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Versican/PG-M G3 domain promotes tumor growth and angiogenesis¹

PENG-SHENG ZHENG^*,, JIANPING WEN^*, LEE CYN ANG, WANG SHENG^*,, ALICIA VILORIA-PETIT^*,, YELINA WANG^*,, YAOJIONG WU^*,, ROBERT S. KERBEL^*, and BURTON B. YANG^*,,2

^* Sunnybrook & Women’s College Health Sciences Centre,
Division of Neuropathology, University of Western Ontario,
Department of Medical Biophysics, and
Department of Laboratory Medicine and Pathobiology, University of Toronto

² Correspondence: S110, Research Building, Sunnybrook & Women’s College Health Sciences Centre, 2075 Bayview Avenue, Toronto M4N 3M5 Canada. E-mail: burton.yang{at}sw.ca

SPECIFIC AIMS

Expression of the extracellular proteoglycan versican is elevated in a variety of human tumors, and this study aimed to investigate the potential mechanisms by which versican may affect tumor growth and angiogenesis.

PRINCIPAL FINDINGS

1. Versican G3 construct promotes tumorigenesis and angiogenesis
We examined versican expression in patients with high grade astrocytoma using the monoclonal antibody 2B1 that recognizes an epitope at the C-terminal G3 domain of versican. Tissue lysate was subjected to SDS-PAGE on 4% gel followed by Western blot analysis. Proteoglycan of high molecular mass was detected as expected (Fig. 1 A). However, when the lysate was analyzed on 10% gel, a protein band with an apparent molecular mass of 48 kDa was observed. This result suggests that versican is cleaved in astrocytoma. Analysis of cell extracts from the astrocytoma cell line U87 and melanoma cell line A2058 also detected G3-containing bands. To test the potential role of the versican G3 domain in tumorigenesis, U87 cells were transfected with a versican G3 construct (Fig. 1B ). In colony formation assay, G3-expressing cells formed much larger colonies than did the vector-transfected cells (Fig. 1C ). When these cells were implanted into CD1 strain nude mice, G3-transfected cells formed larger tumors than did control vector-transfected cells (Fig. 1D ). Difference in tumor sizes between the G3- and vector-transfected cells became very significant at three weeks and reached a 10-fold difference six weeks after tumor grafting. These results indicated that G3 expression greatly promoted tumor growth. Immunocytochemistry with anti-CD31 antibody indicated that the tumors formed by G3-transfected cells contained more and larger blood vessels than those formed by vector-transfected cells, implying that the G3 product played a role in blood vessel formation.

View larger version (59K): [in this window] [in a new window]   Figure 1. Expression of G3 enhances colony formation, tumor growth and angiogenesis.A) Human astrocytoma tissues (patient samples 1 and 2) and tumor cell lines A2058 and U87 were lysed, and equal amounts of proteins were analyzed on Western blot in non-reducing conditions in gels containing 4% and 10% acrylamide probed with 2B1. Versican fragments were detected in all samples (arrows). B) Structure of versican and G3 construct are shown on the upper panel. Expression of the G3 construct in U87 cells is shown on the lower panel. C) In colony formation assays performed in soft agarose gel, G3-expressing cells formed larger colonies than the vector-transfected cells. (n=4; **P<0.01). A typical photograph of each is shown (lower panel, scale bar, 100 µ). D) G3- and vector-transfected U87 cells were introduced into nude mice at 107 cells per injection site. Two weeks after injection, the G3-expressing cells formed significantly larger tumors than cells transfected with a control vector (n= 0; *P<0.05; **P<0.01). Insert shows the sizes of the tumors at six weeks.

2. G3 enhances endothelial cell activities and G3 promotes expression of fibronectin and vascular endothelial growth factor (VEGF)
The effects of the G3 product on endothelial cell functions were investigated. In a cell attachment assay, coating with G3-containing medium enhanced attachment of endothelial cells. G3-containing medium enhanced cell proliferation and migration. In wound healing assays, treatment with G3-containing medium greatly enhanced endothelial cell migration to the wounding areas, as compared to control medium. These results demonstrated the effects of G3 on a number of endothelial cell activities which are known to be relevant and contribute to angiogenesis.

We next explored the possibility that G3 might affect the expression of other matrix molecules and other molecules regulating angiogenesis. RT-PCR analyses showed that the levels of fibronectin and VEGF mRNA were clearly higher in G3-expressing cells than in vector-transfected cells. VEGF isoforms showed different levels of enhancement of expression. Analysis of culture medium and cell lysates from G3- and vector-transfected U87 cells on Western blot and ELISA indicated that fibronectin expression was greatly elevated in G3-transfected cells compared to vector-transfected cells. Expression of fibronectin and VEGF was also assessed in sections from tumors originating from G3- and vector-transfected cells. Expression of fibronectin and VEGF was clearly higher in tumors formed by G3-transfected cells than those formed by vector-transfected cells.

