Pancreatic ductal adenocarcinoma (PDAC) is the seventh leading cause of cancer- related deaths worldwide. This pathology is asymptomatic at early stages and there are no specific symptoms, so patients are typically diagnosed when an unresectable tumour caused by the presence of metastases is shown. The high mortality rate of PDAC has also been attributable to the presence of a small subset of cells with very aggressive features called pancreatic cancer stem cells (PCSCs). Although PCSCs play a key role in driving patient poor outcomes and resistance to targeted therapies, little is known about the regulation of their signalling and metabolic pathways. A complete molecular characterization of PCSC biology is fundamental to make an impact on PDAC. Therefore, the aim of this PhD thesis was to obtain an in-depth characterization of PCSCs from a molecular point of view to detect cellular, metabolic and signalling dysfunctions implicated in pathophysiology of PDAC, possibly suggesting new therapeutic targets. My PhD project was carried out in the Proteomic and Mass Spectrometry Laboratory of Biotechnology Department at University of Verona under the guidance of Prof. Daniela Cecconi and in other laboratories thanks to established collaborations. PCSCs and PDAC cell cultures were obtained in collaboration with the research group of Prof. Marta Palmieri (Dept. of Neuroscience, Biomedicine and Movement of the University of Verona); proteomic analysis was done in collaboration with the research group of Prof. Emiliano Marengo (Dept. of Sciences and Technological Innovation, University of Eastern Piedmont); and lipidomic analysis was performed in collaboration with Prof. Michael J.O. Wakelam and Dr Andrea F. Lopez-Clavijo during my three-months placement abroad at the Lipidomics facility of the Babraham Institute in Cambridge (UK). During my PhD project, four PCSC lines (i.e., PaCa3, PaCa44, MiaPaCa2, and PC1J) and relative parental (P) cells were analysed and compared. The four P cell lines were cultured and used to obtain PCSCs. Morphological evaluation by microscopy of PCSC tumour spheres and their relative P cells was performed. Then, the expression of stem and quiescence related markers were investigated in PaCa3, PaCa44, MiaPaCa2, and PC1J CSCs by qPCR and immunoblotting analyses. Subsequently, proteomics and lipidomics analyses were performed to evaluate possible alterations in proteome and lipidome of PCSCs. Omics data were subjected to bioinformatic analysis and evaluated further in-depth by western blotting to confirm protein modulation, confocal fluorescence microscopy to detect lipid droplets, and colorimetric enzymatic kit to assess free fatty acid levels. The integration of proteomics and lipidomics data suggested that PCSCs displayed common modulations, while significant differences were detected compared to their counterpart. The downregulation of L-lactate dehydrogenase A chain (LDHA) and its phosphorylated form (p-LDHA), and the upregulation of the trifunctional enzyme subunit alpha (HADHA) involved in cardiolipin remodelling were in common among all four PCSC lines. Bioinformatic analysis also revealed that upregulated proteins of PCSCs were mainly involved in lipid-related pathways, such as fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Lipidomics analysis supported these results, indicating an increase of long and very long FAs, which are products of fatty acid elongase-5 predicted as an active gene of PaCa3, PaCa44, MiaPaCa2, and PC1J CSCs. In accordance with HADHA upregulation, PCSCs were also characterized by the induction of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. In addition, lipidomic analysis also suggested the induction of phosphoinositide pathway in PCSCs. In conclusion, the results obtained in the present PhD thesis support the potential of a multi-omics approach and provide a better understanding of lipid metabolism in PCSCs. The findings obtained indicated the importance of FA elongation and highlight for the first time the involvement of cardiolipin remodelling in PCSCs. Altogether this study provides novel information for the development of promising new therapies against PDAC.

