Therapeutic targeting of ROCK reverses EMT in lung cancer cells by impeding TAZ in Hippo signalling pathway.

Aberrant regulation of the Hippo signalling is one of the pivotal phenomena in tumorigenesis. Among several upstream regulators, Rho-ROCK1/2 plays an important role in dysregulating Hippo pathway by nuclear translocation of YAP/TAZ with concomitant EMT and proliferation. In this study, A549 and NCI-H460 cells were selected by analysing gene expression profiles of Hippo pathway. Repurposed drug Lomitapide was identified to target ROCK1/2 based on in silico docking and MD simulation. Experimental validations revealed that Lomitapide at IC50, reduced TAZ expression by 1.4 and 1.8-fold in A549 and NCI-H460. Further, a reduction of around 1.4 and 2-fold in cytosolic and nuclear TAZ protein confers its cytosolic degradation, inhibition of nuclear translocation and Hippo pathway reactivation. Upon Lomitapide treatment 1.5-fold reduction of phospho- cofilin validates its interaction with ROCK1/2. Reduction of cMYC by 2.2 and 3.3-fold and, Cyclin D1 by 2 and 3.3-fold (YAP/TAZ downstream targets) in the respective cell lines further concurs with reactivation of Hippo pathway through ROCK inhibition. At a lower IC50 than the controlled drug Fasudil, Lomitapide also induced increase in cellular ROS, mitochondrial membrane depolarization, G1/S phase cell cycle arrest with increase in apoptotic cell population. Drug treatment resulted into reversal of the EMT, evident by upregulation of epithelial markers and downregulation of mesenchymal markers, through western blotting, qRT-PCR and immunofluorescence studies. Reduction in migration, invasion, colony and sphere forming abilities also supported the annihilation of cancer progression. Overall, the therapeutic potency of Lomitapide was established to target Hippo pathway through disrupting ROCK activity in EMT dynamics.