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Perlecan Knockdown in Metastatic Prostate Cancer Cells Reduces Heparin-binding Growth Factor Responses in vitro and Tumor Growth in vivo

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Abstract

Perlecan (Pln) is a major heparan sulfate proteoglycan (HSPG) of extracellular matrices and bone marrow stroma. Pln, via glycosaminoglycans in domains I and V, acts as a co-receptor for delivery of heparin binding growth factors (HBGFs) that support cancer growth and vascularization. Specifically, glycosaminoglycans bind HBGFs and activate HBGF receptors, including those for FGF-2 and VEGF-A. The contribution of Pln to prostate cancer growth was tested using a ribozyme approach to knockdown Pln expression levels. Transfection into the androgen-independent, bone targeted prostate cancer line, C4-2B, and efficient stable knockdown of Pln was demonstrated by quantitative PCR, immunohistochemistry and immunoblotting. Three individually isolated subclones with 75–80% knockdown in Pln mRNA, protein expression and secretion into ECM were used to study in vitro growth responses to FGF-2 and VEGF-A. While cells with normal Pln levels responded to both HBGFs, knockdown cells responded poorly. All lines responded to serum growth factors and IGF-I. Anchorage-independent growth assays showed reduced colony size and cohesiveness by all Pln deficient subclones compared to parental C4-2B cells. In vivo effects of Pln knockdown were measured by inoculating knockdown and control ribozyme transfected cell lines into athymic mice. A reduced growth rate, smaller tumor size, diminished vascularization and failure to elevate serum PSA characterized mice bearing Pln knockdown C4-2B cells. Poor vascularization correlated with reduced levels of VEGF-A secreted by Pln knockdown lines. We conclude that Pln is an essential ECM component involved in growth responses of metastatic prostate cancer cells to HBGFs deposited in local and metastatic microenvironment.

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Abbreviations

: ECM:

extracellular matrix

FBS:

fetal bovine serum

FGF-2:

basic fibroblast growth factor

FGF-BP:

fibroblast growth factor binding protein

HBGF(R):

heparin binding growth factor (receptor)

HSPG:

heparan sulfate proteoglycan

PBS:

phosphate buffered saline

Pln:

perlecan

SCID:

severe combined immunodeficient

VEGF-A:

vascular endothelial growth factor

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Authors and Affiliations

  1. Department of Biological Sciences, University of Delaware, Newark, Delaware, 19716, USA

    Cristiana Savorè, Chu Zhang, Caroline Muir, Riting Liu, Jeffrey Wyrwa, Daniel D. Carson & Mary C. Farach-Carson

  2. Molecular Urology and Therapeutics Program, Department of Urology and Winship Cancer Institute, School of Medicine, Emory University, Atlanta, Georgia, USA

    Jun Shu, Haiyen E. Zhau & Leland W.K. Chung

Authors
  1. Cristiana Savorè
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  2. Chu Zhang
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  3. Caroline Muir
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  4. Riting Liu
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  5. Jeffrey Wyrwa
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  6. Jun Shu
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  7. Haiyen E. Zhau
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  8. Leland W.K. Chung
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  9. Daniel D. Carson
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  10. Mary C. Farach-Carson
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Corresponding author

Correspondence to Mary C. Farach-Carson.

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Savorè, C., Zhang, C., Muir, C. et al. Perlecan Knockdown in Metastatic Prostate Cancer Cells Reduces Heparin-binding Growth Factor Responses in vitro and Tumor Growth in vivo. Clin Exp Metastasis 22, 377–390 (2005). https://doi.org/10.1007/s10585-005-2339-3

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  • DOI: https://doi.org/10.1007/s10585-005-2339-3

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