Abstract 3915: Melatonin-tamoxifen hybrid ligands and their effects on breast cancer
Authors: Hasan Mahmud , Marzouk Mohamed Akmal , Adhikari Saugat , Wright Thomas , Miller Benton , Peckich Brianna , Yingling Spencer , Stratford Robert , Zlotos Darius , Cavanaugh Jane , Witt-Enderby Paula 
Published: August 2018

Abstract
Background: A dual melatonin and tamoxifen hybrid ligand (HL) was developed and shown to display uterine protective actions while co-administration of the same doses of melatonin and tamoxifen unlinked increased uterine weight (US Patent 8785501). The anti-cancer actions of this HL (C5) and 4 others (C2, C4, C9 and C15)—which differed only in their number of carbon linkers—were further tested in 5 phenotypically diverse breast cancer (BC) cell lines—MCF-7 (ER+/PR+), tamoxifen resistant MCF-7 (TamR), MMC (HER2+) and triple negatives (MDA-MB-231 with a raf-1 mutation and BT549 with PTEN mutation) for their effects on cell proliferation, migration, protein expression and binding affinity to melatonin receptors (MT1Rs) and estrogen receptors (ERs). Analysis of HL effects on pERK1/2, pERK5, NF-kB, Runx2, and β1-integrin expression in each BC line and use of the MEK1/2 inhibitor (PD98059) or the MEK5 inhibitor (Bix02189) in the presence of the HLs helped elucidate their mechanism of action. Also, we determined the pharmacokinetic parameters of C4 and C5 HL using both in vitro and in vivo models.
Results: All HLs demonstrated concentration-dependent inhibition of 2-[125I]-melatonin binding to MT1Rs, whereas C5 HL also showed equal binding affinity like melatonin. Only C4 and C5 HLs demonstrated equal affinity as tamoxifen or 4-OH-tamoxifen to ERs, and no concentration dependent inhibition of [125I]-estradiol/[3H]-estradiol binding occurred for C2, C9, and C15 HLs. All HLs were screened for their effects on BC cell proliferation and migration and it was determined that C4 and C5 demonstrated superior potency and efficacy compared to the other HLs (C2, C9, C15) and to co-exposure to melatonin and tamoxifen or 4-OH-tamoxifen (unlinked) for all BC cell lines including TamR MCF-7 cells. Acute (15 min) exposure to C4 and C5 HL increased pERK1/2 activity in MCF-7 cells and C5 HL increased NF-КB protein expression in MCF-7 and MDA-231 cells vs vehicle. Inhibition of MEK1/2 or MEK5 alone inhibited MMC, MDA-231 and BT549 cell migration. The addition of PD98059 enhanced C4-mediated inhibition of MCF-7 and BT549 cell proliferation and enhanced C5-mediated inhibition of MCF-7 cell proliferation and migration and BT549 cell migration. The addition of Bix02189 enhanced C4-mediated inhibition of MMC and BT549 cell proliferation and enhanced C5-mediated inhibition of MCF-7 and BT549 cell migration. Co-administration of Bix02189 and C4 HL blocked MDA-231 cell migration. C4 and C5 HLs displayed similar CYP-mediated loss profiles as tamoxifen in both mouse and human liver microsomes; including, the rate of metabolism being faster in mice vs human. Moreover, C4 HL showed better oral bioavailability than C5 HL in C57BL/6J female mice.
Conclusions: Novel melatonin-tamoxifen HLs linked by 4 or 5 carbons show promise as anti-cancer drugs in phenotypically diverse BCs including triple negative and tamoxifen resistant and may display less uterotropic effects.
Citation Format: Mahmud Hasan, Mohamed Akmal Marzouk, Saugat Adhikari, Thomas Wright, Benton Miller, Brianna Peckich, Spencer Yingling, Robert Stratford, Darius Zlotos, Jane Cavanaugh, Paula Witt-Enderby. Melatonin-tamoxifen hybrid ligands and their effects on breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3915.