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The Role of the Microenvironment in Mammary Gland Development and Cancer

Kornelia Polyak1,2,3 and Raghu Kalluri4,5

1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115 2Department of Medicine, Brigham and Women’s Hospital, Boston, Massachusetts 02115 3Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115 4Division of Matrix Biology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115 5Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 Correspondence: [email protected]

The mammary gland is composed of a diverse arrayof cell types that form intricate interaction networks essential for its normal development and physiologic function. Abnormalities in these interactions play an important role throughout different stages of tumorigenesis. Branching ducts and alveoli are lined by an inner layer of secretory luminal epithelial cells that produce milk during lactation and are surrounded by contractile myoepithelial cells and basement membrane. The surrounding stroma comprised of extracellular matrix and various cell types including fibroblasts, endothelial cells, and infiltrating leukocytes not only provides a scaffold for the organ, but also regulates mammary epithelial cell function via paracrine, physical, and hormonal interactions. With rare exceptions breast tumors initiate in the epithelial compartment and in their initial phases are confined to the ducts but this barrier brakes down with invasive progression because of a combination of signals emitted by tumor epithelial and various stromal cells. In this article, we overview the impor- tance of cellular interactions and microenvironmental signals in mammary gland develop- ment and cancer.

he mammary gland is composed of a combi- microenvironment composed of extracellular Tnation of multiple cell types that together matrix (ECM) and various stromal cell types form complex interaction networks required (e.g., endothelial cells, fibroblasts, myofibro- for the proper development and functioning of blasts, and leukocytes). Large amount of data the organ. The branching milk ducts are formed suggest that cell-cell and cell-microenvironment by an outer myoepithelial cell layer producing interactions modify the proliferation, survival, the basement membrane (BM) and an inner polarity, differentiation, and invasive capacity luminal epithelial cell layer producing milk dur- of mammary epithelial cells. However, the mol- ing lactation. The ducts are surrounded by the ecular mechanisms underlying these effects are

Editors: Mina J. Bissell, Kornelia Polyak, and Jeffrey Rosen Additional Perspectives on Mammary Gland Biology available at www.cshperspectives.org Copyright # 2010 Cold Spring Harbor Laboratory Press; all rights reserved. Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a003244

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K. Polyak and R. Kalluri

poorly understood. The purification and com- differentiation of epithelial cells, and mammary prehensive characterization of each cell type epithelial cells form salivary gland–like struc- comprising normal and neoplastic human tures when placed on top of salivary gland breast tissue combined with hypothesis testing mesenchyme (Sakakura et al. 1979). This in cell culture and animal models are likely to differentiation-inducing effect of embryonic improve our understanding of the role these mesenchyme is so pronounced that it is able cells play in the normal functioning of the mam- to alter the phenotype of mammary carcinoma mary gland and in breast tumorigenesis. In this cells to a more benign differentiated state (De- article, we overview cellular and microenviron- Cosse et al. 1973, 1975). This could potentially mental interactions that play important roles be explained by the up-regulation of embryonic in the normal functioning of the mammary programs in the tumor cells and then their nor- gland and their abnormalities in breast cancer. malization in response to mesenchymal-derived differentiation inducing signals. Indeed, more recent studies have shown that the embryonic THE ROLE OF THE MICROENVIRONMENT IN morphogen Nodal is overexpressed in highly MAMMARY GLAND DEVELOPMENT AND metastatic breast cancer cells and in melano- FUNCTION mas. Nodal expression, and consequently the Contrary to that of most organs, the develop- invasive phenotype of the cancer cells, can be ment of the mammary gland mostly occurs down-regulated by placing the cells into human postnatally and it is only completed in adult- embryonic stem cell (hESC) microenvironment hood and some aspects of mammary epithelial (Postovit et al. 2008). cell differentiation even require the completion The importance of propercellularcontext for of a full-term pregnancy, lactation, and involu- normal mammary epithelial cell differentiation tion cycle. The mammary gland is also unique was also shown by the observation that cells as it is continuously remodeled following pub- plated on plastic tissue culture dishes displayed erty because of the cyclical influence of repro- very different phenotype and were not able to ductive hormones. Most of our knowledge of differentiate into milk producing mammary mammary gland development has been derived epithelium in contrast to cells plated in three- from observations made in mice and interpo- dimensional (3D) reconstituted basement lated for humans despite the well-known differ- membrane (Matrigel) cultures (Petersen et al. ences between human and mouse mammary 1992; Howlett and Bissell 1993). More recent gland development and function. Studies ad- follow up studies have shown that transition dressing human mammary gland development from monolayer to 3D cultures dramatically in- have been limited to the structural and immu- fluences the epigenetic programs of the cells, par- nohistochemical analyses of a limited number ticularly histone modification patterns (Le Beyec of samples collected at different stages of fetal, et al. 2007). Specifically, plating mammary epi- infantile, childhood, and pubertal development thelial cells in laminin-rich extracellular matrix (Anbazhagan et al. 1998; Osin et al. 1998; Nac- (Matrigel) induces rapid deacetylation of his- carato et al. 2000; Jolicoeur et al. 2003). tones H3 and H4 at the global level accompa- The mammary gland is derived from the nied with decreased overall transcription. ectoderm and in the human embryo the breast However, in the normal mammary gland bud arises as a result of proliferation of basal the majority of luminal epithelial cells are not cells of the epidermis because of factors secreted in direct contact with the basement membrane, by mesenchymal cells present in the breast bud but rather lay on top of myoepithelial cells (Anbazhagan et al. 1998). Mammary epithelial (Fig. 1). Myoepithelial cells influence the differ- cells remain responsive to signals emitted by entiation, polarity, proliferation, and invasion/ embryonic mesenchyme even to adulthood, migration of adjacent luminal epithelial cells. but only in nulliparous mice. In fact, signals They also produce the basement membrane emitted by embryonic mesenchyme dictate the (BM) that forms a physical barrier separating

