EWS-FLI1 induces developmental abnormalities and accelerates sarcoma formation in a transgenic mouse model

Abstract

Ewing's sarcoma is characterized by the t(11;22)(q24:q12) reciprocal translocation. To study the effects of the fusion gene EWS-FLI1 on development and tumor formation, a transgenic mouse model was created. A strategy of conditional expression was used to limit the potentially deleterious effects of EWS-FLI1 to certain tissues. In the absence of Cre recombinase, EWS-FLI1 was not expressed in the EWS-FLI1 transgenic mice, and they had a normal phenotype. When crossed to the Prx1-Cre transgenic mouse, which expresses Cre recombinase in the primitive mesenchymal cells of the embryonic limb bud, the EF mice were noted to have a number of developmental defects of the limbs. These included shortening of the limbs, muscle atrophy, cartilage dysplasia, and immature bone. By itself, EWS-FLI1 did not induce the formation of tumors in the EF transgenic mice. However, in the setting of p53 deletion, EWS-FLI1 accelerated the formation of sarcomas from a median time of 50 to 21 weeks. Furthermore, EWS-FLI1 altered the type of tumor that formed. Conditional deletion of p53 in mesenchymal cells (Prx1-Cre p53(lox/lox)) produced osteosarcomas as the predominant tumor. The presence of EWS-FLI1 shifted the tumor phenotype to a poorly differentiated sarcoma. The results taken together suggest that EWS-FLI1 inhibits normal limb development and accelerates the formation of poorly differentiated sarcomas.

Fig. 1

Cloning scheme and characterization of the type 1 EWS-FLI1 transgenic construct. A, Cre-lox mediated recombination was the strategy used for creating the conditional allele. The DNA construct for the EWS-FLI1 transgenic mouse is shown. The CLE2 plasmid was generated by inserting loxP sites into the EcoR1 restriction sites of pCX-GFP. The CAG promoter consists of the CMV enhancer and the chick β actin core promoter. The gene for enhanced green fluorescent protein (EGFP) is flanked by loxP sites. In the presence of Cre recombinase, the EGFP cassette is excised, thereby enabling the EWS-FLI1 gene to be expressed. B, Quantitative PCR was performed to analyze mouse tail DNAs for the copy number of EWS-FLI1 of each transgenic line. The copy number was determined by comparison against wild-type genomic DNA that had been spiked with 1, 5, or 10 copies of the EWS-FLI1 transgene per genome equivalent DNA.

Fig. 2

After crossing to the Prx1-Cre transgenic mice, abnormalities are observed in the limbs of double-positive offspring carrying both the Prx1-Cre and EWS-FLI1 genes. A. The newborn control pup has normal limbs (arrow). B. The EF-a newborn pup has shortened limbs but a normal crown-rump length. C. The EF-b newborn pup has curved arthrogrypotic-like limbs with six digits in all limbs. D. The EF-c embryo at E14.5 shows severe abnormality of the limbs, with no discernible formation of joints, bones, hands, or feet. The EF-c line does not produce viable pups when crossed to Prx1-Cre.

Fig. 3

The EF transgenic mice exhibit multiple abnormalities of the limbs when crossed to Prx1-Cre. The effects are generally more severe for the EF-a mouse line. A (i–iii), Faxitron X-rays of adult mice at 6 weeks show marked shortening of the long bones of the EF-a mice (panel ii) and duplication of digits in the EF-b mice (white arrows, panel iii) when compared to normal mice (panel i). Histologically, a number of aberrations were found. B (i–iii), Skeletal muscles of the quadriceps were stained with trichrome and shown at low-power magnification (black bar = 1000 microns). The muscles of the EF-a mice (panel ii) and EF-b mice (panel iii) showed attenuation of the muscle fiber diameter and decrease in overall muscle mass compared to normal control (panel i). Degeneration of individual fibers and central location of nuclei can be observed. An increase in endomysial fibrosis is observed in the EF mice compared to normal mice. C(i–iii), Toluidine blue stain of the articular cartilage of the knee showed dysplastic and degenerative features (articular chondromatosis) in the EF-a mice (panel ii) and EF-b mice (panel iii) compared to normal mice (panel i). There is clustering of cells in multiple lacunae and loss of columnar arrangement of chondrocytes. D(i–iii), Hematoxylin and eosin staining of cortical femoral bone under polarized light demonstrates well-organized trabecular bone in the normal mouse (panel i) but an abundance of poorly organized, immature, woven bone (blue arrow) in the EF-a mouse (panel ii). A scant amount of mature lamellar bone (black arrow) is seen. The EF-b mouse (panel iii) also shows woven bone and impaired maturation but to a lesser extent than the EF-a mouse.

Fig. 4

In a model of sarcoma formation based upon conditional deletion of p53 (Prx1-Cre p53^lox/lox), the presence of EWS-FLI1 altered the phenotype of the sarcoma and accelerated the time to onset of sarcomas. A, X-ray of the tibia shows exuberant periosteal bone formation in a mouse with EWS-FLI1. B, In most tumors arising in mice carrying EWS-FLI1, a poorly differentiated sarcoma was found with large, pleomorphic nuclei, scant cytoplasm, and compact cells (hematoxylin and esosin). C, In two cases, a small amount of osteoid was observed (arrow), and these cases the tumors were classified as osteosarcoma. The amount of osteoid was markedly less than typical cases of osteosarcoma, which were the predominant tumor type in mice without EWS-FLI1. D, Survival curves of mice bearing the genotypes EWS-FLI1 Prx1-Cre p53^lox/lox (p53-del + EF) and Prx1-Cre p53^lox/lox (p53-del) showed significantly worse survival when EWS-FLI1 was present (p<0.0001). The median survival was 21 weeks with EWS-FLI1 compared to 50 weeks without EWS-FLI1.

Fig. 5

A, Western blot analysis showed expression of EWS-FLI1 and several of its functional targets, including manic fringe, c-myc, p21^WAF1and TGF-β RII in bone (“Tg bone”) compared to the Ewing’s sarcoma cell line TC-71 and expression in three separate tumor specimens (genotype Prx1-Cre p53^lox/lox EWS-FLI1). All of the tumors were from the EF-a transgenic mouse line. B, Immunohistochemistry with EWS-FLI-1 antibody demonstrated diffuse staining of cells from a tumor (Prx1-Cre p53^lox/lox EWS-FLI1), but little staining in muscle or spleen from the same mouse. C, Immunohistochemistry demonstrated staining of cells for neuron-specific enolase (NSE) from a tumor expressing EWS-FLI1 (right hand panels, genotype Prx1-Cre p53^lox/lox EWS-FLI1 [EF-a]). The staining for NSE, can be observed in patchy areas and sporadic cells. In comparison, there is no staining for NSE in sarcomas obtained by conditional knock-out of p53 without the presence of EWS-FLI1 (left-hand panels, genotype Prx1-Cre p53^lox/lox). D, The expression of mRNA is shown for various specimens. qRT–PCR analyses showed a greater level of mRNA expression of EWS-FLI-1 in the tumor specimen compared to normal bone, muscle, and spleen from the same animal. Each sample was normalized against levels of GAPDH and each symbol denotes the mean value of a sample analyzed in triplicate.