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

The human microbiota is a complex ecosystem that colonizes body surfaces and interacts with host organ systems, especially the immune system. Since the composition of this ecosystem depends on a variety of internal and external factors, each individual harbors a unique set of microbes. These differences in microbiota composition make individuals either more or less susceptible to various diseases, including cancer. Specific microbes are associated with cancer etiology and pathogenesis and several mechanisms of how they drive the typical hallmarks of cancer were recently identified. Although most microbes reside in the distal gut, they can influence cancer initiation and progression in distant tissues, as well as modulate the outcomes of established cancer therapies. Here, we describe the mechanisms by which microbes influence carcinogenesis and discuss their current and potential future applications in cancer diagnostics and management.

• Keywords
• Fecal microbiota transplantation, Gut microbiota, Hallmarks of cancer, Tumor microenvironment,
• Publication type
• Journal Article MeSH
• Review MeSH

Wnt signaling plays an important role in the self-renewal, fate-commitment and survival of the neural stem/progenitor cells (NS/PCs) of the adult central nervous system (CNS). Ischemic stroke impairs the proper functioning of the CNS and, therefore, active Wnt signaling may prevent, ameliorate, or even reverse the negative effects of ischemic brain injury. In this review, we provide the current knowledge of Wnt signaling in the adult CNS, its status in diverse cell types, and the Wnt pathway's impact on the properties of NS/PCs and glial cells in the context of ischemic injury. Finally, we summarize promising strategies that might be considered for stroke therapy, and we outline possible future directions of the field.

• Keywords
• Wnt signaling, adult neurogenesis, central nervous system, glia, ischemia, neural stem/progenitor cell, stroke, subgranular zone, subventricular zone,
• MeSH
• Cell Differentiation genetics   MeSH
• Molecular Targeted Therapy methods   trends   MeSH
• Adult MeSH
• Brain Ischemia genetics   metabolism   pathology   physiopathology   MeSH
• Humans MeSH
• Brain cytology   pathology   physiology   MeSH
• Neural Stem Cells pathology   physiology   MeSH
• Neurogenesis physiology   MeSH
• Neuroglia pathology   physiology   MeSH
• Wnt Signaling Pathway genetics   physiology   MeSH
• Ischemic Attack, Transient genetics   metabolism   pathology   therapy   MeSH
• Health MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review 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

The first step in the development of human colorectal cancer is aberrant activation of the Wnt signaling pathway. Wnt signaling hyperactivation is predominantly caused by loss-of-function mutations in the adenomatous polyposis coli (APC) gene that encodes the pathway negative regulator. In order to identify genes affected by the Apc loss, we performed expression profiling of intestinal epithelium isolated from mice harboring a conditional Apc allele. The gene encoding transcriptional factor msh homeobox 1 (Msx1) displayed robust upregulation upon Apc inactivation. Histological analysis of the Apc-deficient epithelium revealed that in the small intestine, the Msx1 protein was localized exclusively in ectopic crypts, i.e., in pockets of proliferating cells abnormally positioned on the villi. Ablation of the Msx1 gene leads to the disappearance of ectopic crypts and loss of differentiated cells. Moreover, tumors arising from Msx1-deficient cells display altered morphology reminiscent of villous adenomas. In human tumor specimens, MSX1 displayed significantly increased expression in colonic neoplasia with a descending tendency during the lesion progression towards colorectal carcinoma. In summary, the results indicate that Msx1 represents a novel marker of intestinal tumorigenesis. In addition, we described the previously unknown relationship between the Msx1-dependent formation of ectopic crypts and cell differentiation.

• MeSH
• beta Catenin metabolism   MeSH
• Cell Differentiation MeSH
• Colorectal Neoplasms genetics   pathology   MeSH
• Humans MeSH
• Mice, Knockout MeSH
• Colonic Neoplasms genetics   pathology   MeSH
• Adenomatous Polyposis Coli Protein genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Wnt Signaling Pathway MeSH
• Gene Expression Profiling MeSH
• Intestinal Mucosa metabolism   pathology   MeSH
• Intestine, Small pathology   MeSH
• MSX1 Transcription Factor genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans 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
• APC protein, human MeSH Browser
• beta Catenin MeSH
• MSX1 protein, human MeSH Browser
• Msx1 protein, mouse MeSH Browser
• Adenomatous Polyposis Coli Protein MeSH
• MSX1 Transcription Factor MeSH

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

Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is produced in a broad spectrum of mouse embryonic and adult tissues and its deficiency results in embryonal or perinatal lethality. The LGR4 function was mainly related to its potentiation of canonical Wnt signaling; however, several recent studies associate LGR4 with additional signaling pathways. To obtain a suitable tool for studying the signaling properties of Lgr4, we generated a tagged variant of the Lgr4 receptor using gene targeting in the mouse oocyte. The modified Lgr4 allele expresses the Lgr4 protein fused with a triple hemagglutinin (3HA) tag located at the extracellular part of the protein. The allele is fully functional, enabling tracking of Lgr4 expression in the mouse tissues. We also show that via surface labeling, the 3HA tag allows direct isolation and analysis of living Lgr4-positive cells obtained from the small intestinal crypts. Finally, the HA tag-specific antibody can be employed to characterize the biochemical features of Lgr4 and to identify possible biding partners of the protein in cells derived from various mouse tissues.

• Keywords
• Genome editing, Hemagglutinin tag, Knock-in, R-spondin, TALENs, Wnt signaling,
• MeSH
• Alleles MeSH
• Gene Targeting methods   MeSH
• Hemagglutinins genetics   MeSH
• Humans MeSH
• Mice MeSH
• Oocytes metabolism   MeSH
• Receptors, G-Protein-Coupled genetics   MeSH
• Wnt Signaling Pathway genetics   MeSH
• Pregnancy MeSH
• Tissue Distribution MeSH
• Protein Binding genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Pregnancy MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Hemagglutinins MeSH
• LGR4 protein, mouse MeSH Browser
• Receptors, G-Protein-Coupled MeSH

T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) proteins (TCFs) from the High Mobility Group (HMG) box family act as the main downstream effectors of the Wnt signaling pathway. The mammalian TCF/LEF family comprises four nuclear factors designated TCF7, LEF1, TCF7L1, and TCF7L2 (also known as TCF1, LEF1, TCF3, and TCF4, respectively). The proteins display common structural features and are often expressed in overlapping patterns implying their redundancy. Such redundancy was indeed observed in gene targeting studies; however, individual family members also exhibit unique features that are not recapitulated by the related proteins. In the present viewpoint, we summarized our current knowledge about the specific features of individual TCFs, namely structural-functional studies, posttranslational modifications, interacting partners, and phenotypes obtained upon gene targeting in the mouse. In addition, we employed several publicly available databases and web tools to evaluate the expression patterns and production of gene-specific isoforms of the TCF/LEF family members in human cells and tissues.

• Keywords
• Fantom5, GTEx, Wnt signaling, splicing isoforms, the Cancer Genome Atlas,
• 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