Fig 2: Med1, an important node associated with leptin signaling network and obesity-associated signature in breast cancer, is upregulated via miR-205 in hyperleptinemic state.A Protein-protein interaction landscape of Med1 and mediator complex subunits and associated proteins is presented. B An integrated network of proteins involved in leptin signaling network and Med1 is presented. In total, ~70 proteins are represented, 9 of which serve as nodes in the Med1-leptin interaction network. Blue circles represent high confidence leptin-signaling proteins. Pink and purple circles represent additional proteins identified in Med1 protein-protein interaction network. To avoid the overcrowded network, only selected proteins are represented. C Obesity-related gene signature was queried in Med1 ChIP-seq data. 48 out of 75 genes upregulated in breast tumors in obese patients are potential targets of Med1. A summary of Med1 associated proteins that associate with leptin is presented in Supplemental Table S1. D Kaplan–Meier plots of breast cancer survival in relation to mean Med1 (PPARBP) expression (p = 2.6E–03). E MCF7 and BT474 cells were treated with 100 ng/ml leptin (L) for various time intervals as indicated and immunoblotted for Med1 expression. F, G MCF7 cells derived tumors from high-fat-diet (HFD) fed obese mice and normal-diet (ND) fed lean mice and treated with 4-hydroxytamoxifen (T) or vehicle (C) were subjected to immunohistochemical (IHC) using Med1 antibody (Scale bars: 50 µm) and immunoblot analysis using Cathepsin D (CATD), cMyc, Cyclin D1, TFF1, and Med1, antibodies. Actin was used as loading control in immunoblot analysis. H The miRNA target pairing sequences were analyzed by bioinformatics tools. I MCF7 cells were transfected with miR-205 mimic and Med1 expression was examined using real-time PCR. J Kaplan–Meier plots of breast cancer survival in relation to mean miR-205 expression (p = 3.6E–03). K Expression of miR-205 was examined in MCF7 cells treated with 100 ng/ml leptin (L) and/or 1 µM 4-hydroxytamoxifen (T) as indicated. (L) Expression of miR-205 was examined in MCF7 cells-derived tumors from high-fat-diet (HFD) fed obese mice and normal-diet (ND) fed lean mice and treated with 4-hydroxytamoxifen (T) or vehicle (C). M MCF7 cells were transfected with miR-205-mimic and anti-miR-205 followed by treatment with 100 ng/ml leptin (L). Med1, TFF1 and CATD expression was examined using quantitative real-time PCR. ***p < 0.0005, **p < 0.005, *p < 0.05. Data are means ± SD from three experiments.

Fig 3: Proteomic analysis of mammary tumors treated with tamoxifen combination therapy. (A) Western blot image detecting ER and TFF1 in HCI-011 (top) and BCM-15057 (bottom) mammary tumors following tamoxifen combination treatment, and quantification of densitometry of the HCI-011 (top) and BCM-15057 (bottom) blots is shown in (B). The uncropped blots are shown in Figure S3. Representative images of IHC and quantification of PR, aurora kinase A, and aurora kinase B are shown for HCI-011 (C,E) and BCM-15057 (D,F). Images of IHC have been taken at 400× magnification.