ABSTRACT
Oncogenic alterations underlying B-cell acute lymphoblastic leukemia (B-ALL) in adults remain incompletely elucidated. To uncover novel oncogenic drivers, we performed RNA sequencing and whole-genome analyses in a large cohort of unresolved B-ALL. We identified a novel subtype characterized by a distinct gene expression signature and the unique association of 2 genomic microdeletions. The 17q21.31 microdeletion resulted in a UBTF::ATXN7L3 fusion transcript encoding a chimeric protein. The 13q12.2 deletion resulted in monoallelic ectopic expression of the homeobox transcription factor CDX2, located 138 kb in cis from the deletion. Using 4C-sequencing and CRISPR interference experiments, we elucidated the mechanism of CDX2 cis-deregulation, involving PAN3 enhancer hijacking. CDX2/UBTF ALL (n = 26) harbored a distinct pattern of additional alterations including 1q gain and CXCR4 activating mutations. Within adult patients with Ph- B-ALL enrolled in GRAALL trials, patients with CDX2/UBTF ALL (n = 17/723, 2.4%) were young (median age, 31 years) and dramatically enriched in females (male/female ratio, 0.2, P = .002). They commonly presented with a pro-B phenotype ALL and moderate blast cell infiltration. They had poor response to treatment including a higher risk of failure to first induction course (19% vs 3%, P = .017) and higher post-induction minimal residual disease (MRD) levels (MRD ≥ 10-4, 93% vs 46%, P < .001). This early resistance to treatment translated into a significantly higher cumulative incidence of relapse (75.0% vs 32.4%, P = .004) in univariate and multivariate analyses. In conclusion, we discovered a novel B-ALL entity defined by the unique combination of CDX2 cis-deregulation and UBTF::ATXN7L3 fusion, representing a high-risk disease in young adults.
Subject(s)
CDX2 Transcription Factor , Pol1 Transcription Initiation Complex Proteins , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Transcription Factors , Adult , CDX2 Transcription Factor/genetics , Female , Genes, Homeobox , Humans , Male , Neoplasm, Residual/genetics , Oncogene Proteins, Fusion , Pol1 Transcription Initiation Complex Proteins/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Transcription Factors/geneticsABSTRACT
Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.
Subject(s)
Endoderm/growth & development , Intestinal Mucosa/growth & development , Intestine, Small/growth & development , Animals , CDX2 Transcription Factor , Cell Differentiation/genetics , Cells, Cultured , Female , Gastric Mucosa/growth & development , Gene Knockout Techniques , Homeodomain Proteins/biosynthesis , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Morphogenesis/genetics , SOXB1 Transcription Factors/biosynthesis , Stem Cells/physiology , Transcription Factors/biosynthesis , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Cdx2, a gene of the paraHox cluster, encodes a homeodomain transcription factor that plays numerous roles in embryonic development and in homeostasis of the adult intestine. Whereas Cdx2 exerts a tumor suppressor function in the gut, its abnormal ectopic expression in acute leukemia is associated to a pro-oncogenic function. To try to understand this duality, we have hypothesized that Cdx2 may interact with different protein partners in the two tissues and set up experiments to identify them by tandem affinity purification. We show here that Cdx2 interacts with the Ku heterodimer specifically in intestinal cells, but not in leukemia cells, via its homeodomain. Ku proteins do not affect Cdx2 transcriptional activity. However, Cdx2 inhibits in vivo and in vitro the DNA repair activity mediated by Ku proteins in intestinal cells. Whereas Cdx2 does not affect the recruitment of Ku proteins and DNA-PKcs into the DNA repair complex, it inhibits DNA-PKcs activity. Thus, we report here a new function of Cdx2, acting as an inhibitor of the DNA repair machinery, that may contribute to its tumor suppressor function specifically in the gut.
