Identification of the receptor tyrosine kinase AXL in breast cancer as a target for the human miR-34a microRNA

Abstract

The identification of molecular features that contribute to the progression of breast cancer can provide valuable insight into the pathogenesis of this disease. Deregulated microRNA expression represents one type of molecular event that has been associated with many different human cancers. In order to identify a miRNA/mRNA regulatory 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 triple-negative (MDA-MB-231), ER+ (MCF7), and HER-2 over expressed (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 over expression experiments with a miR-34a mimic in two independent triple-negative breast cancer cell lines. In reporter assays, miR-34a binds to its putative target site within the AXL 3′UTR to inhibit 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.

Negative feedback regulation of AXL by miR-34a modulates apoptosis in lung cancer cells

“…We further identified an AXL regulation feedback loop that involves ELK1-mediated upregulation of miR-34a expression via the JNK pathway and that subsequently represses AXL expression to induce G 1 arrest and apoptosis. Although several studies have shown that miR-34a negatively regulates AXL expression (Mackiewicz et al 2011;Mudduluru et al 2011;Boysen et al 2014), here we report that AXL can regulate miR-34a expression through the JNK/ELK1 pathway. Our findings suggest that the relationship between miR-34a and AXL may play a critical role in the maintaining of balanced AXL expression and may provide new insights into its role in tumor progression.…”

“…Their promoters are often silenced by CpG methylation in numerous types of cancers during tumorigenesis and may therefore represent tumor suppressor genes (Lodygin et al 2008;Vogt et al 2011). Several studies have reported relationships between AXL and miR-34a in colon carcinomas (Kaller et al 2011), lung cancer (Lee et al 2011), breast cancer (Mackiewicz et al 2011), and leukemia (Boysen et al 2014). However, how the microRNAs are modulated by the AXL signaling pathway is largely unclear, and the mechanisms underlying how the miR-34 family regulates AXL expression are in need of further study.…”

Passenger strand loading in overexpression experiments using microRNA mimics

“…We noted that the miRNA mimics used in these datasets were biased to a few manufacturers, which limits the extent of the comparison. An example of a study where off-target effects were detected is Mackiewicz et al, 11 where mimics for hsa-miR34a were used (Fig. 3).…”

miR-204 regulates the biological behavior of breast cancer MCF-7 cells by directly targeting FOXA1

“…miRNAs play a vital role in breast cancer as they are reported to be involved in tumor progression, particularly in blocking apoptosis and promoting uncontrolled cell disruption (7). For example, miR-155, miR-373 and miR-520c have each been shown to be upregulated, and to act as oncogenic miRNAs in breast cancer (8)(9)(10), whereas miR-34, miR-200c and miR-205 have been shown to be downregulated and to act as tumor suppressors in breast cancer (11)(12)(13)(14). Li et al (15) demonstrated that the expression of miR-204 was expressed at markedly lower levels in breast cancer tissues than in adjacent normal breast tissues.…”