**6. Tumor- or carcinoma-associated fibroblasts: A bad myofibroblastic influence**

Fibroblasts are the main cellular component of the stroma and responsible for producing the mesenchymal (i.e., interstitial) ECM. These cells have been described as non-epithelial, noninflammatory and non-vascular semi-differentiated connective tissue cells (Tarin & Croft, 1969). They are best known for their role in maintaining the tissue's integrity while they become quickly activated (e.g., myofibroblastic) and can modify the plasticity of the resident's tissue under conditions that alter the homeostatic equilibrium such as during wound healing, organogenesis, cancer and other pathological and inflammatory conditions (Kalluri & Zeisberg, 2006) . In fact, fibroblasts are known as tissue remodelers capable of renovating ECMs while, at the same time, facilitating access to ECM stored growth factors, such as transforming growth factor-beta (TGF-b), through a tightly regulated release and activation of matrix digestive enzymes such as matrix metalloproteinases (MMPs) (Jodele et al., 2006).

The fibroblastic cell population, known as carcinoma-associated fibroblasts (CAFs) or tumor-associated fibroblasts (Barsky et al., 1984), presents a myofibroblastic phenotype that is very similar to the one observed in activated fibroblasts during wound healing (Barsky et al., 1984). CAFs are the main stromal cell component of solid epithelial carcinomas (Shao et al., 2000). In addition to a characteristic, high proliferation rate and increased ECM deposition, the development of contractile cell features affects the physico-chemical characteristics of TA-ECM (Tomasek et al., 2002; Butcher et al., 2009; Cukierman & Bassi, 2010). Interestingly, CAFs are capable of establishing interactions with inflammatory, endothelial, and tumor cells by means of cytokines/chemokines secretions such as interleukin (IL)-1, IL-6, CXCL-8, stromal derived factor-1 (SDF-1), also known as CXCL-12, and the monocyte chemotactic protein (MCPs/CCLs) among others (Silzle et al., 2004; Mishra et al., 2011). In an effort to find a discrete set of CAF specific markers, proteins such as - and -SMA (Brouty-Boye et al., 1991; Kunz-Schughart & Knuechel, 2002b; Desmouliere et al., 2004; Xouri & Christian, 2010) specific isoforms of the actin binding protein palladin, (Ronty et al., 2006; Goicoechea et al., 2010; Gupta et al., 2011) as well as the intermediate filament proteins vimentin and desmin (Schmid et al., 1982) have been suggested. Furthermore, the specific breast cancer microenvironmental niche has been shown to contain increased levels of expression of ECM stabilizing (e.g., cross-linking) enzymes such as prolyl-4 hydroxylase (Orimo et al., 2005) and lysyl oxidase (Chang et al., 2005; Levental et al., 2009; Barry-Hamilton et al., 2010). Additional proteins have been shown to be specifically overexpressed at the tumor-associated stroma such as fibroblast activation protein (LeBeau et al., 2009; Lee, 2011), endosialin (Becker et al., 2008; Christian et al., 2008) S100A4 (Ambartsumian et al., 1996; Ryan et al., 2003; Katoh et al., 2010), and a plethora of MMPs, among others (Rasanen & Vaheri, 2010). In fact, some of these have already been proposed to serve as stromal monitoring or prognostic markers (Erkan et al., 2008; Gupta et al., 2011).

Nevertheless, this hardly consistent signature of myofibroblastic markers strongly suggests that the tumor stroma is a heterogeneous milieu (Sugimoto et al., 2006). The variety of

activated fibroblasts influence the local microenvironment to promote invasion (Orimo et