Subject(s)
Colonic Neoplasms/genetics , DNA End-Joining Repair , Homeodomain Proteins/metabolism , Leukemia/genetics , Tumor Suppressor Proteins/metabolism , Antigens, Nuclear/metabolism , CDX2 Transcription Factor , Cell Line, Tumor , Cell Survival , Colonic Neoplasms/metabolism , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Etoposide/toxicity , Homeodomain Proteins/chemistry , Homeodomain Proteins/physiology , Humans , Ku Autoantigen , Leukemia/metabolism , Protein Interaction Domains and Motifs , Transcription, Genetic , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/physiologyABSTRACT
BACKGROUND & AIMS: The intestine-specific homeobox transcription factor Cdx2 is an important determinant of intestinal identity in the embryonic endoderm and regulates the balance between proliferation and differentiation in the adult intestinal epithelium. Human colon tumors often lose Cdx2 expression, and heterozygous inactivation of Cdx2 in mice increases colon tumorigenesis. We sought to identify Cdx2 target genes to determine how it contributes to intestinal homeostasis. METHODS: We used expression profiling analysis to identify genes that are regulated by Cdx2 in colon cancer cells lines. Regulation and function of a potential target gene were further investigated using various cell assays. RESULTS: In colon cancer cell lines, Cdx2 directly regulated the transcription of the gene that encodes the protocadherin Mucdhl. Mucdhl localized to the apex of differentiated cells in the intestinal epithelium, and its expression was reduced in most human colon tumors. Overexpression of Mucdhl inhibited low-density proliferation of colon cancer cells and reduced tumor formation in nude mice. One isoform of Mucdhl interacted with ß-catenin and inhibited its transcriptional activity. CONCLUSIONS: The transcription factor Cdx2 activates expression of the protocadherin Mucdhl, which interacts with ß-catenin and regulates activities of intestinal cells. Loss of Cdx2 expression in colon cancer cells might reduce expression of Mucdhl and thereby lead to tumor formation.
Subject(s)
Cadherins/metabolism , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Colonic Neoplasms/metabolism , Homeodomain Proteins/metabolism , beta Catenin/metabolism , Animals , CDX2 Transcription Factor , Caco-2 Cells , Cadherin Related Proteins , Cadherins/genetics , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Down-Regulation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , HCT116 Cells , HT29 Cells , Homeodomain Proteins/genetics , Homeostasis , Humans , Mice , Mice, Nude , Neoplasm Transplantation , RNA Interference , Transcription, Genetic , Transfection , Tumor Burden , beta Catenin/geneticsABSTRACT
Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. Tissue-specific inactivation of Taf4 in murine intestinal epithelium during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells. In adults, Taf4 loss impacted the stem cell compartment and associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge. Taf4 inactivation ex vivo in enteroids prevented budding formation and maintenance and caused broad chromatin remodeling and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). Treatment of Taf4-mutant enteroids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Taf4 loss also led to increased PRC2 activity in cells of adult crypts associated with modification of the immune/inflammatory microenvironment that potentiated Apc-driven tumorigenesis. Our results reveal a novel function of Taf4 in antagonizing PRC2-mediated repression of the stem cell gene expression program to assure normal development, homeostasis, and immune-microenvironment of the intestinal epithelium.
Subject(s)
Drosophila Proteins , Stem Cells , Mice , Animals , Cell Differentiation/genetics , Stem Cells/metabolism , Transcription Factor TFIID/genetics , Intestinal Mucosa/metabolism , Drosophila Proteins/metabolism , Polycomb Repressive Complex 2/metabolism , Epigenesis, GeneticABSTRACT
BACKGROUND AND AIMS: Intestinal metaplasia (IM) is a gastric preneoplastic lesion that appears following Helicobacter pylori infection and confers an increased risk for development of cancer. It is induced by gastric expression of the intestine-specific transcription factor CDX2. The regulatory mechanisms involved in triggering and maintaining gastric CDX2 expression have not been fully elucidated. The Cdx2(+/-) mouse develops intestinal polyps with gastric differentiation and total loss of Cdx2 expression in the absence of structural loss of the second allele, suggesting a regulatory defect. This putative haplo-insufficiency, together with the apparent stability of IM, led to the hypothesis that CDX2 regulates its own expression through an autoregulatory loop in both contexts. METHODS: Gastrointestinal cell lines were co-transfected with wild-type or mutated Cdx2 promoter constructs and CDX2 expression vector for luciferase assays. Transfection experiments were also used to assess endogenous CDX2 autoregulation, evaluated by RT-PCR, qPCR and western blotting. Chromatin immunoprecipitation was performed in a cell line, mouse ileum and human IM. RESULTS: CDX2 binds to and transactivates its own promoter and positively regulates its expression in gastrointestinal human carcinoma cell lines. Furthermore, CDX2 is bound to its promoter in the mouse ileum and in human gastric IM, providing a major contribution to understanding the relevance of this autoregulatory pathway in vivo. CONCLUSION: The results of this study demonstrate another layer of complexity in CDX2 regulation by an effective autoregulatory loop which may have a major impact on the stability of human IM, possibly resulting in the inevitable progression of the gastric carcinogenesis pathway.
