The Hippo signalling pathway is a fundamental regulator of cellular growth, and its aberrant regulation stands as a pivotal phenomenon of tumorigenesis. Central to this dysfunction is the Rho-ROCK1/2 axis, an upstream modulator that facilitates the nuclear translocation of YAP/TAZ. This mechanism bypasses standard inhibitory signals, simultaneously driving epithelial-mesenchymal transition (EMT) and accelerated cell proliferation. Consequently, the Rho-ROCK1/2-YAP/TAZ interplay represents a critical molecular bridge linking mechanical signalling to the aggressive phenotypic shifts characteristic of malignant progression.

This study employed a preliminary in silico screening and selection of Lomitapide from an FDA-approved drug library, which was validated in vitro on EMT-induced A549 and NCI-H460 lung cancer cell lines. The mechanistic validation of lomitapide in targeting ROCK1/2 and therefore YAP/TAZ has been done through immunoblotting and immunocytochemistry of the downstream target proteins. The role of Hippo signalling in EMT was further studied by analysing the expression of EMT markers in treated cells. Moreover, the efficacy of the drug was analysed through ROS generation, apoptosis and mitochondrial membrane depolarisation using flow cytometry.

Repurposed drug lomitapide was identified to target ROCK1/2 based on in silico docking and MD simulation results. Experimental validations revealed that lomitapide at IC50 reduced TAZ expression in A549 and NCI-H460, conferring its cytosolic degradation, which was further confirmed by diminished expression of nuclear TAZ compared to untreated control. Upon lomitapide treatment, notable reduction of phospho-cofilin (downstream target of ROCK) validates its interaction with ROCK1/2. Reduction of cMYC and Cyclin D1 (YAP/TAZ downstream targets) in the respective cell lines further concurs with modulation of Hippo pathway through ROCK inhibition. Lomitapide treatment resulted into reversal of the EMT, evident by the upregulation of epithelial and downregulation of mesenchymal markers. Nonetheless, at a lower IC50 than the controlled drug fasudil, lomitapide induced apoptosis along with a significant increase in cellular ROS, mitochondrial membrane depolarisation and G1/S phase cell cycle arrest.

The therapeutic potency of lomitapide was established to target Hippo pathway through disrupting ROCK activity in EMT dynamics. In LUAD cells, lomitapide effectively depleted TAZ, its oncogenic target, and reversed EMT. The treatment also triggered ROS-driven intrinsic apoptosis and cell cycle arrest. By modulating the Hippo pathway via the ROCK axis, lomitapide emerges as a robust alternative to the traditional downstream inhibitors in lung cancer therapy.

Department of Biotechnology, Department of Biosciences and Bioengineering, Param Ishan Computing Facility, Centre for Nanotechnology, IIT Guwahati