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Leukocytes

Fibroblasts

Myoepithelial cell

Luminal epithelial cell

Endothelial cells

Figure 1. Schematic view of a normal mammary duct and lobule. Black line indicates the basement membrane. In the ducts myoepithelial cells form a nearly complete layer around the luminal epithelial cells, whereas in the alveoli the myoepithelial layer is more fenestrated and some luminal epithelial cells can be in direct contact with the basement membrane. Yellow arrows indicate potential cell–cell and cell–matrix interactions.

the epithelial and stromal compartments. Lu- basement membrane molecules such as laminin minal epithelial cells display apical-basal polar- 5 (Zahir et al. 2003). These results suggest that ity as shown by the localization of sialomucin cells that are embedded in 3D environment can (MUC1), epithelial specific antigen (ESA), and produce unique BM components that some- occludin on the apical membrane and integrin times resemble the in vivo conditions. b4 on the basolateral membrane. This polarity Besides myoepithelial cells, the effects of is observed in cell culture when luminal epi- numerous other stromal cell types have also thelial cells are grown in Matrigel but not in been tested in cell culture assays. For example, collagen I (Gudjonsson et al. 2002). However, co-culture of endothelial cells with MCF-7 coculture with normal myoepithelial cells re- breast cancer cells leads to apoptosis of endothe- stores luminal epithelial cell polarity even in lial cells and such apoptosis requires cell–cell collagen cultures and this is at least in part contacts. Although the functional impact of mediated by laminin-1 secreted by the myoepi- such studies remains to be elucidated, these thelial cells. Other studies have suggested that experiments suggest that cancer cells can impact 3D culture of luminal epithelial cells in Matri- the integrity of the tumor endothelium (Kebers gel can induce the production of non-Matrigel et al. 1998). Coculture studies using different

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K. Polyak and R. Kalluri

mesenchymal cells and MCF10A and preneo- long noted the prognostic value of certain his- plastic MCF10AT1-EIII8 mammary epithelial topathological features of breast tumors, in- cells showed that fibroblasts derived from nor- cluding lymphocytic infiltration, fibrosis, and mal reduction mammoplasty inhibit or retard angio- and lymphangiogenesis (Fig. 2). The the morphological conversion and growth of contribution of host factors as shown by dif- MCF10A and EIII8 cells, whereas tumor derived ferences in tumor initiation, progression, and fibroblasts evoke ductal-alveolar morphogene- angiogenesis depending on variability in germ- sis of both cell types (Shekhar et al. 2001). line genotypes also support a role for nonepi- Recently, Caveolin-1 deficient (Cav1-/-) mam- thelial cells in carcinogenesis (Rohan et al. mary stromal fibroblasts were shown to mimic 2000; Hunter 2004). In fact, cancer is a systemic the effects of human breast cancer associated disease involving the whole body, because tu- fibroblasts as they show similar profile of RB/ mor cells perturb the homeostasis of multiple E2F-regulated genes that are up-regulated and organs not only that of those where they reside. confer a poor prognosis with enhanced epi- For example, the hematopoietic system and thelial-mesenchymal transition (EMT) (Sotgia blood clotting cascade appear to be different et al. 2009). between normal individuals and cancer patients (Bluff et al. 2008; Psaila and Lyden 2009). Fur- thermore, primary tumors can influence (pro- ABNORMALITIES IN THE mote or inhibit depending on the specific MICROENVIRONMENT DURING tumor) the growth of cancer cells at distant TUMOR PROGRESSION metastatic sites via hormonal factors (Fig. 3). Early studies showed that the normal mammary This was shown in a xenograft model whereas microenvironment is capable of “reverting” the MDA-MB-231 cells injected into one side of malignant phenotype of breast cancer cells by the mouse were able to “instigate” the growth inducing a more differentiated state (DeCosse of a less tumorigenic cell line injected to the et al. 1973, 1975), suggesting that cancer cells other side (McAllister et al. 2008). can only thrive in an abnormal environment Because metastases are responsible for in which they evolved. Pathologists have also most cancer-related deaths, understanding the

