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.
Highlights
Breast cancer (BC) is the most frequently diagnosed cancer among women worldwide
Analysis of metastasis area revealed that the most affected brain region both at 7 and 10 days was the cranial hippocampus, immediately followed by the striatum, while the least affected one was the cerebellum (Fig. 1G). It revealed that the tumoral area in the lungs was similar to that of the least affected brain region. These results show that 4T1 cells in this mouse model preferentially metastasize to the brain; this is a good in vivo model for the study of peripheral events associated with brain miRNAs and myocyte enhancer factor 2C (MEF2C) in breast cancer brain metastasis metastasization
An analysis of the number of cells with nuclear staining per metastasis for the different timepoints showed that there was a significant increase of the percentage of cells with nuclear staining of approximately 29% between 3 and 10 days (P < 0.005) and of 32% between 7 and 10 days (P < 0.01) with no significant differences between 3 and 7 days (Fig. 5D). These results showed that there is a nuclear translocation of MEF2C with metastasis enlargement, indicating that MEF2C is more active in later stages of the metastatic development and pointing to this transcription factor as a new player in BC brain metastasis (BCBM) and a potential target for modulation
Summary
Breast cancer (BC) is the most frequently diagnosed cancer among women worldwide. It represents about 12% of all new cancer cases and 25% of cancers in women, being one of the leading causes of female death from cancer, according to the International Agency for Research on Cancer (Ferlay et al, 2015; Torre et al, 2016). The BBB and the crosstalk between malignant and brain cells favor brain metastasis (Wilhelm et al, 2019). This renders the brain a sanctuary against antitumor strategies, hindering the treatment of brain metastases with the available current therapies (Kotecki et al, 2018)
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