Hsp90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin Prevents Synovial Sarcoma Proliferation via Apoptosis in In vitro Models

Synovial sarcoma accounts for ∼7% to 10% of soft tissue malignancies and is associated with significant morbidity and mortality (1). Approximately 50% of patients will die from metastatic disease within 10 years of diagnosis despite treatment; the efficacy of conventional chemotherapeutics is controversial, although a recent report has suggested that combination doxorubicin/ifosfamide therapy offers a modest survival advantage in advanced cases (2, 3).

A translocation between SYT on chromosome 18 and SSX1 or SSX2 on chromosome X is characteristic of synovial sarcoma and is the only apparent cytogenetic abnormality in one third of cases (4). The biological roles of SYT and SSX are poorly understood; studies suggest that SYT functions as a transcriptional activator and SSX as a transcriptional repressor (4), although neither binds DNA directly. This implies that the SYT-SSX fusion protein combines these opposing activities and contributes to the pathogenesis of synovial sarcoma by dysregulating transcriptional patterns (4, 5). Expression profiling studies have identified a number of genes that are highly expressed in synovial sarcoma, including known oncogenes (6–10). Epidermal growth factor receptor (EGFR) is expressed at the protein level in synovial sarcoma (11) but is not associated with gene amplification (12). Protein level expression of fibroblast growth factor receptor 3 (FGFR3), c-kit (KIT), and human epidermal growth factor receptor 2 (HER2) has also been reported in synovial sarcoma (11, 13–15). Specific inhibitors for a number of these oncoproteins exist, some of which are in clinical use or clinical trials (16–21) and are of interest as potential systemic therapies for synovial sarcoma; gefitinib (Iressa, ZD1839) is already the subject of a synovial sarcoma clinical trial (European Organization for Research and Treatment of Cancer protocol 62022). Recent research has also shown that small molecule inhibitors of FGFR can prevent the proliferation of synovial sarcoma cultures stimulated with exogenous FGF (22).

Another therapeutic strategy is to employ an inhibitor of multiple receptor tyrosine kinases (RTK). Heat shock protein 90 (Hsp90) has numerous cellular functions, including promotion of proper protein folding (23), and stabilizes a number of oncogenic proteins, many of which are overexpressed in cancers (24). Small molecule inhibitors of Hsp90 like the geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) prevent this stabilizing effect (25, 26), exhibiting anticancer effects and causing degradation of Hsp90 client proteins including RTKs such as EGFR (27). These observations have led to clinical trials of 17-AAG in several adult solid tumors that have shown its safety and therapeutic properties (28, 29). The potential for 17-AAG activity specifically against synovial sarcoma, however, remains unexplored.

The involvement of RTK overexpression in the pathogenesis of various malignancies has been well established. Considering this, the overexpression of RTKs observed in synovial sarcoma suggests that one or more of these receptors is involved in the development and growth of this malignancy. As such, inhibiting activation of one or more of these receptors may prevent synovial sarcoma proliferation and induce apoptosis, suggesting potential new systemic treatments for this disease. Here we show that drugs developed to specifically inhibit EGFR, insulin-like growth factor receptor (IGF-1R), FGFR1 and FGFR3, HER2, and KIT do not significantly prevent the proliferation of two synovial sarcoma cell lines, whereas treatment with 17-AAG induces degradation of each of these RTKs while concurrently inhibiting proliferation and inducing apoptosis in synovial sarcoma monolayer and spheroid models.

Reagents. 17-AAG was provided by the Developmental Therapeutics Branch of the National Cancer Agency (Bethesda, MD). The EGFR inhibitor gefitinib (Iressa, ZD1839; ref. 17) was provided by AstraZeneca PLC (London, United Kingdom). The IGF-1R inhibitor NVP-AEW541 (18) was provided by Novartis-Pharma AG (Basel, Switzerland). The FGFR3 inhibiting antibody PRO-001 (16) was provided by ProChon Biotech Ltd. (Rehovot, Israel). The FGFR1 and FGFR3 inhibitor SU5402 (19) was purchased from Calbiochem UK (Merck Biosciences, Nottingham, United Kingdom), the HER2 inhibitor trastuzumab (Herceptin; ref. 20) was purchased from Genentech (South San Francisco, CA), and the KIT/platelet-derived growth factor receptor inhibitor imatinib mesylate (Gleevec; ref. 21) was purchased from Novartis-Pharma. All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise specified. 17-AAG, radicicol, gefitinib, SU5402, NVP-AEW541, and camptothecin were dissolved to the appropriate 1,000× stock concentrations in DMSO. Imatinib mesylate, trastuzumab, and PRO-001 were diluted to the appropriate 1,000× stock concentrations in PBS. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was dissolved in PBS to a stock concentration of 5 mg/mL and filter sterilized. RPMI 1640, fetal bovine serum (FBS), and trypsin were purchased from Life Technologies (Invitrogen, Mississauga, Ontario, Canada).

