A b s t r ac t Background Resistance to endocrine therapy in breast cancer is associated with activation of the mammalian target of rapamycin (mTOR) intracellular signaling pathway. In early studies, the mTOR inhibitor everolimus added to endocrine therapy showed antitumor activity. Methods In this phase 3, randomized trial, we compared everolimus and exemestane versus exemestane and placebo (randomly assigned in a 2:1 ratio) in 724 patients with hormone-receptor–positive advanced breast cancer who had recurrence or progression while receiving previous therapy with a nonsteroidal aromatase inhibitor in the adjuvant setting or to treat advanced disease (or both). The primary end point was progression-free survival. Secondary end points included survival, response rate, and safety. A preplanned interim analysis was performed by an independent data and safety monitoring committee after 359 progression-free survival events were observed. Results Baseline characteristics were well balanced between the two study groups. The median age was 62 years, 56% had visceral involvement, and 84% had hormone-sensitive disease. Previous therapy included letrozole or anastrozole (100%), tamoxifen (48%), fulvestrant (16%), and chemotherapy (68%). The most common grade 3 or 4 adverse events were stomatitis (8% in the everolimus-plus-exemestane group vs. 1% in the placebo-plus-exemestane group), anemia (6% vs. <1%), dyspnea (4% vs. 1%), hyperglycemia (4% vs. <1%), fatigue (4% vs. 1%), and pneumonitis (3% vs. 0%). At the interim analysis, median progression-free survival was 6.9 months with everolimus plus exemestane and 2.8 months with placebo plus exemestane, according to assessments by local investigators (hazard ratio for progression or death, 0.43; 95% confi dence interval [CI], 0.35 to 0.54; P<0.001). Median progression-free survival was 10.6 months and 4.1 months, respectively, according to central assessment (hazard ratio, 0.36; 95% CI, 0.27 to 0.47; P<0.001). Conclusions Everolimus combined with an aromatase inhibitor improved progression-free survival in patients with hormone-receptor–positive advanced breast cancer previously treated with nonsteroidal aromatase inhibitors. (Funded by Novartis; BOLERO-2 ClinicalTrials .gov number, NCT00863655.)

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Everolimus in Postmenopausal Hormone-Receptor–Positive Advanced Breast Cancer

Recent molecular studies have expanded the biological contexts in which topoisomerase II (TOP2) has crucial functions, including DNA replication, transcription and chromosome segregation. Although the biological functions of TOP2 are important for ensuring genomic integrity, the ability to interfere with TOP2 and generate enzyme-mediated DNA damage is an effective strategy for cancer chemotherapy. The molecular tools that have allowed an understanding of the biological functions of TOP2 are also being applied to understanding the details of drug action. These studies promise refined targeting of TOP2 as an effective anticancer strategy.

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Targeting DNA topoisomerase II in cancer chemotherapy

The efficacy of endocrine therapies (such as tamoxifen) in breast cancer is limited by intrinsic and acquired therapeutic resistance. What do we know about the genetic lesions and molecular processes that determine endocrine resistance in the clinic, and how can we use this to improve therapy? Endocrine therapies targeting oestrogen action (anti-oestrogens, such as tamoxifen, and aromatase inhibitors) decrease mortality from breast cancer, but their efficacy is limited by intrinsic and acquired therapeutic resistance. Candidate molecular biomarkers and gene expression signatures of tamoxifen response emphasize the importance of deregulation of proliferation and survival signalling in endocrine resistance. However, definition of the specific genetic lesions and molecular processes that determine clinical endocrine resistance is incomplete. The development of large-scale computational and genetic approaches offers the promise of identifying the mediators of endocrine resistance that may be exploited as potential therapeutic targets and biomarkers of response in the clinic.

