Nuclear expression of FLT1 and its ligand PGF in FUS-DDIT3 carrying myxoid liposarcomas suggests the existence of an intracrine signaling loop
The FUS-DDIT3 fusion oncogene encodes an abnormal transcription factor that has a causative role in the development of myxoid/round-cell liposarcomas (MLS/RCLS). We have previously identified FLT1 (VEGFR1) as a candidate downstream target gene of FUS-DDIT3. The aim of this study was to investigate expression of FLT1 and its ligands in MLS cells.
HT1080 human fibrosarcoma cells were transiently transfected with FUS-DDIT3-GFP variant constructs and FLT1 expression was measured by quantitative real-time PCR. In addition, FLT1, PGF, VEGFA and VEGFB expression was measured in MLS/RCLS cell lines, MLS/RCLS tumors and in normal adiopocytes. We analyzed nine cases of MLS/RCLS and one cell line xenografted in mice for FLT1 protein expression using immunohistochemistry. MLS/RCLS cell lines were also analyzed for FLT1 by immunofluorescence and western blot. MLS/RCLS cell lines were additionally treated with FLT1 tyrosine kinase inhibitors and assayed for alterations in proliferation rate.
FLT1 expression was dramatically increased in transfected cells stably expressing FUS-DDIT3 and present at high levels in cell lines derived from MLS. The FLT1 protein showed a strong nuclear expression in cells of MLS tissue as well as in cultured MLS cells, which was confirmed by cellular fractionation. Tissue array analysis showed a nuclear expression of the FLT1 protein also in several other tumor and normal cell types including normal adipocytes. The FLT1 ligand coding gene PGF was highly expressed in cultured MLS cells compared to normal adipocytes while the other ligand genes VEGFA and VEGFB were expressed to lower levels. A more heterogeneous expression pattern of these genes were observed in tumor samples. No changes in proliferation rate of MLS cells were detected at concentrations for which the kinase inhibitors have shown specific inhibition of FLT1.
Our results imply that FLT1 is induced as an indirect downstream effect of FUS-DDIT3 expression in MLS. This could be a consequence of the ability of FUS-DDIT3 to hijack parts of normal adipose tissue development and reprogram primary cells to a liposarcoma-like phenotype. The findings of nuclear FLT1 protein and expression of corresponding ligands in MLS and normal tissues may have implications for tissue homeostasis and tumor development through auto- or intracrine signaling.
KeywordsHT1080 Cell Placental Growth Factor Myxoid Liposarcoma FLT1 Ligand Normal Cell Type
CCAAT/enhancer-binding protein homologous protein
DNA-damage-inducible transcript 3
Fms-like tyrosine kinase 1
fusion (involved in t(12; 16) in malignant liposarcoma)
Growth arrest and DNA-damage-inducible protein 153
green fluorescent protein
placental growth factor
quantitative polymerase chain reaction
short interfering RNA
tyrosine kinase inhibitor
translocated in liposarcoma
vascular endothelial growth factor receptor 1
Myxoid/round-cell liposarcoma (MLS/RCLS) is one of the most common forms of liposarcoma and accounts for about 40% of all cases . The tumor cells are characterized by the FET family  FUS-DDIT3 fusion oncogene (also called TLS-CHOP) present in more than 90% of cases [3, 4, 5] or the EWS-DDIT3 found in a minority of cases . The causative role of FUS-DDIT3 in the initiation of MLS/RCLS and its role for the MLS-specific tumor morphology have been demonstrated in transgenic mice, xenografts and with FUS-DDIT3 carrying mesenchymal stem cells [7, 8, 9].
FUS-DDIT3 encodes a protein consisting of the N-terminal half of the FUS protein juxtaposed to the DNA-binding basic leucine zipper transcription factor DDIT3 (also known as CHOP or GADD153) [4, 5]. The FUS-DDIT3 protein acts as an abnormal transcription factor  and the development of myxoid liposarcomas is thus regarded as a consequence of deregulated FUS-DDIT3 target genes [8, 9, 11]. In this study, we have investigated the expression of the putative FUS-DDIT3 target gene FLT1 and its encoded receptor tyrosine kinase in MLS cells.
