Correction: Antón-García et al. TGFß1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation. Cancers 2023, 15, 588.

In the original publication [...].

Transcriptional activity mediated by ß-CATENIN and TCF/LEF family members is completely dispensable for survival and propagation of multiple human colorectal cancer cell lines.

Unrestrained transcriptional activity of ß-CATENIN and its binding partner TCF7L2 frequently underlies colorectal tumor initiation and is considered an obligatory oncogenic driver throughout intestinal carcinogenesis. Yet, the TCF7L2 gene carries inactivating mutations in about 10% of colorectal tumors and is non-essential in colorectal cancer (CRC) cell lines. To determine whether CRC cells acquire TCF7L2-independence through cancer-specific compensation by other T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) family members, or rather lose addiction to ß-CATENIN/TCF7L2-driven gene expression altogether, we generated multiple CRC cell lines entirely negative for TCF/LEF or ß-CATENIN expression. Survival of these cells and the ability to propagate them demonstrate their complete ß-CATENIN- and TCF/LEF-independence. Nonetheless, one ß-CATENIN-deficient cell line eventually became senescent, and absence of TCF/LEF proteins and ß-CATENIN consistently impaired CRC cell proliferation, reminiscent of mitogenic effects of WNT/ß-CATENIN signaling in the healthy intestine. Despite this common phenotype, ß-CATENIN-deficient cells exhibited highly cell-line-specific gene expression changes with little overlap between ß-CATENIN- and TCF7L2-dependent transcriptomes. Apparently, ß-CATENIN and TCF7L2 independently control sizeable fractions of their target genes. The observed divergence of ß-CATENIN and TCF7L2 transcriptional programs, and the finding that neither ß-CATENIN nor TCF/LEF activity is strictly required for CRC cell survival has important implications when evaluating these factors as potential drug targets.

TGFß1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation.

Epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, the main cause of cancer-related mortality. Growing evidence that SNAIL and ZEB transcription factors, typically portrayed as master regulators of EMT, may be dispensable for this process, led us to re-investigate its mechanistic underpinnings. For this, we used an unbiased computational approach that integrated time-resolved analyses of chromatin structure and differential gene expression, to predict transcriptional regulators of TGFß1-inducible EMT in the MCF10A mammary epithelial cell line model. Bioinformatic analyses indicated comparatively minor contributions of SNAIL proteins and ZEB1 to TGFß1-induced EMT, whereas the AP-1 subunit JUNB was anticipated to have a much larger impact. CRISPR/Cas9-mediated loss-of-function studies confirmed that TGFß1-induced EMT proceeded independently of SNAIL proteins and ZEB1. In contrast, JUNB was necessary and sufficient for EMT in MCF10A cells, but not in A549 lung cancer cells, indicating cell-type-specificity of JUNB EMT-regulatory capacity. Nonetheless, the JUNB-dependence of EMT-associated transcriptional reprogramming in MCF10A cells allowed to define a gene expression signature which was regulated by TGFß1 in diverse cellular backgrounds, showed positively correlated expression with TGFß signaling in multiple cancer transcriptomes, and was predictive of patient survival in several cancer types. Altogether, our findings provide novel mechanistic insights into the context-dependent control of TGFß1-driven EMT and thereby may lead to improved diagnostic and therapeutic options.

Canonical TGFß signaling induces collective invasion in colorectal carcinogenesis through a Snail1- and Zeb1-independent partial EMT.

