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.

• Klíčová slova
• Antimicrobial peptides, Colorectal cancer, Intestinal cell lineage, Intestinal crypt, Paneth cells, Single-cell transcriptomics,
• MeSH
• alfa-defensiny metabolismus   MeSH
• buněčná diferenciace MeSH
• lidé MeSH
• myši MeSH
• nádory tračníku * patologie   genetika   mikrobiologie   metabolismus   MeSH
• organoidy metabolismus   MeSH
• Panethovy buňky metabolismus   MeSH
• pohárkové buňky metabolismus   MeSH
• signální dráha Wnt MeSH
• střevní mikroflóra * MeSH
• tenké střevo * metabolismus   patologie   mikrobiologie   MeSH
• transkripční faktor 4 * metabolismus   genetika   MeSH
• transkriptom * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alfa-defensiny MeSH
• Tcf4 protein, mouse MeSH Prohlížeč
• transkripční faktor 4 * MeSH

Proto-oncogene KRAS, GTPase (KRAS) is one of the most intensively studied oncogenes in cancer research. Although several mouse models allow for regulated expression of mutant KRAS, selective isolation and analysis of transforming or tumor cells that produce the KRAS oncogene remains a challenge. In our study, we present a knock-in model of oncogenic variant KRASG12D that enables the "activation" of KRASG12D expression together with production of red fluorescent protein tdTomato. Both proteins are expressed from the endogenous Kras locus after recombination of a transcriptional stop box in the genomic DNA by the enzyme flippase (Flp). We have demonstrated the functionality of the allele termed RedRas (abbreviated KrasRR) under in vitro conditions with mouse embryonic fibroblasts and organoids and in vivo in the lung and colon epithelium. After recombination with adenoviral vectors carrying the Flp gene, the KrasRR allele itself triggers formation of lung adenomas. In the colon epithelium, it causes the progression of adenomas that are triggered by the loss of tumor suppressor adenomatous polyposis coli (APC). Importantly, cells in which recombination has successfully occurred can be visualized and isolated using the fluorescence emitted by tdTomato. Furthermore, we show that KRASG12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the KRASG12D function is effectively suppressed by specific inhibitor MRTX1133.

• Klíčová slova
• Colon cancer, Gene targeting, Intestinal organoids, KRAS oncogene, Lung cancer, MRTX1133 inhibitor,
• MeSH
• červený fluorescenční protein * MeSH
• DNA-nukleotidyltransferasy genetika   metabolismus   MeSH
• genový knockin MeSH
• luminescentní proteiny * genetika   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• myši transgenní MeSH
• myši MeSH
• nádory plic genetika   patologie   MeSH
• protoonkogenní proteiny p21(ras) * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• červený fluorescenční protein * MeSH
• DNA-nukleotidyltransferasy MeSH
• Hras protein, mouse MeSH Prohlížeč
• luminescentní proteiny * MeSH
• protoonkogenní proteiny p21(ras) * MeSH

Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), a Wnt pathway member, has been previously recognised as a stem cell marker in numerous epithelial tissues. In this study, we used Lgr5-EGFP-CreERT2 mice to analyse the distribution of LGR5-positive cells during craniofacial development. LGR5 expressing cells were primarily located in the mesenchyme adjacent to the craniofacial epithelial structures undergoing folding, such as the nasopharyngeal duct, lingual groove, and vomeronasal organ. To follow the fate of LGR5-positive cells, we performed lineage tracing using an inducible Cre knock-in allele in combination with Rosa26-tdTomato reporter mice. The slight expansion of LGR5-positive cells was found around the vomeronasal organ, in the nasal cavity, and around the epithelium in the lingual groove. However, most LGR5 expressing cells remained in their original location, possibly supporting their signalling function for adjacent epithelium rather than exerting their role as progenitor cells for the craniofacial structures. Moreover, Lgr5 knockout mice displayed distinct defects in LGR5-positive areas, especially in the reduction of the nasopharyngeal duct, the alteration of the palatal shelves shape, abnormal epithelial folding in the lingual groove area, and the disruption of salivary gland development. The latter defect manifested as an atypical number and localisation of the glandular ducts. The gene expression of several Wnt pathway members (Rspo1-3, Axin2) was altered in Lgr5-deficient animals. However, the difference was not found in sorted EGFP-positive cells obtained from Lgr5 +/+ and Lgr5 -/- animals. Expression profiling of LGR5-positive cells revealed the expression of several markers of mesenchymal cells, antagonists, as well as agonists, of Wnt signalling, and molecules associated with the basal membrane. Therefore, LGR5-positive cells in the craniofacial area represent a very specific population of mesenchymal cells adjacent to the epithelium undergoing folding or groove formation. Our results indicate a possible novel role of LGR5 in the regulation of morphogenetic processes during the formation of complex epithelial structures in the craniofacial areas, a role which is not related to the stem cell properties of LGR5-positive cells as was previously defined for various epithelial tissues.

