PIK3CA/PTEN mutations and Akt activation as markers of sensitivity to allosteric mTOR inhibitors

Abstract

Purpose: We sought to determine whether phosphoinositide 3-kinase (PI3K) pathway mutation or activation state and rapamycin-induced feedback loop activation of Akt is associated with rapamycin sensitivity or resistance.

Experimental design: Cancer cell lines were tested for rapamycin sensitivity, Akt phosphorylation, and mTOR target inhibition. Mice injected with breast or neuroendocrine cancer cells and patients with neuroendocrine tumor (NET) were treated with rapalogs and Akt phosphorylation was assessed.

Results: Thirty-one cell lines were rapamycin sensitive (RS) and 12 were relatively rapamycin resistant (RR; IC(50) > 100 nmol/L). Cells with PIK3CA and/or PTEN mutations were more likely to be RS (P = 0.0123). Akt phosphorylation (S473 and T308) was significantly higher in RS cells (P < 0.0001). Rapamycin led to a significantly greater pathway inhibition and greater increase in p-Akt T308 (P < 0.0001) and p-Akt S473 (P = 0.0009) in RS cells. Rapamycin and everolimus significantly increased Akt phosphorylation but inhibited growth in an in vivo NET model (BON). In patients with NETs treated with everolimus and octreotide, progression-free survival correlated with p-Akt T308 in pretreatment (R = 0.4762, P = 0.0533) and on-treatment tumor biopsies (R = 0.6041, P = 0.0102). Patients who had a documented partial response were more likely to have an increase in p-Akt T308 with treatment compared with nonresponders (P = 0.0146).

Conclusion: PIK3CA/PTEN genomic aberrations and high p-Akt levels are associated with rapamycin sensitivity in vitro. Rapamycin-mediated Akt activation is greater in RS cells, with a similar observation in patients with clinical responses on exploratory biomarker analysis; thus feedback loop activation of Akt is not a marker of resistance but rather may function as an indicator of rapamycin activity.

Figure 1

Rapamycin sensitivity in cancer cell lines. A. Rapamycin responses were determined by treating 43 cell lines (in triplicate) with six concentrations based on a 10-fold dilution series (range 0–1000 nM). Cell growth was measured 5 days later using SRB assay. PI3K and PTEN mutation status of cell lines are listed underneath. Green circle = wild-type, red circle = mutation/deletion or frameshift, empty circle = not defined. B. Rapamycin sensitivity by PIK3CA and PTEN status. Y-axis represents % of cell lines with each genetic status that are rapamycin sensitive. Rapamycin sensitivity was compared between each group with two-sided Fisher’s Exact test. WT, wild-type; MT, mutant.

Figure 2

Rapamycin sensitivity and baseline Akt activation. All cell lines were cultured in the presence of vehicle only and collected after 2, 24 and 72 hours of culture. A. Using functional proteomics analysis, log transformed p-Akt S473 and p-Akt T308 baseline expression and their change in three time points for RS and RR cell lines are shown. B. A protein signature of PI3K pathway activity and rapamycin sensitivity. Heat map showing pre-ordered 43 cancer cell lines and 17 PI3K hierarchically clustered pathway proteins. Bar at the left of the heat map indicate whether a cell line was RS (green) or RR (red). Proteins’ data were derived from log transformation and median centering for each gene. Red, high expression; green, low expression according to z scores). C. Log transformed p-Akt S473 and p-Akt T308 baseline phosphorylation in PIK3CA/PTEN wild-type, PIK3CA or PTEN mutant cell lines are displayed. The mid-line of each box is at the median for the data represented by that box. The upper and lower edges of each box mark the 75th and 25th percentiles of the data respectively. The upper and lower bars mark the 95th and 5th percentiles of the data respectively. D. Log transformed p-Akt S473 and p-Akt T308 baseline phosphorylation in PIK3CA/PTEN wild-type, PIK3CA kinase domain mutant or other domain mutant cell lines are displayed. The mid-line of each box is at the median for the data represented by that box. The upper and lower edges of each box mark the 75th and 25th percentiles of the data respectively. The upper and lower bars mark the 95th and 5th percentiles of the data respectively.

Figure 3

Feedback loop activation of Akt. A. Cell lines were treated with DMSO or 100 nM rapamycin for 24 hours. Western blotting was performed with antibodies to p-Akt T308, p-Akt S473 and total Akt. B. Cell lines were treated with DMSO or 1, 10 or 100 nM rapamycin and harvested after 2, 24 or 72-hours. Changes in expression of p-Akt S473 and p-Akt T308 in response to rapamycin treatment was analyzed using RPPA. Expression levels at three time points are shown in log scale (RS, green; RR, red; median expression for all cell lines, black line).

Figure 4

Rapamycin-mediated Akt activation in vivo. A. Mice with established MCF7 tumor xenografts received 15 mg/kg rapamycin once a week by intraperitoneal injection. Mice were sacrificed 24 hours after last rapamycin injection. Protein was extracted from xenografts and p-S6 S235/236, p-S6 S240/244 and p-Akt T308 expression in DMSO and rapamycin treated xenografts was analyzed using RPPA. The mid-line of each box is at the median for the data represented by that box. The upper and lower edges of each box mark the 75th and 25th percentiles of the data respectively. The upper and lower bars mark the 95th and 5th percentiles of the data respectively (left and middle panels). The tumor volumes were measured using caliper and presented as the mean ± SE (right panel). B. Mice with established BON tumor xenografts received 15 mg/kg rapamycin once a week by intraperitoneal injection. p-S6 S235/236, p-S6 S240/244 and p-Akt T308 expression in DMSO and rapamycin treated xenografts was analyzed using RPPA. The mid-line of each box is at the median for the data represented by that box. The upper and lower edges of each box mark the 75th and 25th percentiles of the data respectively. The upper and lower bars mark the 95th and 5th percentiles of the data respectively (left and middle panels). The tumor volumes were measured using caliper and presented as the mean ± SE (right panel). C. Mice with established BON tumor xenografts received 10 mg/kg everolimus five days a week by oral gavage for three weeks. Mice were sacrificed 24 hours after last everolimus dose. Protein was extracted from xenografts and p-S6 S240/244, p-Akt-S473 and total Akt expression in vehicle and everolimus treated xenografts were analyzed using MSD assay. p-Akt S473 or p-S6 S240/244 to total Akt ratio is calculated and normalized to vehicle. Data were presented as mean ± SE (left and middle panels). The tumor volumes were measured using caliper and presented as the mean ± SE (right panel).

Figure 5

p-Akt as a biomarker in neuroendocrine tumor patients. A. Pre-treatment and On-treatment p-Akt levels on FNAs were assessed by RPPA. The correlation between p-Akt T308 expression and PFS was analyzed. B. Pre-treatment and On-treatment p-S6 S235/236 and p-S6 S240/244 levels were assessed by RPPA. P values show Pre- and On-treatment comparisons. C. Changes in p-Akt T308 levels in response to everolimus treatment in partial response vs. stable or progressive disease groups were analyzed using RPPA. P value shows comparison of p-Akt T308 levels in patients who demonstrated a partial disease vs. stable or progressive disease.