Subject(s)
Homeodomain Proteins/metabolism , Precancerous Conditions/metabolism , Stomach Neoplasms/metabolism , Stomach/pathology , Adenocarcinoma/metabolism , Animals , CDX2 Transcription Factor , Gene Expression Regulation/genetics , Homeodomain Proteins/genetics , Homeostasis/genetics , Homeostasis/physiology , Humans , Ileum/metabolism , Metaplasia/metabolism , Mice , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Point Mutation , Precancerous Conditions/genetics , Promoter Regions, Genetic/genetics , Stomach Neoplasms/genetics , Transfection , Tumor Cells, CulturedABSTRACT
Most patients affected with colorectal cancers (CRC) are treated with 5-fluorouracil (5-FU)-based chemotherapy but its efficacy is often hampered by resistance mechanisms linked to tumor heterogeneity. A better understanding of the molecular determinants involved in chemoresistance is critical for precision medicine and therapeutic progress. Caudal type homeobox 2 (CDX2) is a master regulator of intestinal identity and acts as tumor suppressor in the colon. Here, using a translational approach, we examined the role of CDX2 in CRC chemoresistance. Unexpectedly, we discovered that the prognosis value of CDX2 for disease-free survival of patients affected with CRC is lost upon chemotherapy and that CDX2 expression enhances resistance of colon cancer cells towards 5-FU. At the molecular level, we found that CDX2 expression correlates with higher levels of genes regulating the bioavailability of 5-FU through efflux (ABCC11) and catabolism (DPYD) in patients affected with CRC and CRC cell lines. We further showed that CDX2 directly regulates the expression of ABCC11 and that the inhibition of ABCC11 improves 5-FU-sensitivity of CDX2-expressing colon cancer cells. Thus, this study illustrates how biological functions are hijacked in CRC cells and reveals the therapeutic interest of CDX2/ABCC11/DPYD to improve systemic chemotherapy in CRC.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Colorectal Neoplasms/drug therapy , Fluorouracil/pharmacology , Adult , Aged , Aged, 80 and over , Antimetabolites, Antineoplastic/chemistry , Antimetabolites, Antineoplastic/therapeutic use , CDX2 Transcription Factor/genetics , CDX2 Transcription Factor/metabolism , Cell Line, Tumor/drug effects , Cohort Studies , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Disease-Free Survival , Drug Resistance, Neoplasm/drug effects , Female , Fluorouracil/chemistry , Fluorouracil/therapeutic use , France , Gene Expression Regulation, Neoplastic/drug effects , Humans , Male , Middle Aged , Young AdultABSTRACT
Whether a gene involved in distinct tissue or cell functions exerts a core of common molecular activities is a relevant topic in evolutionary, developmental, and pathological perspectives. Here, we addressed this question by focusing on the transcription factor and regulator of chromatin accessibility encoded by the Cdx2 homeobox gene that plays important functions during embryonic development and in adult diseases. By integrating RNAseq data in mouse embryogenesis, we unveiled a core set of common genes whose expression is responsive to the CDX2 homeoprotein during trophectoderm formation, posterior body elongation and intestinal specification. ChIPseq data analysis also identified a set of common chromosomal regions targeted by CDX2 at these three developmental steps. The transcriptional core set of genes was then validated with transgenic mouse models of loss or gain of function of Cdx2. Finally, based on human cancer data, we highlight the relevance of these results by displaying a significant number of human orthologous genes to the core set of mouse CDX2-responsive genes exhibiting an altered expression along with CDX2 in human malignancies.
ABSTRACT
The intestine-specific caudal-related homeobox gene-2 (CDX2) homeobox gene, while being a tumor suppressor in the gut, is ectopically expressed in a large proportion of acute leukemia and is associated with poor prognosis. Here, we report that turning on human CDX2 expression in the hematopoietic lineage of mice induces acute monoblastic leukemia, characterized by the decrease in erythroid and lymphoid cells at the benefit of immature monocytic and granulocytic cells. One of the highly stimulated genes in leukemic bone marrow cells was BMP and activin membrane-bound inhibitor (Bambi), an inhibitor of transforming growth factor-ß (TGF-ß) signaling. The CDX2 protein was shown to bind to and activate the transcription of the human BAMBI promoter. Moreover, in a leukemic cell line established from CDX2-expressing mice, reducing the levels of CDX2 or Bambi stimulated the TGF-ß-dependent expression of Cd11b, a marker of monocyte maturation. Taken together, this work demonstrates the strong oncogenic potential of the homeobox gene CDX2 in the hematopoietic lineage, in contrast with its physiological tumor suppressor activity exerted in the gut. It also reveals, through BAMBI and TGF-ß signaling, the involvement of CDX2 in the perturbation of the interactions between leukemia cells and their microenvironment.
