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1.
Nat Commun ; 7: 10916, 2016 Mar 09.
Article in English | MEDLINE | ID: mdl-26956214

ABSTRACT

Intestinal tumour formation is generally thought to occur following mutational events in the stem cell pool. However, active NF-κB signalling additionally facilitates malignant transformation of differentiated cells. We hypothesized that genes shared between NF-κB and intestinal stem cell (ISCs) signatures might identify common pathways that are required for malignant growth. Here, we find that the NF-κB target Bcl-2, an anti-apoptotic gene, is specifically expressed in ISCs in both mice and humans. Bcl-2 is dispensable in homeostasis and, although involved in protecting ISCs from radiation-induced damage, it is non-essential in tissue regeneration. Bcl-2 is upregulated in adenomas, and its loss or inhibition impairs outgrowth of oncogenic clones, because Bcl-2 alleviates apoptotic priming in epithelial cells following Apc loss. Furthermore, Bcl-2 expression in differentiated epithelial cells renders these cells amenable to clonogenic outgrowth. Collectively, our results indicate that Bcl-2 is required for efficient intestinal transformation following Apc-loss and constitutes a potential chemoprevention target.


Subject(s)
Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Intestinal Mucosa/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , Cell Differentiation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Colorectal Neoplasms/genetics , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Female , Humans , Intestines/pathology , Male , Mice , Mice, Inbred C57BL , NF-kappa B/genetics , NF-kappa B/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Stem Cells/cytology , Stem Cells/metabolism
2.
Mol Cancer Ther ; 14(11): 2527-40, 2015 Nov.
Article in English | MEDLINE | ID: mdl-26351325

ABSTRACT

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4(+) malignancies.


Subject(s)
Antibodies/pharmacology , Antibody Specificity/immunology , Bone Morphogenetic Proteins/antagonists & inhibitors , Camelids, New World/immunology , Neoplasms/drug therapy , Animals , Antibodies/immunology , Antibodies/metabolism , Antibody Affinity/immunology , Blotting, Western , Bone Morphogenetic Protein 2/chemistry , Bone Morphogenetic Protein 2/immunology , Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein 4/chemistry , Bone Morphogenetic Protein 4/immunology , Bone Morphogenetic Protein 4/metabolism , Bone Morphogenetic Proteins/immunology , Bone Morphogenetic Proteins/metabolism , Cell Line , HT29 Cells , Humans , Mice , Models, Molecular , Neoplasms/immunology , Neoplasms/metabolism , Protein Binding/immunology , Protein Structure, Tertiary
3.
Nature ; 521(7550): 43-7, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25924068

ABSTRACT

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.


Subject(s)
Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Intestines/pathology , Mutation/genetics , Organoids/metabolism , Organoids/pathology , Stem Cells/pathology , Aneuploidy , Animals , CRISPR-Cas Systems , Child , Child, Preschool , Colorectal Neoplasms/metabolism , Female , Genes, APC , Genes, p53/genetics , Heterografts , Humans , Imidazoles , Intercellular Signaling Peptides and Proteins/metabolism , Intestinal Mucosa/metabolism , Mice , Middle Aged , Mutagenesis, Site-Directed , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Transplantation , Piperazines , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Smad4 Protein/deficiency , Stem Cell Niche/physiology , Stem Cells/metabolism
4.
FASEB J ; 29(5): 2070-80, 2015 May.
Article in English | MEDLINE | ID: mdl-25648995

ABSTRACT

Histone deacetylases (HDACs) are posttranslational modifiers that deacetylate proteins. Despite their crucial role in numerous biological processes, the use of broad-range HDAC inhibitors (HDACi), has shown clinical efficacy. However, undesired side effects highlight the necessity to better understand the biology of different HDACs and target the relevant HDACs. Using a novel mouse model, in which HDAC1 and HDAC2 can be simultaneously deleted in the intestine of adult mice, we show that the simultaneous deletion of HDAC1 and HDAC2 leads to a rapid loss of intestinal homeostasis. Importantly, this deletion cannot be sustained, and 8 days after initial ablation, stem cells that have escaped HDAC1 or HDAC2 deletion swiftly repopulate the intestinal lining. In vitro ablation of HDAC1 and HDAC2 using intestinal organoid cultures resulted in a down-regulation of multiple intestinal stem cell markers and functional loss of clonogenic capacity. Importantly, treatment of wild-type organoids with class I-specific HDACi MS-275 also induced a similar loss of stemness, providing a possible rationale for the gastrointestinal side effects often observed in HDACi-treated patients. In conclusion, these data show that HDAC1 and HDAC2 have a redundant function and are essential to maintain intestinal homeostasis.


