Nicholas J. Brady

Macrophages: Regulators of the Inflammatory Microenvironment during Mammary Gland Development and Breast Cancer

Macrophages are critical mediators of inflammation and important regulators of developmental processes. As a key phagocytic cell type, macrophages evolved as part of the innate immune system to engulf and process cell debris and pathogens. Macrophages produce factors that act directly on their microenvironment and also bridge innate immune responses to the adaptive immune system. Resident macrophages are important for acting as sensors for tissue damage and maintaining tissue homeostasis. It is now well-established that macrophages are an integral component of the breast tumor microenvironment, where they contribute to tumor growth and progression, likely through many of the mechanisms that are utilized during normal wound healing responses. Because macrophages contribute to normal mammary gland development and breast cancer growth and progression, this review will discuss both resident mammary gland macrophages and tumor-associated macrophages with an emphasis on describing how macrophages interact with their surrounding environment during normal development and in the context of cancer.

Activation of the FGFR–STAT3 Pathway in Breast Cancer Cells Induces a Hyaluronan-Rich Microenvironment That Licenses Tumor Formation

Aberrant activation of fibroblast growth factor receptors (FGFR) contributes to breast cancer growth, progression, and therapeutic resistance. Because of the complex nature of the FGF/FGFR axis, and the numerous effects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanisms through which aberrant FGFR activity contributes to breast cancer are not completely understood. We show here that FGFR activation induces accumulation of hyaluronan within the extracellular matrix and that blocking hyaluronan synthesis decreases proliferation, migration, and therapeutic resistance. Furthermore, FGFR-mediated hyaluronan accumulation requires activation of the STAT3 pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent hyaluronan synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and hyaluronan levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and hyaluronan synthesis is more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, these studies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces a protumorigenic microenvironment.

The FGF/FGF receptor axis as a therapeutic target in breast cancer

FGF receptor (FGFR) signaling is a vital component of both embryonic and postnatal mammary gland development, which has prompted researchers to investigate both its relevance to breast cancer and its potential as a therapeutic target. Deregulated FGFR signaling during breast cancer occurs through various mechanisms, including amplification of the receptor genes, aberrant ligand expression, receptor mutations and translocations. Recent experimental outcomes involving both animal models and human breast cancer cell lines have led to the initiation of multiple early clinical trials investigating the safety and efficacy of small-molecule FGFR inhibitors. In this article, the authors review both the most recent discoveries and the need for further investigation of the mechanisms through which FGF/FGFR signaling has emerged as an oncogenic driver.

Epiregulin contributes to breast tumorigenesis through regulating matrix metalloproteinase 1 and promoting cell survival

BackgroundThe epidermal growth factor (EGF) family of ligands has been implicated in promoting breast cancer initiation, growth and progression. The contributions of EGF family ligands and their receptors to breast cancer are complex, and the specific mechanisms through which different ligands regulate breast tumor initiation and growth are not well-defined. These studies focus on the EGF family member epiregulin (EREG) as a mediator of early stage breast tumorigenesis.MethodsEREG expression levels were assessed in both cell lines and human samples of ductal carcinoma in situ (DCIS) using quantitative RT-PCR, ELISA and immunohistochemistry. Gene knock-down approaches using shRNA-based strategies were used to determine the requirement of EREG for growth of MCF10DCIS cells in vivo, and for identifying mechanisms through which EREG promotes tumor cell survival. Experiments were performed using a combination of two-dimensional culture, three-dimensional culture and tumor growth in vivo.ResultsIn comparison with other EGF family members, EREG was induced in MCF10DCIS cells compared with MCF10A and MCF10AT cells and its expression was partially regulated by fibroblast growth factor receptor (FGFR) activity. Reduced EREG expression in MCF10DCIS cells led to decreased tumor growth in vivo, which was associated with reduced cell survival. Furthermore, treatment of MCF10A cells with exogenous EREG enhanced cell survival both in three-dimensional culture and in response to chemotherapeutic agents. Examination of EREG-induced signaling pathways demonstrated that EREG promoted survival of MCF10A cells through regulating expression of matrix metalloproteinase-1 (MMP-1). To determine the relevance of these findings in human tumors, samples of DCIS were analyzed for EREG and MMP-1 expression. EREG was induced in DCIS lesions compared to normal breast epithelium, and EREG and MMP-1 were correlated in a subset of DCIS samples.ConclusionsTogether, these studies lead to identification of a novel pathway involving EREG and MMP-1 that contributes to the formation of early stage breast cancer. Understanding these complex pathways could ultimately lead to the development of novel biomarkers of neoplastic progression and/or new therapeutic strategies for patients with early stage cancer.

