Abstract GS1-06: FGFR1 associates with gene promoters and regulates gene transcription: Implications for endocrine resistance in ER+/FGFR1-amplified breast cancer
Authors: Servetto Alberto , Kollipara Rahul , Formisano Luigi , Lin Chang-Ching , Lee Kyung-min , Sudhan Dhivya R , Hanker Ariella B , Chatterjee Sumanta , Guerrero-Zotano Angel , Gonzalez-Ericsson Paula , Mendiratta Saurabh , Akamatsu Hiroaki , James Nicholas , Kittler Ralf , Arteaga Carlos L 
Published: February 2021

Abstract
Background: FGFR1 amplification occurs in ~ 15% of ER+ breast cancers. In these tumors, nuclear FGFR1 has been shown to interact with DNA, but its role in transcriptional regulation is unclear. Thus, we investigated the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Results: FGFR1 ChIP-Seq detected 4,412 DNA binding sites in CAMA1 ER+/FGFR1-amplified breast cancer cells cultured in estrogen-free conditions. Of these binding sites, 67% were enriched at promoter regions. ChIP-qPCR confirmed FGFR1 binding to several promoter regions in a second ER+/FGFR1-amplified cell line, MDA-MB-134, and a patient derived xenograft, HCI-011. To determine the nuclear FGFR1 interactome, we performed FLAG immunoprecipitation of mixed nuclear and chromatin fractions of CAMA1 cells transduced with a 3XFLAG-FGFR1 plasmid, followed by mass spectrometry (MS) of FLAG antibody pulldowns. MS revealed RNA Polymerase II subunits among the top nuclear FGFR1 interacting proteins. FGFR1 mainly bound Pol II phosphorylated on Ser5 (Pol II S5P), a marker of transcription initiation, in CAMA1, MDA-MB-134 and HCI-011 cell extracts. Pol II S5P ChIP-Seq revealed that 2,867/4,412 (65%) FGFR1 peaks were shared with Pol II S5P. ChIP-Seq also showed that 95% of FGFR1 peaks overlapped with both H3K4me3 and H3K27ac, markers of active transcription. Consistent with these results, RNA-Seq of CAMA1 cells showed that expression of FGFR1-bound genes was markedly higher than non FGFR1-bound genes (p&lt;0.0001), suggesting that FGFR1 binds to actively transcribed genes. In addition to Pol II, MS detected FOXA1 among FGFR1 interacting proteins. ChIP-Seq analysis revealed FOXA1 enriched at FGFR1-bound loci. siRNA-mediated FOXA1 knockdown reduced FGFR1 distribution to several genomic loci in CAMA1 cells, as measured by FGFR1 ChIP-Seq, suggesting that FOXA1 mediates FGFR1 recruitment to chromatin. We next transduced MCF-7 cells with an FGFR1(SP-)(NLS) plasmid, where the NLS sequence forces nuclear import of the resulting protein. To determine the role of FGFR1 on transcriptional regulation, we used Binding and Expression Target Analysis (BETA), integrating FGFR1 ChIP-Seq and RNA-Seq results from MCF7FGFR1(SP-)(NLS) vs MCF7EV cells. This analysis predicted a direct role for genomic-bound FGFR1 in activating gene expression (p=8.01e-6). MCF7FGFR1(SP-)(NLS) cells were markedly less sensitive to fulvestrant compared to control cells. Gene Set Enrichment Analysis (GSEA) of the 1,009 genes upregulated in MCF7FGFR1(SP-)(NLS) cells and bound by FGFR1 at a genomic level revealed a strong enrichment of estrogen response early (q=2.2e-44) and late (q=6.4e-33) genes, suggesting that nuclear FGFR1 induces an ERα-associated transcriptional profile that may contribute to endocrine resistance. Finally, an expression signature associated with nuclear FGFR1 correlated with endocrine resistance in three cohorts of patients with ER+ breast cancer treated with aromatase inhibitors. We next studied the effect of growth factor stimulation on FGFR1 transcriptional function. Stimulation with FGF2 enhanced nuclear FGFR1 import in CAMA1 cells, as well as FGFR1-Pol II S5P association. Notably, these effects were not abrogated by treatment with the FGFR1 inhibitor erdafitinib. ChIP-Seq revealed that erdafitinib did not impair the FGFR1 genomic distribution. These results do not support a causal link between the FGFR1 activated TK and the receptor’s activity in the nucleus. Conclusions: We have demonstrated a role for nuclear FGFR1 in transcriptional regulation in breast cancer. FGFR1-induced gene expression contributes to endocrine resistance and is not affected by FGFR TKIs. These findings provide a rationale for developing treatment strategies to inhibit nuclear FGFR1 in ER+/FGFR1-amplified breast cancer.
Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Chang-Ching Lin, Kyung-min Lee, Dhivya R Sudhan, Ariella B Hanker, Sumanta Chatterjee, Angel Guerrero-Zotano, Paula Gonzalez-Ericsson, Saurabh Mendiratta, Hiroaki Akamatsu, Nicholas James, Ralf Kittler, Carlos L Arteaga. FGFR1 associates with gene promoters and regulates gene transcription: Implications for endocrine resistance in ER+/FGFR1-amplified breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr GS1-06.