BACKGROUND: The canonical Wnt signaling pathway controls the continuous renewal of the intestinal epithelium and the specification of epithelial cell lineages. Tcf4, a nuclear mediator of Wnt signaling, is essential for the differentiation and maintenance of Paneth cells in the small intestine. Its deficiency is associated with reduced expression of key α-defensins, highlighting its role in host-microbe interactions. However, the exact function of Tcf4 in specifying the secretory lineage and its contribution to antimicrobial peptide production remain incompletely understood. Remarkably, α-defensin expression has also been detected in human colon adenomas, where aberrant Wnt signaling is a hallmark. This raises important questions: What is the role of these Paneth-like cells in tumor biology, and how does Tcf4 influence their identity and function? METHODS: We investigated cell specification in small intestinal crypts and colon tumors using conditional Tcf7l2 deletion, cell type-specific Cre recombinases, and reporter alleles in mice. Transcriptomic (single-cell and bulk RNA sequencing) and histological analyses were performed and complemented by microbiome profiling, antibiotic treatment, and intestinal organoids to functionally validate the main findings. RESULTS: The inactivation of Tcf4 depletes Paneth cells and antimicrobial peptides, disrupting the gut microbiota balance. In secretory progenitors, loss of Tcf4 shifts differentiation toward goblet cells. In the small intestine, alternative secretory progenitors produce Wnt ligands to support stem cells and epithelial renewal in the absence of Paneth cells. In colon tumors, Paneth-like cells form a tumor cell population, express Wnt ligands, and require Tcf4 for their identity. Loss of Tcf4 redirects their differentiation toward goblet cells. CONCLUSIONS: Tcf4 controls the balance between Paneth and goblet cells and is essential for antimicrobial peptide production in the small intestine. In colon adenomas, Paneth-like tumor cells drive antimicrobial gene expression and provide Wnt3 ligands, which may have implications for cancer therapy.

• Keywords
• Antimicrobial peptides, Colorectal cancer, Intestinal cell lineage, Intestinal crypt, Paneth cells, Single-cell transcriptomics,
• MeSH
• alpha-Defensins metabolism   MeSH
• Cell Differentiation MeSH
• Humans MeSH
• Mice MeSH
• Colonic Neoplasms * pathology   genetics   microbiology   metabolism   MeSH
• Organoids metabolism   MeSH
• Paneth Cells metabolism   MeSH
• Goblet Cells metabolism   MeSH
• Wnt Signaling Pathway MeSH
• Gastrointestinal Microbiome * MeSH
• Intestine, Small * metabolism   pathology   microbiology   MeSH
• Transcription Factor 4 * metabolism   genetics   MeSH
• Transcriptome * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• alpha-Defensins MeSH
• Tcf4 protein, mouse MeSH Browser
• Transcription Factor 4 * MeSH

Trophoblastic cell surface antigen 2 (TROP2) is a membrane glycoprotein overexpressed in many solid tumors with a poor prognosis, including intestinal neoplasms. In our study, we show that TROP2 is expressed in preneoplastic lesions, and its expression is maintained in most colorectal cancers (CRC). High TROP2 positivity correlated with lymph node metastases and poor tumor differentiation and was a negative prognostic factor. To investigate the role of TROP2 in intestinal tumors, we analyzed two mouse models with conditional disruption of the adenomatous polyposis coli (Apc) tumor-suppressor gene, human adenocarcinoma samples, patient-derived organoids, and TROP2-deficient tumor cells. We found that Trop2 is produced early after Apc inactivation and its expression is associated with the transcription of genes involved in epithelial-mesenchymal transition, the regulation of migration, invasiveness, and extracellular matrix remodeling. A functionally similar group of genes was also enriched in TROP2-positive cells from human CRC samples. To decipher the driving mechanism of TROP2 expression, we analyzed its promoter. In human cells, this promoter was activated by β-catenin and additionally by the Yes1-associated transcriptional regulator (YAP). The regulation of TROP2 expression by active YAP was verified by YAP knockdown in CRC cells. Our results suggest a possible link between aberrantly activated Wnt/β-catenin signaling, YAP, and TROP2 expression.

