Loss of SS18-SSX1 inhibits viability and induces apoptosis in synovial sarcoma

Abstract

Background: Most synovial sarcomas contain a chromosomal translocation t(X;18), which results in the formation of an oncoprotein SS18-SSX critical to the viability of synovial sarcoma.

Questions/purposes: We (1) established and characterized three novel synovial sarcoma cell lines and asked (2) whether inhibition of SS18-SSX1 decreases cell viability in these cell lines; and (3) whether reduction in viability after SS18-SSX1 knockdown is caused by apoptosis. After identifying a specific posttranscriptional splice variant in our cell lines, we asked (4) whether this provides a survival benefit in synovial sarcoma.

Methods: Cells lines were characterized. SS18-SSX1 knockdown was achieved using a shRNA system. Cell viability was assessed by WST-1 analysis and apoptosis examined by caspase-3 activity.

Results: We confirmed the SS18-SSX1 translocation in all cell lines and identified a consistent splicing variant. We achieved successful knockdown of SS18-SSX1 and with this saw a significant reduction in cell viability. Decreased viability was a result of increased apoptosis. Reintroduction of the exon 8 sequence into cells reduced cell viability in all cell lines.

Conclusions: We confirmed the presence of the SS18-SSX1 translocation in our cell lines and its importance in the survival of synovial sarcoma. We have also demonstrated that reduction in cell viability is related to an increase in apoptosis. In addition, we have identified a potential mediator of SS18-SSX function in exon 8.

Clinical relevance: SS18-SSX represents a tumor-specific target in synovial sarcoma. Exploitation of SS18-SSX and its protein partners will allow us to develop potent tumor-specific therapeutic agents.

Fig. 1

SS18-SSX1 is an intrinsically disordered protein. The IUPRed algorithm predicts disorder at 1.0 and an ordered protein at 0. By this algorithm, SS18-SSX1 is predicted to be almost entirely disordered.

Fig. 2A–B

Characterization of human synovial sarcoma cell lines GUSS-1, GUSS-2, and GUSS-3. We performed reverse transcription–PCR on total RNA from each of our synovial sarcoma cell lines using the SS18, SSX (consensus), SSX1, and SSX2 primers. The PCR products were then separated by electrophoresis on an agarose gel and visualized with ethidium bromide. All cell lines are of the SS18-SSX1 subtype (A). Protein lysates from each were analyzed by Western blot for SS18-SSX1 expression. All cell lines express SS18-SSX1 protein (B). G1 = GUSS-1; G2 = GUSS-2; G3 = GUSS-3 prechemotherapy; G3b = GUSS-3 postchemotherapy; IgG = negative IgG control. Actin was used as a protein concentration loading control.

Fig. 3A–B

Identification of splicing variant with exon 8 deletion. Fresh PCR product corresponding to the SS18-SSX1 sequence from all of our cell lines was sent for sequencing analysis. The highlighted portion represents a 93 base pair deletion in the expected coding sequence for SS18-SSX1, which corresponds to the deletion of exon-8 in the SS18 domain of the fusion protein (A). Genomic DNA was isolated from synovial sarcoma cell lines and simplified by PCR with primers designed to flank the exon-8 sequence. Exon-8 was present in the genomic DNA of all three cell lines (B). cDNA from GUSS-3, which lacks the exon-8 sequence, was used as a negative control.

Fig. 4A–C

Successful reduction of SS18-SSX1 protein by shRNA. Protein lysates were extracted from synovial sarcoma cell lines transfected with anti-SS18-SSX shRNAs. (A) GUSS-1; (B) GUSS-2; (C) GUSS-3. U = untreated cells; VC = empty vector control; SC = shRNA scramble control; T = treated cells. Numbers to the right of the panel quantify percent reduction in protein expression by densitometry analysis. Variable knockdown of SYT-SSX was seen with various shRNA constructs, but > 90% reduction was seen in all three cell lines with shRNAs 1, 5, and 6.

Fig. 5

Reduction of SS18-SSX reduces cell viability. Cell lines were treated with shRNAs against SS18-SSX and cell viability was examined by WST-1 assay. We saw a significant reduction in cell viability in all three cell lines with each of the shRNA constructs.

Fig. 6A–B

SS18-SSX knockdown induces apoptosis. Caspase-3 activity was measured after disruption of SS18-SSX (A). We saw a significant fold increase in caspase-3 activity in all three cell lines with each of the shRNA constructs. Untreated cells (U), empty vector (VC), and scramble shRNA (SC) were used as negative controls. These results depict the average of three separate experiments, each run in triplicate (n = 9). Total cell lysates were analyzed by Western blot analysis for full-length PARP and cleaved PARP. Cells treated with anti-SS18-SSX shRNA expressed cleaved PARP, whereas untreated cells did not. G1 = GUSS-1; G2 = GUSS-2; G3 = GUSS-3.

Fig. 7A–B

Reinsertion of exon-8 into synovial sarcoma cell lines reduces cell viability without affecting SS18-SSX expression. Synovial sarcoma cell lines were transfected with a pcDNA4TO vector containing the exon-8 sequence. Cell viability was examined by WST-1 analysis. We saw a significant reduction in cell viability after the exon-8 peptide was reintroduced into the cell (A). SS18-SSX expression was examined by Western blot analysis (B). There was no change in SS18-SSX expression after reinsertion of exon-8 into the cell lines. Actin was used as a protein loading control. S1 = GUSS-1; S2 = GUSS-2; S3 = GUSS-3. Positive FLAG expression confirms the presence of exon-8. U = untreated cells; VC = empty vector control. A depicts the results of three separate experiments each run in triplicate (n = 9).