Local invasion is the initial step towards metastasis, the main cause of cancer mortality. In human colorectal cancer (CRC), malignant cells predominantly invade as cohesive collectives and may undergo partial epithelial-mesenchymal transition (pEMT) at the invasive front. How this particular mode of stromal infiltration is generated is unknown. Here we investigated the impact of oncogenic transformation and the microenvironment on tumor cell invasion using genetically engineered organoids as CRC models. We found that inactivation of the Apc tumor suppressor combined with expression of oncogenic KrasG12D and dominant-negative Trp53R172H did not cell-autonomously induce invasion in vitro. However, oncogenic transformation primed organoids for activation of a collective invasion program upon exposure to the prototypical microenvironmental factor TGFß1. Execution of this program co-depended on a permissive extracellular matrix which was further actively remodeled by invading organoids. Although organoids shed some epithelial properties particularly at the invasive edge, TGFß1-stimulated organoids largely maintained epithelial gene expression while additionally implementing a mesenchymal transcription pattern, resulting in a pEMT phenotype that did not progress to a fully mesenchymal state. Notably, while TGFß1 induced pEMT and promoted collective invasion, it abrogated self-renewal capacity of TKA organoids which correlated with the downregulation of intestinal stem cell (ISC) marker genes. Mechanistically, induction of the non-progressive pEMT required canonical TGFß signaling mediated by Smad transcription factors (TFs), whereas the EMT master regulators Snail1 and Zeb1 were dispensable. Gene expression profiling provided further evidence for pEMT of TGFß1-treated organoids and showed that their transcriptomes resemble those of human poor prognosis CMS4 cancers which likewise exhibit pEMT features. We propose that collective invasion in colorectal carcinogenesis is triggered by microenvironmental stimuli through activation of a novel, transcription-mediated form of non-progressive pEMT independently of classical EMT regulators.

SMAD4 mutations do not preclude epithelial-mesenchymal transition in colorectal cancer.

Transforming growth factor beta (TGFß) superfamily signaling is a prime inducer of epithelial-mesenchymal transitions (EMT) that foster cancer cell invasion and metastasis, a major cause of cancer-related deaths. Yet, TGFß signaling is frequently inactivated in human tumor entities including colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD) with a high proportion of mutations incapacitating SMAD4, which codes for a transcription factor (TF) central to canonical TGFß and bone morphogenetic protein (BMP) signaling. Beyond its role in initiating EMT, SMAD4 was reported to crucially contribute to subsequent gene regulatory events during EMT execution. It is therefore widely assumed that SMAD4-mutant (SMAD4mut) cancer cells are unable to undergo EMT. Here, we scrutinized this notion and probed for potential SMAD4-independent EMT execution using SMAD4mut CRC cell lines. We show that SMAD4mut cells exhibit morphological changes, become invasive, and regulate EMT marker genes upon induction of the EMT-TF SNAIL1. Furthermore, SNAIL1-induced EMT in SMAD4mut cells was found to be entirely independent of TGFß/BMP receptor activity. Global assessment of the SNAIL1-dependent transcriptome confirmed the manifestation of an EMT gene regulatory program in SMAD4mut cells highly related to established EMT signatures. Finally, analyses of human tumor transcriptomes showed that SMAD4 mutations are not underrepresented in mesenchymal tumor samples and that expression patterns of EMT-associated genes are similar in SMAD4mut and SMAD4 wild-type (SMAD4wt) cases. Altogether, our findings suggest that alternative TFs take over the gene regulatory functions of SMAD4 downstream of EMT-TFs, arguing for considerable plasticity of gene regulatory networks operating in EMT execution. Further, they establish that EMT is not categorically precluded in SMAD4mut tumors, which is relevant for their diagnostic and therapeutic evaluation.

Canonical BMP Signaling Executes Epithelial-Mesenchymal Transition Downstream of SNAIL1.

Epithelial-mesenchymal transition (EMT) is a pivotal process in development and disease. In carcinogenesis, various signaling pathways are known to trigger EMT by inducing the expression of EMT transcription factors (EMT-TFs) like SNAIL1, ultimately promoting invasion, metastasis and chemoresistance. However, how EMT is executed downstream of EMT-TFs is incompletely understood. Here, using human colorectal cancer (CRC) and mammary cell line models of EMT, we demonstrate that SNAIL1 critically relies on bone morphogenetic protein (BMP) signaling for EMT execution. This activity requires the transcription factor SMAD4 common to BMP/TGFß pathways, but is TGFß signaling-independent. Further, we define a signature of BMP-dependent genes in the EMT-transcriptome, which orchestrate EMT-induced invasiveness, and are found to be regulated in human CRC transcriptomes and in developmental EMT processes. Collectively, our findings substantially augment the knowledge of mechanistic routes whereby EMT can be effectuated, which is relevant for the conceptual understanding and therapeutic targeting of EMT processes.

Loss of the nuclear Wnt pathway effector TCF7L2 promotes migration and invasion of human colorectal cancer cells.

