From transformation to metastasis: deconstructing the extracellular matrix in breast cancer.

The extracellular matrix (ECM) is a guiding force that regulates various developmental stages of the breast. In addition to providing structural support for the cells, it mediates epithelial-stromal communication and provides cues for cell survival, proliferation, and differentiation. Perturbations in ECM architecture profoundly influence breast tumor progression and metastasis. Understanding how a dysregulated ECM can facilitate malignant transformation is crucial to designing treatments to effectively target the tumor microenvironment. Here, we address the contribution of ECM mechanics to breast cancer progression, metastasis, and treatment resistance and discuss potential therapeutic strategies targeting the ECM.

The extracellular matrix modulates the hallmarks of cancer.

The extracellular matrix regulates tissue development and homeostasis, and its dysregulation contributes to neoplastic progression. The extracellular matrix serves not only as the scaffold upon which tissues are organized but provides critical biochemical and biomechanical cues that direct cell growth, survival, migration and differentiation and modulate vascular development and immune function. Thus, while genetic modifications in tumor cells undoubtedly initiate and drive malignancy, cancer progresses within a dynamically evolving extracellular matrix that modulates virtually every behavioral facet of the tumor cells and cancer-associated stromal cells. Hanahan and Weinberg defined the hallmarks of cancer to encompass key biological capabilities that are acquired and essential for the development, growth and dissemination of all human cancers. These capabilities include sustained proliferation, evasion of growth suppression, death resistance, replicative immortality, induced angiogenesis, initiation of invasion, dysregulation of cellular energetics, avoidance of immune destruction and chronic inflammation. Here, we argue that biophysical and biochemical cues from the tumor-associated extracellular matrix influence each of these cancer hallmarks and are therefore critical for malignancy. We suggest that the success of cancer prevention and therapy programs requires an intimate understanding of the reciprocal feedback between the evolving extracellular matrix, the tumor cells and its cancer-associated cellular stroma.

Stromal matrix metalloproteinase 2 regulates collagen expression and promotes the outgrowth of experimental metastases.

Breast cancer survival rates decrease from 99% for patients with local disease to 25% for those with distant metastases. Matrix metalloproteinases (MMPs), including MMP2, are associated with metastatic progression. We found that loss of host MMP2 reduces the proliferation of experimental metastases in the lungs and identified fibroblasts in tumour-bearing lungs as the major source of MMP2. In vitro, spheroidal mammary tumour growth was increased by co-culture with control fibroblasts isolated from tumour-bearing lungs, but not when fibroblasts with stable Mmp2 knockdown were used. This result prompted us to assess whether MMP2 was responsible for a tumour-proliferative, activated fibroblast phenotype. To test this, we evaluated: (a) fibroblasts from wild-type tumour-bearing lungs, with or without shRNA-mediated MMP2 knockdown; and (b) normal, quiescent fibroblasts isolated from either WT or Mmp2(-/-) mice. Quantitative PCR revealed that Mmp2 knockdown attenuated expression of two markers of activation (α-smooth muscle actin and vimentin), but there was minimal expression in quiescent WT or Mmp2(-/-) fibroblasts, as expected. Placing quiescent fibroblasts under activating conditions led to increases in activation-associated transcripts in WT but not Mmp2(-/-) fibroblasts. Additionally, Mmp2 knockdown fibroblasts showed significantly decreased expression of the matrix transcripts collagen I, collagen IV and fibronectin. Addition of active TGFß was sufficient to rescue the MMP2-dependent collagen I and IV expression, while MMP2-induced collagen expression was blocked by the addition of TGFß1-neutralizing antibody. Gene expression data in stromal cells of human breast cancers reveal that MMP2 expression is also positively correlated with activation and matrix transcripts. Thus, we present a model whereby MMP2 production in tumour fibroblasts is important for TGFß1 activity and subsequent activation of fibroblasts to a matrix-producing, proliferation-supportive phenotype. Overall, our results reveal a previously undefined role for MMP2 in metastatic outgrowth mediated by fibroblasts, and extend the mechanisms by which MMPs contribute to tumour progression.

