Interleukin-6 in the Breast Tumor Microenvironment

Greater than 200,000 new cases of breast cancer cases were diagnosed in 2010 in the United States, with approximately 40,000 women succumbing to the disease (www.cancer.gov). Globally, an estimated 1.38 million new cases of breast cancer were diagnosed in 2008, with greater than 450,000 women succumbing to the disease (Jemal et al., 2011). Despite our improved understanding of breast carcinogenesis, breast cancer remains the second most commonly diagnosed cancer in women behind non-melanoma skin cancer and the second leading cause of death in women behind lung cancer. These epidemiological statistics highlight the overwhelming clinical dilemma of breast cancer and emphasize the need for novel therapeutic targets and prevention strategies. Countless studies in the fields of mammary gland development and breast cancer have led to an appreciation of a breast tumor microenvironment that actively contributes to the heterogeneous nature of breast cancer. The current review will focus on the impact of IL-6 and STAT3 activation in the breast tumor microenvironment and subsequently present rationale for targeting the IL6/STAT3 signaling pathway in this setting. IL-6 is a quintessential pleiotropic cytokine produced by a diverse number of cell populations, most of which can localize to the breast tumor microenvironment. Excessive IL-6 has been demonstrated in primary breast tumors and breast cancer patient sera and is associated with poor clinical outcomes in breast cancer. These clinical associations are corroborated by emerging preclinical data revealing that IL-6 is a potent growth factor and promotes an epithelial-mesenchymal (EMT) phenotype in breast cancer cells to indicate that IL-6 in the breast tumor microenvironment is clinically relevant. Numerous clinical reports have now demonstrated the safety and efficacy of IL-6 signaling antagonists in multiple diseases, which supports future investigations of these therapies in breast cancer. Estrogen receptor-alpha (ER┙) is a latent cytoplasmic ligand-activated transcription factor utilized by clinicians to subclassify the heterogeneous disease of breast cancer. ER┙-positive breast cancer incidence increases up to age 51, the mean age of menopause, and continues to increase until age 80. Conversely, ER┙-negative breast cancer incidence plateaus and even slightly decreases at age 51, while demonstrating an increase prior to age 50 comparable to that of ER┙-positive disease. This discrepancy between the two incidence rates at menopause produces an inflection in the incidence rate of all breast cancer cases which has been termed Clemmesen’s hook (Anderson and Matsuno, 2006). Whereas the prevalence of


The breast tumor microenvironment
A normal epithelial tissue can undergo hyperplasia and acquire tumorigenic properties that promote the development of a benign, non-invasive solid tumor known as carcinoma in situ.Normal epithelial tissues and non-invasive carcinoma in situ tumors are separated from a supportive stromal compartment by an intact basement membrane.Ultimately, carcinoma in situ can progress to a malignant, invasive carcinoma, the most common form of human cancer.The panoply of published investigations between the fields of mammary gland development and breast cancer has led to an appreciation for a supportive non-epithelial mammary stroma that mechanically and biologically restrains tumorigenesis.However, tumors of the breast and other epithelial tissues obviously overcome these growth restraints and exploit this stroma to sculpt a vastly divergent tumor stroma.Tumor stroma is generally divided into four main components: tumor vasculature, inflammatory leukocytes, extracellular matrix (ECM) and soluble growth factors, and fibroblasts.Malignant carcinoma cells and tumor stromal cells bi-directionally communicate with one another through paracrine signaling and intercellular contacts in a disorganized ECM to constitute a tumor microenvironment.Tumor-associated fibroblasts (TAF), the predominant stromal cell population within the tumor microenvironment, acquire and sustain an "activated" phenotype that promotes tumor progression (Rasanen and Vaheri, 2010).TAF are capable of enhancing breast tumor growth and metastasis by means of promoting angiogenesis (Orimo et al., 2005), epithelial-mesenchymal transition (EMT) (Martin et al., 2010;Radisky et al., 2005), and progressive genetic instability (Kurose et al., 2001;Moinfar et al., 2000).In contrast, a normal mammary microenvironment can act in a dominant manner to inhibit tumor growth and "revert" the malignant phenotype of breast cancer cells (Kenny and Bissell, 2003).While resident breast tissue fibroblasts can inhabit breast tumors as TAF, breast tumors also recruit distant cell populations that engraft within the breast tumor microenvironment where they actively contribute as TAF.For example, mesenchymal stem cells (MSC), a bone marrow-derived stromal cell population, home to breast cancer cell xenograft tumors and persist as TAF (Spaeth et al., 2009).