The transcription factor TCF7L2 is indispensable for intestinal tissue homeostasis where it transmits mitogenic Wnt/ß-Catenin signals in stem and progenitor cells, from which intestinal tumors arise. Yet, TCF7L2 belongs to the most frequently mutated genes in colorectal cancer (CRC), and tumor-suppressive functions of TCF7L2 were proposed. This apparent paradox warrants to clarify the role of TCF7L2 in colorectal carcinogenesis. Here, we investigated TCF7L2 dependence/independence of CRC cells and the cellular and molecular consequences of TCF7L2 loss-of-function. By genome editing we achieved complete TCF7L2 inactivation in several CRC cell lines without loss of viability, showing that CRC cells have widely lost the strict requirement for TCF7L2. TCF7L2 deficiency impaired G1/S progression, reminiscent of the physiological role of TCF7L2. In addition, TCF7L2-negative cells exhibited morphological changes, enhanced migration, invasion, and collagen adhesion, albeit the severity of the phenotypic alterations manifested in a cell-line-specific fashion. To provide a molecular framework for the observed cellular changes, we performed global transcriptome profiling and identified gene-regulatory networks in which TCF7L2 positively regulates the proto-oncogene MYC, while repressing the cell cycle inhibitors CDKN2C/CDKN2D. Consistent with its function in curbing cell motility and invasion, TCF7L2 directly suppresses the pro-metastatic transcription factor RUNX2 and impinges on the expression of cell adhesion molecules. Altogether, we conclude that the proliferation-stimulating activity of TCF7L2 persists in CRC cells. In addition, TCF7L2 acts as invasion suppressor. Despite its negative impact on cell cycle progression, TCF7L2 loss-of-function may thereby increase malignancy, which could explain why TCF7L2 is mutated in a sizeable fraction of colorectal tumors.

SNAIL1 employs ß-Catenin-LEF1 complexes to control colorectal cancer cell invasion and proliferation.

The transcription factor SNAIL1 is a master regulator of epithelial-to-mesenchymal transition (EMT), a process entailing massive gene expression changes. To better understand SNAIL1-induced transcriptional reprogramming we performed time-resolved transcriptome analysis upon conditional SNAIL1 expression in colorectal cancer cells. Gene set variation analyses indicated that SNAIL1 strongly affected features related to cell cycle and Wnt/ß-Catenin signalling. This correlated with upregulation of LEF1, a nuclear binding partner of ß-Catenin. Likewise, transcriptomes of cell lines and colorectal cancers, including poor-prognosis mesenchymal tumours, exhibit positively correlated SNAI1 and LEF1 expression, and elevated LEF1 levels parallel increased patient mortality. To delineate the functional contribution of LEF1 to SNAIL1-induced EMT, we used the CRISPR/Cas9 system to knock-out LEF1 in colorectal cancer cells, and to engineer cells that express LEF1 mutants unable to interact with ß-Catenin. Both complete LEF1-deficiency and prevention of the ß-Catenin-LEF1 interaction impaired the ability of SNAIL1 to elicit expression of an alternative set of Wnt/ß-catenin targets, and to promote cancer cell invasion. Conversely, overexpression of wildtype, but not of mutant LEF1, stimulated alternative Wnt/ß-Catenin target gene expression, and caused cell-cycle arrest. Moreover, like SNAIL1, LEF1 retarded tumour growth in xenotransplantations. Thus, LEF1 phenocopies SNAIL1 with respect to several critical aspects of EMT. Indeed, comparative transcriptomics suggested that 35% of SNAIL1-induced transcriptional changes are attributable to LEF1. However, LEF1 did not autonomously induce EMT. Rather, LEF1 appears to be a strictly ß-Catenin-dependent downstream effector of SNAIL1. Apparently, SNAIL1 employs ß-Catenin-LEF1 complexes to redirect Wnt/ß-Catenin pathway activity towards pro-invasive and anti-proliferative gene expression.

Genome-wide mapping of DNA-binding sites identifies stemness-related genes as directly repressed targets of SNAIL1 in colorectal cancer cells.

