Optimisation of the conventional tripartite of pancreatic cancer (PC) treatment have led to significant improvements in mortality, however further knowledge of the underlying molecular processes is still required. Transcript profiling of mRNA expression of over 44K genes with microarray technology demonstrated upregulation of secreted frizzled related protein 4 (sFRP4) and ??-catenin in PC compared to normal pancreata. Their pathway ??? Wnt signalling is integral to transcriptional regulation and aberrations in these molecules are critical in the development of many human malignancies. Immunohistochemistry protocols were evaluated by two independent blinded examiners for antigen expression differences associated with survival patterns in 140 patients with biopsy verified PC and a subset of 23 normal pancreata with substantial observer agreement (kappa value 0.6-0.8). A retrospective cohort was identified from 6 Sydney hospitals between 1972-2003 and archival formalin fixed tissue was collected together with clinicopathological data. Three manual stepwise regression models were fitted for overall, disease-specific and relapse-free survival to determine the value of significant prognostic variables in risk stratification. The models were fitted in a logical order using a careful strategy with step by step interpretation of the results. Immunohistochemistry demonstrated increased sFRP4 membranous expression (&gt 10%) in 49/95 PC specimens and this correlated with improved overall survival (HR:0.99;95%CI:0.97-6.40;LRchi2=134.75; 1df; ??&lt 0.001). Increased sFRP4 cytoplasmic staining (&gt 2/3) in 46/85 patients increased the disease-specific survival (HR:0.52;95%CI:0.31-0.89;LR test statistic =248.40;1df;??&lt 0.001). Increasing ??-catenin membranous expression (&lt _60%) in 26/116 patients was associated with an increased risk of overall death (HR:3.18;95%CI:1.14-8.89;LR test statistic =4.61;1df,??&lt 0.05). Increasing cytoplasmic expression in 65/114 patients was protective and was associated with prolonged survival on univariate, but not multivariate analysis (Disease specific survival HR:0.75;95%CI:0.56-1.00;logrank chi2=3.91;1df; ??=0.05). Increased nuclear ??-catenin expression in 65/114 patients was associated with prolonged survival (disease-specific HR:0.92;95%CI:0.83-1.02; LR test statistic= 49.72;1df;??&lt 0.001). At the conclusion, 12 patients (8.6%) remained alive, 122 died of their disease (68 males versus 54 females). They were followed for a median of 8.7 months (range 1.0-131.3) months. The median age was 66.5 years (range 34.4-96.0, standard deviation 10.9) years. Pancreatic resection was achieved in 79 patients with 46.8% achieving RO resection. The 30 day post-operative mortality was 2.1%. The overall 1 year survival rate was (33.7% ; 95%CI: 25.78-33.79) with a 5 year survival of (2.87%, 95%CI: 2.83-6.01) and a median survival of (8.90 months; 95%CI: 7.5-10.2). The median disease-specific survival was (9.40; 95%CI: 7.9-10.5 months) and the median time to relapse was 1.2 months (95%CI 1.0-1.2 months). A central tenet of contemporary cancer research is that an understanding of the genetic and molecular abnormalities that accompany the development and progression of cancer is critical to further advances in diagnosis, treatment and eventual prevention. High throughput tissue microarrays were used to study expression of two novel tumour markers in a cohort of pancreatic cancer patients and identified sFRP4 and ??-catenin as potential novel prognostic markers.

Cancer cell metabolism is reorganized around the needs of proliferating cells, particularly the management of organic metabolites and the balance of redox state. Here, we show that pancreatic cancer requires exogenous sources of cysteine for tumor growth and maintenance due to its critical role in redox balance. Using a multidisciplinary approach, we find that cancer cells rely on imported cystine (oxidized cysteine) to detoxify lipid reactive oxygen species (ROS) and avert ferroptosis, a form of non-apoptotic cell death. Cystine–derived glutathione was necessary for this protection, but its depletion was not sufficient to induce ferroptosis. Correspondingly, genetic inactivation of system xc–, the cystine/glutamate antiporter, in established pancreatic tumors induced stabilization or regression, extending survival in an autochthonous mouse model. We observed distinctive lesions of non-apoptotic cell death that may represent an in vivo manifestation of ferroptosis, highlighting a novel, cancer-specific dependency on a potentially druggable membrane channel.

