Abstract
Background
Melanoma has the highest tropism of any cancer to metastasize to the brain, and 40% of late-stage patients develop brain metastasis. Invasion, survival, and progression of tumors is dependent on the support of the surrounding microenvironment; therefore, modulation of neighboring cells is a key factor in metastasis. Extracellular vesicles (EVs) are important in cell-to-cell signalling, shuttling proteins, RNA and DNA to alter the surroundings into a favorable tumor microenvironment. Our aims were to investigate the role of melanoma brain metastasis (MBM) derived EVs in MBM development to find possible contributing mechanisms to cancer progression for eventual therapeutic targeting.
Material and Methods
MBM-EVs isolated via sequential ultracentrifugation were injected into mice as a pre-treatment prior to intracardial injection of MBM cells. EVs were co-cultured with normal human astrocytes (NHA) to investigate phenot ypic changes. MiRNA sequencing was performed on EVs collected from MBM cells and compared to NHA and melanocytes to determine a candidate miRNA for targeting. In situ hybridization was utilized to evaluate the level of miRNA in clinical patient MBM samples. Functional in vivo validation was performed by injecting miRNA knockout MBM cells into mice. Sequencing of NHA in the presence or absence of target miRNA mimic was used to determine downstream targets.
Results
Mice primed with EVs had a significant increase in MBM tumor burden, compared to non-primed mice. Co-culture with MBM-EVs resulted in NHA activation in vitro, with increased proliferation, invasion, cytokine production, and upregulation of GFAP. MiR-146a was highly upregulated in MBM EVs, and miR-146a mimics activated NHA. Patient samples had a significant increase in miR-146a expression, compared to healthy brain controls. MiR-146a knockdown in MBM mice models reduced MBM burden and prolonged animal survival. Sequ encing of NHA determined NUMB, an inhibitor of the Notch signalling pathway, as a target of miR-146a. Numb and other downstream Notch proteins expression was significantly altered in NHA in the presence of both MBM-EVs and miR-146a.
Conclusion
In conclusion, EVs are important regulators of MBM and establish tumor-supporting reactive astrocytes by delivery of miR-146a. MiR-146a alters Notch signalling in astrocytes via inhibition of the tumor suppressor gene NUMB. Elevated miR-146a levels in patients suggests a potential clinical intervention is possible via miR-146a targeting.
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Δημοσίευση σχολίου
Medicine by Alexandros G. Sfakianakis,Anapafseos 5 Agios Nikolaos 72100 Crete Greece,00302841026182,00306932607174,alsfakia@gmail.com,