Role of TGF-ß signaling in generation of CD39+CD73+ myeloid cells in tumors.

There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-ß type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-ß signaling. In this study, we tested the hypothesis that TGF-ß drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-ß receptor in mammary epithelium, an increased level of TGF-ß protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFßRII(WT) control tumors with intact TGF-ß signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-ß signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-ß signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-ß signaling can directly induce the generation of CD39(+)CD73(+) TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment.

Disruption of bone morphogenetic protein receptor 2 (BMPR2) in mammary tumors promotes metastases through cell autonomous and paracrine mediators.

Bone morphogenetic proteins (BMPs) are members of the TGF-ß superfamily of signaling molecules. BMPs can elicit a wide range of effects in many cell types and have previously been shown to induce growth inhibition in carcinoma cells as well as normal epithelia. Recently, it has been demonstrated that BMP4 and BMP7 are overexpressed in human breast cancers and may have tumor suppressive and promoting effects. We sought to determine whether disruption of the BMP receptor 2 (BMPR2) would alter mammary tumor progression in mice that express the Polyoma middle T antigen. Mice expressing Polyoma middle T antigen under the mouse mammary tumor virus promoter were combined with mice that have doxycycline-inducible expression of a dominant-negative (DN) BMPR2. We did not observe any differences in tumor latency. However, mice expressing the BMPR2-DN had a fivefold increase in lung metastases. We characterized several cell autonomous changes and found that BMPR2-DN-expressing tumor cells had higher rates of proliferation. We also identified unique changes in inflammatory cells and secreted chemokines/cytokines that accompanied BMPR2-DN-expressing tumors. By immunohistochemistry, it was found that BMPR2-DN primary tumors and metastases had an altered reactive stroma, indicating specific changes in the tumor microenvironment. Among the changes we discovered were increased myeloid derived suppressor cells and the chemokine CCL9. BMP was shown to directly regulate CCL9 expression. We conclude that BMPR2 has tumor-suppressive function in mammary epithelia and microenvironment and that disruption can accelerate mammary carcinoma metastases.

Transforming growth factor beta receptor type III is a tumor promoter in mesenchymal-stem like triple negative breast cancer.

INTRODUCTION: There is a major need to better understand the molecular basis of triple negative breast cancer (TNBC) in order to develop effective therapeutic strategies. Using gene expression data from 587 TNBC patients we previously identified six subtypes of the disease, among which a mesenchymal-stem like (MSL) subtype. The MSL subtype has significantly higher expression of the transforming growth factor beta (TGF-ß) pathway-associated genes relative to other subtypes, including the TGF-ß receptor type III (TßRIII). We hypothesize that TßRIII is tumor promoter in mesenchymal-stem like TNBC cells. METHODS: Representative MSL cell lines SUM159, MDA-MB-231 and MDA-MB-157 were used to study the roles of TßRIII in the MSL subtype. We stably expressed short hairpin RNAs specific to TßRIII (TßRIII-KD). These cells were then used for xenograft tumor studies in vivo; and migration, invasion, proliferation and three dimensional culture studies in vitro. Furthermore, we utilized human gene expression datasets to examine TßRIII expression patterns across all TNBC subtypes. RESULTS: TßRIII was the most differentially expressed TGF-ß signaling gene in the MSL subtype. Silencing TßRIII expression in MSL cell lines significantly decreased cell motility and invasion. In addition, when TßRIII-KD cells were grown in a three dimensional (3D) culture system or nude mice, there was a loss of invasive protrusions and a significant decrease in xenograft tumor growth, respectively. In pursuit of the mechanistic underpinnings for the observed TßRIII-dependent phenotypes, we discovered that integrin-α2 was expressed at higher level in MSL cells after TßRIII-KD. Stable knockdown of integrin-α2 in TßRIII-KD MSL cells rescued the ability of the MSL cells to migrate and invade at the same level as MSL control cells. CONCLUSIONS: We have found that TßRIII is required for migration and invasion in vitro and xenograft growth in vivo. We also show that TßRIII-KD elevates expression of integrin-α2, which is required for the reduced migration and invasion, as determined by siRNA knockdown studies of both TßRIII and integrin-α2. Overall, our results indicate a potential mechanism in which TßRIII modulates integrin-α2 expression to effect MSL cell migration, invasion, and tumorigenicity.

