Dataset Information

Mechanistic Model of Signaling Dynamics Across an Epithelial Mesenchymal Transition.

ABSTRACT: Intracellular signaling pathways are at the core of cellular information processing. The states of these pathways and their inputs determine signaling dynamics and drive cell function. Within a cancerous tumor, many combinations of cell states and microenvironments can lead to dramatic variations in responses to treatment. Network rewiring has been thought to underlie these context-dependent differences in signaling; however, from a biochemical standpoint, rewiring of signaling networks should not be a prerequisite for heterogeneity in responses to stimuli. Here we address this conundrum by analyzing an in vitro model of the epithelial mesenchymal transition (EMT), a biological program implicated in increased tumor invasiveness, heterogeneity, and drug resistance. We used mass cytometry to measure EGF signaling dynamics in the ERK and AKT signaling pathways before and after induction of EMT in Py2T murine breast cancer cells. Analysis of the data with standard network inference methods suggested EMT-dependent network rewiring. In contrast, use of a modeling approach that adequately accounts for single-cell variation demonstrated that a single reaction-based pathway model with constant structure and near-constant parameters is sufficient to represent differences in EGF signaling across EMT. This result indicates that rewiring of the signaling network is not necessary for heterogeneous responses to a signal and that unifying reaction-based models should be employed for characterization of signaling in heterogeneous environments, such as cancer.

SUBMITTER: Wade JD

PROVIDER: S-EPMC7733964 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Mechanistic Model of Signaling Dynamics Across an Epithelial Mesenchymal Transition.

Wade James D JD Lun Xiao-Kang XK Zivanovic Nevena N Voit Eberhard O EO Bodenmiller Bernd B

Frontiers in physiology 20201130

Intracellular signaling pathways are at the core of cellular information processing. The states of these pathways and their inputs determine signaling dynamics and drive cell function. Within a cancerous tumor, many combinations of cell states and microenvironments can lead to dramatic variations in responses to treatment. Network rewiring has been thought to underlie these context-dependent differences in signaling; however, from a biochemical standpoint, rewiring of signaling networks should n ...[more]

PMID: 33329028

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Project description:BACKGROUND:Chronic obstructive pulmonary disease (COPD) is characterized by airflow limitation that is progressive and not fully reversible. Cigarette smoking is one of the most commonly and important risk factors for COPD, which contributes to airway remodeling, the outstanding pathological changes in COPD. One potential mechanism which might be important for airway remodeling is the process called epithelial-mesenchymal transition (EMT). However, the underlying molecular mechanisms of EMT in CS-induced COPD are still poorly understood. METHODS:Two Gene Expression Omnibus (GEO) datasets (GSE108134 and GSE5058) were combined to identify the key genes involved in COPD. Then, single-gene analysis of Lyn was performed. Lyn expression was confirmed in patients with COPD. 16HBE cells were treated with cigarette smoking extracts (CSE). Wild type (WT) C57BL/6?J mice and Lyn+/+ transgenic mice were exposed to CSE to establish CS-exposed model. Pathological changes were observed by hematoxylin-eosin staining. The expression levels of EMT markers were examined by using western blot and immunofluorescence. The expression and phosphorylation levels of Lyn and Smad2/3 were detected as well. RESULTS:The gain of mesenchymal markers vimentin and ?-SMA with a concomitant loss of E-cadherin was observed in both in vivo and in vitro studies. Meanwhile, cigarette smoking extracts (CSE) induced EMT in 16HBE cells in a time- and dose- dependent manner. Furthermore, by analyzing GEO datasets and using molecular methods, we explored a kinase, Lyn, its expression correlated with the expression of E-cadherin, vimentin and ?-SMA in CS-exposed model. Moreover, we found that EMT induced by CSE was regulated by activated Lyn through phosphorylation of Smad2/3. CONCLUSIONS:In summary, we found that Lyn regulates epithelial-mesenchymal transition in CS-exposed model through Smad2/3 signaling. As a kinase, Lyn is "druggable", and might provide a therapeutic opportunity for targeting EMT. Therefore, our research might provide a new method to treat COPD by targeting Lyn kinase specifically.