At the molecular level, epithelial-to-mesenchymal transition (EMT) necessitates extensive transcriptional reprogramming which is orchestrated by a small group of gene-regulatory factors that include the zinc-finger DNA-binding protein SNAIL1. Although SNAIL1 is a well-known master regulator of EMT, knowledge of its immediate target genes is incomplete. Here, we used ChIP-seq to identify genes directly regulated by SNAIL1 in colorectal adenocarcinoma cells. When comparing the genomic distribution of SNAIL1 to that of the intestinal stem cell (ISC) transcription factors ASCL2 and TCF7L2, we observed a significant overlap. Furthermore, SNAIL1 ChIP-seq peaks are associated with a substantial fraction of ISC signature genes. In two colorectal cancer cell lines, we verified that SNAIL1 decreases ISC marker expression. Likewise, SNAIL1 directly represses the proto-oncogene MYB, and the long noncoding RNA (lncRNA) WiNTRLINC1, a recently described regulator of ASCL2. SNAIL1 targets multiple regulatory elements at the MYB and WiNTRLINC1 loci, and displaces ASCL2 and TCF7L2 from their binding regions at a MYB downstream regulatory element. Correlation analyses and expression profiling showed antiparallel expression of SNAIL1 and MYB in colorectal and breast cancer cell lines and tumor transcriptomes, suggesting that SNAIL1 controls MYB expression in different tissues. MYB loss-of-function attenuated proliferation and impaired clonogenicity in two- and three-dimensional cell cultures. Therefore, SNAIL1-mediated downregulation of MYB and ISC markers like WiNTRLINC1 likely contributes to the decrease in proliferation known to be associated with EMT, while simultaneously abrogating stemness features of colorectal cancer cells. Apparently, the relationship between EMT and stemness varies in different tumor entities.

SNAIL1-mediated downregulation of FOXA proteins facilitates the inactivation of transcriptional enhancer elements at key epithelial genes in colorectal cancer cells.

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.

ZEB1 is neither sufficient nor required for epithelial-mesenchymal transition in LS174T colorectal cancer cells.

Epithelial-mesenchymal transition (EMT) is implicated in metastases formation and acquired therapy resistance in several tumor entities. The two transcription factors SNAIL1 and ZEB1 are thought to be master regulators of EMT and to form a core regulatory network required for EMT-associated transcriptional reprogramming. Yet, inducible EMT models show the sequential upregulation first of SNAIL1 and only subsequently of ZEB1. Therefore, SNAIL1 and ZEB1 might be differentially needed for the onset and propagation of EMT. Here we used LS174T colorectal adenocarcinoma cells which do not express endogenous EMT-inducing transcription factors, to investigate whether ZEB1 is an obligatory downstream mediator of Snail1-induced EMT, and to test whether ZEB1 could elicit an EMT in a background of naïve epithelial cells by itself. However, CRISPR/Cas9-mediated knockout of ZEB1 did not affect the ability of ectopically expressed Snail1 to trigger a complete EMT in ZEB1-deficient LS174T cells. In contrast to Snail1, ectopic ZEB1 had only minor effects on cell morphology and invasive growth in three-dimensional spheroid cultures. In agreement with this, expression of ZEB1 did not lead to repression of epithelial marker genes, and mesenchymal markers were not upregulated by ZEB1. Likewise, ectopic ZEB1 expression did not confer increased chemoresistance. We conclude that ZEB1 is neither required nor sufficient for EMT in LS174T colorectal cancer cells.

Enhancer decommissioning by Snail1-induced competitive displacement of TCF7L2 and down-regulation of transcriptional activators results in EPHB2 silencing.

Transcriptional silencing is a major cause for the inactivation of tumor suppressor genes, however, the underlying mechanisms are only poorly understood. The EPHB2 gene encodes a receptor tyrosine kinase that controls epithelial cell migration and allocation in intestinal crypts. Through its ability to restrict cell spreading, EPHB2 functions as a tumor suppressor in colorectal cancer whose expression is frequently lost as tumors progress to the carcinoma stage. Previously we reported that EPHB2 expression depends on a transcriptional enhancer whose activity is diminished in EPHB2 non-expressing cells. Here we investigated the mechanisms that lead to EPHB2 enhancer inactivation. We show that expression of EPHB2 and SNAIL1 - an inducer of epithelial-mesenchymal transition (EMT) - is anti-correlated in colorectal cancer cell lines and tumors. In a cellular model of Snail1-induced EMT, we observe that features of active chromatin at the EPHB2 enhancer are diminished upon expression of murine Snail1. We identify the transcription factors FOXA1, MYB, CDX2 and TCF7L2 as EPHB2 enhancer factors and demonstrate that Snail1 indirectly inactivates the EPHB2 enhancer by downregulation of FOXA1 and MYB. In addition, Snail1 induces the expression of Lymphoid enhancer factor 1 (LEF1) which competitively displaces TCF7L2 from the EPHB2 enhancer. In contrast to TCF7L2, however, LEF1 appears to repress the EPHB2 enhancer. Our findings underscore the importance of transcriptional enhancers for gene regulation under physiological and pathological conditions and show that SNAIL1 employs a combinatorial mechanism to inactivate the EPHB2 enhancer based on activator deprivation and competitive displacement of transcription factors.

