Marc P. Stemmler

The p300/CBP acetyltransferases function as transcriptional coactivators of β‐catenin in vertebrates

Wnt growth factors regulate a variety of developmental processes by altering specific gene expression patterns. In vertebrates β‐catenin acts as transcriptional activator, which is needed to overcome target gene repression by Groucho/TLE proteins, and to permit promoter activation as the final consequence of Wnt signaling. However, the molecular mechanisms of transcriptional activation by β‐catenin are only poorly understood. Here we demonstrate that the closely related acetyltransferases p300 and CBP potentiate β‐catenin‐mediated activation of the siamois promoter, a known Wnt target. β‐catenin and p300 also synergize to stimulate a synthetic reporter gene construct, whereas activation of the cyclin D1 promoter by β‐catenin is refractory to p300 stimulation. Axis formation and activation of the β‐catenin target genes siamois and Xnr‐3 in Xenopus embryos are sensitive to the E1A oncoprotein, a known inhibitor of p300/CBP. The C‐terminus of β‐catenin interacts directly with a region overlapping the CH‐3 domain of p300. p300 could participate in alleviating promoter repression imposed by chromatin structure and in recruiting the basal transcription machinery to promoters of particular Wnt target genes.

The EMT-activator Zeb1 is a key factor for cell plasticity and promotes metastasis in pancreatic cancer

Metastasis is the major cause of cancer-associated death. Partial activation of the epithelial-to-mesenchymal transition program (partial EMT) was considered a major driver of tumour progression from initiation to metastasis. However, the role of EMT in promoting metastasis has recently been challenged, in particular concerning effects of the Snail and Twist EMT transcription factors (EMT-TFs) in pancreatic cancer. In contrast, we show here that in the same pancreatic cancer model, driven by Pdx1-cre-mediated activation of mutant Kras and p53 (KPC model), the EMT-TF Zeb1 is a key factor for the formation of precursor lesions, invasion and notably metastasis. Depletion of Zeb1 suppresses stemness, colonization capacity and in particular phenotypic/metabolic plasticity of tumour cells, probably causing the observed in vivo effects. Accordingly, we conclude that different EMT-TFs have complementary subfunctions in driving pancreatic tumour metastasis. Therapeutic strategies should consider these potential specificities of EMT-TFs to target these factors simultaneously.

Identification of a Promoter-specific Transcriptional Activation Domain at the C Terminus of the Wnt Effector Protein T-cell Factor 4

Wnt growth factors control numerous cell fate decisions in development by altering specific gene expression patterns through the activity of heterodimeric transcriptional activators. These consist of β-catenin and one of the four members of the T-cell factor (TCF) family of DNA-binding proteins. How can the Wnt/β-catenin pathway control various sets of target genes in distinct cellular settings with such a limited number of nuclear effectors? Here we asked whether different TCF proteins could perform specific, nonredundant functions at natural β-catenin/TCF-regulated promoters. We found that TCF4E but not LEF1 supported β-catenin-dependent activation of the Cdx1promoter, whereas LEF1 specifically activated the Siamoispromoter. Deletion of a C-terminal domain of TCF4E preventedCdx1 promoter induction. A chimeric protein consisting of LEF1 and the C terminus of TCF4E was fully functional. Therefore, the TCF4E C terminus harbors a promoter-specific transactivation domain. This domain influences the DNA binding properties of TCF4 and additionally mediates an interaction with the transcriptional coactivator p300. Apparently, the C terminus of TCF4E cooperates with β-catenin and p300 to form a specialized transcription factor complex that specifically supports the activation of the Cdx1promoter.

The Epithelial Cell Adhesion Molecule EpCAM Is Required for Epithelial Morphogenesis and Integrity during Zebrafish Epiboly and Skin Development

The aberrant expression of the transmembrane protein EpCAM is associated with tumor progression, affecting different cellular processes such as cell–cell adhesion, migration, proliferation, differentiation, signaling, and invasion. However, the in vivo function of EpCAM still remains elusive due to the lack of genetic loss-of-function studies. Here, we describe epcam (tacstd) null mutants in zebrafish. Maternal-zygotic mutants display compromised basal protrusive activity and epithelial morphogenesis in cells of the enveloping layer (EVL) during epiboly. In partial redundancy with E-cadherin (Ecad), EpCAM made by EVL cells is further required for cell–cell adhesion within the EVL and, possibly, for proper attachment of underlying deep cells to the inner surface of the EVL, thereby also affecting deep cell epiboly movements. During later development, EpCAM per se becomes indispensable for epithelial integrity within the periderm of the skin, secondarily leading to disrupted morphology of the underlying basal epidermis and moderate hyper-proliferation of skin cells. On the molecular level, EVL cells of epcam mutant embryos display reduced levels of membranous Ecad, accompanied by an enrichment of tight junction proteins and a basal extension of apical junction complexes (AJCs). Our data suggest that EpCAM acts as a partner of E-cadherin to control adhesiveness and integrity as well as plasticity and morphogenesis within simple epithelia. In addition, EpCAM is required for the interaction of the epithelia with underlying cell layers.

