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More than 90% of human myxoid liposarcoma (MLS) cases are associated with the chromosomal translocation, which creates a chimeric oncogene comprising part of the TLS (translocated in liposarcoma) gene (also known as FUS (fused in Ewing’s sarcoma)) and part of the CHOP (CCAAT/enhancer binding protein (C/EBP) homologous protein) gene (also called DDIT3 (DNA damage-inducible transcript 3) and GADD153 (growth arrest- and DNA damage-inducible gene 153)) (Crozat et al, 1993; Rabbitts et al, 1993; Powers et al, 2010). The resultant fusion gene TLS–CHOP encodes the N-terminal half of TLS fused to complete sequence of CHOP (Powers et al, 2010; Figure 1A). TLS-CHOP protein is considered to function as an abnormal transcription factor (Kuroda et al, 1999; Pérez-Mancera et al, 2008; Andersson et al, 2010). The definitive TLS–CHOP function for MLS development, however, is unclear.

Figure 1
figure 1

Repression of TLS–CHOP expression by TLS–CHOP siRNA in MLS-derived cells inhibits cell growth. (A) Schematic structures of various types of TLS–CHOP fusion gene. Grey and open boxes represent exons of the TLS and CHOP genes, respectively. The target site of TLS–CHOP siRNA and the hybridisation sites of TLS–CHOP detection primers are also shown. (B) Detection of TLS–CHOP transcripts in MLS-derived cell lines. PCR with TLS–CHOP detection primers was performed using cDNAs synthesised from total RNAs of MLS-derived cells. The PCR products were fractionated by electrophoresis on a 2% agarose gel. Types of TLS–CHOP were determined by direct sequencing of the PCR products. (C) Reduction of TLS–CHOP transcript in 1955/91 and 2645/94 cells by TLS–CHOP siRNA. In all, 72 h after siRNA transfection, total RNA from the cells was extracted and subjected to real-time PCR analysis. Data were normalised to a minimum mRNA level that was arbitrarily set to 1 in the graphical presentation. (D) Western blot analysis of total cell extracts from 1955/91 and 2645/94 cells 48 h after siRNA transfection. α-Tubulin is shown as a loading control. (E) TLS–CHOP siRNA inhibits cell growth of MLS-derived cells. 1955/91 and 2645/94 cells were transfected with TLS–CHOP siRNA or negative control siRNA. Then, the cells in 12-well culture plates were counted at several time points using a haemocytometer. Bars, SD. (F) Representative phase-contrast images of 1955/91 and 2645/94 cells at 72 h after siRNA transfection.

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Melanoma differentiation-associated gene 7 (MDA-7)/interleukin-24 (IL-24) protein is expressed in cells of the immune system and normal human melanocytes (Jiang et al, 1995; Wolk et al, 2002). Exogenous expression of MDA-7/IL-24 induces growth arrest and apoptotic cell death in various human malignant cells (Dash et al, 2010; Rahmani et al, 2010).

In this report, we have found a novel pathway of TLS–CHOP with MDA-7/IL-24 repression.

Materials and methods

Cell culture

The MLS-derived cell lines, 1955/91 and 2645/94, were kindly provided from Professor David Ron (University of Cambridge), and were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich Corporation, St Louis, MO, USA) supplemented with 10% foetal bovine serum. Cell quantification was performed as previously described (Oikawa et al, 2004).

Small interfering RNA transfection

Small interfering RNA (siRNA) transfection (1 μ M final concentration) was performed as previously described (Oikawa et al, 2004). The nucleotide sequences of the chemically synthesised double-stranded siRNAs are as follows: TLS–CHOP siRNA, 5′-GGAAGUGUAUCUUCAUACAdTdT-3′; MDA-7/IL-24 siRNA, 5′-GUGGAUGGGUGCUUAGUAAdTdT-3′; and negative control siRNA, 5′-AUCCGCGCGAUAGUACGUAdTdT-3′.

Detection of TLS–CHOP variants and quantitative real-time PCR analysis

RNA isolation and first-strand cDNA synthesis were performed as previously described (Oikawa et al, 2008a). For detection of TLS–CHOP variants, we performed PCR analysis with TLS–CHOP detection primers 5′-CTTATGGCCAGAGCCAGAAC-3′ and 5′-AAGGCAATGACTCAGCTGCC-3′. The amplification products were sequenced with ABI PRISM 310 Genetic analyser (Applied Biosystems, Foster City, CA, USA). Real-time PCR analysis was performed as previously described (Oikawa et al, 2008b) using TLS–CHOP-specific primers 5′-ATGAACGGCTCAAGCAGGAA-3′ and 5′-TGGTGCAGATTCACCATTCG-3′, and MDA-7/IL-24-specific primers 5′-GTTTTCCATCAGAGACAGTG-3′ and 5′-GTAGAATTTCTGCATCCAGG-3′. The TLS–CHOP and MDA-7/IL-24 mRNA levels were normalised to β-actin signals (Oikawa et al, 2004). We performed real-time PCR analysis in duplicate.

Microarray analysis

Cells were transfected with the TLS–CHOP or negative control siRNAs, and were incubated for 72 h. Biotin-labelled complementary RNA (cRNA) was then generated from 1 μg of total RNA of the cells using CodeLink iExpress Expression Assay Reagent Kit (GE Healthcare UK Ltd, Buckinghamshire, UK), and was hybridised to CodeLink Human Whole Genome Bioarray (GE Healthcare) using iAmplify cRNA Preparation and Hybridisation Reagents Kit (GE Healthcare) according to Expression Bioarray System User Guide ver. 2.0. The array slides were incubated for 21 h at 37 °C with shaking, and were scanned with a DNA microarray scanner G2505A (Agilent Technologies, Inc., Santa Clara, CA, USA). The scanned images were analysed and median normalised using CodeLink Expression Analysis Version 4.1.0.29054 (GE Healthcare). The data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE33616.

