University of Central Florida College of Medicine Orlando, FL
Michael W. Rohr, BA1, Spencer Lessans, BS1, Jihad Aljabban, MD, BSc2, Dexter Hadley, MD, PhD1, Deborah Altomare, PhD1; 1University of Central Florida College of Medicine, Orlando, FL; 2University of Wisconsin Hospital and Clinics, Madison, WI
Introduction: Pancreatic cancer (PC) is one of the deadliest cancers worldwide, having a 5-year survival rate of ~10%. Patients with PC typically present with late stage disease (80-90%) and tumor cells have a propensity to metastasize and resist conventional chemotherapy. Underlying this phenotype is the early downregulation of cell adhesion proteins, especially those comprising the N-CAM complex. Fibroblast Growth Factor Receptor 4 (FGFR4) is major component of the N-CAM complex, but few studies have assessed its role in PC. In this emerging study, we investigate FGFR4’s putative role in mediating PC cell migration and chemoresistance. Methods: Transcriptomic data from TCGA, GTEx, and GEO were analyzed using STARGEO, a validated RNAseq meta-analysis tool developed in-house, to assess FGFR4 expression and prognostication in PC. Ingenuity Pathway Analysis was then used to predict FGFR4-associated functions and upstream regulators in PC. A panel of PC cells was used to determine expression of FGFR4 and the effects of its inhibition by BLU9931 on epithelial-mesenchymal transition (EMT), viability, gemcitabine sensitivity, and migration. Results: Meta-analysis of over 500 samples revealed significant FGFR4 downregulation in PC which corresponded with negative prognostic markers, including disease-free survival and a hypoxia metagene score. Expression of FGFR4 and associated genes were predictive of inhibited tumor cell migration, invasion, and angiogenesis, in opposition to cell adhesion. Additionally, activated upstream regulators included multiple PC tumor suppressors (TS) whereas inhibited regulators were mainly oncogenes. In vitro, FGFR4 was highly expressed in PANC1 and L3.6PL, but not Mia-PaCa2 and HPAC, cells. Inhibition of FGFR4 by BLU9931 and growth on collagen reduced FGFR4 activity and induced EMT in PANC1 cells. FGFR4 inhibition also enhanced PC cell viability, resistance to gemcitabine, and migration. Discussion: FGFR4 is a well-known cancer oncogene and drives tumorigenesis in an autocrine-paracrine manner. However, recent evidence points to FGFR4 having context-dependent TS activity which we show occurs in PC. Low expression of FGFR4 was found in PC and was associated with poor prognosis and inhibition of FGFR4 by BLU9931 induced PC cell EMT and chemoresistance. Future studies are needed to test if early PC metastasis could be suppressed by therapeutic FGFR4 agonism. We also caution long-term use of clinical FGFR4 inhibitors for treating cancer as they may inadvertently enhance PC risk.
Figure 1. Bioinformatics predicts FGFR4 as a suppressor of PC tumorigenesis. (A) Transcriptomic meta-analysis (STARGEO) of FGFR4 expression in PC. Results expressed as the mean difference (MD) between average tumor and normal pancreatic tissue expression with meta values (Q, I^2, Z, and P) generated by random effects modeling. Study weights were determined using the inverse variance method. (B) Kaplan-Meier plots representing univariate analyses of FGFR4 expression relating to overall and disease-free survival. Expression cutoffs were determined by P-value optimization and analyzed by Mantel-Cox log-rank test. Data from TCGA-Firehose Legacy. (C) FGFR4 expression plotted as a function of PC hypoxia metagene score using three different classifications. This score correlates FGFR4 expression with hypoxia-associated transcriptome clusters in PC; higher scores correspond to greater tumor hypoxia and worse prognosis and vice versa. Significance determined by Pearson correlation analysis. (D & E) Ingenuity Pathway Analysis (IPA) prediction FGFR4 and closely associated genes (FDR<0.05) in a PC cohort stratified by (D) functional networks, and (E) upstream regulators. All associations were significant (P<0.05), and activity was predicted by IPA based on network overlap and directionality of experimental log ratios for each gene. Score > 0 (red) predicts activation while score < 0 (blue) predicts inhibition
Figure 2. Inhibition of FGFR4 promotes PC cell EMT and gemcitabine chemoresistance in vitro. (A) FGFR4 RNA and protein expression was assessed in a panel of four PC cell lines via qRT-PCR and western blot, respectively. Modified FGFR4 represents the glycosylated (active) form while unmodified FGFR4 is not glycosylated (inactive). (B) The effects of chemical inhibition of FGFR4 by BLU9931 (specific antagonist) on vimentin (EMT marker) was assessed in PANC1 cells grown on plastic or collagen. (C) The effects of BLU9931 on PC cell viability was tested by MTS assay, revealing dose-dependent elevation of cell viability only in FGFR4-expressing cells (PANC1 and L3.6PL). (D) The effects of FGFR4 inhibition on chemoresistance was assessed by treating PC cells with increasing concentration of gemcitabine in the presence or absence of 5 µM BLU9931 for 48 hrs; cell viability was analyzed as before. All results are from three independent experiments and significance was determined using Student’s T-test. *P<0.05, **P<0.01, ***P<0.001.
Figure 3. FGFR4 negatively regulates PC cell migration in vitro. FGFR4’s impact on cell migratory ability was assessed in (A) PANC1 and (B) HPAC cells by wound-healing assay in the presence of DMSO vehicle (control) or 5 µM BLU9931. Wound closure by cellular migration was documented by phase-contrast imaging over a period of 48 hrs and quantitated by gap area closure via ImageJ.
Disclosures: Michael Rohr indicated no relevant financial relationships. Spencer Lessans indicated no relevant financial relationships. Jihad Aljabban indicated no relevant financial relationships. Dexter Hadley indicated no relevant financial relationships. Deborah Altomare indicated no relevant financial relationships.