SOX10, a novel HMG-box-containing tumor suppressor, inhibits growth and metastasis of digestive cancers by suppressing the Wnt/β-catenin pathway.

SOX10 was identified as a methylated gene in our previous cancer methylome study. Here we further analyzed its epigenetic inactivation, biological functions and related cell signaling in digestive cancers (colorectal, gastric and esophageal cancers) in detail. SOX10 expression was decreased in multiple digestive cancer cell lines as well as primary tumors due to its promoter methylation. Pharmacologic or genetic demethylation reversed SOX10 silencing. Ectopic expression of SOX10in SOX10-deficient cancer cells inhibits their proliferation, tumorigenicity, and metastatic potentials in vitro and in vivo. SOX10 also suppressed the epithelial to mesenchymal transition (EMT) and stemness properties of digestive tumor cells. Mechanistically, SOX10 competes with TCF4 to bind β-catenin and transrepresses its downstream target genes via its own DNA-binding property. SOX10 mutations that disrupt the SOX10-β-catenin interaction partially prevented tumor suppression. SOX10is thus a commonly inactivated tumor suppressor that antagonizes Wnt/β-catenin signaling in cancer cells from different digestive tissues.

virus-containing pellet was dissolved in DMEM, divided into aliquots, and stored at −80°C.
The infectious titer was determined by counting the blue-stained colonies after crystal violet staining in 293T cells.

Semiquantitative reverse transcription-PCR and qRT-PCR
Total RNA was isolated with the NucleoSpin RNA II Kit (MACHEREY-NAGEL, Düren, Germany) with genomic DNA removed by RNase-Free DNase included. Reverse transcription using random hexamer and reverse transcription-PCR using Go-Taq (Promega, Madison, WI) were done as previously with GAPDH as an endogenous control. Reverse transcription-PCR was done for 28 or 32 cycles for SOX10, but only 23 cycles for GAPDH.
Quantitative RT-PCR was performed with ABI 7500 system (Applied Biosystems, Darmstadt, Germany). Relative expression of genes was calculated and expressed as 2 -△△ CT. The primers used in this study are listed in the Supplementary information, Table S1.

DNA Bisulfite treatment and methylation analysis
DNA bisulfite treatment, MSP, and BGS were carried out as described [1,2]. MSP was done for 40 cycles using AmpliTaq Gold and hot start. MSP primers were tested previously for not amplifying any unbisulfited DNA and the specificity of MSP was further confirmed by direct sequencing of some PCR products. The PCR products were subcloned into the pGEM-Teasy vector (Promega), with 6 to 8 colonies randomly chosen and sequenced. The primers used in this study are listed in the Supplementary information, Table S1.

5-Aza-2'-deoxycytidine and Trichostatin A Treatment
Tumor cells were allowed to grow overnight. The culture medium was then replaced with fresh medium containing 5-aza-2′-deoxycytidine (Aza) at a final concentration of 5 to 10 μmol/L (Sigma-Aldrich).Cells were allowed to grow for 72 hours with change of Aza-containing medium every 24 hours; some were further treated with the HDAC inhibitor trichostatin A (TSA) for an additional 24 hours. Cells were then harvested for DNA and RNA extractions.

Tissue Samples
For IHC, tissue samples as well as corresponding adjacent non-tumor tissues were obtained from Chinese PLA General Hospital and Luoyang 150 Hospital. The use of all the human samples and the experimental procedures for this study were reviewed and approved by the hospital ethics committees. Informed consent was obtained from all subjects included.
The specimens were snap-frozen in liquid nitrogen and stored at -80°C for molecular analyses.
The remaining tissue specimens were fixed in 10% formalin and embedded in paraffin for routine histologic examination. All the paraffin-embedded tissue specimens were diagnosed and reconfirmed by two experienced pathologists.

