CXCR4/YY1 inhibition impairs VEGF network and angiogenesis during malignancy

Abstract

Tumor growth requires neoangiogenesis. VEGF is the most potent proangiogenic factor. Dysregulation of hypoxia-inducible factor (HIF) or cytokine stimuli such as those involving the chemokine receptor 4/stromal-derived cell factor 1 (CXCR4/SDF-1) axis are the major cause of ectopic overexpression of VEGF in tumors. Although the CXCR4/SDF-1 pathway is well characterized, the transcription factors executing the effector function of this signaling are poorly understood. The multifunctional Yin Yang 1 (YY1) protein is highly expressed in different types of cancers and may regulate some cancer-related genes. The network involving CXCR4/YY1 and neoangiogenesis could play a major role in cancer progression. In this study we have shown that YY1 forms an active complex with HIF-1alpha at VEGF gene promoters and increases VEGF transcription and expression observed by RT-PCR, ELISA, and Western blot using two different antibodies against VEGFB. Long-term treatment with T22 peptide (a CXCR4/SDF-1 inhibitor) and YY1 silencing can reduce in vivo systemic neoangiogenesis (P < 0.01 and P < 0.05 vs. control, respectively) during metastasis. Moreover, using an in vitro angiogenesis assay, we observed that YY1 silencing led to a 60% reduction in branches (P < 0.01) and tube length (P < 0.02) and a 75% reduction in tube area (P < 0.001) compared with control cells. A similar reduction was observed using T22 peptide. We demonstrated that T22 peptide determines YY1 cytoplasmic accumulation by reducing its phosphorylation via down-regulation of AKT, identifying a crosstalk mechanism involving CXCR4/YY1. Thus, YY1 may represent a crucial molecular target for antiangiogenic therapy during cancer progression.

Fig. 1.

The systemic angiogenic pathway responds to different treatments by producing vascular tissue within angioreactors lumen. (A) MRI assessment of lung and lymph node metastases. Coronal (a–f,) and sagittal (g) postcontrast T1-weighted images are shown. Lung metastases (*) and lymph node metastases (arrows) are well demonstrated on the T1-weighted images. (B) Representative images of angioreactors recovered 13 wk after implantation in SaOS mouse group (control) (a), SaOS mouse group after treatment with 100 nM of T22 peptide (b), shYY1 mouse group (c), and shYY1/T22– peptide treated mouse group (d). Mouse groups in a and c were treated with scrambled peptide. (C) Vessels were recovered from angioreactors and photographed: vessel mass from control SaOS-bearing mice (a), T22 peptide-treated SaOS-bearing mice (b), shYY1-bearing mice (c), and T22 peptide-treated shYY1-bearing mice (d). (D) Lectin fluorescence quantification of vessels 13 wk after implantation. Immunofluorescence intensity was measured and expressed relative to control mice inoculated with SaOS cells. Data are mean + SD; n = 5 independent mice per group with two angioreactors each. *P < 0.01 and ^#P < 0.05 vs. SaOS.

Fig. 2.

In vitro angiogenesis assay: the effect of treatment with T22 peptide and shYY1 silencing. (A) HAEC/fibroblast monolayer was cultured on Matrigel for 24 h; then SaOS or shYY1 cells were added to the culture with Opti-MEM medium without VEGF and basic fibroblast growth factor for 24 h (details are given in SI Materials and Methods). Cells were photographed, at different time points. (a) SaOS cells after 2 h of coculture. (b) SaOS coculture after 8 h. (c) SaOS coculture after 24 h. (d) SaOS coculture after 48 h. (e) SaOS coculture treated with T22 peptide after 24 h. (f) shYY1 cells plated on HAEC/fibroblast monolayer after 2 h. (g) shYY1 coculture after 24 h. (h) shYY1 cells cocultured after 48 h. (i) shYY1 coculture treated with T22 peptide after 24 h. (l) HAEC cells after 48 h. (B) HAEC cells (1 × 10⁴/well) were plated on 24-well Matrigel-coated plates. After 2 h, 1 × 10⁴ SaOS or shYY1 cells were stratified and cultured in minimum medium for 24 h, with or without 100 nM T22 peptide. Then cells were photographed, and tube formation was quantified with ImageJ analysis software. (a) SaOS cells. (b) SaOS cells treated with T22 peptide. (c) shYY1 cells. (d) shYY1 cells treated with T22 peptide. (e) HAEC cells in minimum medium without VEGF after 24 h. (f) HAEC cells treated with T22 peptide. (g) CD34 staining of SaOS coculture. (h) CD34 staining of shYY1 coculture. (i) SaOS cells and (l) shYY1 cocultures stained with CD34 (20× magnification). (C) HAEC cells were plated on Matrigel. Then SaOS or shYY1 cells were added with Opti-MEM medium without VEGF for 24 h. Four fields at 20× magnification were photographed, and tube formation was quantified with image analysis software, as described in Material and Methods. Tube junctions (a), area (b), and length (c), were quantified in at least four fields per sample and graphed as the mean ± SD, *P < 0.01, ^#P < 0.02, and ^§P < 0.001 vs. SaOS.

Fig. 3.