Subject(s)
CDX2 Transcription Factor/genetics , Leukemia, Monocytic, Acute/genetics , Transforming Growth Factor beta/antagonists & inhibitors , Animals , CD11b Antigen/genetics , Cell Lineage , Humans , Leukemia, Monocytic, Acute/pathology , Membrane Proteins/genetics , Mice , Signal Transduction , Tumor MicroenvironmentABSTRACT
Cadherins form a large and pleiotropic superfamily of membranous proteins sharing Ca2+-binding repeats. While the importance of classic cadherins such as E- or N-cadherin for tumorigenesis is acknowledged, there is much less information about other cadherins that are merely considered as tissue-specific adhesion molecules. Here, we focused on the atypical cadherin MUCDHL that stood out for its unusual features and unique function in the gut. Analyses of transcriptomic data sets (n > 250) established that MUCDHL mRNA levels are down-regulated in colorectal tumors. Importantly, the decrease of MUCDHL expression is more pronounced in the worst-prognosis subset of tumors and is associated with decreased survival. Molecular characterization of the tumors indicated a negative correlation with proliferation-related processes (e.g., nucleic acid metabolism, DNA replication). Functional genomic studies showed that the loss of MUCDHL enhanced tumor incidence and burden in intestinal tumor-prone mice. Extensive structure/function analyses revealed that the mode of action of MUCDHL goes beyond membrane sequestration of ß-catenin and targets through its extracellular domain key oncogenic signaling pathways (e.g., EGFR, AKT). Beyond MUCDHL, this study illustrates how the loss of a gene critical for the morphological and functional features of mature cells contributes to tumorigenesis by dysregulating oncogenic pathways.
Subject(s)
Cadherins/metabolism , Colonic Neoplasms/metabolism , Signal Transduction , Tumor Suppressor Proteins/metabolism , Caco-2 Cells , Cadherin Related Proteins , Cadherins/genetics , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , HEK293 Cells , Humans , Tumor Suppressor Proteins/geneticsABSTRACT
BACKGROUND & AIMS: The Cdx2 homeobox gene exerts multiple functions including trophectoderm specification, antero-posterior patterning, and determination of intestinal identity. The aim of this study was to map genomic regions that regulate the transcription of Cdx2, with a particular interest in the gut. METHODS: Genomic fragments covering 13 kilobase (kb) of the mouse Cdx2 locus were analyzed in transgenic mice and in cell assays. RESULTS: No fragment was active in the trophectoderm. Fragments containing the first intron and extending up to -5-kb upstream of the transcription start site became active posteriorly at gastrulation and then inactive at midgestation in every tissue including the endoderm. Specific persistence of activity in the intestinal endoderm/epithelium beyond midgestation requires extending the genomic fragment up to -9 kb. We identified a 250-base pair segment around -8.5-kb binding and responding to endodermal factors, with a stimulatory effect exerted synergistically by HNF4alpha, GATA6, Tcf4, and beta-catenin. These factors were able to activate endogenous expression of Cdx2 in nonintestinal Hela cells. CONCLUSIONS: Multiple regulatory regions control the complex developmental pattern of Cdx2, including far upstream sequences required for the persistence of gene expression specifically in the gut epithelium throughout life. Cooperation between HNF4alpha, GATA6, beta-catenin, and Tcf4 contributes to the intestine-specific expression of Cdx2.
