Canine and human sarcomas exhibit predominant FGFR1 expression and impaired viability after inhibition of signaling

Abstract

Fibroblast growth factor receptors (FGFRs) are important in malignant progression of several human epithelial tumors. However, little is known about FGFRs in canine or human soft tissue sarcomas. Thus, our aim was to investigate expression of FGFRs and their involvement in cell survival in sarcomas of both species. FGFR1-4 and FGFRL1 transcripts as well as IIIb/IIIc splice variants of FGFR1-3 were evaluated in 3 canine- and 6 human sarcoma cell lines and 19 spontaneous canine sarcomas by SYBRqPCR. FGFR1 protein expression was assessed by immunohistochemistry. Growth inhibitory effects of FGFR1 inhibitor PD166866 and dominant negative recombinant FGFR adenoviral expression constructs (dnFGFR) on tumor cell lines were analyzed. Profiling of multiple FGFR transcripts detected comparable co-expression in most of human and canine sarcoma cell lines and canine tumor specimens. This indicates existence of closely related regulation mechanisms for FGFR expression in sarcomas of both species. FGFR1 with splice variant IIIc was consistently expressed with highest transcript levels. In 88% of the spontaneous tumor samples a heterogeneous FGFR1 protein expression was observed. Significant growth inhibition and cell death was seen after infection with dnFGFR1 in canine and human sarcoma cells, but not with dnFGFR3 and 4. PD166866 showed selective cytotoxicity with IC50 values between 12.1 and 26.4 μM. FGFR1 inhibition blocked ligand-induced tyrosine phosphorylation of ERK1/2 mitogen-activated protein kinase isoforms. This study emphasizes the important role FGFR1, especially splice variant IIIc, likely plays in sarcomas. Inhibitory small molecules could be of potential use for targeted therapy in aggressive sarcomas of both species.

Epithelial splicing regulatory protein 1 and 2 paralogues correlate with splice signatures and favorable outcome in human colorectal cancer

“…Reverse transcription (RT) was performed with 1 μg total RNA for complementary DNA synthesis using RevertAid First Strand cDNA synthesis kit (Fermentas, Germany) with the RevertAid Premium Reverse Transcriptase at 55°C. Aliquots of cDNA corresponding to 40 ng of RNA and GoTaq qPCR Master Mix (Promega, WI, USA) were applied for quantitative real-time PCR (qPCR) with 200nM of both forward and reverse primers [39]. In brief, transcript levels of ESRP1, ESRP2, FGFR1-3 IIIb and IIIc, and reference gene 36B4 were measured in triplicates.…”

“…For efficiency rates standard curves were generated with reamplification of diluted qPCR products as the starting point and four consecutive 1:2 dilutions. Primer details and qPCR efficiencies used for RQ calculations are described in Supplementary Table S2 or published [39]. …”

Telomere Transcripts Target Telomerase in Human Cancer Cells

“…SW480 and YT-BO were grown in RPMI-1640. Canine tumor cell lines originated from soft tissue sarcomas (MBSa, CoFSa, and PSTS), and were obtained from Marlene Hauck and grown as described [19]. …”

“…For recombinant TERRA expression the following primers were used: exo-5′_f (5′-CGATCCCCGGGTACCGAG-3′), exo-5′_r (5′-CCCCAACCCCAACCGGAAT-3′), exo-3′_f (5′-TTAGGGTTAGGGTTCGGAAT-3′), exo-3′_r (5′-AAGTGGAATTGTCGATCTGATA-3′), T7mod (5′-TAATACGACTCACTATAGGGAGAC-3′). Reference 36B4 gene primers for human and canine are described [19]. …”

“…Cell viability rates were determined as recommended using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Easy4U, Biomedica, Vienna, Austria) as described [19]. In brief, aliquots of 2 × 10 3 cells were seeded into 96-well plates in 100 μL cell line specific growth medium.…”