Elsevier

Cellular Signalling

Volume 28, Issue 8, August 2016, Pages 861-870
Cellular Signalling

Autocrine TGF-β/ZEB/microRNA-200 signal transduction drives epithelial-mesenchymal transition: Kinetic models predict minimal drug dose to inhibit metastasis

https://doi.org/10.1016/j.cellsig.2016.03.002Get rights and content

Highlights

  • Kinetic models are able to describe complex molecular experiments such as the protocol to induce autocrine TGF-b/ZEB/microRNA-200 signal transduction during epithelial-mesenchymal transition

  • The validated kinetic models are applied for the design of experiments in cancer therapy by predicting conditions to induce a mesenchymal-epithelial transition.

  • Steady state properties of the kinetic models and data from tumor-derived cell lines of individual patients can be combined to set up a calibration curve such that the minimal amount of an inhibitor to induce a mesenchymal-epithelial transition can be determined.

  • This result could be of significant interest for optimizing treatment protocols in the clinics.

Abstract

The epithelial-mesenchymal transition (EMT) is the crucial step that cancer cells must pass before they can undergo metastasis. The transition requires the activity of complex functional networks that downregulate properties of the epithelial phenotype and upregulate characteristics of the mesenchymal phenotype. The networks frequently include reciprocal repressions between transcription factors (TFs) driving the EMT and microRNAs (miRs) inducing the reverse process, termed mesenchymal-epithelial transition (MET).

In this work we develop four kinetic models that are based on experimental data and hypotheses describing how autocrine transforming growth factor-β (TGF-β) signal transduction induces and maintains an EMT by upregulating the TFs ZEB1 and ZEB2 which repress the expression of the miR-200b/c family members. After successful model calibration we validate our models by predicting requirements for the maintenance of the mesenchymal steady state which agree with experimental data.

Finally, we apply our validated kinetic models for the design of experiments in cancer therapy. We demonstrate how steady state properties of the kinetic models, combined with data from tumor-derived cell lines of individual patients, can predict the minimal amount of an inhibitor to induce a MET.

Introduction

The epithelial-mesenchymal transition (EMT) is an important step during embryogenesis, tissue fibrosis and in cancer progression. It regulates the phenotypic change from proliferating epithelial cancer cells to invading mesenchymal cancer cells [1]. The underlying biochemical regulation is very similar despite the distinct characters of these biological processes [2]. Understanding the EMT has received increasing attention as demonstrated by recent reviews summarizing important new findings [1], [2], [3], [4], [5], [6]. In the following the focus will be on the EMT during cancer progression.

Soluble growth factors can trigger EMT [2], where transforming growth factor (TGF)-β1 (in the following noted as TGF-β) is considered as the major inducer [4], [5], [7]. The activated signaling pathways lead to upregulation of several transcription factors (TFs), including Snail, Slug, Zeb1 and Zeb2, which downregulate the transmembrane adhesion molecule E-cadherin and the tight-junction protein ZO1 and thus trigger a break of tight and adherent junctions between densely packed epithelial cells [4], [5], [8]. This goes along with the upregulation of the mesenchymal markers vimentin and N-cadherin [4].

The regulatory networks, inducing an EMT, include TFs and microRNAs (miRs). The TFs and the miRs interact via a reciprocal repression loop. The TFs drive the EMT and miRs, induce the reverse process, termed mesenchymal-epithelial transition (MET) [3], [9]. A prominent example is the interaction between the TFs Zeb1,2 and their opponents, the members of the miR-200 family.

A systematic experimental investigation of the reciprocal repression between Zeb1/2 and miR-200 has been performed by the lab of G.J. Goodall [9], [10], [11]. They used Madine-Darby canine kidney (MDCK) cells, a polarized epithelial cell line. These cells are frequently applied to study EMT, because they can simply be converted into migratory fibroblasts by incubation with conditioned medium from cultured fibroblasts [12].

Experiments of the Goodall lab with MDCK cells showed that all five members of the miR-200 family are downregulated in response to TGF-β stimulation of the cells [9]. Contrary, enforced expression of miR-200 alone is sufficient to prevent a TGF-β-induced EMT. Changes in expression levels of the miRs and TFs tightly control the EMT as well as the MET [9].

Experimental results presented in [10] revealed further details of this regulation by identifying the promoter location of the miR clusters 200b, and 200c. The promoter region can be accessed in epithelial MDCK cells to induce expression of the miRs. In mesenchymal cells, promotor activity is repressed by the TFs Zeb1,2 which bind to a conserved pair of ZEB type E-box elements located close to the transcriptional start site.

Another detail was contributed by [13]. Experiments demonstrated that ZEB1 directly suppresses transcription of miR-200 family members miR-141 and miR-200c. Closing the loop [13] also revealed that Zeb1/2 are the dominant targets downregulated by these miRs.