Subject(s)
Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Intestines/embryology , Intestines/physiology , Transcription Factors/genetics , Transcription Factors/metabolism , Age Factors , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Blastocyst/cytology , Blastocyst/physiology , CDX2 Transcription Factor , Cecum/embryology , Cecum/physiology , Cell Line , Endoderm/embryology , Endoderm/physiology , GATA6 Transcription Factor/genetics , GATA6 Transcription Factor/metabolism , Genomics , HeLa Cells , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Humans , Intestines/cytology , Lac Operon , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Stomach/embryology , Stomach/physiology , TCF Transcription Factors/genetics , TCF Transcription Factors/metabolism , Transcription Factor 4 , Transfection , Trophoblasts/cytology , Trophoblasts/physiology , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
TCF and SOX proteins belong to the high mobility group box transcription factor family. Whereas TCFs, the transcriptional effectors of the Wnt pathway, have been widely implicated in the development, homeostasis and disease of the intestine epithelium, little is known about the function of the SOX proteins in this tissue. Here, we identified SOX9 in a SOX expression screening in the mouse fetal intestine. We report that the SOX9 protein is expressed in the intestinal epithelium in a pattern characteristic of Wnt targets. We provide in vitro and in vivo evidence that a bipartite beta-catenin/TCF4 transcription factor, the effector of the Wnt signaling pathway, is required for SOX9 expression in epithelial cells. Finally, in colon epithelium-derived cells, SOX9 transcriptionally represses the CDX2 and MUC2 genes, normally expressed in the mature villus cells of the intestinal epithelium, and may therefore contribute to the Wnt-dependent maintenance of a progenitor cell phenotype.
Subject(s)
High Mobility Group Proteins/metabolism , Homeodomain Proteins/metabolism , Mucins/metabolism , Transcription Factors/metabolism , Animals , Blotting, Northern , Blotting, Western , CDX2 Transcription Factor , Carcinoma/metabolism , Cell Differentiation , Cell Line, Tumor , Cell Nucleus/metabolism , Colonic Neoplasms/metabolism , Cytoskeletal Proteins/metabolism , DNA/metabolism , Epithelium/metabolism , Gene Expression Regulation, Neoplastic , Humans , Image Processing, Computer-Assisted , Immunohistochemistry , Intestinal Mucosa/metabolism , Mice , Microscopy, Fluorescence , Mucin-2 , Neoplasm Transplantation , Phenotype , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , SOX9 Transcription Factor , Signal Transduction , Stem Cells/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Transfection , beta CateninABSTRACT
We have previously reported that the CDX1 homeoprotein interacts with the TATA-box binding protein (TBP) on the promoter of the glucose-6-phosphatase (G6Pase) gene. We show here that CDX1 interacts with TBP via the homeodomain and that the transcriptional activity additionally requires the N-terminal domain upstream of the homeodomain. CDX1 interacting with TBP is connected to members of the TFIID and Mediator complexes, two major elements of the general transcriptional machinery. Transcription luciferase assays performed using an altered-specificity mutant of TBP provide evidence for the functionality of the interaction between CDX1 and TBP. Unlike CDX1, CDX2 does not interact with TBP nor does it transactivate the G6Pase promoter. Swapping experiments between the domains of CDX1 and CDX2 indicate that, despite opposite functional effects of the homeoproteins on the G6Pase promoter, the N-terminal domains and homeodomains of both CDX1 and CDX2 have the intrinsic ability to activate transcription and to interact with TBP. However, the carboxy domains define the specificity of CDX1 and CDX2. Thus, intra-molecular interactions control the activity and partner recruitment of CDX1 and CDX2, leading to different molecular functions.
Subject(s)
Homeodomain Proteins/metabolism , TATA-Box Binding Protein/metabolism , Trans-Activators/metabolism , Transcriptional Activation , Binding Sites , CDX2 Transcription Factor , Cell Line , Glucose-6-Phosphatase/genetics , Homeodomain Proteins/chemistry , Humans , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Trans-Activators/chemistryABSTRACT
Head dysgenesis is a major cause of fetal demise and craniofacial malformation. Although mutations in genes of the head ontogenetic program have been reported, many cases remain unexplained. Head dysgenesis has also been related to trisomy or amplification of the chromosomal region overlapping the CDX2 homeobox gene, a master element of the trunk ontogenetic program. Hence, we investigated the repercussion on head morphogenesis of the imbalance between the head and trunk ontogenetic programs, by means of ectopic rostral expression of CDX2 at gastrulation. This caused severe malformations affecting the forebrain and optic structures, and also the frontonasal process associated with defects in neural crest cells colonization. These malformations are the result of the downregulation of genes of the head program together with the abnormal induction of trunk program genes. Together, these data indicate that the imbalance between the anterior and posterior ontogenetic programs in embryos is a new possible cause of head dysgenesis during human development, linked to defects in setting up anterior neuroectodermal structures.
