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.

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.

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.

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.

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.