Subject(s)
Histone Deacetylase 1/physiology , Histone Deacetylase 2/physiology , Homeostasis/physiology , Intestines/cytology , Stem Cells/cytology , Animals , Benzamides/pharmacology , Biomarkers/metabolism , Blotting, Western , Cell Differentiation , Cells, Cultured , Female , Flow Cytometry , Fluorescent Antibody Technique , Histone Deacetylase 1/antagonists & inhibitors , Histone Deacetylase 2/antagonists & inhibitors , Histone Deacetylase Inhibitors/pharmacology , Homeostasis/drug effects , Humans , Immunoenzyme Techniques , Intestines/drug effects , Intestines/enzymology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Organ Culture Techniques , Pyridines/pharmacology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/drug effects , Stem Cells/enzymology
5.
Mol Cancer ; 12(1): 126, 2013 Oct 21.
Article in English | MEDLINE | ID: mdl-24144042

ABSTRACT

BACKGROUND: Colon cancer stem cells are shown to be the self-renewing cells within a tumor that give rise to all lineages of more differentiated tumor cells. In this respect they are remarkably similar to their non-malignant counterparts that orchestrate the intestinal lining. This suggests that, despite the numerous genetic aberrations and morphological changes that have occurred during cancer initiation and progression, a remnant homeostatic regulation persists. FINDINGS: Using a number of human and mouse intestinal-derived organoid cultures from normal, adenoma and cancerous tissues, we show here that Notch signals coordinate self-renewal and lineage determination not only in normal, but also at the adenoma and carcinoma stage in both mice and humans. Moreover, the Wnt pathway, which carries activating mutations in virtually all colon cancers, is not as previously predicted constitutively active in adenomas and carcinomas, but still displays a heterogeneous activity pattern that determined stemness in all stages of disease. CONCLUSION: These data for the first time provide a comprehensive overview of Wnt and Notch-mediated signaling in the different stages of the adenoma-carcinoma sequence and demonstrates that these morphogenic pathways, despite mutations, remain crucial determinants of both architecture and hierarchy in normal and malignant intestinal tissue.


Subject(s)
Adenocarcinoma/metabolism , Adenoma/metabolism , Cell Transformation, Neoplastic/metabolism , Colonic Neoplasms/metabolism , Neoplastic Stem Cells/physiology , Signal Transduction , Adenocarcinoma/pathology , Adenoma/pathology , Animals , Cell Line, Tumor , Cell Proliferation , Colonic Neoplasms/pathology , Homeostasis , Humans , Mice , Mice, Transgenic , Receptors, Notch/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism
6.
Methods Mol Biol ; 1035: 247-59, 2013.
Article in English | MEDLINE | ID: mdl-23959997

ABSTRACT

The design of tissue culture conditions that faithfully reproduce the characteristics of cells in their native environment remains one of the main challenges of cancer stem cell (CSC) biology. Here we describe a detailed methodology for the isolation and expansion of both human colon CSCs and mouse intestinal adenoma together with a brief differentiation and coculture method that proved to be valuable to study the concept of CSCs plasticity.


Subject(s)
Neoplastic Stem Cells/physiology , Adenoma/pathology , Animals , Cell Dedifferentiation , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Cell Separation , Cells, Cultured , Coculture Techniques , Colonic Neoplasms/pathology , Culture Media, Conditioned , Feeder Cells , Humans , Mice
7.
Sci Rep ; 2: 271, 2012.
Article in English | MEDLINE | ID: mdl-22355783

ABSTRACT

The epithelial lining of the intestine is characterized by an immense cellular turn-over ascertaining an extensive regenerative capacity. Multiple reports suggest that besides the local intestinal stem cell pool, circulating cells of bone marrow origin (BMDCs) contribute to this process by fusing with the epithelial lineage. However, the functional relevance of these observations is unknown. In the present study we employ a model system in which we cannot only detect cell fusion but also examine the functional importance of this process in vivo. Our results indicate that fusion between BMDCs and intestinal epithelial cells is an extremely rare event under physiological conditions. More importantly, by employing a system in which fusion-derived cells can be specifically deleted after extensive tissue damage, we present evidence that cell fusion is not relevant for tissue regeneration. Our data decisively demonstrates that intestinal epithelial homeostasis and regeneration is not dependent on cell fusion involving BMDCs.


Subject(s)
Bone Marrow Cells/cytology , Cell Fusion , Homeostasis , Intestinal Mucosa/cytology , Animals , Base Sequence , Bone Marrow Transplantation , DNA Primers , Epithelial Cells/cytology , Flow Cytometry , In Situ Hybridization, Fluorescence , Mice , Models, Animal , Polymerase Chain Reaction
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