Downregulation of circulating miR 802‐5p and miR 194‐5p and upregulation of brain MEF2C along breast cancer brain metastasization

Abstract

Breast cancer brain metastases (BCBMs) have been underinvestigated despite their high incidence and poor outcome. MicroRNAs (miRNAs), and particularly circulating miRNAs, regulate multiple cellular functions, and their deregulation has been reported in different types of cancer and metastasis. However, their signature in plasma along brain metastasis development and their relevant targets remain undetermined. Here, we used a mouse model of BCBM and next‐generation sequencing (NGS) to establish the alterations in circulating miRNAs during brain metastasis formation and development. We further performed bioinformatics analysis to identify their targets with relevance in the metastatic process. We additionally analyzed human resected brain metastasis samples of breast cancer patients for target expression validation. Breast cancer cells were injected in the carotid artery of mice to preferentially induce metastasis in the brain, and samples were collected at different timepoints (5 h, 3, 7, and 10 days) to follow metastasis development in the brain and in peripheral organs. Metastases were detected from 7 days onwards, mainly in the brain. NGS revealed a deregulation of circulating miRNA profile during BCBM progression, rising from 18% at 3 days to 30% at 10 days following malignant cells’ injection. Work was focused on those altered prior to metastasis detection, among which were miR‐802‐5p and miR‐194‐5p, whose downregulation was validated by qPCR. Using targetscan and diana tools, the transcription factor myocyte enhancer factor 2C (MEF2C) was identified as a target for both miRNAs, and its expression was increasingly observed in malignant cells along brain metastasis development. Its upregulation was also observed in peritumoral astrocytes pointing to a role of MEF2C in the crosstalk between tumor cells and astrocytes. MEF2C expression was also observed in human BCBM, validating the observation in mouse. Collectively, downregulation of circulating miR‐802‐5p and miR‐194‐5p appears as a precocious event in BCBM and MEF2C emerges as a new player in brain metastasis development.

Myocyte Enhancer Factor 2C as a New Player in Human Breast Cancer Brain Metastases

“…BCBM were analyzed for MEF2C expression in metastatic cells based on double labeling with pan Cytokeratin, as well as for its nuclear translocation [ 23 ]. The MEF2C expression pattern was related to clinical data, and results were presented as a percentage of cases with a certain MEF2C expression pattern (cytosol/nucleus) as a function of the metastases size (1–≤3 and 3–6 cm diameter) and number (solitary or multiple).…”

“…Previous studies from our laboratory using a mouse model of brain metastases development from triple negative breast cancer (TNBC) revealed that microRNAs 802-5p and 194-5p are downregulated in plasma prior to detection of BM from BC and that MEF2C is a target of both miRNAs. Such studies further revealed that MEF2C is expressed in BCCs extravasating into the brain and in well-established metastases and that it is increasingly translocated into the nucleus as tumorigenesis progresses [ 23 ]. Due to MEF2C function as a TF, it is conceivable that this translocation culminates in higher activation of MEF2C function to promote the transcription of its target genes, such as vascular endothelial growth factor (VEGF) [ 18 , 24 ].…”

Understanding Patterns of Brain Metastasis in Triple-Negative Breast Cancer and Exploring Potential Therapeutic Targets

“…The authors found that the downregulation of miR-802-5p and miR-195-5p was a precocious event in TNBCBM, and that the transcription factor myocyte enhancer factor 2C (MEF2C) was a target of the two miRNAs, while MEF2C expression in TNBC cells was increased with BM development. 100 …”

miRNAs in Health and Disease: A Focus on the Breast Cancer Metastatic Cascade towards the Brain

“…Finally, it was recently demonstrated the downregulation of miR-802-5p and of miR-194-5p in plasma prior to the detection of brain metastases, in a mouse model of preferential formation of brain metastases from BC [ 207 ]. Thus, miR-802-5p and of miR-194-5p appear as early biomarkers of BCBM, suggesting that their determination in liquid biopsies may constitute a new strategy for identification of BC patients at risk of developing brain metastases, or of those patients with micrometastases not suitable to be detected with currently available approaches.…”

“…Importantly, these observations coming out from the analysis of mouse brain sections were validated by analysis of resected brain metastases from BC patients. Hence, these results point to miR-802-5p and of miR-194-5p as tumor suppressors and to its target, MEF2C, as a new player in BCBM formation [ 207 ]. Another recent study revealed that miR-4428 and miR-4480 in serum samples could significantly distinguish BC patients with and without brain metastases, pointing to these miRNAs as predictive biomarkers of brain metastases in BC patients [ 208 ].…”