Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the United States. Genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with increased risk for CRC. A molecular understanding of the functional consequences of this genetic variation has been complicated because each GWAS SNP is a surrogate for hundreds of other SNPs, most of which are located in non-coding regions. Here we use genomic and epigenomic information to test the hypothesis that the GWAS SNPs and/or correlated SNPs are in elements that regulate gene expression, and identify 23 promoters and 28 enhancers. Using gene expression data from normal and tumour cells, we identify 66 putative target genes of the risk-associated enhancers (10 of which were also identified by promoter SNPs). Employing CRISPR nucleases, we delete one risk-associated enhancer and identify genes showing altered expression. We suggest that similar studies be performed to characterize all CRC risk-associated enhancers.

Intravital microscopy is a powerful technique to observe dynamic processes with single-cell resolution in live animals. No intravital window has been developed for imaging the colon due to its anatomic location and motility, although the colon is a key organ where the majority of microbiota reside and common diseases such as inflammatory bowel disease, functional gastrointestinal disorders, and colon cancer occur. Here we describe an intravital murine colonic window with a stabilizing ferromagnetic scaffold for chronic imaging, minimizing motion artifacts while maximizing long-term survival by preventing colonic obstruction. Using this setup, we image fluorescently-labeled stem cells, bacteria, and immune cells in live animal colons. Furthermore, we image nerve activity via calcium imaging in real time to demonstrate that electrical sacral nerve stimulation can activate colonic enteric neurons. The simple implantable apparatus enables visualization of live processes in the colon, which will open the window to a broad range of studies.

Cancers of the same tissue-type but in anatomically distinct locations exhibit different molecular dependencies for tumorigenesis. Proximal and distal colon cancers exemplify such characteristics, with BRAFV600E predominantly occurring in proximal colon cancers along with increased DNA methylation phenotype. Using mouse colon organoids, here we show that proximal and distal colon stem cells have distinct transcriptional programs that regulate stemness and differentiation. We identify that the homeobox transcription factor, CDX2, which is silenced by DNA methylation in proximal colon cancers, is a key mediator of the differential transcriptional programs. Cdx2-mediated proximal colon-specific transcriptional program concurrently is tumor suppressive, and Cdx2 loss sufficiently creates permissive state for BRAFV600E-driven transformation. Human proximal colon cancers with CDX2 downregulation showed similar transcriptional program as in mouse proximal organoids with Cdx2 loss. Developmental transcription factors, such as CDX2, are thus critical in maintaining tissue-location specific transcriptional programs that create tissue-type origin specific dependencies for tumor development.

Colon cancers are composed of phenotypically heterogeneous tumor cell subpopulations with variable expression of putative stem cell and differentiation antigens. While in normal colonic mucosa, clonal repopulation occurs along differentiation gradients from crypt base toward crypt apex, the clonal architecture of colon cancer and the relevance of tumor cell subpopulations for clonal outgrowth are poorly understood. Using a multicolor lineage tracing approach in colon cancer xenografts that reflect primary colon cancer architecture, we here demonstrate that clonal outgrowth is mainly driven by tumor cells located at the leading tumor edge with clonal axis formation toward the tumor center. While our findings are compatible with lineage outgrowth in a cancer stem cell model, they suggest that in colorectal cancer tumor cell position may be more important for clonal outgrowth than tumor cell phenotype.

Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression.

The spindle assembly checkpoint (SAC) acts as a molecular safeguard in ensuring faithful chromosome transmission during mitosis, which is regulated by a complex interplay between phosphatases and kinases including PLK1. Adenomatous polyposis coli (APC) germline mutations cause aneuploidy and are responsible for familial adenomatous polyposis (FAP). Here we study the role of PLK1 in colon cancer cells with chromosomal instability promoted by APC truncation (APC-ΔC). The expression of APC-ΔC in colon cells reduces the accumulation of mitotic cells upon PLK1 inhibition, accelerates mitotic exit and increases the survival of cells with enhanced chromosomal abnormalities. The inhibition of PLK1 in mitotic, APC-∆C-expressing cells reduces the kinetochore levels of Aurora B and hampers the recruitment of SAC component suggesting a compromised mitotic checkpoint. Furthermore, Plk1 inhibition (RNAi, pharmacological compounds) promotes the development of adenomatous polyps in two independent Apc Min/+ mouse models. High PLK1 expression increases the survival of colon cancer patients expressing a truncated APC significantly.