Monolayer and spheroid cell culture. The monophasic synovial sarcoma cell line Fuji (30) and the biphasic synovial sarcoma cell line SYO-1 (31) were kindly provided by Kazuo Nagashima (Hokkaido University School of Medicine) and Akira Kawai (National Cancer Centre Hospital, Tokyo), respectively. The presence of t(X;18) in the synovial sarcoma cell lines was confirmed by diagnostic cytogenetic karyotyping, reverse transcription-PCR, and fluorescence in situ hybridization analysis. The MCF-7 and SKOV-3 cell lines were obtained from the American Type Culture Collection (Manassas, VA). All monolayer cell cultures were grown on untreated tissue culture vessels in RPMI 1640 supplemented with 10% FBS under standard incubation conditions (37°C, 95% humidity, 5% CO₂), with the exception of MCF-7, which was cultured in DMEM supplemented with 10% FBS. All spheroid cell cultures were grown on tissue culture vessels coated with 500 μL of 1.4% agar in RPMI 1640 supplemented with 5% FBS and overlayed with RPMI 1640 supplemented with 5% FBS under standard incubation conditions.

Spheroid cell cultures were produced by trypsinization of confluent monolayer cultures to single cell suspensions. These suspensions were adjusted to 1 × 10⁴ cells per mL and 500 μL aliquots (5 × 10³ cells) were transferred to each well of an agar-coated 24-well plate. The plate was then incubated for 24 hours with gentle rocking and a further 48 hours without rocking to promote formation of a single spheroid per well. Single spheroids were then removed in a minimal amount of medium and combined, four per well, in 500 μL fresh medium overlaying fresh agar-coated 24-well plates for subsequent experimentation.

Proliferation assays. Monolayer culture proliferation was assessed by measuring the reduction of MTT. Confluent monolayer cultures were trypsinized and replated at 2 × 10⁴ cells per well (∼20% confluence), in triplicate, in 48-well plates. These cultures were grown to 50% confluence, at which time the medium was replaced with media containing the final concentration of vehicle control (0.1% DMSO), 17-AAG or doxorubicin (for comparative purposes). MTT was added to a final concentration of 1 mg/mL per well at each time point and incubated under standard conditions for 2 hours, the medium removed and an equal volume of DMSO added. Dissolved MTT formazan for each vessel well was transferred to a 96-well plate in triplicate and the absorbance measured at 570 nm in a PowerWaveX enzyme-linked immunoadsorbent assay plate reader (Bio-Tek Instruments, Inc., Winooski, VT). The average reading for each vessel well was used to determine the overall mean for each treatment time point.

Flow cytometry. To determine the levels of apoptosis in monolayer culture, synovial sarcoma cell lines were cultured and treated with test or control (0.1% DMSO) compound. At predetermined times, treated cultures were trypsinized to a single cell suspension and stained with Annexin V (BD Biosciences-PharMingen, Mississauga, Ontario, Canada), according to the manufacturer's instructions. The cells were then analyzed on an EPICS MXL flow cytometer (Beckman-Coulter, Mississauga, Ontario, Canada). Treated spheroid cultures were prepared for flow cytometric analysis as described previously (32), with the following modifications. Treated spheroids were transferred to a single microfuge tube in a minimal amount of media. Trypsin was added and spheroids disaggregated by gentle pipetting. The resulting cell suspension was filtered to remove any residual cell aggregates and stained with Annexin V and propidium iodide as described above.

Cell block preparation. Formalin-fixed, paraffin-embedded spheroid cell blocks for histologic analysis were produced as described previously (33) with the following modifications. Eight to 10 spheroids were removed from culture for each treatment group in a minimal amount of medium and combined in a microfuge tube. Reconstituted normal plasma (HemosIL; Instrumentation Laboratory, Lexington, MA) was then added and the spheroids resuspended by gentle agitation. Thrombin was then added and the mixture gently agitated to maintain the spheroids in suspension until clotting occurred. The spheroid cell blocks were then formalin fixed, processed, and stained with H&E using standard techniques.

