Anti-tumor actions of novel tamoxifen-melatonin drug conjugates on estrogen-resistant tumors and actions on tumor-and uterine-expressed ESR1 in SCID_BEIGE mice

Background : Often, prolonged use of tamoxifen, an FDA approved SERM to treat ER+ breast cancer (BC), has untoward effects on the uterus and can lead to tamoxifen resistance. Novel tamoxifen-melatonin drug conjugates, C1-C5, with varying CH2- spacer lengths were developed (US Patent No. 8,785,501 B2) to offset tamoxifen-mediated tumor resistance and uterotropic actions. Two candidate compounds, C4 and C5, demonstrated anti-BC actions in MCF7, TNBC, and tamoxifen-resistant MCF7 cells (doi:10.1124/mol.119.116202). Methods: The goal of this study was to evaluate C4- and C5-mediated actions on estrogen resistant BC in vivo and ESR1 levels in the uterus in vivo using SCID/BEIGE mouse xenografts and western blot analyses. Results: Using CCK-8 assays, C4- and C5-mediated potency and efficacy to inhibit proliferation of ESR1 mutant (MCF7-luc Y537S, MCF7-luc D538G, MCF7-E380Q) and parental (MCF7-luc parental, MCF7-PE parental) lines revealed MCF7-lucD538G to be most sensitive to C4 (IC 50 = 3.8μM; 43% inhibition) and C5 (IC 50 =1.2μM; 26% inhibition). Preliminary testing using MCF7-lucD538G or parental cells bilaterally implanted into fat pads of SCID/BEIGE mice given additional estrogen or not demonstrated 60% to 90% greater tumor volumes compared to parental controls by 3 weeks, suggesting estrogen-independence. MCF7-luc D538G cells (5x10 6 ), were bilaterally implanted in the mammary pads of SCID/BEIGE mice and grown for 28 days in the absence of estrogen to allow for tumor formation. Next, treatments with C4 or C5 (1mg/kg/mouse/day, sc,), DMSO (10% in HP-beta-CD), tamoxifen (5mg 60-day release pellet implanted), fulvestrant (200mg/kg/week, sc) began and continued for 28 days. Significant tumor inhibition occurred with C4 and C5 vs DMSO- and tamoxifen-treated mice; and similar tumor-inhibiting effects were observed when compared to fulvestrant (% change from baseline: DMSO=211%; tamoxifen=169%; C4=134%; C5=95%; fulvestrant=96%, n=5/group). Analysis of ESR1 levels in tumor and uterine tissue demonstrated increases in levels in tamoxifen-treated mice and no changes in C4-, C5- and fulvestrant-treated mice vs control. ESR1 stability assays were conducted in wildtype MCF7 cells exposed to vehicle (DMSO), and 10μM each of tamoxifen, C4 or C5 for 24h, followed by a withdrawal period (0, 6, 12, 24h) using media containing cycloheximide (100μg/mL). Increases in the half-life (T 1/2 ) of ESR1 were observed in cells exposed to tamoxifen (T 1/2 =13.2h) or C4 (T 1/2 =12.6h) while decreases were observed in MCF7 cells exposed to C5 (T 1/2 =5.1h) compared to DMSO-treated (T 1/2 =9.6h) and no treatment cells ((T 1/2 =7.4h). Conclusion: Our findings indicate that tamoxifen-melatonin drug conjugates may be a viable BC treatment option for estrogen-resistant and potentially other resistant cancers while offering uterine protection.