SMAD4 is dispensable for execution of epithelial-mesenchymal transition downstream of SNAIL1 in colorectal cancer cells
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PROVIDER: PRJNA717772 | ENA |
REPOSITORIES: ENA
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Project description:Transforming growth factor beta (TGFβ) 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, TGFβ 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 TGFβ 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 TGFβ/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 cases. Altogether, our findings reveal considerable plasticity of gene regulatory networks operating in EMT execution and establish that EMT is not categorically precluded in SMAD4mut tumors, which is relevant for their diagnostic and therapeutic evaluation. To identify genes regulated during EMT execution in HT29 cells, two clonal HT29 cell populations (4F5 and 3C2) overexpressing Snail1-HA in a doxycycline (Dox)-inducible manner, as well as control cells were treated with Dox for different periods of time.
2021-11-29 | GSE169735 | GEO
Project description: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/TGFβ pathways, but is TGFβ 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 during EMT in vivo. 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. To identify BMP-dependent genes regulated during EMT, LS174T cells overexpressing Snail1-HA in a doxycycline (Dox)-inducible manner were treated with Dox and inhibitors of BMP signaling (LDN193189/mNoggin) or DMSO as control for different periods of time.
2020-04-22 | GSE143297 | GEO
Project description:Canonical BMP signaling executes epithelial-mesenchymal transition downstream of SNAIL1
| PRJNA599935 | ENA
Project description:Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy characterized by a median survival rate of approximately six months. While genetic profiling has uncovered common mutations in PDAC, developing targeted therapeutic strategies remains challenging. SMAD4 is frequently mutated or deleted in 30-55% of PDAC patients and correlates with poor survival rates. Such mutations frequently result in loss-of-function, thereby disrupting normal cell cycle regulation and contributing to tumorigenesis. Therefore, translating SMAD4 genotype into actionable targets are highly desired for therapeutic innovation in PDAC. In this study, we performed a SMAD4-focused oncogenic protein-protein interaction (oncoPPI) network mapping and revealed a direct physical interaction between SMAD4 and NFATc1. We found that SMAD4 interacts with NFATc1 in a TGF-independent and NFATc1 phosphorylation-dependent manner. Further, SMAD4 sequesters NFATc1 in cytoplasm and inhibits NFATc1 transcriptional activity. In PDAC cells, SMAD4-loss releases its inhibitory activity on NFATc1, activates NFATc1 transcriptional activity which drives STAT3 mRNA and protein upregulation. Pharmacological profiling identified multiple STAT3 inhibitors selectively inhibit the growth of SMAD4-loss PDAC cells. These results suggested a rewired SMAD4-NFATc1-STAT3 axis and targeting STAT3 as a potential therapeutic strategy in SMAD4-loss PDAC.
2026-02-02 | PXD067749 | Pride
Project description:The transcription factor SNAIL1 is a master regulator of epithelial-to-mesenchymal transition, 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. Bioinformatic analyses indicated that SNAIL1 strongly affected Wnt/β-Catenin pathway activity. This correlated with upregulation of LEF1, a nuclear binding partner of β-Catenin. Several tumour entities, including aggressive mesenchymal colorectal cancers, exhibit positively correlated LEF1 and SNAIL1 expression, and elevated LEF1 levels parallel increased colorectal cancer patient mortality. Comparative gene expression profiling suggested that 35% of Snail1-induced transcriptional changes are attributable to LEF1. LEF1 stimulates Wnt/β-Catenin pathway feedback inhibitor expression, causes cell-cycle arrest in vitro, and retards xenograft tumour growth. Conversely, LEF1-deficiency and preventing the β-Catenin-LEF1 interaction impaired the ability of SNAIL1 to alter Wnt/β-catenin target gene expression and to induce cancer cell invasion. Although LEF1 did not autonomously induce epithelial-mesenchymal transition, LEF1 is a critical factor acting downstream of SNAIL1. Apparently, SNAIL1 employs LEF1 as alternative effector to redirect Wnt/β-catenin pathway activity towards anti-proliferative and pro-invasive gene expression.