Sequencing of epidermal growth factor receptor. Sixteen formalin-fixed paraffin embedded synovial sarcoma tumor samples of both monophasic and biphasic subtypes were obtained from Vancouver General Hospital. Genomic DNA was extracted from the paraffin-embedded synovial sarcoma tumor samples by xylene deparaffinization, overnight Proteinase K digestion at 56°C, and ethanol precipitation. Genomic DNA was prepared from the Fuji and SYO-1 cell lines using the DNeasy tissue kit (Qiagen, Mississauga, Ontario, Canada) according to manufacturer's instructions. Previously described primers were used to PCR amplify exons 18, 19, and 21 of the EGFR gene (34) and the PCR products were sequenced using the Big Dye Terminator kit v3.1 and an ABI 377 DNA sequencer (Applied Biosystems, Foster City, CA) following the manufacturer's instructions.

Immunoblot analysis. All primary antibodies were purchased from Cell Signaling Technologies (Beverly, MA) with the exception of anti-EGFR (Stressgen Biotechnologies, Victoria, British Columbia, Canada), anti-β-actin, anti-HER2, and anti-FGFR3 (Abcam, Cambridge, United Kingdom). Anti-mouse and anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Stressgen. Total cellular lysates of treated SYO-1 and Fuji monolayer cultures were prepared and immunoblotting done according to Cell Signaling Technologies protocols. Synovial sarcoma cultures treated for 24 hours with 1 μmol/L camptothecin, an apoptosis-inducing drug, were included in the caspase 3 immunoblots as positive apoptosis controls. Sample protein concentrations were determined by bicinchoninic acid assay (Pierce, Rockford, IL), and 30 μg of total protein per sample were resolved on 7.5% (RTKs) or 75 μg of total protein was resolved on 12.5% (caspase 3) acrylamide gels by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA) using standard methods. Immunoblots were visualized using the enhanced chemiluminescence detection kit (GE Healthcare, Piscataway, NY) according to manufacturer's instructions.

Statistical analysis. IC₅₀ values were calculated using Sigmaplot v9.0 (Systat, Point Richmond, CA).

The effect of specific receptor tyrosine kinase inhibitors on synovial sarcoma monolayer culture. The overexpression of RTKs in synovial sarcoma suggests that one or more of these pathways may play a role in the pathogenesis of this disease as has been shown in other cancers. To investigate the possibility that inhibiting specific RTKs will prevent the proliferation of synovial sarcoma, we obtained small molecule inhibitors to RTKs for which the receptor itself or activating components of the pathway are expressed: EGFR (gefitinib), FGFR1 and FGFR3 (SU5402), IGF-1R (NVP-AEW541), and KIT (imatinib mesylate). We also obtained humanized inhibitory antibodies to HER2 (trastuzumab) and FGFR3 (PRO-001). These inhibitors were tested for their ability to prevent the proliferation of SYO-1 and Fuji monolayer cultures.

To assess the effect of gefitinib on synovial sarcoma, monolayer Fuji and SYO-1 cultures were exposed to varying concentrations of gefitinib and compared with vehicle-treated control cultures (Fig. 1A). The gefitinib IC₅₀ was 265.1 μmol/L for Fuji and 266.4 μmol/L for SYO-1. These concentrations are significantly higher than those described to inhibit the proliferation of gefitinib-sensitive cell lines (35). Activating mutations in EGFR affecting the kinase domain have recently been identified to bestow sensitivity in non–small cell lung cancer to gefitinib (34). We sought similar sensitizing mutations in exons 18, 19, and 21 of EGFR in each synovial sarcoma cell line and 16 archival synovial sarcoma tumor specimens that strongly expressed the EGFR protein (11). No such mutations were found.

Expression levels of IGF-1R are variable in synovial sarcoma and those tumors exhibiting relatively high levels of IGF-1R display a more aggressive phenotype (36). Our previous gene expression profiling data found the IGF-1R ligand IGF2 to be highly expressed in synovial sarcoma (6). We examined the effect of the IGF-1R inhibitor NVP-AEW541 on both monolayer synovial sarcoma models and found an IC₅₀ of 19.8 μmol/L for Fuji and 29.4 μmol/L for SYO-1 (Fig. 1B). These IC₅₀ values are 10- to 50-fold higher than that described to inhibit IGF1R phosphorylation in cell-free assays and prevent proliferation of NIH 3T3 cells overexpressing human IGF-1R (18).