Attenuated transforming growth factor beta signaling promotes metastasis in a model of HER2 mammary carcinogenesis.

INTRODUCTION: Transforming growth factor beta (TGFß) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGFß receptor (TßRII) for signaling. Previously, we have shown that deletion of TßRII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGFß signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGFß signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression. METHODS: All studies were performed on MMTV-Neu mice with and without dominant-negative TßRII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer. RESULTS: Attenuated TGFß signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients. CONCLUSION: Our studies provide insights into a novel mechanism by which epithelial TGFß signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGFß pathway in vivo during tumor progression remain controversial. The targeting of TGFß signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGFß signaling.

Lack of transforming growth factor-ß signaling promotes collective cancer cell invasion through tumor-stromal crosstalk.

INTRODUCTION: Transforming growth factor beta (TGF-ß) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-ß signaling regulates fibroblast recruitment and activation. Concurrently, TGF-ß signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-ß signaling to tumorigenesis, yet the role of TGF-ß in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-ß is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion. METHODS: Fluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TßRII KO) or TßRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-ß signaling loss. RESULTS: Intravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-ß signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TßRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TßRII KO carcinoma cells was identified by E-cadherin+/p120+/ß-catenin+ clusters. TßRII KO tumors also exhibited a twofold greater metastasis than TßRIIfl/fl tumors, attributed to enhanced extravasation ability. In TßRII KO tumor epithelium compared with TßRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-ß-insensitive mammary cancer. CONCLUSION: Fibroblast-stimulated carcinoma cells utilize TGF-ß signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-ß signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-ß signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.

Compartment resolved proteomics reveals a dynamic matrisome in a biomechanically driven model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a severe fibrotic component that compromises treatment, alters the immune cell profile and contributes to patient mortality. It has been shown that early on in this process, dynamic changes in tissue biomechanics play an integral role in supporting pancreatic cancer development and progression. Despite the acknowledgement of its importance, a granular view of how stromal composition changes during the course of PDAC progression remains largely unknown. To mimic the quasi-mesenchymal phenotype and pronounced desmoplastic response observed clinically, we utilized a genetically engineered mouse model of PDAC that is driven by a KrasG12D mutation and loss of Tgfbr2 expression. Application of compartment resolved proteomics revealed that PDAC progression in this KTC model is associated with dynamic stromal alterations that are indicative of a wound healing program. We identified an early provisional matricellular fibrosis that was accompanied by markers of macrophage activation and infiltration, consistent with the inflammatory phase of wound healing. At 20 weeks a proliferative phenotype was observed with increased fibroblast markers, further collagen deposition and loss of basement membrane and native cell markers.

TGF-ß, Bone Morphogenetic Protein, and Activin Signaling and the Tumor Microenvironment.

The cellular and noncellular components surrounding the tumor cells influence many aspects of tumor progression. Transforming growth factor ß (TGF-ß), bone morphogenetic proteins (BMPs), and activins have been shown to regulate the phenotype and functions of the microenvironment and are attractive targets to attenuate protumorigenic microenvironmental changes. Given the pleiotropic nature of the cytokines involved, a full understanding of their effects on numerous cell types in many contexts is necessary for proper clinical intervention. In this review, we will explore the various effects of TGF-ß, BMP, and activin signaling on stromal phenotypes known to associate with cancer progression. We will summarize these findings in the context of their tumor suppressive or promoting effects, as well as the molecular changes that these cytokines induce to influence stromal phenotypes.

Development of Aggressive Pancreatic Ductal Adenocarcinomas Depends on Granulocyte Colony Stimulating Factor Secretion in Carcinoma Cells.

