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. 2015 Jan 12;27(1):109-22.
doi: 10.1016/j.ccell.2014.11.008. Epub 2014 Dec 24.

Feedback suppression of PI3Kα signaling in PTEN-mutated tumors is relieved by selective inhibition of PI3Kβ

Sarit Schwartz  1 , John Wongvipat  2 , Cath B Trigwell  3 , Urs Hancox  3 , Brett S Carver  2 , Vanessa Rodrik-Outmezguine  1 , Marie Will  1 , Paige Yellen  1 , Elisa de Stanchina  1 , José Baselga  2 , Howard I Scher  4 , Simon T Barry  3 , Charles L Sawyers  2 , Sarat Chandarlapaty  5 , Neal Rosen  6
Affiliations

Feedback suppression of PI3Kα signaling in PTEN-mutated tumors is relieved by selective inhibition of PI3Kβ

Sarit Schwartz et al. Cancer Cell. .

Abstract

In PTEN-mutated tumors, we show that PI3Kα activity is suppressed and PI3K signaling is driven by PI3Kβ. A selective inhibitor of PI3Kβ inhibits the Akt/mTOR pathway in these tumors but not in those driven by receptor tyrosine kinases. However, inhibition of PI3Kβ only transiently inhibits Akt/mTOR signaling because it relieves feedback inhibition of IGF1R and other receptors and thus causes activation of PI3Kα and a rebound in downstream signaling. This rebound is suppressed and tumor growth inhibition enhanced with combined inhibition of PI3Kα and PI3Kβ. In PTEN-deficient models of prostate cancer, this effective inhibition of PI3K causes marked activation of androgen receptor activity. Combined inhibition of both PI3K isoforms and androgen receptor results in major tumor regressions.

