miR-149 suppresses breast cancer metastasis by blocking paracrine interactions with macrophages

Abstract

Triple-negative breast cancer (TNBC) constitutes 20% of all breast cancer cases. TNBC is defined by the lack of estrogen and progesterone receptors, and HER2 overexpression and is characterized by its frequent recurrence and high metastatic potential. The crosstalk between cancer cells and the surrounding tumor microenvironment (TME) plays a crucial role in fostering or restraining tumor progression and metastasis. Tumor-associated macrophages (TAMs) are the predominant cellular component of the TME in several solid-cancer entities, including mammary tumors. In response to microenvironmental stimuli, TAMs polarize towards tumoricidal M1 or tumor-promoting M2 activated cells. In breast cancer, malignant cells recruit and educate macrophages into an M2 phenotype that supports the metastatic spread of cancer cells. Breast cancer progression and metastasis are driven by the epidermal growth factor (EGF) and colony stimulatory factor 1 (CSF1) positive paracrine loop established between macrophages and cancer cells. However, the molecular mechanisms involved in establishing and maintaining these signaling loops are still poorly understood. Dysregulation of microRNAs (miRNAs), a class of short non-coding RNAs that regulate gene expression at the post-transcriptional level, has been implicated in tumor progression by remodeling the TME composition and inducing its transition into a tumor-supportive state. Considering the critical role of the TME in promoting tumor progression, a deeper understanding of the role of miRNAs in TME modulation is fundamental for developing more efficient therapies. This thesis focuses on understanding whether miR-149 downregulation in TNBC is involved in the modulation of TAMs, which may result in a suitable TME required for cancer cell invasion and subsequent metastasis. Here, CSF1 was validated as a direct target of miR-149. In silico analysis of TCGA clinical data demonstrated that in lymph node-positive TNBC tissues low miR-149 expression correlated with high CSF1 expression and M2-macrophage infiltration as well with reduced patient survival. Functional analyses using 3D co-cultures and Transwell assays demonstrated that by directly targeting CSF1, miR-149 overexpression in TNBC cell lines inhibited the interaction between macrophages and the tumor cells. In macrophages co-cultured with MDA-MB-231 cells expressing miR-149 the expression levels of two EGF receptor ligands, EGF and amphiregulin, were strongly reduced, resulting in reduced EGF receptor activation in the cancer cells. Moreover, using an in vivo mouse model, lung metastases developing from orthotopic MDA-MB-231 tumors were reduced by 75% by the ectopic miR-149 expression, and this was associated with impaired M2-macrophage infiltration of the primary tumors. Furthermore, the inhibition of DNA-methyltransferases in TNBC cell lines showed that miR-149 expression is epigenetically silenced by DNA methylation mechanisms. Taken together, the experimental studies performed in this thesis suggest that in TNBC miR-149 downregulation functionally contributes to breast tumor progression by recruiting macrophages to the tumor and facilitating CSF1 and EGF receptor crosstalk between cancer cells and macrophages.