Although we had previously seen no KIT expression (11), others have reported KIT expression in synovial sarcoma (13). This implies that imatinib, which inhibits both KIT and platelet-derived growth factor receptor (21), may inhibit synovial sarcoma growth. We found an imatinib IC₅₀ of 43.5 μmol/L for Fuji and 6.4 μmol/L for SYO-1 (Fig. 1C). These IC₅₀ values are significantly higher than those reported for imatinib-sensitive cell lines (21, 37, 38).

We tested several other RTK inhibitors on the monolayer Fuji and SYO-1 models. The FGFR1 and FGFR3 inhibitor SU5402 did not inhibit the proliferation of SYO-1 and Fuji monolayer cultures at concentrations shown to inhibit proliferation and induce apoptosis in cell lines sensitive to this inhibitor without FGF supplementation (39). In addition, we tested inhibitory humanized antibodies targeting FGFR3 (PRO-001) and HER2 (trastuzumab) and saw no proliferation inhibition by MTT assay in either cell line (data not shown).

17-Allylamino-17-demethoxygeldanamycin inhibits the proliferation of synovial sarcoma monolayer cultures. The Hsp90 inhibitor 17-AAG has been shown to induce degradation of multiple RTKs, many of which are expressed in synovial sarcoma. To assess the effect of 17-AAG on synovial sarcoma, Fuji and SYO-1 were grown in monolayer culture and exposed to varying concentrations of 17-AAG, with doxorubicin-treated cultures included for comparison and vehicle-treated cultures included as controls. 17-AAG significantly inhibits cell proliferation in both the Fuji and SYO-1 cell lines in a dose- and time-dependent manner (Fig. 2A). The 17-AAG IC₅₀ values for synovial sarcoma monolayer models treated for 72 hours were both 0.038 μmol/L. For comparative purposes, we repeated this assay on MCF-7 cells, which have been identified as being relatively sensitive to Hsp90 inhibitors, and SKOV-3 cells, which are relatively resistant (40, 41). The IC₅₀ values for MCF-7 and SKOV-3 were ∼11- and 209-fold higher, respectively, than the IC₅₀ values for the synovial sarcoma cell lines (Table 1). This shows that the synovial sarcoma cell lines are particularly sensitive to 17-AAG. Importantly, significant synovial sarcoma growth inhibition occurs below serum concentrations achievable in humans (1.6-3.0 μmol/L; refs. 29, 42) and 17-AAG is more effective at inhibiting synovial sarcoma growth than doxorubicin at equimolar concentrations. Treatment of each synovial sarcoma cell line with radicicol, another Hsp90 inhibitor (43), produced similar results (data not shown), further supporting that Hsp90 inhibition prevents proliferation in these synovial sarcoma cell lines.

17-Allylamino-17-demethoxygeldanamycin inhibits monolayer synovial sarcoma proliferation by inducing apoptosis. To determine the nature of the 17-AAG-induced growth inhibition on the synovial sarcoma monolayer models, we assessed the levels of apoptosis by immunoblot analysis of caspase 3 activation and by flow cytometric measurement of Annexin V binding. The results of these experiments show that 17-AAG treatment causes activation of caspase 3 (Fig. 2B) and increased staining of SYO-1 monolayer cells with Annexin V before staining with propidium iodide (Fig. 2C), each of which indicates induction of apoptosis. Similar results were observed when the Annexin V staining assay was repeated on Fuji monolayer cultures. Induction of apoptosis occurs in a time scale consistent with that observed for proliferation inhibition in the monolayer MTT assays, showing that 17-AAG is inhibiting synovial sarcoma monolayer proliferation by inducing apoptosis. Similar results were also obtained with radicicol-treated Fuji and SYO-1 monolayer cultures (data not shown), suggesting that Hsp90 inhibition induces apoptosis in these synovial sarcoma models. Treatment with doxorubicin resulted in apoptosis as well but to a lesser extent (Fig. 2C).

