., 1995, Voorzanger, et al., 1996). However, contradictory studies suggested that elevated levels of IL-6 might contribute to breast cancer progression (Karczewska, et al., 2000, Kurebayashi, 2000). Initial analyses regarding IL-1b indicated that its levels were significantly higher in invasive carcinoma than in ductal carcinoma *in situ* or in benign lesions, implying that elevated levels of IL-1b are directly correlated with a more advanced disease (Jin, et al., 1997). Of interest is the fact that the two cytokines (IL-6 and IL-1b) and TNF-a 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. Cyclooxygenase (COX)-2, an inducible enzyme with expression regulated by NF-kb, mediates tumorigenesis. COX-2, the inducible isoform of prostaglandin H synthase has been implicated in the growth and progression of a variety of human cancers, and its expression can be induced by various growth factors, cytokines, oncogenes, and other tumor factors. IL-1 has been reported to upregulate COX-2 expression in human colorectal cancer cells via multiple signalling pathways (Liu, et al., 2003). COX-2 is expressed at an intermediate or high level in epithelial cells of invasive breast cancers (Chang, et al., 2005, Half, et al., 2002). Expression of COX-2 in breast cancer correlates with poor prognosis, and COX-2 enzyme inhibitors reduce breast cancer incidence in humans. COX-2 overexpression has also been found in the mammary gland of transgenic mice induced mammary cancer (Kundu &

which is promotion of tumor-related processes (Garg & Aggarwal, 2002).

inducing cancer (Komori, et al., 1993).

Fulton, 2002).

Hypoxia is also an important cellular stressor that triggers a survival program by which cells attempt to adapt to the new environment. This primarily involves adaptation of metabolism and/or stimulation of oxygen delivery. These cell-rescuing mechanisms can be conducted rapidly by a transcription factor that reacts to hypoxic conditions, the hypoxiainducible factor-1 (HIF-1a) (Semenza & Wang, 1992). HIF-1a stimulates processes such as angiogenesis, glycolysis, and erythropoiesis (Jiang, et al., 1996) by activating genes that are responsible for these processes. Cancer cells are able to survive and proliferate in extreme microenvironmental conditions and show changes in oncogenes and tumor suppressor genes. Hypoxia and HIF-1a have been implicated in carcinogenesis and in clinical behaviour of tumors. Upregulation of HIF-1a was noted during breast carcinogenesis (Bos, et al., 2001) especially in the poorly differentiated pathway. Hypoxia is related to poor response to therapy in various cancer types. In invasive breast cancer, high HIF-1a concentrations were associated with poor survival in lymph node-negative patients (Bos, et al., 2003). As prognosis in breast cancer is closely related to proliferation rate (van Diest & Baak, 1991) and poorly differentiated tumors usually exhibit high proliferation and HIF-1a overexpression, the prognostic value of HIF-1a might well be explained by a close association between HIF-1a and proliferation. Additionally, HIF-1a has shown to be a master regulator for surviving hypoxia interacting with cell cycle-related proteins. High concentrations of HIF-1a are associated with overexpression of p53 and markers of proliferation during the late SG2 phase of the cell cycle (Bos, et al., 2004).
