The Role of Vegf Family in Angiogenesis, Tumor Growth and Metastasis

Tumor growth is dependent on angiogenesis, and cells in tumor tissues produce various angiogenic factors to induce neovascularization. Among tumor-derived angiogenic factors, members of the vascular endothelial growth factor (VEGF) family are most frequently and highly expressed in various solid tumors. VEGF-A, the prototype of VEGF, is the most powerful pro-angiogenic factor that binds to VEGF receptor-1 (VEGFR-1, also called FMSRelated Tyrosine Kinase-1/Flt-1) and VEGFR-2 (also called Kinase Insert Domain Receptor/KDR or Fetal Liver Kinase -1/Flk-1). While the VEGFR-2-transduced angiogenic signals, pathways, and functions are well characterized, the VEGFR-1-mediated functions are poorly understood. The angiogenic functions of placental growth factor (PlGF), which is a specific VEGFR-1-binding ligand, remain controversial. The role of VEGF-B in tumor angiogenesis is still unclear. In addition, the two other VEGF family members, VEGF-C and VEGF-D are the major lymphangiogenic factors that contribute to lymphatic metastasis. The work contained in this thesis aimed to study the role of VEGF family members in angiogenesis, tumor growth and metastasis. Our work shows that PlGF exhibits a duality in modulation of angiogenesis and tumor growth in a VEGF-A-dependent manner. This is noted when the tumor cell-derived PlGF sensitizes the tumor to the anti-angiogenic and anti-tumor effects of anti-VEGF drugs. We also noted that anti-VEGF treatment induces various vascular alterations in mouse healthy tissues. Additionally, we revealed the collaborative interaction between FGF-2 and VEGF-C in promotion of lymphangiogenesis and metastasis. In paper I, using two independent tumor models, we show that PlGF modulated tumor growth, angiogenesis, and vascular remodeling through a VEGF-dependent mechanism in either a positive or a negative manner. In the VEGF-A positive model, PlGF inhibited tumor growth and angiogenesis, leading to normalized tumor vasculature with dilated vessel lumens, infrequent vascular branches and increased perivascular cell coverage. Surprisingly, in the VEGF-A negative model, overexpression of PlGF resulted in the opposite phenotype to that seen in the VEGF-A positive model, namely accelerated tumor growth rates and abundant chaotic tumor vessels. Our data uncovered the molecular mechanisms underlying the complex interplay between PlGF and VEGF-A. These findings have conceptual implications for anti-angiogenic cancer therapy. In paper II, we show that tumors from humans and mice with high levels of expression of PlGF were hypersensitive to anti-VEGF-A and anti-VEGFR-2 therapies. We then validated this finding with a loss-of-function experiment using PLGF shRNA in a human choriocarcinoma cell line. Down-regulation of PlGF significantly accelerated tumor growth rate and led to resistance to anti-VEGF drugs. We also show that VEGFR-2 and VEGFR-1 neutralizing antibodies displayed opposing effects on tumor growth and angiogenesis. These findings demonstrate that tumor-derived PlGF negatively modulates tumor angiogenesis and sensitizes treatment effect of anti-VEGF drugs in VEGF-A positive tumors, PlGF level in VEGF-A positive tumor may potentially be a predictive marker of anti-VEGF cancer therapy. In paper III, we investigated vascular alteration in various organs after systemic treatment with anti-VEGF-A, anti-VEGFR-1 and anti-VEGFR-2 neutralizing antibodies. This study provides functional and structural mechanisms for anti-VEGF drug-induced adverse effects in patients. In paper IV, we looked into the role of fibroblast growth factor-2 (FGF-2) and VEGF-C on angiogenesis, lymphangiogenesis and tumor metastasis. The results showed that FGF-2 and VEGF-C could both separately and collaboratively promote angiogenesis and lymphangiogenesis in the cornea of the mouse and in the mouse tumor tissue, resulting in pulmonary and lymph node metastases in animal models. By blocking VEGFR-3 and FGF receptor-1 (FGFR-1), we also revealed the fact that VEGFR-3-induced lymphatic endothelial cell (LEC) tip formation is a necessity for FGF-2-FGFR-1 signaling stimulated lymphangiogenesis. This study suggests that combined targeting of FGF-2 and VEGF-C might be an effective approach for cancer therapy and prevention of metastasis. LIST OF SCIENTIFIC PAPERS I. Xiaojuan Yang, Yin Zhang, Yunlong Yang, Sharon Lim, Ziquan Cao, Janusz Rak, Yihai Cao. Vascular endothelial growth factor-dependent spatiotemporal dual roles of placental growth factor in modulation of angiogenesis and tumor growth. Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):13932-7 II. Eva-Maria Eleonora Hedlund, Xiaojuan Yang, Yin Zhang, Yunlong Yang, Masabumi Shibuya, Weide Zhong, Baocun Sun, Yizhi Liu, Kayoko Hosaka, Yihai Cao. Tumor cell-derived placental growth factor sensitizes antiangiogenic and antitumor effects of anti-VEGF drugs. Proc Natl Acad Sci U S A. 2013 Jan 8;110(2):654-9. III. Yunlong Yang*, Yin Zhang*, Ziquan Cao*, Hong Ji, Xiaojuan Yang, Hideki Iwamoto, Eric Wahlberg, Toste Länne, Baocun Sun, Yihai Cao. Anti-VEGFand anti-VEGF receptor-induced vascular alteration in mouse healthy tissues. Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):12018-23 IV. Renhai Cao*, Hong Ji*, Ninghan Feng, Yin Zhang, Xiaojuan Yang, Patrik Andersson, Yuping Sun, Katerina Tritsaris, Anker Jon Hansend, Steen Dissing, Yihai Cao. Collaborative interplay between FGF-2 and VEGF-C promotes lymphangiogenesis and metastasis. Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):15894-9