Abstract 3474: Connective tissue growth factor promotes tumor angiogenesis by modulating multiple angiogenic factors via binding to cystine knot motifs

Abstract

Abstract Introduction Connective tissue growth factor (CTGF), a member of the CCN (Cyr61/CTGF/Nov) protein family, may function as both a positive and negative regulator of angiogenesis. However, the mechanism(s) underlying the regulatory behavior of CTGF is poorly understood. Here, we have undertaken a multipronged approach to provide insight into how CTGF enhances tumor angiogenesis. Methods Lewis lung carcinomas (LLC) cells were subcutaneously injected in syngeneic transgenic mice expressing green fluorescent protein (GFP) under the control of CTGF promoter (CTGFp-GFP). Recombinant lentivirus was generated to overexpress FLAG tagged CTGF while recombinant adenovirus expressing CTGF shRNA was used to knock down CTGF expression in LLCs. The levels of CTGF mRNA were examined by RT-PCR analysis. Tumor sizes were measured within two weeks and intratumoral microvessel density was quantified by immunochemical detection of Meca-32+ blood vessels. In addition, CTGF binding proteins were identified using the yeast two-hybrid approach. Immunoprecipitation assay was performed to verify these interactions. In vitro angiogenesis activity was indicated by tubule length formation by human umbilical vein endothelial cells (HUVECs). The migratory activity was measured in Boyden chamber assay. Activation of downstream signal effectors including cell division cycle 42 GTP binding protein (Cdc42) and extracellular signal-regulated kinase (ERK)1/2 was detected in Western blotting analysis. Results CTGF was expressed in Meca-32+ endothelial cells of intratumoral vessels growing in CTGFp-GFP transgenic mice. CTGF expression was also highly induced in hypoxic cultured LLC cells. Ectopic expression of CTGF promoted LLC tumor growth (n=3) and the microvessel density. Knocking down CTGF expression in LLC tumors (n=3) inhibited the growth of LLC tumors and decreased the microvessel density compared to those with scramble shRNA. Several proangiogenic factors were identified and shown to bind CTGF via cystine knot motifs. The factors include vascular endothelial growth factor (VEGF)-A, von Willebrand factor (vWF), platelet derived growth factor (PDGF)-B and Slit3. The combination of CTGF and Slit3 together resulted in an increased tubule length and an enhanced activation of Cdc42. Elevated cell migration and enhanced activation of ERK1/2 were observed when NIH3T3 fibroblasts were cultured with both CTGF and PDGF-B proteins. Although CTGF has been shown to bind to VEGF-A and inhibits VEGF-A mediated angiogenesis, our findings demonstrate that CTGF functions in concert with angiogenic factors Slit3 and PDGF-B to promote angiogenesis. Conclusion This study shows a critical role for CTGF in LLC tumor angiogenesis. CTGF binding to cystine knot motifs of key angiogenic factors represent a novel mechanism to regulate angiogenesis through PDGF-B and Slit3 signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3474. doi:10.1158/1538-7445.AM2011-3474