Normal DCIS IDC Metastatic

Leukocytes Myoepithelial cell Cancer cell Cells of distant organs

Fibroblasts Luminal epithelial cell Myofibroblast Endothelial cells

Figure 2. Microenvironmental alterations during tumor progression. Schematic view of normal breast, DCIS (ductal carcinoma in situ), IDC (invasive ductal carcinoma), and metastatic breast carcinoma. Normal breast ducts are composed of the basement membrane and an inner layer of luminal epithelial sitting on an outer layer of myoepithelial cells. Cells composing the stroma include various leukocytes, fibroblasts, myofibroblasts, and endothelial cells. In DCIS, the myoepithelial cells are epigenetically and phenotypically altered and their number decreases potentially because of signals coming from the tumor epithelial and various stromal cells. The number of stromal fibroblasts, myofibroblasts, lymphocytes, and endothelial cells increases as tumors progress, although the degree of this is tumor subtype-specific.

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Primary invasive Distant metastases

breast tumor 1 4 2 3 16 9 6 5 10 4 9 1 7 10 18 17 10 8 11 12 12 13 14 20 15 19 21

22

“self-seeding” “premetastatic niche”

Figure 3. Systemic effects of breast tumors. Tumor cells shed into the circulation and migrate to various organs. At the same time signals emitted by cells within the primary tumor mobilize bone marrow-derived mesenchymal stem cells (BMD-MSCs). These BMD-MSCs are the recruited to the primary tumor and also to other organs such as lungs and brain creating a premetastatic niche prior to the arrival of the disseminated cancer cells. The interaction between cancer cells and BMD-MSCs within the primary tumor enhances the growth, survival, motility, invasive and metastatic capacity of tumor cells via paracrine interactions. In addition, BMD-MSCs can differentiate into myofibroblasts and other stromal cell types that further support the growth and progression of the tumor. Disseminated cancer cells preferentially grow at sites where BMD- MSCs are localized forming distant metastases. Disseminated tumor cells can also return to the original primary tumor and promote its growth and further metastatic spread in a process called “self-seeding.”

systemic effects of tumors and factors that reg- lung cancer cells resulted in preferential me- ulate the ability of disseminated cancer cells tastasis formation at sites favored by mela- to grow at distant sites are of primary impor- noma and not by lung tumors (Kaplan et al. tance. In line with the “seed and soil” model 2005). of metastasis formation (Fidler 2003), the con- A related observation was described by cept of “premetastatic niche” has recently been Charlotte Kuperwasser’s group whereas bone- formulated by David Lyden and colleagues, marrow derived progenitors promoted the proposing that bone marrow-derived hemato- outgrowth of estrogen receptor negative (ER-) poietic progenitors may preferentially localize breast tumors in an estrogen dependent manner to future sites of metastases and “prepare” (Gupta et al. 2007). Specifically, the authors them for the arrival and outgrowth of dissemi- found that xenografts injected into postpartum nated cancer cells (Kaplan et al. 2005; Kerbel mice grew faster than those injected into nul- et al. 2008; Psaila and Lyden 2009). Although liparous mice and this was because of the pro- the identity of factors that regulate the distri- angiogenic effects of estrogen and parturition. bution of bone marrow-derived hematopoi- Subsequent experiments showed that estrogen etic progenitors is still unclear, it appears to promoted the immobilization of bone marrow- be serum-derived and tumor-specific as trans- derived stromal progenitors, which then colon- fer of serum from melanoma bearing mice to ized to sites of tumor injection and enhanced healthy mouse and subsequent injection of angiogenesis.

