The myxoid/round cell liposarcoma fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype in transfected human fibrosarcoma cells

Abstract

Myxoid/round cell liposarcoma (MLS/RCLS) is the most common subtype of liposarcoma. Most MLS/RCLS carry a t(12;16) translocation, resulting in a FUS-DDIT3 fusion gene. We investigated the role of the FUS-DDIT3 fusion in the development of MLS/RCLS in FUS-DDIT3- and DDIT3-transfected human HT1080 sarcoma cells. Cells expressing FUS-DDIT3 and DDIT3 grew as liposarcomas in severe combined immunodeficient mice and exhibited a capillary network morphology that was similar to networks of MLS/RCLS. Microarray-based comparison of HT1080, the transfected cells, and an MLS/RCLS-derived cell line showed that the FUS-DDIT3- and DDIT3-transfected variants shifted toward an MLS/RCLS-like expression pattern. DDIT3-transfected cells responded in vitro to adipogenic factors by accumulation of fat and transformation to a lipoblast-like morphology. In conclusion, because the fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype when expressed in a primitive sarcoma cell line, MLS/RCLS may develop from cell types other than preadipocytes. This may explain the preferential occurrence of MLS/RCLS in nonadipose tissues. In addition, development of lipoblasts and the typical MLS/RCLS capillary network could be an effect of the DDIT3 transcription factor partner of the fusion oncogene.

Figure 1

Western blot analysis of transfected HT1080 cells and MLS 2645-94 from which the FUS-DDIT3 fusion cDNA was cloned. Sample origin is indicated for each lane. A mix of anti-DDIT3 and anti-FUS antibodies were used to detect all variant proteins. 1, the position of the recombinant FUS-DDIT3EGFP protein; 2, the normal FUS protein present in all cells; 3, the recombinant N-terminal part of FUS (FUSa) linked to EGFP; 4, the native FUS-DDIT3 protein of the MLS cells from which the fusion transcript was cloned; and 5, the recombinant DDIT3EGFP protein. Note that the EGFP tag adds around 30 kd in size to the recombinant proteins. Size markers in kilodaltons.

Figure 2

A: Microphotographs of sections from SCID mouse tumors of HT1080 cells (left column) and the pFUS-DDIT3EGFP- and pDDIT3EGFP-transfected variants (middle and right columns, respectively). Note irregularly sized lipoblasts in the transfected tumors. Original magnifications, ×400 and ×600. B: Anti-D34 staining of capillaries in SCID mouse tumors from HT1080 and the two transfected variants. Note thin branching capillary vessels in the transfected tumors. Microphotographs captured at ×200 magnification. C: In vitro adipogenesis of HT1080 cells and transfected variants. The upper line of photomicrographs shows the indicated cell lines after adipogenic treatment. Control cultured cells are shown in the lower series of photomicrographs. Accumulated lipids are stained red (Oil Red O staining). Original magnification, ×740.

Figure 3

Number of genes that were at least three times up-regulated (↑) and down-regulated (↓) in DDIT3- and FUS-DDIT3-expressing HT1080 cells as compared by microarray analysis with control HT1080 cells. Shaded parts of bars indicate number of genes that are identical with genes up- or down-regulated in MLS 402-91 cells. Total number of genes that were represented on the CHIP was 23,707.