The myxoid liposarcoma cell lines MLS 402-91, MLS 1765-92, MLS 2645-94 [3, 11] and human fibrosarcoma cell line HT1080 were kept frozen in liquid nitrogen or cultured at 37°C and 5% CO2 in RPMI 1640 medium with HEPES buffer supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 μg/ml streptomycin and 10% fetal bovine serum (Invitrogen). Cell lines HT1080 FUSA-GFP, HT1080 DDIT3-GFP and HT1080 FUS-DDIT3-GFP were generated by plasmid transfection of HT1080 fibrosarcoma cells as described elsewhere . G418 (200 μg/ml) was constantly added to cell lines HT1080 FUSA-GFP, HT1080 DDIT3-GFP and HT1080 FUS-DDIT3-GFP to ensure stable expression of GFP constructs in the cell population. In a growth inhibition assay, FLT1-blocking antibody AF 321 (R&D systems) was added to MLS cells precultured in 4% fetal bovine serum for 14 hours in 96 well plates as described . The cultures were visually analyzed by light microscopy after 72 hours of incubation. Flt-1 siRNA (sc-29319), PGF siRNA (sc-44027) and control siRNA-A (sc-37007) were transfected into cells using the siRNA Transfection Reagent (sc-29528, Santa Cruz Biotechnology) according to instructions supplied by the manufacturer.
Quantitative real-time PCR analysis
Total RNA was prepared with the RNeasy lipid tissue kit (Qiagen) from an abdominal subcutaneous adipose tissue biopsies of healthy individuals and from isolated adipocytes as previously described . Acid guanidinium thiocyanate-phenol-chloroform extraction was used to isolate total RNA in representative tumor tissue from patients diagnosed with myxoid liposarcoma. Total RNA of cultured cells was isolated using QIAshredder and RNeasy Mini Kit (Qiagen). RNA concentrations were measured with the NanoDrop ND-1000 spectrophotometer. cDNA was generated using a QuantiTect Reverse transcription kit (Qiagen) or alternatively using oligo dT primers and Superscript III reverse transcriptase (Invitrogen). Real-time PCR was performed using a 7500 Fast real-time PCR system (Applied Biosystems) with SYBR Green detection (Qiagen). Formation of expected PCR products were confirmed by agarose gel electrophoresis and melt curve analysis. Gene expression data was normalized against ACTB and GAPDH expression by geometric averaging . Independent samples t-test was used to compare FLT1 expression between stably transfected cells. The following primer pairs were used for quantitative real-time PCR (5'-3'): FLT1_F TCCCTTATGATGCCAGCAAGT, FLT1_R CCAAAAGCCCCTCTTCCAA; PGF_F GTTCAGCCCATCCTGTGTCT, PGF_R AACGTGCTGAGAGAACGTCA; VEGFA_F CATCCTGTGTGCCCCTGA, VEGFA_R TTGTCTTGCTCTATCTTTCTTTGG; VEGFB_F AGTGCTGTGAAGCCAGACA, VEGFB_R GGAGTGGGATGGGTGATG; ACTB_F GCCGTCTTCCCCTCCATC, ACTB_R GCCTCGTCGCCCACATAG; GAPDH_F TCAGCCGCATCTTCTTTTG, GAPDH_R GACTCCGACCTTCACCTTC.
Immunohistochemistry and immunofluorescence
Formalin-fixed paraffin embedded tissue samples were obtained, prepared and analyzed according to routine pathology protocols as part of clinical diagnostics at the Sahlgrenska University Hospital, Gothenburg, Sweden. Sections were mounted on glass slides, deparaffinated in xylol and stained as previously described  with primary antibodies for FLT1 (C-17, sc-316, Santa Cruz Biotechnology) and VEGF receptor 1 (ab2350, Abcam). Human normal organs and various cancers tissue arrays (Super Bio Chips) containing 59 and 60 core biopsies per slide were stained as above. Angiosarcoma tissue sections were used as positive controls for the FLT1 staining. Negative controls were performed by omitting primary antibodies. For immunofluorescence studies, cells were grown in flaskettes, rinsed in phosphate buffered saline (PBS) and fixed for 20 minutes in 4% formaldehyde in PBS. Fixed cells were stained with antiserum for FLT1 (C-17, sc-316, Santa Cruz Biotechnology) and PGF (ab9542, Abcam) and visualized using goat anti-rabbit Cy3-conjugated secondary antibodies (Dako). Slides were mounted using Prolong Gold antifade with DAPI (Molecular Probes) and allowed to cure overnight. Cellular fluorescence was imaged using a Zeiss LSM510 META confocal microscope system.
