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Thesis Format

Alternative Format

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Di Guglielmo, Gianni, M

Abstract

Transforming growth factor-β (TGFβ) signalling regulates growth, proliferation, immunity, and development. Although TGFβ typically antagonizes tumour formation, tumour cells often acquire mutations within the TGFβ signalling pathway that activate epithelial-mesenchymal transition (EMT). During EMT, epithelial tumour cells lose epithelial-like properties and acquire mesenchymal-like characteristics, which allows tumour cells to detach from the primary tumour and establish metastatic colonies. In addition to EMT, TGFβ augments tumourigenesis by increasing the degradation of damaged macromolecules and organelles via autophagy. Autophagy contributes to radiotherapy and chemotherapy resistance by mitigating the damages inflicted on tumour cells. Currently, there is a growing interest in the relationship between TGFβ signalling and protein degradative pathways as inhibitors of both autophagy and proteasomes block TGFβ-dependent EMT. Therefore, understanding pro-tumourigenic TGFβ signalling and its relationship with degradative processes has become a topic of interest for identifying novel therapeutic targets.

Since the mechanism of TGFβ-dependent autophagy activation was unknown, this work utilized immunoblotting and fluorescence microscopy of non-small cell lung cancer (NSCLC) cells that stably expressed a green fluorescent protein and red fluorescent protein conjugated to microtubule-associated protein light chain 3 (LC3) and LC3 with a glycine deletion, respectively, to elucidate TGFβ signalling branches that activate autophagy. Indeed, both Smad-dependent and -independent TGFβ signalling, activate autophagy by increasing the proportion of active uncoordinated 51-like autophagy activating kinase 1. This work also demonstrated that impeding autophagy using small interfering RNA targeting autophagy-related genes as well as pharmacological inhibitors including chloroquine, spautin-1, and ULK-101 blocked TGFβ receptor endocytosis, Smad phosphorylation, Smad nuclear translocation, EMT, and cell migration. Likewise, proteasome inhibitors, such as MG132 and lactacystin, promoted lysosomal-targeting of TGFβRII and dampened TGFβ-induced R-Smad phosphorylation, R-Smad nuclear translocation, and EMT. Since protein 62/sequestosome 1 (p62/SQSTM1) delivers cargo to both degradative pathways, its expression was silenced using small interfering RNA. Silencing p62/SQSTM1 disrupted TGFβ signalling and promoted EMT; however, there were no changes to autophagy.

In conclusion, this work discovered that altering autophagy or proteasome activity attenuated TGFβ signalling and blocked the pro-tumourigenic properties of TGFβ in NSCLC.

Summary for Lay Audience

Lung cancer is the leading cause of cancer mortality worldwide and mutations in transforming growth factor beta (TGFβ) signalling have been implicated in cancer development. TGFβ promotes epithelial-mesenchymal transition (EMT), which allows cancer cells to become invasive and spread throughout the body. Furthermore, TGFβ activates autophagy, a mechanism for cells to degrade damaged or non-essential cellular components. In cancer, TGFβ-dependent autophagy protects tumour cells from chemotherapeutic drugs and primes cells for invasion. The bulk of protein degradation is facilitated by autophagy and several degradative organelles called proteasomes. Given that recent reports suggest that inhibiting autophagy and proteasome activity blocks EMT, there is a need to explore the role of degradative pathways in TGFβ-dependent tumourigenesis.

In this work, using a model to measure autophagic degradation in combination with known inhibitors and activators of autophagy, I verified that TGFβ activates autophagy and outlined the mechanism of TGFβ-dependent autophagy in lung cancer cell lines. I observed that blocking autophagy disrupts TGFβ-dependent tumourigenesis by decreasing TGFβ signalling, EMT, and cell migration. More specifically, autophagy inhibition blocks the internalization of TGFβ receptors and therefore prevents the activation of downstream signalling molecules that TGFβ relies on to propagate its signalling. Proteasome inhibitors also blocked TGFβ signalling, EMT, and cell migration. However, because proteasomes and autophagy compensate for one another, proteasome inhibitors activated autophagy, which lead to rapid degradation of TGFβ receptors.

Since both autophagy and proteasome inhibitors disrupted pro-tumourigenic TGFβ signalling, I next targeted protein 62/sequestosome 1 (p62/SQSTM1) because it delivers cellular components to both degradative pathways. I observed that decreasing p62/SQSTM1 activity had no effect on autophagy, blocked TGFβ signalling, and activated EMT. Therefore, using this methodology more work is needed to identify a target protein or pathway that regulates autophagy, proteasomes, EMT, and TGFβ signalling.

In conclusion, this work identified that inhibiting proteasomes and autophagy impeded pro-tumourigenic TGFβ signalling. This study has the potential to define the relationship between TGFβ, autophagy, proteasomes, and cancer invasion, which is essential to understanding how tumours that can selectively eliminate damaged proteins and organelles can evade chemotherapeutic toxicities.

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