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. 2013 Jul 1;19(13):3533-44.
doi: 10.1158/1078-0432.CCR-12-3815. Epub 2013 May 14.

PI3K pathway dependencies in endometrioid endometrial cancer cell lines

Britta Weigelt  1 , Patricia H Warne, Maryou B Lambros, Jorge S Reis-Filho, Julian Downward
Affiliations

PI3K pathway dependencies in endometrioid endometrial cancer cell lines

Britta Weigelt et al. Clin Cancer Res. .

Abstract

Purpose: Endometrioid endometrial cancers (EEC) frequently harbor coexisting mutations in phosphoinositide 3-kinase (PI3K) pathway genes, including PTEN, PIK3CA, PIK3R1, and KRAS. We sought to define the genetic determinants of PI3K pathway inhibitor response in EEC cells, and whether PTEN-mutant EEC cell lines rely on p110β signaling for survival.

Experimental design: Twenty-four human EEC cell lines were characterized for their mutation profile and activation state of PI3K and mitogen-activated protein kinase (MAPK) signaling pathway proteins. Cells were treated with pan-class I PI3K, p110α, and p110β isoform-specific, allosteric mTOR, mTOR kinase, dual PI3K/mTOR, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and RAF inhibitors. RNA interference (RNAi) was used to assess effects of KRAS silencing in EEC cells.

Results: EEC cell lines harboring PIK3CA and PTEN mutations were selectively sensitive to the pan-class I PI3K inhibitor GDC-0941 and allosteric mTOR inhibitor temsirolimus, respectively. Subsets of EEC cells with concurrent PIK3CA and/or PTEN and KRAS mutations were sensitive to PI3K pathway inhibition, and only 2 of 6 KRAS-mutant cell lines showed response to MEK inhibition. KRAS RNAi silencing did not induce apoptosis in KRAS-mutant EEC cells. PTEN-mutant EEC cell lines were resistant to the p110β inhibitors GSK2636771 and AZD6482, and only in combination with the p110α selective inhibitor A66 was a decrease in cell viability observed.

Conclusions: Targeted pan-PI3K and mTOR inhibition in EEC cells may be most effective in PIK3CA- and PTEN-mutant tumors, respectively, even in a subset of EECs concurrently harboring KRAS mutations. Inhibition of p110β alone may not be sufficient to sensitize PTEN-mutant EEC cells and combination with other targeted agents may be required.

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Figures

Figure 1
Figure 1. Associations between PTEN, PIK3CA, PIK3R1 and RAS mutations and AKT, rpS6 and ERK activation in EEC cells
A, AKT and phospho-AKT(Ser473), B, rpS6 and phosphor-rpS6(Ser235/236), and C, ERK and phospho-ERK(Thr202/Tyr204) were quantified using quantitative infrared western blotting (LI-COR; see Supplementary Fig. 3), and phospho-/total protein ratios ordered by increasing levels of activation. Mutational profiles of each cell line are shown below the graph where columns represent individual cell lines, rows represent genes, and colored boxes the presence of a mutation.
Figure 2
Figure 2. Response of EEC cells to PI3K and RAF/MEK/ERK pathway inhibitors and association with mutation patterns
A, surviving fractions of 50% (SF50s) of 24 EEC cell lines treated for 72h with serial dilutions of the pan-class I PI3K inhibitor GDC-0941 relative to untreated cells were determined using the CellTiter-Blue assay, ordered from lowest to highest (mean of at least three independent experiments in triplicate ± SEM). Rows below the chart represent genes, and colored boxes the presence of a mutation. B, SF60s of the allosteric mTOR inhibitor Temsirolimus. C, SF50s of the MEK inhibitor PD0325901. D, SF50s of the RAF inhibitor AZD628. E, EEC cell lines HEC-1-B, HEC-50B, HEC-59, KLE and HEC-1-A were treated for 4h with 1μM of the MEK inhibitor PD0325901, the RAF inhibitor AZD628, the allosteric mTOR inhibitor Temsirolimus, the mTOR kinase inhibitor AZD8055, the PI3K/mTOR inhibitor PF-04691502 and the PI3K inhibitor GDC-0941. Whole-cell lysates were analyzed by western blotting for total and phosphorylated levels of AKT, rpS6, and ERK1/2 on the same membrane, and detected by near infrared two-color detection (LI-COR; Odyssey).
Figure 3
Figure 3. KRAS dependency in lung and EEC cell lines harboring activating KRAS mutations
A, cell viability was determined 96h post siRNA-mediated KRAS silencing using a pool of four siRNA duplexes targeting KRAS (SMARTpool) in two KRAS-mutant lung, six KRAS-mutant and six KRAS wild-type EEC cell lines. Cell viability is presented for each cell line relative to its non-targeting siRNA pool #2 (“scrambled”) control, set to 100% (mean of at least three independent experiments in triplicate ± SD). B, apoptosis induction assessed 96h post transfection, depicted as ratio of Caspase-3/7 activation determined using the Apo-ONE assay over cell viability determined using CellTiter-Blue in KRAS-mutant lung cancer (striped bars), KRAS-mutant (open bars) and KRAS wild-type (checked bars) EEC cell lines (mean of at least two independent experiments in triplicate ± SEM). Apoptosis induction following siRNA-mediated silencing of KRAS ablation (red bars), and of the positive controls UBB (yellow bars) and PLK1 (blue bars) is presented for each cell line relative to its non-targeting siRNA pool #2 (i.e. scrambled) control (set to 1). Mut: mutant, wt: wild-type.
Figure 4
Figure 4. p110α and p110β expression, and p110β dependency in EEC cells
A, whole-cell lysates of two PTEN wild-type and six PTEN-mutant EEC cell lines were analyzed by western blotting for p110α and p110β, and α-Tubulin as loading control. B, cell viability was determined after 72h of treatment with indicated concentrations of the p110β selective inhibitors GSK2636771 (left) and AZD6482 (right) in three p110β-reliant breast and prostate cancer cell lines (black circles), seven PTEN wild-type EEC cells (gray boxes), and 17 PTEN-mutant EEC cells (black triangles). Mann-Whitney U, two-tailed, *P<0.05. C, PTEN-deficient p110β-reliant HCC70 breast and PC3 prostate cancer cell lines were treated for 4h with indicated concentrations of the pan-class I PI3K inhibitor GDC-0941, p110α inhibitor A66, p110β inhibitor GSK2636771 and the p110β inhibitor AZD6482. Whole-cell lysates were analyzed by western blotting for total and phosphorylated levels of AKT(Ser473) and AKT(Thr308), and detected by near infrared two-color detection (LI-COR; Odyssey); D, EEC cell lines HEC-50B (PTEN wt) and HEC-116 (PTEN mut) were treated for 4h with indicated concentrations of the pan-class I PI3K inhibitor GDC-0941, p110α inhibitor A66, p110β inhibitor GSK2636771 and the p110β inhibitor AZD6482, alone or in combination. Whole-cell lysates were analyzed by western blotting for total and phosphorylated levels of AKT(Ser473) and AKT(Thr308), and detected by near infrared two-color detection. E, cell viability was determined after 72h of treatment with indicated concentration of the p110β selective inhibitors GSK2636771 (left) and AZD6482 (right), alone (blue) or in combination with the p110α inhibitor A66 (red), in three p110β-reliant breast and prostate cancer cell lines (black), and five PTEN-mutant EEC cells. Mut: mutant, wt: wild-type.
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