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The role of bone marrow-derived mesen- comprehensive gene expression profiles were chymal stem cells (BM-MSCs) in promoting analyzed by SAGE (Serial Analysis of Gene Ex- distant metastasis formation has also been de- pression), whereas genetic changes were de- scribed in a recent study by Bob Weinberg’s tected by cDNA array CGH (Comprehensive laboratory, whereas coinjection of poorly meta- Genomic Hybridization) and SNP (Single static breast cancer cells with BM-MSCs pro- Nucleotide Polymorphism) arrays. Using this moted lung metastases (Karnoub et al. 2007). approach the authors concluded that gene Follow up experiments showed that this meta- expression changes occur in all cell types during stasis promoting-effect is mediated by CCL5 breast tumor progression, but clonally selected induced in MSCs following their interaction genetic alterations are restricted to tumor epi- with breast cancer cells. Thus, inhibitors of thelial cells (Allinen et al. 2004). Interestingly, CCR5, the signaling receptor for CCL5, might the comparison of myoepithelial cells from nor- be useful for the prevention of distant metas- mal breast tissue and DCIS yielded the highest tases. number of consistently differentially expressed Despite these convincing observations im- genes and a significant fraction of these encoded plicating a role for microenvironmental and sys- for secreted proteins and cell surface recep- temic alterations in breast tumorigenesis, our tors suggesting intensive autocrine/paracrine understanding of the genes and pathways me- regulatory loops. Many of the genes reflect- diating cellular interactions and paracrine ing normal myoepithelial cell differentiation regulatory networks among various cell types (SMA, OXTR) were absent or dramatically in normal and neoplastic breast tissue is still downregulated in DCIS-associated myoepithe- limited. Numerous studies have analyzed the lial cells implying perturbed myoepithelial cell expression of selected candidate genes in nor- differentiation. mal and neoplastic primary human breast tissue Independent studies by Dennis Sgroi’s samples by immunohistochemistry or mRNA group using a different approach (laser capture in situ hybridization and found up-regulation microdissection and array-based profiling) re- of invasion and angiogenesis related genes ached similar conclusions as they detected gene (e.g., MMPs and TIMPs) and growth factors expression changes associated with tumor pro- (e.g., HGF and CTGF) in tumor-associated gression both in the stromal and epithelial com- fibroblasts, endothelial cells, and myofibroblasts partment when comparing normal breast, in (Vrana et al. 1996; Guidi et al. 1997; Brown et al. situ and invasive breast carcinomas (Ma et al. 1999; Nielsen et al. 2001; Bisson et al. 2003). 2009). The gene expression signature of epithe- Some of these proinvasive and proangiogenic lial cells correlated with tumor grade, but not genes showed increased expression in ductal with histologic stage. Genes up-regulated in carcinomas in situ (DCIS)-associated myoepi- tumor-associated stroma included many ECM- thelial cells as well, implying the potential role related molecules and the expression of MMP1, for these changes in the gradual brake down of MMP2, and MMP14 was significantly higher the BM separating epithelial and stromal cells. in invasive compared with in situ tumors sug- To analyze molecular alterations in cells gesting a potential role for these matrix metallo- composing the microenvironment in a com- proteases in promoting invasive progression. prehensive and unbiased way, Allinen et al. One of the key basement membrane-degrading purified and characterized the genome-wide MMPs, MMP14, was also shown to be up-regu- gene expression and genetic profiles of all lated in DCIS-associated myoepithelial cells and major cell types (luminal epithelial, myoepithe- in tumor-associated myofibroblasts (Hu et al. lial, and endothelial cells, infiltrating leukocytes, 2008). fibroblasts, and myofibroblasts) from normal The dramatic changes in gene expression breast tissue and in situ and invasive breast patterns but lack of clonally selected somatic carcinomas. Each cell type was purified using genetic alterations in the tumor-associated myo- cell type-specific cell surface markers and their epithelial and stromal cells suggested potential