Cancer-associated inflammation
Although highly characterized for their protective capacity against infection, inflammatory leukocytes also reside within the tumor microenvironment.In fact, various immune cells are capable of eliminating transformed cells and thus preventing tumorigenesis in a process termed immunosurveillance (Dunn et al., 2004).Whereas acute inflammation may prevent tumorigenesis by promoting an immune response directed against transformed cells, chronic inflammation promotes tumorigenesis.Rudolf Virchow is credited with making the seminal link between chronic inflammation and cancer by noting that human tumor biopsies were often infiltrated with inflammatory cells (Balkwill and Mantovani, 2001).Leukocytes can be detected in non-malignant tumors and carcinomas, including breast cancer (DeNardo and Coussens, 2007), which suggests an ongoing antitumor immune response.Despite the infiltration of leukocytes such as cytotoxic T-cells and NK-cells, the persistence of a tumor demonstrates immune evasion and highlights the local and systemic immune suppressive state of the tumor microenvironment and the tumor-bearing host, respectively.

Interleukin-6: A quintessential pleiotropic cytokine
Interleukin-6 (IL-6) is an inflammation-associated cytokine and major inducer of C-reactive protein (CRP) throughout the acute phase inflammatory response.IL6 gene expression is nuclear factor-kappaB (NF-κB)-dependent (Chauhan et al., 1996) and produces a 26 kDa IL-6 protein product.First characterized as a T-cell-derived factor that induced proliferation, differentiation, and immunoglobulin production in B-cells, IL-6 was originally named B-cell stimulating factor-2 (BSF-2).It was later thought to be a novel interferon (IFN-2 ) due to studies demonstrating the ability of IL-6 to activate signal transducer and activator of transcription 3 (STAT3) (Kishimoto, 2006).Complementary DNA encoding the human IL-6 gene was subsequently cloned, and human IL-6 transgenic mice demonstrated a polyclonal IgG1 plasmacytosis phenotype (Suematsu et al., 1989).Next, IL-6 knockout (IL-6 -/-) mice were generated and characterized.IL-6 -/-mice underwent normal development, but adult animals exhibited reduced numbers of peripheral T-cells and impaired antiviral cytotoxic Tcell activity (Kopf et al., 1994).In addition, IL-6 is a critical factor during hematopoiesis and subsequent lymphocyte differentiation and activation.Multiple diverse cell populations including fibroblasts, T and B-cells, monocytes, macrophages, endothelial cells, keratinocytes, astrocytes, and smooth muscle cells all have the potential to produce constitutive or inducible IL-6 (Kishimoto, 2006).Depending on cellular context, IL-6 can signal through multiple kinase-dependent proliferation and anti-apoptosis pathways including the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol-triphosphate kinase (PI-3K)/Akt pathway, and perhaps the most commonly evaluated in breast cancer, the Janus kinase (JAK)/signal transducer and activator of transcription-3 (STAT3) pathway (Hodge et al., 2005).To do so, a plasma membrane-associated IL-6 receptor (IL-6R/CD126) homodimer first ligates two soluble IL-6 molecules, which leads to gp130 (CD130) homodimer ligation.Whereas IL-6R is only expressed on hepatocytes, osteoclasts, and most immune cells under normal physiological conditions, gp130 is a ubiquitous and promiscuous receptor involved in multiple cytokine signaling pathways (e.g., IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neurotrophic factor (CNTF)) (Rose-John et al., 2006).To initiate classical JAK/STAT3 signal transduction, JAK are recruited to the intracellular domain of the gp130 receptor where they bind and autophosphorylate.Subsequent gp130 phosphorylation via activated JAK offers docking sites for STAT3 and other receptorassociated proteins.Once bound to the intracellular domain of gp130, STAT3 is specifically phosphorylated (pSTAT3) by adjacent JAK on a C-terminal tyrosine residue (Y705), which grants its disengagement from the receptor.Dissociation of pSTAT3 Y705 from gp130 facilitates its homodimerization within the cytoplasm, and the pSTAT3 Y705 homodimer translocates to the nucleus.There, pSTAT3 Y705 binds to specific promoters whereby it initiates the transcription of multiple downstream target genes (Clevenger, 2004).Under normal physiological conditions, an inhibitory feedback loop maintains rapid and transient STAT3 activation.Following activation in normal cells, STAT3 induces suppressors of cytokine signaling (SOCS) and protein inhibitors of activated STATs (PIAS) expression.While SOCS-1 specifically inhibits JAK function, SOCS-3 binds the IL-6R complex to inhibit IL-6 signal transduction.PIAS-3 directly interacts with STAT3 to inhibit all STAT3 target gene expression (Kishimoto, 2006).In contrast, many human cancers, including breast cancer, exhibit constitutive STAT3 activity.Recent studies have demonstrated that unphosphorylated STAT3 (U-STAT3) accumulates in tumor cells with constitutively active www.intechopen.comInterleukin-6 in the Breast Tumor Microenvironment 169 STAT3 where it forms a complex with NF-κB to activate a subset of NF-κB target genes (Yang and Stark, 2008).Alternatively, IL-6 trans-signaling describes an IL-6 signaling pathway whereby an IL-6 soluble receptor (IL-6sR) binds IL-6 and subsequently ligates gp130 to stimulate STAT3 activation in cells that only express gp130.IL-6sR is naturally produced by either proteolytic cleavage of the membrane-bound IL-6R or alternative splicing of IL-6R mRNA (Rose-John et al., 2006).Whereas IL-6 serum levels continue to increase with age, levels of serum IL-6sR rise until approximately age 70 at which time they gradually decline (Giuliani et al., 2001).Furthermore, IL-6sR expression has been demonstrated in human breast cancer cell lines (Crichton et al., 1996;Oh et al., 1996;Singh et al., 1995), suggesting that IL-6 trans-signaling mediates the effects of IL-6 in breast cancer cells.In contrast, an endogenous soluble gp130 (sgp130) specifically antagonizes IL-6 trans-signaling by exclusively ligating the IL-6/IL-6sR complex, thus having no effect on cells that express the membrane-bound IL-6R (Rose-John et al., 2006) (Figure 1).

Excessive IL-6 in human breast cancer
Aberrantly elevated IL-6 is associated with a poor prognosis in breast cancer (Bachelot et al., 2003;Salgado et al., 2003;Zhang and Adachi, 1999).Human breast tumors produce more IL-6 when compared to matched healthy breast tissue, and tumor IL-6 levels concurrently increase with tumor grade.In addition, increased serum IL-6 has been demonstrated in breast cancer patients compared to normal donors and correlates with advanced breast tumor stage (Kozlowski et al., 2003) and increased number of metastatic sites (Salgado et al., 2003).Furthermore, a single nucleotide polymorphism (SNP) exists at position -174 in the IL-6 gene promoter region, noted as IL-6 (-174 G>C), with the following allele frequency in a Caucasion population: 36% G/G, 44% G/C, and 18% C/C.An inflammatory stimulus such as Salmonella typhii vaccination induced higher serum IL-6 in those individuals with the G/G allele (Bennermo et al., 2004).Although the IL-6 (-174 G>C) SNP is not associated with increased risk of developing breast cancer (Gonzalez-Zuloeta Ladd et al., 2006;Litovkin et al., 2007;Yu et al., 2009b), it is significantly associated with disease-free and overall survival in breast cancer patients (DeMichele et al., 2003).ER is expressed in luminal subtype breast tumors (Perou et al., 2000) and therefore associated with improved patient survival (Buyse et al., 2006;Sorlie et al., 2001).A clear and well-characterized inverse correlation exists between breast cancer ER status and IL-6.In fact, ER directly binds to NF-κB, thus preventing transactivation of IL6 gene expression (Galien and Garcia, 1997), which demonstrates a direct mechanism for such a correlation.Furthermore, ER -negative human breast tumors produce more IL-6 than tumors that express ER (Chavey et al., 2007), and IL-6 serum levels are higher in ER -negative breast cancer patients compared to ER -positive patients (Jiang et al., 2000).Likewise, ERnegative breast cancer cell lines produce autocrine IL-6 whereas ER -positive breast cancer cell lines do not (Sasser et al., 2007).Therefore, this strongly suggests that ER -negative breast cancer cells would exploit both paracrine (i.e., stromal cell-derived) and autocrine IL-6 signaling, whereas ER -positive breast cancer cells could only utilize paracrine IL-6 signaling.In addition, ER -negative breast cancer patients, whose tumors produce more IL-6 than those that express ER (Chavey et al., 2007), showed no difference in survival between the G/G allele (higher inducible serum IL-6) and any C allele (lower inducible serum IL-6) at the IL-6 (-174 G>C) promoter SNP.In contrast, ER -positive breast cancer patients with any C allele at the IL-6 (-174 G>C) promoter SNP demonstrated improved disease-free and overall survival compared to those with the G/G allele (DeMichele et al., 2003).

