Obesity prevalence is >39% in the USA. Obesity increases estrone synthesis in fat; and both obesity and estrone correlate with increased postmenopausal ER+ breast cancer risk and mortality. We aim to elucidate how estrone links obesity, inflammation and breast cancer. Obese fat is chronically inflamed via NFB activation. We found breast fat inflammation increases with obesity, after menopause and surrounding cancers. Breast cancer:adipocyte contact upregulates proinflammatory cytokines that stimulate NFB- and estrone:ER-dependent cancer stem cell (CSC) expansion. We showed the dominant premenopausal estradiol (E2) and postmenopausal estrone (E1) regulate different genes. While E2-bound ER inhibits NFB, we showed E1-bound ER is an NFB co-activator and induces gene profiles of inflammation, EMT, CSC expansion and metastasis, while E2 did not. Finally, E1: ER caused more cytokine gene induction, stem cell expansion, and ER+ tumor growth and metastasis than E2 in vivo. While our in vivo data suggest E1 driven ER-NFB co-targets promote tumor progression, little is known about how E1-bound ER and NFB interact at chromatin, and how their co-regulators differ from those of E2-ER. Up to 30% of metastatic ER+ BC develop activating ESR1 mutations. We found while 3D growth of MCF7 controls is greater with E1 than E2; both stimulate 3D growth of ER mutant MCF7 lines equally. Further, both ER mutants direct greater NFB activation upon either E1 or E2 treatment, suggesting that the altered mutant ER conformation might direct greater ER-p65B activation. We hypothesize that E1-ER target gene selection, co-regulators, and expression differ from those of E2:ER and that therapy-induced ER mutants will permit pro-inflammatory, oncogenic, and prometastatic ER-NFB target genes, normally activated only by E1-bound ER-WT, to be indiscriminately activated by either E1 or E2 in cancers. Aim 1 will test how E1 and E2 driven gene expression differs, comparing ChIPseq of ER, p65B and FOXA1 and correlating these with gene expression profiles in ER+ cancer lines and organoids stimulated by E1 vs E2, +/- NFB. Aim 2 To identify co-regulators unique to E1 and E2-liganded ER that mediate gene induction or repression, we carry out i) ChIP-Mass Spec in cells treated with E1 or E2, +/- NFB activation; and ii) Gradient-Seq to separate euchromatin from heterochromatin followed by ChIPseq and ChIP-Mass Spec to identify the E1- vs E2-liganded ER interactome in transactivator versus repressor complexes. Aim 3 will test if both E1 and E2 i) cause greater ER:p65 binding and ii) preferential activation of oncogenic ER mutant: B co-target genes normally activated by E1-liganded ER-WT +/-NFB in vitro, and ii) activate a more E1-ER-like oncogenic genes profile in ERY537S BC xenografts and PDX than in ER-WT BC tumors in vivo. This will inform how ER target gene changes during the shift from high E2 to high E1 after menopause might promote breast cancer development and may...