Fibroblast growth factor receptor 1 (FGFR1) as a therapeutic target in adenoid cystic carcinoma of the lacrimal gland

Abstract

Identification of molecular targets is the first step in developing efficacious therapeutic strategies for tumors. A tumors' biological response to perturbagens yields important information on the molecular determinants for tumor growth. The aim of this study was to characterize the response of adenoid cystic carcinoma of the lacrimal gland (LGACC) to intra-arterial cytoreductive chemotherapy (IACC) in order to identify novel targets to enhance therapy. We performed high-throughput proteomic analysis on paired samples from pre-IACC diagnostic biopsies and post-IACC excised tumor samples from 6 LGACC patients. This proteomic analysis provides a comprehensive landscape of the cellular compartments contained within the excised tumors. Interestingly, we found a strong upregulation across the fibroblast growth factor (FGF) signaling pathway, with FGF receptor 1 (FGFR1) exhibiting a consistent and significant upregulation in all post-IACC samples. We thus evaluated the therapeutic efficacy of a novel FGFR1 selective inhibitor, AZD4547, in combination with cisplatin on LGACC cells in-vitro. The combination index (CI) value (<0.895) demonstrated synergistic effect of AZD4547 and cisplatin in inhibiting cell growth and viability (p<0.02), with a differential response seen in post-IACC cultures when compared to pre-IACC cultures. The combination approach showed synergy of the drugs in the migration assay. Western blot analysis indicated a significant upregulation of cleaved caspase-3 and downregulation the expression of FGFR1 (p<0.05) with the combination treatment as compared to either agent independently. Our findings demonstrate that FGFR1 inhibition potentiates the cytoreductive effects of cisplatin and suggest a potential therapeutic benefit of using AZD4547 in the management of LGACC.

Keywords: FGFR; adenoid cystic carcinoma; lacrimal gland; precision medicine; targeted therapy.

Figure 1. Proteomic profile of LGACC tumor's response to IACC protocol

(A) Representative image of L-series array immunoblot readout for pre-, and post-IACC paired samples from 2 individual LGACC patients. (B) Log fold representation of paired proteomic readout for the top 300 differentially expressed protein for all 6 individual patients showing enriched (>1.0), and depleted (<1.0) proteins in post-IACC samples. (C) Bar graph of statistically significant enriched proteins in aggregated post/pre-IACC data for all 6 patients († proteins with statistically significant enrichment in all 6 individual paired patient specimens). (p<0.05).

Figure 2. Apoptotic and stem/progenitor phenotypes present in post-IACC tumor samples

(A) Bar graph for apoptotic cell marker cluster identified as enriched in aggregate post/pre-IACC data in our proteomic data (statistically significant, p<0.05) (B) Bar graph of lacrimal gland stem/progenitor cell markers identified as enriched in aggregate post/pre-IACC samples in our proteomic data (statistically significant, p<0.05). (C) Immunohistochemical staining representative for apoptotic markers cleaved caspase 3 (cCas3), Poly (ADP-ribose) polymerase-1 (PARP-1), p16, and p53 in post-IACC LGACC tumors. (D) Immunofluorescence image for terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay in post-IACC LGACC specimens. (micron bar = 200 μm).

Figure 3. Fibroblast growth factor (FGF) signaling is upregulated following IACC in LGACC tumors

(A) Bar graph of normalized quantitative fold expression of fibroblast growth factor (FGF) signaling family members in aggregate post/pre-IACC LGACC tumors identified in proteomic screening (statistically significant, p<0.05). (B) Immunofluorescence images of paired pre- and post-IACC LGACC samples probed for FGFR1 (red), and counterstained for filamentous actin (green), and nuclei (blue). (C) Representative immunohistochemical staining image for FGFR1 in a post-IACC LGACC tumor specimen. (micron bars: 20X=200 μm; 40X=100 μm).

Figure 4. Morphology and characterization of LGACC cell cultures

(A) Morphology of established LGACC cell lines from primary tumors from pre- and –post IACC samples cultured in serum free human mammary epithelial cell media (B) Characterization of LGACC cells by immunocytochemistry using probes for markers e-cadherin (E-CAD), platelet-derived growth factor receptor (PDGFR), cytokeratin-5 (Ck5), low affinity neurotrophin receptor (p75), and fibroblast growth factor receptor 1 (FGFR1). (C) Growth curve over a 10 day period for pre- and post-IACC cell cultures in control or FGFR1 inhibitor-supplemented media using AZD4547 (2μM). (micron bar = 200 μm).

Figure 5. Synergistic effect of AZD4547 and cisplatin on LGACC cell viability

(A) Dose-dependent viability curves showing the half-maximal effective dose (EC₅₀) for cisplatin and AZD4547. (B) Isobologram of synergistic cooperation between cisplatin and AZD4547 on LGACC cell viability (points plotted) over a 72 hrs. period. (C) Western blot analysis of LGACC cells following treatment with cisplatin, AZD4547, and their combination. (D) The quantitative expression of indicated proteins as determined by densitometric analysis. Data are calculated from triplicate experiments and presented as mean±SD (^**p<0.05; ^***p<0.01 compared with control; ^#p<0.05 wrt to cisplatin; ⁺p<0.05 wrt to AZD4547).

Figure 6. Synergistic effect of AZD4547 and cisplatin on LGACC cell migration and proliferation

(A) Representative images of migration assay results for LGACC cells after 48 hrs. of control, cisplatin, AZD4547, or combinatorial treatment. (B) Quantitative analysis of gap bridging percentage in migration assay of LGACC cells under different treatments. (C) Growth curve analysis of LGACC cells over 7 days of control, cisplatin, AZD4547, or combinatorial treatment. Data presented as mean±SD (^*p<0.05; ^**p<0.01; ^***p<0.001 compared with control; ^#p<0.01 wrt to cisplatin; ⁺p<0.01 wrt to AZD4547).