IL-6 promotes breast cancer cell growth
Stromal fibroblasts isolated from multiple types of tumors (i.e., TAF) or cancers (i.e., CAF) are now appreciated as influential players in cancer progression and metastasis (Orimo and Weinberg, 2006).CAF derived from multiple cancer types, including murine mammary cancers, exhibit an activated, proinflammatory phenotype with increased IL-6 production (Erez et al., 2010).Furthermore, work from our laboratory has demonstated that fibroblasts isolated from breast tissue and common sites of breast cancer metastasis such as bone and lung enhance the growth of breast cancer cells in an IL-6-dependent manner, and IL-6 is the major fibroblast-derived soluble factor that induced STAT3 activation in breast cancer cells (Sasser et al., 2007;Studebaker et al., 2008).MDA-MB-231 breast cancer cells are commonly utilized to model triple negative breast cancer and produce autocrine IL-6.MDA-MB-231 cells expressing a dominant negative isoform of gp130 lacked constitutively active STAT3 and exhibited impaired tumorigenicity in an orthotopic xenograft model (Selander et al., 2004), thus suggesting that IL-6 may drive tumor progression in this model.In addition, STAT3 is estimated to be constitutively activated in more than half of primary breast cancers due to IL-6 signaling (Berishaj et al., 2007).
Mesenchymal stem cells (MSC) are a bone marrow-derived fibroblast cell population that can be recruited to the breast tumor stroma, acquire a TAF phenotype, and produce high levels of IL-6.MSC enhance the growth of ER -positive breast cancer cells, which do not express IL-6 or activated STAT3.In contrast, MSC have no effect on IL-6-producing ERnegative breast cancer cells, which express constitutively activated STAT3.Moreover, ERpositive breast cancer cells orthotopically co-injected with MSC or MSC conditioned medium and ER -positive breast cancer cells that ectopically express IL-6 demonstrate enhanced xenograft tumor growth in the absence of exogenous 17 -estradiol (Sasser et al., 2007).Similar differential growth enhancement was demonstrated in vivo with ER -positive and ER -negative breast cancer cells co-injected with MSC, which also promoted metastasis (Karnoub et al., 2007).Interestingly, IL-6 has been reported to facilitate the recruitment of MSC to hypoxic breast tumor microenvironments (Rattigan et al., 2010).Likewise, IL-6 secreted from breast cancer cells has been shown to contribute to a recently characterized phenomenon termed "self-seeding" in which aggressive circulating tumor cells engraft within their original xenograft tumor (Kim et al., 2009).MSC have also been shown to mediate the self-renewal capacity of breast cancer stem cells, in part, through a reciprocal IL-6 loop (Liu et al., 2010).Taken together, preceding evidence strongly suggests that IL-6 promotes breast cancer cell growth by activating STAT3, which culminates with the upregulation of proliferative oncogenes such as c-Myc and cyclin D1 and and growth factors such as IL-6, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) (Yu et al., 2009a).