Gene replacement reveals a specific role for E-cadherin in the formation of a functional trophectoderm

During mammalian embryogenesis the trophectoderm represents the first epithelial structure formed. The cell adhesion molecule E-cadherin is ultimately necessary for the transition from compacted morula to the formation of the blastocyst to ensure correct establishment of adhesion junctions in the trophectoderm. Here, we analyzed to what extent E-cadherin confers unique adhesion and signaling properties in trophectoderm formation in vivo. Using a gene replacement approach, we introduced N-cadherin cDNA into the E-cadherin genomic locus. We show that the expression of N-cadherin driven from the E-cadherin locus reflects the expression pattern of endogenous E-cadherin. Heterozygous mice co-expressing E- and N-cadherin are vital and show normal embryonic development. Interestingly, N-cadherin homozygous mutant embryos phenocopy E-cadherin-null mutant embryos. Upon removal of the maternal E-cadherin, we demonstrate that N-cadherin is able to provide sufficient cellular adhesion to mediate morula compaction, but is insufficient for the subsequent formation of a fully polarized functional trophectoderm. When ES cells were isolated from N-cadherin homozygous mutant embryos and teratomas were produced, these ES cells differentiated into a large variety of tissue-like structures. Importantly, different epithelial-like structures expressing N-cadherin were formed, including respiratory epithelia, squamous epithelia with signs of keratinization and secretory epithelia with goblet cells. Thus, N-cadherin can maintain epithelia in differentiating ES cells, but not during the formation of the trophectoderm. Our results point to a specific and unique function for E-cadherin during mouse preimplantation development.

ZEB1-associated drug resistance in cancer cells is reversed by the class I HDAC inhibitor mocetinostat

Therapy resistance is a major clinical problem in cancer medicine and crucial for disease relapse and progression. Therefore, the clinical need to overcome it, particularly for aggressive tumors such as pancreatic cancer, is very high. Aberrant activation of an epithelial–mesenchymal transition (EMT) and an associated cancer stem cell phenotype are considered a major cause of therapy resistance. Particularly, the EMT‐activator ZEB1 was shown to confer stemness and resistance. We applied a systematic, stepwise strategy to interfere with ZEB1 function, aiming to overcome drug resistance. This led to the identification of both its target gene miR‐203 as a major drug sensitizer and subsequently the class I HDAC inhibitor mocetinostat as epigenetic drug to interfere with ZEB1 function, restore miR‐203 expression, repress stemness properties, and induce sensitivity against chemotherapy. Thereby, mocetinostat turned out to be more effective than other HDAC inhibitors, such as SAHA, indicating the relevance of the screening strategy. Our data encourage the application of mechanism‐based combinations of selected epigenetic drugs with standard chemotherapy for the rational treatment of aggressive solid tumors, such as pancreatic cancer.

E-cadherin intron 2 contains cis-regulatory elements essential for gene expression

Cadherin-mediated cell-cell adhesion plays important roles in mouse embryonic development, and changes in cadherin expression are often linked to morphogenetic events. For proper embryonic development and organ formation, the expression of E-cadherin must be tightly regulated. Dysregulated expression during tumorigenesis confers invasiveness and metastasis. Except for the E-box motifs in the E-cadherin promoter, little is known about the existence and location of cis-regulatory elements controlling E-cadherin gene expression. We have examined putative cis-regulatory elements in the E-cadherin gene and we show a pivotal role for intron 2 in activating transcription. Upon deleting the genomic intron 2 entirely, the E-cadherin locus becomes completely inactive in embryonic stem cells and during early embryonic development. Later in development, from E11.5 onwards, the locus is activated only weakly in the absence of intron 2 sequences. We demonstrate that in differentiated epithelia, intron 2 sequences are required both to initiate transcriptional activation and additionally to maintain E-cadherin expression. Detailed analysis also revealed that expression in the yolk sac is intron 2 independent, whereas expression in the lens and the salivary glands absolutely relies on cis-regulatory sequences of intron 2. Taken together, our findings reveal a complex mechanism of gene regulation, with a vital role for the large intron 2.

