Figure 5 from Endocrine Therapy Synergizes with SMAC Mimetics to Potentiate Antigen Presentation and Tumor Regression in Hormone Receptor–Positive Breast Cancer
Authors: Hermida-Prado Francisco , Xie Yingtian , Sherman Shira , Nagy Zsuzsanna , Russo Douglas , Akhshi Tara , Chu Zhengtao , Feit Avery , Campisi Marco , Chen Minyue , Nardone Agostina , Guarducci Cristina , Lim Klothilda , Font-Tello Alba , Lee Irene , García-Pedrero Juana , Cañadas Israel , Agudo Judith , Huang Ying , Sella Tal , Jin Qingchun , Tayob Nabihah , Mittendorf Elizabeth A. , Tolaney Sara M. , Qiu Xintao , Long Henry , Symmans William F. , Lin Jia-Ren , Santagata Sandro , Bedrosian Isabelle , Yardley Denise A. , Mayer Ingrid A. , Richardson Edward T. , Oliveira Giacomo , Wu Catherine J. , Schuster Eugene F. , Dowsett Mitch , Welm Alana L. , Barbie David , Metzger Otto , Jeselsohn Rinath 
Published: October 2023

<p>NF-κB pathway activation via RelA phosphorylation and binding is enhanced in hormone-deprived conditions. <b>A,</b> Immunoblot of whole-cell lysates for the NF-κB subunits, RelA and RelB, and ER in MCF7 cells grown with E2 10 nmol/L or in HD conditions in response to IFNγ 10 ng/mL stimulation. <b>B,</b> Whole-cell lysate immunoblots of ER, RelA, and phospho-RelA (Ser536) in MCF7 cells. Hormone-deprived cells were stimulated with E2 (10 nmol/L) for 4 days. Protein was extracted every 24 hours. GAPDH was used as a loading control. <b>C,</b> Tornado plots of RelA binding sites in HD cells or treated with 10 nmol/L E2 with or without IFNγ stimulation (10 ng/mL for 1 hour). <b>D,</b> Volcano plot showing differential expression from RNA-seq of MCF7 cells in HD conditions versus E2-treated conditions. Blue dots (True), genes that are differentially expressed based on RNA-seq and predicted to be regulated by RelA based on RelA ChIP-seq and BETA minus analysis. Orange dots (false), genes that are differentially expressed between HD and E2 conditions but not predicted to be regulated by RelA based on the RelA ChIP-seq data. The <i>P</i> value represents the significance of the association between RelA ChIP-seq and RNA-seq up HD (<i>P</i> = 1.9 E−11) or down in HD (<i>P</i> = 0.156) compared with E2-stimulated cells without IFNγ based on BETA basic. <b>E,</b> GSVA of the HD_RelA gene set (541 genes) in primary ER<sup>+</sup> breast cancers pre- and post-neoadjuvant treatment with an AI. <b>F</b> and <b>G,</b> RNA-seq differential expression after RelA KO compared with control. Volcano plot highlighting genes differentially expressed between RelA KOs and RelA WT cells grown in HD conditions (<b>F</b>), treated with fulvestrant (10 nmol/L; <b>G</b>), or grown in E2 conditions (<b>H</b>) for 72 hours and stimulated with IFNγ (10 ng/mL) for the last 6 hours (h). <i>n</i>, number of genes differentially expressed. <b>I,</b> RelA ChIP-seq tracks showing examples of RelA peaks at the promoter region of IFNγ-associated genes in MCF7 cells grown in the presence of E2 or in HD conditions and stimulated ± IFNγ (10 ng/mL) for one hour. <b>J,</b> mRNA expression levels of ICAM1, HLA-A, TAP1, B2M, and CXCL10 in E2 and HD conditions without and with RELA silencing KO after 6 hours of IFNγ stimulation. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001. Error bars, mean ± SD of at least two replicates per each KO. Two-way ANOVA.</p>