IL-6 promotes epithelial-mesenchymal transition in breast cancer cells
Normal polarized epithelial cells exhibit 'cobblestone' homophilic morphology and express E-cadherin, which is required for epithelial cell polarization, phenotype, and consequent homeostasis (Jeanes et al., 2008).E-cadherin is a key prognostic molecular biomarker clinically utilized to predict the metastatic propensity of breast cancer.Whereas very few studies have failed to demonstrate E-cadherin as an independent prognostic biomarker in breast cancer patients (Lipponen et al., 1994;Parker et al., 2001), the overwhelming majority of relevant studies have revealed E-cadherin as one of the strongest predictors of patient survival.Specifically, impaired E-cadherin expression in human breast tumors correlates with enhanced invasiveness, metastatic potential (Oka et al., 1993), and decreased breast cancer patient survival (Heimann and Hellman, 2000;Pedersen et al., 2002).While appropriate E-cadherin function is essential to the maintenance of epithelial cell morphology, phenotype, and homeostasis, regulation of E-cadherin expression is of equal importance.CDH1, the gene that encodes E-cadherin, is located on human chromosome 16q22.1 (Rakha et al., 2006) and is susceptible to inactivation by promoter hypermethylation, somatic mutation, or aberrant overexpression of repressive transcription factors including Twist, Snail, and Slug among others (Hirohashi, 1998).Likewise, E-cadherin loss of function can arise due to extracellular domain-specific proteolytic cleavage.Although uncommon, germline mutations of CDH1 predispose individuals to hereditary diffuse gastric cancer (HDGC) syndrome, and a proportion of these patients present with other cancers, including breast cancer (Guilford, 1999).E-cadherin was initially termed uvomorulin in mice and L-CAM in chicks following its discovery as a 120 kDa calcium-dependent trypsin-labile cell surface glycoprotein required for intercellular adhesion in mouse blastomeres (Hyafil et al., 1981) and chick embryos (Brackenbury et al., 1981).It now represents the best studied member of the cadherin family of tissue-specific homophilic intercellular adhesion molecules.E-cadherin knockout studies have demonstrated early embryonic lethality due to impaired maintenance of epithelial polarity and failure to form an intact epithelium in E-cadherin -/-embryos (Larue et al., 1994).E-cadherin is localized on the cell surface of epithelial cells, and each E-cadherin protein consists of an amino-terminal extracellular domain, a single-pass transmembrane segment, and a carboxy-terminal intracellular domain.Five calcium-binding repeated subunits comprise an extracellular domain that promotes homophilic interaction to ultimately form anti-parallel trans-E-cadherin dimers between adjacent cells (Guilford, 1999).The intracellular domain is comprised of a juxtamembrane p120-catenin binding subdomain and a C-terminal beta ( )-catenin binding subdomain.-catenin, a potent transcription factor, binds E-cadherin and alpha ( )-catenin subsequently binds -catenin.Although contentious (Weis and Nelson, 2006), it is generally acknowledged that -catenin interacts with F-actin and thereby, facilitates the linkage of E-cadherin to the cytoskeleton.This E-cadherincatenin-actin complex localizes to epithelial intercellular junctions called adherens junctions and is critical to epithelial cell adhesion, polarity, and morphology (Hartsock and Nelson, 2008).Furthermore, E-cadherin sequesters -catenin at the cell surface as one means to inhibit -catenin nuclear translocation and consequent expression of -catenin responsive genes (Perez-Moreno et al., 2003).Another prominent role of E-cadherin is that of an invasion/metastasis suppressor protein.Upon loss of E-cadherin and subsequent dissociation of adherens junctions, epithelial cells acquire enhanced invasive capability (Behrens et al., 1989).MDA-MB-231 cells, an ER -negative breast cancer cell line, lack E-cadherin, whereas MCF-7 cells, an ER -positive breast cancer cell line express high levels of E-cadherin (Kenny et al., 2007), and MDA-MB-231 cells exhibit enhanced invasive capability compared to MCF-7 cells (Sommers et al., 1991).Naturally, E-cadherin expression and consequent invasive capacity regulate the propensity of breast cancer metastasis.Multiple signaling pathways are activated following loss of E-cadherin protein, which promote transformed human breast epithelial cell metastasis