A multi-omics approach identifies the pivotal role of cardiolipin remodelling, alpha subunit of the mitochondrial trifunctional protein and long chain fatty acids in stem cells of pancreatic cancer

DI CARLO, CLAUDIA
2021

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the seventh leading cause of cancer- related deaths worldwide. This pathology is asymptomatic at early stages and there are no specific symptoms, so patients are typically diagnosed when an unresectable tumour caused by the presence of metastases is shown. The high mortality rate of PDAC has also been attributable to the presence of a small subset of cells with very aggressive features called pancreatic cancer stem cells (PCSCs). Although PCSCs play a key role in driving patient poor outcomes and resistance to targeted therapies, little is known about the regulation of their signalling and metabolic pathways. A complete molecular characterization of PCSC biology is fundamental to make an impact on PDAC. Therefore, the aim of this PhD thesis was to obtain an in-depth characterization of PCSCs from a molecular point of view to detect cellular, metabolic and signalling dysfunctions implicated in pathophysiology of PDAC, possibly suggesting new therapeutic targets. My PhD project was carried out in the Proteomic and Mass Spectrometry Laboratory of Biotechnology Department at University of Verona under the guidance of Prof. Daniela Cecconi and in other laboratories thanks to established collaborations. PCSCs and PDAC cell cultures were obtained in collaboration with the research group of Prof. Marta Palmieri (Dept. of Neuroscience, Biomedicine and Movement of the University of Verona); proteomic analysis was done in collaboration with the research group of Prof. Emiliano Marengo (Dept. of Sciences and Technological Innovation, University of Eastern Piedmont); and lipidomic analysis was performed in collaboration with Prof. Michael J.O. Wakelam and Dr Andrea F. Lopez-Clavijo during my three-months placement abroad at the Lipidomics facility of the Babraham Institute in Cambridge (UK). During my PhD project, four PCSC lines (i.e., PaCa3, PaCa44, MiaPaCa2, and PC1J) and relative parental (P) cells were analysed and compared. The four P cell lines were cultured and used to obtain PCSCs. Morphological evaluation by microscopy of PCSC tumour spheres and their relative P cells was performed. Then, the expression of stem and quiescence related markers were investigated in PaCa3, PaCa44, MiaPaCa2, and PC1J CSCs by qPCR and immunoblotting analyses. Subsequently, proteomics and lipidomics analyses were performed to evaluate possible alterations in proteome and lipidome of PCSCs. Omics data were subjected to bioinformatic analysis and evaluated further in-depth by western blotting to confirm protein modulation, confocal fluorescence microscopy to detect lipid droplets, and colorimetric enzymatic kit to assess free fatty acid levels. The integration of proteomics and lipidomics data suggested that PCSCs displayed common modulations, while significant differences were detected compared to their counterpart. The downregulation of L-lactate dehydrogenase A chain (LDHA) and its phosphorylated form (p-LDHA), and the upregulation of the trifunctional enzyme subunit alpha (HADHA) involved in cardiolipin remodelling were in common among all four PCSC lines. Bioinformatic analysis also revealed that upregulated proteins of PCSCs were mainly involved in lipid-related pathways, such as fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Lipidomics analysis supported these results, indicating an increase of long and very long FAs, which are products of fatty acid elongase-5 predicted as an active gene of PaCa3, PaCa44, MiaPaCa2, and PC1J CSCs. In accordance with HADHA upregulation, PCSCs were also characterized by the induction of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. In addition, lipidomic analysis also suggested the induction of phosphoinositide pathway in PCSCs. In conclusion, the results obtained in the present PhD thesis support the potential of a multi-omics approach and provide a better understanding of lipid metabolism in PCSCs. The findings obtained indicated the importance of FA elongation and highlight for the first time the involvement of cardiolipin remodelling in PCSCs. Altogether this study provides novel information for the development of promising new therapies against PDAC.
2021
Inglese
Pancreatic cancer; Pancreatic cancer stem cells; Proteomics; Lipidomics; Mass spectrometry
138
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14242/182746
Il codice NBN di questa tesi è URN:NBN:IT:UNIVR-182746