Single-cell multi-omics reveals insights into differentiation of rare cell types in mucinous colorectal cancer.

Neuroendocrine cells have been implicated in therapeutic resistance and worse overall survival in many cancer types. Mucinous colorectal cancer (mCRC) is uniquely enriched for enteroendocrine cells (EECs), the neuroendocrine cell of the normal colon epithelium, as compared to non-mucinous CRC. Therefore, targeting EEC differentiation may have clinical value in mCRC. Here, single cell multi-omics was used to uncover epigenetic alterations that accompany EEC differentiation, identify STAT3 as a novel regulator of EEC specification, and discover a rare cancer-specific cell type with enteric neuron-like characteristics. Further experiments demonstrated that lysine-specific demethylase 1 (LSD1) and CoREST2 mediate STAT3 demethylation and regulate STAT3 chromatin binding. Knockdown of CoREST2 in an orthotopic xenograft mouse model resulted in decreased primary tumor growth and lung metastases. In culmination, these results provide rationale for new LSD1 inhibitors that target the interaction between LSD1 with STAT3 or CoREST2, which may improve clinical outcomes for patients with mCRC.

Single-cell profiling reveals the impact of genetic alterations on the differentiation of inflammation-induced colon tumors.

Genetic mutations and chronic inflammation of the colon contribute to the development of colorectal cancer (CRC). Using a murine model of inflammation-induced colon tumorigenesis, we determined how genetic mutations alter colon tumor cell differentiation. Inflammation induced by enterotoxigenic Bacteroides fragilis (ETBF) colonization of multiple intestinal neoplasia (Min ApcΔ716/+ ) mice triggers loss of heterozygosity of Apc causing colon tumor formation. Here, we report that the addition of BRAF V600E mutation ( BRAF FV600E Lgr5 tm1(Cre/ERT2)Cle Min ApcΔ716/+ , BLM) or knocking out Msh2 ( Msh2 LoxP/LoxP Vil1-cre Min ApcΔ716/+ , MSH2KO) in the Min model altered colon tumor differentiation. Using single cell RNA-sequencing, we uncovered the differences between BLM, Min, and MSH2KO tumors at a single cell resolution. BLM tumors showed an increase in differentiated tumor epithelial cell lineages and a reduction in the stem cell population. In contrast, MSH2KO tumors were characterized by an increased stem cell population that had higher WNT signaling activity compared to Min tumors. Additionally, comparative analysis of single-cell transcriptomics revealed that BLM tumors had higher expression of transcription factors that drive differentiation, such as Cdx2, than Min tumors. Using RNA velocity, we were able to identify additional potential regulators of BLM tumor differentiation such as NDRG1. The role of CDX2 and NDRG1 as putative regulators for BLM tumor cell differentiation was verified using organoids derived from BLM tumors. Our results demonstrate the critical connections between genetic mutations and cell differentiation in inflammation-induced colon tumorigenesis. Understanding such roles will deepen our understanding of inflammation-associated colon cancer.

Activation of AKT induces EZH2-mediated ß-catenin trimethylation in colorectal cancer.

Colorectal cancer (CRC) develops in part through the deregulation of different signaling pathways, including activation of the WNT/ß-catenin and PI3K/AKT pathways. Additionally, the lysine methyltransferase enhancer of zeste homologue 2 (EZH2) is commonly overexpressed in CRC. EZH2 canonically represses gene transcription by trimethylating lysine 27 of histone H3, but also has non-histone substrates. Here, we demonstrated that in CRC, active AKT phosphorylated EZH2 on serine 21. Phosphorylation of EZH2 by AKT induced EZH2 to interact with and methylate ß-catenin at lysine 49, which increased ß-catenin's binding to the chromatin. Additionally, EZH2-mediated ß-catenin trimethylation induced ß-catenin to interact with TCF1 and RNA polymerase II and resulted in dramatic gains in genomic regions with ß-catenin occupancy. EZH2 catalytic inhibition decreased stemness but increased migratory phenotypes of CRC cells with active AKT. Overall, we demonstrated that EZH2 modulates AKT-induced changes in gene expression through the AKT/EZH2/ß-catenin axis in CRC.

