An Enhancer-Driven Stem Cell-Like Program Mediated by SOX9 Blocks Intestinal Differentiation in Colorectal Cancer

Abstract

Background and aims: Genomic alterations that encourage stem cell activity and hinder proper maturation are central to the development of colorectal cancer (CRC). Key molecular mediators that promote these malignant properties require further elucidation to galvanize translational advances. We therefore aimed to characterize a key factor that blocks intestinal differentiation, define its transcriptional and epigenetic program, and provide preclinical evidence for therapeutic targeting in CRC.

Methods: Intestinal tissue from transgenic mice and patients were analyzed by means of histopathology and immunostaining. Human CRC cells and neoplastic murine organoids were genetically manipulated for functional studies. Gene expression profiling was obtained through RNA sequencing. Histone modifications and transcription factor binding were determined with the use of chromatin immunoprecipitation sequencing.

Results: We demonstrate that SRY-box transcription factor 9 (SOX9) promotes CRC by activating a stem cell-like program that hinders intestinal differentiation. Intestinal adenomas and colorectal adenocarcinomas from mouse models and patients demonstrate ectopic and elevated expression of SOX9. Functional experiments indicate a requirement for SOX9 in human CRC cell lines and engineered neoplastic organoids. Disrupting SOX9 activity impairs primary CRC tumor growth by inducing intestinal differentiation. By binding to genome wide enhancers, SOX9 directly activates genes associated with Paneth and stem cell activity, including prominin 1 (PROM1). SOX9 up-regulates PROM1 via a Wnt-responsive intronic enhancer. A pentaspan transmembrane protein, PROM1 uses its first intracellular domain to support stem cell signaling, at least in part through SOX9, reinforcing a PROM1-SOX9 positive feedback loop.

Conclusions: These studies establish SOX9 as a central regulator of an enhancer-driven stem cell-like program and carry important implications for developing therapeutics directed at overcoming differentiation defects in CRC.

Keywords: Colorectal Cancer; Differentiation Block; PROM1; SOX9.

Figure 1.

SOX9 is overexpressed in mouse models and human tissue of intestinal adenomas and colorectal cancer (CRC). (A) mRNA expression of Wnt target genes in Apc^WT and Apc^KO intestines according to quantitative reverse-transcription polymerase chain reaction (qRT-PCR); mean ± SD. (B) Sox9 mRNA expression in intestinal lesions from Apc shRNA mice ± K-ras mutation ± doxycycline (Dox); mean ± SD Student’s t test P values. (C) Representative histopathology of intestinal lesions (hematoxylin and eosin [H&E], td-RFP immunohistochemistry [IHC], Sox9 IHC) from Lgr5-Apc^f/f;td mice treated with tamoxifen 28 days before harvesting tissue; scale bar = 100 μm. (D) SOX9 IHC in human specimens. Immunostaining was quantified as H-score (left). Paired lesions were analyzed (right). AdCa, adenocarcinoma; HGD, high-grade dysplasia; LGD, low-grade dysplasia; NAT, normal adjacent tissue. (E, F) Representative histopathology (H&E, SOX9 IHC) of human adenomas on the same specimen slide; scale bars = 1 μm, 5 μm, and 20 μm. (G) Kaplan-Meier curves indicating disease-free survival of patients with CRC (left) and non–microsatellite instability (MSI) CRC (right) with indicated expression of SOX9.

Figure 2.

SOX9 is required for CRC proliferation and primary tumor growth. (A, B) Dependency for SOX9 CRISPR knockout (KO) plotted against SOX9 mRNA expression (left) and CTNNB1 CRISPR dependency (right); blue regression line; Pearson’s correlation P value. (C) SOX9 and β-actin expression in HT-115 control and SOX9 KD according to immunoblot. Adherent proliferation by CellTiterGlo. Area of ultra-low-attachment colonies; mean SD; Student’s t test P values. (D) Immunoblot (SOX9, vinculin) and colony formation of Dox-inducible HT-29 non-targeting control (NTC), short hairpin (sh) RNA#1 or shRNA#5 ± 0.5 μg/mL Dox. (E) Soft-agar colony formation assay of COLO-205 control and SOX9 shRNA; mean ± SD of 3 cell culture replicates and representative phase-contrast images; Student’s t test P values. (F) Primary xenograft tumor growth of HT-29 control or SOX9 shRNA; mean ± SD (n = 5); Student’s t test P value (*P < 0.05). (G) Proportions of wild-type (WT)/heterozygous (Het), Het, and homozygous SOX9 inactivation in single-cell clones in 3 CRC Cas9/SOX9 short guide (sg) RNAs. Proliferation by CellTiterGlo. (H) LS180 nucleofected with Cas9/SOX9 sgRNA complex, then amplicon sequenced to quantify in-frame and frameshift indels. Abbreviations as in Figure 1.

Figure 3.

SOX9 blocks intestinal differentiation in human CRC. (A) Sox9 and Krt20 IHC in normal mouse small intestines; scale bar = 100 μm. (B) Immunoblot (V5-tagged, KRT20 GAPDH) of indicated LS180 ± 0.5 μg/mL Dox. (C) SOX9, KRT20, MUC2, and CDX2 mRNA expression in indicated LS180 ± 0.5 μg/mL DOX according to RT-PCR; mean ± SD; Student’s t test: ***P < 0.005, ****P < 0.001. (D) Immunoblot (SOX9, KRT20, GAPDH) of indicated HT-115 ± 0.25 μg/μL Dox. (E) Immunoblot (E-cadherin, vimentin, GAPDH) in indicated COLO-205 Cas9/SOX9 sgRNA single-cell clones; phase-contrast images; scale bar = 100 μm. Abbreviations as in Figures 1 and 2.