A self-enforcing CD44s/ZEB1 feedback loop maintains EMT and stemness properties in cancer cells

Invasion and metastasis of carcinomas are often activated by induction of aberrant epithelial–mesenchymal transition (EMT). This is mainly driven by the transcription factor ZEB1, promoting tumor‐initiating capacity correlated with increased expression of the putative stem cell marker CD44. However, the direct link between ZEB1, CD44 and tumourigenesis is still enigmatic. Remarkably, EMT‐induced repression of ESRP1 controls alternative splicing of CD44, causing a shift in the expression from the variant CD44v to the standard CD44s isoform. We analyzed whether CD44 and ZEB1 regulate each other and show that ZEB1 controls CD44s splicing by repression of ESRP1 in breast and pancreatic cancer. Intriguingly, CD44s itself activates the expression of ZEB1, resulting in a self‐sustaining ZEB1 and CD44s expression. Activation of this novel CD44s‐ZEB1 regulatory loop has functional impact on tumor cells, as evident by increased tumor‐sphere initiation capacity, drug‐resistance and tumor recurrence. In summary, we identified a self‐enforcing feedback loop that employs CD44s to activate ZEB1 expression. This renders tumor cell stemness independent of external stimuli, as ZEB1 downregulates ESRP1, further promoting CD44s isoform synthesis.

A nonneural epithelial domain of embryonic cranial neural folds gives rise to ectomesenchyme

The neural crest is generally believed to be the embryonic source of skeletogenic mesenchyme (ectomesenchyme) in the vertebrate head and other derivatives, including pigment cells and neurons and glia of the peripheral nervous system. Although classical transplantation experiments leading to this conclusion assumed that embryonic neural folds were homogeneous epithelia, we reported that embryonic cranial neural folds contain spatially and phenotypically distinct domains, including a lateral nonneural domain with cells that coexpress E-cadherin and PDGFRα and a thickened mediodorsal neuroepithelial domain where these proteins are reduced or absent. We now show that Wnt1-Cre is expressed in the lateral nonneural epithelium of rostral neural folds and that cells coexpressing Cre-recombinase and PDGFRα delaminate precociously from some of this nonneural epithelium. We also show that ectomesenchymal cells exhibit β-galactosidase activity in embryos heterozygous for an Ecad-lacZ reporter knock- in allele. We conclude that a lateral nonneural domain of the neural fold epithelium, which we call “metablast,” is a source of ectomesenchyme distinct from the neural crest. We suggest that closer analysis of the origin of ectomesenchyme might help to understand (i) the molecular-genetic regulation of development of both neural crest and ectomesenchyme lineages; (ii) the early developmental origin of skeletogenic and connective tissue mesenchyme in the vertebrate head; and (iii) the presumed origin of head and branchial arch skeletal and connective tissue structures during vertebrate evolution.

Analysis of regulatory elements of E‐cadherin with reporter gene constructs in transgenic mouse embryos

Proper regulation of E‐cadherin–mediated cell adhesion is important during early embryonic development and in organogenesis. In mice, E‐cadherin is expressed from the fertilized egg onward and becomes down‐regulated during gastrulation in mesoderm and its derivatives, but its expression is maintained in all epithelia. E‐cadherin promoter analyses led to the identification of binding sites for two transcriptional repressors, Snail and SIP1, which are able to mediate down‐regulation in vitro, but little is known about the regulatory elements that govern E‐cadherin transcriptional activity in vivo. Here, we compared the developmentally regulated expression of a series of lacZ‐reporter transgenes fused to different sequences of the murine E‐cadherin gene between −6 kb, including the promoter, and +16 kb, covering one third of intron 2. Four different segments with distinct regulatory properties were identified. The promoter fragment from +0.1 to −1.5 kb remains inactive in most cases but occasionally induces ectopic expression in mesodermal tissues, although it contains binding sites for the repressors Snail and SIP1. This promoter fragment also lacks positive elements needed for the activation of transcription in ectoderm and endoderm. Sequences from −1.5 to −6 kb harbor regulatory elements for brain‐specific expression and, in addition, insulator or silencer elements, because they are consistently inactive in the mesoderm. Only if sequences from +0.1 to +11 kb are combined with the promoter fragments is E‐cadherin–specific transgene expression observed in endoderm and certain epithelia. Sequences between +11 and +16 kb contain cis‐active elements that generally enhance transcription. Our analyses show that E‐cadherin expression is governed by a complex interplay of multiple regulatory regions dispersed throughout large parts of the locus. Developmental Dynamics 227:238–245, 2003.