Effect of T22 peptide and YY1 silencing on VEGF expression. (A) Media (100 μL/sample) from cultured cells were analyzed by a specific VEGFR2 inhibition assay. Data are presented as percentage of control activation. The mean ± SD of data from three independent experiments is shown. ^§P < 0.001 vs. SaOS. (B) Representative Western blots of total protein extracts from SaOS cells and shYY1 cells, untreated or treated with T22 peptide, revealed with VEGFA, -B and -C antibodies. (C) Real-time PCR quantification of VEGF transcripts performed on total RNA extracts from untreated SaOS cells, SaOS cells treated with T22 peptide, untreated shYY1 cells, and shYY1 cells treated with T22 peptide and normalized with GAPDH. SaOS transcripts were considered equal to 1, and the relative fold of the other transcripts was reported. Data shown are the mean ± SD from three independent experiments. *P < 0.01, ^#P < 0.05, and ^§P < 0.001 vs. SaOS.

Fig. 4.

YY1 is a positive regulator of VEGF transcription. (A) VEGF promoter activity. SaOS and shYY1 cells, untreated or treated with T22 peptide, were transiently cotransfected with VEGF luciferase constructs and PRL-SV40. The amount of transfected DNA was maintained at 1.5 μg/well by the addition of the appropriated amount of the empty vector. After 48 h, cells were harvested for dual-luciferase assays. The relative promoter activity (fold) was the ratio of luciferase (Firefly/Renilla) value relative to SaOS cell value. Data represent the mean of three experiments ± SD. (a) VEGFA promoter activity. Treatment with T22 peptide and YY1 silencing reduced luciferase activity by only 15%, and differences were not significant compared with SaOS (^#P > 0.05). HIF reduced VEGFA promoter activity by 80% compared with SaOS (^§HIFα vs. SaOS, P < 0.001). (b) Luciferase assays of VEGFB regulatory element. VEGFB promoter activity was reduced by 50% in SaOS cells and in shYY1 cells after treatment with T22 peptide (^§P < 0.001 vs. SaOS); treatment with T22 peptide and YY1 silencing had no additive effect. HIF reduced luciferase activity by 80% (^§P < 0.001 vs. SaOS). (c) Luciferase assays of VEGFC regulatory element. T22 peptide in SaOS cells reduced luciferase activity and YY1 silencing by 70% (*P < 0.01 vs. SaOS). Treatment with T22 peptide and YY1 silencing had no additive effect. HIF reduced luciferase activity by 80% (*P < 0.01 vs. SaOS). (B) (a) VEGFA, -B, and -C 5′ flanking regions of the VEGF transcription start site and hypothetical binding sites for YY1, Sp1, and TFIID. Nucleotide positions relative to the transcription start site are shown above the gene. (b) ChIP assays performed on VEGFA, -B, and -C regulatory elements. Chromatin from SaOS cells was immunoprecipitated with YY1, Sp1, and TFIID antibodies and amplified with primers spanning VEGF regulatory element binding sites. (C) Occupancy of YY1 at the VEGFA, -B, and -C binding sites was analyzed by ChIP in SaOS and shYY1 cells untreated or treated with T22 peptide. ChIP was performed using an antibody against YY1α or a nonspecific control. Immunoprecipitated DNA was quantified by real-time PCR using the indicated primer sets; GAPDH primers were used as control. Data are reported as the fold enrichment in the YY1 immunoprecipitation relative to the control. The means ± SD of data from three independent experiments are shown (*P <0.01 vs. SaOS). (D) Western blots of protein extracts from the indicated cells revealed with HIF1α (a). Protein extracts immunoprecipitated with HIF1α antibodies and revealed with YY1 (b). YY1 immunoprecipitates revealed with HIF1α antibodies (c).

Fig. 5.

T22 peptide blocks YY1 activity by impairing its serine phosphorylation via AKT. (A and B) Western blots of total protein extracts from SaOS and shYY1cells treated with T22 peptide at different time points revealed with specific antibodies, as indicated. Tubulin was used as loading control. (C) VEGFA protein expression in SaOS cells after treatment with T22 peptide and LY294002 inhibitor as indicated in figure. (D) Total protein extracts from SaOS cells untreated or treated with 100 nM T22 peptide and LY294002 inhibitor were immunoprecipitated with YY1 and immunoblotted with p-serine antibodies or immunoprecipitated with p-serine and immunoblotted with YY1. (E) Immunofluorescence of YY1 protein in SaOS cells and in SaOS cells treated with AKT inhibitor for 15 min (20× magnification, confocal microscope). DAPI was used for nuclear staining. (a) SaOS cells stained with YY1 antibodies. (b) SaOS nuclei stained with DAPI. (c) Merge. (d) SaOS cells treated with LY294002 for 15 min (Materials and Methods) stained with YY1 antibodies. (e) SaOS nuclei stained with DAPI. (f) Merge. (F) Immunofluorescence of YY1 protein in untreated SaOS cells and in SaOS cells treated with T22 peptide for 4 h (20× magnification). (a) SaOS cells stained with YY1 antibodies. (b) SaOS nuclei stained with DAPI. (c) Merge. (d) SaOS cells stained with YY1 antibodies after treatment with T22 peptide. (e) SaOS nuclei stained with DAPI. (f) Merge.