The survival rate for pancreatic ductal adenocarcinoma (PDAC) remains low. More therapeutic options to treat this disease are needed, for the current standard of care is ineffective. Using an animal model of aggressive PDAC (Kras/p48TGFßRIIKO), we discovered an effect of TGFß signaling in regulation of G-CSF secretion in pancreatic epithelium. Elevated concentrations of G-CSF in PDAC promoted differentiation of Ly6G+ cells from progenitors, stimulated IL10 secretion from myeloid cells, and decreased T-cell proliferation via upregulation of Arg, iNOS, VEGF, IL6, and IL1b from CD11b+ cells. Deletion of csf3 in PDAC cells or use of a G-CSF-blocking antibody decreased tumor growth. Anti-G-CSF treatment in combination with the DNA synthesis inhibitor gemcitabine reduced tumor size, increased the number of infiltrating T cells, and decreased the number of Ly6G+ cells more effectively than gemcitabine alone. Human analysis of human datasets from The Cancer Genome Atlas and tissue microarrays correlated with observations from our mouse model experiments, especially in patients with grade 1, stage II disease. We propose that in aggressive PDAC, elevated G-CSF contributes to tumor progression through promoting increases in infiltration of neutrophil-like cells with high immunosuppressive activity. Such a mechanism provides an avenue for a neoadjuvant therapeutic approach for this devastating disease. Cancer Immunol Res; 5(9); 718-29. ©2017 AACR.

A bulky glycocalyx fosters metastasis formation by promoting G1 cell cycle progression.

Metastasis depends upon cancer cell growth and survival within the metastatic niche. Tumors which remodel their glycocalyces, by overexpressing bulky glycoproteins like mucins, exhibit a higher predisposition to metastasize, but the role of mucins in oncogenesis remains poorly understood. Here we report that a bulky glycocalyx promotes the expansion of disseminated tumor cells in vivo by fostering integrin adhesion assembly to permit G1 cell cycle progression. We engineered tumor cells to display glycocalyces of various thicknesses by coating them with synthetic mucin-mimetic glycopolymers. Cells adorned with longer glycopolymers showed increased metastatic potential, enhanced cell cycle progression, and greater levels of integrin-FAK mechanosignaling and Akt signaling in a syngeneic mouse model of metastasis. These effects were mirrored by expression of the ectodomain of cancer-associated mucin MUC1. These findings functionally link mucinous proteins with tumor aggression, and offer a new view of the cancer glycocalyx as a major driver of disease progression.

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome.

TßRIII Expression in Human Breast Cancer Stroma and the Role of Soluble TßRIII in Breast Cancer Associated Fibroblasts.

The TGF-ß pathway plays a major role in tumor progression through regulation of epithelial and stromal cell signaling. Dysfunction of the pathway can lead to carcinoma progression and metastasis. To gain insight into the stromal role of the TGF-ß pathway in breast cancer, we performed laser capture microdissection (LCM) from breast cancer patients and reduction mammoplasty patients. Microdissected tumor stroma and normal breast stroma were examined for gene expression. Expression of the TGF-ß type III receptor (TGFBR3) was greatly decreased in the tumor stroma compared to control healthy breast tissue. These results demonstrated a 44-fold decrease in TGFBR3 mRNA in tumor stroma in comparison to control tissue. We investigated publicly available databases, and have identified that TGFBR3 mRNA levels are decreased in tumor stroma. We next investigated fibroblast cell lines derived from cancerous and normal breast tissue and found that in addition to mRNA levels, TßRIII protein levels were significantly reduced. Having previously identified that cancer-associated fibroblasts secrete greater levels of tumor promoting cytokines, we investigated the consequences of soluble-TßRIII (sTßRIII) on fibroblasts. Fibroblast conditioned medium was analyzed for 102 human secreted cytokines and distinct changes in response to sTßRIII were observed. Next, we used the fibroblast-conditioned medium to stimulate human monocyte cell line THP-1. These results indicate a distinct transcriptional response depending on sTßRIII treatment and whether it was derived from normal or cancerous breast tissue. We conclude that the effect of TßRIII has distinct roles not only in cancer-associated fibroblasts but that sTßRIII has distinct paracrine functions in the tumor microenvironment.

