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The thrombin inhibitor Argatroban reduces breast cancer malignancy and metastasis via osteopontin-dependent and osteopontin-independent mechanisms

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Abstract

Osteopontin (OPN) has been clinically and experimentally associated with breast cancer metastasis. Proteolytic cleavage of OPN by thrombin has been reported to increase its biologic activity. The purpose of this study was to determine if inhibition of thrombin could reduce the malignancy-promoting effects of OPN on breast cancer cell behavior in vitro and in vivo. MDA-MB-468 human breast cancer cells were stably transfected to overexpress OPN (468-OPN) or a control vector (468-CON) and compared for functional differences in malignant/metastatic behavior in response to treatment with the thrombin-specific inhibitor Argatroban. Western blot analysis revealed that both 468-CON and 468-OPN cells produce thrombin and the thrombin-related protein tissue factor, and express very low levels of thrombin receptor (PAR-1). In vitro assays demonstrated that Argatroban treatment (25 μg/ml) of 468-OPN cells resulted in decreased cell growth, colony-forming ability, adhesion, and migration relative to untreated controls (P < 0.05), but did not have a significant effect on 468-CON cells. Following mammary fat pad injection, treatment with Argatroban (9 mg/kg/day) increased the in vivo tumor latency of both 468-CON and 468-OPN cells, and reduced primary tumor growth of 468-OPN cells (relative to untreated controls; P < 0.05). Furthermore, Argatroban treatment significantly decreased lymphatic metastasis of both 468-CON (P < 0.04) and 468-OPN (P < 0.01) cells relative to untreated controls. These novel findings indicate that inhibition of thrombin can reduce malignant and metastatic behavior of MDA-MB-468 breast cancer cells using both OPN-dependent and OPN-independent mechanisms, and suggest that thrombin inhibitors such as Argatroban may hold potential as therapeutic agents to combat breast cancer progression.

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Abbreviations

ANOVA:

Analysis of variance

BSA:

Bovine serum albumin

EDTA:

Ethylenediaminetetraacetic acid

EGFR:

Epidermal growth factor receptor

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

H&E:

Hematoxylin and eosin

HGF:

Hepatocyte growth factor

HPF:

High-powered field

HRP:

Horseradish peroxidase

LN:

Lymph node

LMWH:

Low molecular weight heparin

LVI:

Lymphovascular invasion

MEM:

Minimum essential media

OPN:

Osteopontin

PAR:

Protease-activated receptor

PE:

Phycoerythrin

PBS:

Phosphate-buffered saline

PMSF:

Phenylmethylsulfonyl fluoride

RGB:

Red green blue

RGD:

Arginine–Glycine–Aspartate

SDS:

Sodium dodecyl sulfate

TBST:

Tris-buffered saline + 0.05% Tween-20

TF:

Tissue factor

TGF-α:

Transforming growth factor α

VN:

Vitronectin

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Acknowledgements

We thank Michael Keeney and the London Health Sciences Centre Flow Cytometry Unit for their help in carrying out and analyzing the flow cytometry studies. We also thank Michael Keeney, Ian Chin-Yee, and Anargyros Xenocostas for helpful discussions and intellectual input. This work was supported in part by grants from the London Regional Cancer Program and the National Research Council of Canada (to A.L.A.) and by grant #04-MAY-00089 from the Ontario Cancer Research Network (to A.F.C. and A.B.T.). B.D.H. is the recipient of a Translational Breast Cancer Scholarship through the London Regional Cancer Program. A.F.C. is the recipient of a Canada Research Chair in Oncology. A.L.A. is supported by the Imperial Oil Foundation.

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Authors and Affiliations

  1. London Regional Cancer Program, London Health Sciences Centre, 790 Commissioners Road East, London, ON, Canada, N6A 4L6

    Erika B. Schulze, Benjamin D. Hedley, David Goodale, Carl O. Postenka, Waleed Al-Katib, Alan B. Tuck, Ann F. Chambers & Alison L. Allan

  2. Department of Oncology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

    Benjamin D. Hedley, Alan B. Tuck, Ann F. Chambers & Alison L. Allan

  3. Department of Medical Biophysics, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

    Benjamin D. Hedley & Ann F. Chambers

  4. Department of Pathology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

    Alan B. Tuck & Ann F. Chambers

  5. Department of Anatomy & Cell Biology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

    Alison L. Allan

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  1. Erika B. Schulze
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Correspondence to Alison L. Allan.

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Schulze, E.B., Hedley, B.D., Goodale, D. et al. The thrombin inhibitor Argatroban reduces breast cancer malignancy and metastasis via osteopontin-dependent and osteopontin-independent mechanisms. Breast Cancer Res Treat 112, 243–254 (2008). https://doi.org/10.1007/s10549-007-9865-4

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  • DOI: https://doi.org/10.1007/s10549-007-9865-4

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