Project description:DCLK1 and Lgr5 have recently been identified as markers of quiescent and cycling stem cells in the small intestinal crypts, respectively. Epithelial-mesenchymal transition (EMT) is a key development program that is often activated during cancer invasion and metastasis, and also imparts a self-renewal capability to disseminating cancer cells. Utilizing the Citrobacter rodentium (CR)-induced transmissible murine colonic hyperplasia (TMCH) model, we observed a relative decrease in DCLK1 expression in the colonic crypts, with significant shift towards stromal staining at peak (12 days post infection) hyperplasia, whereas staining for Lgr5 and Msi-1 increased several fold. When hyperplasia was regressing (days 20-34), an expansion of DCLK1+ve cells in the CR-infected crypts compared with that seen in uninfected control was recorded. Purified colonic crypt cells exhibiting epigenetic modulation of the transforming growth factor-? (TGF?), Wnt and Notch pathways on 12 or 34 days post infection formed monolayers in vitro, and underwent trans-differentiation into fibroblast-like cells that stained positive for vimentin, fibronectin and DCLK1. These cells when trypsinized and regrown in soft agar, formed colonospheres/organoids that developed into crypt-like structures (colonoids) in Matrigel and stained positive for DCLK1. Mice exhibiting 12 or 34 days of TMCH were given azoxymethane once for 8?h (Gp1) or weekly for 3 weeks (Gp2), and subjected to crypt isolation. Crypt cells from Gp1 animals formed monolayers as well as colonospheres in soft agar and nodules/tumors in nude mice. Crypt cells isolated from Gp2 animals failed to form the monolayers, but developed into colonospheres in soft agar and nodules/tumors in nude mice. Thus, both hyperplasia and increased presence of DCLK1+ve cells promote cellular transformation in response to a second hit. The TMCH model, therefore, provides an excellent template to study how alterations in intestinal stem cells promote trans-differentiation, crypt regeneration or colon carcinogenesis following bacterial infection.

Project description:BackgroundThe epithelial-mesenchymal transition (EMT) is a de-differentiation process required for wound healing and development. In tumors of epithelial origin aberrant induction of EMT contributes to cancer progression and metastasis. Studies have begun to implicate epigenetic reprogramming in EMT; however, the relationship between reprogramming and the coordination of cellular processes is largely unexplored. We have previously developed a system to study EMT in a canonical non-small cell lung cancer (NSCLC) model. In this system we have shown that the induction of EMT results in constitutive NF-κB activity. We hypothesized a role for chromatin remodeling in the sustained deregulation of cellular signaling pathways.ResultsWe mapped sixteen histone modifications and two variants for epithelial and mesenchymal states. Combinatorial patterns of epigenetic changes were quantified at gene and enhancer loci. We found a distinct chromatin signature among genes in well-established EMT pathways. Strikingly, these genes are only a small minority of those that are differentially expressed. At putative enhancers of genes with the 'EMT-signature' we observed highly coordinated epigenetic activation or repression. Furthermore, enhancers that are activated are bound by a set of transcription factors that is distinct from those that bind repressed enhancers. Upregulated genes with the 'EMT-signature' are upstream regulators of NF-κB, but are also bound by NF-κB at their promoters and enhancers. These results suggest a chromatin-mediated positive feedback as a likely mechanism for sustained NF-κB activation.ConclusionsThere is highly specific epigenetic regulation at genes and enhancers across several pathways critical to EMT. The sites of these changes in chromatin state implicate several inducible transcription factors with critical roles in EMT (NF-κB, AP-1 and MYC) as targets of this reprogramming. Furthermore, we find evidence that suggests that these transcription factors are in chromatin-mediated transcriptional feedback loops that regulate critical EMT genes. In sum, we establish an important link between chromatin remodeling and shifts in cellular reprogramming.

Dataset Information

Mechanistic Model of Signaling Dynamics Across an Epithelial Mesenchymal Transition.

Publications

Mechanistic Model of Signaling Dynamics Across an Epithelial Mesenchymal Transition.

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