in a xenograft model.Interestingly, Twist, a transcriptional repressor of CDH1, is induced upon loss of E-cadherin and is necessary for metastasis in this model.Furthermore, the E-cadherin binding partner, -catenin, was shown to be necessary but not sufficient for the EMT phenotype induced following loss of E-cadherin (Onder et al., 2008).Ectopic expression of murine E-cadherin in highly metastatic human MDA-MB-231 cells significantly reduced osteolytic bone metastases in a murine intracardiac dissemination model (Mbalaviele et al., 1996).Likewise, aberrant cytoplasmic or diminished to negative E-cadherin immunostaining patterns are commonly detected in invasive poorly differentiated breast carcinomas compared to noninvasive welldifferentiated breast carcinomas and are associated with increased probability of breast carcinoma metastasis (Oka et al., 1993).The finding that distant metastases often express E-cadherin even in patients which exhibit primary breast carinomas which lack Ecadherin suggests that ultimate re-expression may be necessary for colonization of secondary tissues (Kowalski et al., 2003;Saha et al., 2007).Loss of E-cadherin is a prerequisite for epithelial-mesenchymal transition (EMT), a highly conserved process which exemplifies the aberrant activation of an embryonic gene expression program during carcinoma progression.EMT is critical for multiple steps of developmental metazoan cellular morphogenesis as demonstrated in well-characterized www.intechopen.comInterleukin-6 in the Breast Tumor Microenvironment 173 Drosophila and Xenopus models.Throughout embryonic development, EMT whereby epithelial cells give rise to more motile mesenchymal cells is essential for mesoderm and neural crest formation.Importantly, this is a transient process and mesenchymal-epithelial transition (MET) allows for cellular reversion (Yang and Weinberg, 2008).Whereas EMT has been extensively studied for its essential role in embryogenesis, the concept of EMT-like cellular changes in human cancers has gained acceptance as a major mechanism to promote primary tumor cell invasion and subsequent tumor metastasis.A carcinoma cell must first detach from the primary tumor and invade through the basement membrane into the underlying tissue parenchyma to initiate the metastasic cascade.Although cancer-associated EMT was considered a controversial notion even in recent years (Tarin et al., 2005), it has been demonstrated in multiple human carcinomas, including breast cancer (Cheng et al., 2008;Heimann and Hellman, 2000;Moody et al., 2005;Sarrio et al., 2008), and is now recongnized as a putative mediator of tumor metastasis.An EMT phenotype including impaired E-cadherin expression with concominant induction of Vimentin, Alpha-smooth-muscle-actin, and/or N-cadherin is associated with the basal breast cancer subtype, suggesting that EMT may promote characteristic aggressiveness in these tumors and contribute to poor breast cancer patient survival (Sarrio et al., 2008).Likewise, relatively noninvasive ER -positive MCF-7 cells express E-cadherin, consistent with a characteristic epithelial phenotype, and are classified as luminal subtype, whereas highly invasive ER -negative MDA-MB-231 cells lack E-cadherin and are classified as basal subtype (Blick et al., 2008).Furthermore, ER directly correlates with E-cadherin in primary human breast tumors (Ye et al., 2010).While EMT may enhance carcinoma cell invasion and subsequent dissemination which would increase metastatic potential, it is not synonymous with metastasis in all models.For example, Lou, et al. demonstrated that EMT alone was insufficient for spontaneous murine mammary carcinoma metastasis (Lou et al., 2008).Yet, Weinberg and colleagues described the promotion of metastasis with loss of E-cadherin and a consequent EMT phenotype in transformed human breast epithelial cells (Onder et al., 2008).Our laboratory has previously demonstrated that exogenous IL-6 exposure induced an EMT phenotype in a panel of human ER -positive breast cancer cells, which included E-cadherin repression and concomitant induction of Vimentin, N-cadherin, Snail, and Twist.In addition, ectopic expression of IL-6 in ER -positive MCF-7 breast cancer cells promoted an EMT phenotype and enhanced invasiveness.Likewise, MCF-7 cells with ectopic Twist expression exhibit an EMT phenotype (Mironchik et al., 2005), autocrine IL-6 production, and constitutive STAT3 activation (Sullivan et al., 2009).

