Abstract PD6-2: FoxA1 gene amplification in ER+ breast cancer mediates endocrine resistance by increasing IL-8
Authors: Fu Xiaoyong , Jeselsohn Rinath , Hollingsworth Emporia F , Lopez-Terrada Dolores , Creighton Chad J , Nardone Agostina , Shea Martin , Heiser Laura M , Anur Pavana , Wang Nicholas , Grasso Catie , Spellman Paul , Gutierrez Carolina , Rimawi Mothaffar F , Hilsenbeck Susan G , Gray Joe W , Brown Myles , Osborne C K , Schiff Rachel 
Published: May 2015

Abstract
Background: ER transcriptional programming is associated with fundamental changes when endocrine resistance develops. The Forkhead transcription factor, FoxA1, is a pioneer factor for ER-DNA binding. We hypothesize that FoxA1 plays a critical role in ER transcriptional reprogramming in endocrine resistance by augmenting itself and the specific downstream effectors. Methods: Next generation sequencing was applied to characterize a panel of endocrine-resistant (Endo-R) cell models. Genomic PCR amplification and FISH assays were developed to measure FoxA1 copy number gain (CNG). Q-RT-PCR, Western blots, IHC, ELISA, and cytokine arrays were used to determine the levels of FoxA1 and IL-8 in cell culture and in vivo xenograft tumors. Effects of gene knockdown (ER, FoxA1, or IL-8) or inducible FoxA1 overexpression on ER and growth factor receptor (GFR) downstream signaling were determined by cell growth and Western blots. ER and FoxA1 binding at the IL-8 gene locus was measured by ChIP-qPCR. ChIP-seq analysis was integrated with RNA-seq data. Kaplan-Meier analysis evaluated the predictive role of FoxA1in ER+ breast tumors. Results: Exome-seq revealed that FoxA1 is the most highly amplified gene in TamR vs. P cells from two independent MCF7 models. Genomic PCR and FISH also indicate FoxA1 CNG in Endo-R models of ZR75-1 and BT474. Increased FoxA1 expression was found in multiple Endo-R cells and in MCF7L Endo-R xenograft tumors. Cytokines, especially IL-8, are more highly expressed in multiple Endo-R cell models, similar to our previous microarray data from MCF7 Endo-R xenograft tumors. FoxA1 forced overexpression significantly induced IL-8 expression in MCF7L-P cells. It also activated multiple GFR downstream signaling pathways, and conferred endocrine resistance. Conversely, knockdown of either FoxA1 or ER significantly decreased IL-8 levels in TamR cells, and inhibited cell growth in both P and TamR cells. Knockdown of IL-8 in TamR cells substantially inhibited GFR downstream signaling, and was more cytotoxic than in P cells. A novel FoxA1-binding site (10 kb at 5’UTR of IL-8) recruited more FoxA1 and p300 in MCF7L-TamR than -P cells. ChIP-seq shows a general enhancement of FoxA1 binding around the genes (within 20 kb) that are differentially expressed in TamR vs. P cells. We identified a FoxA1 CNG-associated gene signature from TCGA breast tumors that predicts worse relapse-free survival (RFS) in Tam-treated ER+ tumors (from Loi et al). Meta-analysis showed that FoxA1 mRNA levels in the top 25th percentile predict worse RFS in ER+ patients treated with Tam (N=615), but not in systemically untreated patients (N=500). FoxA1 CNG and overexpression in clinical specimens by using our newly developed FISH and IHC assays are currently being investigated. Conclusions: FoxA1 gene amplification was enriched in two independent MCF7 Tam-R cell models. Clonal selection of FoxA1 gene amplification may occur and lead to endocrine resistance. High levels of FoxA1 may mediate endocrine resistance by directly inducing IL-8. The data suggest that IL-8 signaling is a component of a cytokine loop controlled by the FoxA1/ER transcriptional reprogramming, which might be exploited in therapeutics to overcome endocrine resistance.
Citation Format: Xiaoyong Fu, Rinath Jeselsohn, Emporia F Hollingsworth, Dolores Lopez-Terrada, Chad J Creighton, Agostina Nardone, Martin Shea, Laura M Heiser, Pavana Anur, Nicholas Wang, Catie Grasso, Paul Spellman, Carolina Gutierrez, Mothaffar F Rimawi, Susan G Hilsenbeck, Joe W Gray, Myles Brown, C K Osborne, Rachel Schiff. FoxA1 gene amplification in ER+ breast cancer mediates endocrine resistance by increasing IL-8 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-2.