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Figures

Figure 1
Figure 1. PI3Kα inhibition in PTEN mutant models leads to a transient inhibition of the Akt pathway
(A, B) LNCaP (A) and BT-549 (B) cells were treated with 250 nM AZD8186 as indicated and the effect on the downstream signaling was analyzed. (C) The fold change in P-Akt (T308), P-Akt (S473) and P-PRAS40 levels following AZD8186 treatment was determined by densitometric analysis of the bands in (A) and (B), respectively. (D, E) LNCaP (D) and BT-549 (E) cells were treated with 250 nM AZD8186 for the indicated times. Lysates were immunoblotted with indicated antibodies (left panels). After 4 (BT-549 cells) or 6 (LNCaP cells) hours of AZD8186 treatment, AZD8186 was re-added to the cells (right panels) for the indicated times and lysates were immunoblotted with the indicated antibodies. See also Figure S1 and Table S1.
Figure 2
Figure 2. Reactivation of Akt signaling following PI3Kα inhibition is PI3Kβ-dependent
(A) LNCaP cells were treated with AZD8186 (250 nM), BYL719 (2.5 μM) or the combination. Cells were collected at indicated times and the effect on Akt signaling and apoptotic markers was analyzed by immunoblotting. (B) LNCaP cells were treated with AZD8186 for various times. PI3Kβ was immunoprecipitated from cell lysate and PI3K activity assay was performed. The product, PI3P, was resolved by thin-layer chromatography and detected by autoradiography. A lane with a purified PI3Kβ was used as a positive control and a lane with IgG was used as a negative control for the assay (left panel). Whole cell lysates and immunoprecipitates were blotted with anti-PI3Kβ (left panel). The change in PI3Kβ activity was determined by densitometric analysis of the bands (right panel). (C) Cell growth of LNCaP cells treated as indicated was monitored with cell counter. The data represent mean ± SD of triplicate samples. (D) Apoptotic cell death of LNCaP cells treated as indicated for 48 hr was quantified by Annexin V staining. The data represent mean ± SD of duplicate samples. See also Figure S2.
Figure 3
Figure 3. PI3Kβ activation is IGF1R dependent
(A) Expression and/or phosphorylation of several RTKs in LNCaP cells treated with 250 nM AZD8186 for the indicated times was examined by immunoblot analysis. (B) The phosphorylation of IGF1R (Y1131) in LNCaP cells treated with 250 nM AZD8186 for the indicated times was quantified by sandwich ELISA kit. The data represent mean ± SD of triplicate samples. (C) LNCaP cells were treated with 250 nM AZD8186 and collected at various time points. Immunoblotting analysis was performed as indicated. The change in the expression of IRS1 was determined by densitometric analysis of the bands. (D) LNCaP cells were treated with AZD8186 (250 nM), OSI-906 (2 μM), or the combination for the indicated times. Immunoblotting of lysates was used to examine the effect on downstream signaling. (E) LNCaP cells were treated with AZD8186, BYL719, OSI-906, or the combinations as indicated. Apoptotic cell death was quantified by Annexin V staining after 48 hours of treatment. Data are the mean percentage of Annexin V-positive cells ± SD. (F) LNCaP cells were treated with AZD8186 for various times. At the indicated time cells were stimulated with IGF-1 (100 ng/ml) for 10 min. Immunoblotting analysis was used to assay the effect on downstream signaling. See also Figure S3.
Figure 4
Figure 4. Inhibition of the PI3K pathway in mice bearing LNCaP tumors
(A) Mice (n=3 mice/time point) bearing LNCaP tumors were randomized to receive vehicle, 75 mg/kg AZD8186, 25 mg/kg BYL719 or the combination of both, and treated for the specified times. The tumors were analyzed by immunoblotting to assay downstream signaling. (B) Mice bearing LNCaP tumors were randomized to receive vehicle, AZD8186 75 mg/kg twice per day, BYL719 25 mg/kg once per day, or the combination for 25 days. Tumor size was measured twice per week. The results are presented as the mean tumor volume ± SEM (n = 5 mice/group). See also Figure S4 and Table S2.
Figure 5
Figure 5. Combined inhibition of PI3Kα, PI3Kβ and AR potently inhibits LNCaP tumors in vitro and in vivo
(A) LNCaP cell were treated as indicated and expression of NKX3.1 normalized to GAPDH was determined using qRTPCR. Error bars represent the SD of triplicate samples. (B, C) LNCaP cell were treated as indicated and cell growth was monitored with cell counter (B, error bars represent the SD of triplicate samples) or apoptotic cell death was quantified by Annexin V staining after 2 days of treatment (C, data are the mean percentage of Annexin V- positive cells ± SD of two independent experiments). Levene's test for equality of variance was followed by an unpaired, two-tailed Student t-test. (D) Castrated mice bearing LNCaP tumors were randomized to vehicle, AZD8186 (75 mg/kg) twice per day, BYL719 (25 mg/kg) once per day, MDV3100 (30 mg/kg) once per day or the different combinations for 3 weeks. Tumor size was measured twice per week. The results are presented as the mean tumor volume ± SEM (n = 5 mice/group). (E) Representative images of tumors following 3 weeks of treatment as in (D) Scale bar determined using the diameter of the 10 cm plate as a scale, represents 1 cm. See also Figure S5 and Table S3.
Figure 6
Figure 6. Triple therapy of PI3Kα, PI3Kβ and androgen receptor inhibitors potently inhibits Ptenlox/lox prostate tumors in vivo
(A) Intact mice were treated with vehicle and castrated PB-Cre;Ptenlox/lox mice were treated with AZD8186 (75 mg/kg) twice per day alone, AZD8186 in combination with either BYL719 (25 mg/kg) once per day or MDV3100 (30 mg/kg) once per day, or the triple combination. Representative Magnetic Resonance Imaging (MRI) images of mice at initiation (T0) and completion of study (30 days, T30) are shown at the left and the waterfall plot depicting proportional change in tumor tumor volume of treated mice (n=4 per group) is shown at the right. (B) Representative images of histological and immunohistochemical staining for P-Akt (S473), AR and ki67 of tumors from mice treated as in (A). Scale bar represents 50 μm.
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