El barrio: “la periferia de la periferia”

STAT3 is a major member of STAT family and one of the most established intracellular signalling molecules. It represents as a central effector of the local inflammatory response in cells and was first named acute phase response factor as it was reported to regulate the expression of many genes involved in the acute phase response to tissue injury and infection (Pensa et al., 2009). Since then, STAT3 has been found to be activated by tyrosine phosphorylation (pY705) in response to IL-6 and other inflammatory cytokines (Berishaj et al., 2007) (Figure 1.7). The activity of STAT3 is also controlled by phosphorylation at serine residue (pS727) within the transactivation domain of the protein by members of the mitogen-activated protein kinases (MAPK) and c-Jun N-terminal kinase families (Decker, 2000).

Several line of evidence from different human cell types and murine implement STAT3 as an oncogene because it up-regulate target gens involved in proliferation (cyclin D1, c-Myc, c-Fos and MEK5), angiogenesis (VEGF MMP-2, MMP-10, MMP-1 and KIF-8) and apoptosis (BCL-2, BCL-XL, MCL-1, and survivin), reflecting the role of STAT3 in cell cycle, cell invasion, angiogenesis and cell survival ( Niu et al., 2001;Buettner et al., 2002; Yu et al., 2004; Alvarez et al., 2005; Hsieh et al., 2005; Germain and Frank, 2007).

Early studies, in model systems, have shown a direct role of STAT3 in oncogenesis using a constitutively active STAT3 mutant, which transforms fibroblasts in culture and allows the transformed cells to form tumours (Bromberg et al., 1999). STAT3 inhibition is associated with anti-tumour effect and suppression of proliferation of tumour cells with activated STAT3 (Rivat et al., 2004; Gao et al., 2005; Xi et al., 2005), supporting direct contribution of STAT3 in tumours pathogenesis, rather than serving only as a marker of tumorigenesis. Inhibiting STAT3 in tumours can also induce apoptosis in addition to cell cycle arrest (Niu

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65 et al., 1999; Niu et al., 2001). In contrast, activated STAT3 in human multiple myeloma cell supported survival of tumour cells and prevented apoptosis by upregulating expression of the anti-apoptotic protein BCL-XL (Catlett-Falcone et al., 1999).

The role of STAT3 in cancer progression is further supported by its regulatory role on cell migration. STAT3 can affect the migration of tumour cells not only via its transcriptional regulation of genes involved in cell migration but also through its transcription- independent interaction with focal adhesion molecules. Phosphorylated STAT3 was found to localise to the migrating protrusions and focal adhesions in migrating cells (Silver et al., 2004; Jia et al., 2005). STAT3 also can inhibit the tubulin binding protein Stathmin which promotes microtubule depolymerisation (Ng et al., 2006).

Based on its role in regulating IL6-JAK/STAT3-dependent inflammation and immunity, studies have identified STAT3 as an important molecule in regulating immune responses in the tumour microenvironment (Catlett-Falcone et al., 1999; Yu et al., 2007; Grivennikov et al., 2009; Mantovani et al., 2008; Kortylewski et al., 2009; Wang et al., 2009; Yu et al., 2009). STAT3 is implicated in mediating pro-tumour immune responses and inducing pathways underlying cancer inflammation (Heinrich et al., 2003; Wang et al., 2009; Yu et al., 2009). It is also an important activator of many genes that are crucial for immunosuppression (Bronte-Tinkew et al., 2009; Ernst et al., 2008). STAT3 on the one hand promotes pro-oncogenic inflammatory pathways, including nuclear factor-κB (NF-κB) and IL-6-JAK pathways, while on the other hand opposes STAT1- and NF-κB-mediated TH1 anti-tumour immune responses (Yu et al., 2007; Yu et al., 2009).

In innate immune cells, STAT3 signalling is required for the immunosuppressive and tumour-promoting effects of myeloid-derived suppressor cells and tumour associated macrophages (Cheng et al., 2003; Kortylewski et al., 2005; Kortylewski et al., 2009).

