Carcinogenesis Advance Access originally published online on January 22, 2008
Carcinogenesis 2008 29(4):696-703; doi:10.1093/carcin/bgn019
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Cyr61/CCN1 and CTGF/CCN2 mediate the proangiogenic 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, SC 29425, USA
4 Present address: Cytopathology Unit, University of Rochester Medical Center, 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; Email: hsut{at}musc.edu
The von Hippel–Lindau (VHL) protein serves as a negative regulator of hypoxia-inducible factor (HIF)-
subunits. 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 proangiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor upregulated in VHL-mutant cells, the efficacy of antiangiogenic 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 with VHL wild-type cells, VHL-mutant RCC cells demonstrated a significantly increased proangiogenic activity, which correlated with increased secretion of cysteine-rich 61 (Cyr61)/cysteine-rich 61-connective tissue growth factor-nephroblastoma overexpressed (CCN) 1, connective tissue growth factor (CTGF)/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNA interference knockdown experiments. Importantly, the proangiogenic 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 RCC tissue samples showed that increased expression of these proteins correlates with the loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple proangiogenic factors that function in parallel pathways.
Abbreviations: CCN, cysteine-rich 61-connective tissue growth factor-nephroblastoma overexpressed; cRCC, clear cell renal cell carcinoma; CTGF, connective tissue growth factor; Cyr61, cysteine-rich 61; EGFP, enhanced green fluorescence protein; HDMEC, human dermal microvascular endothelial cell; HEK, human embryonic kidney; HIF, hypoxia-inducible factor; IHC, immunohistochemistry; PBS, phosphate buffered saline; RCC, renal cell carcinoma; shRNA, short-hairpin RNA; siRNA, short interfering RNA; TGF-β, transforming growth factor-β; VEGF, vascular endothelial growth factor; VHL, von Hippel–Lindau; WISP, Wnt-induced secreted protein; 3-D, three-dimensional
Received June 29, 2007; revised December 28, 2007; accepted January 8, 2008.