Fibroblasts are the main cellular component of the stroma and responsible for producing the mesenchymal (i.e., interstitial) ECM. These cells have been described as non-epithelial, noninflammatory and non-vascular semi-differentiated connective tissue cells (Tarin & Croft, 1969). They are best known for their role in maintaining the tissue's integrity while they become quickly activated (e.g., myofibroblastic) and can modify the plasticity of the resident's tissue under conditions that alter the homeostatic equilibrium such as during wound healing, organogenesis, cancer and other pathological and inflammatory conditions (Kalluri & Zeisberg, 2006) . In fact, fibroblasts are known as tissue remodelers capable of renovating ECMs while, at the same time, facilitating access to ECM stored growth factors, such as transforming growth factor-beta (TGF-b), through a tightly regulated release and activation of matrix digestive enzymes such as matrix metalloproteinases (MMPs) (Jodele et

The fibroblastic cell population, known as carcinoma-associated fibroblasts (CAFs) or tumor-associated fibroblasts (Barsky et al., 1984), presents a myofibroblastic phenotype that is very similar to the one observed in activated fibroblasts during wound healing (Barsky et al., 1984). CAFs are the main stromal cell component of solid epithelial carcinomas (Shao et al., 2000). In addition to a characteristic, high proliferation rate and increased ECM deposition, the development of contractile cell features affects the physico-chemical characteristics of TA-ECM (Tomasek et al., 2002; Butcher et al., 2009; Cukierman & Bassi, 2010). Interestingly, CAFs are capable of establishing interactions with inflammatory, endothelial, and tumor cells by means of cytokines/chemokines secretions such as interleukin (IL)-1, IL-6, CXCL-8, stromal derived factor-1 (SDF-1), also known as CXCL-12, and the monocyte chemotactic protein (MCPs/CCLs) among others (Silzle et al., 2004; Mishra et al., 2011). In an effort to find a discrete set of CAF specific markers, proteins such as - and -SMA (Brouty-Boye et al., 1991; Kunz-Schughart & Knuechel, 2002b; Desmouliere et al., 2004; Xouri & Christian, 2010) specific isoforms of the actin binding protein palladin, (Ronty et al., 2006; Goicoechea et al., 2010; Gupta et al., 2011) as well as the intermediate filament proteins vimentin and desmin (Schmid et al., 1982) have been suggested. Furthermore, the specific breast cancer microenvironmental niche has been shown to contain increased levels of expression of ECM stabilizing (e.g., cross-linking) enzymes such as prolyl-4 hydroxylase (Orimo et al., 2005) and lysyl oxidase (Chang et al., 2005; Levental et al., 2009; Barry-Hamilton et al., 2010). Additional proteins have been shown to be specifically overexpressed at the tumor-associated stroma such as fibroblast activation protein (LeBeau et al., 2009; Lee, 2011), endosialin (Becker et al., 2008; Christian et al., 2008) S100A4 (Ambartsumian et al., 1996; Ryan et al., 2003; Katoh et al., 2010), and a plethora of MMPs, among others (Rasanen & Vaheri, 2010). In fact, some of these have already been proposed to serve as stromal monitoring or prognostic markers (Erkan et al., 2008; Gupta et

Nevertheless, this hardly consistent signature of myofibroblastic markers strongly suggests that the tumor stroma is a heterogeneous milieu (Sugimoto et al., 2006). The variety of

**6. Tumor- or carcinoma-associated fibroblasts: A bad myofibroblastic** 

al., 2005; Hu et al., 2008).

**influence** 

al., 2006).

al., 2011).

myofibroblastic phenotypes is also suggestive of the eliciting of different roles played by these cell populations at the tumor stroma. Interestingly, this heterogeneity could have been originated (i.e., differentiated) by the multiple cell lineages known to produce myofibroblastic CAFs. These are: local fibroblasts (Kalluri & Zeisberg, 2006), bone marrow recruited mesenchymal cells (Ishii et al., 2003; Goldstein et al., 2010), as well as endothelial and tumor (i.e., epithelial) cells (Petersen et al., 2003; Kalluri & Zeisberg, 2006; Zeisberg et al., 2007), among others. In all these cases, TGF-β has been closely associated with tumorinduced myofibroblastic activation or differentiation (Zeisberg et al., 2007; Hinz, 2010; Taylor et al., 2010). The myofibroblastic differentiation is a complex and not yet fully understood process that is believed to play a central role during breast tumorigenesis (Cukierman, 2004; McAllister & Weinberg, 2010). Even though a plethora of molecules has been implicated in regulating fibroblastic activation, the specific desmoplastic response in breast cancer is believed to be driven by four main groups of inducers; i) growth factors, ii) TA-ECM, iii) acute inflammation and iv) microenvironmental stress denoted by nutrient and oxygen depravation as well as low pH.


