Construction and Experimental Validation of a Petri net Model of Wnt/β-catenin Signaling

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Abstract

Abstract The Wnt/β-catenin signaling pathway is important for multiple developmental processes and tissue maintenance in adults. Consequently, deregulated signaling is involved in a range of human diseases including cancer and developmental defects. A better understanding of the intricate regulatory mechanism and effect of physiological (active) and pathophysiological (hyperactive) WNT signaling is important for predicting treatment response and developing novel therapies. The constitutively expressed CTNNB1 (commonly and hereafter referred to as β-catenin) is degraded by a destruction complex, composed of amongst other AXIN1 and GSK3. The destruction complex is inhibited during active signaling leading to β-catenin stabilization and induction of β-catenin/TCF target genes. In this study we investigated the mechanism and effect of β-catenin stabilization during active and hyperactive WNT signaling in a combined in silico and in vitro approach. We constructed a Petri net model of Wnt/β-catenin signaling including main players from the plasma membrane (WNT ligands and receptors), cytoplasmic effectors and the downstream negative feedback target gene AXIN2 . We simulated the model with active (i.e. WNT stimulation) and hyperactive (i.e. GSK3 inhibition) signaling, which led to the following observations: 1) A dose- and time-dependent response was observed for both WNT stimulation and GSK3 inhibition. 2) The Wnt-pathway activity was 2-fold higher for GSK3 inhibition compared to WNT stimulation. Both of these observations were corroborated by TCF/LEF luciferase reporter assays. Using this experimentally validated model we simulated the effect of the negative feedback regulator AXIN2 upon WNT stimulation and observed an attenuated β-catenin stabilization. We furthermore simulated the effect of APC inactivating mutations, yielding a stabilization of β-catenin levels comparable to the Wnt-pathway activities observed in colorectal and breast cancer. Our model can be used for further investigation and viable predictions of the role of Wnt/β-catenin signaling in oncogenesis and development. Author Summary Deregulated Wnt/β-catenin signaling is implicated in cancer and developmental defects. In this study we combined in silico and in vitro efforts to investigate the behavior of physiological and pathophysiological WNT signaling. We created a model of Wnt/β-catenin signaling that describes the core interactions: receptor activation, inhibition of downstream effectors and an important negative feedback mechanism. Simulations with the model demonstrated the expected dose- and time-dependent response for both conditions, and the Wnt-pathway activity was significantly higher for pathophysiological compared to physiological signaling. These observations were experimentally validated, which allowed us to investigate and predict the effect of the negative feedback and an inactivating cancer mutation on the Wnt-pathway activity. Our model provides mechanistic insight on the different conditions and can easily be extended and used to answer other questions on Wnt/β-catenin signaling in the area of cancer research and regenerative medicine.

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