Mathematical modelling suggests a differential impact of ß-transducin repeat-containing protein paralogues on Wnt/ß-catenin signalling dynamics.

The Wnt/ß-catenin signalling pathway is involved in the regulation of a multitude of cellular processes by controlling the concentration of the transcriptional regulator ß-catenin. Proteasomal degradation of ß-catenin is mediated by two ß-transducin repeat-containing protein paralogues, homologous to Slimb protein (HOS) and F-box/WD repeat-containing protein 1A (FWD1), which are functionally interchangeable and thereby considered to function redundantly in the pathway. HOS and FWD1 are both regulated by Wnt/ß-catenin signalling, albeit in opposite directions, thus establishing interlocked negative and positive feedback loops. The functional relevance of the opposite regulation of HOS and FWD1 by Wnt/ß-catenin signalling in conjunction with their redundant activities in proteasomal degradation of ß-catenin remains unresolved. Using a detailed ordinary differential equation model, we investigated the specific influence of each individual feedback mechanism and their combination on Wnt/ß-catenin signal transduction under wild-type and cancerous conditions. We found that, under wild-type conditions, the signalling dynamics are predominantly affected by the HOS feedback as a result of a higher concentration of HOS than FWD1. Transcriptional up-regulation of FWD1 by other signalling pathways reduced the impact of the HOS feedback. The opposite regulation of HOS and FWD1 expression by Wnt/ß-catenin signalling allows the FWD1 feedback to be employed as a compensation mechanism against aberrant pathway activation as a result of a reduced HOS concentration. By contrast, the FWD1 feedback provides no protection against aberrant activation in adenomatous polyposis coli protein mutant cancer cells.

SNAIL1 combines competitive displacement of ASCL2 and epigenetic mechanisms to rapidly silence the EPHB3 tumor suppressor in colorectal cancer.

EPHB3 is a critical cellular guidance factor in the intestinal epithelium and an important tumor suppressor in colorectal cancer (CRC) whose expression is frequently lost at the adenoma-carcinoma transition when tumor cells become invasive. The molecular mechanisms underlying EPHB3 silencing are incompletely understood. Here we show that EPHB3 expression is anti-correlated with inducers of epithelial-mesenchymal transition (EMT) in primary tumors and CRC cells. In vitro, SNAIL1 and SNAIL2, but not ZEB1, repress EPHB3 reporter constructs and compete with the stem cell factor ASCL2 for binding to an E-box motif. At the endogenous EPHB3 locus, SNAIL1 triggers the displacement of ASCL2, p300 and the Wnt pathway effector TCF7L2 and engages corepressor complexes containing HDACs and the histone demethylase LSD1 to collapse active chromatin structure, resulting in rapid downregulation of EPHB3. Beyond its impact on EPHB3, SNAIL1 deregulates markers of intestinal identity and stemness and in vitro forces CRC cells to undergo EMT with altered morphology, increased motility and invasiveness. In xenotransplants, SNAIL1 expression abrogated tumor cell palisading and led to focal loss of tumor encapsulation and the appearance of areas with tumor cells displaying a migratory phenotype. These changes were accompanied by loss of EPHB3 and CDH1 expression. Intriguingly, SNAIL1-induced phenotypic changes of CRC cells are significantly impaired by sustained EPHB3 expression both in vitro and in vivo. Altogether, our results identify EPHB3 as a novel target of SNAIL1 and suggest that disabling EPHB3 signaling is an important aspect to eliminate a roadblock at the onset of EMT processes.

Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma-carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/ß-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

Acetylation of human TCF4 (TCF7L2) proteins attenuates inhibition by the HBP1 repressor and induces a conformational change in the TCF4::DNA complex.