Novel Insights Into the Role of Inflammation in Promoting Breast Cancer Development 143

iv. It is well known that as tumors progress increased regions of nutrient deprivation, low pH and low oxygen tension (hypoxia) are evident. Under these hypoxic stress conditions, breast cancer tissues are known to up-regulate the expression of hypoxiainducible family (HIF) genes such as HIF-1 (Chen, C.L. et al., 2010). HIF proteins are known to participate in many cellular events such as angiogenesis, through the induction of vascular endothelium growth factor (VEGF), angiopoietin-2, PDFG and FGF (Allen & Louise Jones, 2011) which in turn can also activate stromal myofibroblastic differentiation in breast cancers (Shao et al., 2000). Finally, other molecules known to be induced by HIF-1 in carcinomas (and other fibrotic conditions) are the above mentioned ECM-cross-linkers (i.e., LOX) which have been associated with aggressive breast tumorigenesis (Chang et al., 2005; Levental et al., 2009; Barry-

**7. Fibroblasts as moderators of signals at the tumor microenvironment** 

At the tumor microenvironment, intercellular communications resemble a social network emitting signals (either static or diffusible molecules) that in turn are collected, processed and emitted to additional cells. Using this analogy, it seems that CAFs play a decisive role during cancer progression acting as microenvironment signals moderators that sense extracellular signals and, after intracellular processing, emit new ones that in turn modulate both stromal and neoplastic neighbouring cells' behaviours (Bhowmick et al., 2004). In fact during cancer progression, CAFs constitute a very important source of the exogenous stimulants such as the above-mentioned TGF- (Kalluri & Zeisberg, 2006). To this end, using an elegant humanized stromal reconstruction model of human breast cancer in mouse, Kuperwasser *et al* demonstrated that CAFs facilitate tumor development in a fibroblastic TGF-- and HGF-dependent manner (Kuperwasser et al., 2004). Additionally, recent findings have demonstrated that epigenetic changes induced by mesenchymal cells on breast cancer cells that are regulated by the TGF-/TGF- R/Smad2 signalling axis provoke the silencing of critical epithelial genes resulting in the pro-tumorigenic EMT process (Papageorgis et al., 2010). To this end, in support of the above proposed vicious cycle effect, it is interesting to note that following quiescent fibroblasts transdifferentiation into CAFs, these cells support an invasive phenotype of mammary carcinomas where they secrete inflammatory cytokines (Powell et al., 1999; Buckley et al., 2001; Silzle et al., 2004) thus activating NF-b and promoting EMT as well as promoting aggressiveness of breast cancer cells (Sullivan et al., 2009; Wu et al., 2009). An ever more complicated interplay between CAFs, cytokines and neoplastic cells has recently been proposed in breast cancers where,

tumor free survival rate (Toullec et al., 2010).

Hamilton et al., 2010).

supporting tumor progression (Yang et al., 2010). In addition, this same factor plays an additional important stromal role in inducing the expression of NADPH oxidase family protein, Nox4 (Bondi et al., 2010). Nox4 is a potent regulator of reactive oxygen species (ROS) (Barnes & Gorin, 2011) and has been shown to induce the accumulation of ROS in damaged tissues while transactivation of fibroblasts into myofibroblasts (Cucoranu et al., 2005; Rocic & Lucchesi, 2005). In breast cancer, the oxidative stress present at the tumor stroma is also considered to be an inductor for myofibroblastic differentiation, as recently shown in a JunD deficient mouse model, where the absence of this transcription factor allowed the accumulation of Ras-mediated production of ROS with the subsequent conversion of fibroblasts into myofibroblasts and shortening of the