Immunohistochemistry and Scoring
The expressions of SOX10 protein in the specimens were detected by Dako REAL™ EnVision™ Detection System (DAKO) as described previously [3]. Briefly, sections were cut at 4μm and dried at 60 • C. The sections were deparaffinized in xylene, hydrated through graded alcohols and rinsed in distilled water. Heat-induced epitope retrieval was performed in a pressure cooker in 10 mM citrate buffer (pH 6.0) for 5 min. Sections were cooled for 30 min and then were rinsed in PBS. Endogenous peroxidase activity was blocked by hydrogen peroxide treatment. The antibody against SOX10 was diluted at 1:100 and incubated overnight at 4°C. Secondary detection was accomplished using horseradish peroxidase-labelled secondary anti-mouse antibody and staining was visualized using the peroxide substrate solution diaminobenzidine followed by light nuclear counterstaining with hematoxylin. For antibody control, one set of samples was incubated with non-immune mouse IgG (1:100) instead of primary antibody as negative control and four melanoma tissue specimens as positive control.
Evaluation of SOX10 staining was independently performed by two experienced pathologists. The intensity of SOX10 immunostaining was semiquantitatively estimated according to the signal intensity and distribution. Briefly, a mean percentage of positive tumor cells was determined in at least five areas ×400 magnification and assigned to one of the five following categories: 0, <5%; 1, 5-25%; 2, 25-50%; 3, 50-75% and 4, >75%. The intensity of immunostaining was scored as follows: 1, weak; 2, moderate and 3, intense. The percentage of positive tumor cells and the staining intensity were multiplied to produce a weighted score for each case. Tissues with immunohistochemical scoring ≤2 were considered as negative, 3-12 as positive.

Western blot
Total cell lysate was prepared in 1×SDS lysis buffer (62.5 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue). Proteins at the same amount were separated by 10% SDS-PAGE and transferred onto PVDF membranes. After probing with antibodies, the signals were visualized by Immobilon TM Western Chemiluminescence HRP Substrate (Millipore Corporation).

Colony formation assay and anchorage-independent growth assay
For colony formation assay, cells were cultured overnight in a 10 cm plate (1.0 × 10 4 per well) and infected with LV-GFP or LV-SOX10. After 14 days, surviving colonies were stained with gentian violet after methanol fixation and visible colonies (≥50 cells) were counted.
For anchorage-independent growth assay, the cells in single-cell suspension were plated in 0.3% agarose over a 0.7% agarose bottom layer at a density of 1.0 × 10 4 cells per well in 6-well plates and incubated for 14 days and stained. Colonies with a diameter greater than 100 μm were counted. Each experiment was done in triplicate and repeated for three times.

Wound-healing assay
For wound-healing assay, the cells were first seeded in 6-well culture plates. A wound was made in the confluent monolayer with a plastic pipette tip and the migration of the cells at the wound front was photographed using an inverted microscope at indicated times after the scratch.

Transwell migration and invasion assay
Transwell migration assays were quantified in vitro using Transwell chambers with polycarbonate membrane filters (8 μm pore size; Corning) according to the manufacturer's instructions. In brief, the lower chamber was filled with 0.6 ml medium containing 20% fetal bovine serum, and 0.2ml of medium that contained 1×10 5 cells under serum-starving conditions was plated in the upper chamber and incubated at 37°C. Then cells that had not migrated were removed from the upper face of filters using cotton swabs. The cells that migrated through the membrane and attached to the bottom of the membrane were fixed and stained with crystal violet. Images of five random fields were captured from each membrane and the number of migratory cells was counted, and the extent of migration was expressed as the average number of cells per microscopic field at a magnification of 100. All experiments were performed for three times. Similar inserts coated with Matrigel (BD Biosciences) were used to determine invasive potential in invasion assay. Two independent investigators were blinded when reading the migration and invasion assays.

Dual-Luciferase reporter assay
Cells were transiently transfected with luciferase reporters as indicated and Renilla vector