The members of the TCF/LEF family of DNA-binding proteins are components of diverse gene regulatory networks. As nuclear effectors of Wnt/ß-catenin signaling they act as assembly platforms for multimeric transcription complexes that either repress or activate gene expression. Previously, it was shown that several aspects of TCF/LEF protein function are regulated by post-translational modification. The association of TCF/LEF family members with acetyltransferases and deacetylases prompted us to investigate whether vertebrate TCF/LEF proteins are subject to acetylation. Through co-expression with p300 and CBP and subsequent analyses using mass spectrometry and immunodetection with anti-acetyl-lysine antibodies we show that TCF4 can be acetylated at lysine K150 by CBP. K150 acetylation is restricted to TCF4E splice variants and requires the simultaneous presence of ß-catenin and the unique TCF4E C-terminus. To examine the functional consequences of K150 acetylation we substituted K150 with amino acids representing the non-acetylated and acetylated states. Reporter gene assays based on Wnt/ß-catenin-responsive promoter regions did not indicate a general role of K150 acetylation in transactivation by TCF4E. However, in the presence of CBP, non-acetylatable TCF4E with a K150R substitution was more susceptible to inhibition by the HBP-1 repressor protein compared to wild-type TCF4E. Acetylation of K150 using a bacterial expression system or amino acid substitutions at K150 alter the electrophoretic properties of TCF4E::DNA complexes. This result suggests that K150 acetylation leads to a conformational change that may also represent the mechanism whereby acetylated TCF4E acquires resistance against HBP1. In summary, TCF4 not only recruits acetyltransferases but is also a substrate for these enzymes. The fact that acetylation affects only a subset of TCF4 splice variants and is mediated preferentially by CBP suggests that the conditional acetylation of TCF4E is a novel regulatory mechanism that diversifies the transcriptional output of Wnt/ß-catenin signaling in response to changing intracellular signaling milieus.

Modeling Wnt/ß-Catenin Target Gene Expression in APC and Wnt Gradients Under Wild Type and Mutant Conditions.

The Wnt/ß-catenin pathway is involved in the regulation of a multitude of physiological processes by controlling the differential expression of target genes. In certain tissues such as the adult liver, the Wnt/ß-catenin pathway can attain different levels of activity due to gradients of Wnt ligands and/or intracellular pathway components like APC. How graded pathway activity is converted into regionally distinct patterns of Wnt/ß-catenin target gene expression is largely unknown. Here, we apply a mathematical modeling approach to investigate the impact of different regulatory mechanisms on target gene expression within Wnt or APC concentration gradients. We develop a minimal model of Wnt/ß-catenin signal transduction and combine it with various mechanisms of target gene regulation. In particular, the effects of activation, inhibition, and an incoherent feedforward loop (iFFL) are compared. To specify activation kinetics, we analyze experimental data that quantify the response of ß-catenin/TCF reporter constructs to different Wnt concentrations, and demonstrate that the induction of these constructs occurs in a cooperative manner with Hill coefficients between 2 and 5. In summary, our study shows that the combination of specific gene regulatory mechanisms with a time-independent gradient of Wnt or APC is sufficient to generate distinct target gene expression patterns as have been experimentally observed in liver. We find that cooperative gene activation in combination with a TCF feedback can establish sharp borders of target gene expression in Wnt or APC gradients. In contrast, the iFFL renders gene expression independent of gradients of the upstream signaling components. Our subsequent analysis of carcinogenic pathway mutations reveals that their impact on gene expression is determined by the gene regulatory mechanism and the APC concentration of the cell in which the mutation occurs.

Intrinsic properties of Tcf1 and Tcf4 splice variants determine cell-type-specific Wnt/ß-catenin target gene expression.