Tissue mechanics promote IDH1-dependent HIF1α-tenascin C feedback to regulate glioblastoma aggression.

Increased overall survival for patients with glioma brain tumours is associated with mutations in the metabolic regulator isocitrate dehydrogenase 1 (IDH1). Gliomas develop within a mechanically challenged microenvironment that is characterized by a dense extracellular matrix (ECM) that compromises vascular integrity to induce hypoxia and activate HIF1α. We found that glioma aggression and patient prognosis correlate with HIF1α levels and the stiffness of a tenascin C (TNC)-enriched ECM. Gain- and loss-of-function xenograft manipulations demonstrated that a mutant IDH1 restricts glioma aggression by reducing HIF1α-dependent TNC expression to decrease ECM stiffness and mechanosignalling. Recurrent IDH1-mutant patient gliomas had a stiffer TNC-enriched ECM that our studies attributed to reduced miR-203 suppression of HIF1α and TNC mediated via a tension-dependent positive feedback loop. Thus, our work suggests that elevated ECM stiffness can independently foster glioblastoma aggression and contribute to glioblastoma recurrence via bypassing the protective activity of IDH1 mutational status.

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression.

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-ß (TGF-ß) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-ß signaling and elevated ß1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-ß signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype.

Deletion of the BMP receptor BMPR1a impairs mammary tumor formation and metastasis.

Bone Morphogenetic Proteins (BMPs) are secreted cytokines/growth factors belonging to the Transforming Growth Factor ß (TGFß) family. BMP ligands have been shown to be overexpressed in human breast cancers. Normal and cancerous breast tissue display active BMP signaling as indicated by phosphorylated Smads 1, 5 and 9. We combined mice expressing the MMTV.PyMT oncogene with mice having conditional knockout (cKO) of BMP receptor type 1a (BMPR1a) using whey acidic protein (WAP)-Cre and found this deletion resulted in delayed tumor onset and significantly extended survival. Immunofluorescence staining revealed that cKO tumors co-expressed Keratin 5 and mesenchymal cell markers such as Vimentin. This indicates that epithelial-to-mesenchymal (EMT)-like transitions occurred in cKO tumors. We performed microarray analysis on these tumors and found changes that support EMT-like changes. We established primary tumor cell lines and found that BMPR1a cKO had slower growth in vitro and in vivo upon implantation. cKO tumor cells had reduced migration in vitro. We analyzed human databases from TCGA and survival data from microarrays to confirm BMPR1a tumor promoting functions, and found that high BMPR1a gene expression correlates with decreased survival regardless of molecular breast cancer subtype. In conclusion, the data indicate that BMP signaling through BMPR1a functions as a tumor promoter.

BMPR2 loss in fibroblasts promotes mammary carcinoma metastasis via increased inflammation.

Bone Morphogenetic Protein (BMP) receptors mediate a diverse range of signals to regulate both development and disease. BMP activity has been linked to both tumor promoting and suppressive functions in both tumor cells and their surrounding microenvironment. We sought to investigate the requirement for BMPR2 in stromal fibroblasts during mammary tumor formation and metastasis. We utilized FSP1 (Fibroblast Specific Protein-1) promoter driven Cre to genetically delete BMPR2 in mice expressing the MMTV.PyVmT mammary carcinoma oncogene. We found that abrogation of stromal BMPR2 expression via FSP1 driven Cre resulted in increased tumor metastasis. Additionally, similar to epithelial BMPR2 abrogation, stromal loss of BMPR2 results in increased inflammatory cell infiltration. We proceeded to isolate and establish fibroblast cell lines without BMPR2 and found a cell autonomous increase in inflammatory cytokine secretion. Fibroblasts were co-implanted with syngeneic tumor cells and resulted in accelerated tumor growth and increased metastasis when fibroblasts lacked BMPR2. We observed that the loss of BMPR2 results in increased chemokine expression, which facilitates inflammation by a sustained increase in myeloid cells. The chemokines increased in BMPR2 deleted cells correlated with poor outcome in human breast cancer patients. We conclude that BMPR2 has tumor suppressive functions in the stroma by regulating inflammation.