LSD1 and Aberrant DNA Methylation Mediate Persistence of Enteroendocrine Progenitors That Support BRAF-Mutant Colorectal Cancer.

Despite the connection of secretory cells, including goblet and enteroendocrine (EEC) cells, to distinct mucus-containing colorectal cancer histologic subtypes, their role in colorectal cancer progression has been underexplored. Here, our analysis of The Cancer Genome Atlas (TCGA) and single-cell RNA-sequencing data demonstrates that EEC progenitor cells are enriched in BRAF-mutant colorectal cancer patient tumors, cell lines, and patient-derived organoids. In BRAF-mutant colorectal cancer, EEC progenitors were blocked from differentiating further by DNA methylation and silencing of NEUROD1, a key gene required for differentiation of intermediate EECs. Mechanistically, secretory cells and the factors they secrete, such as trefoil factor 3, promoted colony formation and activation of cell survival pathways in the entire cell population. Lysine-specific demethylase 1 (LSD1) was identified as a critical regulator of secretory cell specification in vitro and in a colon orthotopic xenograft model, where LSD1 loss blocks formation of EEC progenitors and reduces tumor growth and metastasis. These findings reveal an important role for EEC progenitors in supporting colorectal cancer. SIGNIFICANCE: This study establishes enteroendocrine progenitors as a targetable population that promotes BRAF-mutant colorectal cancer and can be blocked by LSD1 inhibition to suppress tumor growth.

Dietary Oligosaccharides Attenuate Stress-Induced Disruptions in Immune Reactivity and Microbial B-Vitamin Metabolism.

Background: Exposure to stressful stimuli dysregulates inflammatory processes and alters the gut microbiota. Prebiotics, including long-chain fermentable fibers and milk oligosaccharides, have the potential to limit inflammation through modulation of the gut microbiota. To determine whether prebiotics attenuate stress-induced inflammation and microbiota perturbations, mice were fed either a control diet or a diet supplemented with galactooligosaccharides, polydextrose and sialyllactose (GOS+PDX+SL) or sialyllactose (SL) for 2 weeks prior to and during a 6-day exposure to a social disruption stressor. Spleens were collected for immunoreactivity assays. Colon contents were examined for stressor- and diet- induced changes in the gut microbiome and metabolome through 16S rRNA gene sequencing, shotgun metagenomic sequencing and UPLC-MS/MS. Results: Stress increased circulating IL-6 and enhanced splenocyte immunoreactivity to an ex vivo LPS challenge. Diets containing GOS+PDX+SL or SL alone attenuated these responses. Stress exposure resulted in large changes to the gut metabolome, including robust shifts in amino acids, peptides, nucleotides/nucleosides, tryptophan metabolites, and B vitamins. Multiple B vitamins were inversely associated with IL-6 and were augmented in mice fed either GOS+PDX+SL or SL diets. Stressed mice exhibited distinct microbial communities with lower abundances of Lactobacillus spp. and higher abundances of Bacteroides spp. Diet supplementation with GOS+PDX+SL, but not SL alone, orthogonally altered the microbiome and enhanced the growth of Bifidobacterium spp. Metagenome-assembled genomes (MAGs) from mice fed the GOS+PDX+SL diet unveiled genes in a Bifidobacterium MAG for de novo B vitamin synthesis. B vitamers directly attenuated the stressor-induced exacerbation of cytokine production in LPS-stimulated splenocytes. Conclusions: Overall, these data indicate that colonic metabolites, including B vitamins, are responsive to psychosocial stress. Dietary prebiotics reestablish colonic B vitamins and limit stress-induced inflammation.