RAS interaction with PI3K p110α is required for tumor-induced angiogenesis

Abstract

Direct interaction of RAS with the PI3K p110α subunit mediates RAS-driven tumor development: however, it is not clear how p110α/RAS-dependant signaling mediates interactions between tumors and host tissues. Here, using a murine tumor cell transfer model, we demonstrated that disruption of the interaction between RAS and p110α within host tissue reduced tumor growth and tumor-induced angiogenesis, leading to improved survival of tumor-bearing mice, even when this interaction was intact in the transferred tumor. Furthermore, functional interaction of RAS with p110α in host tissue was required for efficient establishment and growth of metastatic tumors. Inhibition of RAS and p110α interaction prevented proper VEGF-A and FGF-2 signaling, which are required for efficient angiogenesis. Additionally, disruption of the RAS and p110α interaction altered the nature of tumor-associated macrophages, inducing expression of markers typical for macrophage populations with reduced tumor-promoting capacity. Together, these results indicate that a functional RAS interaction with PI3K p110α in host tissue is required for the establishment of a growth-permissive environment for the tumor, particularly for tumor-induced angiogenesis. Targeting the interaction of RAS with PI3K has the potential to impair tumor formation by altering the tumor-host relationship, in addition to previously described tumor cell-autonomous effects.

Figure 6. Interaction of RAS with PI3K and the immune system.

B16F10 subcutaneous tumors were collected after 14 days of injection. (A and B) Paraffin sections stained with F4/80 antibody. Arrows in A indicate typical macrophages. (C) Quantification of F4/80⁺ cells in the viable tumor area. n = 5 per group. (D) Total mRNA from whole tumor samples was isolated, and Vegfa expression was analyzed (n = 4 per genotype, pooled in groups of 2). (E) Unsorted tumor cells were plated overnight in a petri dish, and supernatant was collected for ELISA analysis of secreted VEGF-A. n = 3. Scale bars: 100 μm (A, top, and B); 50 μm (A, bottom).

Figure 5. Functional interaction of RAS with PI3K is needed for efficient angiogenesis.

(A and B) Angiogenesis induced by Matrigel plugs containing VEGF-A (A) or FGF-2 (B). Paraffin-embedded sections were stained for endomucin. Dashed outlines denote the Matrigel plug. (C) Matrigel plugs were quantified from paraffin-embedded sections stained for endomucin. n = 8. (D) The ability of endothelial cells to form capillary-like structures was analyzed in a tubulogenesis assay. Cells were treated with VEGF-A (40 ng/ml) or FGF-2 (10 ng/ml). (E) Matrigel plug sections stained for Ki67. (F) Quantification of E using NIS-Elements program. n = 5 per group. Scale bars: 100 μm.

Figure 4. Disruption of the interaction of RAS with PI3K reduces tumor-induced angiogenesis.

B16F10 subcutaneous tumors were collected after 14 days of injection. (A) Paraffin sections were stained with endomucin and SMA specific antibodies. (B and C) Endomucin-stained sections were subjected to quantitative analysis using Leica’s Digital Pathology capture and analysis platform to analyze vessel density (B) and total vessel area (C). Mann-Whitney statistical analysis was performed (n = 5 per group). (D) Lung sections from experimental metastasis assays were also collected, and paraffin sections were stained with endomucin and nestin specific antibodies. Representative single nodules for each genotype are shown. Arrows denote preexisting, nesting-negative vessels in the periphery of the nodules. Scale bars: 100 μm (A, right, and D); 500 μm (A, middle); 1,000 μm (A, left).

Figure 3. Disruption of the interaction of RAS with PI3K in the host reduces metastasis.

(A) Luciferase activity of B16F10 cells injected in the tail vein was measured ex vivo 21 days after injection. Bars represent the median. Mann-Whitney nonparametric statistical analysis was performed. (B) H&E staining of lung sections. Arrows indicate diffuse nodules in the periphery of the lung. Scale bars: 1,000 μm (left); 100 μm (right). (C) Number of tumor foci per whole lung. (D) Tumor area, expressed as a percentage of total lung area. (E) Average foci area, expressed as a percentage of lung area. (C–E) Mann-Whitney statistical analysis was performed. n = 6 per group.

Figure 2. Disruption of the interaction of RAS with PI3K in the host reduces tumor growth.

B16F10 (A–C) and LLC1 (D–F) cells were injected into the indicated mice. (A and D) Tumor volumes, measured by caliper over time. n = 11 (A); 6 (D). (B and E) Tumor volume 18 (B) or 21 (E) days after cell injection. Horizontal bars represent the median. Mann-Whitney nonparametric statistical analysis was performed. (C and F) Overall survival. The endpoint of the analysis was set in 2 cm of length in any direction, as stated in the approved Animal Project License. Kaplan-Meier survival curves are shown. Mantel-Cox (Log-rank) statistical analysis was performed. n = 6 (C); 6 (F, WT/– and MUT/–); 10 (F, WT/fl and MUT/fl).

Figure 1. Generation of a conditional RBD mutant Pik3ca mouse model.

(A) In order to assess the degree of recombination of the floxed Pik3ca allele, different tissues from a group of mice were collected no earlier than 21 days after tamoxifen food was withdrawn. DNA extracts were prepared and subjected to PCR analysis for detection of the floxed Pik3ca allele (upper band, 500 bp; lower band, 400 bp). (B–E) Primary MEFs were obtained from tamoxifen-untreated mice containing either the RBD mutant or the WT Pik3ca allele. Cells were treated with vehicle or tamoxifen (1 μM) for 16 hours followed by 24 hours in complete, tamoxifen-free media. Subsequently, cells were starved for 16 hours and treated with EGF (20 ng/ml) (B and C), PDGF (20 ng/ml) (D and E), or vehicle for 10 minutes. Protein extracts were prepared and subjected to Western blot analysis (B and D). Fluorescent secondary antibodies were used, and subsequent quantification in a LI-COR system was performed (C and E). A representative experiment is shown.