Abstract PD1-05: Targeting the FRA1-dependent transcriptional nexus in high FOXA1-driven endocrine-resistant and metastatic breast cancer
Authors: Liu Chia Chia , Qin Lanfang , De Angelis Carmine , Nanda Sarmistha , Pereira Resel , Shea Martin J. , Nardone Agostina , Jeselsohn Rinath , Cohen Ofir , Wagle Nikhil , Liu Zhijie , Rimawi Mothaffar F. , Osborne C. Kent , Schiff Rachel , Fu Xiaoyong 
Published: March 2022

Abstract
Background: Aberrant activation of the pioneer transcription factor (TF) FOXA1 contributes to endocrine resistance and metastasis in ER+ breast cancer (BC) by promoting genome-wide enhancer and transcriptional reprogramming that engages the AP-1 TF complex. Identification of central transcriptional nexuses in this deregulated network is key for developing new therapeutic interventions focusing on transcriptional programs. We previously identified FRA1, among the AP-1 components, as the top super-enhancer target forming a FOXA1/FRA1-centered transcriptional axis to activate genes enriched in luminal B-subtype BC and ER+ metastases. To further dissect the FOXA1/FRA1-centered transcriptional axis and its potential therapeutic role, we employed integrative multi-omics data analysis and functional studies targeting FRA1 using our MCF7-parental (P) and FOXA1-amplifed tamoxifen-resistant (TamR) preclinical models. Methods: RNA-seq data were obtained in MCF7-P and TamR cells with FOXA1 and/or AP-1 (FRA1 and c-Jun) perturbation via overexpression and/or si/shRNA knockdown (KD). Differential gene expression was analyzed using DESeq2 or the limma-voom R package. ChIP-seq-based genome-wide FOXA1 binding sites were further refined by intersection with promoter-tethered regions (PTRs) denoting Hi-C-mapped chromatin looping. Genomic binding of FOXA1/c-Jun and H3K27ac modification at target gene loci were aligned and visualized by IGV. Significance of gene set enrichment was determined using a chi-square test adjusted for multiple comparisons. Clinical relevance was examined using an RNA-seq dataset of ER+ metastatic BC (SABCS19-GS2-02). Clonogenicity, soft agar, and wound-healing assays were performed using MCF7-TamR cell derivatives engineered for doxycycline (Dox)-inducible KD of FRA1 or NS shRNA. Significance of the KD effects in functional assays was determined using a linear mixed-effects model. Results: We found that FRA1 and the two embryonic TFs SOX9 and KLF4 that harbor FOXA1-bound PTRs were commonly down-regulated in TamR cells upon KD of FOXA1, FRA1, or c-Jun. We observed increased c-Jun binding at the FOXA1-bound super-enhancer and the PTR looping to the FRA1 gene locus in TamR vs. P cells, suggesting a feed-forward mechanism by which FRA1 transcription is strengthened by the AP-1-engaged super-enhancer in TamR cells. Among the FOXA1-activated genes in TamR cells, a subset of secretory protein-encoding genes was more enriched in the genes commonly dependent on both FRA1 and c-Jun, vs. the genes depending on either FRA1 or c-Jun alone. This FOXA1/FRA1/c-Jun-activated secretome is enriched for multiple biological processes engaged in tumor metastasis and associated microenvironmental niches, and was upregulated in clinical ER+ metastases vs. primary tumors. A larger proportion of this secretome, including CXCL8 and S100P, also relies on ER preferentially in TamR vs. P cells (40% vs. 16%, respectively). FRA1 KD using two different shRNA sequences in TamR cells reduced SOX9 and KLF4 expression levels, and significantly diminished clonogenicity, soft agar colony formation, and wound healing, compared to the control NS KD. Conclusions: Our integrative bioinformatics analyses reveal a feed-forward mechanism on FRA1 activation in amplifying high-FOXA1-induced transcriptional reprogramming in endocrine resistance. A prometastatic secretome activated by FRA1/c-Jun may represent a main transcriptional output of the FOXA1/FRA1-dependent nexus in promoting ER+ disease progression. These identified key transcriptional nexuses may present network hotspots susceptible to therapeutic interventions in which FRA1 inhibition could be used as a new transcriptional program-oriented therapy to treat advanced ER+ BC.
Citation Format: Chia Chia Liu, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Resel Pereira, Martin J. Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Zhijie Liu, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Xiaoyong Fu. Targeting the FRA1-dependent transcriptional nexus in high FOXA1-driven endocrine-resistant and metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-05.