ADAM17 in tumor associated leukocytes regulates inflammatory mediators and promotes mammary tumor formation

The presence of inflammatory cells within the tumor microenvironment has been tightly linked to mammary tumor formation and progression. Specifically, interactions between tumor cells and infiltrating macrophages can contribute to the generation of a pro-tumorigenic microenvironment. Understanding the complex mechanisms that drive tumor cell-macrophage cross-talk will ultimately lead to the development of approaches to prevent or treat early stage breast cancers. As described here, we demonstrate that the cell surface protease a disintegrin and metalloproteinase 17 (ADAM17) is expressed by macrophages in mammary tumors and contributes to regulating the expression of pro-inflammatory mediators, including inflammatory cytokines and the inflammatory mediator cyclooxygenase-2 (Cox-2). Furthermore, we demonstrate that ADAM17 is expressed on leukocytes, including macrophages, within polyoma middle T (PyMT)-derived mammary tumors. Genetic deletion of ADAM17 in leukocytes resulted in decreased onset of mammary tumor growth, which was associated with reduced expression of the Cox-2 within the tumor. These findings demonstrate that ADAM17 regulates key inflammatory mediators in macrophages and that leukocyte-specific ADAM17 is an important promoter of mammary tumor initiation. Understanding the mechanisms associated with early stage tumorigenesis has implications for the development of preventive and/or treatment strategies for early stage breast cancers.

STAT5 deletion in macrophages alters ductal elongation and branching during mammary gland development

Macrophages are required for proper mammary gland development and maintaining tissue homeostasis. However, the mechanisms by which macrophages regulate this process remain unclear. Here, we identify STAT5 as an important regulator of macrophage function in the developing mammary gland. Analysis of mammary glands from mice with STAT5-deficient macrophages demonstrates delayed ductal elongation, enhanced ductal branching and increased epithelial proliferation. Further analysis reveals that STAT5 deletion in macrophages leads to enhanced expression of proliferative factors such as Cyp19a1/aromatase and IL-6. Mechanistic studies demonstrate that STAT5 binds directly to the Cyp19a1 promoter in macrophages to suppress gene expression and that loss of STAT5 results in enhanced stromal expression of aromatase. Finally, we demonstrate that STAT5 deletion in macrophages cooperates with oncogenic initiation in mammary epithelium to accelerate the formation of estrogen receptor (ER)-positive hyperplasias. These studies establish the importance of STAT5 in macrophages during ductal morphogenesis in the mammary gland and demonstrate that altering STAT5 function in macrophages can affect the development of tissue-specific disease.

Breast cancer cell-derived fibroblast growth factors enhance osteoclast activity and contribute to the formation of metastatic lesions

Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been implicated in promoting breast cancer growth and progression. While the autocrine effects of FGFR activation in tumor cells have been extensively studied, little is known about the effects of tumor cell-derived FGFs on cells in the microenvironment. Because FGF signaling has been implicated in the regulation of bone formation and osteoclast differentiation, we hypothesized that tumor cell-derived FGFs are capable of modulating osteoclast function and contributing to growth of metastatic lesions in the bone. Initial studies examining FGFR expression during osteoclast differentiation revealed increased expression of FGFR1 in osteoclasts during differentiation. Therefore, studies were performed to determine whether tumor cell-derived FGFs are capable of promoting osteoclast differentiation and activity. Using both non-transformed and transformed cell lines, we demonstrate that breast cancer cells express a number of FGF ligands that are known to activate FGFR1. Furthermore our results demonstrate that inhibition of FGFR activity using the clinically relevant inhibitor BGJ398 leads to reduced osteoclast differentiation and activity in vitro. Treatment of mice injected with tumor cells into the femurs with BGJ398 leads to reduced osteoclast activity and bone destruction. Together, these studies demonstrate that tumor cell-derived FGFs enhance osteoclast function and contribute to the formation of metastatic lesions in breast cancer.

Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models are Promoted by PELP1/AIB1 Complexes

Proline, glutamic acid, leucine-rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, interacting partners unique to cytoplasmic PELP1 and the mechanisms by which these interactions promote oncogenic PELP1 signaling were sought. AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) was identified as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor–positive (ER+) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e., activation) of AIB1 at Thr24, enhanced ALDH+ tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and downregulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, limited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line. Furthermore, in vivo syngeneic tumor studies revealed that PELP1 knockdown resulted in increased survival of tumor-bearing mice as compared with mice injected with control cells. Implications: These data demonstrate that cytoplasmic PELP1/AIB1–containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression. Mol Cancer Res; 16(4); 707–19. ©2018 AACR.

Abstract B64: Macrophage-specific deletion of STAT5 disrupts normal mammary gland development and accelerates mammary tumorigenesis