Cellular nuclear and cytoplasmic fractions were isolated using a PARIS kit (Ambion) according to the instructions by the manufacturer. Protein concentrations in fractions were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce) and diluted for equal loading on gels. Samples were mixed with 4X LDS sample buffer (Invitrogen), 10% 0.5 M dithiothreitol and run on NuPage 4-12% Bis-Tris gels (Invitrogen). Proteins were blotted onto PVDF membranes (Immobilon) and probed with antibodies for FLT1 (C-17, sc-316, Santa Cruz Biotechnology), Lamin A (133A2, ab8980, Abcam) and GAPDH (mAbcam 9484, Abcam). Bands were visualized by horseradish peroxidase-conjugated secondary antibodies by chemiluminscent detection (SuperSignal West Dura Extended Duration Substrate, Pierce). Chemiluminscent membranes were imaged using a LAS-4000 imaging system (Fujifilm).
Tumor cells were seeded out in wells of black walled/clear bottom 96-well plates (BD Falcon) and allowed to adhere overnight. The following day, VEGFR tyrosine kinase inhibitors (TKI) II or III (Calbiochem) were added in a concentration range of 0 to 10 μM. After 72 hours, cells were subjected to multiple freeze-thaw lyses at -80°C and 37°C, respectively, where after cell proliferation relative to untreated control cells was assayed with a CyQuant Cell Proliferation Assay Kit (Molecular Probes) using a Wallac 1420 multilabel counter (PerkinElmer). Independent samples t-test was used to evaluate differences between wells.
Results and Discussion
The nuclear localization of FLT1 in MLS/RCLS was surprising since the protein is commonly reported a cytoplasmic or plasma membrane bound protein . To further probe the cellular distribution of the FLT1 protein, we prepared nuclear and cytoplasmic extracts from the MLS-derived cell line MLS 402-91. Western blot analysis of these extracts with an FLT1 specific antibody showed that anti-FLT1 reactive proteins were enriched in the nuclear fraction (Figure 2c). The western blot results thus confirmed the nuclear localization of FLT1 protein in MLS cells. The migration difference detected between nuclear and cytoplasmic FLT1 (Figure 2c) could be due to different ionic strengths of the extraction buffers used for fractionation but may well be a result of post-translational modifications of nuclear FLT1 proteins.
We conclude that upregulation of FLT1 in MLS/RCLS is an indirect downstream effect of the liposarcoma fusion oncogene FUS-DDIT3 and that FLT1 is expressed as a nuclear protein both in MLS/RCLS lipoblasts and in normal adipocytes. The nuclear expression of FLT1 in adipocytes and other normal cell types suggest that this receptor tyrosine kinase has cell type specific functions in the nuclear compartment. Most likely these involve maintenance of cell lineage through phosphorylation of target proteins residing in the nucleus. The expression of FLT1 ligand genes PGF, VEGFA and VEGFB in MLS/RCLS cells and in normal adipocytes further indicates the presence of possible autocrine or intracrine loops. The importance of FLT1, PGF, VEGFA and VEGFB expression and their functions in MLS/RCLS tumor development remain unclear but our results point out this receptor/ligand system as operational in MLS/RCLS and as such a potential target for intervention in this tumor type.
Thanks to Dr. Margareta Jernås and Dr. Lena Carlsson (University of Gothenburg) for normal adipocyte RNA. This work was supported by grants from the Swedish Cancer Society, Assar Gabrielssons Research Foundation and the Johan Jansson Foundation for Cancer Research.
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