Western blot analysis

Western blot analysis was performed as previously described (Oikawa et al, 2002). Anti-TLS–CHOP monoclonal antibody (clone 14) was previously generated (Oikawa et al, 2006). Monoclonal anti-α-tubulin antibody clone B-5-1-2 (T-5168; Sigma) was purchased.

Plasmid construction and transfection

To create an MDA-7/IL-24 expression vector, cDNA fragment containing the complete coding region of MDA-7/IL-24 was amplified by PCR using the primers 5′-GCGCGGATCCGAGATGAATTTTCAACAGAG-3′ and 5′-GGCCAAGCTTCCTGGTCTAGACATTCAGAG-3′, and inserted into the mammalian expression vector, pcDNA3.1(−) (Invitrogen, Carlsbad, CA, USA). Plasmid transfection was performed using Lipofectamine 2000 reagent (Invitrogen) and Opti-MEM I Reduced-Serum Medium (Invitrogen).

Results

TLS–CHOP knockdown represses cell growth of MLS-derived cell lines

First, we examined the activity of the three newly designed effective siRNAs that target different positions of TLS–CHOP in a preliminary experiment (Supplementary Figure 1), and selected the most effective siRNA among them (hereafter termed TLS–CHOP siRNA) for use in subsequent experiments. The TLS–CHOP siRNA targets exon 2 of the CHOP gene (Figure 1A). Although types 4 and 11 of TLS–CHOP variants do not have the target region, TLS–CHOP in over 80% of MLS is type 1 or 2. We confirmed that the two MLS-derived cell lines, 1955/91 and 2645/94, carries type 1 and type 2, respectively (Figure 1B). TLS-CHOP knockdown by the siRNA inhibited cell growth and induced cell death in both cell lines (Figure 1C–F). On the other hand, a non-targeting negative control siRNA did not affect cell growth, indicating that the effects of TLS–CHOP siRNA are not by off-target effects.

TLS–CHOP knockdown induces MDA-7/IL-24 expression in MLS cells

Next, we compared mRNA expression profiles of both 1955/91 and 2645/94 cells transfected with TLS–CHOP siRNA or negative control siRNA by microarray analysis (see Materials and Methods). We found that several dozen genes showed at least two-fold differential expression by TLS–CHOP siRNA (Table 1). Among the genes, we focused on the MDA-7/IL-24 gene because it encodes an anticancer cytokine (Dash et al, 2010). TLS-CHOP siRNA induced a significant increase in the expression of MDA-7/IL-24 in both cell lines (Table 1; Figure 2B). Thus, to confirm that MDA-7/IL-24 is important for growth arrest by TLS–CHOP knockdown, we prepared MDA-7/IL-24 siRNA and performed double transfection with both TLS–CHOP and MDA-7/IL-24 siRNAs into 1955/91 cells. MDA-7/IL-24 knockdown cancelled the growth inhibitory effects by TLS–CHOP siRNA alone (Figure 2A and B).

Table 1 Differential expression probes between MLS cells treated with TLS–CHOP and negative control siRNAs
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Figure 2
figure 2

Growth arrest of MLS cells by TLS–CHOP siRNA is caused by MDA-7/IL-24 expression. (A) Representative phase-contrast images (upper panels) and cell numbers (lower panel) of 1955/91 cells at 72 h after transfection with TLS–CHOP siRNA and/or MDA-7/IL-24 siRNA, or negative control siRNA. (B) Induction of MDA-7/IL-24 expression in 1955/91 cells by TLS–CHOP siRNA. In all, 72 h after siRNA transfection, total RNA and protein samples were prepared from the cells and subjected to real-time PCR and western blot analysis, respectively. Left panel shows MDA-7/IL-24 mRNA level. Data were normalised to the mRNA level of non-treated cells that was arbitrarily set to 1 in the graphical presentation. Right panel shows western blot analysis of TLS–CHOP expression. α-Tubulin is shown as a loading control. (C) Ectopic expression of MDA-7/IL-24 in MLS cells represses cell growth. 1955/91 and 2645/94 cells were transfected with expression vector. Then, the cells in 12-well culture plates were counted at several time points using a haemocytometer. Bars, SD.

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Overexpression of MDA-7/IL-24 represses MLS cell growth

MDA-7/IL-24 displays nearly ubiquitous cancer-specific toxicity (Dash et al, 2010; Rahmani et al, 2010). To confirm that MDA-7/IL-24 is also toxic for MLS, we transfected 1955/91 and 2645/94 cells with an MDA-7/IL-24 expression vector MDA-7/IL-24-pcDNA3.1(−) or a control vector pcDNA3.1(−). As shown in Figure 2C, MDA-7/IL-24-pcDNA(3.1) transfection represses the growth of the cells.

Discussion

We have demonstrated that TLS–CHOP knockdown in MLS cells represses cell growth (Figure 1C–E), suggesting that TLS–CHOP plays an essential role for growth of MLS cells. Furthermore, our results suggest that TLS–CHOP may become a promising molecular target for MLS treatment.

TLS-CHOP knockdown in MLS cells induced increased expression of an anticancer cytokine MDA-7/IL-24 (Table 1; Figure 2B). Thus, we consider that although the cancerous characteristics of MLS cells have potential to induce MDA-7/IL-24 expression, TLS–CHOP represses it and contributes to maintain the tumour growth.

In conclusion, we have revealed a novel pathway involving repression of MDA-7/IL-24 expression for tumourigenesis and/or growth of MLS. We believe that our results will contribute understanding of molecular function of the chimeric oncoprotein and development of a novel molecular therapy for cancers.