A Macrophage-Dominant PI3K Isoform Controls Hypoxia-Induced HIF1α and HIF2α Stability and Tumor Growth, Angiogenesis, and Metastasis

PI3K/AKT signaling axis control HIFα/VEGF axis. A and B, BMDMs from WT animals were treated with different concentrations of PI3K inhibitors (500 nmol/L PF4691502, PI-103, BKM120 and 25 μmol/L SF1126) followed by hypoxia for 4 hours for Western blots or 24 hours for real-time PCR studies. These macrophages were either used for lysate preparation (nuclear extracts for HIFα or WCE for pAKT and AKT) and Western blot analysis (A) or RNA and real-time PCR for VEGF (B). C and D, WT BMDMs were treated with different concentrations of SF2523 (10, 20, and 50 μmol/L) followed by hypoxia for 4 hours for Western blot analyses (C) or 24 hours for RNA isolation and real-time PCR for VEGF (D). E and F, BMDMs from WT animals were transfected with either 5 μg of myrAKT plasmid or 100 nmol/L control siRNA or 100 nmol/L siRNA against AKT1/2 using Amaxa Mouse Macrophage Nucleofaction kit (Lonza). Thirty-six hours after transfection, hypoxia was given to these transfected BMDMs for 4 hours followed by cell lysate preparation and Western blot analysis. Graphs in B and D represent mean ± SEM (n = 3–4). Statistical significance is assessed by 2-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Experiments were repeated 3 to 4 times with similar results.

Specific p110γ and p110δ isoform of PI3K control HIFα/VEGF axis in macrophages. A and B, BMDM from WT animals were treated with 100 nmol/L of GDC0941or TGX221or Cal101 or AS605240 followed by hypoxia for 4 hours for Western blot analysis or 24 hours for RNA isolation and real-time PCR analysis. C and D, BMDMs from WT animals were transfected with either 100 nmol/L control siRNA or siRNA against p110 α or β or δ or γ isoform using Amaxa Mouse Macrophage Nucleofaction kit (Lonza). Thirty-six hours after transfection, hypoxia was given to these siRNA-transfected BMDMs for 4 hours for Western blot analysis (C) or 24 hours for RNA isolation and real-time PCR analysis (D). E, Western blot analysis of HIF1α and HIF2α from WT and p110γ^−/− BMDMs kept in hypoxic (1% O₂) conditions for 4 hours followed by preparation of nuclear extracts. Each lane in figure represents lysate prepared from an individual mouse. F, relative VEGF expression in WT, p110 γ^−/− BMDMs treated with or without 100 nmol/L AS605240 and kept in hypoxia (1% O₂) for 24 hours, followed by RNA isolation and real-time PCR studies. Graphs in B, D, and F represent mean ± SEM (n = 4–5) for 3 independent experiments. Statistical significance is assessed by 2-sample t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Hypoxic degradation of HIF1α and HIF2α by PTEN/PI3K/AKT pathway occurs via the 26S proteasome. A, Western blot analysis of HIF1α and HIF2α from BMDMs from PTEN^MC-WT and PTEN^MC-KO treated with 10 μmol/L MG132 and kept in hypoxic conditions for 4 hours. B–D, WT BMDMs were treated with 10 μmol/L proteasome inhibitor MG132 for 5 minutes before being pulsed with the pan-PI3K inhibitors, 25 μmol/L SF1126, and 500 nmol/L PF4691502 (B), 100 nmol/L AS605240 (C), and 10 μmol/L SF2523 (D) followed by normoxia (21% O₂) or hypoxia (1% O₂) for 4 hours. Nuclear extracts were prepared for HIFα. Western blot analyses are as described before. Experiments were repeated 3 to 4 times with similar results.

Expression of p110γ isoform in macrophages promotes tumor growth and angiogenesis. A, tumor growth of LLC cells inoculated subcutaneously in WT and p110γ^−/− mice (n = 6–8). B, left, representative immunofluorescent staining of tumor vasculature by CD31 (green) and counterstain by DAPI (blue) on frozen tumor sections of LLC tumors implanted subcutaneously in WT and p110γ^−/− mice. Right, reduced microvascular density (MVD) in tumors isolated from p110γ^−/− animals as compared with WT animals. MVD was determined by counting the number of microvessels per high-power field (HPF) in the section with an antibody reactive to CD31. Microvessels were counted blindly in 5 to 10 randomly chosen fields, and data are representative of 3 independent experiments with 4 to 5 mice. *, P < 0.05 versus WT. C, relative expression levels of specific genes required for tumor growth in macrophages sorted from LLC tumors implanted into WT and p110γ^−/− animals. D, left, representative photograph of pulmonary metastatic foci produced 15 days after intravenous injection of B16F10 cells in WT and p110γ^−/− mice. Right, number of experimental pulmonary B16F10 metastases in lungs counted under dissecting microscope. E, expression of specific tumor-promoting genes in the BMDMs isolated from WT and p110γ^−/− mice and cultured in MCSF in vitro. Graphs present mean ± SEM of 7 mice for A, B, and D and 3 to 4 mice for C. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus WT, as determined using 2-sample t test.

SF1126, a pan-PI3K inhibitor blocks tumor growth, angiogenesis, metastasis and reduces the expression of proangiogenic factors by TAMs. A, tumor volume of LLC inoculated subcutaneously in WT mice treated with or without 50 mg/kg SF1126 3 times a week. Treatment was started 10 days after inoculation of LLC cells and was continued until tumors were harvested (n = 6–8). B, MVD was scored by manual counting of CD31-positive vessels in subcutaneous LLC tumor sections was reduced in tumors isolated from WT animals treated with SF1126. Right, reduced microvascular density in tumors isolated from untreated or SF1126-treated animals as confirmed by quantification of CD31-positive area in high-power field (HPF) C, relative expression levels of specific genes required for tumor growth in macrophages sorted from LLC tumors implanted into WT mice and treated with 50 mg/kg of SF1126, 3 times a week until tumors were harvested. D, left, representative photograph of pulmonary metastatic foci produced in WT animals injected with B16F10 melanoma and treated with daily dose of 50 mg/kg SF1126 for 15 days. Right, number of experimental pulmonary B16F10 metastases in lungs. E, figure shows representative lung hematoxylin and eosin (H&E) sections of B16 metastases from WT mice treated with or without 50 mg/kg SF1126. Arrows point to metastatic foci. F, Kaplan–Meier survival curve for B16 metastasized mice treated with or without 50 mg/kg SF1126 daily. Data are representative of 2 to 3 independent experiments. Graphs present mean ± SEM of 8 to 10 mice for A, B, and D–F and 3 to 4 mice for C. *, P < 0.05 and **, P < 0.01 versus WT, as determined using 2-sample t test.