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

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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 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 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.

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Acknowledgments

This research was supported by the Intramural Research Program (Center for Cancer Research, NCI) of the NIH. The authors thank Drs Stan Lipkowitz, Thomas Reid, Scott Martin, Philip Lorenzi, Amanda Hummon, Michael Difilippantonio, and Paul Meltzer for their useful comments and suggestions. The authors also thank Robert Cornelison, Diane Palmieri, Sarah Anzick, Kristen Gehlhaus, Tamara Jones, Lihui Ou, and Brady Wahlberg for their technical assistance. The authors greatly appreciate the gesture extended by Ashish Lal and Judy Lieberman (Harvard Medical School) in sharing their unpublished data on miR-34a/AXL targeting with us. Finally, the authors gratefully acknowledge the technical assistance that Subu Yerramilli (Qiagen) provided to us in regard to the qRT-PCR analysis of miR-34a primary, precursor, and mature transcripts.

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No potential conflicts of interest are disclosed.

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Authors and Affiliations

  1. Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bldg. 37, Rm. 6128, NCI, NIH, Bethesda, MD, 20892, USA

    Mark Mackiewicz, Konrad Huppi, Jason J. Pitt & Natasha J. Caplen

  2. Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA

    Tiffany H. Dorsey & Stefan Ambs

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  1. Mark Mackiewicz
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  2. Konrad Huppi
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  3. Jason J. Pitt
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  5. Stefan Ambs
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  6. Natasha J. Caplen
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Correspondence to Natasha J. Caplen.

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Mackiewicz, M., Huppi, K., Pitt, J.J. et al. Identification of the receptor tyrosine kinase AXL in breast cancer as a target for the human miR-34a microRNA. Breast Cancer Res Treat 130, 663–679 (2011). https://doi.org/10.1007/s10549-011-1690-0

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