Silencing SOX2 Induced Mesenchymal-Epithelial Transition and Its Expression Predicts Liver and Lymph Node Metastasis of CRC Patients

<div><p>SOX2 is an important stem cell marker and plays important roles in development and carcinogenesis. However, the role of SOX2 in Epithelial-Mesenchymal Transition has not been investigated. We demonstrated, for the first time, that SOX2 is involved in the Epithelial-Mesenchymal Transition (EMT) process as knock downof SOX2 in colorectal cancer (CRC) SW620 cells induced a Mesenchymal-Epithelial Transition (MET) process with recognized changes in the expression of key genes involved in the EMT process including E-cadherin and vimentin. In addition, we provided a link between SOX2 activity and the WNT pathway by showing that knock down of SOX2 reduced the WNT pathway activity in colorectal cancer (CRC) cells. We further demonstrated that SOX2 is involved in cell migration and invasion <em>in vitro</em> and in metastasis <em>in vivo</em> for CRC cells, and that the process might be mediated through the MMP2 activity. Finally, an IHC analysis of 44 cases of colorectal cancer patients suggested that SOX2 is a prognosis marker for metastasis of colorectal cancers.</p> </div

Targeted Re-Sequencing Identified rs3106189 at the 5′ UTR of TAPBP and rs1052918 at the 3′ UTR of TCF3 to Be Associated with the Overall Survival of Colorectal Cancer Patients

<div><p>Recent studies have demonstrated the power of deep re-sequencing of the whole genome or exome in understanding cancer genomes. However, targeted capture of selected genomic whole gene-body regions, rather than the whole exome, have several advantages: 1) the genes can be selected based on biology or a hypothesis; 2) mutations in promoter and intronic regions, which have important regulatory roles, can be investigated; and 3) less expensive than whole genome or whole exome sequencing. Therefore, we designed custom high-density oligonucleotide microarrays (NimbleGen Inc.) to capture approximately 1.7 Mb target regions comprising the genomic regions of 28 genes related to colorectal cancer including genes belonging to the WNT signaling pathway, as well as important transcription factors or colon-specific genes that are over expressed in colorectal cancer (CRC). The 1.7 Mb targeted regions were sequenced with a coverage ranged from 32× to 45× for the 28 genes. We identified a total of 2342 sequence variations in the CRC and corresponding adjacent normal tissues. Among them, 738 were novel sequence variations based on comparisons with the SNP database (dbSNP135). We validated 56 of 66 SNPs in a separate cohort of 30 CRC tissues using Sequenom MassARRAY iPLEX Platform, suggesting a validation rate of at least 85% (56/66). We found 15 missense mutations among the exonic variations, 21 synonymous SNPs that were predicted to change the exonic splicing motifs, 31 UTR SNPs that were predicted to occur at the transcription factor binding sites, 20 intronic SNPs located near the splicing sites, 43 SNPs in conserved transcription factor binding sites and 32 in CpG islands. Finally, we determined that rs3106189, localized to the 5′ UTR of antigen presenting tapasin binding protein (TAPBP), and rs1052918, localized to the 3′ UTR of transcription factor 3 (TCF3), were associated with overall survival of CRC patients.</p></div

The expression of key genes related to the EMT process and the cell morphology changed in response to SOX2 knock down.

<p>(A) A microarray analysis of the expression changes of genes related to epithelial and mesenchymal cells between SOX2 knock down and mock knock down SW620 colorectal cancer cells. (B) Quantitative real time PCR validation of expression changes in (A). GAPDH was used for normalization. The data are reported as mean ± SEM. (C) Western blot analysis of the expression of SOX2 in SW620-mock and SW620-shRNA-SOX2 cells. (D) Cell morphology changes between SW620-mock cells and SW620-shRNA-SOX2 cells. The SW620-mock cells maintained a spindle and fibroblastoid morphology while the SW620-shRNA-SOX2 cell showed an epithelioid spreading morphology. Magnifications: the top panel, 100×; the middle panel, 200×; the bottom panel: 400×.</p

SOX2 is involved in the WNT signaling pathway in colorectal cancer cells.

