| dc.description.abstract | Autophagy is a highly conserved recycling process carried out ubiquitously in humans that primarily involves both selective and non-selective degradation of cellular materials ranging from small proteins to organelles as a means to provide an alternate nutrient source. This process is generally regarded as a survival pathway by resisting cell-mediated death (i.e. apoptosis) and sustaining cellular resources under stressful conditions (i.e. hypoxia, and glucose and nutrient starvation). Unfortunately, PDAC is a devastating cancer subtype that is closely linked with a highly aggressive and, metastatic and chemoresistant phenotype. As an intricately regulated pathway with a highly complex upstream canonical network, mutations in the PDAC genome often dysregulate homeostatic autophagy. As a result of its markedly rapid growth and progression, PDAC tumours are frequently associated with intense metabolic and mechanical stressors that significantly contribute to the augmented autophagy in PDAC. Hence, autophagy has been comprehensively linked to PDAC progression and could be a key mechanism responsible for poor therapeutic efficacy in this disease. While therapeutic, diagnostic and prognostic advancements in the treatment and management of PDAC have been at the forefront of recent research, patient survival rate has only marginally improved over the last 20 years. Addressing the treatment of PDAC, the inhibition of autophagy is a therapeutic strategy that has been trialled in the past with only the late-stage inhibitors, Chloroquine and Hydroxychloroquine, reaching clinical trials. While these agents are shown to synergise with other currently used PDAC chemotherapeutics, they have limited potency and are known to induce various adverse effects. As a fundamental pathway involved in the survival and progression of PDAC, the development and validation of novel therapies targeting autophagy could be pivotal in overcoming chemoresistant PDAC.
In the first results chapter of this thesis (i.e. Chapter 3), we demonstrate a novel chemotherapeutic strategy involving the potent suppression of the two central autophagy initiation complexes, primarily coordinated by ULK1 and Beclin-1, and the synergistic combination with Gemcitabine in both parent and chemoresistant PDAC cell lines. Initially, the targeted gene silencing of either ULK1 or BECN1 induced a counteractive cellular response to upregulate the expression of the other. After the development of two Gemcitabine-resistant PDAC cellular models from parent PANC-1 and MIA PaCa-2 cells, we proceeded to assess the effectiveness of two ULK1 complex inhibitors (i.e.
MRT68921 and SBI-0206965) and two Beclin-1/VPS34 complex inhibitors (i.e. SAR405 and Spautin-1) with Gemcitabine treatment on proliferation, apoptosis and migration.
Our results demonstrated that the targeted inhibition of either autophagic initiation complex increased PDAC chemosensitivity to Gemcitabine in the parent and resistant cell lines. The simultaneous inhibition of both the ULK1 complex and Beclin-1/VPS34 complex also resulted in a synergistic anti-proliferative effect on both PDAC phenotypes and were typically more effective in the resistant cells. We determined that SAR405 and MRT68921 were the most potent autophagy inhibitors individually, in combination with each other and in combination with Gemcitabine. Combining these concepts together, we employed these drugs as a triple combination treatment and observed a strong synergism in the resistant PDAC cells. Considering the importance of stress-induced autophagy in PDAC, serum and glucose starvation conditions were commissioned to more accurately mimic the stressful tumour microenvironment associated with PDAC. The same treatment strategy was performed in the PDAC cells after autophagy was confirmed to be upregulated from nutrient or glucose starvation. The combination composed of Gemcitabine, SAR405 and MRT68921 demonstrated a potent and synergistic anti-proliferative effect in the metabolically stressed parent and resistant PDAC cells.
Next, the metabolic stress conditions (i.e. serum and glucose starvation) were applied together to further stress the PDAC cells and allow for a detailed assessment of the drugs on apoptosis. This study demonstrated that SAR405 and MRT68921 can effectively induce apoptosis individually and promote Gemcitabine-induced apoptosis in the parent and resistant PDAC cells. Moreover, SAR405, SBI-0206965 and MRT68921 were highly effective at limiting PDAC migration individually and in combination with Gemcitabine, which was unable to affect migration independently. Considering the synergy between Gemcitabine, SAR405 and MRT68921, an in vivo assessment was performed using a subcutaneous xenograft model with both parent and resistant PDAC. Although, the triple combination treatment was only significantly effective at reducing tumour weight and size in the tumours from parent PDAC, while a statistically non-significant decrease was observed in the chemoresistant tumours. Finally, we observed that high levels of p62, which is a marker of dysfunctional autophagy, was associated with improved patient overall survival and was an independent prognostic factor. Collectively, these results demonstrate the importance of autophagy activity in PDAC cells and indicate that its inhibition is an effective treatment strategy at overcoming chemoresistant PDAC.
In Chapter 4, we examined the mechanisms of acquired chemoresistance using Gemcitabine-resistant PDAC cell models. The underlying mutational composition is unique to each PDAC patient. As a result, the modern pitfalls of chemotherapy in PDAC are often associated with chemoresistant mechanisms responsible for reduced therapeutic efficacy. Understanding the intricate cellular mechanisms in acquired chemoresistance is vital for optimising PDAC treatment. By investigating the transcriptome, proteome, and metabolome of Gemcitabine-resistant PDAC cells, we highlighted novel insights into this network and identified AMPK as a crucial upstream regulator of acquired chemoresistance.
We observed that Gemcitabine-resistant PANC-1 and MIA PaCa-2 cells elicit significantly upregulated autophagic activity, major upstream dysregulation to the MAPK and PI3K/AKT pathways and a hyperactivated metabolism when compared to the parent cells. These networks are all linked through AMPK, which was shown to be activated overall and specifically from the catalytic subunit AMPKα2. We also identified RASSF1 in both PANC-1 and MIA PaCa-2 resistant cells as an activated upstream regulator of autophagy. As RASSF1 is a known autophagy regulator in the literature, we propose it as a vital protein in PDAC-acquired chemoresistance. Emphasising this, we demonstrated that reduced AMPKα2 levels increased the sensitivity of resistant PDAC cells to Gemcitabine. Finally, AMPKα2 was shown to be pharmacologically inhibited by SBI-0206965 as a potential autophagy targeting therapy in chemoresistant PDAC.
In the final results chapter (i.e. Chapter 5), we assessed the potential of a novel biomarker panel at predicting chemoresponse in PDAC patients. The currently available biomarkers in PDAC are limited and ineffective at predicting and managing patient response to neoadjuvant chemotherapy (NAC). Improving these biomarkers could help personalise the clinical therapeutic strategy for patients based on their response to NAC and improve survival outcomes. This chapter demonstrates that a promising biomarker panel in serum composed of the existing CA19-9 and the novel PDAC marker CA-125 could be used as a predictive tool in NAC-treated PDAC patients. The panel was confirmed to significantly predict PDAC patient’s response to NAC. In all aspects of its assessment, it was superior to the currently used CA19-9. Thus, implicating further validation of this biomarker panel as a valuable clinical aide.
In conclusion, this thesis has demonstrated the value of autophagic inhibition in PDAC and its use as a viable therapy against chemoresistant PDAC through the nomination of an effective combination regime. Moreover, we identified that AMPK and its major downstream effector pathway (i.e. autophagy) are activated in Gemcitabine resistant PDAC. Finally, we propose a novel biomarker panel that can predict patient response to NAC. Collectively, this thesis proposes novel and promising strategies across various stages of PDAC treatment that can be used to improve patient survival outcomes. | en_AU |
