A novel melatonin, estrogen, and progesterone hormone therapy was developed as a safe bio-identical alternative hormone therapy for menopausal women based on the Women’s Health Initiative findings that PremPro™ increased breast cancer risk and mortality of all types of breast cancer in postmenopausal women. For HER2 breast cancer, melatonin, estrogen, and progesterone delayed tumor onset and reduced tumor incidence in neu female mice. For other breast cancers, its actions are unknown. In this study, melatonin, estrogen, and progesterone hormone therapy were assessed in human ER+ (MCF-7) and triple negative breast cancer (MDA-MB-231) cells, and found to decrease proliferation and migration of both breast cancer lines. Inhibition of MEK1/2 and 5 using PD98059 and BIX02189, respectively, inhibited proliferation and migration in MDA-MB-231 cells and proliferation in MCF-7 cells; however, when combined with melatonin, estrogen, and progesterone, BIX02189 blocked melatonin, estrogen, and progesterone–mediated inhibition of migration in MCF-7 cells and induced Elf-5. For MDA-MB-231 cells, BIX02189 combined with melatonin, estrogen, and progesterone inhibited proliferation and increased pERK1/2 and β1-INTEGRIN; levels of pERK5 remained low/nearly absent in both breast cancer lines. These findings demonstrate novel anti-cancer actions of melatonin, estrogen, and progesterone in ER+ and triple negative breast cancer cells through intricate MEK1/2- and MEK5-associated signaling cascades that favor anti-proliferation and anti-migration.

A novel melatonin, estrogen (E2), and progesterone (P4) hormone therapy (MEPT) was developed as a safe bio‐identical alternative hormone therapy for menopausal women based on the Women’s Health Initiative findings that PremPro™ increased breast cancer (BC) risk and mortality of all types of BC in postmenopausal women. MEPT delayed tumor onset and reduced tumor incidence in Neu female mice (Dodda et al., 2019; Front Oncol). For other BCs, its actions are currently unknown. In this study, MEPT’s anti‐cancer actions (cell viability, migration) were tested in MCF‐7 (human ER+/PR+) and MDA‐MB‐231 (human TNBC with constitutively active ras mutation) using equivalent concentrations of melatonin (1nM), E2 (50pM), and P4 (10nM) observed in serum after an oral dose that produced anti‐tumor actions in neu mice (Dodda et al 2019; Front Oncol). Potential mechanisms underlying MEPT’s anti‐cancer actions were assessed by western blot (pERK1/2, pERK5, NF‐kB, RUNX2, β1‐INTEGRIN, RANKL, and ELF‐5) using MEK1/2 (PD98059) and MEK5 (Bix02189) inhibitors and radioligand binding using 2‐[125I]‐iodomelatonin or [3H]estradiol binding for melatonin and estrogen receptor expression, respectively. For MCF‐7 cells, BIX02189 combined with MEPT blocked MEPT‐mediated inhibition of migration and MEPT‐mediated ELF5 induction in MCF‐7 cells (vs. vehicle) and decreased pERK5 (vs. Bix02189) suggesting that MEK5, through pERK1/2 and ELF5, were involved in the anti‐cancer actions of MEPT in MCF‐7 cells. For MDA‐MB‐231 cells, although PD98059 and Bix02189 alone inhibited viability and migration, when added in combination with MEPT, no further blockade or enhancement occurred suggesting that MEK1/2 and MEK5 were not involved in MEPT’s anti‐cancer actions in MDA‐MB‐231 cells. MEPT’s differential actions on MCF‐7 and MDA‐MB‐231 cells were most likely attributed to unique melatonin/melatonin receptor actions within each cell. Both MCF‐7 and MDA‐MB‐231 cells express high‐affinity melatonin receptors (MCF‐7: Kd = 13 pM; Bmax = 20.55 fmol/mg protein vs. MDA‐231: Kd = 1.7 nM; Bmax = 60.09 fmol/mg protein) but only in MCF‐7 cells were [3H]‐estradiol binding sites detected (MCF‐7: Kd = 2.2 nM; Bmax = 8.6 pmol/mg protein) and stimulatory actions of E2 or P4 on MCF‐7 cell proliferation observed. These findings demonstrate novel anti‐cancer actions of MEPT in ER+ BC and TNBC cells through distinct mechanisms. Future studies assessing MEPT’s actions against ER+ BC and TNBC in vivo are warranted. Support or Funding Information This study was funded by the Marie‐Clement Rodier, C.S.Sp. Endowed Chair.