SOX9 suppresses colon cancer via inhibiting epithelial-mesenchymal transition and SOX2 induction

The Wnt/β-catenin pathway regulates expression of the SOX9 gene, which encodes SRY-box transcription factor 9, a differentiation factor and potential β-catenin regulator. Because APC tumor suppressor defects in ~80% of colorectal cancers (CRCs) activate the Wnt/β-catenin pathway, we studied SOX9 inactivation in CRC biology. Compared to effects of Apc inactivation in mouse colon tumors, combined Apc and Sox9 inactivation instigated more invasive tumors with epithelial-mesenchymal transition (EMT) and SOX2 stem cell factor upregulation. In an independent mouse CRC model with combined Apc, Kras, and Trp53 defects, Sox9 inactivation promoted SOX2 induction and distant metastases. About 20% of 171 human CRCs showed loss of SOX9 protein expression, which correlated with higher tumor grade. In an independent group of 376 CRC patients, low SOX9 gene expression was linked to poor survival, earlier age at diagnosis, and increased lymph node involvement. SOX9 expression reductions in human CRC were linked to promoter methylation. EMT pathway gene expression changes were prominent in human CRCs with low SOX9 expression and in a mouse cancer model with high SOX2 expression. Our results indicate SOX9 has tumor suppressor function in CRC; its loss may promote progression, invasion, and poor prognosis by enhancing EMT and stem cell phenotypes. To identify genes regulated by SOX9, we investigated gene expression profiles in mouse colon tissues and organoids using RNA sequencing (RNA-seq). The mouse colon tissues were obtained from five different groups of mice, each with specific genotypes in the Sox9 and Apc genes: 1) control mice: these are littermates without Cre or Apc/Sox9 floxed alleles; 2) CDX2P-CreERT2 Sox9flox/flox (abbreviated as S) mice; 3) CDX2P-CreERT2 Apcflox/flox (abbreviated as A) mice; 4) CDX2P-CreERT2 Apcflox/flox Sox9flox/+ (abbreviated as AShet) mice; 5) CDX2P-CreERT2 Apcflox/flox Sox9flox/flox (abbreviated as AS) mice. Each group consisted of six replicate mice except AShet group (which has 7 replicates), resulting in a total of 31 mice being analyzed in the study. The mice in each group were treated with tamoxifen (TAM) to induce deletions in the Apc and Sox9 genes in the colon. Total RNAs were collected from proximal colon tissues of these mice after 30-40 days post TAM treatment for RNA-seq analysis. Paired comparisons of gene expression profiles (e.g., AS versus A, AS versus AShet, and S versus control) were performed using the RNA-seq data obtained from these RNA samples. We also performed gene expression profiling analysis using RNA-seq data for organoids derived from the colon of A mice or AS mice that have been treated with TAM to induce gene inactivation in Apc and/or Sox9. The study aimed to compare gene expression between the four organoids derived from four TAM-treated AS mice and the four control organoids from four TAM-treated A mice.