A novel oncogenic pathway by TLS-CHOP involving repression of MDA-7/IL-24 expression

Abstract

Background: Translocated in liposarcoma-CCAAT/enhancer binding protein homologous protein (TLS-CHOP) (also known as FUS-DDIT3) chimeric oncoprotein is found in the majority of human myxoid liposarcoma (MLS), but its molecular function remains unclear.

Methods: We knockdowned TLS-CHOP expression in MLS-derived cell lines by a specific small interfering RNA, and analysed the gene expression profiles with microarray.

Results: TLS-CHOP knockdown inhibited growth of MLS cells, and induced an anticancer cytokine, melanoma differentiation-associated gene 7 (MDA-7)/interleukin-24 (IL-24) expression. However, double knockdown of TLS-CHOP and MDA-7/IL-24 did not inhibit MLS cell growth.

Conclusion: Repression of MDA-7/IL-24 expression by TLS-CHOP is required for MLS tumour growth, and TLS-CHOP may become a promising therapeutic target for MLS treatment.

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.

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.