that as the substrate stiffness increases, fibroblastic cells change exhibiting three discrete phenotypic switch stages: normal or naive fibroblasts, intermediate or protomyofibroblastic and activated myofibroblastic (Hinz, 2010). The phenotype transition induced by the increased tension in the substratum is also accompanied by the maturation or elongation of focal adhesions, together with cytoskeletal changes known to build-up contractile stress fibers (Hinz, 2010). Interestingly, studies of normal breast revealed a relatively limp tissue composition (0.15 kPa, expressed in *E* values of a Young modulus) compared to the stiffer and highly desmoplastic ~4 kPa tissue that has been affected by breast cancer (Butcher et al., 2009). The altered (i.e., myofibroblastic) phenotype of fibroblasts is linked to the stiffer ECM during tumor progression as these cells are responsible for the production of the TA-ECM (Cukierman & Bassi, 2010). Indeed increments of mammographic density, suggesting excessive collagen deposition, have been associated with higher risk in breast cancer (Boyd et al., 1998). Moreover, increases in cross-linked collagen due to over expression of LOX together with patterned linearization of the TA-ECM and specific ECM receptor, integrin, clustering and enhanced phosphoinositide 3-kinase (PI3K) activity, have all been correlated with breast cancer progression (Levental et al., 2009). Additionally, it has been shown that the interstitial ECM can function as a reservoir for diffusible molecules, such as the above-mentioned TGF- which is secreted by both stromal and tumor cells in its inactivated form (Wipff & Hinz, 2008), but can be both activated and released due to the intrinsic myofibroblastic forces that increase the tension of TA-ECM's fibrils (Wipff et al., 2007; Tenney & Discher, 2009).

iii. Recently, an inflammatory microenvironment has been suggested as the seventh hallmark of cancer (Colotta et al., 2009). This cancer hallmark is also believed to play an important role in desmoplasia as a fibroblast phenotypic-switch activator. To this end, it has been demonstrated that stromal inflammatory responses that result from wounding can trigger tumorigenesis (Arwert et al., 2010). The importance of an inflammatory component has also been suggested for the breast cancer stroma (Hu & Polyak, 2008), and its repercussion in inducing or promoting cancer aggressiveness and metastasis has been highlighted in numerous occasions (Pantschenko et al., 2003; Elaraj et al., 2006; Valdivia-Silva et al., 2009; Franco-Barraza et al., 2010; Goldberg & Schwertfeger, 2010). However, our current knowledge regarding fibroblastic responses to inflammatory cytokines in breast cancer remains relatively modest. Work conducted at the Polyak laboratory suggested that cytokines could participate in triggering a fibroblast phenotypic switch at the breast cancer microenvironment (Hu et al., 2009). This work and the work of others has opened up the possibility of targeting inflammatory cytokines for the treatment of neoplasias as in the case of COX-2 and arachidonic acid inhibitors (Chen, X. et al., 2006; Hu et al., 2009). In fact, in the kidney, it has been shown that collagen I regulates COX-2 expression in a pro-proliferative type of response (Alique et al., 2011). Interestingly, CAFs are known to promote inflammation in an NF b dependent manner, suggesting a vicious cycle between inflammation and stromal activation during tumorigenesis (Erez et al., 2010). Moreover, it has been shown that CAFs effectively suppress anti-tumor inflammation while, at the same time, maintaining acute inflammatory (pro-tumor) conditions (Kraman et al., 2010).