17-Allylamino-17-demethoxygeldanamycin induces apoptosis in synovial sarcoma spheroid cultures. Spheroid cultures have been shown to more closely resemble in vivo tumor biology and are intrinsically more resistant to chemotherapeutic agents than monolayer cultures as a consequence of cell-cell interactions and of limited drug diffusion (32, 44–46). As such, spheroids represent a more stringent evaluation of the growth inhibitory effects of a particular drug. We used spheroid cultures generated from each synovial sarcoma cell line to assess the ability of 17-AAG to induce apoptosis under these more rigorous conditions. Vehicle- (0.1% DMSO), 17-AAG-, and doxorubicin-treated Fuji and SYO-1 spheroids were formalin fixed and paraffin embedded. H&E-stained sections of representative Fuji spheroid cell blocks are presented in Fig. 3. For each cell line model, apoptosis and necrosis is widespread in the 17-AAG-treated spheroids compared with the more focal apoptosis observed in doxorubicin-treated spheroids and the lack of apoptosis in the untreated spheroids. We confirmed these results by disaggregating Fuji and SYO-1 spheroids treated with or without 17-AAG and determining the proportion of apoptotic cells by Annexin V flow cytometry. 17-AAG induces significant levels of apoptosis in both SYO-1 and Fuji spheroids and to a much greater extent than equimolar concentrations of doxorubicin. Similar results were obtained for radicicol-treated Fuji and SYO-1 spheroids (data not shown).

17-Allylamino-17-demethoxygeldanamycin induces degradation of receptor tyrosine kinases. 17-AAG is well known to promote RTK degradation, which could explain the 17-AAG-associated induction of apoptosis in the synovial sarcoma cell lines. We examined the effect of 17-AAG treatment on EGFR, HER2, FGFR3, IGF-1R, and KIT in SYO-1 and Fuji monolayer cultures (Fig. 4). All of these RTKs were expressed in the cell lines at levels detectable by Western blot, with the exception of IGF-1R in Fuji cells which was undetectable. We found that 24 hours of treatment with 17-AAG caused a dose-dependent reduction in the levels of each of these RTKs in both synovial sarcoma cell lines. HER2 and FGFR3 were reduced to extremely low levels by 5 μmol/L 17-AAG.

These experiments show that the proliferation of two synovial sarcoma monolayer models is not significantly inhibited by the RTK-specific inhibitors gefitinib, NVP-AEW541, imatinib, SU5402, PRO-001, and trastuzumab, whereas synovial sarcoma proliferation is inhibited by 17-AAG. 17-AAG-mediated inhibition of proliferation in monolayer models occurs by induction of apoptosis and this is also observed in more stringent spheroid synovial sarcoma models. 17-AAG-induced inhibition of proliferation and induction of apoptosis occurs at concentrations achievable in humans and is greater than that observed for doxorubicin, which is presently used to treat synovial sarcoma.

This study was undertaken in an effort to identify an effective systemic therapy for synovial sarcoma, which is desperately needed. With a view to expediting the progression of a potential treatment from the laboratory to clinical use, we chose to focus our initial investigations on drugs that have completed phase I clinical trials and are presently in clinical use or being tested in at least phase II trials, so that pharmacology, toxicity, dosing, and side effects are known. We found that gefitinib does not inhibit synovial sarcoma proliferation at the maximum concentrations achievable in human serum (756 ng/mL or 1.7 μmol/L; ref. 17), although it does at higher concentrations. Similar results were obtained with the IGF-1R inhibitor AEW541 and the KIT inhibitor imatinib mesylate. This led us to assess the effect of 17-AAG, an inhibitor of multiple RTKs, on the synovial sarcoma models. We show that 17-AAG inhibits proliferation by inducing apoptosis in monolayer synovial sarcoma cell lines at concentrations that are achievable in human serum and which correlate with inhibitory concentrations observed in other preclinical cancer models (42). Furthermore, 17-AAG was more cytotoxic in monolayer cultures than doxorubicin, which is presently used to treat synovial sarcoma. In vitro monolayer assay conditions differ greatly from those found in patients, however, and it is well known that cell lines can significantly differ from the tumors from which they were derived (47) and tend to be more susceptible to chemotherapeutic agents. Thus, the results we have observed in monolayer cell culture assays may not accurately predict the antitumor effect of 17-AAG and its efficacy relative to doxorubicin in a clinical setting. Of note, the inhibition of synovial sarcoma culture proliferation was greater than that observed for cell lines previously described as Hsp90 sensitive or resistant, suggesting that synovial sarcoma is particularly susceptible to 17-AAG.

Three-dimensional cancer models, particularly spheroid cell cultures, have been shown to more closely approximate in vivo tumor biology and to be more resistant to chemotherapeutic agents than monolayer cultures. We assessed the effect of 17-AAG on synovial sarcoma spheroid cultures to obtain a more accurate prediction of the potential clinical effectiveness of 17-AAG. 17-AAG was observed to induce high levels of apoptosis in spheroid culture, similar to that observed in monolayer culture; in contrast, the cytotoxic effect of doxorubicin was moderate in spheroid culture. Unlike monolayer cultures, spheroids include a basal level of necrotic cells, the result of a complex interplay among culture conditions, nutrient diffusion, and cell-cell signaling (45, 46); however, the levels of apoptosis observed in the 17-AAG-treated spheroids are much higher than this inherent baseline. Because endogenous necrosis is located in the spheroid center, we have presented the peripheral portions of each spheroid in images of the cell block sections.

