Loss of amphiregulin reduces myoepithelial cell coverage of mammary ducts and alters breast tumor growth.

BACKGROUND: Amphiregulin (AREG), a ligand of the epidermal growth factor receptor, is not only essential for proper mammary ductal development, but also associated with breast cancer proliferation and growth. In the absence of AREG, mammary ductal growth is stunted and fails to expand. Furthermore, suppression of AREG expression in estrogen receptor-positive breast tumor cells inhibits in-vitro and in-vivo growth. METHODS: We crossed AREG-null (AREG-/-) mice with the murine luminal B breast cancer model, MMTV-PyMT (PyMT), to generate spontaneous breast tumors that lack AREG (AREG-/- PyMT). We evaluated tumor growth, cytokeratin-8 (K8)-positive luminal cells, cytokeratin-14 (K14)-positive myoepithelial cells, and expression of AREG, Ki67, and PyMT. Primary myoepithelial cells from nontumor-bearing AREG+/+ mice underwent fluorescence-activated cell sorting and were adapted to culture for in-vitro coculture studies with AT-3 cells, a cell line derived from C57Bl/6 PyMT mammary tumors. RESULTS: Intriguingly, PyMT-induced lesions progress more rapidly in AREG-/- mice than in AREG+/+ mice. Quantification of K8+ luminal and K14+ myoepithelial cells in non-PyMT AREG-/- mammary glands showed fewer K14+ cells and a thinner myoepithelial layer. Study of AT-3 cells indicated that coculture with myoepithelial cells or exposure to AREG, epidermal growth factor, or basic fibroblast growth factor can suppress PyMT expression. Late-stage AREG-/- PyMT tumors are significantly less solid in structure, with more areas of papillary and cystic growth. Papillary areas appear to be both less proliferative and less necrotic. In The Cancer Genome Atlas database, luminal-B invasive papillary carcinomas have lower AREG expression than luminal B invasive ductal carcinomas. CONCLUSIONS: Our study has revealed a previously unknown role of AREG in myoepithelial cell development and PyMT expression. AREG expression is essential for proper myoepithelial coverage of mammary ducts. Both AREG and myoepithelial cells can suppress PyMT expression. We find that lower AREG expression is associated with invasive papillary breast cancer in both the MMTV-PyMT model and human breast cancer.

A novel neuregulin - jagged1 paracrine loop in breast cancer transendothelial migration.

BACKGROUND: The interaction of breast cancer cells with other cells in the tumor microenvironment plays an important role in metastasis. Invasion and intravasation, two critical steps in the metastatic process, are influenced by these interactions. Macrophages are of particular interest when it comes to studying tumor cell invasiveness. Previous studies have shown that there is paracrine loop signaling between breast cancer cells and macrophages involving colony stimulating factor 1 (CSF-1) produced by tumor cells and epidermal growth factor (EGF) production by macrophages. In this paper, we identify a novel paracrine loop between tumor cells and macrophages involving neuregulin (NRG1) and notch signaling. METHODS: The aim of this study was to determine the role of NRG1, a ligand of the ErbB3 receptor, in macrophage stimulation of tumor cell transendothelial migration and intravasation. We used fluorescence-activated cell sorting (FACS) and western blot to determine ErbB3 and NRG1 expression, respectively. An in vitro transendothelial migration (iTEM) assay was used to examine the effects of short hairpin (sh)RNA targeting NRG1 in tumor cells and clustered regularly interspaced short palindromic repeats (CRISPR) knockout of jagged 1 (JAG1) in macrophages. Orthotopic xenograft injections in mice were used to confirm results in vivo. RESULTS: In our system, macrophages were the primary cells showing expression of ErbB3, and a blocking antibody against ErbB3 resulted in a significant decrease in macrophage-induced transendothelial migration of breast cancer cells. Stimulation of macrophages with NRG1 upregulated mRNA and protein expression of JAG1, a ligand of the Notch receptor, and JAG1 production by macrophages was important for transendothelial migration of tumor cells. CONCLUSIONS: This study demonstrates that stimulation of macrophages by tumor cell NRG1 can enhance transendothelial migration and intravasation. We also demonstrate that this effect is due to induction of macrophage JAG1, an important ligand of the Notch signaling pathway.

Tumor cell intravasation.

The process of entering the bloodstream, intravasation, is a necessary step in the development of distant metastases. The focus of this review is on the pathways and molecules that have been identified as being important based on current in vitro and in vivo assays for intravasation. Properties of the vasculature which are important for intravasation include microvessel density and also diameter of the vasculature, with increased intravasation correlating with increased vessel diameter in some tumors. TGFB signaling can enhance intravasation at least in part through induction of EMT, and we discuss other TGFB target genes that are important for intravasation. In addition to TGFB signaling, a number of studies have demonstrated that activation of EGF receptor family members stimulates intravasation, with downstream signaling through PI3K, N-WASP, RhoA, and WASP to induce invadopodia. With respect to proteases, there is strong evidence for contributions by uPA/uPAR, while the roles of MMPs in intravasation may be more tumor specific. Other cells including macrophages, fibroblasts, neutrophils, and platelets can also play a role in enhancing tumor cell intravasation. The technology is now available to interrogate the expression patterns of circulating tumor cells, which will provide an important reality check for the model systems being used. With a better understanding of the mechanisms underlying intravasation, the goal is to provide new opportunities for improving prognosis as well as potentially developing new treatments.