As established before, the cytokine/growth factor TGF- imparts a pleiotropic and decisive role in the promotion of the desmoplastic tumor microenvironment thus

ECM's fibrils (Wipff et al., 2007; Tenney & Discher, 2009).

iii. Recently, an inflammatory microenvironment has been suggested as the seventh hallmark of cancer (Colotta et al., 2009). This cancer hallmark is also believed to play an important role in desmoplasia as a fibroblast phenotypic-switch activator. To this end, it has been demonstrated that stromal inflammatory responses that result from wounding can trigger tumorigenesis (Arwert et al., 2010). The importance of an inflammatory component has also been suggested for the breast cancer stroma (Hu & Polyak, 2008), and its repercussion in inducing or promoting cancer aggressiveness and metastasis has been highlighted in numerous occasions (Pantschenko et al., 2003; Elaraj et al., 2006; Valdivia-Silva et al., 2009; Franco-Barraza et al., 2010; Goldberg & Schwertfeger, 2010). However, our current knowledge regarding fibroblastic responses to inflammatory cytokines in breast cancer remains relatively modest. Work conducted at the Polyak laboratory suggested that cytokines could participate in triggering a fibroblast phenotypic switch at the breast cancer microenvironment (Hu et al., 2009). This work and the work of others has opened up the possibility of targeting inflammatory cytokines for the treatment of neoplasias as in the case of COX-2 and arachidonic acid inhibitors (Chen, X. et al., 2006; Hu et al., 2009). In fact, in the kidney, it has been shown that collagen I regulates COX-2 expression in a pro-proliferative type of response (Alique et al., 2011). Interestingly, CAFs are known to promote inflammation in an NF b dependent manner, suggesting a vicious cycle between inflammation and stromal activation during tumorigenesis (Erez et al., 2010). Moreover, it has been shown that CAFs effectively suppress anti-tumor inflammation while, at the same time,

maintaining acute inflammatory (pro-tumor) conditions (Kraman et al., 2010).

As established before, the cytokine/growth factor TGF- imparts a pleiotropic and decisive role in the promotion of the desmoplastic tumor microenvironment thus

that as the substrate stiffness increases, fibroblastic cells change exhibiting three discrete phenotypic switch stages: normal or naive fibroblasts, intermediate or protomyofibroblastic and activated myofibroblastic (Hinz, 2010). The phenotype transition induced by the increased tension in the substratum is also accompanied by the maturation or elongation of focal adhesions, together with cytoskeletal changes known to build-up contractile stress fibers (Hinz, 2010). Interestingly, studies of normal breast revealed a relatively limp tissue composition (0.15 kPa, expressed in *E* values of a Young modulus) compared to the stiffer and highly desmoplastic ~4 kPa tissue that has been affected by breast cancer (Butcher et al., 2009). The altered (i.e., myofibroblastic) phenotype of fibroblasts is linked to the stiffer ECM during tumor progression as these cells are responsible for the production of the TA-ECM (Cukierman & Bassi, 2010). Indeed increments of mammographic density, suggesting excessive collagen deposition, have been associated with higher risk in breast cancer (Boyd et al., 1998). Moreover, increases in cross-linked collagen due to over expression of LOX together with patterned linearization of the TA-ECM and specific ECM receptor, integrin, clustering and enhanced phosphoinositide 3-kinase (PI3K) activity, have all been correlated with breast cancer progression (Levental et al., 2009). Additionally, it has been shown that the interstitial ECM can function as a reservoir for diffusible molecules, such as the above-mentioned TGF- which is secreted by both stromal and tumor cells in its inactivated form (Wipff & Hinz, 2008), but can be both activated and released due to the intrinsic myofibroblastic forces that increase the tension of TA-

supporting tumor progression (Yang et al., 2010). In addition, this same factor plays an additional important stromal role in inducing the expression of NADPH oxidase family protein, Nox4 (Bondi et al., 2010). Nox4 is a potent regulator of reactive oxygen species (ROS) (Barnes & Gorin, 2011) and has been shown to induce the accumulation of ROS in damaged tissues while transactivation of fibroblasts into myofibroblasts (Cucoranu et al., 2005; Rocic & Lucchesi, 2005). In breast cancer, the oxidative stress present at the tumor stroma is also considered to be an inductor for myofibroblastic differentiation, as recently shown in a JunD deficient mouse model, where the absence of this transcription factor allowed the accumulation of Ras-mediated production of ROS with the subsequent conversion of fibroblasts into myofibroblasts and shortening of the tumor free survival rate (Toullec et al., 2010).

