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.

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.

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.

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.

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.

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.

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 [...].

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.

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.

Alternative splicing of Tcf7l2 transcripts generates protein variants with differential promoter-binding and transcriptional activation properties at Wnt/beta-catenin targets.

Alternative splicing can produce multiple protein products with variable domain composition from a single gene. The mouse Tcf7l2 gene is subject to alternative splicing. It encodes TCF4, a member of the T-cell factor (TCF) family of DNA-binding proteins and a nuclear interaction partner of beta-catenin which performs essential functions in Wnt growth factor signalling. Multiple TCF4 isoforms, potentially exhibiting cell-type-specific distribution and differing in gene regulatory properties, could strongly influence tissue-specific Wnt responses. Therefore, we have examined mouse Tcf7l2 splice variants in neonatal tissues, embryonic stem cells and neural progenitors. By polymerase chain reaction amplification, cloning and sequencing, we identify a large number of alternatively spliced transcripts and report a highly flexible combinatorial repertoire of alternative exons. Many, but not all of the variants exhibit a broad tissue distribution. Moreover, two functionally equivalent versions of the C-clamp, thought to represent an auxiliary DNA-binding domain, were identified. Depending upon promoter context and precise domain composition, TCF4 isoforms exhibit strikingly different transactivation potentials at natural Wnt/beta-catenin target promoters. However, differences in C-clamp-mediated DNA binding can only partially explain functional differences among TCF4 variants. Still, the cell-type-specific complement of TCF4 isoforms is likely to be a major determinant for the context-dependent transcriptional output of Wnt/beta-catenin signalling.

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 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.

4-Aminoethylamino-emodin--a novel potent inhibitor of GSK-3beta--acts as an insulin-sensitizer avoiding downstream effects of activated beta-catenin.

Glycogen synthase kinase-3beta (GSK-3beta) is a key target and effector of downstream insulin signalling. Using comparative protein kinase assays and molecular docking studies we characterize the emodin-derivative 4-[N-2-(aminoethyl)-amino]-emodin (L4) as a sensitive and potent inhibitor of GSK-3beta with peculiar features. Compound L4 shows a low cytotoxic potential compared to other GSK-3beta inhibitors determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and cellular ATP levels. Physiologically, L4 acts as an insulin-sensitizing agent that is able to enhance hepatocellular glycogen and fatty acid biosynthesis. These functions are particularly stimulated in the presence of elevated concentrations of glucose and in synergy with the hormone action at moderate but not high insulin levels. In contrast to other low molecular weight GSK-3beta inhibitors (SB216763 and LiCl) or Wnt-3alpha-conditioned medium, however, L4 does not induce reporter and target genes of activated beta-catenin such as TOPflash, Axin2 and glutamine synthetase. Moreover, when present together with SB216763 or LiCl, L4 counteracts expression of TOPflash or induction of glutamine synthetase by these inhibitors. Because L4 slightly activates beta-catenin on its own, these results suggest that a downstream molecular step essential for activation of gene transcription by beta-catenin is also inhibited by L4. It is concluded that L4 represents a potent insulin-sensitizing agent favouring physiological effects of insulin mediated by GSK-3beta inhibition but avoiding hazardous effects such as activation of beta-catenin-dependent gene expression which may lead to aberrant induction of cell proliferation and cancer.

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.

Canonical Wnt signaling controls proliferation of retinal stem/progenitor cells in postembryonic Xenopus eyes.

Vertebrate retinal stem cells, which reside quiescently within the ciliary margin, may offer a possibility for treatment of degenerative retinopathies. The highly proliferative retinal precursor cells in Xenopus eyes are confined to the most peripheral region, called the ciliary marginal zone (CMZ). Although the canonical Wnt pathway has been implicated in the developing retina of different species, little is known about its involvement in postembryonic retinas. Using a green fluorescent protein-based Wnt-responsive reporter, we show that in transgenic Xenopus tadpoles, the canonical Wnt signaling is activated in the postembryonic CMZ. To further investigate the functional implications of this, we generated transgenic, hormone-inducible canonical Wnt pathway activating and repressing systems, which are directed to specifically intersect at the nuclear endpoint of transcriptional Wnt target gene activation. We found that postembryonic induction of the canonical Wnt pathway in transgenic retinas resulted in increased proliferation in the CMZ compartment. This is most likely due to delayed cell cycle exit, as inferred from a pulse-chase experiment on 5-bromo-2'-deoxyuridine-labeled retinal precursors. Conversely, repression of the canonical Wnt pathway inhibited proliferation of CMZ cells. Neither activation nor repression of the Wnt pathway affected the differentiated cells in the central retina. We conclude that even at postembryonic stages, the canonical Wnt signaling pathway continues to have a major function in promoting proliferation and maintaining retinal stem cells. These findings may contribute to the eventual design of vertebrate, stem cell-based retinal therapies. Disclosure of potential conflicts of interest is found at the end of this article.

The microphthalmia-associated transcription factor Mitf interacts with beta-catenin to determine target gene expression.

Commitment to the melanocyte lineage is characterized by the onset of expression of the microphthalmia-associated transcription factor (Mitf). This transcription factor plays a fundamental role in melanocyte development and maintenance and seems to be crucial for the survival of malignant melanocytes. Furthermore, Mitf has been shown to be involved in cell cycle regulation and to play important functions in self-renewal and maintenance of melanocyte stem cells. Although little is known about how Mitf regulates these various processes, one possibility is that Mitf interacts with other regulators. Here we show that Mitf can interact directly with beta-catenin, the key mediator of the canonical Wnt signaling pathway. The Wnt signaling pathway plays a critical role in melanocyte development and is intimately involved in triggering melanocyte stem cell proliferation. Significantly, constitutive activation of this pathway is a feature of a number of cancers including malignant melanoma. Here we show that Mitf can redirect beta-catenin transcriptional activity away from canonical Wnt signaling-regulated genes toward Mitf-specific target promoters to activate transcription. Thus, by a feedback mechanism, Mitf can diversify the output of canonical Wnt signaling to enhance the repertoire of genes regulated by beta-catenin. Our results reveal a novel mechanism by which Wnt signaling and beta-catenin activate gene expression, with significant implications for our understanding of both melanocyte development and melanoma.

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.

Oncogenic transformation by beta-catenin: deletion analysis and characterization of selected target genes.

Genetic analysis of beta-catenin-induced oncogenic transformation in chicken embryo fibroblasts (CEF) revealed the following prerequisites for oncogenicity: (1) removal of the N terminal phosphorylation sites targeted by glycogen synthase kinase 3beta (GSK3beta), (2) retention of the N terminal transactivation domain, and (3) retention of the armadillo repeats. The C terminal transactivation domain played an ancillary role in the transformation of CEF. There was a rough correlation between the transforming activity of various beta-catenin constructs and their transactivation of the TOPFLASH reporter. Expression levels of the candidate target genes of beta-catenin-LEF, cyclin D1 and myc were not correlated with each other or with the transforming activity of beta-catenin constructs. A new target gene, coding for inositol hexakisphosphate kinase 2 (IP6K2) was identified. Its expression showed concordance with the transforming activity of beta-catenin constructs.