Abstract
BackgroundFibroblast growth factors FGF-1 and FGF-2 are often upregulated in tumors, but tightly bound to heparan sulphate proteoglycans of the extracellular matrix (ECM). One mechanism of their bioactivation relies on the FGF-binding protein (FGF-BP) which, upon reversible binding to FGF-1 or -2, leads to their release from the ECM. FGF-BP increases tumorigenicity and is highly expressed in tumors like colon carcinoma. In this paper, we analyse cellular and molecular consequences of RNAi-mediated FGF-BP knockdown in colon carcinoma, and explore the therapeutic effects of the nanoparticle-mediated delivery of small interfering RNAs (siRNAs) for FGF-BP targeting.ResultsEmploying stable RNAi cells, we establish a dose-dependence of cell proliferation on FGF-BP expression levels. Decreased proliferation is mirrored by alterations in cell cycle distribution and upregulation of p21, which is relevant for mediating FGF-BP effects. While inhibition of proliferation is mainly associated with reduced Akt and increased GSK3β activation, antibody array-based analyses also reveal other alterations in MAPK signalling. Additionally, we demonstrate induction of apoptosis, mediated through caspase-3/7 activation, and alterations in redox status upon FGF-BP knockdown. These effects are based on the upregulation of Bad, Bax and HIF-1α, and the downregulation of catalase. In a therapeutic FGF-BP knockdown approach based on RNAi, we employ polymer-based nanoparticles for the in vivo delivery of siRNAs into established wildtype colon carcinoma xenografts. We show that the systemic treatment of mice leads to the inhibition of tumor growth based on FGF-BP knockdown.ConclusionsFGF-BP is integrated in a complex network of cytoprotective effects, and represents a promising therapeutic target for RNAi-based knockdown approaches.
Highlights
Fibroblast growth factors FGF-1 and FGF-2 are often upregulated in tumors, but tightly bound to heparan sulphate proteoglycans of the extracellular matrix (ECM)
RNAi-mediated FGF-binding protein (FGF-BP) knockdown exerts ‘gene dosedependent’ anti-proliferative effects in colon carcinoma cells in vitro LS174T cells were stably mass transfected with shRNA expression plasmids and, upon generation of G418-resistant cells, clonal selection was performed through limited dilution
The analysis of FGF-BP expression levels in various clonal cell lines by Quantitative Reverse transcription (RT)-PCR (qRT-PCR) demonstrated the functionality of all three shRNAs as compared to control-shRNA or non-transfected cells, with residual mRNA levels in clonal cell lines being between 20% and > 90%
Summary
Fibroblast growth factors FGF-1 and FGF-2 are often upregulated in tumors, but tightly bound to heparan sulphate proteoglycans of the extracellular matrix (ECM). One mechanism of their bioactivation relies on the FGF-binding protein (FGF-BP) which, upon reversible binding to FGF-1 or -2, leads to their release from the ECM. FGF-1 (acidic FGF, aFGF) and FGF-2 (basic FGF, bFGF) are the beststudied members and are often upregulated in tumors Since both are tightly bound to heparan sulphate proteoglycans of the extracellular matrix (ECM), their bioactivation in terms of release from the ECM is FGF-BP was first isolated from the supernatants of A431 epidermoid carcinoma cells and termed HBp17 [2]. Upregulation of FGF-BP can occur, among others, by TPA through Krüppel-like factor 5 (KLF-5) [18,19], DMBA [11], Wnt/ b-catenin signalling [12], HPV16 E6 [20], androgen receptor activation [21] or EGF [22], while FGF-BP downregulation has been described for retinoids [23,24,25], TGF-b [26] or p53wt overexpression [27]
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