Resistance to Endocrine Therapy in Estrogen Receptor-Positive (ER+) Breast Cancer Is Dependent upon Phosphatidylinositol-3 Kinase (PI3K) Signaling.
Authors: Miller T. , Fox E. , Gonzalez-Angulo A. , Hennessy B. , Mills G. , McKinley E. , Manning H. , Arteaga C. 
Published: December 2010

Abstract
ER+ breast cancers typically respond to treatment with aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To model AI resistance, we cultured four ER+, hormone-dependent human breast cancer cell lines under hormone-depleted conditions for several months until hormone-independent populations emerged (termed long-term estrogen-deprived, LTED). LTED cells outgrew parental counterparts under hormone-depleted conditions. While two LTED lines showed increased ER levels and response to estradiol, two LTED lines showed the opposite. Therefore, ER+ breast cancer cells did not consistently overcome hormone deprivation by increasing sensitivity to low estrogen levels.We analyzed protein lysate arrays with 625 antibodies against signaling proteins to discover common mechanisms of hormone-independent growth. All LTED lines showed increased phosphorylation of p70S6K, an mTOR substrate, compared to parental controls. An siRNA library screen targeting 779 kinases revealed that downregulation of kinases linked to the phosphatidylinositol-3 kinase (PI3K) signaling pathway (serum/glucocorticoid-regulated kinase 1; insulin receptor; p110α/PI3K) inhibited the growth of MCF-7/LTED but not parental MCF-7 cells under hormone-depleted conditions. Immunoblot analysis for P-AKT and P-p70S6K showed that PI3K/AKT/mTOR signaling was increased in all LTED lines. Pathway analysis of gene expression microarray data showed significant alteration of genes involved in insulin-like growth factor-I (IGF-I) signaling in 3/4 LTED lines. Receptor tyrosine kinase array analysis revealed increased activation of receptors for IGF-I (IGF-IR) and/or insulin (InsR) in 3/4 LTED lines. These receptors transduce signals to potently activate PI3K. Treatment with the IGF-IR/InsR kinase inhibitor AEW541 decreased P-AKT in 3/4 LTED lines. Treatment with inhibitors of IGF-IR/InsR (AEW541), PI3K/mTOR (BEZ235), or mTOR (RAD001) suppressed the monolayer and anchorage-independent growth of 3/4, 4/4, and 4/4 LTED lines, respectively, and prevented the emergence of hormone-independent cells. Treatment with PI3K and mTOR inhibitors induced apoptosis in 3/4 LTED lines. Finally, we analyzed levels of PI3K pathway markers (P-AKT, P-S6, P-GSK3α/β) in primary tumor lysates from 65 ER+ breast cancer patients using reverse-phase protein arrays. Hierarchical clustering yielded two groups of tumors with high or low PI3K activation. Following adjuvant anastrozole therapy, PI3K-high patients had significantly shorter disease-free survival compared to PI3K-low patients. With a median follow-up of 9.4 months, recurrence rates were 16% and 0%, respectively (p&lt;0.05). These data suggest that upon the acquisition of hormone independence and resistance to endocrine therapy, ER+ breast cancer cells increase their dependence on the PI3K pathway. Thus, resistance to endocrine therapy may be abrogated by combination therapies targeting both the ER and PI3K pathways, which may sometimes be achieved by blocking IGF-IR/InsR signaling upstream of PI3K.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 403.