2019-08-15 | GSE115716 | GEO
Project description:Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here that Snail1 plays also a key role in tumor associated fibroblasts since is necessary for enhancement by these cells on epithelial cells tumor invasion. Snail1 expression in fibroblast requires signals derived from tumor cells such as TGF-b; reciprocally, in fibroblasts Snail1 organizes a complex program that favors collective invasion of epithelial cells at least in part by the secretion of diffusible signaling molecules, such as prostaglandin E2. The capability of human or murine tumor-derived cancer associated fibroblasts to promote tumor invasion is associated to Snail1 expression and obliterated by Snail1 depletion. In vivo experiments show that tumor cells co-transplanted with Snail1 depleted fibroblasts show lower invasion than those xenografted with control fibroblasts. Finally Snail1 depletion in mice prevents the formation of breast tumors and decreased their invasion. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to its effect in EMT but dependent on its expression in stromal fibroblasts where it orchestrates its activation and the crosstalk with epithelial cells.
2016-12-22 | GSE74056 | GEO
Project description:Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here that Snail1 plays also a key role in tumor associated fibroblasts since is necessary for enhancement by these cells on epithelial cells tumor invasion. Snail1 expression in fibroblast requires signals derived from tumor cells such as TGF-b; reciprocally, in fibroblasts Snail1 organizes a complex program that favors collective invasion of epithelial cells at least in part by the secretion of diffusible signaling molecules, such as prostaglandin E2. The capability of human or murine tumor-derived cancer associated fibroblasts to promote tumor invasion is associated to Snail1 expression and obliterated by Snail1 depletion. In vivo experiments show that tumor cells co-transplanted with Snail1 depleted fibroblasts show lower invasion than those xenografted with control fibroblasts. Finally Snail1 depletion in mice prevents the formation of breast tumors and decreased their invasion. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to its effect in EMT but dependent on its expression in stromal fibroblasts where it orchestrates its activation and the crosstalk with epithelial cells.
2016-12-22 | GSE74057 | GEO
Project description:Snail1 is a transcriptional repressor required for a correct embryonic development. In cancer, Snail1 promotes the epithelial to mesenchymal transition in tumorigenic epithelial cells. In this work, we have analyzed the control of Snail1 in the differentiation of the 3T3-L1 cell line derived from murine embryo cells. The activation by snail of 3T3-L1 induced typical markers of cancer-activated fibroblasts as S100A4 or CD44. We generated 3T3-L1 cells stably over expressing Snail1 (3T3L1/Snail1) and control (3T3-L1/control) cells. We used SILAC quantitative approach to identify and characterize protein alterations induced by Snail1. Cells were fractionated in 5 subcellular fractions. The nuclear fraction of the cells was separated by 10% SDS-PAGE. Gels with forward and reverse experiments were stained with Coomassie Blue and cut into 18 slices prior to reduction, alkylation and digestion with trypsin. Tryptic peptides were scanned and fragmented with a linear ion trap-Orbitrap Velos (ThermoScientific). We identified a total of 3108 proteins, with 2572 quantified proteins, and 565 proteins modulated >1.5-fold by Snail1 overexpression. Among them, we found interesting up-regulated proteins associated to early differentiation of adipogenesis (C/EBPβ) and down-regulated proteins implicated in the final stages of differentiation to adipocytes (Fatty acid-binding protein or Fatty acid synthase). We also observed as down-regulated proteins important mediators of PPARγ pathway. We also observed downregulation of proteins implicated in mTOR, SRC and JAK/STAT pathway. We validated these proteomics data by western blot and qPCR in 3T3-L1 cells and other types of fibroblasts with capable to differentiate to terminal mesenchymal phenotypes, as well as in mesenchymal stem cells (MSC). This work provided insight into novel proteins with potential roles in the regulation of differentiation of the 3T3-L1 and MSCs as Nr2F6, ASC-1, Prrx1 or Cbx6. These candidates are down regulated due to the overexpression of Snail1 in 3T3-L1 cells. We next investigated the potential binding of Snail1 to promoter of these candidates. In silico analysis with MatInspector program revealed various putative E-box consensus motifs for Snail1. We performed ChIP and Luciferase assay to validate Snail1 binds to different E-box motifs of our candidates. Additionally, we analyzed the ability to prevent the differentiation to adipocytes of the 3T3-L1 cells using siRNAs. This work provided insight into novel proteins with potential roles in the regulation of differentiation to adipocytes of the 3T3-L1 and mMSC cells as Nr2F6, ASC-1, Prrx1 or Cbx6 controlled by Snail1.
