ETV4 and ETV5 drive synovial sarcoma through cell cycle and DUX4 embryonic pathway control
Joanna DeSalvo, Yuguang Ban, Luyuan Li, Xiaodian Sun, Zhijie Jiang, Darcy A. Kerr, Mahsa Khanlari, Maria Boulina, Mario R. Capecchi, Juha M. Partanen, Lin Chen, Tadashi Kondo, David M. Ornitz, Jonathan C. Trent, Josiane E. Eid
Joanna DeSalvo, Yuguang Ban, Luyuan Li, Xiaodian Sun, Zhijie Jiang, Darcy A. Kerr, Mahsa Khanlari, Maria Boulina, Mario R. Capecchi, Juha M. Partanen, Lin Chen, Tadashi Kondo, David M. Ornitz, Jonathan C. Trent, Josiane E. Eid
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Research Article Oncology

ETV4 and ETV5 drive synovial sarcoma through cell cycle and DUX4 embryonic pathway control

Abstract

Synovial sarcoma is an aggressive malignancy with no effective treatments for patients with metastasis. The synovial sarcoma fusion SS18-SSX, which recruits the SWI/SNF-BAF chromatin remodeling and polycomb repressive complexes, results in epigenetic activation of FGF receptor (FGFR) signaling. In genetic FGFR-knockout models, culture, and xenograft synovial sarcoma models treated with the FGFR inhibitor BGJ398, we show that FGFR1, FGFR2, and FGFR3 were crucial for tumor growth. Transcriptome analyses of BGJ398-treated cells and histological and expression analyses of mouse and human synovial sarcoma tumors revealed prevalent expression of two ETS factors and FGFR targets, ETV4 and ETV5. We further demonstrate that ETV4 and ETV5 acted as drivers of synovial sarcoma growth, most likely through control of the cell cycle. Upon ETV4 and ETV5 knockdown, we observed a striking upregulation of DUX4 and its transcriptional targets that activate the zygotic genome and drive the atrophy program in facioscapulohumeral dystrophy patients. In addition to demonstrating the importance of inhibiting all three FGFRs, the current findings reveal potential nodes of attack for the cancer with the discovery of ETV4 and ETV5 as appropriate biomarkers and molecular targets, and activation of the embryonic DUX4 pathway as a promising approach to block synovial sarcoma tumors.

Authors

Joanna DeSalvo, Yuguang Ban, Luyuan Li, Xiaodian Sun, Zhijie Jiang, Darcy A. Kerr, Mahsa Khanlari, Maria Boulina, Mario R. Capecchi, Juha M. Partanen, Lin Chen, Tadashi Kondo, David M. Ornitz, Jonathan C. Trent, Josiane E. Eid

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Figure 9

Intersection of ETV4/5 and the DUX4 pathway in SS cells.

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Intersection of ETV4/5 and the DUX4 pathway in SS cells. (A) Immunoblots...
(A) Immunoblots show expression of the designated proteins in SYO-1 and HS-SY-II cells expressing control, shETV4-2, and shETV5-2. Numbers represent relative protein signals. Protein intensities were adjusted to actin as described in Statistics. (B) Immunoblots show ETV5 and DUX4 levels in SYO-1 cells expressing shETV5 and shDUX4 vectors. Numbers display relative band intensities compared with actin. (C) Bar graph compares effects of cotransduced shETV5 and shDUX4 (shETV5-2–shDUX4-1; shETV5-2–shDUX4-2), shETV5-2, and control vectors on SYO-1 cell growth. Growth was measured by absorbance at 570 nm. The 10 overlaid dots in each graph represent independent values from 2 SRB assays performed in 5 replicates. Error bars indicate SEM. P values compare mean absorbance between the shETV5 groups and the control group (*P < 0.0003), and between the shETV5-shDUX4 groups and the shETV5-control group (**P < 0.00001). (D and E) Fold growth of SYO-1 and HS-SY-II cells expressing lentiviral DUX4 over uninduced cells. Time points indicate days after DUX4 induction with 1 μg/mL doxycycline. The 20 overlaid dots in every graph represent values from 2 SRB assays performed in 10 replicates each. Error bars indicate SEM. P values compare mean growth on day 2 and day 3 against average growth on day 1. *P < 0.00001. (F) RT-PCR of gene targets in SYO-1 cells expressing inducible DUX4. Results are shown from 2 independent experiments (Exp. 1 and 2), comparing induced with uninduced cells. DX, doxycycline. Numbers represent ratios of band intensity in induced over uninduced cells. Measurements were adjusted using GAPDH signal as described in Statistics. PCR primers are described in Supplemental Methods. (G) Immunoblot shows proteins activated upon DUX4 induction (+DX) in SYO-1 and HS-SY-II cells. Actin is loading control. ANOVA F ratios and corresponding P values shown on top of the graphs in C–E demonstrate significant variation of means among the 5 and 3 groups, respectively.