Author Manuscript Published OnlineFirst on November 27, 2017; DOI: 10.1158/0008-5472.CAN-17-1153 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 1 ER alpha binding by transcription factors NFIB and YBX1 enables FGFR2 2 signalling to modulate estrogen responsiveness in breast cancer 3 4 Thomas M. Campbell1, Mauro A. A. Castro2, Kelin Gonçalves de Oliveira2, Bruce A. J. 5 Ponder1 and Kerstin B. Meyer1,3* 6 7 *Corresponding author: [email protected] 8 1Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing 9 Centre, Robinson Way, Cambridge CB2 0RE, UK. 10 2Bioinformatics and Systems Biology Lab, Federal University of Paraná (UFPR), 11 Polytechnic Center, Rua Alcides Vieira Arcoverde, 1225 Curitiba, PR 81520-260, 12 Brazil. 13 3Current address: Wellcome Trust Sanger Institute, Wellcome Genome Campus, 14 Hinxton, Cambridge CB10 1SA, UK. 15 [email protected] 16 [email protected] 17 [email protected] 18 [email protected] 19 [email protected] 20 21 Running title: NFIB and YBX1 regulate ESR1 in breast cancer 22 5 keywords: Breast cancer, ESR1, NFIB, YBX1, FGFR2 1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 27, 2017; DOI: 10.1158/0008-5472.CAN-17-1153 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 23 Abstract 24 Two opposing clusters of transcription factors (TF) have been associated with the 25 differential risks of estrogen receptor positive or negative breast cancers, but the 26 mechanisms underlying the opposing functions of the two clusters are undefined. In 27 this study, we identified NFIB and YBX1 as novel interactors of the estrogen receptor 28 (ESR1). NFIB and YBX1 are both risk TF associated with progression of ESR1-negative 29 disease. Notably, they both interacted with the ESR1-FOXA1 complex and inhibited 30 the transactivational potential of ESR1. Moreover, signaling through FGFR2, a known 31 risk factor in breast cancer development, augmented these interactions and further 32 repressed ESR1 target gene expression. We therefore show that members of two 33 opposing clusters of risk associated with ESR1 positive and negative breast cancer 34 can physically interact. We postulate that this interaction forms a toggle between 35 two developmental pathways affected by FGFR2 signaling, possibly offering a 36 junction to exploit therapeutically. 37 2 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 27, 2017; DOI: 10.1158/0008-5472.CAN-17-1153 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 38 Introduction 39 The estrogen receptor (ESR1) is the key driver and therapeutic target of breast 40 cancer [1] and plays a critical role in determining the risk of developing this disease 41 [2-4]. Using a systems biology approach we have examined transcriptional networks 42 in breast cancer affecting ESR1 activity and have identified two distinct and opposing 43 clusters of transcription factors (TFs) associated with enhanced breast cancer risk 44 [5]. The “cluster 1” risk TFs are associated with estrogen receptor-positive (ER+) 45 breast cancer risk and comprise TFs such as ESR1, FOXA1 and GATA3 whereas the 46 “cluster 2” risk TFs appear to be associated with estrogen receptor-negative (ER-), 47 basal-like breast cancer (BLBC). Two of the TFs located in the cluster associated with 48 ER- disease are NFIB and YBX1. Here we examine the molecular mechanisms 49 underlying the opposing functions of the two groups of TFs by studying protein- 50 protein interactions between TFs and their functional consequences. We also 51 examine the effect of cell signalling, in particular by fibroblast growth factor receptor 52 2 (FGFR2), on the relative activity of the two groups of TFs. 53 The nuclear factor I (NFI) family of TFs consists of four members, NFIA, NFIB, 54 NFIC and NFIX, which can all bind DNA as homo- or heterodimers [6]. They are 55 particularly important during developmental stages [7, 8], and NFIB is crucial for 56 normal lung and brain development [9]. NFIB commonly has an increased copy 57 number in small cell lung cancer (SCLC), indicating a role as an oncogene [10]. In 58 BLBC, both copy number and expression levels of NFIB are also increased [11, 12]. In 59 addition, NFIB is important in the regulation of expression of mammary gland- 60 specific genes, specifically those associated with lactation such as Whey acidic 61 protein (WAP) and α-lactalbumin [13]. NFIB has been shown to modulate androgen 3 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 27, 2017; DOI: 10.1158/0008-5472.CAN-17-1153 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 