Identification of therapeutical targets and novel drug candidates for pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is currently the 7th leading cause of cancer-related death, and it is projected to become the 2nd by the end of 2030. The major factors contributing to the poor prognosis of PDAC are late diagnosis, early onset of metastases, development of resistance to conventional therapy, and frequent tumour relapse. Increasing evidence suggests that these traits are mainly driven by pancreatic cancer stem cells (PCSCs). This small subset of cells is characterized by self-renewal properties, anchorage-independent growth, long-term proliferation, and radio- and chemo-resistance. Currently, intending to develop innovative targeted therapy against PCSCs that can help to eradicate PDAC, studies focused on the different mechanisms involved in the maintenance of their stemness features, as well as on their metabolism and epigenetics. Nevertheless, despite efforts, there is still an urgent need to explore innovative therapeutic approaches. Therefore, the objective of this Ph.D. thesis involved a comprehensive range of analyses, including proteomics, epiproteomics, biochemical, molecular biology, and cellular assays, on an in vitro model of PDAC cells (i.e., parental and PCSCs), to identify potential therapeutic targets and innovative drug candidates. The Ph.D. project was carried out at the Proteomic and Mass Spectrometry Laboratory of the Biotechnology Department at the University of Verona under the supervision of Prof. Daniela Cecconi. This project involved some collaborations, specifically: PDAC cell lines were provided by the research group of Prof. Ilaria Dando (University of Verona); epiproteomic analyses were performed in collaboration with Prof. Tiziana Bonaldi’s group (European Institute of Oncology IRCCS, Milan) supported by EPIC-XS, project number 0000382/ 0000221, funded by the Horizon 2020 program of the European Union; proteomic analyses were done in collaboration with Prof. Emilio Marengo and Prof. Marcello Manfredi (University of Eastern Piedmont, Novara) and at the Mass Spectrometry platform of “Centro Piattaforme Tecnologiche” of the University of Verona; and TPP-substituted pentadecyl fatty acid (named SS4) was provided by Prof. Michael Murray from the Sydney Pharmacy School (Sydney, Australia) and Prof. Tristan Rawling of the University of Technology Sydney (Ultimo, Australia). The first part of this project involved a combined epiproteome and proteome analysis aimed at the identification of new potential therapeutic targets. The precise quantification of histone post-translational modifications (hPTMs) through epi-proteomic analysis, was first performed, serving as an excellent strategy to enhance our comprehension of PCSC biology. To accomplish this aim, the analysis was performed on three different biological replicates of: PANC-1 and PaCa3 PDAC cell lines obtained from parental (P) cells; their relative PCSCs grown up to 2, 4, and 8 weeks; and adherent cells (Ad) obtained from 4 weeks PCSCs re-cultured in "P cell medium" for periods extending up to 10 days and 2 months. Extracted histones were mixed with an equal amount of a super-SILAC mix and then analysed using an ultra-nanoflow HPLC system connected online to a Q Exactive HF Orbitrap MS. The epiproteomic profiling revealed remarkable alterations in hPTMs within PCSCs, impacting processes such as quiescence, apoptosis, chemoresistance, and epithelial-mesenchymal transition (EMT). A total proteome study was then conducted on the PANC-1 cell line with a label-free (SWATH-MS) strategy through a micro-LC 5600+ TripleTOF MS system. The comparative proteome analysis of PANC-1 cells enabled the detection of dysregulated proteins in PCSCs, exerting a specific influence on identified hPTMs and thereby presenting potential therapeutic targets. Notably, among such proteins it was possible to find histone methyltransferases like SHMT2 and DPY30, epigenetic regulators such as H1.0 and RBBP4, and enzymes engaged in one-carbon metabolism like PHGDH and AHCY, all impacting histone methylation patterns. The second task of this study focused on exploring the potential use of the hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit alpha (HADHA), crucial for β-oxidation of fatty acids and cardiolipin remodelling, as a potential therapeutic target for PDAC. In this part of the project, a siRNA approach to reduce HADHA expression at the mRNA level was first tested. However, HADHA silencing by the optimized siRNA protocol slightly reduced its expression in PCSCs. Subsequently, an optimized CRISPR/Cas9 protocol was successfully established generating a stable PANC-1 HADHA knockout (KO) cell line. Although the results obtained are currently in the pilot stages, they indicate that HADHA does not affect the ability of PCSCs to form spheroids in vitro. Once the absence of off-targets is confirmed, the obtained HADHA KO clone could represent a fundamental starting point to verify the effect of the gene editing on PCSC proliferation, migration, and chemoresistance. In the third part of this research, the effectiveness of mitochondrial targeting was evaluated as a strategy to reduce the viability of P and PCSCs. Indeed, recent studies demonstrated that energetic metabolism and mitochondrial function play a fundamental role in phenotype maintenance and spreading of PCSCs. In this context, mitochondria-impairing agents can be used to hamper PCSC propagation. Among them, triphenyl-phosphonium (TPP)-based compounds are known to accumulate in mitochondria and act as potent inhibitors of mitochondrial function selectively targeting cancer cells and PCSCs. In addition, several fatty acids (FAs) have been found to modulate the viability of cancer cells exhibiting anticancer activity, at least in part, by targeting the mitochondrion. Therefore, considering the strategic importance of TPP-based compounds to target PCSCs, in the third part of this Ph.D. project, the anticancer effects of the novel TPP-substituted pentadecyl fatty acid, named SS4, were tested in an in vitro model of PDAC cells (P and PCSCs). The obtained results indicate that SS4 successfully reduced PDAC cell proliferation probably by modulating mitochondrial processes, stimulating the production of reactive oxygen species (ROS), and enhancing the endoplasmic reticulum (ER) stress. In conclusion, the present work carries the potential to provide new insights into the advancement of enhanced PDAC anticancer therapies, specifically addressing the existing challenge of their limited effectiveness against PCSCs.