Targeting the Wnt pathway in synovial sarcoma models

Abstract

Synovial sarcoma is an aggressive soft-tissue malignancy of children and young adults, with no effective systemic therapies. Its specific oncogene, SYT-SSX (SS18-SSX), drives sarcoma initiation and development. The exact mechanism of SYT-SSX oncogenic function remains unknown. In an SYT-SSX2 transgenic model, we show that a constitutive Wnt/β-catenin signal is aberrantly activated by SYT-SSX2, and inhibition of Wnt signaling through the genetic loss of β-catenin blocks synovial sarcoma tumor formation. In a combination of cell-based and synovial sarcoma tumor xenograft models, we show that inhibition of the Wnt cascade through coreceptor blockade and the use of small-molecule CK1α activators arrests synovial sarcoma tumor growth. We find that upregulation of the Wnt/β-catenin cascade by SYT-SSX2 correlates with its nuclear reprogramming function. These studies reveal the central role of Wnt/β-catenin signaling in SYT-SSX2-induced sarcoma genesis, and open new venues for the development of effective synovial sarcoma curative agents.

Significance: Synovial sarcoma is an aggressive soft-tissue cancer that afflicts children and young adults, and for which there is no effective treatment. The current studies provide critical insight into our understanding of the pathogenesis of SYT–SSX-dependent synovial sarcoma and pave the way for the development of effective therapeutic agents for the treatment of the disease in humans.

Figure 1. β-catenin knockout inhibits SS tumor growth in SYT-SSX2 transgenic mice

(A) Histogram represents total number of tumors in SG1 (SSM2⁺/Myf5-Cre⁺), SG2 (SSM2⁺/B-CAT^fl+/−/Myf5-Cre⁺), and SG3 (SSM2⁺/B-CAT^fl+/+/Myf5-Cre⁺) mice. (B) Dot plot tabulating number of tumors in the SG1, SG2, and SG3 mice, with median value bar. P values compare difference significance between each pair of the experimental groups. (C) Left column: hematoxylin and eosin (H&E) staining of tumors (black arrows: blue nuclei) in the intercostal muscle (pink fibers) of SG1 (upper row, SSM2⁺) and one of the three SG3 (lower row, SSM2⁺/B-CAT^fl+/+) mice that developed tumors. Middle column: control mouse IgG immunostaining. Right column: β-catenin immunostaining. Control IgG and β-catenin stainings were performed on serial tumor sections present on the same slide. Scale bars of all images are 99μm. (D) β-catenin immunofluorescent staining (red) in tumors from SG1 (SSM2⁺) and SG3 (SSM2⁺/B-CAT^fl+/+) mice. Mouse IgG served as negative control. DAPI (blue) stains nuclei. Merged blue and red images show β-catenin nuclear localization.

Figure 2. Integrity of the rib musculature in SYT-SSX2 transgenic mice

(A) H&E staining (left and middle images) of an intercostal SG2 tumor (black arrow, pink muscle fibers) near the rib cartilage (black arrowheads). Right image: β-catenin immunostaining in the SG2 tumor. Scale bars on middle and right images are 99μm. (B) H&E staining of the rib musculature. Upper row, left and middle images: SG2 tumor (black arrow, blue nuclei), infiltrating muscle fibers (pink) near the rib bone/cartilage. Upper right image: normal rib musculature in a tumor-free SG3 mouse. Lower row: normal rib musculature in a β-catenin-null knockout mouse (left image), a heterozygous β-catenin knockout mouse (middle image), and a SYT-SSX2/BCAT double mutant (right image). Black arrowheads denote cartilage/bone junction. Excepting the upper left image, all displayed scale bars are 99μm.

Figure 3. DKK1 attenuates β-catenin nuclear localization and signaling

(A) Immunostaining of C2C12 myoblasts transduced with empty retroviral vectors: POZ and LZRS, and those expressing SYT-SSX2 and DKK1. DKK1 (green) and β-catenin (red) were detected with a specific polyclonal and monoclonal antibody, respectively. Numbers represent average percent of SYT-SSX2/DKK1-espressing cells, exhibiting nuclear β-catenin, +/− standard deviations (n=3). 500 nuclei were counted for each vector. (B) TOP-FLASH activity in SYT-SSX2-expressing C2C12 cells transduced with incremental amounts of DKK1 cDNA. Histogram represents fold activation over Vector. Error bars are standard deviations (n=3). P values were calculated relative to SYT-SSX2.

Figure 4. DKK1 inhibits SS tumor growth in nude mice

(A) Histogram represents ratios of TOP-FLASH (T) activity in mSS cells transfected with increasing amounts of DKK1 cDNA, over empty vector. Errors bars are standard deviation (n=3) P values were calculated relative to empty vector (T, 0). FOP-FLASH (F) was baseline activity. (B) Dot plot comparing tumor volumes in naïve, LZRS vector-, and LZRS-DKK1-infected mSS cells, with median value bars. Dunn’s post-hoc test showed significant difference between DKK1 and Naïve or Vector SS tumors. Naïve and Vector were not significantly different. Inset: immunoblot showing stable DKK1 expression in mSS cells implanted in nude mice. Equivalent amounts (100 μg) of protein lysate were loaded in each lane. Doublet reflects DKK1post-translational modification. (C) H&E staining of Naïve, Vector, or DKK1 tumors. Arrows indicate hypocellular centers. Scale bars on all images are 99μm. (D) Ki-67 (green, white arrowhead) immunoflurescent staining in Vector and DKK1 tumors. DAPI staining: double arrowheads reveal paucity of nuclei in the hypocellular centers of DKK1 tumors. Right histogram compares Ki-67 expression (counted visually) in the Vector- and DKK1 tumors. Error bars indicate standard deviations (n=3). P value was calculated relative to Vector SS.