Biological determinants of endocrine resistance in breast cancer

http://kitto.cm.utexas.edu/courses/ch395g/fall2009/MOL190/LoPiccolo.pdf

Targeting the PI3K/Akt/mTOR pathway: effective combinations and clinical considerations

Purpose Cross-talk between signal transduction pathways likely contributes to hormone resistance in metastatic breast cancer (mBC). Everolimus, an oral inhibitor of the mammalian target of rapamycin, has restored sensitivity in endocrine-resistance models and shown anticancer activity in early-phase mBC clinical trials. This analysis evaluated efficacy and safety of everolimus in combination with tamoxifen in patients with mBC resistant to aromatase inhibitors (AIs). Patients and Methods This open-label, phase II study randomly assigned postmenopausal women with hormone receptor–positive, human epidermal growth factor receptor 2–negative, AI-resistant mBC to tamoxifen 20 mg/d plus everolimus 10 mg/d (n = 54) or tamoxifen 20 mg/d alone (n = 57). Randomization was stratified by primary and secondary hormone resistance. Primary end point was clinical benefit rate (CBR), defined as the percentage of all patients with a complete or partial response or stable disease at 6 months. No formal statistical compariso...

Randomized Phase II Trial of Everolimus in Combination With Tamoxifen in Patients With Hormone Receptor–Positive, Human Epidermal Growth Factor Receptor 2–Negative Metastatic Breast Cancer With Prior Exposure to Aromatase Inhibitors: A GINECO Study

Purpose: RAD001 (everolimus), a mammalian target of rapamycin (mTOR) pathway inhibitor in phase II clinical trials in oncology, exerts potent antiproliferative/antitumor activities. Many breast cancers are dependent for proliferation on estrogens synthesized from androgens (i.e., androstenedione) by aromatase. Letrozole (Femara) is an aromatase inhibitor used for treatment of postmenopausal women with hormone-dependent breast cancers. The role of the mTOR pathway in estrogen-driven proliferation and effects of combining RAD001 and letrozole were examined in vitro in two breast cancer models. Experimental Design: The role of the mTOR pathway in estrogen response was evaluated in aromatase-expressing MCF7/Aro breast cancer cells by immunoblotting. Effects of RAD001 and letrozole (alone and in combination) on the proliferation and survival of MCF7/Aro and T47D/Aro cells were evaluated using proliferation assays, flow cytometry, immunoblotting, and apoptosis analyses. Results: Treatment of MCF7/Aro cells with estradiol or androstenedione caused modulation of the mTOR pathway, a phenomenon reversed by letrozole or RAD001. In MCF7/Aro and T47D/Aro cells, both agents inhibited androstenedione-induced proliferation; however, in combination, this was significantly augmented ( P 1 progression and a significant decrease in cell viability ( P P Conclusion: mTOR signaling is required for estrogen-induced breast tumor cell proliferation. Moreover, RAD001-letrozole combinations can act in a synergistic manner to inhibit proliferation and trigger apoptotic cell death. This combination holds promise for the treatment of hormone-dependent breast cancers.

https://clincancerres.aacrjournals.org/content/clincanres/11/14/5319.full.pdf

Dual Inhibition of mTOR and Estrogen Receptor Signaling In vitro Induces Cell Death in Models of Breast Cancer