Previous reports have suggested that some probiotics inhibit tumorigenesis and cancer progression. However, the molecules involved have not yet been identified. Here, we show that the culture supernatant of Lactobacillus casei ATCC334 has a strong tumour-suppressive effect on colon cancer cells. Using mass spectrometry, we identify ferrichrome as a tumour-suppressive molecule produced by L. casei ATCC334. The tumour-suppressive effect of ferrichrome is greater than that of cisplatin and 5-fluorouracil, and ferrichrome has less of an effect on non-cancerous intestinal cells than either of those agents. A transcriptome analysis reveals that ferrichrome treatment induces apoptosis, which is mediated by the activation of c-jun N-terminal kinase (JNK). Western blotting indicates that the induction of apoptosis by ferrichrome is reduced by the inhibition of the JNK signalling pathway. This we demonstrate that probiotic-derived ferrichrome exerts a tumour-suppressive effect via the JNK signalling pathway.

Intestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.

Wilms tumor gene on the X chromosome (WTX) is a putative tumor suppressor gene in Wilms tumor, but its expression and functions in other tumors are unclear. Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in women and the second leading cause in men in the United States. We demonstrated that WTX frequently lost in CRC which was highly correlated with cell proliferation, tumor invasion and metastasis. Mechanistically, WTX loss disrupts the interaction between RhoGDIα and CDC42 by losing of the binding with RhoGDIα and triggers the activation of CDC42 and its downstream cascades, which promotes CRC development and liver metastasis. The aberrant upregulation of miR-20a/miR-106a were identified as the reason of WTX loss in CRC both in vivo and in vitro. These study defined the mechanism how miR-20a/miR-106a-mediated WTX loss regulates CRC progression and metastasis, and provided a potential therapeutic target for preventing CRC progression.

The colonic epithelial turnover is driven by crypt-base stem cells that express the R-spondin receptor Lgr5. Signals that regulate epithelial regeneration upon stem cell injury are largely unknown. Here, we explore the dynamics of Wnt signaling in the colon. We identify two populations of cells with active Wnt signaling: highly proliferative Lgr5+/Axin2+ cells, as well as secretory Lgr5-/Axin2+ cells. Upon Lgr5+ cell depletion, these cells are recruited to contribute to crypt regeneration. Chemical injury induced by DSS leads to a loss of both Lgr5+ cells and Axin2+ cells and epithelial regeneration is driven by Axin2- cells, including differentiated Krt20+ surface enterocytes. Regeneration requires stromal Rspo3, which is present at increased levels upon injury and reprograms Lgr5- but Lgr4+ differentiated cells. In contrast, depletion of stromal Rspo3 impairs crypt regeneration, even upon mild injury. We demonstrate that Rspo3 is essential for epithelial repair via induction of Wnt signaling in differentiated cells.

The gastrointestinal tract is a challenging anatomical target for diagnostic and therapeutic procedures for bleeding, polyps and cancerous growths. Advanced endoscopes that combine imaging and therapies within the gastrointestinal tract provide an advantage over stand-alone diagnostic or therapeutic devices. However, current multimodal endoscopes lack the spatial resolution necessary to detect and treat small cancers and other abnormalities. Here we present a multifunctional endoscope-based interventional system that integrates transparent bioelectronics with theranostic nanoparticles, which are photoactivated within highly localized space near tumours or benign growths. These advanced electronics and nanoparticles collectively enable optical fluorescence-based mapping, electrical impedance and pH sensing, contact/temperature monitoring, radio frequency ablation and localized photo/chemotherapy, as the basis of a closed-loop solution for colon cancer treatment. In vitro, ex vivo and in vivo experiments highlight the utility of this technology for accurate detection, delineation and rapid targeted therapy of colon cancer or precancerous lesions.

Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.

The physiological homeostasis of gut mucosal barrier is maintained by both genetic and environmental factors and its impairment leads to pathogenesis such as inflammatory bowel disease. A cytokine like molecule, FAM3D (mouse Fam3D), is highly expressed in mouse gastrointestinal tract. Here, we demonstrate that deficiency in Fam3D is associated with impaired integrity of colonic mucosa, increased epithelial hyper-proliferation, reduced anti-microbial peptide production and increased sensitivity to chemically induced colitis associated with high incidence of cancer. Pretreatment of Fam3D-/- mice with antibiotics significantly reduces the severity of chemically induced colitis and wild type (WT) mice co-housed with Fam3D-/- mice phenocopy Fam3D-deficiency showing increased sensitivity to colitis and skewed composition of fecal microbiota. An initial equilibrium of microbiota in cohoused WT and Fam3D-/- mice is followed by an increasing divergence of the bacterial composition after separation. These results demonstrate the essential role of Fam3D in colon homeostasis, protection against inflammation associated cancer and normal microbiota composition.

SARI, also called as BATF2, belongs to the BATF family and has been implicated in cancer cell growth inhibition. However, the role and mechanism of SARI in tumour angiogenesis are elusive. Here we demonstrate that SARI deficiency facilitates AOM/DSS-induced colonic tumorigenesis in mice. We show that SARI is a novel inhibitor of colon tumour growth and angiogenesis in mice. Antibody array and HUVEC-related assays indicate that VEGF has an essential role in SARI-controlled inhibition of angiogenesis. Furthermore, Co-IP/PAGE/mass spectrometry indicates that SARI directly targets ceruloplasmin (Cp), and induces protease degradation of Cp, thereby inhibiting the activity of the HIF-1α/VEGF axis. Tissue microarray results indicate that SARI expression inversely correlates with poor clinical outcomes in colon cancer patients. Collectively, our results indicate that SARI is a potential target for therapy by inhibiting angiogenesis through the reduction of VEGF expression and is a prognostic indicator for patients with colon cancer.

Enteric nervous system neuropathy causes a wide range of severe gut motility disorders. Cell replacement of lost neurons using enteric neural stem cells (ENSC) is a possible therapy for these life-limiting disorders. Here we show rescue of gut motility after ENSC transplantation in a mouse model of human enteric neuropathy, the neuronal nitric oxide synthase (nNOS-/-) deficient mouse model, which displays slow transit in the colon. We further show that transplantation of ENSC into the colon rescues impaired colonic motility with formation of extensive networks of transplanted cells, including the development of nNOS+ neurons and subsequent restoration of nitrergic responses. Moreover, post-transplantation non-cell-autonomous mechanisms restore the numbers of interstitial cells of Cajal that are reduced in the nNOS-/- colon. These results provide the first direct evidence that ENSC transplantation can modulate the enteric neuromuscular syncytium to restore function, at the organ level, in a dysmotile gastrointestinal disease model.

Uncontrolled canonical Wnt signalling supports colon epithelial tumour expansion and malignant transformation. Understanding the regulatory mechanisms involved is crucial for elucidating the pathogenesis of and will provide new therapeutic targets for colon cancer. Epsins are ubiquitin-binding adaptor proteins upregulated in several human cancers; however, the involvement of epsins in colon cancer is unknown. Here we show that loss of intestinal epithelial epsins protects against colon cancer by significantly reducing the stability of the crucial Wnt signalling effector, dishevelled (Dvl2), and impairing Wnt signalling. Consistently, epsins and Dvl2 are correspondingly upregulated in colon cancer. Mechanistically, epsin binds Dvl2 via its epsin N-terminal homology domain and ubiquitin-interacting motifs and prohibits Dvl2 polyubiquitination and degradation. Our findings reveal an unconventional role for epsins in stabilizing Dvl2 and potentiating Wnt signalling in colon cancer cells to ensure robust colon cancer progression. The pro-carcinogenic role of Epsins suggests that they are potential therapeutic targets to combat colon cancer.

