Abstract A27: Micromanaging the microenvironment: The role of microRNA in metastatic mammary tumor-associated macrophages
Authors: Mathsyaraja Haritha , Thies Katie , Taffany David A. , Ostrowski Michael C. 
Published: August 2014

Abstract
Metastasis is the primary cause of mortality in breast cancer patients. Tumor associated macrophages (TAMs) are active collaborators in mediating several steps of the metastatic cascade. There are numerous reports of microRNA deregulation in tumor cells but the role of microRNA in the tumor microenvironment remains poorly understood. The aim of this study was to elucidate the role of TAM derived microRNA in promoting metastatic mammary tumor progression.
We utilized orthotopic tail vein injection models in mice to recapitulate metastatic progression. Global microRNA expression analysis was performed on macrophages flow sorted from lung metastases at different time points post-injection in order to identify differentially expressed microRNA. We employed both loss-of-function and gain-of-function approaches to demonstrate the functional significance of the microRNA in mediating pro-tumor processes. In addition, in vitro as well as in vivo assays with macrophages were used to determine the mode of regulation of the microRNA and identify their mRNA targets.
We observed that the expression of several microRNA, including miR-21 and miR-29ab, was significantly elevated in TAMs during metastatic mammary tumor progression in the lung. Selective ablation of the endonuclease Dicer in macrophages resulted in decreased tumor cell proliferation and reduced angiogenesis. Conversely, over-expression of miR-21 and miR-29 in macrophages led to accelerated angiogenesis and increased matrix metalloproteinase activity in matrigel plug assays. We confirmed that miR-29 mediates its pro-angiogenic effects through the repression of anti-angiogenic targets such as Sparc and Col4a2. We further determined that CSF1, a factor crucial for macrophage survival, activates the expression of these microRNA through its downstream effector Ets2. Deletion of Ets2 specifically in TAMs led to a decrease in expression of the microRNA. In addition, we demonstrated that Ets2 directly binds to the promoters of the genes encoding the microRNA in bone marrow derived macrophages from tumor bearing mice. Preliminary studies indicate that TAMs derived from the lungs of tumor bearing mice treated with a specific inhibitor targeting the receptor for CSF1 (CSF1R) display lower levels of the microRNA. Currently, we are continuing these inhibitor studies and beginning to examine the relevance of our findings in patients suffering from invasive breast cancer.
The field of non-coding RNA, especially in the context of the tumor stroma, is still young and largely unexplored. A number of microRNA are upregulated in TAMs during metastatic progression, strongly indicating that they are key players in host involvement in cancer. Intriguingly, these microRNA appear to be regulated by the CSF1-Ets2 pathway. It is well established that CSF1 plays a major role in the recruitment of TAMs to the metastatic site. We have uncovered an additional novel function of the CSF1-Ets2 signaling axis in mediating metastatic tumor progression by activating the expression of microRNA in TAMs that regulate pro-tumorigenic processes such as angiogenesis and matrix remodeling. Therefore, inhibitors targeting signaling through the CSF1 receptor might be beneficial in terms of developing holistic treatment strategies for metastatic breast cancer. In addition, several of these microRNA are seen to be elevated in human invasive breast cancer. Hence, TAM derived microRNA expression signatures are likely to prove useful in terms of prognosis as well.
Citation Format: Haritha Mathsyaraja, Katie Thies, David A. Taffany, Michael C. Ostrowski. Micromanaging the microenvironment: The role of microRNA in metastatic mammary tumor-associated macrophages. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A27.