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Silencing of miR-148a in cancer-associated fibroblasts results in WNT10B-mediated stimulation of tumor cell motility

Oncogene volume 32, pages 3246–3253 (2013)Cite this article

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Abstract

The tumor microenvironment has an important role in cancer progression. Here we show that miR-148a is downregulated in 15 out of 16 samples (94%) of cancer-associated fibroblasts (CAFs) compared with matched normal tissue fibroblasts (NFs) established from patients with endometrial cancer. Laser-capture microdissection of stromal cells from normal tissue and endometrial cancer confirmed this observation. Treatment of cells with 5-aza-deoxycytidine stimulated the expression of miR-148a in the majority of CAFs implicating DNA methylation in the regulation of miR-148a expression. Investigation of miR-148a function in fibroblasts demonstrated that conditioned media (CM) from CAFs overexpressing miR-148a significantly impaired the migration of five endometrial cancer cell lines without affecting their growth rates in co-culture experiments. Among predicted miR-148a target genes are two WNT family members, WNT1 and WNT10B. Activation of the WNT/β-catenin pathway in CAFs was confirmed by microarray analysis of gene expression and increased activity of the SuperTOPFlash luciferase reporter. We found elevated levels of WNT10B protein in CAFs and its level decreased when miR-148a was re-introduced by lentiviral infection. The 3′-UTR of WNT10B, cloned downstream of luciferase cDNA, suppressed luciferase activity when co-expressed with miR-148a indicating that WNT10B is a direct target of miR-148a. In contrast to the effect of miR-148a, WNT10B stimulated migration of endometrial cancer cell lines. Our findings have defined a molecular mechanism in the tumor microenvironment that is a novel target for cancer therapy.

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Acknowledgements

We thank Julie Oliver, Tracy Litzi, and Shelley Hoover for help with laser capture microdissection, Dr Xin Wei Wang (NCI) for the WNT10B expression vector and Dr Randall Moon (University of Washington) for the SuperTOPFlash and SuperFOPFlash constructs. This work was supported by the Intramural Research Program of National Institutes of Health, National Cancer Institute, and Center for Cancer Research.

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Authors and Affiliations

  1. Cancer and Cell Biology Branch, Bethesda, MD, USA

    O Aprelikova, J Palla, B Hibler, X Yu & J Niederhuber

  2. Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, USA

    Y E Greer & J S Rubin

  3. SAIC Inc, Frederick, MD, USA

    M Yi & R Stephens

  4. United States Military Cancer Institute, Walter Reed Army Medical Center, Washington DC, USA

    G L Maxwell

  5. University of Virginia, Charlottesville, VA, USA

    A Jazaeri

  6. Department of Obstetrics Gynecology and Reproductive Biology, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA

    J I Risinger

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Aprelikova, O., Palla, J., Hibler, B. et al. Silencing of miR-148a in cancer-associated fibroblasts results in WNT10B-mediated stimulation of tumor cell motility. Oncogene 32, 3246–3253 (2013). https://doi.org/10.1038/onc.2012.351

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