Requirement for interaction of PI3-kinase p110α with RAS in lung tumor maintenance

Abstract

RAS proteins directly activate PI3-kinases. Mice bearing a germline mutation in the RAS binding domain of the p110α subunit of PI3-kinse are resistant to the development of RAS-driven tumors. However, it is unknown whether interaction of RAS with PI3-kinase is required in established tumors. The need for RAS interaction with p110α in the maintenance of mutant Kras-driven lung tumors was explored using an inducible mouse model. In established tumors, removal of the ability of p110α to interact with RAS causes long-term tumor stasis and partial regression. This is a tumor cell-autonomous effect, which is improved significantly by combination with MEK inhibition. Total removal of p110α expression or activity has comparable effects, albeit with greater toxicities.

Figure 1

Expression of p110α-RBD Induces Tumor Regression in Early-Stage Tumors (A) Schematic representation of experimental conditions. (B) Representative images of 4-week-old and 16-week-old mouse lungs treated and untreated with tamoxifen (TX). Graph showing tumor number on the pleural surface of lungs. Four-week-old group: Pik3ca^WT/flox n = 7 mice, Pik3ca^RBD/flox n = 11; 16-week-old group: Pik3ca^WT/flox n = 9, Pik3ca^RBD/flox n = 12, Pik3ca^WT/- n = 10, Pik3ca^RBD/- n = 11 mice. (C) Representative H&E-stained lung sections from 4-week-old and 16-week-old mice. Representation of tumor burden (tumor area as a percentage of total lung area) in 4-week-old mice and in 16-week-old mice 12 weeks after tamoxifen treatment. (D) Representation of average tumor size from the same group of mice mentioned above. (E) Quantification of TUNEL positive tumors in 16 weeks old Pik3ca^RBD/− and Pik3ca^WT/− mice. (F) Analysis of the proliferative state of Pik3ca^WT/− and Pik3ca^RBD/− tumors using phospho-histone H3 staining. Error bars indicate mean ± SEM (significance using Student’s t test: ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001). See also Figure S1.

Figure 2

Expression of p110α-RBD in Established Tumors Reduces Tumor Burden (A) Schematic representation of mouse treatment and micro-CT scanning intervals. (B) Ten-week-old mice were left untreated or were treated with tamoxifen for 2 weeks and the effect on tumor volume was assessed by micro-CT analysis over a 6-week period. Tumor volumes are plotted relative to the initial volume at the start of treatment. Black lines represent tumors from Pik3ca^WT/− mice and red lines represent tumors from Pik3ca^RBD/− mice. Green lines represent tumors from tamoxifen-treated Pik3ca^WT/WT mice that express Cre recombinase following treatment but do not have loxP sites flanking the Pik3ca gene. Pik3ca^WT/flox n = 4 mice, Pik3ca^RBD/flox n = 4, Pik3ca^WT/- n = 6, Pik3ca^RBD/- n = 4, Pik3ca^WT/WT n = 3 mice. (C) Quantification of TUNEL-positive tumors following tamoxifen treatment for 11 days. Pik3ca^WT/− n = 5 mice, Pik3ca^RBD/− n = 7 mice (D) Quantification of tumor multiplicity on the pleural surface of lungs. Representative images of lungs from Pik3ca^WT/flox, Pik3ca^RBD/flox, Pik3ca^WT/−, and Pik3ca^RBD/− mice at 16 weeks of age are shown. (E) Quantification of tumor burden (tumor area as a percentage of total lung area) in mice 6 weeks after commencement of tamoxifen treatment. Representative images of histological sections stained with H&E in Pik3ca^WT/flox, Pik3ca^RBD/flox, Pik3ca^WT/−, and Pik3ca^RBD/− mice at 16 weeks of age are shown. Error bars indicate mean ± SEM (significance using Student’s t test: ^∗p < 0.05 ^∗∗p < 0.01). See also Figure S2.

Figure 3

p110α-RBD Mutation Inhibits Tumor Progression (A) Schematic representation of mouse treatment and micro-CT scanning intervals. (B) The long-term effect of Pik3ca^RBD expression on tumor volume was assessed using micro-CT analysis. Lungs were scanned before tamoxifen treatment and at 3, 9, and 16 weeks after commencement of treatment. Tumor volumes are plotted relative to the initial volume at the start of treatment. Black lines represent tumors from Pik3ca^WT/− mice and red lines represent tumors from Pik3ca^RBD/− mice. Pik3ca^WT/− n = 3 mice, Pik3ca^RBD/− n = 3 mice. (C) Representative tomographs from micro-CT analysis of Pik3ca^WT/− and Pik3ca^RBD/− mice before tamoxifen treatment and 16 weeks later. Representative micro-CT images from each genotype 30 weeks after treatment are shown on the right. (D) Histological quantification of tumor burden, tumor size, and tumor number either 16 weeks or >32 weeks after the commencement of tamoxifen treatment. Representative histological images of lungs from Pik3ca^WT/− and Pik3ca^RBD/− mice are shown. (E) Western blot analysis of AKT phosphorylation in tumors extracted from the lungs of Pik3ca^WT/− and Pik3ca^RBD/− mice 15 weeks after commencement of tamoxifen treatment. Error bars indicate mean ± SEM (significance using Student’s t test: ^∗p < 0.05 ^∗∗p < 0.01). See also Figure S3.