<p>(A) Immunofluorescence images of SW620-mock and SW620-shRNA-SOX2 cells stained with anti-beta-catenin antibody. Secondary antibody was conjugated with CY3. Cell nuclei were stained with Hoechest 33258. The top 6 panels are in 100× magnification and the lower 6 panels are in 400× magnification. (B) Western blot analysis of beta-catenin expression using cytoplasmic and nuclear fractions of SW620-mock and SW620-shRNA-SOX2 cells. Alpha-tubulin and Lamin B were used as loading controls for the cytoplasmic and nuclear fractions respectively. The right panel showed the band intensities (left panel) quantified by the Quantity One software (Bio-Rad). (C) LEF1/TCF1-luciferase activity in SW620-mock and SW620-shRNA-SOX2 cells. (D) Quantitative real time PCR (upper panel) and western blot (lower panel) analyses of LEF1 in SW620-mock and SW620-shRNA-SOX2 cells. GAPDH was used as a loading control. (E) Quantitative real time PCR analysis of c-Myc gene in SW620-mock and SW620-shRNA-SOX2 cells. (F) Quantitative real time PCR analysis of cyclin D1 expression in SW620-mock and SW620-shRNA-SOX2 cells.</p

SOX2 is involved in cell migration and invasion in colorectal cancer cells.

<p>(A) Left panel, photographs of SW620-shRNA-SOX2 and SW620-mock cells using the Transwell migration assays; right panel, quantification data (mean ± SD) of the cells that migrated to the lower chambers. (B) Photographs (left panel) and the quantification results (mean ± SD) (right panel) of the cells that migrated to the lower chambers for SW620-shRNA-SOX2 and SW620-mock cells using the Transwell cell invasion assays. (C) Gelatin zymography assay for gelatinases (matrix metalloproteinases MMP2 and MMP9) in SW620-shRNA-SOX2 and SW620-mock cells.</p

SOX2 knock down reduced <i>in vivo</i> metastasis of colorectal cancer cells in mice.

<p>(A) HE staining of mice lung tissue containing metastasis. (B) HE staining of mice liver tissue containing metastasis. (C) HE staining of mice lung tissue without metastasis. (D) HE staining of mice liver tissue without metastasis. (E) SOX2 staining of the lung metastasis with the arrow indicating strong staining of SOX2 in the invasive frontal regions. (F). SOX2 staining of the mouse lung tissue without metastasis. All images are in 100×.</p

Immunohistochemical (IHC) staining of SOX2 in CRC tissues.

<p>(A–D) IHC images of SOX2-positve colorectal cancer tissues. Arrows indicate patches of strong SOX2 staining could indicate active tumor invasion fronts of the tumor capsules. (E–F) IHC images of SOX2-negative colorectal cancer tissues. All images are in 100×. (G) Statistical analysis of the relationship of SOX2 IHC staining with tumor stages, lymph node or liver metastases.</p

Silencing SOX2 induced a MET process in colorectal cancer cells.

<p>(A) Western blot analysis of expression of E-cadherin and vimentin in SW620-mock and SW620-shRNA-SOX2 cells. GAPDH was used as a loading control. The right panel showed the right panel quantified by the Quantity One software (Bio-Rad). GAPDH was used for normalization. (B) Immunofluorescence images of SW620-mock and SW620-shRNA-SOX2 cells stained with antibodies against E-cadherin, vimentin and SOX2. Secondary antibody was conjugated with FITC and Cy3. Cell nuclei were stained with Hoechest 33258. (C) Semi-quantitative RT-PCR analysis of EMT-related regulatory factors. GAPDH was used as a loading control.</p

Colorectal cancer overall survival in relationship to SNPs.

<p>(A) Kaplan-Meier plot for rs3106189 localized to the 5′ UTR of TAPBP. (B) Kaplan-Meier plot for rs1052918 localized to the 3′ UTR of the TCF3. Y-axis, CRC survival probability; X-axis, months from surgery. Blue lines are homozygous wildtype (wild), green are homozygous variant (var), red are heterozygous variant (het).</p

List of synonymous SNPs with ESE/ESS motifs changed.

a<p>Tissue samples with SNP detected by NGS. CRC is the colorectal cancer tissue, and CRN is the colorectal cancer adjacent normal tissue.</p>b<p>“+”indicates “validated” and “−” indicated “not tested” by Sequenom.</p