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66 STAT3 also mediates T regulatory cell expansion, an important negative regulator of TH1- type CD4+ T-lymphocytes and CD8+ T-lymphocytes (Kortylewski et al., 2005; Matsumura et al., 2007; Kortylewski et al., 2009) in tumours and is central for the development of TH17 T-lymphocytes (Chen et al., 2006) which can promote tumour growth (Wang et al., 2009; Yu et al., 2009). Thus, STAT3 plays a critical role in determining the nature of cancer-associated inflammation.

STAT3 plays an important role in the normal development of the mammary gland. STAT3 mRNA levels are high in the mammary epithelium of adult virgins and remain elevated till early pregnancy (Philp et al., 1996, Watson, 2001). It is necessary for the cell death that occurs during the involution and remodelling process after cessation of lactation (Pensa et al., 2009). At weaning, an apoptotic program mediated by STAT3 is initiated to clear the mammary gland of its excess cellular burden. During the first phase of involution, levels of ph-STAT3 are elevated in the mammary gland, and this rapid activation of STAT3 is essential for involution to proceed (Chapman et al., 1999; Humphreys et al., 2002). During the second phase of involution, STAT3 induces an immune response and modulates macrophages and mast cells into an alternate state required for to clear epithelial cells (Hughes et al., 2012; Kreuzaler et al., 2011).

However, STAT3 activation can also promote breast cancer formation and progression (Burke et al., 2001; Garcia et al., 2001). Constitutive activation of STAT3 is required for enhancing transformation or blocking apoptosis in breast cancer cell lines and tissues (Bromberg and Darnell, 2000; Garcia et al., 1997; Page et al., 2000; Garcia et al., 2001). In mice model, constitutive activation of STAT3C allele could enhance the rat Neu oncogene tumourigenic activity and trigger development of earlier onset tumour with more aggressive features and metastatic potential than wild-type mice (Barbieri et al., 2010). STAT3 has also been shown to promote epithelial-to-mesenchymal transition and cell

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67 invasion in breast cancer tissue (Sullivan et al., 2009). In addition, inhibition of STAT3 with various pharmacological agents suppresses tumour growth, recurrence and invasion in breast cancer cell lines as well as in a human-xenograft model (Yang et al., 2012; Zhang et al., 2012).

In addition, STAT3 activation is frequently observed in breast tissue and more than 60% of primary tumours display constitutive pY705 STAT3 (Alvarez et al., 2005). Previous studies of ph-STAT3 in invasive breast cancers showed positive correlations between increased levels of ph-STAT3 expression and metastasis to regional lymph nodes (Hsieh et al 2005), Her-2 positivity, surviving, and incomplete response to neoadjuvant chemotherapy (Diaz et al., 2006); all are poor prognostic features. STAT3 is also serine phosphorylated in about 60% of breast tumours and is associated with ER-ve tumours, increased stage and increased tumour size (Yeh et al., 2006).

However, very little is known about the prognostic value of STAT3 in breast cancer, and this value remains controversial. Tissue microarray studies have reported significant relationship between ph-STAT3 and improved survival in 346 node negative breast cancers (Dolled-Filhart et al., 2003), 721 patients with low grade tumours (Sato et al., 2011), and 125 node positive breast cancers (Sonnenblick et al., 2012). In contrast, automated aassessment of ph-STAT3 levels in more than 900 specimens, found negative association between ph-STAT3 and survival (Charpin et al., 2009) consistent with findings of Sheen-Chen and colleagues (Sheen-Chen et al., 2008), whereas a study of 571 breast cancers documented no association between STAT3 expression and survival (Yamashita et al., 2006).

It is clear that conclusion about the prognostic value of STAT3 as well as STAT1 in breast cancer cannot be made due to rarity and inconsistency of evidence available. The

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68 mechanisms behind the relationship between STAT1 and STAT3 and cancer survival have yet to be clarified.

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