3. Complex formed by versican, fibronectin and VEGF enhances endothelial cell adhesion, proliferation and migration
We investigated the existence of the binding activities of versican, fibronectin and VEGF in grafted mouse tumors and human astrocytomas, and also investigated the possibility of the formation of a complex of these three molecules. Immunoprecipitation experiments confirmed the binding and formation of the complex in grafted animal tumors, and agarose gel electrophoresis provided evidence of co-migration of these three molecules in samples from human brain tumors. The interaction of versican, fibronectin and VEGF was further confirmed by co-immunoprecipitation of samples prepared from grafted animal tumors and human brain tumors. Immunostaining showed a high expression of these three molecules in tumors grafted with G3-transfected U87 cells and in human astrocytoma tissues. Finally, we examined whether the formation of the complex affected endothelial cell activities. Combinations of fibronectin, VEGF, and purified G3 were tested. In all cases, the simultaneous presence of fibronectin, VEGF, and purified G3 promoted endothelial cell adhesion (Fig. 2 A), proliferation (Fig. 2B ), and migration (Fig. 2C ) when compared to treatments containing none, one, or two of these molecules. Results demonstrated an additive effect of these components of the putative complex. Removal of the complex with anti-fibronectin antibody reduced significantly the effect of G3-containing medium on endothelial cell adhesion (Fig. 2D ), proliferation (Fig. 2E ), and migration (Fig. 2F ). These results suggest that this complex may play important roles in modulating endothelial cell activities. This may be the molecular basis by which G3 promotes angiogenesis and at least in part, tumor growth.

View larger version (44K): [in this window] [in a new window]   Figure 2. The role of versican-fibronectin-VEGF complex in endothelial cell adhesion, growth and migration. A)Tissue culture plates were coated with different combinations of fibronectin (10 µg/mL), VEGF (10 ng/mL) and purified G3 product (1 ng/µL) as indicated, followed by inoculation of endothelial cells for attachment assay. Purified G3 product is shown as an insert. Cell attachment was significantly enhanced when the plates were simultaneously coated with fibronectin, VEGF and purified G3 product. In the presence of all three molecules, the level of cell attachment was the highest (*P<0.05, **P<0.01, analyzed with t-test). B) Endothelial cells were seeded on 24-well tissue culture plates, to which fibronectin, VEGF and purified G3 were added individually or in combination. Cell proliferation reached the highest level in the presence of all three molecules. C) A cell motility study was also performed on cell culture insert in the presence or absence of different combinations of the above three molecules. Similar results were obtained. When G3-containing medium was pre-treated with antifibronectin polyclonal antibody, its effect on cell attachment (D), growth (E) and migration (F) significantly decreased compared to the medium pre-treated with protein-A beads alone (**P<0.01).

CONCLUSIONS AND SIGNIFICANCE

Versican is one of the extracellular proteoglycans containing an N-terminal G1 domain, a glycosaminoglycan attachment region and a C-terminus G3 domain. This proteoglycan is highly expressed in the early stages of tissue development and its expression decreases after tissue maturation. Its expression is elevated during wound repair, and tumor formation and growth. In human tumors, versican is detected in the interstitial tissues at the invasive margins of breast carcinoma, and in the elastic tissues associated with tumor invasion. Increased versican immunostaining is also detected in tumor blood vessels. However, the effect of versican on tumorigenesis has not yet been understood. To investigate the role of versican in brain tumor formation, we examined versican expression in patients with high-grade astrocytoma. Unexpectedly, we detected high levels of versican G3-containing fragments in tumor tissues. To investigate if this endogenous versican G3-containing fragment affects tumor growth directly, we exogenously expressed a versican G3 construct in the human U87 astrocytoma cell line. Expression of this construct in U87 cells enhanced tumor growth and angiogenesis in nude mice greatly ( 10-fold). This is the first direct evidence that the versican G3 domain plays a role in tumorigenesis. Another novel aspect of our work is the observation that G3-expressing cells and tumors formed by these cells expressed very high levels of fibronectin and VEGF. Furthermore, the G3 domain directly interacted with fibronectin and formed a complex together with VEGF. In the presence of these three molecules, endothelial cell adhesion, proliferation, and migration were found to be significantly enhanced. Removal of the complexes reversed these processes. All of our results were consistent, and we conclude that expression of a versican G3 construct enhanced angiogenesis and promoted tumor growth by modulating endothelial cell activities favoring angiogenesis (Fig. 3 ). G3 stimulated the expression of fibronectin and VEGF and formed a complex with these two molecules. The complex promotes angiogenesis-associated activities in endothelial cells. Further studies are required to characterize the subdomains regulating formation of the ternary complex. This may provide useful information for development of anti-tumor therapies. Finally, while we have stressed the effects of versican G3 fragments on promoting tumor growth through a host mechanism involving angiogenesis, it is possible that a direct effect on tumor cells through enhanced cell survival mechanisms could also be making a contribution. This conclusion is based on the enhanced ability of versican G3-transfected tumor cells to form larger colonies in soft agarose. Our findings suggest a new avenue for development of anticancer and antiangiogenic therapies based on targeting versican G3 fragments.

View larger version (28K): [in this window] [in a new window]   Figure 3. Schematic diagram of versican’s role in tumorigenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0545fje; doi: 10.1096/fj.03-0545fje

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