Purpose: Despite adjuvant endocrine therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Experimental Design: Mimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant endocrine therapy. Results: Dormant breast cancer cells exhibited upregulated 5' adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the anti-diabetes AMPKactivating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival.Conclusions: AMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.

Tumor microenvironment (TME) cytokine screening revealed FGF2 as a clinically relevant mechanism of resistance to anti-estrogens, mTORC1 inhibition, and PI3K inhibition in ER+ breast cancer. Shee et al. highlight an underdeveloped aspect of precision oncology: treating patients according to their TME constitution.

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 As another means to reactivate ER , the anti‐estrogen fulvestrant was withdrawn from fulvestrant‐resistant MCF ‐7 cells.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.

Rac GTPases have oncogenic roles in cell growth, survival, and migration. We tested response to the Rac inhibitor EHT1864 in a panel of breast cancer cell lines. EHT1864-induced growth inhibition was associated with dual inhibition of the PI3K/AKT/mTORC1 and MEK/ERK pathways. Breast cancer cells harboring PIK3CA mutations or HER2 overexpression were most sensitive to Rac inhibition, suggesting that such oncogenic alterations link Rac activation with PI3K/AKT/mTORC1 and MEK/ERK signaling. Interestingly, EHT1864 decreased activation of the mTORC1 substrate p70S6K earlier than AKT inhibition, suggesting that Rac may activate mTORC1/p70S6K independently of AKT. Comparison of the growth-inhibitory profile of EHT1864 to 137 other anti-cancer drugs across 656 cancer cell lines revealed significant correlation with the p70S6K inhibitor PF-4708671. We confirmed that Rac complexes contain MEK1/2 and ERK1/2, but also contain p70S6K; these interactions were disrupted by EHT1864. Pharmacokinetic profiles revealed that EHT1864 was present in mouse plasma at concentrations effective in vitro for approximately 1 h after intraperitoneal administration. EHT1864 suppressed growth of HER2+ tumors, and enhanced response to anti-estrogen treatment in ER+ tumors. Further therapeutic development of Rac inhibitors for HER2+ and PIK3CA-mutant cancers is warranted.

Purpose: We hypothesized that integrated analysis of cancer types from different lineages would reveal novel molecularly defined subgroups with unique therapeutic vulnerabilities. On the basis of the molecular similarities between subgroups of breast and ovarian cancers, we analyzed these cancers as a single cohort to test our hypothesis.Experimental Design: Identification of transcriptional subgroups of cancers and drug sensitivity analyses were performed using mined data. Cell line sensitivity to Hsp90 inhibitors (Hsp90i) was tested in vitro. The ability of a transcriptional signature to predict Hsp90i sensitivity was validated using cell lines, and cell line-and patient-derived xenograft (PDX) models. Mechanisms of Hsp90i sensitivity were uncovered using immunoblot and RNAi.Results: Transcriptomic analyses of breast and ovarian cancer cell lines uncovered two mixed subgroups comprised primarily of triple-negative breast and multiple ovarian cancer subtypes. Drug sensitivity analyses revealed that cells of one mixed subgroup are significantly more sensitive to Hsp90i compared with cells from all other cancer lineages evaluated. A gene expression classifier was generated that predicted Hsp90i sensitivity in vitro, and in cell lineand PDXs. Cells from the Hsp90i-sensitive subgroup underwent apoptosis mediated by Hsp90i-induced upregulation of the proapoptotic proteins Bim and PUMA.Conclusions: Our findings identify Hsp90i as a potential therapeutic strategy for a transcriptionally defined subgroup of ovarian and breast cancers. This study demonstrates that gene expression profiles may be useful to identify therapeutic vulnerabilities in tumor types with limited targetable genetic alterations, and to identify molecularly definable cancer subgroups that transcend lineage.