Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor-positive human breast cancer

Abstract

Many breast cancers exhibit a degree of dependence on estrogen for tumor growth. Although several therapies have been developed to treat individuals with estrogen-dependent breast cancers, some tumors show de novo or acquired resistance, rendering them particularly elusive to current therapeutic strategies. Understanding the mechanisms by which these cancers develop resistance would enable the development of new and effective therapeutics. In order to determine mechanisms of escape from hormone dependence in estrogen receptor-positive (ER-positive) breast cancer, we established 4 human breast cancer cell lines after long-term estrogen deprivation (LTED). LTED cells showed variable changes in ER levels and sensitivity to 17beta-estradiol. Proteomic profiling of LTED cells revealed increased phosphorylation of the mammalian target of rapamycin (mTOR) substrates p70S6 kinase and p85S6 kinase as well as the PI3K substrate AKT. Inhibition of PI3K and mTOR induced LTED cell apoptosis and prevented the emergence of hormone-independent cells. Using reverse-phase protein microarrays, we identified a breast tumor protein signature of PI3K pathway activation that predicted poor outcome after adjuvant endocrine therapy in patients. Our data suggest that upon adaptation to hormone deprivation, breast cancer cells rely heavily on PI3K signaling. Our findings also imply that acquired resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both ER and PI3K pathways.

Figure 1. LTED cells exhibit PI3K/mTOR pathway hyperactivation and variable response to E2.

(A) Cells were treated with 10% DCC-FBS with or without 0–1,000 pM E2. Media and drugs were replenished every 2–3 days. Adherent cells were counted after 5–10 days. (B) Cells transfected with luciferase reporter plasmids were treated as in A. Luciferase activities were measured after 16–20 hours. Data are presented as percent parental control (RLU, firefly/Renilla), mean of triplicates ± SD, and were analyzed using 2-way ANOVA. *P < 0.05, **P < 0.01 versus parental cells, Bonferroni post-hoc test corrected for multiple comparisons. (C) Lysates from cells treated with 0%–10% DCC-FBS for 24 hours were analyzed by immunoblotting with the indicated antibodies. (D) Lysates from cells treated with 1% DCC-FBS for 24 hours were analyzed using antibody microarrays. Proteins/phosphoproteins altered at least 1.4-fold in LTED/parental comparisons were compared for overlap, which revealed 16 proteins/phosphoproteins commonly altered across all LTED lines.

Figure 2. PI3K pathway inhibition suppresses hormone-independent cell growth.

(A) Phospho-RTK arrays were probed with lysates from cells treated with 10% DCC-FBS for 24 hours. Signal indicates tyrosine phosphorylation. Blots from each pair of lines are exposure-matched. RTKs are labeled as follows: 1, EGFR; 2, HER2; 3, HER3; 4, HER4; 5, FGFR4; 6, InsR; 7, IGF-IR; 8, Dtk; 9, MSP-R; 10, EphA1; 11, ROR2; 12, FGFR3; 13, Tie-2; 14, EphA4. Corner spots are positive controls. (B) Immunoblot analysis of lysates from cells treated for 24 hours with 10% DCC-FBS and the indicated kinase inhibitors using the indicated antibodies. (C) Cells were treated as in B. Media and drugs were replenished every 2–3 days. Adherent cells were counted after 5–10 days. Data are presented as mean of triplicates ± SD and were analyzed using 2-way ANOVA. *P < 0.05, **P < 0.01 versus control (or as indicated by brackets), Bonferroni post-hoc test corrected for multiple comparisons.

Figure 3. A tumor protein signature of PI3K activation predicts poor disease outcome following adjuvant endocrine therapy.

(A) Lysates from 64 primary human breast tumors were analyzed by RPPA using antibodies against P-AKT_S473, AKT, P-S6_S240/244, P-GSK3α/β_S21/9, GSK3α/β, EGFR, ER, Src, and P-PKCα_S657. Log₂ mean-centered values were hierarchically clustered, which revealed PI3K-high (blue) and -low (red) tumor clusters. (B) Kaplan-Meier recurrence curves of the clusters in A were compared by log-rank test. The number of patients at risk of recurrence at different time points is noted below.