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epigenetic alterations, because stromal cells iso- highlight the importance of microenvironment lated from normal and tumor tissue are known in shaping tumor evolution and progression. to maintain their differences even after pro- Numerous studies using diverse experimental longed cell culture and in xenograft studies systems have shown that the proliferation, sur- (Bissell and Radisky 2001; Tlsty and Hein vival, polarity, differentiation state, and invasive 2001; Bissell et al. 2002; Orimo et al. 2005). capacity of breast cancer cells can be modulated Indeed, a follow up study by Hu et al. analyzing by myoepithelial and various stromal cells global DNA methylation profiles using MSDK (Sternlicht and Barsky 1997; Sternlicht et al. (methylation-specificdigital karyotyping) iden- 1997; Elenbaas and Weinberg 2001; Radisky tified altered DNA methylation patterns not et al. 2001; Tlsty 2001; Tlsty and Hein 2001; only in tumor epithelial but also in DCIS- Bissell et al. 2002; Coussens and Werb 2002; associated myoepithelial cells and stromal Deugnier et al. 2002; Gudjonsson et al. 2002; fibroblasts (Hu et al. 2005). Independent stud- Wiseman and Werb 2002; Kenny and Bissell ies testing the methylation of selected candidate 2003; Shekhar et al. 2003). The diagnostic crit- genes in breast carcinomas also described DNA eria that pathologists use to distinguish in situ methylation differences between normal and from invasive carcinomas is the presence or cancer-associated stroma and the frequency of absence of an intact myoepithelial cell layer these alterations was dependent on tumor confirmed by immunohistochemical analyses subtype (Fiegl et al. 2006). of myoepithelial cell-specific markers includ- The molecular mechanisms responsible ing smooth muscle actin, calponin, and CD10 for the altered epigenetic profiles of cancer- (Damiani et al. 1999). However, it is unknown associated stromal cells and the stage of tumor what leads to the progressive disappearance of progression when these first can be detected the myoepithelial cells in DCIS and how this have not been clarified yet. Prolonged paracrine contributes to invasive progression. stimulation by cancer cells may initiate epige- Building on their prior results demonstrat- netic alterations in the surrounding stromal ing dramatic alterations in myoepithelial cells cells or the differentiation of local or recruited and various stromal cell types in DCIS, Hu circulating stem cells is perturbed by the tumor. and colleagues tested the role of these cell types Alternatively, epigenetic alterations in stromal in the progression of in situ to invasive breast cells might be initiated by chronic inflamma- carcinomas using the MCF10DCIS.com (Miller tion thereby resulting in abnormal microenvir- et al. 2000) xenograft model of human DCIS onment and increased risk of tumor initiation. (Hu et al. 2008). This cell line has the unique Most likely each one of these potential mecha- property that it is able to differentiate into lumi- nisms can occur and their relative contribution nal epithelial and myoepithelial cells when varies depending on tumor type and progres- injected into immunodeficient mice and forms sion stage. tumors with high histologic and molecular resemblance to human comedo DCIS that with time progresses to invasive tumors. Using this THE FUNCTIONAL RELEVANCE OF xenograft model and coinjection experiments MICROENVIRONMENTAL ALTERATIONS with normal myoepithelial cells and various IN BREAST TUMORIGENESIS fibroblasts (e.g., isolated from normal breast, The historically prevailing view of breast tumor breast tumors, and rheumatoid arthritis), the progression is focused on tumor epithelial authors found that normal myoepithelial cells, whereby gradual progression of a tumor cells efficiently suppressed tumor growth and through defined steps is entirely because of transition to IDC whereas all fibroblasts but the accumulation of genetic and epigenetic al- particularly cancer and chronic inflammation- terations that confer progressively malignant associated cells had progression-promoting phenotypes. However, this model has been effects (Hu et al. 2008). Experiments using pri- challenged by multiple recent studies, which mary human breast tissue samples corroborated

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the findings obtained in the xenograft model ANIMAL MODELS and suggest that the key event of in situ to inva- sive breast carcinoma progression is the disap- The importance of microenvironmental alter- pearance of myoepithelial cells resulting from ations in tumorigenesis was dramatically illus- the aberrant differentiation of myoepithelial trated by the demonstration that systemic in- progenitors because of a combination of signals activation of TGFb type II receptor in stromal emitted by tumor epithelial and various stromal fibroblasts led to prostate and gastric epithelial cells (Fig. 4). Because myoepithelial cell differ- neoplasia (Bhowmick et al. 2004), whereas its entiation is critically dependent on contact with inactivation in mammary stromal fibroblasts the basement membrane, which is produced by led to abnormal ductal development and pro- the myoepithelial cells, slight perturbation of moted the growth of transplanted tumors this differentiation process initiates a positive (Cheng et al. 2005). A major function of TGFb feed-back loop that leads to the progressive dis- signaling is the suppression of expression of appearance of differentiated myoepithelial cells. growth factors, cytokines and chemokines In a follow up study, Hu et al. showed that anti- (Bierie and Moses 2006). Conditional knockout inflammatory drugs such as cycloxygenase of Tgfbr2 resulting in loss of expression of the (COX) inhibitors are able to decrease the tumor receptor and thus abrogation of all TGFb sig- progression-promoting effects of fibroblasts by naling, when combined with oncogene acti- delaying the loss of myoepithelial cells (Hu vation or tumor suppressor gene attenuation, et al. 2009) highlighting the potential of cancer accelerates the development of carcinomas and therapeutic and preventative approaches target- enhances tumor progression, including metas- ing epithelial-stromal cell interactions. tasis. Mechanistic studies using the mammary

Barrier evasion

In situ carcinoma Barrier failure

Invasive carcinoma

Figure 4. Hypothetical model depicting two different views of in situ to invasive breast carcinoma transition. Cells are depicted as in Figure 2. In the “barrier evasion” model the tumor epithelial cells disrupt the myoepithelial cell layer, degrade the basement membrane, and migrate into the stroma. In the “barrier failure” model the myoepithelial cells become progressively lost and the basement membrane is disrupted at sites coinciding with areas of leukocytic infiltration and accumulation of myofibroblasts. The loss of the myoepithelial cell layer and basement membrane results in invasive carcinomas where tumor cells can invade surrounding tissues and can migrate to distant organs.

