Original article
FGF-2 is a driving force for chromosomal instability and a stromal factor associated with adverse clinico-pathological features in prostate cancer

https://doi.org/10.1016/j.urolonc.2018.05.020Get rights and content

Highlights

  • FGF-2 drives both numerical and structural chromosomal instability in prostate cancer cells.

  • A high stromal FGF-2 expression is associated with adverse clinico-pathological features.

  • The tumor stroma can shape prostate cancer cell genomes and is hence a promising new source for prognostic biomarkers and drug targets.

Abstract

Background

There is mounting evidence to suggest that stromal cells play an integral role in the progression of prostate cancer (PCa). One of the most frequently altered growth factors in PCa is fibroblast growth factor-2 (FGF-2). It has previously been proposed that early stages of PCa are characterized by a primarily exogenous, that is, stromal cell-derived FGF-2 production, whereas advanced tumors rely more on an autocrine FGF-2 production. Prostate cancer progression is characterized by an increase of genomic instability including aneuploidy and structural chromosomal alterations. Herein, we address 2 problems that have not been comprehensively answered. First, we ask whether exogenous FGF-2 can directly drive genomic instability to promote PCa progression. Second, we investigate whether and to what extent stromal FGF-2 expression is maintained in advanced PCa and whether this influences tumor progression and patient prognosis.

Methods

In vitro experiments to investigate the role of FGF-2 in numerical and structural chromosomal instability were performed using immunofluorescence microscopy, fluorescence in situ hybridization and single cell electrophoresis. A human patient-derived xenograft mouse model recapitulating osteoblastic PCa bone metastasis was used for in vivo validation experiments. The prognostic role of stromal FGF-2 expression was analyzed using immunohistochemical staining of a tissue microarray with primary tumor specimens from 162 predominantly high-risk patients with PCa.

Results

Our results show that FGF-2 not only rapidly induces mitotic defects and numerical chromosomal imbalances but also an enhanced DNA breakage to promote chromosomal instability. Using the patient-derived xenograft model, we show that a deregulation of the FGF axis results in an increase of mitotic aberrations as well as DNA damage checkpoint activation in vivo. The FGFR inhibitor dovitinib was found to reduce numerical chromosomal instability as well as DNA breakage, thus underscoring the relevance of the FGF axis in promoting genomic instability. An overexpression of tumor cell-associated FGF-2 was detected in 52 of 162 patients (32.1%), whereas a stromal overexpression was found in 27 of 165 patients (16%). Remarkably, a strong stromal FGF-2 expression was associated with a significantly higher clinical stage and higher biochemical recurrence rate. Patients with strong stromal FGF-2 expression also had a significantly worse biochemical recurrence-free survival.

Conclusions

Our results underscore that exogenous FGF-2 can shape PCa cell genomes and that stromal FGF-2 expression is detectable in a sizeable proportion of advanced PCa where it is associated with adverse clinico-pathological features. Our results highlight the impact of the tumor stroma on malignant progression and provide a rationale for a further exploration of components of the tumor stroma as therapeutic targets in PCa.

Introduction

Prostate cancer is a leading cause of cancer-related morbidity and mortality in men in most industrialized countries [1]. Although the precise mechanisms of prostate cancer (PCa) progression and metastasis are still poorly understood, a number of findings suggest that these processes are not strictly tumor cell-specific but also shaped by strong influences of the tumor stroma. One growth factor family that has been extensively studied in this context are fibroblast growth factors (FGFs) [2], [3], [4], [5].

Numerous in vitro and in vivo results underscore a crucial role of FGFs and their receptors in prostate carcinogenesis and tumor progression [3], [6], [7]. The human FGF gene family consists of over 20 members that encode secreted polypeptides. FGFs exert their activities mainly through 4 conserved transmembrane tyrosine kinase receptors (FGFR1–4). Downstream signaling events are complex and mainly consist of activation of the phosphatidylinositol 3-kinase (PI3K)/AKT, mitogen-activated kinase, phospholipase Cγ (PLCγ) and signal transducers and activators of transcription signaling pathways. FGFs are mitogenic, they increase cell survival and migration and stimulate angiogenesis [2].

There is compelling evidence that in particular upregulation of FGF-2 (basic FGF) plays an important role in prostate carcinogenesis [3], [4]. A major source of FGF-2 in PCa is the stromal cell compartment as well as the extracellular matrix [6]. FGF-2 can also be produced by PCa cells to function in an paracrine or autocrine fashion [6]. A knock-out of FGF-2 was found to delay tumor progression in the transgenic adenocarcinoma of the mouse prostate model [8]. In addition, dovitinib, a small molecule multi-kinase inhibitor that exerts antitumoral activities through inhibition of the FGF and PDGF signaling axis, respectively, has shown promising clinical effects in FGFR-driven PCa patient-derived xenograft (PDX) models and in PCa patients with bone metastasis [9].

Prostate cancer progression is characterized by increasing genomic instability including numerical chromosomal aberrations (aneuploidy) as well as structural chromosomal changes [10], [11]. Although aneuploidy is typically a result of mitotic defects, structural chromosomal instability requires DNA breaks that are left unrepaired or repaired in an erroneous fashion [12].

In the present report, we demonstrate that exogenous FGF-2 can provoke numerical chromosomal imbalances as well as DNA breakage both in vitro and in a PDX mouse model. In addition, we show that stroma-associated FGF-2 is a negative prognostic factor in high-risk PCa thus underscoring the important role of the tumor stroma in malignant progression. Our findings highlight that PCa cell genomes can be shaped by factors derived from the tumor stroma and that targeting these processes represents a promising therapeutic option [5].

Section snippets

Cell culture and drug treatments

LNCaP and PC-3 PCa cells were provided by CLS (Eppelheim, Germany) and maintained as recommended by the distributor. For experiments, cells were treated with 10 ng/ml biologically active recombinant FGF-2 (rFGF-2; NovActive, NBC1–21335, Novus Biologicals, Littleton, CO; migrating at 23 kDa and 26 kDa in SDS-PAGE) for 72 hour or dH2O used as solvent control. Cells were treated with 1 µM dovitinib (TKI-258; Selleck Chemicals, Houston, TX) for 72 hour or 0.1% dimethyl sulfoxide (DMSO) as solvent

FGF-2 stimulates centrosome amplification and aberrant mitoses in vitro and in vivo

Centrosomes are the major microtubule-organizing centers in most mammalian cells and aberrant centrosome numbers, which can lead to mitotic defects and chromosome segregation errors, are a common finding in PCa [17], [18]. We have previously shown that FGF-2 can stimulate aberrant centrosome duplication and mitotic defects in vitro [19]. However, whether these alterations can occur in vivo and result in chromosomal imbalances has not been tested. We hence first corroborated the role of FGF-2 in

Discussion

In the present study, we report a number of novel findings centered on FGF-2, a well-known growth factor involved in prostate carcinogenesis. We first confirm and extend our previous findings [19] by showing that FGF-2 can not only disrupt mitotic fidelity by inducing an abnormal duplication of centrosomes but that this process in fact leads to numerical chromosomal imbalances. Furthermore, we show that FGF-2 can provoke DNA breakage thereby contributing to genomic instability in PCa cells

Acknowledgements

We would like to thank Geraldine Rauch for advice on statistics and the tumor documentation team of the Department of Urology for patient data collection. We are grateful to the tissue bank of the National Center for Tumor Diseases Heidelberg for tissue procurement and TMA construction. This work was supported by the Medical Faculty Heidelberg and by the MD Anderson Cancer Center Global Academic Program Sister Institution Network Fund (SINF).

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