62 receptor (AR) target genes in prostate cancer cells via an interaction with FOXA1 [14, 63 15]. An investigation into whether similar modulation of estrogen receptor (ER) 64 occurs in the breast has yet to be carried out. 65 Y-box binding protein 1 (YBX1) is a member of a family of DNA- and RNA- 66 binding proteins with an evolutionarily ancient and conserved cold shock domain. It 67 is a multifunctional protein that certainly does not follow the classical “one protein- 68 one function” rule, but rather has disordered structure suggesting many different 69 functions [16]. It has been extensively studied in cancer and its overexpression is 70 associated with many hallmarks of the disease. It is expressed in many breast cancer 71 cell lines regardless of subtype. However, there are higher levels of phosphorylated 72 YBX1 in BLBC cell lines [17, 18]. YBX1 expression in inversely correlated with ER, PR 73 and HER2 expression and is positively correlated with the MAPK signalling cascade, a 74 pathway important in BLBC [19, 20]. YBX1 is highly expressed in 70% of BLBC cases 75 and many of its target genes are associated with a basal-like signature [18, 20]. 76 Higher expression of YBX1 correlates with poor survival, drug resistance and a high 77 rate of relapse in all subtypes [18, 19, 21-23]. Suppression of YBX1 reduces 2D cell 78 growth and growth in mammospheres [18, 20]. There is also evidence to suggest 79 that YBX1 binds ESR1 in ER+ breast cancer cell nuclei [24, 25]. 80 A locus within the second intron of the FGFR2 (fibroblast growth factor 81 receptor 2) gene is consistently identified as the genetic locus most strongly 82 associated with estrogen receptor-positive (ER+) breast cancer risk by independent 83 genome-wide association studies (GWASs) [26]. We have shown previously that the 84 top three risk single nucleotide polymorphisms (SNPs) [27, 28] act to reduce FGFR2 85 gene expression and enhance the estrogen response [29]. Increased FGFR2 4 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 27, 2017; DOI: 10.1158/0008-5472.CAN-17-1153 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 86 stimulation repressed estrogen signalling in ER+ breast cancer cell lines. However, 87 the underlying molecular mechanism remains unclear. 88 Here, we demonstrate that two members of the cluster 2 TFs, NFIB and YBX1, 89 both physically interact with ESR1, repress its activity, and drive breast cancer cells 90 towards a less estrogen-dependent cancer phenotype. FGFR2 signalling augments 91 this interaction and subsequent repression of ESR1 target gene expression. Our 92 evidence suggests that FGFR2 has wide-ranging effects on driving breast cancer cells 93 towards a more basal-like phenotype and that inhibiting FGFR2 signalling in ER+ 94 breast cancer sensitizes cells to anti-estrogen therapies. 95 5 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 27, 2017; DOI: 10.1158/0008-5472.CAN-17-1153 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 96 Materials and methods 97 Cell culture 98 MCF-7 human breast cancer cells and HeLa cells were cultured in DMEM (Invitrogen) 99 supplemented with 10% FBS and antibiotics. ZR751 human breast cancer cells were 100 cultured in RPMI (Invitrogen) supplemented with 10% FBS and antibiotics. SUM52PE 101 human breast cancer cells were cultured in Ham/F-12 (Invitrogen) supplemented 102 with 10% FBS, 5 g/mL insulin, 1 g/mL hydrocortisone and antibiotics. All cells were 103 maintained at 37°C, 5% CO2, obtained from the CRUK Cambridge Institute collection 104 and authenticated by STR genotyping. 105 106 Quantitative RT-PCR 107 1 g of total RNA was reverse transcribed using the High Capacity cDNA Reverse 108 Transcription Kit (Applied Biosystems) and qRT-PCR performed using cDNA obtained 109 from 10 ng of total RNA. qRT-PCR was performed using a QuantStudio6 system (Life 110 Technologies). Amplification and detection were carried out in 384-well Optical 111 Reaction Plates (Applied Biosystems) with Power SYBR Green Fast 2x qRT-PCR 112 Mastermix (Applied Biosystems). All expression data were normalised to DGUOK 113 expression. The specificity of primers (Supplementary Table 1) was confirmed 114 through generation of single peaks in a melt-curve analysis. Data analysis was 115 performed using the 2-CT method [30]. 116 117 Western immunoblotting 118 Cells were grown in 10 cm Petri dishes, washed in PBS and lysed on ice in RIPA buffer 119 with cOmplete Mini EDTA-free protease inhibitor cocktail (Roche).