• Klíčová slova
• LGR5, craniofacial, epithelial folding, palate, stem cell, tongue, vomeronasal organ,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• APC, EMT, TACSTD2, WNT/β-catenin signaling, colorectal cancer, expression profiling, organoids,
• Publikační typ
• časopisecké články MeSH

Focal adhesion kinase (FAK) is essential for vascular endothelial growth factor-A (VEGFA)/VEGF receptor-2 (VEGFR2)-stimulated angiogenesis and vascular permeability. We have previously noted that presence of the Src homology-2 domain adapter protein B (SHB) is of relevance for VEGFA-stimulated angiogenesis in a FAK-dependent manner. The current study was conducted in order address the temporal dynamics of co-localization between these components in HEK293 and primary lung endothelial cells (EC) by total internal reflection fluorescence microscopy (TIRF). An early (<2.5 min) VEGFA-induced increase in VEGFR2 co-localization with SHB was dependent on tyrosine 1175 in VEGFR2. VEGFA also enhanced SHB co-localization with FAK. FAK co-localization with VEGFR2 was dependent on SHB since it was significantly lower in SHB deficient EC after VEGFA addition. Absence of SHB also resulted in a gradual decline of VEGFR2 co-localization with FAK under basal (prior to VEGFA addition) conditions. A similar basal response was observed with expression of the Y1175F-VEGFR2 mutant in wild type EC. The distribution of focal adhesions in SHB-deficient EC was altered with a primarily perinuclear location. These live cell data implicate SHB as a key component regulating FAK activity in response to VEGFA/VEGFR2.

• Klíčová slova
• FAK, SHB, TIRF, VEGFR2, angiogenesis, focal adhesions,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• endoteliální buňky metabolismus   MeSH
• fokální adhezní tyrosinkinasy genetika   metabolismus   MeSH
• fyziologická neovaskularizace fyziologie   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• myši inbrední BALB C MeSH
• myši knockoutované MeSH
• myši MeSH
• pohyb buněk fyziologie   MeSH
• protoonkogenní proteiny metabolismus   MeSH
• receptor 2 pro vaskulární endoteliální růstový faktor metabolismus   MeSH
• vaskulární endoteliální růstový faktor A genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• fokální adhezní tyrosinkinasy MeSH
• KDR protein, human MeSH Prohlížeč
• Kdr protein, mouse MeSH Prohlížeč
• protoonkogenní proteiny MeSH
• receptor 2 pro vaskulární endoteliální růstový faktor MeSH
• SHB protein, human MeSH Prohlížeč
• vascular endothelial growth factor A, mouse MeSH Prohlížeč
• vaskulární endoteliální růstový faktor A MeSH
• VEGFA protein, human MeSH Prohlížeč

Colorectal cancer (CRC) is a heterogeneous disease that includes both hereditary and sporadic types of tumors. Tumor initiation and growth is driven by mutational or epigenetic changes that alter the function or expression of multiple genes. The genes predominantly encode components of various intracellular signaling cascades. In this review, we present mouse intestinal cancer models that include alterations in the Wnt, Hippo, p53, epidermal growth factor (EGF), and transforming growth factor β (TGFβ) pathways; models of impaired DNA mismatch repair and chemically induced tumorigenesis are included. Based on their molecular biology characteristics and mutational and epigenetic status, human colorectal carcinomas were divided into four so-called consensus molecular subtype (CMS) groups. It was shown subsequently that the CMS classification system could be applied to various cell lines derived from intestinal tumors and tumor-derived organoids. Although the CMS system facilitates characterization of human CRC, individual mouse models were not assigned to some of the CMS groups. Thus, we also indicate the possible assignment of described animal models to the CMS group. This might be helpful for selection of a suitable mouse strain to study a particular type of CRC.

