Breast cancers expressing estrogen receptor α, progesterone receptor, or the human epidermal growth factor receptor 2 (HER2) proto-oncogene account for approximately 90% of cases, and treatment with antiestrogens and HER2-targeted agents has resulted in drastically improved survival in many of these patients. However, de novo or acquired resistance to antiestrogen and HER2-targeted therapies is common, and many tumors will recur or progress despite these treatments. Additionally, the remaining 10% of breast tumors are negative for estrogen receptor α, progesterone receptor, and HER2 (“triple-negative”), and a clinically proven tumor-specific drug target for this group has not yet been identified. Therefore, the identification of new therapeutic targets in breast cancer is of vital clinical importance. Preclinical studies elucidating the mechanisms driving resistance to standard therapies have identified promising targets including cyclin-dependent kinase 4/6, phosphoinositide 3-kinase, poly adenosine diphosphate–ribose polymerase, Src, and histone deacetylase. Herein, we discuss the clinical potential and status of new therapeutic targets in breast cancer.

Estrogens have been shown to elicit anticancer effects against estrogen receptor α ( ER )‐positive breast cancer. We sought to determine the mechanism underlying the therapeutic response. Response to 17β‐estradiol was assessed in ER + breast cancer models with resistance to estrogen deprivation: WHIM 16 patient‐derived xenografts, C7‐2‐ HI and C4‐ HI murine mammary adenocarcinomas, and long‐term estrogen‐deprived MCF ‐7 cells. As another means to reactivate ER , the anti‐estrogen fulvestrant was withdrawn from fulvestrant‐resistant MCF ‐7 cells. Transcriptional, growth, apoptosis, and molecular alterations in response to ER reactivation were measured. 17β‐estradiol treatment and fulvestrant withdrawal induced transcriptional activation of ER , and cells adapted to estrogen deprivation or fulvestrant were hypersensitive to 17β‐estradiol. ER transcriptional response was followed by an unfolded protein response and apoptosis. Such apoptosis was dependent upon the unfolded protein response, p53, and JNK signaling. Anticancer effects were most pronounced in models exhibiting genomic amplification of the gene encoding ER ( ESR 1 ), suggesting that engagement of ER at high levels is cytotoxic. These data indicate that long‐term adaptation to estrogen deprivation or ER inhibition alters sensitivity to ER reactivation. In such adapted cells, 17β‐estradiol treatment and anti‐estrogen withdrawal hyperactivate ER , which drives an unfolded protein response and subsequent growth inhibition and apoptosis. 17β‐estradiol treatment should be considered as a therapeutic option for anti‐estrogen‐resistant disease, particularly in patients with tumors harboring ESR 1 amplification or ER overexpression. Furthermore, therapeutic strategies that enhance an unfolded protein response may increase the therapeutic effects of ER reactivation.

Purpose Phosphatidylinositol 3-kinase (PI3K) inhibitors are being developed for the treatment of estrogen receptor α (ER)-positive breast cancer in combination with anti-estrogens. Understanding the temporal response and pharmacodynamic effects of PI3K inhibition in ER+ breast cancer will provide rationale for treatment scheduling to maximize therapeutic index. Experimental Design Anti-estrogen-sensitive and -resistant ER+ human breast cancer cell lines, and mice bearing PIK3CA-mutant xenografts were treated with the anti-estrogen fulvestrant, the PI3K inhibitor GDC-0941 (pictilisib; varied doses/schedules that provided similar amounts of drug each week), or combinations. Cell viability, signaling pathway inhibition, proliferation, apoptosis, tumor volume, and GDC-0941 concentrations in plasma and tumors were temporally measured. Results Treatment with the combination of fulvestrant and GDC-0941, regardless of dose/schedule, was significantly more effective than single-agent treatments in fulvestrant-resistant tumors. Short-term, complete PI3K inhibition blocked cell growth in vitro more effectively than chronic, incomplete inhibition. Longer-term PI3K inhibition hypersensitized cells to growth factor signaling upon drug withdrawal. Different schedules of GDC-0941 elicited similar tumor responses. While weekly high-dose GDC-0941 with fulvestrant continuously suppressed PI3K signaling for 72 hours, inducing a bolus of apoptosis and inhibiting proliferation, PI3K reactivation upon GDC-0941 washout induced a proliferative burst. Fulvestrant with daily low-dose GDC-0941 metronomically suppressed PI3K for 6–9 hours/day, repeatedly inducing small amounts of apoptosis and temporarily inhibiting proliferation, followed by proliferative rebound compared to fulvestrant alone. Conclusions Continuous and metronomic PI3K inhibition elicit robust anti-cancer effects in ER+, PIK3CA-mutant breast cancer. Clinical exploration of alternate treatment schedules of PI3K inhibitors with anti-estrogens is warranted.

