AbstractIntroduction: Pancreatic ductal adenocarcinoma (PDAC) has very low survival rates that have not changed in over 40 years. Delay in diagnosis and the development of drug-resistance limits the effectiveness of current therapies, making identification of novel early detection and combination treatment strategies, extremely important in order to improve survival rates.
Materials and Methods: An in vitro model of gemcitabine and 5FU resistant PDAC cell lines were generated in the lab by continuous treatment with the drugs. Reverse Phase Protein Array (RPPA) and Liquid Chromatography-Mass Spectrometry (LC-MS) based analysis of protein expression and metabolite levels was carried out in PDAC cell line models on chemotherapy treatment in order to identify upregulated proteins and metabolites. Based on the importance of targeting downstream signalling pathways to KRAS in PDAC and the joint proteomics and metabolomics analysis, AKT was chosen as a target that could potentially enhance chemotherapy effectiveness in PDAC. AKT inhibition was assessed in combination with single chemotherapy agents. Based on these findings and previous research in our lab in Oesophageal Adenocarcinoma (OAC) cell lines, AKT inhibition in combination with anti-apoptotic protein inhibition was assessed. Cell viability was assessed using MTT assays and CI values were generated to assess synergy. Proliferation assays, western blotting, flow cytometry and high content analysis were used to decipher mechanism of action of the combination treatments. Chemoresistant PDAC cell lines were used to assess AKT’s role in acquired resistance and to investigate the efficacy of combined AKT/IAP inhibition.Additionally, to identify the biological pathways implicated in the early development of PDAC, a systematic review was carried out in order to assess the association between protein and metabolite based biomarkers and PDAC risk (published findings included manuscript in final appendix, Chapter 9).
Results: Protein expression analysis using RPPA showed increased expression of several receptor tyrosine kinases (RTKs) and a few downstream mediators to KRAS. In addition, the top dysregulated proteins based on fold change were identified on combination chemotherapy treatment that could serve as potential interesting combination targets. Jagged-1 and Connexin-43 were also found to be highly upregulated in the chemoresistant lines, indicating a role in governing chemoresistance. LC-MS based metabolomics analysis showed upregulation of TCA cycle metabolites and non-essential amino acids in the PDAC cell lines. Links to AKT signalling were identified with the RTKs identified, EGFR, EphA2, IGF all potentially acting through AKT signalling and an extensive literature review also highlighted the potential role of this pathway, downstream of KRAS in regulating the metabolic changes seen. Based on these observations and previous work in our lab in OAC cell lines assessing AKT inhibition, the efficacy of AKT inhibition was assessed. Two allosteric AKT inhibitors were found to synergize with the chemotherapy drugs oxaliplatin, gemcitabine and 5FU but the combination was found to induce only cytostatic/anti-proliferative effects. In order to swing the balance towards cell death, AKT inhibition was assessed in combination with anti-apoptotic proteins and found to be highly synergistic. Mechanism of action studies revealed that both a caspase dependent and independent mechanism of cell death contributed to this synergy. Interestingly, this combination was also effective in reducing cell viability of in vitro chemoresistant lines generated in the lab.
Additionally, our pooled analysis in the form of a systematic review demonstrates that higher serum glucose levels and lower levels of PLP are associated with risk of PDAC and highlights the importance of more prospective studies that are required to identify biomarkers for early detection (published findings included manuscript in final appendix, Chapter 9).
Conclusion: A combined proteomics and metabolomics based approach was used to identify novel treatment strategies for PDAC and identified that combined inhibition of AKT and IAPs could synergistically reduce cell viability in parental and chemo-resistant PDAC cell lines. This suggests that this combination could be used as a second line therapy in chemo refractory PDAC. Our results suggest that the dual targeting of AKT and IAPs could be an effective therapeutic combination in PDAC.
Thesis embargoed until 31st December 2027
|Date of Award||Dec 2022|
|Sponsors||EC/Horizon 2020 Marie Skłodowska-Curie actions|
|Supervisor||Richard Turkington (Supervisor) & Emma Kerr (Supervisor)|
- Pancreatic cancer
- combination treatment
- AKT inhibition
- inhibitor of apoptosis proteins