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The role of TG2 in ECV304-related vasculogenic mimicry

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Abstract

Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry.

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Abbreviations

TG2:

Tissue transglutaminase

TAA:

Tumour angiogenic activity

VEGF:

Vascular endothelial growth factor

FGF:

Fibroblast growth factor

HUVEC:

Human umbilical vein endothelial cells

ECM:

Extracellular matrix

FN:

Fibronectin

DMEM:

Dulbecco’s modified Eagles media

FBS:

Fetal bovine serum

PBS:

Phosphate buffered saline

HRP:

Horseradish peroxidase

DAB:

Diaminobenzidine

FITC:

Fluorescein isothiocyanate

IF:

Immunofluorescence

IHC:

Immunohistochemistry

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Acknowledgments

This work is dedicated to Richard Jones who in his prime sadly died of brain cancer. He was both a dedicated scientist and talented artist.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, UK

    Richard A. Jones & Shakthi Dookie

  2. School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK

    Zhuo Wang & Martin Griffin

Authors
  1. Richard A. Jones
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  2. Zhuo Wang
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  3. Shakthi Dookie
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  4. Martin Griffin
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Correspondence to Martin Griffin.

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Jones, R.A., Wang, Z., Dookie, S. et al. The role of TG2 in ECV304-related vasculogenic mimicry. Amino Acids 44, 89–101 (2013). https://doi.org/10.1007/s00726-011-1214-6

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  • DOI: https://doi.org/10.1007/s00726-011-1214-6

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