No description available.

Pancreatic cancer is an aggressive disease with poor prognosis and a high fatality rate. Gemcitabine, the standard first-line chemotherapy for advanced disease, has negligible effects, necessitating the development of new therapies. We previously demonstrated that deletion of the anti-apoptotic gene E1B19K (AdΔ19K) in a replication-selective adenoviral mutant, caused synergistically-enhanced cell-killing when combined with low-dose DNA-damaging drugs in pancreatic cancer xenograft models. To delineate the cellular pathways targeted by the combination treatment we employed AdΔ19K and gemcitabine or irinotecan, with the goal of identifying cellular factors that are essential for the synergistic cell-killing. We hypothesised that AdΔ19K and DNA-damaging drugs act synergistically to deregulate cell-cycle mechanisms. Pancreatic cancer cell death induced by AdΔ19K and DNA-damaging drugs is apoptotic and time-dependent. AdΔ19K could not block DNA-damage responses (DDR) elicited by the drugs, despite virus-mediated degradation of the DDR factor Mre11. Mre11 siRNA-mediated knockdown augmented the synergistic cell death. Mitotic-index analysis in synchronised cells and immunofluorescence microscopy suggested that AdΔ19K promotes mitotic entry of gemcitabine-treated DNA-damaged cells. Moreover, AdΔ19K inhibited drug-induced accumulation of Claspin, a DDR protein whose degradation is required for checkpoint recovery. Treatment with AdΔ19K and gemcitabine accelerated Claspin degradation, and siRNA-mediated Claspin knockdown enhanced the synergistic cell death. Time-lapse microscopy in histoneH2B mCherry-expressing cells showed that AdΔ19K enhanced gemcitabine-induced mitotic catastrophe, characterised by prolonged mitosis, chromosome missegregation errors, cytokinesis failure and formation of multinucleated cells. Moreover, live-cell imaging revealed that the majority of cells treated with AdΔ19K and gemcitabine die before mitotic entry. 5 These findings suggest that E1B19K-deleted adenoviruses cannot prevent cell-cycle checkpoint responses elicited by DNA-damaging drugs, but enhance drug-induced cell death by downregulating DDR factors, such as Mre11 and Claspin. Additionally, the virus enhances mitotic catastrophe of DNA-damaged cells escaping cell-cycle checkpoints, eventually leading to increased apoptosis. Through these studies cellular pathways and factors involved in the synergistic cell killing were identified, that could be explored in the future to develop improved targeted therapies for pancreatic cancer.

Thesis (M.D.)—Boston University

Genomic sequencing of pancreatic ductal adenocarcinoma (PDAC) tumours has highlighted the existence of wide genetic diversity alongside frequent mutations in KRAS, TP53 and SMAD4. Within this heterogeneity many components of the epigenetic machinery are mutated, including the histone H3 lysine 4 methyltransferases KMT2C and KMT2D, which are frequently subject to mutation and can identify patients with a more favorable prognosis. In this thesis low expression of KMT2C and KMT2D were shown to also define better outcome groups, with median survivals of 15.9 vs 9.2 months (p = 0.029), and 19.9 vs 11.8 months (p = 0.001) respectively. Experiments across eight human pancreatic cell lines following their depletion suggest that this improved outcome may be due to attenuated cell proliferation, with decreased progression of cells from G0/G1 observed upon KMT2D loss. Whole transcriptome analysis of PDAC cell lines following KMT2C or KMT2D knockdown identified 31 and 124 differentially expressed genes respectively, with 19 common to both. Gene set enrichment analysis revealed a significant downregulation of genes relating to cell-cycle pathways, confirmed by interrogation of the International Cancer Genome Consortium and The Cancer Genome Atlas PDAC data series. Furthermore, these experiments highlighted a potential role for NCAPD3, a subunit of the condensin II complex, as a PDAC outcome predictor across four patient gene expression series. Alongside this, Kmt2d depletion in cells derived from murine models of pancreatic cancer led to an increase in their response to the antimetabolites 5-fluorouracil and gemcitabine. Taken together, the studies herein suggest that lower levels of this methyltransferase may mediate the sensitivity of PDAC patients to particular treatments. Altogether, these data suggest a potential therapeutic benefit in targeting these methyltransferases within PDAC, especially in those patients that demonstrate higher KTM2C/D expression.

