Herein, we identified PI3K as a critical hub in the emergence and maintenance of hormone-independent, ER-positive breast cancer cells. We found that acquired hormone-independent (i.e., LTED) breast cancer cell growth was associated with hyperactivation of the IGF-IR/InsR/PI3K/mTOR pathway. Direct inhibition of PI3K effectively suppressed the hormone-independent growth of both estrogen-independent and -dependent cells, whereas inhibition of nodes upstream (RTKs) and downstream (mTOR) of PI3K only partially blocked cell growth. In support of these findings, we identified a protein signature of PI3K pathway activation that was predictive of poor disease outcome following adjuvant endocrine therapy in breast cancer patients.
Although prior reports indicate that MCF-7 cells selected for hormone independence exhibit hypersensitivity to exogenous E2 (6
), we found no such change in MCF-7/LTED cells. The 3 other LTED lines showed an attenuated response to E2 in growth assays (Figure ). In ER transcriptional reporter assays, MCF-7/LTED and HCC-1428/LTED cells showed hyperresponsiveness to E2, whereas the other 2 LTED lines showed the opposite. Such changes in ER transcriptional activity were paralleled by changes in ER levels, which suggests that ER content may be a marker of estrogen sensitivity. Indeed, increased ER levels in breast cancer cells have previously been linked with improved patient outcome following endocrine therapy (21
). Additionally, decreased ER levels following therapy with tamoxifen or AIs have been shown to predict shorter time to recurrence (24
). Importantly, 3 of 4 LTED lines remained ER positive and sensitive to nanomolar concentrations of E2 (Figure A), which suggests that cessation of estrogen deprivation with an AI in patients who progress on this therapy may not always be warranted. Discontinuation of an AI would result in resumption of estrogen production, stimulation of hormone-independent but still hormone-responsive cancer, and protection from an alternative therapy.
We and others previously found that activation of the PI3K pathway (by PTEN
knockdown or HER2
overexpression) promotes antiestrogen resistance and hormone independence in ER-positive breast cancer cells (26
). Herein, we report that intrinsic PI3K signaling was required for the acquisition and maintenance of hormone independence. These findings corroborate the observation that mice bearing MCF-7/aromatase xenografts resistant to the AI letrozole showed modestly inhibited tumor growth when treated with the PI3K inhibitor wortmannin (28
). Moreover, treatment of MCF-7 and MCF-7/LTED cells with the Ras inhibitor farnesylthiosalicylic acid decreased mTOR signaling and hormone-independent growth (29
). We found that RAD001 treatment blocked TORC1, but feedback activation of PI3K/AKT (Figure B) likely hampered its ability to completely inhibit hormone-independent cell growth (Figure C and Supplemental Figures 5 and 6). In support of these findings, neoadjuvant treatment of patients bearing ER-positive tumors with letrozole and RAD001 decreased the fraction of Ki67-positive tumor cells and induced clinical responses more effectively than did letrozole alone (30
). Interestingly, neoadjuvant therapy with letrozole decreased tumor levels of PI3K, P-AKTS473
, and P-mTORS2448
. Because reductions in P-AKTS473
correlate with better response rates and outcome (31
), we speculate that estrogen deprivation may suppress cell growth in part by decreasing PI3K signaling.
Treatment with the PI3K/mTOR inhibitor BEZ235 inhibited growth of all cell lines and induced apoptosis in 3 of 4 LTED and 3 of 4 parental cell lines, thereby preventing the emergence of hormone-independent cells (Figure C and Supplemental Figures 5 and 6). Notably, the 3 parental/LTED cell line pairs most sensitive to BEZ235 were MCF-7, MDA-361, and ZR75-1, which harbor mutations in PIK3CA
(MCF-7 and MDA-361) or PTEN
(ZR75-1; ref. 32
) or gene amplification of HER2
(MDA-361). These mutations result in hyperactivation of the PI3K pathway (33
) and confer sensitivity to PI3K and AKT inhibitors (34
). HCC-1428 cells, which were growth arrested by BEZ235 but did not undergo apoptosis, have no known PI3K pathway mutations. These data suggest that PI3K pathway–activating mutations may generate dependence on this pathway for cell survival. However, PIK3CA
mutations and disease outcome following endocrine therapy are not consistently correlated (30
), which suggests that alternative measurements of PI3K pathway activation as shown herein (Figure ) may provide additional predictive information.
In summary, our findings suggest that patients with hormone receptor–positive tumors exhibiting a high degree of PI3K signaling, and patients who relapse on endocrine therapy, may benefit from therapeutics targeting both the ER and the PI3K pathways. Although directly targeting PI3K and mTOR maximally inhibited hormone-independent cell growth and induced apoptosis, inhibition of signaling kinases upstream (IGF-IR/InsR/ErbBs) and downstream (mTOR) of PI3K also had partial inhibitory effects. Additionally, inhibition of PI3K prevented the emergence of hormone-independent cells, which suggests that early intervention with combined endocrine and PI3K-directed therapies could limit escape from antiestrogens in patients with ER-positive breast cancer.