Purpose: Therapeutic strategies against hormonal receptorpositive (HR þ

Multiple mechanisms exist for endocrine disruption; one nonreceptor-mediated mechanism is via effects on aromatase, an enzyme critical for maintaining the normal in vivo balance of androgens and estrogens. We adapted the AroER tri-screen 96-well assay to 1536-well format to identify potential aromatase inhibitors (AIs) in the U.S. Tox21 10K compound library. In this assay, screening with compound alone identifies estrogen receptor alpha (ERα) agonists, screening in the presence of testosterone (T) identifies AIs and/or ERα antagonists, and screening in the presence of 17β-estradiol (E2) identifies ERα antagonists. Screening the Tox-21 library in the presence of T resulted in finding 302 potential AIs. These compounds, along with 31 known AI actives and inactives, were rescreened using all 3 assay formats. Of the 333 compounds tested, 113 (34%; 63 actives, 50 marginal actives) were considered to be potential AIs independent of cytotoxicity and ER antagonism activity. Structure-activity analysis suggested the presence of both conventional (eg, 1, 2, 4, - triazole class) and novel AI structures. Due to their novel structures, 14 of the 63 potential AI actives, including both drugs and fungicides, were selected for confirmation in the biochemical tritiated water-release aromatase assay. Ten compounds were active in the assay; the remaining 4 were only active in high-throughput screen assay, but with low efficacy. To further characterize these 10 novel AIs, we investigated their binding characteristics. The AroER tri-screen, in high-throughput format, accurately and efficiently identified chemicals in a large and diverse chemical library that selectively interact with aromatase.

Overall, mixtures of kappa and mu opioids might have therapeutic potential for treating pain, particularly when the mixture has a greater ratio of mu to kappa agonist. If adverse effects of each constituent drug are reduced or avoided, then kappa:mu mixtures might be advantageous to mu opioids alone.

The purpose of the study is to define AroER tri-screen's utility for identifying endocrine-disrupting chemicals (EDCs) that target aromatase and/or estrogen receptor (ER), and to measure the total estrogenic activity in biological specimens. ER-positive, aromatase-expressing MCF-7 breast cancer cells were stably transfected with an estrogen responsive element (ERE)-driven luciferase reporter plasmid to yield a new high-throughput screening platform—the AroER tri-screen. AroER tri-screen was capable of identifying estrogen precursors, such as cortodoxone, which function as estrogens through a two-step conversion process in aromatase-expressing tissue. Furthermore, the system proved useful for assessing EDC activity in biologically relevant samples. Estimating these activities is critical because natural estrogens and estrogenic EDCs are important factors in ER-positive breast cancer risk. As our research demonstrates, incorporating functionally active aromatase into the AroER tri-screen produces a powerful and unique tool to (1) identify new EDCs targeting aromatase and/or ER; (2) discover novel EDCs activated by aromatase; and (3) estimate overall estrogenic activities in biological samples as a potential intermediate risk factor for breast cancer.

Background: Up to 10% of total breast cancers are positive for both hormone receptor [HR: estrogen receptor (ER) and/or progesterone receptor (PR)] and HER2. These patients belong mainly to the luminal B subtype, which exhibits resistance to endocrine therapy. Currently, systemic treatment for HR+/HER2+ breast cancer patients involves a combination of chemotherapy and HER2-directed therapy. While these therapies improve outcomes, they are associated with pernicious side effects. Results and Discussion: As demonstrated by studies from a number of laboratories, in HR+/HER2+ cancer, ER is constitutively activated. In other words, the estrogen ligand is not needed for ER activation. The constitutively activated ER, through its non-genomic pathways, can stimulate both HER2 (a feed forward loop) and associated kinases. In patients with HR+/HER2+ tumors, genes in the PI3K and ER pathways have been altered. Activation of PI3K/Akt/mTOR by HER2 overexpression predicts tumor progression in breast cancer [Zhou et al., Clin Cancer Res. 10: 6779 (2004)]. Therefore, PI3k/AKT/mTOR is considered as an attractive therapeutic target for HR+/HER2+ breast cancer. Allosteric inhibitors of mTOR, such as RAD001 (Everlimus), only target mTORC1 but not mTORC2, relieving the negative feedback loop in this pathway, and leading to the activation of AKT. Our results suggest that to effectively treat HR+/HER2+ cancers, both mTORC1 and mTORC2 signaling must be suppressed. In these contexts; MLN0128 (i.e., INK128 or "MLN") is a new ATP-competitive inhibitor of mTOR. It targets both mTORC1 and mTORC2; and it does not interact with FKBP12 (an immunoregulatory protein). Consequently, MLN produces weaker immune-suppressing effects than everolimus. Results from our cell culture experiments reveal that MLN is twenty times more potent than everolimus against ER+/HER2+ cells. Furthermore, while MLN alone has been demonstrated to inhibit the proliferation of ER+/HER2+ cells, we have observed more benefits when it is used as part of a combination. Fulvestrant (ICI) by itself only suppresses the proliferation of ER+/HER2+ cells partially. However, when MLN and ICI are used together, our studies revealed a synergistic effect. In addition, HR+/HER2+ PDX models have been generated to identify novel molecular networks and to examine the in vivo action of new targeted therapies against HR+/HER2+ cancer. We not only have verified the synergistic effect of MLN and ICI combination in vivo, we also identified a set of important genes playing roles in the growth of HR+/HER2+ tumors through RNA-Seq analysis. Conclusion. Due to their synergistic and targeted action, this MLN and ICI combination could provide better clinical outcome and less side-effects to HR+/HER2+ breast cancer patients, compared to the currently available options in the clinics. Citation Format: Shang Victoria Wu, Hannah Lu, Masaya Kai, Noriko Kanaya, Thenhang Luu, Courtney Vito, Laura Kruper, Joanne Mortimer, Shiuan Chen. A preclinical study to demonstrate the utility of fulvestrant and MLN0128 combination against HR+ and HER2+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-11-09.