Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling

Abstract

Background: Dysregulated PI3K/Akt signaling occurs commonly in breast cancers and is due to HER2 amplification, PI3K mutation or PTEN inactivation. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.

Methodology/principal findings: A selective allosteric inhibitor of Akt kinase was used to interrogate a panel of breast cancer cell lines characterized for genetic lesions that activate PI3K/Akt signaling: HER2 amplification or PI3K or PTEN mutations in order to determine the biochemical and biologic consequences of inhibition of this pathway. A variety of molecular techniques and tissue culture and in vivo xenograft models revealed that tumors with mutational activation of Akt signaling were selectively dependent on the pathway. In sensitive cells, pathway inhibition resulted in D-cyclin loss, G1 arrest and induction of apoptosis, whereas cells without pathway activation were unaffected. Most importantly, the drug effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.

Conclusions/significance: These data demonstrate that breast cancers with PI3K mutation or HER2 amplification are selectively dependent on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast and other cancers that are addicted to the pathway including tumors with resistant to Herceptin.

Figure 1. PIK3CA-mutated or HER2-overexpressing breast cancer cells are highly sensitive to AKTi-1/2.

(A) Half-maximal growth inhibitory concentration (IC₅₀) of AKTi-1/2 to a panel of breast cancer cell lines with wild-type (wt) and mutant (mut) PIK3CA, PTEN loss, HER2 and EGFR amplification (amp). Growth inhibition assays and Western blot analysis for expression of EGFR, HER2, Akt1, Akt2, Akt3, pan-Akt, PTEN, β-actin and phosphorylated Akt at Ser473 and Thr308 were performed as described in Materials and Methods. (B) Western blot analysis of Ser473 phosphorylated Akt and pan-Akt in cell lysates of the indicated breast cancer cells after treatment with various concentrations of AKTi-1/2 for 24 h.

Figure 2. AKTi-1/2 inhibits Akt signaling and causes G1 arrest in PIK3CA-mutant but not in PIK3CA-WT breast cancer cells.

(A) HCC1806 and T47D cells were treated with 1 µM AKTi-1/2 for the indicated times and cell lysates were immunoblotted with the indicated antibodies. (B) HCC1806 and T47D cells were treated with 1 µM AKTi-1/2 or DMSO for 24 h. The fractions of cells in G1, S and G2/M were determined by flow cytometry.

Figure 3. Akt inhibition induces G1 arrest and apoptosis in HER2-overexpressing with or without PIK3CA-mutated breast cancer cells.

(A) Akt inhibition causes dephosphorylation of its downstream targets (Foxo1, GSK3α, p70S6K, S6 and 4EBP1), loss of D-cyclin expression, and induction of p27 and PARP cleavage in BT474 and MDA-MB-453 cells treated with 1 µM AKTi-1/2. (B–C) Cells were analyzed by flow cytometry and gated differently to determine fractions of G1, S and G2/M (B) and the fraction of apoptotic cells (sub-G1) (C). (B) Cells were treated with 1 µM AKTi-1/2 or DMSO for 24 h. (C) Cells were treated with the indicated concentrations of AKTi-1/2 for 24 h, 48 h and 72 h. All error bars indicate standard error.

Figure 4. AKTi-1/2 effectively suppresses HER2-overexpressing and PIK3CA-mutated tumor growth in vivo.

(A) AKTi-1/2 inhibits Akt followed by dephosphorylation of its downstream targets (GSK3, Foxo, p70S6K, S6 and 4EBP1), loss of D-cyclin expression, Rb hypophosphrylation and induction of PARP cleavage in BT474 xenograft tumors. Mice with established BT474 xenografts were treated with AKTi-1/2 100 mg/kg for the indicated times. Tumor lysates were immunoblotted with the indicated antibodies. (B) Mice with established BT474 xenografts were treated with AKTi-1/2 50 and 100 mg/kg/day×5 days/week or vehicle only as control. The results represent the mean tumor volume±standard error (n = 5 mice per group) from two independent experiments. *, P<0.005, 50 versus 100 mg/kg AKTi-1/2, and 50 and 100 mg/kg AKTi-1/2 versus control. (C) Representative immunohistochemistry fields of BT474 tumors from mice euthanized 6 h after the final treatment of AKTi-1/2 as in (B). Tumors were excised, and H&E, phosphorylated Akt, Ki67 and TUNEL were assessed by immunohistochemic statining. (D) Mice with established MCF7 xenografts were treated with AKTi-1/2 50 and 100 mg/kg/day×5 days/week or vehicle only as control. The results represent the mean tumor volume±standard error (n = 5 mice per group) from two independent experiments. *, P<0.005, 50 versus 100 mg/kg AKTi-1/2, and 50 and 100 mg/kg AKTi-1/2 versus control.

Figure 5. Herceptin-resistant HER2-overexpressing breast cancer cells retain Akt dependence.

(A) Both Herceptin-sensitive (BT474) and Herceptin-resistant (BT474:EII) HRE2-overexpressing breast cancer cells are sensitive to AKTi-1/2. Growth inhibition assays were performed as described in Figure 1A. All error bars indicate standard error. *, P<0.001, Herceptin and AKTi-1/2 versus control in BT474 cells. **, P = 0.35, Herceptin versus control in BT474:EII cells. ***, P<0.001, AKTi-1/2 versus Herceptin and control in BT474:EII cells. (B) Herceptin has no anti-tumor effects on the Fo5 xenograft model. Mice with established Fo5 tumors were treated with Herceptin 20 mg/kg/day×2 days (Tue/Fri)/week or vehicle only as control. The results represent the mean tumor volume±standard error (n = 5 mice per group) from two independent experiments. *, P = 0.31, Herceptin versus control. (C) AKTi-1/2 demonstrates anti-tumor activity against the Herceptin-resistant Fo5 xenograft model. Mice with established Fo5 tumors were treated with AKTi-1/2 100 mg/kg/day×5 days/week or vehicle only as control. The results represent the mean tumor volume±standard error (n = 5 mice per group) from two independent experiments. *, P<0.001, AKTi-1/2 versus control. (D) Mice with established Fo5 tumors were treated with AKTi-1/2 100 mg/kg for the indicated times. Tumor lysates were immunoblotted with the indicated antibodies.