Subject(s)
CDX2 Transcription Factor/genetics , Craniofacial Abnormalities/genetics , Head/physiopathology , Morphogenesis/genetics , Animals , Craniofacial Abnormalities/physiopathology , Embryonic Development/genetics , Gastrulation/genetics , Gene Expression Regulation, Developmental/genetics , Genes, Homeobox/genetics , Head/growth & development , Humans , Mice , Neural Crest/growth & development , Neural Crest/physiopathology , Prosencephalon/growth & development , Prosencephalon/pathologyABSTRACT
Mechanical properties of the cellular environment are known to influence cell fate. Chromatin de-condensation appears as an early event in cell reprogramming. Whereas the ratio of euchromatin versus heterochromatin can be increased chemically, we report herein for the first time that the ratio can also be increased by purely changing the mechanical properties of the microenvironment by successive 24 h-contact of the cells on a soft substrate alternated with relocation and growth for 7 days on a hard substrate. An initial contact with soft substrate caused massive SW480 cancer cell death by necrosis, whereas approximately 7% of the cells did survived exhibiting a high level of condensed chromatin (21% heterochromatin). However, four consecutive hard/soft cycles elicited a strong chromatin de-condensation (6% heterochromatin) correlating with an increase of cellular survival (approximately 90%). Furthermore, cell survival appeared to be reversible, indicative of an adaptive process rather than an irreversible gene mutation(s). This adaptation process is associated with modifications in gene expression patterns. A completely new approach for chromatin de-condensation, based only on mechanical properties of the microenvironment, without any drug mediation is presented.
Subject(s)
Adaptation, Biological/genetics , Cellular Reprogramming , Chromatin Assembly and Disassembly , Euchromatin/metabolism , Heterochromatin/metabolism , Tumor Microenvironment , Cell Differentiation , Cell Line, Tumor , Cell Movement/genetics , Cell Survival/genetics , Elasticity , Gene Expression Regulation, Neoplastic , HumansABSTRACT
Developmental genes contribute to cancer, as reported for the homeobox gene Cdx2 playing a tumor suppressor role in the gut. In this study, we show that human colon cancers exhibiting the highest reduction in CDX2 expression belong to the serrated subtype with the worst evolution. In mice, mosaic knockout of Cdx2 in the adult intestinal epithelium induces the formation of imperfect gastric-type metaplastic lesions. The metaplastic knockout cells do not spontaneously become tumorigenic. However, they induce profound modifications of the microenvironment that facilitate the tumorigenic evolution of adjacent Cdx2-intact tumor-prone cells at the surface of the lesions through NF-κB activation, induction of inducible nitric oxide synthase, and stochastic loss of function of Apc This study presents a novel paradigm in that metaplastic cells, generally considered as precancerous, can induce tumorigenesis from neighboring nonmetaplastic cells without themselves becoming cancerous. It unveils the novel property of non-cell-autonomous tumor suppressor gene for the Cdx2 gene in the gut.
Subject(s)
CDX2 Transcription Factor/genetics , Carcinogenesis/genetics , Carcinogenesis/pathology , Intestinal Neoplasms/genetics , Intestinal Neoplasms/pathology , Animals , Cecum/pathology , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Heterozygote , Humans , Intestines/pathology , Metaplasia , Mice , NF-kappa B/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Nitric Oxide Synthase Type II/metabolism , Stromal Cells/metabolism , Stromal Cells/pathology , Tumor MicroenvironmentABSTRACT
The expression of the CDX2 gene, a crucial regulator of gut homeostasis, is altered in human colorectal cancers in parallel with de-differentiation. Here, we have investigated the chromosomal status of CDX2 in human sporadic colorectal cancers with the phenotype of chromosomal instability. Allelic imbalance determination showed frequent rearrangements at the CDX2 locus. The rearrangements correlated with CDX2 gene amplification, as assessed by quantitative PCR analysis. However, they were not predictive of the Cdx2 protein pattern. These data suggest that mechanisms other than structural alterations at the CDX2 locus account for the change of expression in colorectal cancers.
