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Laminin α5-derived peptides modulate the properties of metastatic breast tumour cells

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Abstract

The basement membrane protein laminin-511 has been implicated in breast cancer progression and metastasis. To identify peptides from LM-511 that modulate the metastatic properties of breast tumours, we screened laminin alpha 5 chain-derived peptides for their ability to promote adhesion of metastatic mammary carcinoma cells. Two selected adhesive peptides, α5A13b (FHVAYVLIKF) from the LN domain and A5G27 (RLVSYNGIIFFLK) from the LG-globular domain, were further characterised for their inhibitory properties against LM-511 activities in vitro and metastasis in vivo. In vitro, these peptides strongly inhibited LM-511-dependent adhesion and migration of highly metastatic 4T1.2 mammary carcinoma cells. In addition, A5G27 but not α5A13b significantly reduced breast tumour cell proliferation and inhibited laminin-511-induced matrix metalloproteinase-9 expression. Surprisingly, despite its potent inhibitory activity in vitro, A5G27 promoted rather than inhibited 4T1.2 experimental pulmonary metastasis in vivo, regardless of its route of administration. Adhesion of 4T1.2 cells to A5G27 was not inhibited by antibodies directed against α6, β1 or β3 integrins or CD44 but was significantly reduced in the presence of heparin suggesting a role for cell surface glycans. Treatment of the cells with α-l-fucosidase but not neuraminidase or heparinase II also partially inhibited cell adhesion to A5G27 and to LM-511 indicating that these interactions are mediated in part via terminal fucosyl residues. Overall, these results show that LMα5 peptides exhibit distinct functional properties in vitro and in vivo and suggest that interactions between the RLVSYNGIIFFLK sequence present in LM-511 and cell surface glycans may regulate LM-511 metastatic properties in vivo.

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Abbreviations

LM:

Laminin

ECM:

Extracellular matrix

MMP:

Matrix metalloproteinase

SFM:

Serum-free medium

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Acknowledgments

The authors wish to thank Prof. Bruce Kemp, Dr. Rohan Steel and Frosa Katsis (St. Vincent’s Institute of Medical Research, Melbourne, Australia) for validating laminin-derived synthetic peptide sequences, Colin House (Peter MacCallum Cancer Centre, Melbourne, Australia) for help with laminin-511 purification and Joshy George (Peter MacCallum Cancer Centre, Melbourne, Australia) for the statistical analysis. We thank Dr. Hynda K. Kleinman, NIH, USA for providing the initial set of laminin peptides. This work was supported by an Australian National Health & Medical Research Council Research Grant #454564 (NP), National Breast Cancer Foundation fellowship (RLA), and Dora Lush Postgraduate Research Scholarship, National Health and Medical Research Council (NK).

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Authors and Affiliations

  1. Metastasis Research Laboratory, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, VIC, 3002, Australia

    Nicole Kusuma, Robin L. Anderson & Normand Pouliot

  2. Pathology Department, University of Melbourne, Parkville, VIC, Australia

    Robin L. Anderson & Normand Pouliot

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  1. Nicole Kusuma
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  2. Robin L. Anderson
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  3. Normand Pouliot
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Correspondence to Normand Pouliot.

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Supplementary Fig. 1

Effect of A5G27S peptide on LM-511-induced expression of MMP-9 in 4T1.2 cells. 4T1.2 cells (1 × 106) were seeded in uncoated or LM-511-coated 24-well plates. Adherent 4T1.2 cells were incubated in the presence of A5G27S peptide (50 μg/ml) in SFM as described in Materials and Methods. a A representative image of gelatin zymography. b Quantitation of MMP-9 protein levels. The results represent mean fold induction relative to control (SFM)±SD from three independent experiments. **p<0.01 by One-way ANOVA. c Real time RT-qPCR analysis of MMP-9 mRNA. MMP-9 transcript levels were calculated relative to that of GAPDH. Values represent the mean ± SD of three independent replicate cultures. *p<0.05 by One-way ANOVA test. (JPEG 493 kb)

Supplementary Fig. 2

a Adhesion of 4T1.2 cells to A5G27 in the presence of 20 μg/ml integrin function-blocking antibodies. b Adhesion of 4T1.2 cells to A5G27 in the presence of 20 μg/ml CD44 function-blocking antibody. c Flow cytometric analysis of CD44 expression in 4T1.2 cells. Cell surface expression of CD44 was determined by standard flow cytometry as described in Materials and Methods. (JPEG 427 kb)

Supplementary Fig. 3

Effect of neuraminidase on 4T1.2 cell adhesion to A5G27 peptides and LM-511. Adhesion of calcein labelled 4T1.2 cells (4 × 104) to A5G27 or LM-511 coated wells was measured after one hour in the presence of neuraminidase as described in Materials and Methods. Results are expressed as mean percentage adhesion relative to normalised untreated controls (A5G27 or LM-511 alone) of three independent experiments. (JPEG 379 kb)

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Kusuma, N., Anderson, R.L. & Pouliot, N. Laminin α5-derived peptides modulate the properties of metastatic breast tumour cells. Clin Exp Metastasis 28, 909–921 (2011). https://doi.org/10.1007/s10585-011-9422-8

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  • DOI: https://doi.org/10.1007/s10585-011-9422-8

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