Abstract S3-03: Nuclear FGFR1 interaction with estrogen receptor (ER) α is associated with resistance to endocrine therapy in ER+/FGFR1-amplified breast cancer

Abstract Background: Estrogen receptor (ER)-positive breast cancers (BC) initially respond to antiestrogens but eventually become hormone-independent and recur. FGFR1 is amplified in ∼10% of ER+ BC and is associated with early recurrence on antiestrogen therapy. Notably, one third of FGFR1-amplified tumors have simultaneous amplification of CCND1, FGF3, FGF4 and FGF19 on chromosome 11q12-14. Herein, we investigated the mechanisms by which FGFR1 amplification confers resistance to antiestrogen therapy in ER+ BC cells. Results: We performed whole exome sequencing in tumor biopsies from 130 patients with an operable ER+/HER2- BC who had received letrozole for 10-21 days prior to surgery. Tumors were categorized by the natural log (ln) of post-letrozole Ki67 as sensitive (ln ≤1 or ≤2.7% Ki67+ cells; n=68) or resistant (ln ≥2 or ≥7.4%; n=18). We found amplifications in FGFR1 and/or 11q12-14 in 6/11 (55%) resistant tumors compared with 5/34 (15%) in sensitive tumors (p=0.006); all cases were confirmed by FGFR1-fluorescence in situ hydridization (FISH). Resistant tumors with FGFR1 and/or 11q12-14-amplification showed a marked increase in nuclear FGFR1 with letrozole. ER+/FGFR1-amplified CAMA1 and MDA134 cell lines also exhibited co-localization of ER and FGFR1 in the nucleus. Cell proliferation was partially reduced by estrogen deprivation, and FGFR1 siRNA further reduced cell growth in hormone-depleted medium. We generated CAMA1 and MDA134 cells resistant to long-term estrogen deprivation (LTED). These cells exhibited overexpression of FGF3/4/19 and ERα with a concomitant increase in ligand-independent ER transcriptional activity and growth. An ER-FGFR1 interaction was observed in the nucleus and cytosol of CAMA1 parental cells with enhanced interaction in CAMA1 LTED cells. Genetic (with siRNA) and pharmacologic (with lucitinib) inhibition of FGFR1 reduced a) nuclear localization of FGFR1; b) ER transcriptional activity; and c) cell proliferation. Nuclear localization and ER-FGFR1 interaction were disrupted by a kinase-deficient FGFR1. Conversely, addition of FGF3 ligand stimulated ER-FGFR1 interaction and ER transcriptional activity, suggesting FGFR activation can regulate ER function. Inhibition of FGF receptor-specific substrate (FRS2), a principal mediator of FGFR1 signal transduction to the MAPK and PI3K pathways, with siRNA or pharmacologic inhibition of PI3K with buparlisib or MEK with GSK1120212 did not reduce ER transcriptional activity suggesting that, in ER+/FGFR1-amplified cancer cells, ER function is not modulated by FGFR signal transducers. Finally, using chromatin immunoprecipitation (ChIP) we showed that FGFR1 binds directly to estrogen response elements (ERE). This association was reduced with lucitanib. We are currently investigating genes modulated by ER/FGFR1 in ER+ BC and the in vivo anti-tumor efficacy of dual inhibition of FGFR1 and ER in ER+/FGFR1-amplified patient-derived breast cancer xenografts. Conclusions: These data support a critical role of ER and FGFR1 interaction in endocrine resistance in ER+/FGFR1-amplified breast cancer. Targeting of FGFR1 in combination with antiestrogens may abrogate resistance to endocrine therapy in these tumors and is worthy of clinical investigation. Citation Format: Formisano L, Young CD, Bhola NE, Bulen B, Estrada VM, Wagle N, Van Allen E, Red Brewer ML, Jansen VM, Guerrero AL, Giltnane JM, Strcker T, Arteaga CL. Nuclear FGFR1 interaction with estrogen receptor (ER) α is associated with resistance to endocrine therapy in ER+/FGFR1-amplified breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S3-03.</jats:p

Enhanced dimerization drives ligand-independent activity of mutant epidermal growth factor receptor in lung cancer

Mutations within the epidermal growth factor receptor (EGFR/erbB1/Her1) are often associated with tumorigenesis. In particular, a number of EGFR mutants that demonstrate ligand-independent signaling are common in non–small cell lung cancer (NSCLC), including kinase domain mutations L858R (also called L834R) and exon 19 deletions (e.g., ΔL747-P753insS), which collectively make up nearly 90% of mutations in NSCLC. The molecular mechanisms by which these mutations confer constitutive activity remain unresolved. Using multiple subdiffraction-limit imaging modalities, we reveal the altered receptor structure and interaction kinetics of NSCLC-associated EGFR mutants. We applied two-color single quantum dot tracking to quantify receptor dimerization kinetics on living cells and show that, in contrast to wild-type EGFR, mutants are capable of forming stable, ligand-independent dimers. Two-color superresolution localization microscopy confirmed ligand-independent aggregation of EGFR mutants. Live-cell Förster resonance energy transfer measurements revealed that the L858R kinase mutation alters ectodomain structure such that unliganded mutant EGFR adopts an extended, dimerization-competent conformation. Finally, mutation of the putative dimerization arm confirmed a critical role for ectodomain engagement in ligand-independent signaling. These data support a model in which dysregulated activity of NSCLC-associated kinase mutants is driven by coordinated interactions involving both the kinase and extracellular domains that lead to enhanced dimerization