Fig 2: Characterization of palbociclib resistance models developed in ESR1 wild-type and ESR1 mutant (D538G and Y537S) backgrounds. CellTiter-Glo assay, colony formation assay, and western blot analysis of cell cycle proteins of a ESR1wt-PalboS and ESR1wt-PalboR cells, b ESR1mut: D538G-PalboS and ESR1mut: D538G-PalboR cells, and c ESR1mut: Y537S-PalboS and ESR1mut: Y537S-PalboR cells, treated with controls and palbociclib at the indicated doses. d Pathway perturbations in ESR1wt-PalboR vs ESR1wt-PalboS cell lines; red bar represents upregulated genes, blue bar represents downregulated genes

Fig 3: CDK4/6i-resistant cell lines retain ER and ER signaling. a Western blot analysis of ER and downstream ER target genes (GREB1/PR) in the CDK4/6i-sensitive and CDK4/6i-resistant ESR1 wild-type and ESR1 mutant cell lines. PalboS/PalboR, RiboS/RiboR, and AbemaS/AbemaR cells were treated with either control or the indicated CDK4/6i for 24 h. b Table summarizing the changes in ER and ER target genes in the resistant cells when compared to the sensitive cell lines

Fig 4: Upregulation of ERα expression in murine and human BC cells by SFA-stimulated macrophages. E0771 (A–C) and MCF-7 (D–F) BC cells either remained untreated (Cont.) or were incubated (24 h, 37 °C) with medium conditioned by either unstimulated macrophages (usMϕ), or macrophages stimulated (16 h) by vehicle (BSA) alone (v·Mϕ) or by SFA (i.e., palmitic acid) conjugated with fatty acid free-BSA at a 2:1 molar ratio (SFA·Mϕ). (A,D) ERα protein levels were determined by immunoblotting. (B,E) The band intensity was quantified using ImageJ software; intensity ratio for ERα/actin or ERα/GAPDH is shown. (C,F) The expression of mouse (C) and human (F) ESR1 gene, encoding for ERα, was determined by qRT-PCR. The data shown are representative of ≥3 independent experiments; error bars represent ± SD. * p < 0.02; ** p < 0.0015.

Fig 5: Elacestrant inhibits the growth of CDK4/6i-resistant breast cancer cell lines in vitro. a CellTiter-Glo assay of ER+ (wild-type and mutant) CDK4/6i-sensitive and CDK4/6i-resistant breast cancer cells treated with elacestrant at the indicated doses for 7 days. Relative EC50 values were calculated by using a log(inhibitor) vs response curve fit. b Colony formation assay of ER+ (wild-type and mutant) CDK4/6i-sensitive and CDK4/6i-resistant breast cancer cells treated with elacestrant at the indicated dose for 3–5 weeks. c Western blot analysis of ER and downstream ER target genes (GREB1/PR) in the CDK4/6i-sensitive and CDK4/6i-resistant ESR1 wild-type and ESR1 mutant cell lines. CDK4/6i-sensitive, PalboR, RiboR, and AbemaR cells were treated with either control or elacestrant (300 nM). d qRT-PCR of PGR, TFF1, and GREB1 in palbociclib-sensitive and palbociclib-resistant cells treated with elacestrant at the specified doses