COUP-TFII regulates tumor growth and metastasis by modulating tumor angiogenesis

Abstract

Tumor growth depends on nutrients and oxygen supplied by the vasculature through angiogenesis. Here, we show that the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), a member of the nuclear receptor family, is a major angiogenesis regulator within the tumor microenvironment. Conditional ablation of COUP-TFII in adults severely compromised neoangiogenesis and suppressed tumor growth in xenograft mouse models. In addition, tumor growth and tumor metastasis were also impaired in a spontaneous mammary-gland tumor model in the absence of COUP-TFII. We showed that COUP-TFII directly regulates the transcription of Angiopoietin-1 in pericytes to enhance neoangiogenesis. Importantly, provision of Angiopoietin-1 partially restores the angiogenic defects exhibited by the COUP-TFII-deficient mice, which supports the notion that COUP-TFII controls Angiopoietin-1/Tie2 signaling to regulate tumor angiogenesis. Because COUP-TFII has little impact on normal adult physiological function, our results raise an interesting possibility that inhibition of COUP-TFII may offer a therapeutic approach for anticancer intervention.

Fig. 1.

Essential role of COUP-TFII in adult angiogenesis. (A) Control (F/F: COUP-TF^flox/flox) and mutant (Cre/+; F/F: ROSA26^CRE-ERT2/+/COUP-TFII^{flox/flox )} mice were i.p. injected with tamoxifen to induce COUP-TFII deletion at 2 months of age. Matrigel plugs containing PBS or VEGF-A and FGF were implanted s.c. After 14 days, the plugs were removed and photographed. (B) Hemoglobin content of PBS or growth-factor–supplemented Matrigel from control and mutant mice (n = 10) is shown. (C) CD31 whole-mounted immunostaining images of angiogenic factor-containing plugs excised from control and mutant mice.

Fig. 2.

Inhibition of tumor growth in mice lacking COUP-TFII. (A) After COUP-TFII is ablated at 2 months of age, F/F (control) and Cre/+; F/F (mutant) mice were s.c. grafted with B16F10 melanoma cells, and the rate of tumor growth was measured at the indicated time (n = 15). (B) Immunofluorescence for CD31 and SMAα on melanoma tumor sections from control and mutant mice are shown. The graph shows the positive numbers of CD31 and SMAα positive signals per square millimeter (n = 5). (C) H&E staining, TUNEL assay, and Phospho-H3 staining in B16F10 tumor sections from control and mutant mice. Quantitative results of tumor-cell proliferation and apoptosis are shown (Lower). (D) After the melanoma tumors had reached the size of approximately 50 mm³, tamoxifen was injected to induce the COUP-TFII deletion. Tumor volume was measured at the indicated time (n = 15). (E) The tumor-growth rate of LLC in control and mutant mice (n = 15) is shown.

Fig. 3.

COUP-TFII promotes mammary tumorigenesis in vivo. (A) Image of whole thoracic and inguinal mammary glands excised from 18-week-old PyMT; F/F and PyMT; Cre/+; F/F mice. COUP-TFII was deleted at the age of 5 weeks old. (B) Total mammary-tumor burden of control and mutant mice is shown (n = 20). (C) Carmine alum-stained whole-mount mammary glands from control and mutant mice at the age of 4.5 months. The boxed areas were sectioned and subjected to H&E staining (Lower). N, lymph node. (D) Sections were immunostained with CD31, cleaved caspase-3, or Ki-67 antibody. Quantification results show the vessel density, tumor-cell proliferation, and apoptosis (n = 6). (E) The image shows the typical appearance of metastatic foci within the control lung as indicated by the arrows. The number of visible metastatic foci was counted (Right). (F) H&E-stained lung sections were prepared from control and mutant mice. Arrows indicate tumor foci. Microscopic tumors in the lungs were indicated by immunostaining with PyMT antibody. (G) The β-casein mRNA content in lungs from control and mutant mice was quantified by qRT-PCR analysis (n = 6).

Fig. 4.

Ang-1 is directly regulated by COUP-TFII to modulate angiogenesis. (A) In situ hybridization of Ang-1 expression in B16F10 tumor sections from control and mutant mice. (B) qRT-PCR analysis of Ang-1 and Ang-2 expression in the melanoma tumors from control and mutant mice (n = 6). Expression levels were normalized with CD31 transcript. (C) qRT-PCR analysis of Ang-1 transcripts in Movas and C3H10T1/2 fibroblast cells. (D) ChIP analysis showed that COUP-TFII and Sp1 bind to the region containing Sp1-binding sites in Ang-1 promoter (Top). The region in the first intron serves as a control (Middle). (E) Relative luciferase activity of Ang-1 promoter-driven reporter with or without cotransfection of COUP-TFII is shown. (F) Representative photos are shown of Matrigel plugs containing bFGF + VEGF-A or bFGF + VEGF-A + Ang-1 (Left). Hemoglobin content of the plugs was shown (n = 10; Center). Vascularization was visualized by CD31 whole-mount staining (Right).