Regulation of the THRA gene, encoding the thyroid hormone nuclear receptor TRα1, in intestinal lesions.

The THRA gene, encoding the thyroid hormone nuclear receptor TRα1, is expressed in an increasing gradient at the bottom of intestinal crypts, overlapping with high Wnt and Notch activities. Importantly, THRA is upregulated in colorectal cancers, particularly in the high-Wnt molecular subtype. The basis of this specific and/or altered expression pattern has remained unknown. To define the mechanisms controlling THRA transcription and TRα1 expression, we used multiple in vitro and ex vivo approaches. Promoter analysis demonstrated that transcription factors important for crypt homeostasis and altered in colorectal cancers, such as transcription factor 7-like 2 (TCF7L2; Wnt pathway), recombining binding protein suppressor of hairless (RBPJ; Notch pathway), and homeobox protein CDX2 (epithelial cell identity), modulate THRA activity. Specifically, although TCF7L2 and CDX2 stimulated THRA, RBPJ induced its repression. In-depth analysis of the Wnt-dependent increase showed direct regulation of the THRA promoter in cells and of TRα1 expression in murine enteroids. Given our previous results on the control of the Wnt pathway by TRα1, our new results unveil a complex regulatory loop and synergy between these endocrine and epithelial-cell-intrinsic signals. Our work describes, for the first time, the regulation of the THRA gene in specific cell and tumor contexts.

Concurrent CDX2 cis-deregulation and UBTF::ATXN7L3 fusion define a novel high-risk subtype of B-cell ALL.

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.

A Core Response to the CDX2 Homeoprotein During Development and in Pathologies.

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.

CDX2 expression in the hematopoietic lineage promotes leukemogenesis via TGFß inhibition.

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.

Fine-tuning and autoregulation of the intestinal determinant and tumor suppressor homeobox gene CDX2 by alternative splicing.

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.

Distinct mechanisms for opposite functions of homeoproteins Cdx2 and HoxB7 in double-strand break DNA repair in colon cancer cells.

Homeobox genes, involved in embryonic development and tissues homeostasis in adults, are often deregulated in cancer, but their relevance in pathology is far from being fully elucidated. In colon cancers, we report that the homeoproteins HoxB7 and Cdx2 exhibit different heterogeneous patterns, Cdx2 being localized in moderately altered neoplasic glands in contrast to HoxB7 which predominates in poorly-differentiated areas; they are coexpressed in few cancer cells. In human colon cancer cells, both homeoproteins interact with the DNA repair factor KU70/80, but functional studies reveal opposite effects: HoxB7 stimulates DNA repair and cell survival upon etoposide treatment, whereas Cdx2 inhibits both processes. The stimulatory effect of HoxB7 on DNA repair requires the transactivation domain linked to the homeodomain involved in the interaction with KU70/80, whereas the transactivation domain of Cdx2 is dispensable for its inhibitory function, which instead needs the homeodomain to interact with KU70/80 and the C-terminal domain. Thus, HoxB7 and Cdx2 respectively use transcription-dependent and -independent mechanisms to stimulate and inhibit DNA repair. In addition, in cells co-expressing both homeoproteins, Cdx2 lessens DNA repair activity through a novel mechanism of inhibition of the transcriptional function of HoxB7, whereby Cdx2 forms a molecular complex with HoxB7 and prevents it to recognize its target in the chromatin. These results point out the complex interplay between the DSB DNA repair activity and the homeoproteins HoxB7 and Cdx2 in colon cancer cells, making the balance between these factors a determinant and a potential indicator of the efficacy of genotoxic drugs.

Extending the functions of the homeotic transcription factor Cdx2 in the digestive system through nontranscriptional activities.

The homeoprotein encoded by the intestinal-specific Cdx2 gene is a major regulator of gut development and homeostasis, also involved in colon cancer as well as in intestinal-type metaplasias when it is abnormally expressed outside the gut. At the molecular level, structure/function studies have demonstrated that the Cdx2 protein is a transcription factor containing a conserved homeotic DNA-binding domain made of three alpha helixes arranged in a helix-turn-helix motif, preceded by a transcriptional domain and followed by a regulatory domain. The protein interacts with several thousand sites on the chromatin and widely regulates intestinal functions in stem/progenitor cells as well as in mature differentiated cells. Yet, this transcription factor also acts trough original nontranscriptional mechanisms. Indeed, the identification of novel protein partners of Cdx2 and also of a splicing variant revealed unexpected functions in the control of signaling pathways like the Wnt and NF-κB pathways, in double-strand break DNA repair and in premessenger RNA splicing. These novel functions of Cdx2 must be considered to fully understand the complexity of the role of Cdx2 in the healthy intestine and in diseases.

Cdx2 homeoprotein inhibits non-homologous end joining in colon cancer but not in leukemia cells.

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.

CDX2 in congenital gut gastric-type heteroplasia and intestinal-type Meckel diverticula.

