Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Di Guglielmo, John M

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.

Summary for Lay Audience

Lung cancer is the second most common diagnosed cancer world-wide, and the leading cause of cancer-related deaths, with non-small cell lung cancer (NSCLC) making up 85% of all cases. In later stages of lung cancer, a signalling molecule called transforming growth factor-beta (TGF-β) is overexpressed and contributes to tumor progression by activating epithelial-to-mesenchymal transition (EMT). This is a process where tumor cells undergo changes that allow them spread to and invade distant sites of the body, which is associated with poor outcomes for NSCLC patients. TGF-β signalling begins at the cell surface, when TGF-β binds to TGF-β receptor type 2 (TβRII), which then associates with TGF-β receptor type 1 (TβRI) to activate various signalling pathways. Because TβRII initiates TGF-β signalling, its inhibition in diseases like cancer is of interest. Recently, a new class of compounds called b-annulated 1,4-dihydropyridines (1,4-DHPs) was shown to selectively degrade TβRII in heart cells, without affecting the levels of its closest interacting partner, TβRI. Because the effect of 1,4-DHPs has not been investigated in NSCLC, this thesis shows work towards understanding how 1,4-DHPs reduce TβRII levels, determining the effects that they have on TGF-β-dependent processes, and analyzing how genes are affected by these compounds in NSCLC cells. I showed that 1,4-DHPs decreased TβRII protein levels, without affecting TβRI, and that they inhibited TGF-β-dependent EMT and cell migration. Additionally, I discovered that 1,4-DHPs degrade TβRII after it is synthesized but before it reaches the cell surface, with 90 kDa heat shock protein (Hsp90) and its binding partner cell division cycle (Cdc37) potentially playing a role in this mechanism. Finally, analysis of RNA sequencing data of NSCLC cells treated with 1,4-DHPs showed that the compounds reduced expression of TGF-β-related genes, and modulated genes related to cell migration and cell adhesion. The work presented here lays a solid foundation for the potential use of 1,4-DHPs as an effective anti-TGF-β treatment strategy to inhibit the spread and invasion of tumor cells in NSCLC patients in the clinic.

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Available for download on Saturday, December 06, 2025

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