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gland models have shown that a major mecha- mammary glands accelerates the initiation, nism whereby loss or attenuation of TGFb progression and malignant transformation of signaling accelerates tumor progression is mammary epithelial tumors in association increased secretion of chemokines, particularly with massive remodeling of the ECM, innate Cxcl1 and Cxcl5, by the knockout carcinoma immune cell infiltration and increased angio- cells (Bierie et al. 2009). Importantly, global genesis (Trimboli et al. 2009). Loss of PTEN gene expression signatures associated with in stromal fibroblasts leads to increased phos- loss of TGFb signaling within carcinoma cells phorylation and recruitment of Ets2 to target were shown to correlate with poor prognosis, promoters that enhance malignant transforma- particularly in estrogen receptor positive tion. These findings identify the Pten-Ets2 axis human breast cancer (Bierie et al. 2009). In as a critical stroma-specific signaling pathway the mouse models, increased chemokine ex- that suppresses mammary epithelial tumors. pression resulted in recruitment of myeloid- Tumor-associated macrophages are one of derived suppressor cells (MDSCs), also called the most prominent and well-characterized sub- immature myeloid cells (iMCs) to the invasive set of leukocytes that can promote or inhibit edge of the primary carcinoma with resultant tumorigenesis depending on tumor type and enhancement of invasion and metastasis. Sig- progression stage. Multiple animal models have nificantly, inhibition of the cognate receptor tested their roles in mammary tumorigenesis. for Cxcl1 and Cxcl5, Cxcr2 with a small mole- Using animal models of breast cancer metasta- cule inhibitor, resulted in decreased MDSC sis, Jeff Pollard and colleagues showed that a infiltration and decreased pulmonary metasta- population of host macrophages displaying a ses (Yang et al. 2008). distinct phenotype is recruited to extravasating In the recent years, heterogeneity of the stro- pulmonary metastatic breast cancer cells re- mal fibroblasts is being appreciated. FSP1 (also gardless of their species of origin. Ablation of know in the literature as S100A4 protein/mts1 this macrophage population through several protein) was originally identified as a fibroblast- independent means (genetic and chemical) specific transcript in a differential display screen revealed that these macrophages are required between renal epithelial cells and interstitial for efficient metastatic seeding and growth of fibroblasts (Strutz et al. 1995). FSP1 shows spe- breast cancer cells (Qian et al. 2009). In another cific expression in various fibroblast cultures recent paper, using the MMTV-PyMT model and is not expressed in epithelial cells, at least of mammary carcinogenesis, Lisa Coussens in the mouse (Strutz et al. 1995; El-Rifai et al. and colleagues showed that IL-4-expressing 2002). Specificity of FSP1 expression was con- CD4(þ) T lymphocytes indirectly promote firmed in vivo by the observation that dividing invasion and subsequent metastasis of mam- fibroblasts exposed to nucleoside analogues mary adenocarcinomas by directly regulating are also selectively eliminated in transgenic the phenotype and effector function of tumor- mice expressing thymidine kinase under control associated CD11b(þ)Gr1(2)F4/80(þ)mac- of the FSP1 promoter (Iwano et al. 2001). Ex- rophages that in turn enhance metastasis pression of FSP1 is observed in some fibroblasts through activation of epidermal growth factor of the body and it is first observed at embryonic receptor signaling in malignant mammary day 9 (Strutz et al. 1995). Therefore, FSP1 serves epithelial cells (DeNardo et al. 2009). as an excellent marker for one class of fibro- blasts, both in normal and activated state (Kalluri and Neilson 2003) and because of this, CLINICAL RELEVANCE OF MICROENVIRONMENTAL ALTERATIONS FSP1 targeted Cre (cre recombinase) has been IN BREAST TUMORIGENESIS used for the selective elimination of specific genes in fibroblasts. Harol Dvorak’s well-known statement “tumors A recent study showed that genetic inactiva- are wounds that never heal” was substantiated tion of PTEN in stromal fibroblasts of mouse by results of comprehensive molecular profiling

