Identification of the receptor tyrosine kinase AXL in triple negative breast cancer as a novel target for the human miR-34a microRNA (gene expression)

Triple negative breast cancer (TNBC) is histologically characterized by the absence of the hormone receptors estrogen and progesterone, in addition to having a negative immunostain for HER-2. The aggressiveness of this disease and lack of targeted therapeutic options for treatment is of high clinical importance. MicroRNAs are short 21- to 23 nucleotide endogenous non-coding RNAs that regulate gene expression by binding to mRNA transcripts, resulting in either decreased protein translation or mRNA degradation. Dysregulated expression of miRNAs is now a hallmark of many human cancers. In order to identify a miRNA/mRNA interaction that is biologically relevant to the triple negative breast cancer genotype/phenotype, we initially conducted a miRNA profiling experiment to detect differentially expressed miRNAs in cell line models representing the triple negative (MDA-MB-231), ER+ (MCF7), and HER-2 overexpressed (SK-BR-3) histotypes. We identified human miR-34a expression as being >3-fold down (from its median expression value across all cell lines) in MDA-MB-231 cells, and identified AXL as a putative mRNA target using multiple miRNA/target prediction algorithms. The miR-34a/AXL interaction was functionally characterized through ectopic overexpression experiments with a miR-34a mimic. In reporter assays, miR-34a binds to the putative target site within the AXL 3UTR to affect luciferase expression. We also observed degradation of AXL mRNA and decreased AXL protein levels, as well as cell signaling effects on AKT phosphorylation and phenotypic effects on cell migration. Finally, we present an inverse correlative trend in miR-34a and AXL expression for both cell line and patient tumor samples.

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Mackiewicz M, Huppi K, Pitt JJ, Dorsey TH, Ambs S, Caplen NJ