Taken together these results strongly support that the expression of the FUS-DDIT3 oncogene is able to block the adipocyte differentiation program of target mesenchymal progenitor cells

May 27, 2017

d with a retroviral vector expressing C/EBPa. To define whether overexpression of C/ EBPa could rescue adipogenesis in FUS-DDIT3 cells, adipocytic differentiation was induced in FUS-DDIT3 MEFs infected either with empty vector or with the C/EBPa expressing vector. Of interest, the impaired adipocyte differentiation block in FUS-DDIT3 MEFs was not normalized by restoring C/EBPa at day 8 after hormonal induction,, although the retroviral vector was producing C/EBPa correctly as defined by western-blot. FUS-DDIT3 up-regulates expression of eIF4E in liposarcomas Although the inactivation of C/EBPa is not required, itself, for the blockade of adipogenesis in mesenchymal progenitor cells by FUS-DDIT3, however, the C/EBPa isoform ratio shift towards the truncated isoform both in mouse liposarcomas and in human liposarcoma cell lines. It has been previously reported that the control of initiation of translation of C/EBPa and C/ EBPb by the 11906293 eukaryotic translation initiation factors eIF2 and eIF4E is critical for the behavior of preadipocytes 3T3-L1. Thus, high levels of the eukaryotic translation initiation factors produce a shift towards truncated C/EBP isoforms that, in turn, induce a blockade in the terminal differentiation of 3T3-L1 cells. Thus, we next studied the expression level of eIF2a and eIF4E in FUSDDIT3-liposarcomas. Analysis by western-blot of protein lysates showed a dramatic overexpression of both eIF4E and eIF2 in liposarcomas arisen both in FUS-DDIT3 mice and human 19770292 liposarcomas cell lines FUS-DDIT3 positives, explaining the shift towards the truncated p30 isoform of C/ EBPa in liposarcomas. Similarly, the impaired expression of both eIF4E and eIF2 in liposarcomas of CombitTA-FUS-DDIT3 mice was normalized following administration of tetracycline , indicating that the fusion protein is directly responsible for the overexpression of both eIF4E and eIF2 in liposarcoma. Moreover, eIF4E was also strongly upregulated in normal WAT of FUS-DDIT3 transgenic mice, suggesting that overexpression of eIF4E may be one of the first events in the initiation of liposarcomas. Next, we BIX01294 web examined whether this upregulation of eIF4E was a direct effect of FUS-DDIT3, or a consequence of the blockade in adipocyte differentiation in liposarcomas, as it has been previously shown that knock out mice for 4E-BP1, a protein that represses capdependent translation initiation by sequestering eIF4E, evidenced reduced WAT mass. We examined whether FUS-DDIT3 might be directly involved in the control of the eIF4E expression. In order to address this question, we used a vector containing a CAT gene reporter under the control of,2.5 kb proximal promoter region of the murine eIF4E promoter . When Function of FUS-DDIT3 U2OS cells were co-transfected with the reported vector along with the FUS-DDIT3 expression vector, there was a specific increae of the CAT activity compared to the activity with the empty vector, demonstrating that FUS-DDIT3 is involved in the upregulation of eIF4E in liposarcomas. We further investigated which FUS-DDIT3 domain was responsible for the repression of the eIF4E promoter. Using the same system, we proved that neither the co-expression of the domain NH2-FUS nor the co-expression of the DDIT3 domain produced any effect on the transactivation of the eIF4E promoter, indicating that both domains are required for affecting eIF4E expression. These observations establish for the first time the role of FUS-DDIT3 in preventing the development of adip