Therapeutic targeting of the IL-6/STAT3 pathway
IL-6 levels are increased in human breast tumors and breast cancer patient sera, and excessive IL-6, both circulating and within the breast tumor microenvironment, is associated with poor clinical outcomes in breast cancer.STAT3, a critical downstream mediator of IL-6 signaling, is constitutively activated in more than half of human cancers and promotes the expression of proliferative, anti-apoptotic, immune suppressive, and pro-angiogenic target genes, which all potentiate carcinogenesis.Whereas the IL-6 signaling network has been targeted in numerous autoimmune diseases and cancers, this therapeutic strategy has yet to be clinically employed for breast cancer.Increased preclinical reports have revealed novel mechanisms underlying IL-6/STAT3 signaling in breast cancer cells such as enhanced growth, induction of EMT, multidrug resistance, and recruitment of peripheral fibroblasts.Taken together, accumulating preclinical and clinical data emphasize IL-6 as a highly attractive therapeutic target in breast cancer.It is therefore imperative that more work be done to evaluate current therapeutics and develop novel agents that target IL-6/STAT3 signaling in breast cancer models.Multiple strategies could be utilized to target the IL-6/STAT3 pathway, but first and most obvious would be anti-IL-6 neutralizing antibodies.One such anti-IL-6 monoclonal antibody is Siltuximab (CNTO 328).The safety and efficacy of Situximab has been demonstrated in preclinical studies and phase I/II clinical trials of diverse human pathologies and malignancies including Castleman's disease (van Rhee et al., 2010), multiple myeloma (Hunsucker et al., 2011;Voorhees et al., 2007), prostate cancer (Cavarretta et al., 2007;Cavarretta et al., 2008;Dorff et al., 2010;Karkera et al., 2011), renal cell carcinoma (Puchalski et al., 2010;Rossi et al., 2010), non-small cell lung cancer (Song et al., 2010), and ovarian cancer (Guo et al., 2010).Furthermore, IL-6R can be targeted with tocilizumab, an anti-IL-6R monoclonal antibody that has shown promising results in IL-6driven autoimmune diseases (Tanaka et al., 2011) and was recently approved by the FDA for the treatment of rheumatoid arthritis.The promiscuous IL-6 coreceptor, gp130, also has an endogenous soluble form (sgp130) that exclusively inhibits IL-6 trans-signaling, thus preserving classical IL-6 signaling.Therapeutic sgp130 would potentially be more targeted toward breast cancer cells, which generally lack membrane-associated IL-6R and therefore utilize IL-6 trans-signaling through IL-6sR.Recombinant soluble gp130 (sgp130-Fc) has been shown to inhibit murine colon carcinogenesis (Becker et al., 2004), suggesting that it may prove effective in breast cancer as well.Finally, a growing number of nonselective kinase inhibitors and recent focus on specific JAK and STAT3 inhibitor development will provide further insight into the roles of JAK and STAT3 in breast cancer.

Conclusions
Breast cancer is a heterogeneous disease and thus, highly variable across individual patients.This heterogenicity arises not only due to the diversity of genetic and molecular aberrations in primary breast cancer cells but also due to the diversity of cellular populations that inhabit the breast tumor microenvironment.Although IL-6 levels are higher in breast tumors and patient sera, the precise source of this IL-6 remains elusive.Importantly, many breast tumor stromal cells provide a paracrine source of IL-6 for breast cancer cells within the breast tumor microenvironment.In addition, certain clinical subtypes of breast cancers and research models, such as ER -negative primary breast cancers and ER -negative breast cancer cell lines, produce excessive IL-6 (Figure 2).Therefore, ER -negative breast cancer cells may supply the tumor microenvironment with IL-6 by means of autocrine IL-6 production to exacerbate the poor prognosis associated with this clinical subtype.It will be critical to determine the specific cellular source of breast tumor-associated IL-6 to advance our understanding of this pleiotropic cytokine in breast cancer progression and metastasis.Moreover, this knowledge will facilitate the validation and subsequent clinical utility of current and novel targeted antagonists of the IL-6/STAT3 signaling network in breast cancer.