Figure 4

Deficient Tumor Growth in p110α-RBD Mutant Mice Is a Cell-Autonomous Process (A) 3D models showing the growth of transplanted Pik3ca^WT/− and Pik3ca^RBD/− tumor cells in the lung based on micro-CT images. Due to the tumor occupying the complete lobe at 12 weeks after treatment in Pik3ca^WT/− inoculated lungs, modeling was not possible. Representative images of Pik3ca^WT/− and Pik3ca^RBD/− tumor cell inoculated lungs 14 weeks after tamoxifen treatment are shown in the most right panels. (B) Mice were scanned 6 and 12 weeks after tamoxifen treatment. The graph represents the normalized air volume in the lungs at the moment of the scanning. Black lines represent air content in NuNu mice bearing Pik3ca^WT/−-derived tumors and red lines represent air content in mice bearing Pik3ca^RBD/−-derived tumors. Representative tomographs from micro-CT analysis of Pik3ca^WT/−-derived and Pik3ca^RBD/−-derived tumors are shown at the right. (C) Representation of lifespan of NuNu mice bearing either Pik3ca^WT/−-derived or Pik3ca^RBD/−-derived tumors. NuNu mice containing Pik3ca^RBD/−-derived tumors were sacrificed when the last mouse from the control group had to be culled because of breathing problems. (D) Representative images of histological sections stained with H&E in mice inoculated with Pik3ca^WT/−-derived and Pik3ca^RBD/−-derived tumor cells 14 weeks after tamoxifen treatment.

Figure 5

Complete Removal of p110α in Established Tumors Similarly Reduces Tumor Burden (A) Schematic representation of mouse treatment and micro-CT scanning intervals. (B) Western blot analysis of p110α expression in tumors extracted from Pik3ca^flox/flox and Pik3ca^−/− lungs 3 weeks after commencement of tamoxifen treatment. (C) Ten-week-old mice were left untreated or were treated with tamoxifen for 2 weeks and the effect on tumor volume was assessed by micro-CT analysis over a 6-week period. Black lines represent tumors from Pik3ca^flox/flox mice and blue lines represent tumors from Pik3ca^−/− mice. Pik3ca^flox/flox n = 3 animals, Pik3ca^−/− n = 4 animals. (D) Change in tumor volume in Pik3ca^WT/WT, Pik3ca^WT/RBD, Pik3ca^WT/−, Pik3ca^RBD/-, and Pik3ca^−/− mice. Ten-week-old mice were left untreated or were treated with tamoxifen for 2 weeks and the effect on tumor volume was assessed by micro-CT analysis over a 6-week period. Tumor volumes are plotted relative to the initial volume at the start of treatment. (E) Histological quantification of tumor number and tumor burden in mice 6 weeks after commencement of tamoxifen treatment. Representative images of lungs from Pik3ca^flox/flox and Pik3ca^−/− at 16 weeks of age are also shown. (F) The long-term effect of Pik3ca removal on tumor volume was assessed using micro-CT analysis. Lungs were scanned before tamoxifen treatment and at various intervals up to 12 weeks after the commencement of treatment. Black lines represent tumors from Pik3ca^flox/flox mice and blue lines represent tumors from Pik3ca^−/− mice. Pik3ca^flox/flox n = 3 animals, Pik3ca^−/− n = 3 animals. (G) Glucose tolerance test of Pik3ca^flox/flox, Pik3ca^WT/−, Pik3ca^RBD/−, and Pik3ca^−/− mice 3 weeks after the end of tamoxifen treatment. Mice were injected with glucose (1 g/kg) and blood glucose levels were measured at the indicated time points. Glucose levels are plotted relative to the initial value before injection. Pik3ca^flox/flox n = 8 animals, Pik3ca^−/− n = 11 animals, Pik3ca^WT/− n = 6 animals, Pik3ca^RBD/− n = 8 animals. Error bars indicate mean ± SEM (significance using Student’s t test: ^∗p < 0.05). See also Figure S4.

Figure 6

Combined PI3-Kinase Inhibition with MEK Inhibition Induces Tumor Regression (A) Waterfall plot showing tumor response in 10-week-old mice that were treated with tamoxifen for 2 weeks, left untreated for 5 days, and then treated with trametinib for 4 weeks. Each bar represents one tumor and response is shown relative to initial tumor volume before tamoxifen treatment. (B) Representative histological images of lungs from mice, histological quantifications of tumor burden, and tumor number in mice treated as described in (A) are shown. (C) Kras^LA2 mice were treated with GDC0941 (100 mg/kg/day; green bars), BYL719 (50 mg/kg/day; purple bars), trametinib (3 mg/kg/day; blue bars), the combination of GDC0941 or BYL719 with trametinib (full dose for each drug; red and yellow bars, respectively), or vehicle alone (black bars). The waterfall plot represents response on tumor growth after 4 weeks treatment. Each bar represents one tumor and response is shown relative to initial tumor volume before tamoxifen treatment. Representative images of lungs from mice on each treatment are also shown. (D) H&E staining, tumor burden representation, and tumor number assessment from lungs treated with the drugs described in (C). Error bars indicate mean ± SEM (significance using Student’s t test: ^∗p < 0.05 ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001). See also Figure S5.