• Keywords
• APC, EMT, TACSTD2, WNT/β-catenin signaling, colorectal cancer, expression profiling, organoids,
• Publication type
• Journal Article MeSH

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates cell response to genotoxic stress by negatively regulating the tumor suppressor p53 and other targets at chromatin. Mutations in the exon 6 of the PPM1D result in production of a highly stable, C-terminally truncated PPM1D. These gain-of-function PPM1D mutations are present in various human cancers but their role in tumorigenesis remains unresolved. Here we show that truncated PPM1D impairs activation of the cell cycle checkpoints in human non-transformed RPE cells and allows proliferation in the presence of DNA damage. Next, we developed a mouse model by introducing a truncating mutation in the PPM1D locus and tested contribution of the oncogenic PPM1DT allele to colon tumorigenesis. We found that p53 pathway was suppressed in colon stem cells harboring PPM1DT resulting in proliferation advantage under genotoxic stress condition. In addition, truncated PPM1D promoted tumor growth in the colon in Apcmin mice and diminished survival. Moreover, tumor organoids derived from colon of the ApcminPpm1dT/+ mice were less sensitive to 5-fluorouracil when compared to ApcminPpm1d+/+and the sensitivity to 5-fluorouracil was restored by inhibition of PPM1D. Finally, we screened colorectal cancer patients and identified recurrent somatic PPM1D mutations in a fraction of colon adenocarcinomas that are p53 proficient and show defects in mismatch DNA repair. In summary, we provide the first in vivo evidence that truncated PPM1D can promote tumor growth and modulate sensitivity to chemotherapy.

• MeSH
• Chromatin drug effects   MeSH
• Exons genetics   MeSH
• Fluorouracil pharmacology   MeSH
• Carcinogenesis drug effects   MeSH
• Cell Cycle Checkpoints genetics   MeSH
• Humans MeSH
• Mutation genetics   MeSH
• Mice MeSH
• Tumor Suppressor Protein p53 genetics   MeSH
• Colonic Neoplasms drug therapy   genetics   pathology   MeSH
• DNA Repair drug effects   MeSH
• DNA Damage drug effects   MeSH
• Cell Proliferation drug effects   MeSH
• Adenomatous Polyposis Coli Protein genetics   MeSH
• Protein Phosphatase 2C genetics   MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• adenomatous polyposis coli protein, mouse MeSH Browser
• Chromatin MeSH
• Fluorouracil MeSH
• Tumor Suppressor Protein p53 MeSH
• PPM1D protein, human MeSH Browser
• Adenomatous Polyposis Coli Protein MeSH
• Protein Phosphatase 2C MeSH
• Trp53 protein, mouse MeSH Browser

T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.

• Keywords
• TCF7L2, Wnt signaling, colorectal cancer, conditional gene inactivation, epithelium, gut, organoids, tumorigenesis,
• Publication type
• Journal Article MeSH

In this review, we address aspects of Wnt, R-Spondin (RSPO) and Hippo signalling, in both healthy and transformed intestinal epithelium. In intestinal stem cells (ISCs), the Wnt pathway is essential for intestinal crypt formation and renewal, whereas RSPO-mediated signalling mainly affects ISC numbers. In human colorectal cancer (CRC), aberrant Wnt signalling is the driving mechanism initiating this type of neoplasia. The signalling role of the RSPO-binding transmembrane proteins, the leucine-rich-repeat-containing G-protein-coupled receptors (LGRs), is possibly more pleiotropic and not only limited to the enhancement of Wnt signalling. There is growing evidence for multiple crosstalk between Hippo and Wnt/β-catenin signalling. In the ON state, Hippo signalling results in serine/threonine phosphorylation of Yes-associated protein (YAP1) and tafazzin (TAZ), promoting formation of the β-catenin destruction complex. In contrast, YAP1 or TAZ dephosphorylation (and YAP1 methylation) results in β-catenin destruction complex deactivation and β-catenin nuclear localization. In the Hippo OFF state, YAP1 and TAZ are engaged with the nuclear β-catenin and participate in the β-catenin-dependent transcription program. Interestingly, YAP1/TAZ are dispensable for intestinal homeostasis; however, upon Wnt pathway hyperactivation, the proteins together with TEA domain (TEAD) transcription factors drive the transcriptional program essential for intestinal cell transformation. In addition, in many CRC cells, YAP1 phosphorylation by YES proto-oncogene 1 tyrosine kinase (YES1) leads to the formation of a transcriptional complex that includes YAP1, β-catenin and T-box 5 (TBX5) DNA-binding protein. YAP1/β-catenin/T-box 5-mediated transcription is necessary for CRC cell proliferation and survival. Interestingly, dishevelled (DVL) appears to be an important mediator involved in both Wnt and Hippo (YAP1/TAZ) signalling and some of the DVL functions were assigned to the nuclear DVL pool. Wnt ligands can trigger alternative signalling that directly involves some of the Hippo pathway components such as YAP1, TAZ and TEADs. By upregulating Wnt pathway agonists, the alternative Wnt signalling can inhibit the canonical Wnt pathway activity.