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studies as many of the genes up-regulated in different apoptosis-inducing stimuli and this tumor-associated stroma were known to play a resistance is at least in part mediated by the role in wound-healing (Allinen et al. 2004). activation of NF-kB mediated by ligation of Further highlighting the similarities between b4 integrin to the ECM. Thus, disrupting tumor-associated stroma and wound healing integrin-ECM interactions may improve che- response was the demonstration that gene ex- motherapeutic responses. pression signatures initiated in fibroblasts in Abnormal tissue organization and homeo- response to serum stimulation were present in stasis can increase the chance of tumor initia- breast and other solid (but not hematopoietic) tion as illustrated by the increased cancer risk tumors and predicted relapse-free survival associated with chronic inflammation and tis- (Chang et al. 2004). Many genes present in sue injury (Dvorak 1986; Schafer and Werner this “wound-response signature” include ECM- 2008). This link is most clearly recognizable in modifying enzymes such as LOXL2 (lysyl colon, gastric, and hepatocellular carcinomas, oxidase-like 2), PLOD2 (procollagen lysine, 2- but human epidemiological data demonstrat- oxoglutarate 5-dioxygenase 2), and PLAUR ing decreased risk in users of NSAIDs (nonster- (plasminogen activator urokinase receptor). oidal anti-inflammatory drugs) also supports Subsequent follow up study by the same group the relevance of inflammation in breast cancer showed that the wound-response signature out- (Moran 2002). One possible mechanism by performed other known prognostic markers in which chronic inflammation and tissue damage breast cancer for the identification of patients may contribute to tumor initiation is alterations with shorter relapse and distant metastasis-free in the epithelial stem cell niche and expansion survival (Chang et al. 2005). of the stem or progenitor cell pool because of A similar study analyzed tumor stromal factors secreted by inflammatory cells. Because gene signatures by laser-capture microdissect- tumors thought to initiate from cells with ing 53 primary breast tumors and comprehen- stem or progenitor-like properties, increasing sive profiling of the isolated cells (Finak et al. the numbers of these cells might increase the 2008). Using this approach the authors defined probability of tumor initiation by increasing a 26-gene stroma-derived prognostic predictor target size for oncogenic mutations. Further- (SDPP) that classified breast cancer patients more, alterations in the microenvironment alter according to their overall and relapse-free sur- the fitness landscape providing possible growth vival independent of known clinical prognostic advantage for cells with tumor-initiating ge- markers and previously published expression netic or epigenetic changes (Blagosklonny 2002; signatures. However, stromal expression profiles Marusyk and DeGregori 2008; Marusyk and somewhat correlated with breast tumor sub- Polyak 2010). ROS (reactive oxygen species) types that may have influenced the prognostic generated by infiltrating leukocytes (macro- value of this SDPP as basal-like tumors more phages and neutrophils) and resulting from frequently expressed an activated stroma sig- intermittent hypoxia induced by tissue damage, nature. might also lead to increased mutation rates and Microenvironmental alterations in breast alter the epigenetic profiles of epithelial and cancer not only influence tumor progression stromal cells. Indeed, a recent report described and predict prognosis, but they also have major alterations in DNA methylation in hypoxic effect on the efficacy of cancer therapy especially conditions (Pal et al. 2009). targeted therapy aimed at growth factor recep- Inflammation was also shown to be respon- tors and secreted proteins. An elegant study sible for the induction of wound-induced showed the importance of tissue organization tumors in chickens infected with the Rous sar- and interactions with the ECM in defining coma virus (Martins-Green et al. 1994). This apoptotic responses to chemotherapy (Weaver classic study by Mina Bissell’s lab provided a et al. 2002). Specifically, polarized epithelial cells good example for the importance of tissue (normal or malignant) are resistant to several injury in promoting tumor initiation. Despite

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The Microenvironment of the Breast