• Klíčová slova
• carcinoma, consensus molecular subtypes, intestine, oncogenes, signaling cascades, tumor suppressors, tumorigenesis,
• MeSH
• epidermální růstový faktor genetika   MeSH
• geny p53 genetika   MeSH
• karcinogeneze genetika   MeSH
• kolorektální nádory klasifikace   metabolismus   patofyziologie   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nádorová transformace buněk genetika   MeSH
• nádory tračníku genetika   MeSH
• oprava chybného párování bází DNA genetika   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• regulace genové exprese u nádorů genetika   MeSH
• signální dráha Hippo MeSH
• signální dráha Wnt genetika   MeSH
• transformující růstový faktor beta genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• epidermální růstový faktor MeSH
• protein-serin-threoninkinasy MeSH
• transformující růstový faktor beta MeSH

The tamoxifen-responsive conditional Cdh5-CreERT2 is commonly used for endothelial cell specific conditional deletion of loxP-flanked gene sequences. To address the role of endothelial cell Shb gene for B16F10 melanoma immune responses, tamoxifen-injected Cdh5-CreERT2/WT and Cdh5-CreERT2/Shbflox/flox mice received subcutaneous tumor cell injections. We observed a decrease of tumor myeloid cell Shb mRNA in the tamoxifen treated Cdh5-CreERT2/Shbflox/flox mice, which was not present when the mice had undergone a preceding bone marrow transplantation using wild type bone marrow. Differences in CD4+/FoxP3+ Tregs were similarly abolished by a preceding bone marrow transplantation. In ROSA26-mTmG mice, Cdh5-CreERT2 caused detectable floxing in certain bone marrow populations and in spleen cells. Floxing in bone marrow could be detected two months after tamoxifen treatment. In the spleen, however, floxing was undetectable two months after tamoxifen treatment, suggesting that Cdh5-CreERT2 is operating in a non-renewable population of hematopoietic cells in this organ. These data suggest that conditional gene deletion in hematopoietic cells is a potential confounder in experiments attempting to assess the role of endothelial specific effects. A cautious approach to achieve an endothelial-specific phenotype would be to adopt a strategy that includes a preceding bone marrow transplantation.

• MeSH
• CD antigeny genetika   MeSH
• delece genu * MeSH
• endoteliální buňky cytologie   MeSH
• hematopoetické kmenové buňky cytologie   MeSH
• kadheriny genetika   MeSH
• melanom experimentální genetika   imunologie   MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• patologická angiogeneze genetika   MeSH
• proliferace buněk genetika   MeSH
• protoonkogenní proteiny genetika   MeSH
• tamoxifen farmakologie   MeSH
• transplantace hematopoetických kmenových buněk MeSH
• transplantace kostní dřeně MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cadherin 5 MeSH Prohlížeč
• CD antigeny MeSH
• kadheriny MeSH
• protoonkogenní proteiny MeSH
• Shb protein, mouse MeSH Prohlížeč
• tamoxifen 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.

• Klíčová slova
• TCF7L2, Wnt signaling, colorectal cancer, conditional gene inactivation, epithelium, gut, organoids, tumorigenesis,
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Hippo pathway, LGR, R-Spondins, Wnt/β-catenin signalling, YAP1/TAZ, colorectal cancer,
• Publikační typ
• časopisecké články MeSH
• přehledy 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.

• Klíčová slova
• Genome editing, Hemagglutinin tag, Knock-in, R-spondin, TALENs, Wnt signaling,
• MeSH
• alely MeSH
• genový targeting metody   MeSH
• hemaglutininy genetika   MeSH
• lidé MeSH
• myši MeSH
• oocyty metabolismus   MeSH
• receptory spřažené s G-proteiny genetika   MeSH
• signální dráha Wnt genetika   MeSH
• těhotenství MeSH
• tkáňová distribuce MeSH
• vazba proteinů genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hemaglutininy MeSH
• LGR4 protein, mouse MeSH Prohlížeč
• receptory spřažené s G-proteiny MeSH