The mechanisms that determine organ identity along the digestive tract in humans are poorly understood. Here we describe the rare case of a young patient who presented with congenital gastric-type heteroplasia in the midjejunum. The lesions, located along the antimesenteric midline of the gut, were made of histologically and functionally normal gastric epithelium without inflammation or in situ/invasive carcinoma. They resembled the anatomy of the lesions developing in the mouse gut as a result of haploinsufficiency of the Cdx2 homeobox gene. The lesions were devoid of CDX2 but without mutation in the coding sequence or in a cis-regulatory element required for intestine-specific expression. Combining these data with the CDX2 expression pattern established from human embryos and cases of Meckel diverticula, we propose a scenario for this patient's presentation, in which CDX2 was missing at the site of ventral closure during gut morphogenesis, with subsequent default differentiation into gastric instead of intestinal tissue. Altogether, these observations argue in favor of a pivotal role played by CDX2 in determining intestinal identity during human embryonic development, as previously shown experimentally in mice.

Multiple regulatory regions control the complex expression pattern of the mouse Cdx2 homeobox gene.

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.

Multiple-contrast X-ray micro-CT visualization of colon malformations and tumours in situ in living mice.

The development of new therapeutic approaches against colorectal cancer requires preclinical studies in mice. In vivo imaging could greatly facilitate these trials, but the small size of the animals is a major limitation for the direct visualization of intestinal tissue. Here we report a method of in vivo imaging of the mouse intestine based on X-ray micro-computed tomography using multiple contrast agents. This method was validated in the model of non-cancerous polyp-like heteroplasia that spontaneously develops in the caecum area of Cdx2+/- mutant mice and in the model of colon adenocarcinoma induced by administration of the chemical carcinogen azoxymethane. As a simple and non-invasive method, multiple-contrast X-ray micro-computed tomography is appropriate for pre-clinical studies of intestinal diseases in living mice.

Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies.

In Caenorhabditis elegans, the Mex-3 protein is a translational regulator that specifies the posterior blastomere identity in the early embryo and contributes to the maintenance of the germline totipotency. We have now identified a family of four homologous human Mex-3 genes, called hMex-3A to -3D that encode proteins containing two heterogeneous nuclear ribonucleoprotein K homology (KH) domains and one carboxy-terminal RING finger module. The hMex-3 are phosphoproteins that bind RNA through their KH domains and shuttle between the nucleus and the cytoplasm via the CRM1-dependent export pathway. Our analysis further revealed that hMex-3A and hMex-3B, but not hMex-3C, colocalize with both the hDcp1a decapping factor and Argonaute (Ago) proteins in processing bodies (P bodies), recently characterized as centers of mRNA turnover. Taken together, these findings indicate that hMex-3 proteins constitute a novel family of evolutionarily conserved RNA-binding proteins, differentially recruited to P bodies and potentially involved in post-transcriptional regulatory mechanisms.

The microenvironment controls CDX2 homeobox gene expression in colorectal cancer cells.

The homeobox gene CDX2 plays a major role in development, especially in the gut, and it also acts as a tumor suppressor in the adult colon. Using orthotopic and heterotopic xenografts of human primary colorectal tumor cells and cell lines in nude mice, we addressed the effect of the microenvironment on CDX2 expression. In cells expressing CDX2 at a high level in culture, this level was maintained in subcutaneous grafts but was reduced when implanted into the cecum wall. Reciprocally, in cells with low CDX2 expression in culture, the level remained low in grafts into the cecum wall but was stimulated subcutaneously. In vitro co-cultures showed that CDX2 expression was activated in cells grown on layers of skin fibroblasts but not on intestinal fibroblasts. The stimulation was transcriptional, as assessed by transfection experiments with reporter plasmids containing the murine Cdx2 promoter. Together, these data demonstrate experimentally that CDX2 expression is adaptable and strongly dependent on the microenvironment surrounding the tumor cells. We exclude a role of the Notch pathway in this regulation. The regulation of CDX2 by the microenvironment might be relevant during the process of metastatic dissemination when the gene is transiently turned down in invasive cells.

Functional interaction between the homeoprotein CDX1 and the transcriptional machinery containing the TATA-binding protein.

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.

Frequent rearrangements and amplification of the CDX2 homeobox gene in human sporadic colorectal cancers with chromosomal instability.

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.

Phosphorylation of the homeotic tumor suppressor Cdx2 mediates its ubiquitin-dependent proteasome degradation.

The Caudal-related homeodomain transcription factor Cdx2 plays a key role in intestinal cell fate determination. Reduction of Cdx2 expression is a feature of many human colon carcinomas and inactivation of one cdx2 allele facilitates the development of invasive adenocarcinoma in the murine colon. Here, we investigated the post-translational regulation of Cdx2. We showed that various forms of Cdx2 coexist in the intestine and colon cancer cell lines, some of them being phosphorylated forms. We found that cyclin-dependent kinase 2 phosphorylated Cdx2 in vitro and in vivo. Using site-specific mutagenesis, we identified serine 281 as a new key residue for Cdx2 phosphorylation. Intriguingly, serine 281 belongs to a conserved motif of four evenly spaced serines (the 4S motif) similar to the one controlling beta-catenin degradation by the proteasome pathway. A nonphosphorylated mutant Cdx2 lacking the 4S motif (4S>A) exhibited reduced polyubiquitination upon proteasome inhibition and increased stability compared to wild-type Cdx2. In addition, we found that this mutant was less efficient to suppress colony formation than wild-type Cdx2. Thus, our data highlight a novel post-translational mechanism controlling Cdx2 degradation via phosphorylation and polyubiquitination, which may be of importance for intestinal development and cancer.

