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Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both β-catenin dependent and independent Wnt signalling

  • Original Article – Cancer Research
  • Published:
Journal of Cancer Research and Clinical Oncology Aims and scope Submit manuscript

Abstract

Purpose

Wnt signalling has been implicated in breast cancer, and in particular aberrant β-catenin-independent Wnt signalling has been associated with breast cancer metastasis and Tamoxifen resistance. Despite Wnt pathway involvement in many human cancers, attempts to target the pathway therapeutically have been disappointing. The recent discovery that the receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a novel Wnt receptor provides a potential new therapeutic and diagnostic target.

Methods

To clarify the role of ROR2 in breast cancer, we investigated its expression via ROR2 immunohistochemistry in a clinical cohort of breast cancer patients, and via in vitro studies incorporating both overexpression and knock-down of ROR2.

Results

ROR2 was expressed in the majority of breast cancer patients (87 %), including those classed as triple negative. Breast cancer patients expressing ROR2 had a significantly shorter overall survival than those lacking ROR2 expression (P < 0.05). Overexpression of ROR2 in the mammary epithelial cell line, MCF10A, increased both β-catenin-dependent and β-catenin-independent targets and decreased cell adhesion. Knock-down of ROR2 in the breast cancer cell lines, MDA-MB-453 and HCC1143, decreased both β-catenin-dependent and β-catenin-independent targets and increased cell adhesion. Treatment of ROR2-expressing breast cancer cells with the novel berberine derivative, NAX53, significantly inhibited cell proliferation and migration.

Conclusions

This is the first study to report the expression of ROR2 in breast cancer. Breast cancer patients expressing ROR2 had a significantly worse prognosis than those lacking ROR2. ROR2 may regulate both β-catenin-dependent and β-catenin-independent Wnt signalling pathways, and represents a potential diagnostic and therapeutic target.

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Acknowledgments

This study was funded, in part, by a National Health and Medical Research Council (NHMRC) Project Grant (#630458) awarded to CF. NAX53 was kindly provided by Paolo Lombardi, Naxospharma.

Conflict of interest

The authors declare that they have no conflict of interests.

Author information

Authors and Affiliations

  1. Adult Cancer Program, Level 2, Metastasis Research Group, Lowy Cancer Research Centre, Prince of Wales Clinical School, University of New South Wales, UNSW, NSW, 2052, Australia

    C. Henry, A. Quadir, E. Jary, E. Llamosas, D. Kumar, R. L. Ward & C. E. Ford

  2. Faculty of Medicine, School of Medical Science, University of New South Wales, UNSW, NSW, Australia

    N. J. Hawkins

  3. Faculty of Medicine, Translational Cancer Research Network and Prince of Wales Clinical School, University of New South Wales, UNSW, NSW, Australia

    B. Daniels

Authors
  1. C. Henry
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  2. A. Quadir
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  3. N. J. Hawkins
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  4. E. Jary
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  5. E. Llamosas
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  6. D. Kumar
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  7. B. Daniels
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  8. R. L. Ward
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  9. C. E. Ford
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Corresponding author

Correspondence to C. E. Ford.

Additional information

C. Henry and A. Quadir have contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Additional File 1: PDF file. qRT-PCR primer sequences and Antibody details. (DOCX 13 kb)

432_2014_1824_MOESM2_ESM.eps

Additional File 2: PDF file. Supplementary Figure showing results in a second breast cancer cell line. MDA-MB-453. Knock-down of ROR2 in a different TNBC cell line inhibits cell proliferation, migration and Wnt target genes. a: ROR2 knock-down in MDA-MB-453 breast cancer cells significantly inhibits cell proliferation over 24 h (n = 3). b: ROR2 knock-down in MDA-MB-453 breast cancer cells significantly increases cell adhesion to collagen over 24 h (n = 3) c: ROR2 knock-down alters expression of β-catenin-dependent and d: β-catenin-independent Wnt genes. qRT-PCR was performed in triplicate and normalised to three different housekeeping genes (SDHA, HSPCB, RPL13A). Results represent an average of 3 experiments. Error bars represent the standard deviation of the mean.*P < 0.05. E: Densitometric analysis of three separate experiments indicates that ROR2 is decreased, but EMT markers unaltered following knock-down in MDA-MB-453 cells. *P < 0.05. (EPS 4576 kb)

432_2014_1824_MOESM3_ESM.eps

Additional File 3: PDF file. Supplementary Figure showing results of IWP-2 treatment of HCC1143 cell line. Treatment with Wnt inhibitor IWP-2. a: Treatment of HCC1143 cells with IWP-2 did not significantly change cell proliferation over 24 h (n = 3). b: Treatment of HCC1143 cells with IWP-2 did not significantly change cell migration over 24 h (n = 3). c: Representative immunoblots of Wnt and EMT targets altered following IWP-2 treatment of HCC1143 cells. d: Densitometric analysis of three separate experiments indicates Wnt pathway and EMT components are altered at the protein level following treatment with IWP-2. In all graphs, black bars represent the DMSO control, and grey bars represent IWP-2 (5 µM) treatment. (EPS 1880 kb)

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Henry, C., Quadir, A., Hawkins, N.J. et al. Expression of the novel Wnt receptor ROR2 is increased in breast cancer and may regulate both β-catenin dependent and independent Wnt signalling. J Cancer Res Clin Oncol 141, 243–254 (2015). https://doi.org/10.1007/s00432-014-1824-y

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  • DOI: https://doi.org/10.1007/s00432-014-1824-y

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