Subject(s)
Chromosomal Instability , Colorectal Neoplasms/genetics , Gene Amplification , Gene Rearrangement , Genes, Homeobox , Homeodomain Proteins/genetics , Base Sequence , CDX2 Transcription Factor , DNA Primers , HumansABSTRACT
We use high-resolution [Formula: see text] data in multiple experiments to estimate the sources of error during coregistration of images acquired on separate preclinical instruments. In combination with experiments with phantoms, we completed in vivo imaging on mice, aimed at identifying the possible sources of registration errors, caused either by transport of the animal, movement of the animal itself, or methods of coregistration. The same imaging cell was used as a holder for phantoms and animals. For all procedures, rigid coregistration was carried out using a common landmark coregistration system, placed inside the imaging cell. We used the fiducial registration error and the target registration error to analyze the coregistration accuracy. We found that moving an imaging cell between two preclinical devices during a multimodal procedure gives an error of about [Formula: see text] at most. Therefore, it could not be considered a source of coregistration errors. Errors linked to spontaneous movements of the animal increased with time, to nearly 1 mm at most, excepted for body parts that were properly restrained. This work highlights the importance of animal intrinsic movements during a multiacquisition procedure and demonstrates a simple method to identify and quantify the sources of error during coregistration.
ABSTRACT
CXCL12 has been shown to be involved in colon cancer metastasis, but its expression level and molecular mechanisms regulating its expression remain controversial. We thus evaluated CXCL12 expression in a large cohort of colon adenomas and carcinomas, investigated for an epigenetic mechanism controlling its expression and evaluated the impact of CXCL12 levels on cell migration and tumor growth. CXCL12 expression was measured in human colon adenomas and carcinomas with transcriptome array and RT-qPCR. The promoter methylation was analyzed with whole-genome DNA methylation chips and protein expression by immunohistochemistry. We confirm a reduced expression of CXCL12 in 75% of MSS carcinomas and show that the decrease is an early event as already present in adenomas. The methylome analysis shows that the CXCL12 promoter is methylated in only 30% of microsatellite-stable tumors. In vitro, treatments with HDAC inhibitors, butyrate and valproate restored CXCL12 expression in three colon cell lines, increased acetylation of histone H3 within the CXCL12 promoter and inhibited cell migration. In vivo, valproate diminished (65%) the number of intestinal tumors in APC mutant mice, slowed down xenograft tumor growth concomitant to restored CXCL12 expression. Finally we identified loss of PCAF expression in tumor samples and showed that forced expression of PCAF in colon cancer cell lines restored CXCL12 expression. Thus, reduced PCAF expression may participate to CXCL12 promoter hypoacetylation and its subsequent loss of expression. Our study is of potential clinical interest because agents that promote or maintain histone acetylation through HDAC inhibition and/or HAT stimulation, may help to lower colon adenoma/carcinoma incidence, especially in high-risk families, or could be included in therapeutic protocols to treat advanced colon cancer.
Subject(s)
Chemokine CXCL12/biosynthesis , Colonic Neoplasms/pathology , Gene Expression Regulation, Neoplastic/genetics , Histones/genetics , Acetylation , Adenocarcinoma/pathology , Adenoma/pathology , Adult , Aged , Animals , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Chemokine CXCL12/genetics , Colonic Neoplasms/genetics , DNA Methylation , Down-Regulation , Female , Heterografts , Histones/metabolism , Humans , Male , Mice , Mice, Mutant Strains , Middle AgedABSTRACT
On the basis of phylogenetic analyses, we uncovered a variant of the CDX2 homeobox gene, a major regulator of the development and homeostasis of the gut epithelium, also involved in cancer. This variant, miniCDX2, is generated by alternative splicing coupled to alternative translation initiation, and contains the DNA-binding homeodomain but is devoid of transactivation domain. It is predominantly expressed in crypt cells, whereas the CDX2 protein is present in crypt cells but also in differentiated villous cells. Functional studies revealed a dominant-negative effect exerted by miniCDX2 on the transcriptional activity of CDX2, and conversely similar effects regarding several transcription-independent functions of CDX2. In addition, a regulatory role played by the CDX2 and miniCDX2 homeoproteins on their pre-mRNA splicing is displayed, through interactions with splicing factors. Overexpression of miniCDX2 in the duodenal Brunner glands leads to the expansion of the territory of these glands and ultimately to brunneroma. As a whole, this study characterized a new and original variant of the CDX2 homeobox gene. The production of this variant represents not only a novel level of regulation of this gene, but also a novel way to fine-tune its biological activity through the versatile functions exerted by the truncated variant compared to the full-length homeoprotein. This study highlights the relevance of generating protein diversity through alternative splicing in the gut and its diseases.