Down-regulation of the homeodomain factor Cdx2 in colorectal cancer by collagen type I: an active role for the tumor environment in malignant tumor progression.

The homeobox transcription factor Cdx2 specifies intestinal development and homeostasis and is considered a tumor suppressor in colorectal carcinogenesis. However, Cdx2 mutations are rarely found. Invasion of colorectal cancer is characterized by a transient loss of differentiation and nuclear accumulation of the oncoprotein beta-catenin in budding tumor cells. Strikingly, this is reversed in growing metastases, indicating that tumor progression is a dynamic process that is not only driven by genetic alterations but also regulated by the tumor environment. Here we describe a transient loss of Cdx2 in budding tumor cells at the tumor host interface, and reexpression of Cdx2 in metastases. Cell culture experiments show that collagen type I, through beta(1) integrin signaling, triggers a transient transcriptional down-regulation of Cdx2 and its intestine-specific target gene sucrase isomaltase, associated with a loss of differentiation. These data indicate an active role for the tumor environment in malignant tumor progression.

Down-regulation of the tumor suppressor PTEN by the tumor necrosis factor-alpha/nuclear factor-kappaB (NF-kappaB)-inducing kinase/NF-kappaB pathway is linked to a default IkappaB-alpha autoregulatory loop.

The PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor gene affects multiple cellular processes including cell growth, proliferation, and cell migration by antagonizing phosphatidylinositol 3-kinase (PI3K). However, mechanisms by which PTEN expression is regulated have not been studied extensively. Similar to PTEN, tumor necrosis factor-alpha (TNF-alpha) affects a wide spectrum of diseases including inflammatory processes and cancer by acting as a mediator of apoptosis, inflammation, and immunity. In this study, we show that treatment of cancer cell lines with TNF-alpha decreases PTEN expression. In addition, overexpression of TNF-alpha downstream signaling targets, nuclear factor-kappaB (NF-kappaB)-inducing kinase (NIK) and p65 nuclear factor NF-kappaB, lowers PTEN expression, suggesting that TNF-alpha-induced down-regulation of PTEN is mediated through a TNF-alpha/NIK/NF-kappaB pathway. Down-regulation of PTEN by NIK/NF-kappaB results in activation of the PI3K/Akt pathway and augmentation of TNF-alpha-induced PI3K/Akt stimulation. Importantly, we demonstrate that this effect is associated with a lack of an inhibitor of kappaB (IkappaB)-alpha autoregulatory loop. Moreover, these findings suggest the interaction between PI3K/Akt and NF-kappaB via transcriptional regulation of PTEN and offer one possible explanation for increased tumorigenesis in systems in which NF-kappaB is chronically activated. In such a tumor system, these findings suggest a positive feedback loop whereby Akt activation of NF-kappaB further stimulates Akt via down-regulation of the PI3K inhibitor PTEN.

Differential regulation of the glucose-6-phosphatase TATA box by intestine-specific homeodomain proteins CDX1 and CDX2.

Glucose-6-phosphatase (Glc6Pase), the last enzyme of gluconeogenesis, is only expressed in the liver, kidney and small intestine. The expression of the Glc6Pase gene exhibits marked specificities in the three tissues in various situations, but the molecular basis of the tissue specificity is not known. The presence of a consensus binding site of CDX proteins in the minimal Glc6Pase gene promoter has led us to consider the hypothesis that these intestine-specific CDX factors could be involved in the Glc6Pase-specific expression in the small intestine. We first show that the Glc6Pase promoter is active in both hepatic HepG2 and intestinal CaCo2 cells. Using gel shift mobility assay, mutagenesis and competition experiments, we show that both CDX1 and CDX2 can bind the minimal promoter, but only CDX1 can transactivate it. Consistently, intestinal IEC6 cells stably overexpressing CDX1 exhibit induced expression of the Glc6Pase protein. We demonstrate that a TATAAAA sequence, located in position -31/-25 relating to the transcription start site, exhibits separable functions in the preinitiation of transcription and the transactivation by CDX1. Disruption of this site dramatically suppresses both basal transcription and the CDX1 effect. The latter may be restored by inserting a couple of CDX- binding sites in opposite orientation similar to that found in the sucrase-isomaltase promoter. We also report that the specific stimulatory effect of CDX1 on the Glc6Pase TATA-box, compared to CDX2, is related to the fact that CDX1, but not CDX2, can interact with the TATA-binding protein. Together, these data strongly suggest that CDX proteins could play a crucial role in the specific expression of the Glc6Pase gene in the small intestine. They also suggest that CDX transactivation might be essential for intestine gene expression, irrespective of the presence of a functional TATA box.