• Keywords
• Hippo pathway, LGR, R-Spondins, Wnt/β-catenin signalling, YAP1/TAZ, colorectal cancer,
• Publication type
• Journal Article MeSH
• Review MeSH

The Wnt pathway plays a crucial role in self-renewal and differentiation of cells in the adult gut. In the present study, we revealed the functional consequences of inhibition of canonical Wnt signaling in the intestinal epithelium. The study was based on generation of a novel transgenic mouse strain enabling inducible expression of an N-terminally truncated variant of nuclear Wnt effector T cell factor 4 (TCF4). The TCF4 variant acting as a dominant negative (dn) version of wild-type (wt) TCF4 protein decreased transcription of β-catenin-TCF4-responsive genes. Interestingly, suppression of Wnt/β-catenin signaling affected asymmetric division of intestinal stem cells (ISCs) rather than proliferation. ISCs expressing the transgene underwent several rounds of division but lost their clonogenic potential and migrated out of the crypt. Expression profiling of crypt cells revealed that besides ISC-specific markers, the dnTCF4 production downregulated expression levels of epithelial genes produced in other crypt cells including markers of Paneth cells. Additionally, in Apc conditional knockout mice, dnTCF activation efficiently suppressed growth of Apc-deficient tumors. In summary, the generated mouse strain represents a convenient tool to study cell-autonomous inhibition of β-catenin-Tcf-mediated transcription.

• Keywords
• Cre/loxP, TCF/LEF transcription factors, Wnt pathway, gene targeting, gut, β-catenin,
• MeSH
• beta Catenin metabolism   MeSH
• Cell Differentiation MeSH
• Cell Division MeSH
• Transcription, Genetic MeSH
• Stem Cells cytology   metabolism   MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Cell Proliferation MeSH
• Wnt Signaling Pathway * MeSH
• Intestinal Mucosa cytology   metabolism   MeSH
• Intestine, Small cytology   metabolism   MeSH
• Transcription Factor 4 MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors chemistry   genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• beta Catenin MeSH
• Tcf4 protein, mouse MeSH Browser
• Transcription Factor 4 MeSH
• Basic Helix-Loop-Helix Leucine Zipper Transcription Factors MeSH

The regulation and stem cell origin of normal and neoplastic gastric glands are uncertain. Here, we show that Mist1 expression marks quiescent stem cells in the gastric corpus isthmus. Mist1(+) stem cells serve as a cell-of-origin for intestinal-type cancer with the combination of Kras and Apc mutation and for diffuse-type cancer with the loss of E-cadherin. Diffuse-type cancer development is dependent on inflammation mediated by Cxcl12(+) endothelial cells and Cxcr4(+) gastric innate lymphoid cells (ILCs). These cells form the perivascular gastric stem cell niche, and Wnt5a produced from ILCs activates RhoA to inhibit anoikis in the E-cadherin-depleted cells. Targeting Cxcr4, ILCs, or Wnt5a inhibits diffuse-type gastric carcinogenesis, providing targets within the neoplastic gastric stem cell niche.

• MeSH
• Anoikis MeSH
• Cell Lineage MeSH
• Time Factors MeSH
• Chemokine CXCL12 metabolism   MeSH
• Endothelial Cells metabolism   pathology   MeSH
• Epithelial Cells drug effects   metabolism   pathology   MeSH
• Cadherins metabolism   MeSH
• Humans MeSH
• Lymphocytes metabolism   pathology   MeSH
• Cell Communication MeSH
• Mice, Transgenic MeSH
• Mice MeSH
• Cell Transformation, Neoplastic genetics   metabolism   pathology   MeSH
• Cell Line, Tumor MeSH
• Neoplastic Stem Cells drug effects   metabolism   pathology   MeSH
• Tumor Microenvironment * MeSH
• Stomach Neoplasms drug therapy   genetics   metabolism   pathology   MeSH
• Stem Cell Niche * MeSH
• Wnt-5a Protein MeSH
• Wnt Proteins metabolism   MeSH
• Antineoplastic Agents pharmacology   MeSH
• Receptors, CXCR4 metabolism   MeSH
• rho GTP-Binding Proteins metabolism   MeSH
• rhoA GTP-Binding Protein MeSH
• Wnt Signaling Pathway MeSH
• Signal Transduction MeSH
• Cellular Senescence MeSH
• Basic Helix-Loop-Helix Transcription Factors genetics   metabolism   MeSH
• Bone Marrow Transplantation MeSH
• Gastric Mucosa drug effects   metabolism   pathology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• BHLHA15 protein, human MeSH Browser
• Bhlha15 protein, mouse MeSH Browser
• Chemokine CXCL12 MeSH
• Cxcl12 protein, mouse MeSH Browser
• CXCR4 protein, mouse MeSH Browser
• Cadherins MeSH
• Wnt-5a Protein MeSH
• Wnt Proteins MeSH
• Antineoplastic Agents MeSH
• Receptors, CXCR4 MeSH
• rho GTP-Binding Proteins MeSH
• rhoA GTP-Binding Protein MeSH
• RhoA protein, mouse MeSH Browser
• Basic Helix-Loop-Helix Transcription Factors MeSH
• Wnt5a protein, mouse MeSH Browser