Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182

Mohit Sachdeva; Rebecca D Dodd; Zhiqing Huang; Carole Grenier; Yan Ma; Dina C Lev; Diana M Cardona; Susan K Murphy; David G Kirsch

doi:10.1016/j.canlet.2015.08.016

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Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182

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Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182

Mohit Sachdeva, Rebecca D Dodd, Zhiqing Huang, Carole Grenier, Yan Ma, Dina C Lev, Diana M Cardona, Susan K Murphy and David G Kirsch

Cancer letters, Vol.369(1), pp.202-211

12/01/2015

DOI: 10.1016/j.canlet.2015.08.016

PMCID: PMC4600675

PMID: 26314219

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Abstract

•Klf-3 is a novel transcriptional repressor of pro-metastatic miR-182 in sarcomas.•Klf-3 suppresses a myogenic transcription factor MyoD1.•Klf-3 is epigenetically silenced by DNA hypermethylation.•Demethylating agents could potentially be used to modulate miR-182 levels as a therapeutic strategy. Accumulating evidence indicates that microRNAs (miRs) regulate cancer metastasis. We have shown that miR-182 drives sarcoma metastasis in vivo by coordinated regulation of multiple genes. Recently, we also demonstrated that in a subset of primary sarcomas that metastasize to the lung, miR-182 expression is elevated through binding of MyoD1 to the miR-182 promoter. However, it is not known if there are also transcription factors that inhibit miR-182 expression. Defining negative regulators of miR-182 expression may help explain why some sarcomas do not metastasize and may also identify pathways that can modulate miR-182 for therapeutic benefit. Here, we use an in silico screen, chromatin-immunoprecipitation, and luciferase reporter assays to discover that Kruppel like factor-3 (Klf-3) is a novel transcriptional repressor of miR-182. Knockdown of Klf-3 increases miR-182 expression, and stable overexpression of Klf-3, but not a DNA-binding mutant Klf-3, decreases miR-182 levels. Klf-3 expression is downregulated in both primary mouse and human metastatic sarcomas, and Klf-3 levels negatively correlate with miR-182 expression. Interestingly, Klf-3 also negatively regulates MyoD1, suggesting an alternative mechanism for Klf-3 to repress miR-182 expression in addition to direct binding of the miR-182 promoter. Using Methylation Specific PCR (MSP) and pyrosequencing assays, we found that Klf-3 is epigenetically silenced by DNA hypermethylation both in mouse and human sarcoma cells. Finally, we show the DNA methylation inhibitor 5′Azacytidine (Aza) restores Klf-3 expression while reducing miR-182 levels. Thus, our findings suggest that demethylating agents could potentially be used to modulate miR-182 levels as a therapeutic strategy.

Epigenetics

microRNA

Metastasis

Gene regulation

Details

Title: Subtitle: Epigenetic silencing of Kruppel like factor-3 increases expression of pro-metastatic miR-182
Creators: Mohit Sachdeva - Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA
Rebecca D Dodd - Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA
Zhiqing Huang - Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC 27710, USA
Carole Grenier - Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC 27710, USA
Yan Ma - Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA
Dina C Lev - Department of Cancer Biology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA
Diana M Cardona - Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA
Susan K Murphy - Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, NC 27710, USA
David G Kirsch - Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710, USA
Resource Type: Journal article
Publication Details: Cancer letters, Vol.369(1), pp.202-211
DOI: 10.1016/j.canlet.2015.08.016
PMID: 26314219
PMCID: PMC4600675
NLM abbreviation: Cancer Lett
ISSN: 0304-3835
eISSN: 1872-7980
Publisher: Elsevier B.V
Grant note: name: QuadW-AACR Fellowship; DOI: 10.13039/100000048, name: American Cancer Society; DOI: 10.13039/100001545, name: Children's Tumor Foundation; DOI: 10.13039/100000002, name: National Institutes of Health, award: RO1 CA138265; DOI: 10.13039/100000054, name: National Cancer Institute, award: 5P30-CA14236-38
Language: English
Date published: 12/01/2015
Academic Unit: Hematology, Oncology, and Blood & Marrow Transplantation; Internal Medicine
Record Identifier: 9984094615902771

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