Fibroblast growth factor receptor 1 as a putative therapy target in colorectal cancer

Abstract

Background/aims: Resembling a potential therapeutic drug target, fibroblast growth factor receptor 1 (FGFR1) amplification and expression was assessed in 515 human colorectal cancer (CRC) tissue samples, lymph node metastases and CRC cell lines.

Methods: FGFR1 amplification status was determined using fluorescence in situ hybridization. Additionally, we assessed protein levels employing Western blots and immunohistochemistry. The FGFR1 mRNA localization was analyzed using mRNA in situ hybridization. Functional studies employed the FGFR inhibitor NVP-BGJ398.

Results: Of 454 primary CRCs, 24 displayed FGFR1 amplification. 92/94 lymph node metastases presented the same amplification status as the primary tumor. Of 99 investigated tumors, 18 revealed membranous activated pFGFR1 protein. FGFR1 mRNA levels were independent of the amplification status or pFGFR1 protein occurrence. In vitro, a strong antiproliferative effect of NVP-BGJ398 could be detected in cell lines exhibiting high FGFR1 protein.

Conclusion: FGFR1 is a potential therapeutic target in a subset of CRC. FGFR1 protein is likely to represent a central factor limiting the efficacy of FGFR inhibitors. The lack of correlation between its evaluation at genetic/mRNA level and its protein occurrence indicates that the assessment of the receptor at an immunohistochemical level most likely represents a suitable way to assess FGFR1 as a predictive biomarker for patient selection in future clinical trials.

Fig. 1

FISH images of CRC cases with different FGFR1 copy number status: diploid FGFR1 copy number status in CRC sample (a) and corresponding lymph node (b), low-level FGFR1 amplification in CRC sample (c) with corresponding lymph node (d), and high-level FGFR1 amplification in CRC sample (e) with corresponding lymph node (f).

Fig. 2

Immunohistological stainings. a–c Membranous localization of pFGFR1 in CRC: no pFGFR1 (a), heterogeneous localization (b), and high pFGFR1 levels (c). d–f FGFR1 mRNA status: score 0 (d), score 2+ (e), and score 4+ (f).

Fig. 3

Amplification, mRNA, and protein status of FGFR1 in HCD9, HCT116, and SNU-C1 cells. a Relative mRNA levels of FGFR1 in HDC9, SNU-C1 and HCT116 employing qRT-PCR (normalized to β-actin). HDC9 displays the highest levels, followed by HCT116 and SNU-C1. b FISH images of (1) inter- and metaphase nucleus of HDC9 cells harboring no FGFR1 amplification, (2) inter- and metaphase nucleus of the FGFR1-amplified SNU-C1 cells, and (3) inter- and metaphase nucleus of the HCT116 cells with no FGFR1 amplification. c FGFR1 protein levels in HDC9, SNU-C1, and HCT116 cells as assessed by immunoblotting. HDC9 displays the highest amounts, followed by HCT116 and SNU-C1.

Fig. 4

FGFR inhibitor NVP-BGJ398 reduces cell growth correlating with FGFR1 mRNA/protein. Inhibition of HDC9 and HCT116 cell lines using NVP-BGJ398 (48 and 72 h) showed a decrease in cell viability, whereas SNU-C1 cell viability was not affected. HDC9 was the most sensitive cell line followed by HCT116 showing only a response at high concentrations of the inhibitor. Viability (NVP-BGJ398 treatment as compared to DMSO control) was determined via MTT assay as described. Viability of 1.0 means 100% cell survival. The number of viable cells is relative to the untreated control cells at the same time points (48 and 72 h).

Fig. 5

Western blots of pFGFR1/FGFR1 and downstream molecules. HCT116 and HDC9 cells showed a clear downregulation of the activated pFGFR1, whereas total FGFR1 showed no decrease in the treated cell lines. HDC9 cells showed a decrease in the phosphorylation of the downstream molecule pERK. HCT116 showed no change in the activation of pERK and pAkt. The levels of total ERK1/2 and total Akt remained the same after inhibition.

Fig. 6

Immunohistological staining of HDC9. Increase of cleaved caspase-3 in HDC9 cells after inhibition for 48 h with rising amounts of NVP-BGJ398: 0 µ M NVP-BGJ398 (a), 0.5 µ M NVP-BGJ398 (b), and 1 µ M NVP-BGJ398 (c).