the fact that the oncogenic virus was present in gland during different stages of its development the circulation of the chick embryos, tumors and the abnormalities of these in breast tumors, only developed at the site of injection or at translating of these findings into clinical prac- experimentally induced wounds. In subsequent tice will remain a challenge. The redundancy experiments the authors showed that a combi- of cell types and paracrine factors eliciting sim- nation of growth factors including TGFb and ilar effects predicts that blocking any one of acidic and basic FGF are responsible for this these is likely to be therapeutically ineffective. wounding-induced tumor initiation. The similarities between tumor-associated Inflammation and infiltrating leukocytes microenvironmental alterations and those in- are also involved in later stages of tumor pro- duced by physiologic or other pathologic proc- gression by promoting progression to invasion esses such as wound healing and inflammation and metastasis. One mechanism by which this imply that any therapy targeting these may have can occur is the induction of EMT in breast serious and unpredictable side effects. Lack cancer cells by infiltrating T cells (Santisteban of suitable models that faithfully reproduce et al. 2009). Specifically, CD8þ T cells can the normal mammary gland and breast tumor induce the CD44þ/CD24- stem cell-like phe- microenvironments poses a challenge for func- notype of breast cancer cells in co-cultures, tional studies aimed at testing hypotheses built which correlates with increased invasive and based on observations in human tissues, metastatic capacity and resistance to therapeutic thereby hindering the development of novel interventions. preventative and therapeutic interventions A recent report also showed a direct link targeting microenvironmental alterations. between inflammation and breast tumorigene- Deficiencies of currently used cell culture and sis involving an epigenetic switch induced by animal models might also explain their poor Src, NF-kB, microRNAs, and IL6 in tumor predictive value of the in vivo efficacy of thera- epithelial cells leading to increasingly malignant peutic agents. To overcome these deficiencies phenotype (Iliopoulos et al. 2009). The authors improved culture models that better reflect the showed the link between these molecules using physiologic environment and, thus, sensitivity MCF-10A cells as model, but subsequently to therapeutic agents such as 3D epithelial- showed that the activation signature is also stromal cell co-cultures are being developed by present in primary human breast tumors impli- multiple labs (Weaver et al. 2002; Krause et al. cating IL6-STAT3 signaling in breast cancer. 2008). However, despite all these challenges, In summary, cancer progression is not an anti-angiogenic and anti-inflammatory thera- entirely cell-autonomous process. Instead, Dar- pies have shown promise for cancer treatment winian evolution of tumors and resulting clini- and prevention (Ellis and Hicklin 2008), giving cal progression are influenced, and perhaps hope that improved knowledge will facilitate the even driven, by changes that occur in the development of a wider array of similar and tumor microenvironment. Therefore, cancer more successful approaches. risk-stratification, diagnostic, and preventative approaches have to consider the potential con- ACKNOWLEDGMENTS tribution of microenvironmental alterations in tumor initiation and progression, and use this The authors thank members of their lab- knowledge for the design of more efficient ap- oratories for their critical reading of the manu- proaches toward cancer prevention and therapy. script and constructive discussions. Work in the authors’ laboratories is supported by the National Cancer Institute (CA89393, CONCLUDING REMARKS CA116235, CA143233, and CA080111), Depart- Even after deciphering the molecular mecha- ment of Defense (W81XWH-07-1-0294 and nisms underlying cellular and microenviron- W81XWH-09-1-0131), Avon Foundation, Susan mental interactions in the normal mammary G. Komen Breast Cancer Research Foundation,

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The Role of the Microenvironment in Mammary Gland Development and Cancer

Kornelia Polyak and Raghu Kalluri

Cold Spring Harb Perspect Biol published online June 30, 2010

Subject Collection The Mammary Gland as an Experimental Model

On the Role of the Microenvironment in Mammary On How Mammary Gland Reprogramming Gland Development and Cancer Metalloproteinases Couple Form with Function Derek Radisky Bonnie F. Sloane On Using Functional Genetics to Understand On Molecular Mechanisms Guiding Embryonic Breast Cancer Biology Mammary Gland Development Kornelia Polyak Gertraud W. Robinson On Oncogenes and Tumor Suppressor Genes in On Stem Cells in the Human Breast the Mammary Gland Mark A. LaBarge Rushika M. Perera and Nabeel Bardeesy On Leukocytes in Mammary Development and On Murine Mammary Epithelial Stem Cells: Cancer Discovery, Function, and Current Status Cyrus M. Ghajar Jeffrey M. Rosen On Chromatin Remodeling in Mammary Gland On In Vivo Imaging in Cancer Differentiation and Breast Tumorigenesis David Piwnica-Worms Kornelia Polyak On Hormone Action in the Mammary Gland Choosing a Mouse Model: Experimental Biology J.M. Rosen in Context−−The Utility and Limitations of Mouse Models of Breast Cancer Alexander D. Borowsky TGF-β Biology in Mammary Development and Mammary Gland ECM Remodeling, Stiffness, and Breast Cancer Mechanosignaling in Normal Development and Harold Moses and Mary Helen Barcellos-Hoff Tumor Progression Pepper Schedin and Patricia J. Keely

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A Compendium of the Mouse Mammary Tumor Molecular Mechanisms Guiding Embryonic Biologist: From the Initial Observations in the Mammary Gland Development House Mouse to the Development of Genetically Pamela Cowin and John Wysolmerski Engineered Mice Robert D. Cardiff and Nicholas Kenney

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