Differential Effects of EGFL6 on Tumor versus Wound Angiogenesis

Abstract

Angiogenesis inhibitors are important for cancer therapy, but clinically approved anti-angiogenic agents have shown only modest efficacy and can compromise wound healing. This necessitates the development of novel anti-angiogenesis therapies. Here, we show significantly increased EGFL6 expression in tumor versus wound or normal endothelial cells. Using a series of in vitro and in vivo studies with orthotopic and genetically engineered mouse models, we demonstrate the mechanisms by which EGFL6 stimulates tumor angiogenesis. In contrast to its antagonistic effects on tumor angiogenesis, EGFL6 blockage did not affect normal wound healing. These findings have significant implications for development of anti-angiogenesis therapies.

Keywords: chitosan nanoparticles; ovarian cancer; tumor endothelial cells; tumor vasculature; wound healing.

Figure 1. EGFL6 is upregulated in tumor-associated endothelial cells but not in normal ovary and healing wound tissues

a, Human normal ovary, ovarian tumor, and healing wound tissues were dissociated, and isolated endothelial cells and samples were processed for microarray. b, Gene microarray of endothelial cells from normal ovary, healing-wound tissue, and ovarian tumor–associated endothelial cells. c, Expression of EGFL6 in human normal ovary, wound, and ovarian tumor samples. Representative images were taken from different samples. Scale bar =100 µm d, Validation of gene microarray data using q-PCR.(Normal ovary; n = 5, Ovarian tumor; n = 10, Wound; n = 7). Validation Error bars indicates SEM. *p<0.05 vs. Normal. See also Figure S1.

Figure 2. Effect of EGFL6 silencing on tumor growth in orthotopic ovarian cancer mouse models

a, Graph of wound area on mice treated with either control IgG antibody or DC101 (anti-VEGFR2) quantified on days 0 through 15 after a skin excision wound. (n=10 mice per group); error bars indicate SEM. *p<0.05 vs. Control IgG. b, One day after SKOV3ip1 tumor cell injection, a wound was created on the dorsal side of the mice. Animals were treated with either Control siRNA-CH or mEGFL6 siRNA-CH. Graphical depiction of wound areas quantified on days 0 through 15 after skin excision wound. c, Effect of mEGFL6 silencing on tumor growth; representative images of tumor burden. Tumor weights are shown in d and numbers of tumor nodules in e. (n=10 mice per group); error bars indicate SEM. *p<0.05 vs. Control siRNA. f, Hind limb ischemia. After arterial ligation, mice were separated into 3 groups (n = 5 mice per group): normal, ischemia-24 h, and 96 h. Blood flow was monitored before and after femoral artery ligation with use of serial color doppler. At each time point, tissue was harvested and frozen so that immunofluorescence staining could be performed. g, EGFL6 expression was increased in endothelial cells in the ischemic (hypoxic) condition compared with the normal conditions. Error bars indicate SEM. *p<0.05 vs. Normal. See also Figure S2.

Figure 3. TWIST1 induces EGFL6 expression under hypoxia

a, EGFL6 promoter reporter analysis under normoxia and hypoxic conditions. b, TWIST1 ectopic expression increases EGFL6 transcription activity. c, Increase in TWIST1 and EGFL6 expression in hypoxia and CoCl₂ treatment. d, Ectopic expression of TWIST1 increases EGFL6 expression in RF24 cells. e, ChIP analysis of TWIST1 binding to EGFL6 promoter region in hypoxia compared with normoxia. Cross-linked chromatin from RF24 cells incubated in hypoxia chamber for 48 h and immunoprecipitated with TWIST1 or IgG control antibodies. The input and immunoprecipitated DNA was subjected to PCR using primers corresponding to the base pairs upstream of EGFL6 transcription start site. f, EGFL6 gene silencing using siRNA leads to increased cell death in hypoxia condition. (n = 3) **p<0.005, *p<0.05 See also Figure S3.

Figure 4. Treatment of EGFL6 activates PI3K/AKT signaling

a, Expression level change in selected proteins after normalization by RPPA analysis. b and c, Western blotting of EGFL6-mediated activation of PI3K/AKT signaling, Tie2 and EGFR signaling. d, RF24 cells treated with Control (PBS) or EGFL6. Extracts were subjected to anti-Tie2 immunoprecipitation (IP) and integrin α5, αV, β1, and β3 detected by immunoblotting. e, Expression level of pTie2 and pAKT in cytosol and membrane fractions with Control (PBS) and EGFL6 treatment. αβ-tubulin was used as internal control of cytosolic fraction; NaK ATPase was used as membrane marker. f, Silencing of Integrin β1 (ITGB1) and Tie2 using specific siRNAs decreases Tie2 and AKT signaling. g–h, Silencing of Integrin β1 (ITGB1) and Tie2 decreases EGFL6-mediated tube formation (g) and migration (h) in endothelial cells. *p<0.05 vs. Control siRNA. In i–k, RGD blocking peptide decreases Tie2/AKT activation (i), tube formation (j) and migration (k). (n=3) *p<0.05 vs. Control. See also Figure S4.

Figure 5. EGFL6 blocking antibody reduces angiogenesis and tumor growth

a, The binding affinity of recombinant EGFL6 to monoclonal antibody #93 and #135 was measured by Biacore. The dissociation constant (Kd) value of the monoclonal antibodies were calculated to be 1.89 nM (#93) and 2.19 nM (#135). b, Effect of EGFL6 blocking antibodies on Tie2/AKT activation in RF24 cells (n=3). c, Effect of EGFL6 blocking antibodies on wound healing in vivo (n=5 mice/group, #135; 5 mg/kg). d and e, EGFL6 antibodies decreased tube formation and migration of RF24 cells. f, Effect of EGFL6 blocking antibodies on tumor weight and tumor nodules in SKOV3ip1 tumor-bearing mice. Seven days after tumor cell injection, mice were randomly divided into three groups (10 mice/group) to receive therapy: (1) Control IgG Ab, (2) EGFL6 #93, and (3) EGFL6 Ab #135 (5 mg/kg). Antibody treatment was given once a week. g, Effect of targeted EGFL6 on proliferation (Ki67) and microvessel density (CD31). Scale bar = 100µm. The bars in the graphs correspond sequentially to the labeled columns of images on the left. Error bars indicates SEM. *p<0.05 vs. Control IgG. See also Figure S5.

Figure 6. Effect of endothelial-specific EGFL6 knock-out on tumor growth and angiogenesis

a, Tumor growth in Tie2;EGFL6 KO mice (KO) and WT mice. ID8 murine ovarian cancer cells were injected into KO and WT mice (n=10 mice per group). b, Double immunofluorescence staining of CD31 and EGFL6 in ID8 tumor from WT and KO mice. Scale bar = 100 µm. c, Bar graph represents tumor weight. d and e, Proliferation (Ki67) and microvessel density (CD31) counted in WT and KO mice tumor sections. Error bars indicate SEM. Scale bar = 100 µm. *p<0.05 vs. WT mice. f, Representative images of human ovarian cancer vasculature with low or high immunohistochemical staining for EGFL6. Scale bar =200 µm. Representative images were taken from different samples. g, Kaplan-Meier curves of disease-specific mortality of patients whose ovarian vasculature expressed low versus high EGFL6. See also Figure S6 and Table S1.