Figure 5. SSTC-104 and LRP6 depletion effects on β-catenin and SS cells

(A) Left histogram shows growth of SSTC-104-treated SYO-1 cells relative to Vehicle (DMSO) after a 2- or 4-day treatment. Right histogram shows percent of SSTC-104- and Vehicle-treated cells containing nuclear β-catenin. 700 nuclei were counted in each group. Error bars represent standard deviations (n=2). P values were calculated relative to Vehicle. (B) Immunoblot of de-phosphorylated β-catenin in SSTC-104-treated SYO-1 cells. Numbers represent ratios of signal intensities over control (V=vehicle). Tubulin served as loading control. (C) β-catenin immunofluorescent staining (green) in SSTC-104-treated cells. White arrows indicate large cytoplasmic extensions with multiple intercellular junctions. Right image (high magnification) showcases β-catenin at intercellular junctions. DAPI stains nuclei. β-catenin and DAPI images were merged for co-localization. (D) Immunoblot shows LRP6 levels in SYO-1 cells transfected with LRP6 non-targeting (NT) and targeting (Si-LRP6-1 and SI-LRP6-2) siRNAs. Numbers show relative intensities (NT is 1). Tubulin served as loading control. (E) Histogram shows percent of siRNA-transfected SYO-1 cells containing nuclear β-catenin. 500 nuclei were counted in each group. Error bars represent standard deviations (n=2). P values were calculated relative to NT. (F) β-catenin immunofluorescent staining (green) in SYO-1 cells transfected with NT, Si-LRP6-1 and Si-LRP6-2 oligomers. Merged (merge) β-catenin and DAPI images show nuclear (or lack of) β-catenin. White arrowheads in Si-LRP6-1 and Si-LRP6-2 (magnified) images show neurite-like cytoplasmic protrusions connecting cells over long distances at multiple points of contact.

Figure 6. SSTC-104 in vivo effects in SS tumors

(A) The three histograms represent tumor volumes on the 1^st, 4^th and 8^th day of SSTC-104 treatment. Vehicle is 15% DMSO in PBS solution. Error bars denote standard deviations. P values were calculated relative to Vehicle. (B) β-catenin immunostaining in Vehicle-treated (top row; increasing magnification) and SSTC-104-treated (middle and lower row) tumors. Double arrowheads: areas denuded of β-catenin. Single arrowheads: clear nuclei. (C) Immunofluorescent staining of β-catenin (Red) in Vehicle-treated tumors (upper row), and in the hypocellular centers of SSTC-104-treated tumors (lower row, white arrows). DAPI (not shown) and red images were merged (merge). Scale bar is 50μm (D) Histogram shows Ki-67 scores in SSTC-104- and Vehicle-treated tumors. 500 nuclei were counted in each group. Error bars represent standard deviations. P values show significance difference between each pair of groups. Lower image panel: immunofluorescent staining of Ki-67 (green) in SSTC-104- and Vehicle-treated tumors. DAPI stains nuclei. Scale bar is 50μm. (E) Upper histogram shows number of tumors detected in SSTC-104-treated Control and SSM2⁺/Myf5-Cre⁺ mice, and Vehicle-treated SSM2⁺/Myf5-Cre⁺ mice. P value was calculated relative to Vehicle. Error bars represent standard deviations. Lower histogram shows combined tumor volumes as tumor load, in SSM2⁺/Myf5-Cre⁺ mice treated with Vehicle or SSTC-104. Error bars represent standard deviations. P value was calculated relative to Vehicle.

Figure 7. β-catenin nuclear signaling is mediated by SYT-SSX2 N-terminal and C-terminal SSXRD

(A) Schematic of SYT-SSX2 and its deletion mutants. SXdl3 and SXdl9 lack residues 35–55 and 65–78 of the SSXRD, respectively. Lower panel: immunostaining of C2C12 myoblasts transduced with the designated retroviral vectors. POZ is empty vector. Expressed SYT-SSX2-derived proteins are HA (and FLAG)-tagged (green). POZ expresses an irrelevant small peptide that disappears with subcloning SYT-SSX2 and other cDNAs. β-catenin is visualized with a specific monoclonal antibody (red). The red and the green channels were merged (merge) for co-localization. Numbers represent average percent of HA-positive cells with nuclear β-catenin +/− standard deviation (n=3). 500 nuclei were counted for each vector. (B) Inset: FLAG immunoblot showing expression of SYT-SSX2 vectors. Histogram depicts fold increase of TOP-FLASH (T) activity with the SYT-SSX2-derived vectors relative to control vector (POZ). FOP-FLASH (F) was baseline activity; Error bars denote standard deviations; n=3. P values were calculated relative to SYT-SSX2.