Rapid induction of the unfolded protein response and apoptosis by synthetic oestrogen mimic TTC-352 and naturally-occurring oestrogen oestetrol for the treatment of endocrine-resistant breast cancer

Patients with long-term oestrogen-deprived (LTED) breast cancer (BC), after resistance to tamoxifen or aromatase inhibitors develops, can experience tumour regression when treated with oestrogens. Oestrogen’s anti-tumour effect is attributed to apoptosis via the oestrogen receptor (ER). Oestrogen treatment can have unpleasant gynecological, as well as non-gynecological adverse events, and thus the development of safer oestrogenic agents remains a clinical priority. Here, we study synthetic selective oestrogen mimics (SEMs) BMI-135 and TTC-352, and the naturally-occurring oestrogen oestetrol (E4), which are proposed as safer oestrogenic agents compared to 17β-oestradiol (E2), for the treatment of endocrine-resistant BC. TTC-352 and E4 are being evaluated in advanced BC clinical trials. Cell viability assays, real-time polymerase chain reaction, luciferase reporter assays, chromatin immunoprecipitation, immunoblotting, ERE DNA pull downs, mass spectrometry, X-ray crystallography, docking and molecular dynamics simulations, live cell microscopic imaging and analysis, and annexin V staining, were conducted in 11 biologically-different patient-derived BC models and a human endometrial cancer model. Results were compared with the potent full agonist E2, less potent full agonists oestrone and oestriol, the benchmark partial agonist triphenylethylene bisphenol (BPTPE), antagonists or Selective ER modulators 4-hydroxytamoxifen, endoxifen, and raloxifene, and Selective ER Downregulator fulvestrant. This work reports ERα’s regulation and coregulators’ binding profiles with SEMs and E4. It also describes SEMs and E4’s pharmacology as weak full agonists, and anti-tumour molecular mechanisms through the unfolded protein response (UPR) and apoptosis. These studies highlight the phenolic OH of TTC-352’s benzothiophene scaffold and E4’s A ring that yield an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction; sealing ERα’s ligand binding domain (LBD), recruiting E2-enriched coactivators, and triggering rapid ERα-induced UPR and apoptosis, as the basis of its anti-cancer properties. By contrast, BPTPE’s phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction; not sealing ERα’s LBD properly, not recruiting many E2-enriched coactivators, and triggering delayed ERα-induced UPR and apoptosis. Such delay might increase the risk of metastasis, clonal evolution, and acquired resistance. Overall, this work concludes the structural, pharmacological, and mechanistic constituents of an effective oestrogen for the treatment of LTED advanced BC.