Similar to many cancers, pancreatic cancer (PDA) assumes an altered metabolic state that is designed to support the manufacture of macromolecules necessary for cell division. For example, membrane synthesis from lipid precursors is dependent on de novo fatty acid synthesis (dnFAS), a pathway that is upregulated in many cancers. The rate-limiting enzyme of dnFAS is fatty acid synthase (FASN), an enzyme that is overexpressed in cancer but found at very low levels in normal tissues. Here we demonstrate that fatty acid synthase (FASN) inhibition using novel small molecule inhibitor IPI-9119 induces apoptosis in FASN overexpressing epithelial cells of a pre-clinical pancreatic cancer mouse model (KPC). We also provide evidence that FASN inhibition insensitivity in PDA cells is due to flux through scavenging pathways, particularly macropinocytosis. We initially hypothesized that FASN overexpression is a survival strategy for PDA epithelial cells through which they acclimate to a low nutrient microenvironment. Indeed, K-ras driven metabolic reprograming has been shown to increase flux through the dnFAS pathway indicating that PDA cells should rely heavily on FASN activity. Surprisingly we found that PDA cell lines are minimally affected by FASN inhibition and rely chiefly on scavenging pathways. In vitro cells displayed a dependency on lysosomal function, determined using lysosomal inhibitor chloroquine (CQ). Dual therapy with FASN inhibitor, IPI-9119, and lysosomal inhibitor, CQ, increased apoptosis and decreased cell viability in human PDA cell lines and in bulk KPfl/flC PDA tumors. Our findings suggest that mutated epithelial tumor cells overexpress FASN and that these oncogenic cells can be specifically targeted using FASN inhibition. Our findings in regards to scavenging pathways demonstrate that PDA is reliant on lysosomal activity and may utilize both autophagy and macropinocytosis to maintain lipid levels in addition to dnFAS. Dual therapy with a FASN inhibitor and a lysosomal inhibitor induces apoptosis in PDA cell lines regardless of their sensitivity to FASN inhibition alone indicating that this therapeutic strategy, dual treatment with lysosomal inhibitors and FASN inhibitors, should be further developed. Our major conclusions from this work are: (1) that PDA is not solely dependent on FASN activity for lipid maintenance but short-term inhibition results in targeted apoptosis in FASN expressing cells, in vivo. (2) Lipid scavenging pathway macropinocytosis accounts for primary resistance to FASN inhibition in vitro, although autophagy may also contribute. (3) Dual inhibition of FASN and lysosomal function induces apoptosis and decreases proliferation in PDA cell lines and tumors.

No description available.

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Despite the considerable expansion of knowledge in the development of pancreatic cancer, there has been little progress made in facilitating an early diagnosis of this disease and predicting an accurate response to treatment. We aim to translate this knowledge to clinical practice by using a prospective database of precursor cystic lesions in pancreas cancer, assessing the use of over-expressed genes in pancreatic juice as a surrogate marker of these pancreas cancer and finally, downstream of these changes at the genetic level, use metabolomic techniques to look for biomarkers in pancreas cancer in serum. In the first study, we investigate the natural history of pancreatic cystic neoplasms, specifically IPMNs, using a prospectively collected database to examine the profiles and outcomes of main duct IPMN, branch duct IPMN and cystic lesions measuring less than 3 cm in size. A total of 99 patients with suspected pancreatic cystic tumours were enrolled over 3 years. Median follow-up was 24 months (range 0 – 124). Cystic tumours comprised of 13 MD-IPMN, 40 BD-IPMN, 11 MCN and 8 adenocarcinomas among others. The complete cohort showed an overall risk of adenocarcinoma of 8%. Main duct IPMN showed a cumulative risk of 46% with evidence of progression of disease in a further 23%. The associated mortality in MD-IPMN was related to the underlying adenocarcinoma and was 38% in our group. The incidence of adenocarcinoma in branch duct IPMN was 11% with disease progression seen 13.8%. Evidence of extra-pancreatic malignancies was seen in 37.7% of patients with IPMN. In the second study, we explore the feasibility of gene expression profiling from RNA isolated from matched pancreatic juice and tumour tissue in patients with pancreatic cancer and pancreatic cystic tumours. RNA was isolated and Poly(A) PCR was used to globally amplify the RNA. RT-PCR was used to measure expression levels of 18 genes common to both pancreas cancer and pancreatic cystic tumours. Spearman’s rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. One gene out of eighteen, MSLN (p<0.008), showed significant correlation in the expression levels between paired pancreatic juice and tissue samples in pancreas cancer. In the cystic tumour group, only one gene MMP-7 (p<0.01), showed a significant correlation between paired juice and tissue samples. When the whole cohort was analysed for the false discovery rate, these genes did not exhibit statistically significant correlation between the samples. RNA analysis of pancreatic juice is feasible using the Poly(A) cDNA technique and correlation of gene expression is shown to exist, albeit with low sensitivity, indicating its potential use in clinical practice with small tissue and juice samples. In the final study, we performed a literature review on the use of metabolomics in pancreas cancer. We performed metabolic profiling of serum samples from selected cancer patients and noncancerous controls using UPHLC-MS to generate and compare the metabolic profiles in serum samples from a cohort of patients with pancreas cancer, ampullary cancer and endocrine cancer. Thirty nine serum samples (including 19 pancreatic cancers, 9 ampullary cancers and 5 endocrine cancers) and 21 matched HUSERMET controls were analysed using Ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) in both positive and negative ESI modes. The output was generated as a data matrix of mass spectral features with related accurate m/z and retention time pairs. The data was then signal corrected and individual peaks were normalised and the resultant spectra were compared against a metabolite reference library and analysed using univariate and multivariate statistical tests. We found a disparity in the metabolite peaks between the cases and controls on PCA that did not permit the accurate interpretation of the data in the case study set compared to the control set. No obvious reason other than metabolite degradation during storage could account for this difference. PC-DFA analysis of metabolite peaks between pancreas cancer, ampullary cancer and endocrine cancer showed significant difference between endocrine cancers and the other two groups. Significant ESI positive metabolites included those involved in lipid pathways and metabolites involved in glucose metabolism. There is encouraging scope for studies using prospective controls to identify and develop metabolic biomarkers in pancreas cancer.