A role for Hedgehog (Hh) signalling in the development of colorectal cancer (CRC) has been proposed. In CRC and other solid tumours, Hh ligands are upregulated; however, a specific Hh antagonist provided no benefit in a clinical trial. Here we use Hh reporter mice to show that downstream Hh activity is unexpectedly diminished in a mouse model of colitis-associated colon cancer, and that downstream Hh signalling is restricted to the stroma. Functionally, stroma-specific Hh activation in mice markedly reduces the tumour load and blocks progression of advanced neoplasms, partly via the modulation of BMP signalling and restriction of the colonic stem cell signature. By contrast, attenuated Hh signalling accelerates colonic tumourigenesis. In human CRC, downstream Hh activity is similarly reduced and canonical Hh signalling remains predominantly paracrine. Our results suggest that diminished downstream Hh signalling enhances CRC development, and that stromal Hh activation can act as a colonic tumour suppressor.

Our recent work has shown that DCAF1 (also known as VprBP) is overexpressed in colon cancer and phosphorylates histone H2AT120 to drive epigenetic gene inactivation and oncogenic transformation. We have extended these observations by investigating whether DCAF1 also phosphorylates non-histone proteins as an additional mechanism linking its kinase activity to colon cancer development. We now demonstrate that DCAF1 phosphorylates EZH2 at T367 to augment its nuclear stabilization and enzymatic activity in colon cancer cells. Consistent with this mechanistic role, DCAF1-mediated EZH2 phosphorylation leads to elevated levels of H3K27me3 and altered expression of growth regulatory genes in cancer cells. Furthermore, our preclinical studies using organoid and xenograft models revealed that EZH2 requires phosphorylation for its oncogenic function, which may have therapeutic implications for gene reactivation in colon cancer cells. Together, our data define a mechanism underlying DCAF1-driven colonic tumorigenesis by linking DCAF1-mediated EZH2 phosphorylation to EZH2 stability that is crucial for establishing H3K27me3 and gene silencing program.

Gut homeostasis and mucosal immune defense rely on the differential contributions of dendritic cells (DC) and macrophages. Here we show that colonic CX3CR1(+) mononuclear phagocytes are critical inducers of the innate response to Citrobacter rodentium infection. Specifically, the absence of IL-23 expression in macrophages or CD11b(+) DC results in the impairment of IL-22 production and in acute lethality. Highlighting immunopathology as a death cause, infected animals are rescued by the neutralization of IL-12 or IFNγ. Moreover, mice are also protected when the CD103(+) CD11b(-) DC compartment is rendered deficient for IL-12 production. We show that IL-12 production by colonic CD103(+) CD11b(-) DC is repressed by IL-23. Collectively, in addition to its role in inducing IL-22 production, macrophage-derived or CD103(-) CD11b(+) DC-derived IL-23 is required to negatively control the otherwise deleterious production of IL-12 by CD103(+) CD11b(-) DC. Impairment of this critical mononuclear phagocyte crosstalk results in the generation of IFNγ-producing former TH17 cells and fatal immunopathology.

Copper levels are known to be elevated in inflamed and malignant tissues. But the mechanism underlying this selective enrichment has been elusive. In this study, we report a axis by which inflammatory cytokines, such as IL-17, drive cellular copper uptake via the induction of a metalloreductase, STEAP4. IL-17-induced elevated intracellular copper level leads to the activation of an E3-ligase, XIAP, which potentiates IL-17-induced NFκB activation and suppresses the caspase 3 activity. Importantly, this IL-17-induced STEAP4-dependent cellular copper uptake is critical for colon tumor formation in a murine model of colitis-associated tumorigenesis and STEAP4 expression correlates with IL-17 level and XIAP activation in human colon cancer. In summary, this study reveals a IL-17-STEAP4-XIAP axis through which the inflammatory response induces copper uptake, promoting colon tumorigenesis.