In order to discover mechanisms of escape from hormone dependence, we established four ERα-positive human breast cancer cell lines after long-term estrogen-deprivation (LTED). Growth of MCF-7/LTED and HCC-1428/LTED cells was inhibited by the ER downregulator fulvestrant and by ER siRNA, suggesting continued dependence upon estrogen-independent ER signaling. Fulvestrant also prevented the emergence of hormone-independent MCF-7, HCC-1428, and BT-474 cells, but not ZR75- 1, MDA-361, and T47D cells. Gene expression analysis revealed that ER modulates a transcriptional program directed by the E2F proteins, a family of transcription factors derepressed by CDK4/6-mediated phosphorylation of Rb. A set of 1,003 genes with expression altered ≥1.5-fold (P<0.05) by fulvestrant in MCF-7/LTED and HCC-1428/LTED cells was used to query the TRANSFAC database. Five of the top ten sets of genes which share a transcription factor binding motif ± 2 kb of transcription start sites contained E2F motifs (all p<10-12). From these, we derived an expression signature of 24 genes with no cell cycle association. This signature was predictive of response of 48 ER+ primary tumors to 2 weeks of neoadjuvant letrozole as assessed by Ki67 IHC. In a separate set of experiments, we screened a siRNA library targeting 779 kinases and found that CDK4 is required for MCF-7/LTED cell growth. Inhibition of CDK4/6 using the small molecule PD-0332991 or CDK4 siRNA phenocopied the inhibitory effect of fulvestrant on MCF-7/LTED and HCC-1428/LTED cells, implying that the primary growth-promoting effect of ligand-independent ER is activation of the Rb/E2F pathway. CDK4 inhibition also suppressed the hormone-independent growth of 4 other ER+ breast cancer lines. We previously demonstrated that phosphatidylinositol-3 kinase (PI3K) signaling is required for the escape of ER+ breast cancer cells from hormone dependence. Thus, we postulated that treatment with the PI3K inhibitor BKM120 (Novartis) would synergize with fulvestrant to suppress hormone-independent cell growth. Indeed, these drugs exerted a synergistic inhibitory effect in 6/8 ER+ breast cancer cell lines. Finally, we injected MCF-7 cells and 14-day-release 17P-estradiol pellets into female athymic mice. After 19-29 days, mice bearing tumors ≥100 mm3 were randomized to treatment with vehicle, fulvestrant, BKM120, or the combination. While BKM120 and fulvestrant individually slowed tumor growth, the combination induced near-complete tumor regression. Fulvestrant downregulated ER and PR levels, while BKM120 decreased P-AKT-S473, P-AKT-T308, P-S6-S240/244 (by immunoblotting), and [18F]fluorodeoxyglucose (FDG) tumor uptake as measured by PET, indicative of suppressed ER and PI3K signaling, respectively. These findings collectively suggest that 1) a fraction of ER+ breast cancer cells engage ERα in a ligand-independent manner to drive cell growth, 2) pharmacological downregulation of ER may be effective in a fraction of ER+ tumor cells that escape estrogen deprivation, 3) CDK4/6 inhibition suppresses the hormone-independent growth of fulvestrant-sensitive and-insensitive ER+ breast cancer cells, and 4) resistance to endocrine therapy in breast cancer may be abrogated by combination therapies targeting both the ER and PI3K pathways.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD05-05.