T-cell factor (Tcf)/lymphoid-enhancer factor (Lef) proteins are a structurally diverse family of deoxyribonucleic acid-binding proteins that have essential nuclear functions in Wnt/ß-catenin signalling. Expression of Wnt/ß-catenin target genes is highly dependent on context, but the precise role of Tcf/Lef family members in the generation and maintenance of cell-type-specific Wnt/ß-catenin responses is unknown. Herein, we show that induction of a subset of Wnt/ß-catenin targets in embryonic stem cells depends on Tcf1 and Tcf4, whereas other co-expressed Tcf/Lef family members cannot induce these targets. The Tcf1/Tcf4-dependent gene responses to Wnt are primarily if not exclusively mediated by C-clamp-containing Tcf1E and Tcf4E splice variants. A combined knockdown of Tcf1/Tcf4 abrogates Wnt-inducible transcription but does not affect the active chromatin conformation of their targets. Thus, the transcriptionally poised state of Wnt/ß-catenin targets is maintained independent of Tcf/Lef proteins. Conversely, ectopically overexpressed Tcf1E cannot invade silent chromatin and fails to initiate expression of inactive Wnt/ß-catenin targets even if repressive chromatin modifications are abolished. The observed non-redundant functions of Tcf1/Tcf4 isoforms in acute transcriptional activation demonstrated that the cell-type-specific complement of Tcf/Lef proteins is a critical determinant of context-dependent Wnt/ß-catenin responses. Moreover, the apparent inability to cope with chromatin uncovers an intrinsic property of Tcf/Lef proteins that prevents false ectopic induction and ensures spatiotemporal stability of Wnt/ß-catenin target gene expression.

Snapshots of protein dynamics and post-translational modifications in one experiment--beta-catenin and its functions.

ß-catenin plays multiple roles in the canonical Wnt signaling pathway and in cell-cell adhesion complexes. In addition, ß-catenin is a proto-oncogene and activating ß-catenin mutations are relevant in the genesis of colorectal, hepatocellular and other common cancers. Different functions of ß-catenin as transcriptional co-activator or cell adhesion molecule are orchestrated by changes in concentration and phosphorylation as well as its ability to complex with proteins such as cadherins or transcription factors. Detailed quantitative and time-resolved analysis of ß-catenin, based on the evaluation of the changes in the Wnt pathway, enable greater insights into health- and disease-related ß-catenin function. The present paper describes a novel suspension bead array assay panel for ß-catenin, which requires minimal amounts of sample and is able to relatively quantify total ß-catenin, the extent of phosphorylation at multiple sites and the ratio of complexed and free ß-catenin. This is the first study to combine three biochemical methods--sandwich immunoassay, co-immunoprecipitation, and protein-protein interaction assay--in one suspension bead assay panel. The assay was used to measure changes in the concentration of eight different ß-catenin forms in HEK293 cells in a time-resolved manner. In contrast to the general consensus, our study demonstrates an increase in ß-catenin phosphorylated at Ser-45 upon treatment of cells with rWnt3a or a GSK3 inhibition; we also link C-terminal phosphorylation of ß-catenin on Ser-552 and Ser-675 with canonical Wnt signaling.

Class I and III HDACs and loss of active chromatin features contribute to epigenetic silencing of CDX1 and EPHB tumor suppressor genes in colorectal cancer.

Aberrant Wnt/ß-catenin signaling is a driving force during initiation and progression of colorectal cancer. Yet, the Wnt/ß-catenin targets CDX1, EPHB2, EPHB3 and EPHB4 (EPHB2-4) act as tumor suppressors in intestinal epithelial cells and frequently appear to be transcriptionally silenced in carcinomas. The molecular mechanisms which underlie the apparent loss of expression of a subset of Wnt/ß-catenin targets in a background of persistent pathway activity are largely unknown. To gain insight into this, we quantified expression of CDX1 and EPHB2-4 in human tissue specimens of case-matched colorectal normal mucosa, adenoma and invasive carcinoma. In particular EPHB2-4 display biphasic, albeit not strictly coincident, expression profiles with elevated levels in adenomas and decreased transcription in approximately 30% of the corresponding carcinomas. Consistent with their divergent and variable expression we observed considerable heterogeneity among the epigenetic landscapes at CDX1 and EPHB2-4 in a model of colorectal carcinoma cell lines. Unlike the inactive CDX1 locus, EPHB2-4 maintain DNA hypomethylation of their promoter regions in the silent state. A strong reduction of active histone modifications consistently parallels reduced expression of CDX1 and EPHB3 and to some extent of EPHB2. Accordingly, treatment with inhibitors for DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) restored CDX1 and EPHB2-4 expression depending upon epigenetic features at their promoters but also upon cellular background. Overall our findings show that downregulation of CDX1 and EphB receptor genes occurs independently and that different branches of epigenetic control systems including class I and III HDACs contribute to epigenetic silencing of Wnt/ß-catenin targets during colorectal tumorigenesis.