iv. It is well known that as tumors progress increased regions of nutrient deprivation, low pH and low oxygen tension (hypoxia) are evident. Under these hypoxic stress conditions, breast cancer tissues are known to up-regulate the expression of hypoxiainducible family (HIF) genes such as HIF-1 (Chen, C.L. et al., 2010). HIF proteins are known to participate in many cellular events such as angiogenesis, through the induction of vascular endothelium growth factor (VEGF), angiopoietin-2, PDFG and FGF (Allen & Louise Jones, 2011) which in turn can also activate stromal myofibroblastic differentiation in breast cancers (Shao et al., 2000). Finally, other molecules known to be induced by HIF-1 in carcinomas (and other fibrotic conditions) are the above mentioned ECM-cross-linkers (i.e., LOX) which have been associated with aggressive breast tumorigenesis (Chang et al., 2005; Levental et al., 2009; Barry-Hamilton et al., 2010).

### **7. Fibroblasts as moderators of signals at the tumor microenvironment**

At the tumor microenvironment, intercellular communications resemble a social network emitting signals (either static or diffusible molecules) that in turn are collected, processed and emitted to additional cells. Using this analogy, it seems that CAFs play a decisive role during cancer progression acting as microenvironment signals moderators that sense extracellular signals and, after intracellular processing, emit new ones that in turn modulate both stromal and neoplastic neighbouring cells' behaviours (Bhowmick et al., 2004). In fact during cancer progression, CAFs constitute a very important source of the exogenous stimulants such as the above-mentioned TGF- (Kalluri & Zeisberg, 2006). To this end, using an elegant humanized stromal reconstruction model of human breast cancer in mouse, Kuperwasser *et al* demonstrated that CAFs facilitate tumor development in a fibroblastic TGF-- and HGF-dependent manner (Kuperwasser et al., 2004). Additionally, recent findings have demonstrated that epigenetic changes induced by mesenchymal cells on breast cancer cells that are regulated by the TGF-/TGF- R/Smad2 signalling axis provoke the silencing of critical epithelial genes resulting in the pro-tumorigenic EMT process (Papageorgis et al., 2010). To this end, in support of the above proposed vicious cycle effect, it is interesting to note that following quiescent fibroblasts transdifferentiation into CAFs, these cells support an invasive phenotype of mammary carcinomas where they secrete inflammatory cytokines (Powell et al., 1999; Buckley et al., 2001; Silzle et al., 2004) thus activating NF-b and promoting EMT as well as promoting aggressiveness of breast cancer cells (Sullivan et al., 2009; Wu et al., 2009). An ever more complicated interplay between CAFs, cytokines and neoplastic cells has recently been proposed in breast cancers where,

Novel Insights Into the Role of Inflammation in Promoting Breast Cancer Development 145

Tang, 2008), paclitaxel delivered through nanoparticles conjugated to albumin (nabpaclitaxel) are being tested (Vishnu & Roy, 2010; Robert et al., 2011; Volk et al., 2011). Moreover inhibition of the serine protease activity of the CAF specific fibroblast activation protein has been suggested as a therapeutic target in a plethora of cancers including breast (Mersmann et al., 2001). In fact, antibodies against fibroblast activation protein induced a marked decrease in desmoplastic collagen I expression resulting in an increased (up to 70%) increment in chemotherapeutic drugs uptake (Loeffler et al., 2006). Therefore, it is not surprising that fibroblast activation protein has been suggested as a tumor targeting molecule for the delivery of peptide protoxins (amongst others) thus diminishing non-

