2002 - University of Washington Bone and Joint Sources

The Role of Fusion Protein-Induced Alternative Splicing in the
Development of Ewing’s Sarcoma
DAVID D. ODELL, B.S., HOWARD A. CHANSKY, M.D., A. ZIELINSKA-KWIATKOWSKA, M.D.,
AND LIU YANG, PH.D.
Cancers arise when genetic
changes, such as chromosomal
translocations and point
mutations, enable cells to escape the
control mechanisms that usually serve
to limit cell division to periods of
growth or normal replenishment of
cells. A better understanding of the
specific pathways that lead from a
genetic change to a cancerous cell may
yield targets for molecular treatments.
Ewing’s sarcoma is a malignant
neoplasm of bone and soft tissue. A
chromosomal translocation, t(11;22)
that fuses two distinct genes is
associated with a large percentage
(85%) of Ewing’s tumors. The
translocation results in the Ewing’s
sarcoma protein (EWS) being fused to
the DNA binding domain of the ETS
transcription factor Fli-1 (Figure 1).
These fusion proteins retain the N-
terminal portion of normal EWS (WT-
EWS) while the fusion partner Fli-1
replaces the C-terminal portion of WT-
EWS. The N-terminal portion of
normal EWS has been found to bind
the largest subunit of the RNA
polymerase II (Pol II) complex, an
enzyme necessary for transcription of
genes from DNA to messenger RNA
(mRNA). The C-terminal domain of
normal EWS recruits proteins referred
to as serine-arginine (SR) splicing
factors that function in splicing the of
pre-mRNA to mRNA. The EWS/Fli-1
fusion protein retains the ability to bind
RNA Pol II. However, EWS/Fli-1
cannot recruit SR proteins due to the
replacement of its C-terminal domain
by the Fli-1fusion partner.
The EWS/Fli-1 fusion protein was
originally believed to induce cellular
transformation through improper
activation of Fli-1 target genes.
However, new evidence indicates that
the EWS/Fli-1 fusion protein may
interfere with adapter functions of
normal-EWS. In an adenovirus E1A
splicing model, EWS/Fli-1 has been
shown to alter splicing of exogenous
pre-mRNA when compared to ‘normal’
splicing patterns mediated by WT-
EWS. This study was designed to test
the effects of inhibiting expression of
EWS/Fli-1 on patterns of splicing of
molecules implicated in the
development of sarcomas.
RESULTS
Using the Ewing’s sarcoma cell line
SK-N-MC as a model system, a series
of transfections were performed to
integrate a “doxycycline-off” antisense
EWS/Fli-1 construct into the genome
of the Ewing’s cells. In this system, sense
EWS/Fli-1 is expressed in the presence
of doxycycline but upon withdrawal of
doxycycline the antisense EWS/Fli-1
construct is expressed. Antisense EWS/
FLI-1 then inhibits expression of the
‘normal’ EWS/FLI-1 sarcoma protein.
In our genetically engineered Ewing’s
sarcoma cells, induction of antisense
EWS/Fli-1 was monitored through the
co-expression of green fluorescent
protein (GFP) that could be visualized
using laser light (Figure 2). RT-PCR
analysis of EWS/FLI-1 mRNA and
Western blotting of the EWS/Fli-1
fusion protein were used to monitor
EWS/Fli-1 expression in induced and
non-induced Ewing’s sarcoma cells.
The inducible expression of EWS/
Fli-1 antisense RNA proved to be
effective in reducing the quantity of
EWS/Fli-1 fusion protein to a negligible
level after withdrawal of doxycycline
from the culture medium. Comparison
of cellular growth rates indicated that
antisense expression correlated with a
decrease in the proliferation of Ewing’s
sarcoma cells (Figure 3). In addition,
RT-PCR analysis showed that downregulation
of EWS/Fli-1 leads to
changes in mRNA splicing patterns for
the cell surface marker CD44 and the
tumor suppressor Bin-1.
DISCUSSION
The EWS/Fli-1 fusion protein is the
hallmark of Ewing’s tumors, yet little is
Figure 1: The normal structures of the EWS and Fli-1 genes are shown with the point of fusion indicated on the wild-type EWS gene. The N-terminal RNA Pol
II binding domain remains in the fusion protein while the C-terminal splicing factor-recruiting domain is replaced by the Fli-1 DNA binding domain.
30 2002 ORTHOPAEDIC RESEARCH REPORT