Abstract P6-04-02: Integrative cistromic/transcriptomic profiling identifies a high FOXA1/ER-activated pro-metastatic secretome in endocrine-resistant breast cancer
Authors: Fu Xiaoyong , Pereira Resel , De Angelis Carmine , Nanda Sarmistha , Qin Lanfang , Shea Martin J , Mitchell Tamika , Veeraraghavan Jamunarani , Sethunath Vidyalakshmi , Gutierrez Carolina , Győrffy Balázs , Cohen Ofir , Wagle Nikhil , Nardone Agostina , Jeselsohn Rinath , Brown Myles , Rimawi Mothaffar F , Osborne C Kent , Schiff Rachel 
Published: March 2020

Abstract
Background: The estrogen receptor (ER) plays an evolving role in conferring endocrine resistance (EndoR) via altering chromatin binding and transcriptional reprogramming in ER+ metastatic breast cancer (BC). We have recently reported that high levels of FOXA1 (H-FOXA1), a pioneer factor for ER-chromatin binding and function, promote EndoR and metastatic phenotypes in ER+ BC cells. Here we further investigated the interplay between FOXA1 and ER by cistromic and transcriptomic profiling of ER+ BC cells expressing H-FOXA1 and a consequential pro-metastatic secretome associated with poor outcome of ER+ BC.
Methods: FOXA1 and ER cistromes were analyzed by ChIP-seq in MCF7 cells expressing ectopic H-FOXA1 or treated with estrogen or the growth factor EGF. Transcriptomes of ER+ BC cells (MCF7, ZR75-1, and T47D) expressing ectopic H-FOXA1 and our established EndoR cell models (MCF7, ZR75-1, T47D, and 600MPE) were analyzed by RNA-seq. A core gene signature (CGS) and secretome gene sets induced by H-FOXA1 were identified by integrative analyses of cistromic/transcriptomic data and public secretory protein databases. Multivariant proportional hazards modeling and Kaplan-Meier log-rank test were performed to assess prognostic significance of the secretome gene sets using the METABRIC and KMplotter datasets. A transcriptomic dataset of ER+ primary and metastatic BC (The Metastatic Breast Cancer Project) was used to determine secretome expression changes in metastatic vs. primary tumors and the correlation with FOXA1 levels. A tamoxifen-resistant ER+ metastatic mouse model we previously established was used to determine pro-metastatic secretome gene expression.
Results: Ectopic H-FOXA1 expanded and reprogramed the FOXA1 cistrome in ER+ BC cells and induced a CGS shared by multiple H-FOXA1-expressing cancer cell models, including prostate and pancreatic cancer cells. H-FOXA1 redirected an estrogen-independent ER cistrome in ER+ BC cells, but had no effect on the FOXA1 cistrome under short-term treatment with estrogen or EGF. Upregulated CGS was enriched for secretome-encoding genes with variance in ER dependency across multiple EndoR cell models. The H-FOXA1/ER-activated (n=10), but not the ER-repressed or -independent secretome, predicted poor disease-free survival in ER+ BC treated with endocrine therapy (METABRIC, n=1,103). Multivariate analyses further identified each of four secretome genes (ST6GALNAC2, SERPINI1, S100P, and CD55) significantly contributing to overall survival of ER+ BC. The prognostic significance of this secretome signature was also observed in relapse-free survival and distant metastasis-free survival of ER+, but not ER- BC, using the KMplotter dataset. The expression levels of the identified secretome were significantly increased in ER+ metastatic (n=147) vs. primary (n=48) tumors (including 31 pairs) and positively correlated with FOXA1 mRNA levels in metastases. Notably, there was no association between the H-FOXA1/ER-regulated pro-metastatic secretome and the ESR1 mutations. Finally, H-FOXA1-induced EndoR metastatic xenograft tumors were associated with elevated secretome gene expression.
Conclusions: We identified an H-FOXA1/ER-activated pro-metastatic secretome via integrative analyses of the FOXA1/ER cistromes and transcriptomes of our preclinical models expressing H-FOXA1. This secretome signature predicts poor outcome of ER+ BC treated with endocrine therapy and has no correlation with the ESR1 mutations in ER+ metastatic BC. The increased expression of these secretome genes in ER+ metastatic vs. primary tumors suggests continuing interplay of FOXA1 and ER in promoting pro-metastatic transcriptional programs during ER+ disease progression, which warrants further investigation.
Citation Format: Xiaoyong Fu, Resel Pereira, Carmine De Angelis, Sarmistha Nanda, Lanfang Qin, Martin J Shea, Tamika Mitchell, Jamunarani Veeraraghavan, Vidyalakshmi Sethunath, Carolina Gutierrez, Balázs Győrffy, Ofir Cohen, Nikhil Wagle, Agostina Nardone, Rinath Jeselsohn, Myles Brown, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Integrative cistromic/transcriptomic profiling identifies a high FOXA1/ER-activated pro-metastatic secretome in endocrine-resistant breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-02.