Pro-inflammatory molecules have also been used as effective targets. For example TNF antagonists have been shown to have good results preventing disease acceleration in a considerable number of breast cancer patients (Madhusudan et al., 2004; Brown et al., 2008). The SDF-1/CXCR4 chemokine axis has been proposed as a general target for anticancer strategies (Guleng et al., 2005), and recently a compound derived from marine organisms that blocks CXCR4 has been shown effective as well (He et al., 2008). Antibodies blocking the TGF- signalling pathway have been developed and showed promising synergistic effects when added to known chemotherapeutics and, thus, have been regarded as anti angiogenesis-depending tumor stromal agents in breast cancer (Takahashi et al., 2001). Finally, it was recently shown that eliminating pro-tumorigenic macrophages in pancreas causes desmoplastic shrinkage and subsequent tumor stalling (Beatty et al., 2011). We believe that these types of treatments, together with similar novel ones, could provide

A DGAPA Grant IN214611-2 from Universidad Nacional Autonoma de Mexico to EGZ and

Adams, M.;Jones, J.L.;Walker, R.A.;Pringle, J.H. & Bell, S.C. (2002). Changes in tenascin-C

Ali, S. & Lazennec, G. (2007). Chemokines: novel targets for breast cancer metastasis. Cancer

Alique, M.;Calleros, L.;Luengo, A.;Griera, M.;Iniguez, M.A.;Punzon, C.;Fresno,

cells. Am J Physiol Cell Physiol, Vol.300, No.4, pp. C907-918, ISSN 1522-1563 Allavena, P.; Sica, A.; Solinas, G.; Porta, C. & Mantovani, A. (2008). The inflammatory micro-

Critical reviews in oncology/hematology, Vol. 66, No. 1, pp. 1-9, 1040-8428 Allen, M. & Louise Jones, J. (2011). Jekyll and Hyde: the role of the microenvironment on the

isoform expression in invasive and preinvasive breast disease. Cancer research,

M.;Rodriguez-Puyol, M. & Rodriguez-Puyol, D. (2011). Changes in extracellular matrix composition regulate cyclooxygenase-2 expression in human mesangial

environment in tumor progression: The role of tumor-associated macrophages.

progression of cancer. The Journal of pathology, Vol.223, No.2, pp. 162-176, ISSN

tumoral side effect toxicities (LeBeau et al., 2009).

increased hope in the common fight against breast cancers.

CA113451 from NIH/NCI to EC and JFB supported this work.

Vol.62, No.11, pp. 3289-3297, ISSN 0008-5472

metastasis reviews, Vol. 26, No. pp. 401-420,

**9. Acknowledgment** 

1096-9896

**10. References** 

due to the presence of an altered TA-ECM, an integrin-dependent activation of Src family kinases results in the increase of NF-B activity which blocks the production of certain microRNAs such as Let-7. Under these conditions, IL-6 production is promoted resulting in the increased secretion of this pro-tumorigenic cytokine, which in turn induces or promotes a positive feedback in tumor cells (Iliopoulos et al., 2009). Moreover, activated myofibroblastic and cancer cells are known to remodel the stromal ECM by means of increased secretion of MMPs and urokinase-type plasminogen activator (uPA). These enzymes cleave the ECM molecules to release fragments that contain chemotactic properties called matrikines that activate leukocytes to also release inflammatory cytokines (Maquart et al., 2004; Silzle et al., 2004). For example, a special feature of MMP-2, -3 and -9 is that these proteases can increase the availability of IL-1b at the tumor microenvironment by cleavage of the pIL-1b (immature IL-1b) (Schonbeck et al., 1998). Also, analyses of co-cultures containing both breast cancer cells and CAFs have shown increases in stromal MMP-2 and MMP-9 expression (Singer et al., 2002). These observations concur with observations stemming from an immunohistochemical study where tissue arrays of breast cancer patients showed that intratumor stromal fibroblasts express MMP-2, -7, and -14, while fibroblast at the invasive front highly express MMP-9. What is more, this specific profile of stromal MMPs staining was found to be a predictor of future distant metastases occurrences (Del Casar et al., 2009). Another uncovered effect of released MMPs into the tumor stroma is the capacity of these molecules to promote a permissive environment that supports epithelial tumorigenic progression including the promotion of genomic alterations (Radisky, E.S. & Radisky, 2007). In the mammary glands of transgenic mice, the overexpression of MMP-3 has been shown to be sufficient to stimulate myofibroblastic presence, increased fibrosis, epithelial hyperplasia, and development of mammary carcinoma (Thomasset et al., 1998). What is more, mammary epithelial cells exposed to stromal MMP-3 showed activation of a genotoxic metabolic pathway, where the over expression of the spliced variant Rac1b produced DNA-damaging superoxide radicals and induced EMT (Radisky, D.C. et al., 2005). Interestingly, the epithelial genomic alterations induced by stromal MMPs *in vitro,* suggest a possible mechanism to understand the presence of areas with genomic imbalance patterns detected in histologically normal tissues adjacent to the tumor stroma (Ellsworth et al., 2004; Holliday et al., 2009).