Subsequently, it was experimentally shown that autocrine TGF-β signaling is necessary to drive and maintain sustained ZEB expression which induces a stable mesenchymal phenotype [11]. The authors argued that a critical threshold in balance between Zeb1,2 levels and miR-200b,c determines whether cells remain in epithelial state or transit to mesenchymal state. To test this proposition, EMT was induced in MDCK cells by administration of TGF-β which was then removed at different time points (stimulus-removal-experiments).

The experimental results of [11] show that TGF-β removal after 5 days leads to a decrease of ZEB1/2 expression and to a complementary increase of miR-200b/c. Thus, autocrine TGF-β signal transduction is not yet established and the cells revert to the epithelial phenotype. In contrast, TGF-β removal after 8 days maintains the mesenchymal phenotype of the cells. In addition, treating the MDCK-TGF-β cells (remain mesenchymal 35 days after cessation of TGF-β treatment [11]) with the inhibitor of TGF-β receptor 1 (RI) activity SB-505,124 leads to a time-dependent decrease of ZEB1,2 and to an increase of miR-200a,b as shown in [11]. These results demonstrate that autocrine TGF-β signaling is required for maintaining the mesenchymal state in MDCK-TGF cells.

Systems biology approaches, specifically kinetic models, have been successfully employed to gain insights into the complex dynamics of molecular networks, supporting the design of experiments through model predictions [14], [15], [16], [17].

Modeling the complex biochemical processes of EMT is challenging. So far, only few mathematical models describing signal transduction during EMT have been published. An individual-cell-based off-lattice model describes how cell adhesion could be regulated by interactions between E-cadherin and β-catenin [18]. Simulations demonstrated that regulation of soluble β-catenin concentration by local contacts can induce EMT.

The role of different feedback loops between EGF and Wnt signaling to induce downregulation of E-cadherin could be revealed by ordinary differential equation (ODE) modeling [19]. The authors identified a feedback loop composition leading to a switch-like behavior in E-cadherin expression. Another feedback composition with RKIP expression prevents E-cadherin repression and thus EMT.

Kinetic modeling of the reciprocal repression between TFs and miRs during EMT has so far focused on an assumed third stable steady state corresponding to a partial EMT sharing epithelial and mesenchymal properties [20], [21]. The authors argue that such a third steady state is in agreement with collective cell migration. Zhang et al. provided experimental evidence of such a third steady state and they demonstrated that simulations of their mathematical model, based on the assumption of cascading switches, are in agreement with their experimental results [21].

TGF-β signaling in hepatocellular carcinoma EMT was mathematically described by a Boolean model [22]. Model analysis identified feedback motifs that stabilize EMT and predicted activation of Wnt and hedgehog signaling pathways, which could be experimentally validated.

In our work we establish a simple kinetic model dedicated to answer a specific question, formulated in terms of ODEs. The ODEs describe how autocrine TGF-β signal transduction induces an EMT via the ZEB-miR-200 reciprocal repression in MDCK cells. After successful model calibration we validate our model by predicting requirements for the maintenance of the mesenchymal state. Results agree very well with experimental data that were not used for model calibration. Finally, we demonstrate how steady state properties of the kinetic models, combined with data from tumor-derived cell lines of individual patients, can predict the minimal amount of an inhibitor required to induce MET.

Section snippets

Molecular network of autocrine TGF-β/ZEB/miR200 signal transduction

Our approach focuses on a small molecular network which is translated into a small kinetic model describing only one aspect of the EMT: Kinetic model of TGF-β signal transduction predicts minimal drug dose to inhibit MET. Our modeling approach is in agreement with clinical objectives including that only few key parameters have to be measured to start with treatment as early as possible. The experimental data of [11] are appropriate to calibrate our model and to validate our model predictions. A

Model simulations

The results of model calibration are presented in Table S1. A comparison between experimental time series and simulation results of the kinetic models M1-M4 is presented in Fig. 2, Fig. 3, Fig. 4, Fig. 5. All four models could reproduce the experimental findings and only slightly differ in their value of the best fit χ2/N, see Table S2.

The simulations of M1-M4 with TGF-β removal at day five clearly show that ZEB1/2 return to the epithelial state (lower stable steady state) after reaching a

Conclusions

We demonstrated that kinetic models are able to describe complex molecular experiments to study the EMT such as the protocol of the stimulus-removal-experiment presented in [11]. The mathematical description of the TGF-β removal at days five and eight after TGF-β administration enabled that the experimental results presented in [11] could be reproduced by model simulations. In particular, we validated our models by predicting requirements for the maintenance of the mesenchymal steady state

Author contributions

K.R. conceived the study, established the kinetic models, calibrated the models with experimental data, performed model comparison, model simulations and model validation. K.R. wrote a first version of the manuscript. All authors participated in the interpretation of the results. All authors revised the manuscript and read and approved the final manuscript.

Conflict of interests

All authors declare no conflict of interest.

Acknowledgement

We thank Ulf Schmitz for fruitful discussions on miR regulation.

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