**7. 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

Interleukin-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

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 ERα-positive MCF-7 cells express E-cadherin, consistent with a characteristic epithelial phenotype, and are classified as luminal subtype, whereas highly invasive ERα-negative MDA-MB-231 cells lack E-cadherin and are classified as basal subtype (Blick *et al.*, 2008). Furthermore, ERα 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.*,

Our laboratory has previously demonstrated that exogenous IL-6 exposure induced an EMT phenotype in a panel of human ERα-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 ERα-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,

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

and constitutive STAT3 activation (Sullivan *et al.*, 2009).

**8. Therapeutic targeting of the IL-6/STAT3 pathway** 

transition (MET) allows for cellular reversion (Yang and Weinberg, 2008).

2008).

(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 (β)-catenin binding subdomain. β-catenin, a potent transcription factor, binds E-cadherin and alpha (α)-catenin subsequently binds β-catenin. Although contentious (Weis and Nelson, 2006), it is generally acknowledged that α-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 β-catenin at the cell surface as one means to inhibit β-catenin nuclear translocation and consequent expression of β-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 ERα-negative breast cancer cell line, lack E-cadherin, whereas MCF-7 cells, an ERα-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, β-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

(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 (β)-catenin binding subdomain. β-catenin, a potent transcription factor, binds E-cadherin and alpha (α)-catenin subsequently binds β-catenin. Although contentious (Weis and Nelson, 2006), it is generally acknowledged that α-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 β-catenin at the cell surface as one means to inhibit β-catenin nuclear translocation and consequent expression of β-catenin responsive

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 ERα-negative breast cancer cell line, lack E-cadherin, whereas MCF-7 cells, an ERα-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, β-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

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

genes (Perez-Moreno *et al.*, 2003).

secondary tissues (Kowalski *et al.*, 2003; Saha *et al.*, 2007).

*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 ERα-positive MCF-7 cells express E-cadherin, consistent with a characteristic epithelial phenotype, and are classified as luminal subtype, whereas highly invasive ERα-negative MDA-MB-231 cells lack E-cadherin and are classified as basal subtype (Blick *et al.*, 2008). Furthermore, ERα 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 ERα-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 ERα-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).