#### **8. Targeting fibroblasts as an anti-cancer therapy**

Various aspects of the tumor microenvironment have been explored as putative therapeutic targets in the fight against cancer (Andre et al., 2010; Cukierman & Khan, 2010; Allen & Louise Jones, 2011). Since a desmoplastic reaction is an ECM component-rich substratum and some of the TA-ECM components are believed to be specific for discrete types of carcinomas, they constitute a promising basis for therapeutics (i.e., inhibitory functional antibodies). For example, in glioblastoma patients an iodine-131 radiolabeled anti-tenascin-C monoclonal antibody has produced encouraging results in phase II trials (Reardon et al., 2006). Similarly, the development of radioactive or bioactive molecules coupled to antibodies against TA-ECM specific EDB, the L-19 antibody, showed encouraging results when tested in various carcinomas (Kaspar et al., 2006). The TA-ECM has been considered as both a target as well as a means to attract anti-tumoral drugs. For example, as albumin binds efficiently to the TA-ECM protein osteonectin (also known as SPARC), known to be upregulated in a plethora of cancer stromas and often associated with bad prognosis (Tai & Tang, 2008), paclitaxel delivered through nanoparticles conjugated to albumin (nabpaclitaxel) are being tested (Vishnu & Roy, 2010; Robert et al., 2011; Volk et al., 2011). Moreover inhibition of the serine protease activity of the CAF specific fibroblast activation protein has been suggested as a therapeutic target in a plethora of cancers including breast (Mersmann et al., 2001). In fact, antibodies against fibroblast activation protein induced a marked decrease in desmoplastic collagen I expression resulting in an increased (up to 70%) increment in chemotherapeutic drugs uptake (Loeffler et al., 2006). Therefore, it is not surprising that fibroblast activation protein has been suggested as a tumor targeting molecule for the delivery of peptide protoxins (amongst others) thus diminishing nontumoral side effect toxicities (LeBeau et al., 2009).

Pro-inflammatory molecules have also been used as effective targets. For example TNF antagonists have been shown to have good results preventing disease acceleration in a considerable number of breast cancer patients (Madhusudan et al., 2004; Brown et al., 2008). The SDF-1/CXCR4 chemokine axis has been proposed as a general target for anticancer strategies (Guleng et al., 2005), and recently a compound derived from marine organisms that blocks CXCR4 has been shown effective as well (He et al., 2008). Antibodies blocking the TGF- signalling pathway have been developed and showed promising synergistic effects when added to known chemotherapeutics and, thus, have been regarded as anti angiogenesis-depending tumor stromal agents in breast cancer (Takahashi et al., 2001). Finally, it was recently shown that eliminating pro-tumorigenic macrophages in pancreas causes desmoplastic shrinkage and subsequent tumor stalling (Beatty et al., 2011). We believe that these types of treatments, together with similar novel ones, could provide increased hope in the common fight against breast cancers.