Purpose Determine the roles of the phosphatidylinositol 3-kinase (PI3K) isoforms p110α and p110β in PTEN-deficient, estrogen receptor α (ER)-positive breast cancer, and the therapeutic potential of isoform-selective inhibitors. Experimental Design Anti-estrogen-sensitive and -resistant PTEN-deficient, ER+ human breast cancer cell lines, and mice bearing anti-estrogen-resistant xenografts were treated with the anti-estrogen fulvestrant, the p110α inhibitor BYL719, the p110β inhibitor GSK2636771, or combinations. Temporal response to growth factor receptor-initiated signaling, growth, apoptosis, predictive biomarkers, and tumor volumes were measured. Results p110β primed cells for response to growth factor stimulation. While p110β inhibition suppressed cell and tumor growth, dual targeting of p110α/β enhanced apoptosis and provided sustained tumor response. The growth of anti-estrogen-sensitive cells was inhibited by fulvestrant, but fulvestrant inconsistently provided additional therapeutic effects beyond PI3K inhibition alone. Treatment-induced decreases in phosphorylation of AKT and Rb were predictive of therapeutic response. Short-term drug treatment induced tumor cell apoptosis and proliferative arrest to induce tumor regression, while long-term treatment only suppressed proliferation to provide durable regression. Conclusions p110β is the dominant PI3K isoform in PTEN-deficient, ER+ breast cancer cells. Upon p110β inhibition, p110α did not induce significant reactivation of AKT, but combined targeting of p110α/β most effectively induced apoptosis in vitro and in vivo and provided durable tumor regression. Since apoptosis and tumor regression occurred early but not late in the treatment course, and proliferative arrest was maintained throughout treatment, p110α/β inhibitors may be considered short-term cytotoxic agents and long-term cytostatic agents.

Hyperactivation of the PI3K pathway has been implicated in resistance to antiestrogen therapies in estrogen receptor α (ER)-positive breast cancer, prompting the development of therapeutic strategies to inhibit this pathway. Autophagy has tumor-promoting and -suppressing roles and has been broadly implicated in resistance to anticancer therapies, including antiestrogens. Chloroquine (CQ) is an antimalarial and amebicidal drug that inhibits autophagy in mammalian cells and human tumors. Herein, we observed that CQ inhibited proliferation and autophagy in ER breast cancer cells. PI3K inhibition with GDC-0941 (pictilisib) induced autophagy. Inhibition of autophagy using CQ or RNA interference potentiated PI3K inhibitor-induced apoptosis. Combined inhibition of PI3K and autophagy effectively induced mitochondrial membrane depolarization, which required the BH3-only proapoptotic proteins Bim and PUMA. Treatment with GDC-0941, CQ, or the combination, significantly suppressed the growth of ER breast cancer xenografts in mice. In an antiestrogen-resistant xenograft model, GDC-0941 synergized with CQ to provide partial, but durable, tumor regression. These findings warrant clinical evaluation of therapeutic strategies to target ER, PI3K, and autophagy for the treatment of ER breast cancer.-Yang, W., Hosford, S. R., Traphagen, N. A., Shee, K., Demidenko, E., Liu, S., Miller, T. W. Autophagy promotes escape from phosphatidylinositol 3-kinase inhibition in estrogen receptor-positive breast cancer.