2015-01-06 | PXD001529 | Pride
Project description:Tumor growth requires elevated ribosome biogenesis. Targeting ribosomes is an important strategy for cancer therapy. The ribosome inhibitor, homoharringtonine (HHT), is used for the clinical treatment of leukemia, yet it is ineffective for the treatment of solid tumors, the reasons for which remain unclear. Here we show that Snail1, a key factor in the regulation of epithelial-to-mesenchymal transition, plays a pivotal role in cellular surveillance response upon ribotoxic stress. Mechanistically, ribotoxic stress activates the JNK-USP36 signaling to stabilize Snail1 in the nucleolus, which facilitates ribosome biogenesis and tumor cell survival. Furthermore, we show that HHT activates the JNK-USP36-Snail1 axis in solid tumor cells, but not in leukemia cells, resulting in solid tumor cell resistance to HHT. Importantly, a combination of HHT with the inhibition of the JNK-USP36-Snail1 axis synergistically inhibits solid tumor growth. Together, this study provides a rationale for targeting the JNK-USP36-Snail1 axis in ribosome inhibition-based solid tumor therapy.
2023-09-26 | PXD045622 | Pride
Project description:The annual ~36,000 prostate cancer (PCa) deaths represent a large clinical unmet need and a call for deeper understanding of PCa metastasis. Epithelial-mesenchymal-transition (EMT) has been used to model metastatic behaviors in numerous cancers including PCa. One hallmark of EMT is cell cycle suppression, but how EMT impacts PCa proliferation remains unclear primarily due to the lack of appropriate models. Methods: We transiently induced expression of Snail1 (SNAI1), an EMT driver expressed in PCa, at physiological levels in three PCa cells lines, C4-2B, 22Rv1, and DU145. We used RNA-seq, ChIP-Seq, bioinformatics, qRT-PCR, shRNA, and immunoblotting to identify mechanisms of Snail1-driven inhibition of proliferation. Results: Snail1 suppressed proliferation and the G2/M cell cycle progression, without affecting cell death. Mechanistically, Snail1 upregulated expression of CEBPg, ERG1, FOXO1, cyclin G1, p21, stress genes SESN3 and SOD3, apoptotic programmers Puma, Bax, and Noxa, and senescence-related laminB1, and downregulated Ki67, cyclins A2 and B2. ChIP-Seq data identified Snail1 direct binding to p21, cyclin B2 and G1, EGR1, and CEPBg promoters. EGR1 induced FOXO1 and EGR1 was required for Snail1-induced SOD3 and Puma, and suppression of Caspase 3 to prevent apoptosis. The EGR1/FOXO1 axis induced BAX, Noxa, and SESN3. CEBPg was required for Snail1 induction of Lamin B1 to block Snail1-induced senescence. Conclusions: We identified three new major downstream targets of Snail1 that improve our understanding of the role of EMT in limiting stress signaling, apoptosis, and senescence during cell cycle suppression to create a vulnerability for therapeutic targeting.
2026-02-06 | GSE318340 | GEO