Gene expression studies indicate that synovial sarcoma is a disease typified by expression of multiple oncogenes, a number of which have been verified as expressed at the protein level. 17-AAG has been shown to cause the degradation of multiple oncoproteins, which made it appealing to us as a potential systemic therapy for synovial sarcoma. We observed that the level of 17-AAG-induced growth inhibition and apoptosis was similar between each synovial sarcoma cell line tested. We found that both synovial sarcoma cell lines are sensitive to the effects of radicicol, suggesting that the survival of the synovial sarcoma cell lines is dependent on one or more Hsp90 clients. Many oncoproteins are affected by 17-AAG treatment, including kinases involved in signal transduction. In particular, Hsp90 has been shown to be involved in the protein level expression of RTKs. Accordingly, 17-AAG inhibits the growth of malignancies typified by overexpression of RTKs such as EGFR and HER2 and this correlates with degradation of these receptors (27). We have observed by immunoblot that both synovial sarcoma cell lines express HER2, FGFR3, EGFR, and KIT protein whereas IGF-1R is expressed in SYO-1 but undetectable in Fuji, and that dose- and time-dependent degradation of each of these proteins occurs in both synovial sarcoma cell lines upon 17-AAG treatment. Significant depletion of each of these RTKs at the highest 17-AAG dose occurs by the time appreciable levels of each culture have entered apoptosis, suggesting that 17-AAG mediated degradation of these RTKs could be involved in the induction of apoptosis. FGFR3 is a candidate target to explain the effect of 17-AAG on synovial sarcoma. This protein not only has a documented role in promoting mesenchymal growth, but expression profiling studies from ourselves (6) and others (9) have highlighted expression of FGFR3 in synovial sarcoma, and furthermore, recent studies have shown antiproliferative activity of FGFR inhibitors in synovial sarcoma (16, 22). Our data shows FGFR3 degradation is induced by 17-AAG, although we were unable to show growth inhibition by small molecule or antibody inhibitors of FGFR3 in our assays, which differed from the cited studies in that we did not use exogenous FGF ligand supplementation. Similarly, others have shown that media supplementation with stem cell factor increases proliferation in KIT-dependent cell lines (48); however, we did not add exogenous stem cell factor to our assays to promote KIT-dependent growth, which could accentuate the growth-inhibitory effect of imatinib, and our results were similar to those for nonsensitive sarcoma cell lines treated with imatinib without the addition of SCF (37). HER2 is another candidate target to explain the effect of 17-AAG in synovial sarcoma, as it is a well-documented client protein of Hsp90 (25), which we show here to be degraded by 17-AAG in our cell line models. HER2 has been proposed as a therapeutic target in synovial sarcoma (15), although our previous tissue microarray studies found minimal protein expression (11). Our negative findings with trastuzumab and PRO-001 must be also viewed with caution as in vitro assays exclude any potential beneficial interaction with the human immune system and may underestimate the effect of therapeutic antibodies (49). Finally, our synovial sarcoma models carry SSX2 rather than SSX1 translocations, which may influence susceptibility to gefitinib (15).

Many proteins are Hsp90 clients, and Hsp90 itself has other cellular functions apart from promoting proper protein folding (23), making identification of the ultimate target or targets of 17-AAG-induced cytotoxicity in synovial sarcoma difficult. An attractive proposition is that the stability of the SYT-SSX fusion protein may be dependent on Hsp90 and that 17-AAG inhibition of Hsp90 activity will cause degradation of the fusion protein or other proteins that SYT-SSX is dependent upon for activity; we are currently investigating these possibilities.

Presently, more than half of those diagnosed with synovial sarcoma will ultimately die of this malignancy because there is no consistently effective systemic therapy to treat or prevent recurrence. We have provided in vitro evidence that 17-AAG, a clinically applicable drug with known pharmacology and toxicity, inhibits synovial sarcoma proliferation by inducing apoptosis and may prove to be an effective systemic therapy for this deadly disease.

We thank Kazuo Nagashima and Akira Kawai for kindly providing us with synovial sarcoma cell lines and Janine Senz and Cindy Ruttan for technical assistance.