S3-4: ER Downregulation with Fulvestrant in Combination with pan-PI3K Inhibitor BKM120 Synergizes Against ER+/PI3K-Mutant Breast Cancer Xenografts In Vivo.
Authors: Miller TW , Fox EM , Balko JM , Meszoely IM , Sanders ME , Kuba MG , Wagle N , Garraway LA , Maira S-M , Arteaga CL 
Published: June 2012

Abstract
ER+ breast cancers typically respond to aromatase inhibitors (AIs), but a significant fraction exhibit de novo or acquired resistance. To discover mechanisms of resistance to AIs, we maintained four ER+, hormone-dependent human breast cancer cell lines in hormone-depleted conditions for several months until estrogen-independent populations emerged (termed long-term estrogen-deprived, LTED). A siRNA kinome screen identified 42 kinases that suppressed hormone-independent MCF-7/LTED cell growth ≥33% (p&lt;0.05). Pathway analysis revealed phosphatidylinositol 3-kinase (PI3K) as a central hub which integrates signaling from, or emits signaling to many of these 42 kinases. Further, treatment with the PI3K/mTOR dual inhibitor BEZ235 or the pan-PI3K inhibitor BKM120 suppressed LTED cell growth, and prevented the emergence of hormone-independent cells.
Activating mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are present in about 35% of ER+ breast cancers. We investigated whether PIK3CA mutations modify the tumor response to estrogen deprivation with letrozole in a pre-surgical clinical trial (NCT00651976). Twenty-one patients with stage I-II operable ER+/HER2− breast cancer were treated with letrozole for 10–21 days up to 24 h prior to surgery. Core tumor biopsies were obtained before treatment and at the time of surgical resection. Ki67 immunohistochemistry (IHC) was scored in pre- and post-letrozole specimens. The change in natural log of the Ki67 score between pre-and post-treatment samples was stratified by PIK3CA mutant vs. wildtype (WT) status. PIK3CA-mutant tumors exhibited a statistically lower reduction in Ki67 score compared to tumors with WT PIK3CA (p=0.03). These findings suggest that mutational activation of PI3K attenuates the response to estrogen deprivation.
In some LTED cell lines, downregulation of ER with fulvestrant or siRNA inhibited estrogen-independent growth, suggesting that the unliganded ER can maintain proliferation of cells adapted to estrogen deprivation. Prior work suggested that PI3K-ER crosstalk promotes resistance to estrogen deprivation. However, inhibition of PI3K/AKT/TORC1 signaling did not alter ERS167 phosphorylation in LTED cells, and ER downregulation with fulvestrant did not inhibit PI3K/AKT, suggesting that PI3K and ER modulate non-overlapping pathways in cells adapted to estrogen deprivation. Treatment with BKM120 synergized with fulvestrant to inhibit the growth of 6/8 ER+ breast cancer cell lines in estrogen-depleted conditions. Treatment with single-agent fulvestrant or BKM120 in athymic mice devoid of estrogen supplementation partially inhibited the growth of ER+/PI3K-mutant MCF-7 xenografts, while the combination induced near-complete tumor regression. IHC analysis showed increased cleaved caspase-3+ cells in BKM120-treated tumors and decreased Ki67+ cells in fulvestrant-treated tumors, implying that the combination induced tumor regression by both inhibition of tumor cell proliferation and induction of apoptosis. These data suggest that upon progression on an aromatase inhibitor, patients with ER+/PI3K-mutant breast cancer would benefit from a combination of an ER downregulator and a PI3K pathway inhibitor.
Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr S3-4.