Induction of a secreted protein by the myxoid liposarcoma oncogene

Abstract

The TLS-CHOP oncoprotein, found in the majority of human myxoid liposarcomas, consists of a fusion between the transcription factor CHOP/GADD153 and the N terminus of an RNA-binding protein TLS/FUS. Clinical correlation and in vitro transformation assays indicate that the N terminus of TLS plays an important role in oncogenesis by TLS-CHOP. Until now, however, the only activity attributed to the oncoprotein is that of inhibiting the binding of transcription factors of the C/EBP class to certain adipogenic target genes, a function that TLS-CHOP shares with the nononcogenic CHOP protein. Here we report the isolation of a gene, DOL54, that is activated in primary fibroblasts by the expression of TLS-CHOP. DOL54 is expressed in the neoplastic component of human myxoid liposarcomas and increases the tumorigenicity of cells injected in nude mice. Activation of DOL54 requires an intact DNA-binding and dimerization domain in TLS-CHOP, a suitable cellular dimerization partner, and depends on the TLS N terminus. Normal adipocytic differentiation is associated with an early and transient expression of DOL54, and the gene encodes a secreted protein that is tightly associated with the cell surface or extracellular matrix. TLS-CHOP thus leads to the unscheduled expression of a gene that is normally associated with adipocytic differentiation.

Figure 1

A conditional allele expressing TLS-CHOP. (A) Targeting vector and targeted allele of Tls (“m2”) before and after Cre-mediated site-specific recombination. The loxP-flanked Neo cassette and juxtaposed human CHOP coding region represent an in-frame interruption of the Tls gene. Before Cre-mediated recombination, the allele encodes a TLS-Neo fusion protein, whereas after recombination it encodes TLS-CHOP. (B) Genomic PCR analysis of the targeted allele. The 3S and 4AS primers lie outside the targeting vector (horizontal arrows). (C) Site-specific recombination-dependent expression of TLS-CHOP. Shown is a Western blot of whole-cell extracts from embryonic fibroblasts with (m2/+) or without (+/+) the mutant Tls allele that had been transduced with a Cre-expressing retrovirus. Position of the TLS-CHOP protein is indicated by the arrow. TLS serves as a loading marker.

Figure 2

DOL54 expression is induced by TLS-CHOP. (A) Northern blot of total RNA from the MEFs shown in Fig. 1C hybridized to the labeled DOL54 cDNA. (B) Northern blot of RNA from wild-type MEFs transduced with retroviruses expressing the indicated proteins. LZ⁻ and BR⁻ represent deletions of the leucine-zipper dimerization domain and basic-region DNA-binding domains of TLS-CHOP, respectively. VP16-CHOP and C/EBPα-CHOP are fusions of CHOP to the activation domain of herpes-virus VP16 and rat C/EBPα proteins, respectively. The photomicrographs (Lower) are from fixed samples of the transduced cells stained with antiserum to CHOP, serving as a control for the expression of the various fusion proteins.

Figure 3

Activation of DOL54 by TLS-CHOP requires a dimerization partner. (A) Northern blot analysis of MEFs with the mutant Tls allele and having (+/−) or lacking (−/−) a functional C/ebpβ allele. The blot was hybridized with the DOL54 cDNA insert (Upper) or tubulin as loading control (Lower). (B) DOL54 Northern blot of wild-type (+/+) or C/ebpβ−/− MEFs transduced with retroviruses expressing the indicated proteins.

Figure 4

(A) Schematic diagram of the predicted peptide sequence of DOL54. The signal peptide (SP), two somatomedin B-like repeats (Som-B), mucin-homology domain (Muc), and vitronectin-related sequences (Vtr) are indicated. (B) DOL54 immunostaining of fixed but nonpermeabilized CHO cells. The cells (Top) were stably transfected with a full length DOL54 expression plasmid, whereas the cells on the bottom are the parental strain. (C) Northern blot of RNA from human tumor samples hybridized to a DOL54 probe and tubulin loading control. The lowest gel is a human TLS-CHOP reverse transcription–PCR analysis on the same RNA. Note that the tumors positive for TLS-CHOP were also positive for DOL54. The TLS-CHOP mRNA in the positive control MEFs (lane 1) is a hybrid of murine Tls and human CHOP and is not amplified by the PCR primers used here. (D) Tumorigenicity of individual CHO clones expressing DOL54 (mice 3–6) and parental cells that do not express the protein (mice 1 and 2) injected into nude mice and photographed 10 days after injection of 10⁶ cells into each of three subcutaneous sites. The CHO clone injected into mouse no. 5 expressed ≈3-fold lower levels of DOL54 mRNA than the clones injected into mice 3, 4, and 6).

Figure 5

Northern blot analysis of DOL54 expression in mouse tissues and during adipogenesis. (A) Total RNA from the indicated organs of adult mice (Left) or white adipose tissue of young (3 wk) and adult mice (4 mo) (Right) were sequentially hybridized with the DOL54 cDNA and tubulin. (B) Total RNA from MEFs or 3T3-L1 cells differentiating in vitro to adipocytes. Cells were harvested at the indicated time points after being exposed to an in vitro differentiation protocol.