Cancer Letters

Cancer Letters

Volume 372, Issue 1, 1 March 2016, Pages 137-146
Cancer Letters

Original Articles
LMTK3 escapes tumour suppressor miRNAs via sequestration of DDX5

https://doi.org/10.1016/j.canlet.2015.12.026Get rights and content

Highlights

  • LMTK3 regulates a subset of miRNAs, including miR-34a, miR-196a-2 and miR-182.

  • LMTK3 binds to DDX5 and to the pri-miRNA of these three mature miRNAs, thereby promoting their processing from pri-miRNAs to pre-miRNAs.

  • miR-34a and miR-182 bind to the 3'UTR of LMTK3 and inhibit its expression at the post-transcriptional and translational level respectively.

  • Ectopic expression of miR-34a and miR-182 in LMTK3-overexpressed cell lines (MCF7-LMTK3 and MDA-MB-231-LMTK3) inhibits breast cancer proliferation, invasion and migration.

Abstract

Lemur tyrosine kinase-3 (LMTK3) plays an important role in cancer progression and is associated with breast, lung, gastric and colorectal cancer. MicroRNAs (miRNAs) are small endogenous non-coding RNAs that typically repress target genes at post-transcriptional level and have an important role in tumorigenesis. By performing a miRNA expression profile, we identified a subset of miRNAs modulated by LMTK3. We show that LMTK3 induces miR-34a, miR-196-a2 and miR-182 levels by interacting with DEAD-box RNA helicase p68 (DDX5). LMTK3 binds via DDX5 to the pri-miRNA of these three mature miRNAs, thereby sequestrating them from further processing. Ectopic expression of miR-34a and miR-182 in LMTK3-overexpressing cell lines (MCF7-LMTK3 and MDA-MB-231-LMTK3) inhibits breast cancer proliferation, invasion and migration. Interestingly, miR-34a and miR-182 directly bind to the 3'UTR of LMTK3 mRNA and consequently inhibit both its stability and translation, acting as tumour suppressor-like miRNAs. In aggregate, we show that LMTK3 is involved in miRNA biogenesis through modulation of the Microprocessor complex, inducing miRNAs that target LMTK3 itself.

Section snippets

Background

LMTK3 is involved in multiple biological processes, including tumour progression by regulating oestrogen receptor α (ERα) transcription and its stability [1]. Furthermore, high LMTK3 expression has also been associated with poor survival in breast cancer patients [2], while genome-wide studies have revealed that LMTK3 has a crucial role in endocrine resistance via different signalling pathways [3]. We have also demonstrated a role of LMTK3 in breast cancer cell motility, migration, and invasion

Cell culture

Human breast cancer cell lines MCF7 and MDA-MB-231 were purchased from the American Type Culture Collection and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS and 1% penicillin streptomycin and L-glutamine. MCF7 and MDA-MB-231 that stably overexpress LMTK3 were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum (FCS), G418 (500 µg/ml; Invitrogen) and 1% penicillin streptomycin and L-glutamine. All cells were

Overexpression of LMTK3 up-regulates a subset of miRNAs

In order to investigate whether LMTK3 regulates miRNA levels, we used Nanostring nCounter to perform a miRNA expression profile in parental MCF7 cells and MCF7 cells stably overexpressing LMTK3 (MCF7-LMTK3). Fold change assessments (>or <1.5×) and t-tests (P < 0.01) criteria revealed a small number of up- and down-regulated miRNAs in MCF7-LMTK3 cells compared to parental MCF7 (Fig. 1a–c). We further validated most of the significantly modified miRNAs (up- or down-regulated) by performing

Discussion

LMTK3 is abnormally abundant in most breast cancer cell lines. Our recent work revealed that LMTK3 acts as a transcription factor [5], leading to hypothesize its involvement in miRNAs regulation. In this study, using miRNA expression profiling, we identified a subset of miRNAs regulated by LMTK3. Interestingly, miR-34a, miR-196a2 and miR-182 were regulated at the post-transcriptional level in MCF7 cells stably overexpressing LMTK3 (MCF7-LMTK3). Although miR-34a, miR-182 and miR-196a2 belong to

Conflict of interest

The authors declare no competing financial interests.

Authors' contributions

JJ acquired the data and performed most of the experiments. JJ, JS and GG contributed to conception and design. NK, MM performed some of the experiments and RF, NK and MM analysed some of the data. VH and LC provided conceptual advices. JJ, RF, GG and LC wrote the paper. All authors read and approved the final manuscript.

Acknowledgments

We would like to thank Adam Frampton, Jonathan Krell and Joao Nunes for the helpful discussion. This work was supported by Action Against Cancer. We would also like to thank Richard and Evelina Girling and the “kinase group” for their support.

References (54)

  • C. Sellier et al.

    Sequestration of DROSHA and DGCR8 by expanded CGG RNA repeats alters microRNA processing in fragile X-associated tremor/ataxia syndrome

    Cell Rep

    (2013)
  • G. Giamas et al.

    Kinome screening for regulators of the estrogen receptor identifies LMTK3 as a new therapeutic target in breast cancer

    Nat. Med

    (2011)
  • J. Stebbing et al.

    LMTK3 expression in breast cancer: association with tumor phenotype and clinical outcome

    Breast Cancer Res. Treat

    (2012)
  • J. Stebbing et al.

    LMTK3 is implicated in endocrine resistance via multiple signaling pathways

    Oncogene

    (2013)
  • Y. Xu et al.

    The kinase LMTK3 promotes invasion in breast cancer through GRB2-mediated induction of integrin beta(1)

    Sci. Signal

    (2014)
  • A.E. Frampton et al.

    MicroRNAs cooperatively inhibit a network of tumor suppressor genes to promote pancreatic tumor growth and progression

    Gastroenterology

    (2014)
  • F.G. Pinho et al.

    Downregulation of microRNA-515-5p by the estrogen receptor modulates sphingosine kinase 1 and breast cancer cell proliferation

    Cancer Res

    (2013)
  • C.E. Stahlhut Espinosa et al.

    The role of microRNAs in cancer

    Yale J. Biol. Med

    (2006)
  • G.A. Calin et al.

    MicroRNA signatures in human cancers

    Nat. Rev. Cancer

    (2006)
  • X. Cai et al.

    Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs

    RNA

    (2004)
  • Y. Lee et al.

    MicroRNA genes are transcribed by RNA polymerase II

    EMBO J.

    (2004)
  • G.M. Borchert et al.

    RNA polymerase III transcribes human microRNAs

    Nat. Struct. Mol. Biol

    (2006)
  • Y. Zeng et al.

    Sequence requirements for micro RNA processing and function in human cells

    RNA

    (2003)
  • Y.K. Kim et al.

    Processing of intronic microRNAs

    EMBO J.

    (2007)
  • T.P. Chendrimada et al.

    TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing

    Nature

    (2005)
  • A.D. Haase et al.

    TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

    EMBO Rep

    (2005)
  • S. Chakraborty et al.

    Pri-miR-17-92a transcript folds into a tertiary structure and autoregulates its processing

    RNA

    (2012)
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