Figure 4.

SOX9 knockdown promotes intestinal differentiation in organoid models of CRC. (A) mRNA expression heat map of intestinal differentiation (blue) and stem cell (red) markers in control and indicated SOX9 shRNA human colon organoids. (B) Immunoblot (Sox9 and vinculin) of mouse control or indicated Sox9 shRNA Apc^KO Kras^G12D colon organoids. Proliferation by CellTiterGlo; mean ± SD; Student’s t test: ***P < 0.005, ****P < 0.001. (C) Ki67 IHC quantification of indicated fixed mouse Apc^KO Kras^G12D colon organoids; mean ± SD; Student;s t test ***P < 0.005, ****P < 0.001. (D) Sox9, Lgr5, Lrig1, Prom1, Axin2, Ascl2, Krt20 mRNA expression in Apc^KO Kras^G12D colon organoids according to qRT-PCR; mean ± SD; Student’s t test: **P < 0.01, ***P < 0.005, ****P < 0.001. (E) Apc^KO Kras^G12D Sox9 xenograft schematic. Primary tumor xenograft growth curve and day 30 quantification. Representative images of xenograft tumors. (F) Immunoblot (Sox9 and vinculin) of xenograft tumors. (G) Ki67 and Muc2 immunohistochemistry and Alcian blue–periodic acid Schiff (AB-PAS) staining of xenograft tumors; scale bars = 100 μm and 20 μm. Abbreviations as in Figures 1 and 2.

Figure 5.

SOX9 activates an enhancer-driven stem and Paneth cell transcriptional program. (A) Gene expression heat map of indicated LS180 CRC ± Dox. Selected up-regulated (red) and down-regulated (blue) gene sets displayed. (B) Gene ontology (GO Biological Process) of SOX9-bound genes. False discovery rate (FDR) q-value after Bonferroni correction displayed. (C) SOX9-binding distribution among intergenic, intronic, promoter, and exonic regions. (D) Transcription factor–binding similarity (Giggle score) of publicly available vs our V5-SOX9 chromatin immunoprecipitation sequencing (ChIP-seq) data. (E) Integrative Genomics Viewer (IGV) gene track at indicated gene loci of H3K27ac (red) and V5-ChIP (blue) in indicated HT-115; signal track for TCF4 (purple) from published endogenous LS180 ChIP-seq data. GFP, green fluorescent protein; other abbreviations as in Figure 1.

Figure 6.

SOX9 directly activates PROM1 via a Wnt-responsive intronic enhancer. (A) Volcano plot displaying log2 fold change of differentially expressed genes (SOX/GFP) along x-axis (FDR <0.05). Blue points mark genes significantly associated with SOX9-binding or differential H3K27ac. (B) Prom1 mRNA expression in indicated Apc^KO Kras^G12D colon organoids according to qRT-PCR; mean ± SD; Student’s t test: ****P < 0.001. (C) PROM1 mRNA expression in human CRC with WT, Het, or homozygous (Homo) SOX9 mutations. TCGA, The Cancer Genome Atlas. (D) mRNA and protein expression of PROM1 in indicated cell lines ± 0.5 μg/mL Dox. (E) IGV gene track of PROM1 intron1; signal tracks of H3K27ac (red) and V5-ChIP (blue) in indicated conditions; peak scale displayed in top right. (F) PROM1 enhancer reporter assay: phase-contrast and fluorescence images and quantification of HEK293T transiently transfected with indicated plasmids and/or treated with WNT3A. Abbreviations as in Figures 1, 2, and 5.

Figure 7.

PROM1 blocks intestinal differentiation via its first intracellular domain. (A, B) Proliferation and colony formation of indicated control and PROM1 shRNA HT-115 and LS180 ± 0.25 μg/mL Dox; mean ± SD; Student’s t test: ***P < 0.005. (C) Primary tumor xenograft growth curve and weight of NTC or indicated PROM1-knockdown HT-29 cell line; Student’s t test P values. (D) Immunoblot (PROM1, KRT20, and GAPDH) in indicated HT115 cell lines ± 0.5 μg/mL Dox. (E) Immunoblot (KRT20 and vinculin) in indicated LS180 cell lines ± 0.5 μg/mL Dox. (F) Immunoblot (KRT20, PROM1, SOX9, and vinculin) in indicated control or LS180 overexpression cells. (G) Immunoblot (KRT20, PROM1, SOX9, GAPDH, and vinculin) in indicated HT-115 and LS180 cells. (H) Immunoblot (KRT20, PROM1, SOX9, AXIN2, GAPDH, and vinculin) in inducible PROM1 shRNA HT-115 and LS180 cells with indicated Dox treatment. (I) KRT20 mRNA and KRT20, SOX9, vinculin protein expression in NTC or PROM1 shRNA LS180 cells expressing GFP or WT-SOX9 ± 0.5 μg/μL Dox; mean ± SD; Student’s t test: ****P < 0.001. (J) KRT20 mRNA and protein expression in indicated LS180 overexpression. (K) Schematic summarizing the SOX9-PROM1 reinforcing feedback loop. Abbreviations as in Figures 1, 2, and 5.