P70S6K 1 regulation of angiogenesis through VEGF and HIF-1alpha expression

Abstract

The 70kDa ribosomal S6 kinase 1 (p70S6K1), a downstream target of phosphoinositide 3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK), is an important regulator of cell cycle progression, and cell proliferation. Recent studies indicated an important role of p70S6K1 in PTEN-negative and AKT-overexpressing tumors. However, the mechanism of p70S6K1 in tumor angiogenesis remains to be elucidated. In this study, we specifically inhibited p70S6K1 activity in ovarian cancer cells using vector-based small interfering RNA (siRNA) against p70S6K1. We found that knockdown of p70S6K1 significantly decreased VEGF protein expression and VEGF transcriptional activation through the HIF-1alpha binding site at its enhancer region. The expression of p70S6K1 siRNA specifically inhibited HIF-1alpha, but not HIF-1beta protein expression. We also found that p70S6K1 down-regulation inhibited ovarian tumor growth and angiogenesis, and decreased cell proliferation and levels of VEGF and HIF-1alpha expression in tumor tissues. Our results suggest that p70S6K1 is required for tumor growth and angiogenesis through HIF-1alpha and VEGF expression, providing a molecular mechanism of human ovarian cancer mediated by p70S6K1 signaling.

Fig. 1

Knockdown of p70S6K1 inhibited VEGF protein expression and VEGF transcriptional activity. (A) OVCAR-3 cells stably expressing p70S6K1 siRNA (sip70S6K1) or scrambled control (scrambled ctrl) were cultured to 100% confluence. The cells were changed to fresh complete medium. Aliquots of cell supernatant were analyzed for VEGF protein levels by ELISA. (B) Cells were seeded in 12 well plates and cultured to 40–50% confluence, then transfected with VEGF promoter reporter pGL-Stu1, pCMV-β-galactosidase plasmid (β-gal), sip70S6K1 and wild-type HIF-1α plasmids at the concentrations as indicated. The empty vector was added to make total transfected DNA to 1.15 µg. Relative Luc activity was calculated as the ratio of Luc/β-gal activity. (C) The similar experiment was performed using pMAP11wt reporter as described for pGL-Stu1 reporter. (D) The pMAP11 mutant reporter was used to perform the similar experiment. *, indicates the significant difference (p<0.05).

Fig. 1

Knockdown of p70S6K1 inhibited VEGF protein expression and VEGF transcriptional activity. (A) OVCAR-3 cells stably expressing p70S6K1 siRNA (sip70S6K1) or scrambled control (scrambled ctrl) were cultured to 100% confluence. The cells were changed to fresh complete medium. Aliquots of cell supernatant were analyzed for VEGF protein levels by ELISA. (B) Cells were seeded in 12 well plates and cultured to 40–50% confluence, then transfected with VEGF promoter reporter pGL-Stu1, pCMV-β-galactosidase plasmid (β-gal), sip70S6K1 and wild-type HIF-1α plasmids at the concentrations as indicated. The empty vector was added to make total transfected DNA to 1.15 µg. Relative Luc activity was calculated as the ratio of Luc/β-gal activity. (C) The similar experiment was performed using pMAP11wt reporter as described for pGL-Stu1 reporter. (D) The pMAP11 mutant reporter was used to perform the similar experiment. *, indicates the significant difference (p<0.05).

Fig. 2. Sip70S6K1 expression inhibited HIF-1α protein expression in ovarian cancer cells

(A) OVCAR-3 cells and A2780 cells stably expressing sip70S6K1 or scrambled control were seeded in RPMI 1640 medium with 10% FBS for 24 h. The total cellular protein extracts were prepared, and subjected to immunoblotting analysis using specific antibodies against HIF-1α and HIF-1β. (B) The cells were cultured in a 60 mm dish at a density of 5×10⁵ cells/dish for 24 h, followed by the incubation with serum-free medium for 24 h. Cells were switched to the medium in the presence or absence of 100 nM insulin for 6 h as indicated. The total cellular proteins were prepared and subjected to immunoblotting analysis using antibodies against HIF-1α and HIF-1β.

Fig. 3. Sip70S6K1 expression inhibited ovarian tumor angiogenesis and tumor growth in CAM model

(A) Two million of OVCAR-3 cells expressing sip70S6K1 or scrambled control were collected in serum-free medium and mixed with the equal volume of Matrigel. The mixture or the Matrigel alone was placed on the CAM of 9 days old chicken embryos. Angiogenesis of tumors in the CAM was photographed 5 days after implantation. The experiments were repeated twice with five embryos for each group and representative fields were photographed. (B) The relative blood vessel density was determined by measuring the number of blood vessels in a unit area on Day 5. Results are presented as mean ± SD from replicate experiments, and normalized to results obtained with the CAMs implanted by Matrigel alone. (C) The cells were treated and implanted onto CAM as above. The tumors were harvested after incubation for 9 days. The tumors were photographed and weighed. (D) Tumor weight was obtained from ten tumors in different embryos in each treatment. (E) Parts of tumor tissues from chicken embryos were frozen in liquid nitrogen, and total proteins were extracted and analyzed. Immunoblotting analysis was performed using antibodies against PCNA, phospho-p70S6K1 (Thr421/Ser424), total p70S6K, and β-actin. *, indicates the significant difference (p<0.05).

Fig. 3. Sip70S6K1 expression inhibited ovarian tumor angiogenesis and tumor growth in CAM model

(A) Two million of OVCAR-3 cells expressing sip70S6K1 or scrambled control were collected in serum-free medium and mixed with the equal volume of Matrigel. The mixture or the Matrigel alone was placed on the CAM of 9 days old chicken embryos. Angiogenesis of tumors in the CAM was photographed 5 days after implantation. The experiments were repeated twice with five embryos for each group and representative fields were photographed. (B) The relative blood vessel density was determined by measuring the number of blood vessels in a unit area on Day 5. Results are presented as mean ± SD from replicate experiments, and normalized to results obtained with the CAMs implanted by Matrigel alone. (C) The cells were treated and implanted onto CAM as above. The tumors were harvested after incubation for 9 days. The tumors were photographed and weighed. (D) Tumor weight was obtained from ten tumors in different embryos in each treatment. (E) Parts of tumor tissues from chicken embryos were frozen in liquid nitrogen, and total proteins were extracted and analyzed. Immunoblotting analysis was performed using antibodies against PCNA, phospho-p70S6K1 (Thr421/Ser424), total p70S6K, and β-actin. *, indicates the significant difference (p<0.05).