**4. 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-β2) 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 pSTAT3Y705 from gp130 facilitates its homodimerization within the cytoplasm, and the pSTAT3Y705 homodimer translocates to the nucleus. There, pSTAT3Y705 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

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-β2) 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

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 pSTAT3Y705 from gp130 facilitates its homodimerization within the cytoplasm, and the pSTAT3Y705 homodimer translocates to the nucleus. There, pSTAT3Y705 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

**4. Interleukin-6: A quintessential pleiotropic cytokine** 

constitutive or inducible IL-6 (Kishimoto, 2006).

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).

Fig. 1. The IL-6/STAT3 signaling pathway