Gastrointestinal malignancies such as liver and pancreatic cancers are the deadliest due to late detection and drug resistance. Nucleoside analogues, like Gemcitabine, are the conventional therapy despite their little impact on survival and off-target toxicity. A novel class of nucleoside analogues able to evade drug resistance mechanisms has been developed by the Guindon group and biologically screened in our lab. Some of these proprietary molecules were further equipped with a lipoate moiety designed to target cancer cell metabolism. LCB2151 and LCB2179 have emerged as the lead molecules in this class, with an IC50 of 10-15 µM in the Gemcitabine-resistant human pancreatic (Capan-2 & Panc-1) cancer cell lines. The focus of this project is deciphering the cellular mechanisms activated by LCB2151 in these pancreatic cancer lines. A series of biased molecular approaches, like gene expression profiling, and unbiased large throughput proteomic and metabolomics analyses were applied to identify potential targets and affected pathways. Results collectively show that LCB2151 evades drug resistance mechanisms, increases pro-apoptotic markers and impairs mitochondrial respiration as early as 6 hours posttreatment. Furthermore, MS/MS analyses reveal that LCB2151 alters the levels of several metabolites in the central carbon metabolism pathway and identifies the citric acid cycle enzyme α-ketoglutarate dehydrogenase as a potential molecular target of LCB2151. Understanding the exact mechanism of action of our lead molecule along with extensive testing on murine cancer models, will surely pave its way to clinical testing and evaluation.

Imexon is an iminopyrrolidone aziridine which previously has shown activity against a variety of human cancer types, including multiple myeloma and pancreatic adenocarcinoma. Recently, mechanistic studies in the MIA PaCa-2 human pancreatic cancer cell line have demonstrated binding to sulfhydryls, build-up of reactive oxygen species (ROS), perturbations in mitochondrial membrane potential (MMP), and activation of caspases 3, 8 and 9. Because imexon binds sulfhydryls and generates ROS, it was hypothesized that imexon would have considerable activity against pancreatic cancer by promoting oxidative stress in cells which are already oxidatively challenged and in combination with gemcitabine by interacting with key sulfhydryl-dependent enzymes involved with gemcitabine metabolism. In vitro anti-tumor activity of imexon and gemcitabine was evaluated in PANC-1, MIA PaCa-2, MutJ, and BxPC-3 human pancreatic cancer cell lines. Interactions between imexon and gemcitabine were assessed with simultaneous drug exposure at a fixed (imexon: gemcitabine) ratio using median effect analysis. The PANC-1, MutJ, and BxPC-3 cells demonstrated synergy with combination treatment. Severe combined immune deficient (SCID) mice bearing PANC-1 cells treated with imexon and gemcitabine demonstrated tumor growth inhibition and regression. Imexon inhibited ribonucleotide reductase (RNR) at drug concentrations ≥100 μMol. This is similar to the selective RNR inhibitor hydroxyurea, suggesting that imexon may enhance gemcitabine-mediated inhibition of RNR as a mechanism of synergy. An S phase accumulation of PANC-1 cells occurred at ≥300 μMol imexon at 24 hr. This was associated with a ≥2-fold increase of radiolabeled gemcitabine incorporation into PANC-1 DNA at ≥100 μMol imexon. Therefore the mechanisms of synergy between imexon and gemcitabine appear to include: (1) cell cycle arrest in S-phase, and (2) inhibition of RNR. Both actions would increase the uptake of the active metabolite, gemcitabine-triphosphate, (GEM-TP), into DNA. Arresting cells in S-phase would increase the time of cellular incorporation of deoxynucleotides, including GEM-TP, into DNA. Similarly, RNR inhibition reduces the availability of normal deoxynucleotides which compete with GEM-TP for incorporation. Overall, these data demonstrate that imexon is uniquely synergistic with gemcitabine in vitro and in vivo and support the rationale for combining the agents in clinical trials for the treatment of pancreatic cancer.

The human MAL2 protein has been demonstrated to regulate secretion in kidney epithelial cells in a lipid raft-dependent manner.  Further, MAL2 interacts with TPD52, a protein that promotes secretion in rat pancreatic acinar cells.  Two MAL2 homologues have recently been identified within rat pancreatic β-cells: MAL2A and MAL2B.  This suggested that MAL2 and TPD52 might interact with each other to coordinate secretion within both human kidney epithelial cells and rat pancreatic β-cells. I report here that both <i>MAL2A </i>and <i>MAL2B </i>are expressed within rat pancreatic β-cells.  Further, the expression of <i>MAL2B </i>appears to be tightly regulated as sequences within the 5’ UTR of the <i>MAL2B </i>transcript inhibit its translation.  Both MAL2A and MAL2B are associated with lipid rafts.  Attempts were made to identify interactions between TPD52 and the MAL2 proteins.  GST pull-down assays indicated that TPD52 interacts with both MAL2A and MAL2B.  This is consistent with previous observations and suggests that TPD52 does not interact with MAL2A or MAL2B via their N-termini, which are distinct between each MAL2 protein.  However, a novel <i>Xenopus </i>egg extract interaction assay failed to detect an interaction between TPD52 and the MAL2 proteins.  Further, immunohistochemistry indicated that TPD52 did not co-localise and either MAL2A or MAL2B within pancreatic β-cells.  These findings indicate that TPD52 and the MAL2 proteins might participate in overlapping secretion pathways.  Further, these data suggest that any interaction between TPD52 and the MAL2 proteins might be weak or transient.  Finally, initial attempts were made to reduce <i>TPD52 </i>expression within β-cells via RNA silencing in order to elucidate the role of TPD52 in directing secretion.  RNA silencing will also be used to examine the role of MAL2A in coordinating secretion.

No description available.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2008.