Characterizing How beta-annulated 1,4-Dihydropyridines Inhibit TGF-beta Signalling
Abstract
Lung cancer is the leading cause of cancer-related deaths, with non-small cell lung cancer (NSCLC) making up 85% of all cases. Transforming growth factor beta (TGF-β) is a cytokine involved in many cellular processes, including apoptosis and differentiation. However, in later stages of lung cancer, tumor cells aberrantly use TGF-β to fuel epithelial-to-mesenchymal transition (EMT), a distinctive trait of many epithelial-derived tumors and a driver for metastasis and poor NSCLC patient prognoses. TGF-β signalling begins when TGF-β binds to TGF-β receptor type II (TβRII), which recruits the type I receptor (TβRI) to form a signalling complex that activates various signalling pathways. Because TβRII initiates TGF-β signalling, its inhibition in diseases like cancer is of interest. Recently, a novel class of b-annulated 1,4-dihydropyridines (1,4-DHPs) was shown to specifically degrade TβRII in cardiomyocytes, without affecting TβRI. Because effects of 1,4-DHPs on NSCLC has not been investigated, I characterized how 1,4-DHPs inhibit TGF-β signalling and trafficking and analyzed their effects on TGF-β-related functional outcomes in NSCLC cells. Using Western blotting, I observed that 1,4-DHPs significantly decreased TβRII protein levels and TGF-β-dependent signaling, without affecting TβRI. I also determined that 1,4-DHPs inhibited TGF-β-dependent events such as EMT, actin stress fiber formation, and cell migration. Interestingly, I observed that 1,4-DHPs also inhibited cell migration in the absence of TGF-β. Because the 1,4-DHP mechanism is still unknown, this work focused on revealing how 1,4-DHPs induce TβRII degradation. Using techniques such as radioactively labelling TGF-β cell surface receptors, western blotting, and siRNA knockdowns, I discovered that 1,4-DHPs target TβRII prior to trafficking to the cell surface, and that 90 kDa heat shock protein (Hsp90) and its co-chaperone, cell division cycle (Cdc37) may be involved in the 1,4-DHP mechanism. Finally, an RNA sequencing experiment was performed to investigate why 1,4-DHPs inhibited cell migration independent of TGF-β. The results suggest that 1,4-DHPs reduce transcription of TGF-β-related genes, but they also modulate expression levels of cell adhesion- and migration-related genes. The work presented here provides a potential future use of the 1,4-DHPs as an effective anti-TGF-β treatment strategy to inhibit metastasis of tumor cells in NSCLC patients in the clinic.