Fibroblast growth factor receptor 1 (FGFR1) is amplified in 10% of human breast cancers and the ligands for FGFR1 are overexpressed in 60% of triple negative breast cancers. Additionally, amplification of the FGFR1 locus can confer resistance to endocrine-based therapies in patients with estrogen receptor (ER) positive tumors. Previous studies using a mouse model of FGFR1-induced mammary tumorigenesis demonstrated that FGFR1 activation in mammary epithelial cells recruits macrophages to hyperplastic regions where they promote angiogenesis and epithelial cell proliferation. Clinically, patients with increased numbers of tumor-associated macrophages have increased risk of relapse and decreased overall survival. While these previous studies demonstrate a role for macrophages during tumor initiation and progression the specific mechanisms that orchestrate the pro-tumor response are poorly understood. Our work uses numerous in vitro and in vivo models to study the dynamic interactions between mammary epithelial cells and macrophages. In an initial screen, we identified the transcription factor signal transducer and activator of transcription 5 (STAT5) as being rapidly activated in macrophages in response to factors involved in mammary gland development in vitro and have observed phospho-STAT5+ macrophages present in the developing mammary gland in vivo. To assess the role of STAT5 in macrophages during normal mammary gland development, we created a macrophage-specific conditional knockout mouse line with Cre recombinase expression driven by the Csf1r promoter. STAT5-deficient macrophages have significantly increased expression of pro-inflammatory cytokines as well as the estrogen-synthesizing enzyme aromatase. Consistent with increased estrogen production, mice carrying a macrophage-specific deletion of STAT5 (Stat5 fl/fl ; Csf1r-Cre +) show increased epithelial cell proliferation, increased mammary gland branching and impaired ductal elongation at 6 weeks of age compared to littermate controls (Stat5 fl/fl ; Csf1r-Cre -) along with increased expression of canonical ER target genes. Based on these data, we hypothesized that the loss of STAT5 in macrophages would accelerate mammary tumorigenesis. To test this, we crossed the macrophage-specific STAT5 knockout mice with a mouse model of FGFR1-induced mammary tumorigenesis. FGFR1 activation was induced in the mice for 2 weeks beginning at 6 weeks of age. Consistent with previous reports, activation of FGFR1 in mammary epithelial cells results in increased proliferation and lateral bud formation. Surprisingly, mice with STAT5-deficient macrophages show signs of accelerated tumorigenesis, with increased proliferation and numerous instances of ER+ hyperplasia in the first 2 weeks of FGFR1 activation. Future studies will focus on understanding the downstream effects of STAT5 deletion in macrophages and the therapeutic potential of targeting this pathway in vivo. All together, these data demonstrate that STAT5 is a critical factor that allows macrophages to regulate normal mammary gland development. In addition, this work illustrates that reduced STAT5 activity in macrophages can cooperate with a common genetic event in mammary epithelial cells to promote breast cancer initiation. Citation Format: Nicholas J. Brady, Michael A. Farrar, Kathryn L. Schwertfeger. Macrophage-specific deletion of STAT5 disrupts normal mammary gland development and accelerates mammary tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B64.

Abstract S4-01: Macrophage-specific deletion of STAT5 disrupts normal mammary gland development and accelerates mammary tumorigenesis

Fibroblast growth factor receptor 1 (FGFR1) is amplified in 10% of human breast cancers and the ligands for FGFR1 are overexpressed in 60% of triple negative breast cancers. Previous studies using a mouse model of FGFR1-induced mammary tumorigenesis demonstrated that FGFR1 activation in mammary epithelial cells recruits macrophages to hyperplastic regions where they promote angiogenesis and epithelial cell proliferation. Clinically, patients with increased numbers of tumor-associated macrophages have increased risk of relapse and decreased overall survival. While these previous studies demonstrate a role for macrophages during tumor initiation and progression the specific mechanisms that orchestrate the pro-tumor response are poorly understood. Our work uses numerous in vitro and in vivo models to study the dynamic interactions between mammary epithelial cells and macrophages. In an initial screen, we identified the transcription factor signal transducer and activator of transcription 5 (STAT5) as being rapidly activated in macrophages in response to tumor cell-derived factors and modulating pro-tumor functions of macrophages. To assess the role of STAT5 in macrophages during normal mammary gland development, we created a macrophage-specific conditional knockout mouse line with Cre recombinase expression driven by the Csf1r promoter. Mice carrying a macrophage-specific deletion of STAT5 (Stat5 fl/fl ; Csf1r-Cre +) show increased epithelial cell proliferation and impaired ductal elongation at 6 weeks of age compared to littermate controls (Stat5 fl/fl ; Csf1r-Cre -). Based on these data, we hypothesized that the loss of STAT5 in macrophages would accelerate mammary tumorigenesis. To test this, we crossed the macrophage-specific STAT5 knockout mice with a mouse model of FGFR1-induced mammary tumorigenesis. FGFR1 activation was induced in the mice for 2 or 4 weeks beginning at 6 weeks of age. Consistent with previous reports, activation of FGFR1 in mammary epithelial cells results in increased proliferation and lateral bud formation. Surprisingly, mice with STAT5-deficient macrophages show signs of accelerated tumorigenesis, with increased proliferation and numerous instances of hyperplasia in the first 2 weeks of FGFR1 activation. Future studies will focus on understanding the downstream effects of STAT5 deletion in macrophages and the therapeutic potential of targeting this pathway in vivo. All together, these data demonstrate that STAT5 is a critical factor that allows macrophages to regulate normal mammary gland development. In addition, this work illustrates that the loss of STAT5 in macrophages can cooperate with a common genetic event in mammary epithelial cells during breast cancer initiation. Citation Format: Nicholas J Brady, Michael A Farrar, Kathryn L Schwertfeger. Macrophage-specific deletion of STAT5 disrupts normal mammary gland development and accelerates mammary tumorigenesis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S4-01.