Carcinogenesis Advance Access published online on January 22, 2008
Carcinogenesis, doi:10.1093/carcin/bgn019
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Cyr61/CCN1 and CTGF/CCN2 mediate the pro-angiogenic activity of VHL mutant renal carcinoma cells
1 Department of Pathology and Laboratory Medicine
2 Department of Medicine, Division of Rheumatology
3 Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29425, USA
4 Present address: University of Rochester Medical Center, Cytopathology Unit, 601 Elmwood Avenue, Box 626, Rochester, NY 14642, USA
* To whom correspondence should be addressed. Tel: +1 843 792 0638; Fax: +1 843 792 5002; E-mail: hsut{at}musc.edu
The von Hippel-Lindau (VHL) protein serves as a negative regulator of hypoxia inducible factor-alpha subunit (HIF-
). Since HIF regulates critical angiogenic factors such as vascular endothelial growth factor (VEGF) and lesions in VHL gene are present in a majority of the highly vascularized renal cell carcinoma (RCC), it is believed that deregulation of the VHL-HIF pathway is crucial for the pro-angiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor up-regulated in VHL mutant cells, the efficacy of anti-angiogenic therapy specifically targeting VEGF signaling remains modest. In this study we developed a three-dimensional in vitro assay to evaluate the ability of RCC cells to promote cord formation by the primary human dermal microvascular endothelial cells (HDMECs). Compared to VHL wild-type cells, VHL mutant RCC cells demonstrated a significantly increased pro-angiogenic activity, which correlated with increased secretion of Cyr61/CCN1, CTGF/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNAi knock-down experiments. Importantly, the pro-angiogenic activities conferred by the CCN proteins and VEGF are additive, suggesting non-overlapping functions. Expression of the CCN proteins is at least partly dependent on the HIF-2 function, the dominant HIF- isoform expressed in RCC. Finally, immunohistochemical staining of Cyr61/CCN1 and CTGF/CCN2 in renal cell carcinoma tissue samples showed that increased expression of these proteins correlates with loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple pro-angiogenic factors that function in parallel pathways.
Received June 29, 2007; revised December 28, 2007; accepted January 8, 2008.