Topoisomerase II poisons are in clinical use as anti-cancer therapy for decades and work by stabilizing the enzyme-induced DNA breaks. In contrast, catalytic inhibitors block the enzyme before DNA scission. Although several catalytic inhibitors of topoisomerase II have been described, preclinical concepts for exploiting their anti-proliferative activity based on molecular characteristics of the tumor cell have only recently started to emerge. Topoisomerase II is an ATPase and uses the energy derived from ATP hydrolysis to orchestrate the movement of the DNA double strands along the enzyme. Thus, interfering with ATPase function with low molecular weight inhibitors that target the nucleotide binding pocket should profoundly affect cells that are committed to undergo mitosis. Here we describe the discovery and characterization of a novel purine diamine analogue as a potent ATP-competitive catalytic inhibitor of topoisomerase II. Quinoline aminopurine compound 1 (QAP 1) inhibited topoisomerase II ATPase activity and decatenation reaction at sub-micromolar concentrations, targeted both topoisomerase II alpha and beta in cell free assays and, using a quantitative cell-based assay and a chromosome segregation assay, displayed catalytic enzyme inhibition in cells. In agreement with recent hypothesis, we show that BRCA1 mutant breast cancer cells have increased sensitivity to QAP 1. The results obtained with QAP 1 demonstrate that potent and selective catalytic inhibition of human topoisomerase II function with an ATP-competitive inhibitor is feasible. Our data suggest that further drug discovery efforts on ATP-competitive catalytic inhibitors are warranted and that such drugs could potentially be developed as anti-cancer therapy for tumors that bear the appropriate combination of molecular alterations.

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Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue.

5620 Fatty acid synthase (FAS) is a 250-270 kd cytosolic multifunctional polypeptide required for the conversion of dietary carbohydrates to fatty acids. While high levels of FAS expression compared to normal tissue have been observed in several cancers, including breast, prostate, colon and lung carcinoma, there is little information regarding gliomas. For the first time, we show high levels of FAS expression associated with glioma cell lines. Western blotting revealed higher FAS expression (4-20 fold increase) in U251, U138, NSB-19, and U373 human glioma cell lines and in C6 rat glioma cells compared to normal rat astrocytes. Cerulenin-mediated inhibition of lipid synthesis was determined using 14C labeled acetic acid. After labeling, total lipids were extracted and counted for 14C by scintillation counting. Endogenous fatty acid synthesis in cerulenin-treated glioma cells decreased by 49%, 49.7% and 51.1% at 2 hours in NSB-19, U251, and C6 cells, respectively. Cell cycle analysis, using flow cytometry, demonstrated that 3 μg/mL of cerulenin resulted in S-phase cell arrest 24h after treatment; the number of G0/G1 and G2/M cells was decreased Further, treatment with cerulenin resulted in dose- and time-dependent decreases in glioma cell viability, assessed using the MTT assay. The mean IC50 values for U251 were 4.5 μg/mL and 2 μg/mL at 24 and 96h, respectively. For NSB-19 glioma cells, that expressed lower levels of FAS protein, the mean IC50 values were 5.5 μg/mL and 2.7 μg/mL at 24 and 96h, respectively. Cerulenin also reduced glioma cell clonogenic survival; incubating NSB-19 cells with 3μg/mL cerulenin reduced clonogenic cell survival 97% compared to controls. Increased apoptotic cell death and activated caspase-3 expression was observed in U251 and NSB-19 cells treated with cerulenin. To test whether FAS inhibition led to selective glioma cell cytotoxicity, primary rat astrocytes that express little FAS were incubated with 0.5-7.5 μg/mL cerulenin for 24h; no decrease in cell viability was observed. These data demonstrate that :(1) human and rat glioma cell lines express high levels of FAS compared with normal brain astrocytes; (2) FAS inhibition is selectively cytotoxic to glioma cells; normal astrocytes are unaffected; and (3) cerulenin mediated cytotoxicity is due in part to apoptotic cell death and cell cycle arrest. These findings suggest that FAS might be a novel target for anti-glioma therapy. (Supported by NIH Grant CA82722)

The mTOR pathway in estrogen response: A potential for combining the rapamycin derivative RAD001 with the aromatase inhibitor Letrozole (Femara®) in breast carcinoma

Joëlle Rudloff

Papers

Dual Inhibition of mTOR and Estrogen Receptor Signaling In vitro Induces Cell Death in Models of Breast Cancer

Catalytic inhibition of topoisomerase II by a novel rationally designed ATP-competitive purine analogue.

The mTOR pathway in estrogen response: A potential for combining the rapamycin derivative RAD001 with the aromatase inhibitor Letrozole (Femara®) in breast carcinoma