Pancreatic cancer (PC) is the fourth leading cause of cancer death in Western societies. Despite significant progress in understanding the molecular pathology of PC and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility. Affymetrix Genechipfi oligonucleotide microarrays were used to interrogate mRNA expression of PC and normal pancreas to identify molecular pathways dysregulated in PC. Analysis of these data identified altered expression of numerous components of the S100 Calcium Binding Protein Family, Retinoic Acid signalling pathway and the HOX transcriptional network in PC compared to normal pancreas. These pathways were assessed using immunohistochemistry (IHC) and in-situ hybridisation (ISH) in a cohort of patients with PC. Increased protein expression, of S100A2, S100A6 and S100P was observed in 43%, 60% and 48% of PC respectively. Expression of S100A2 was associated with a poor outcome (p = 0.009), whilst increased expression of S100A6 (p = 0.0008) and S100P (p = 0.0005) were associated with an improved outcome. Additionally, S100A2 expression was identified as an independent marker of outcome in resected tumours. Aberrant expression of retinoic acid signalling components was demonstrated in PC cell lines using semi-quantitative RT-PCR. ISH demonstrated expression of Retinoic Acid Induced 3 (RAI3), an orphan G protein coupled receptor normally expressed in the fetal lung, in 68% of PC, and this co-segregated with an improved overall survival (p = 0.026).Ectopic protein expression of HOXB2, a transcription factor normally expressed in the developing hindbrain and modulated by retinoic acid, was observed in 15% of early PanIN lesions and 38% of PC specimens. Expression of HOXB2 was associated with non-resectable tumours and was an independent predictor of poor survival in resected tumours. Suppression of HOXB2 protein expression using small interfering RNA, resulted in epithelioid trans-differentiation in the Panc-1 PC cell line, however no alteration in proliferation rates were observed compared to controls. This thesis has shown that transcript profiling and tissue validation has identified potential markers of early diagnosis and outcome in PC. Furthermore, pathways and molecules previously thought to be associated with normal human development have been implicated to play a role in the development and progression of PC. Further analyses of these markers will determine any potential role in future diagnostic and therapeutic strategies.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of unmet need with a 5-year survival following diagnosis of 3% with limited surgical, radiotherapy and chemotherapy treatment options. Central to PDAC tumorigenesis is acquisition of an oncogenic Kras mutation to drive acinar-to-ductal metaplasia and progression to PDAC that is potentiated by NF-kB deregulation. However, PDAC requires the additional loss of tumour suppressors such as p53, SMAD4 or p16. ASPP family members ASPP2 and iASPP regulate both p53 and NF-kB, and are classified as a tumour suppressor and oncogene respectively. However, the precise roles of ASPP2 and iASPP in pancreatic cancer are unknown. In this thesis I demonstrate that ASPP2 suppresses metastasis and iASPP suppresses the pro-inflammatory tumour microenvironment. In a mouse model of PDAC development, ASPP2-deficiency does not alter metaplasia, PanIN progression or primary PDAC onset. However, median survival due to metastasis is significantly reduced in an ASPP2-deficient PDAC model. I demonstrate ASPP2-deficient PDAC can result in increased squamous differentiation defined histologically or via increased p63 expression. I propose ASPP2 is a key suppressor &Delta;Np63 and the squamous PDAC subtype in vivo. Conversely, iASPP is a putative oncogene and high expression in cancer associates with poor prognosis. However, in a mouse model of PDAC, loss of iASPP accelerates PDAC onset and metastasis. I demonstrate that iASPP is a functional tumour suppressor of a pro-inflammatory phenotype in response to oncogenic Kras and pancreatitis. I propose ASPP2- and iASPP-deficient mouse models of PDAC represent in vivo the squamous and immunogenic subtypes of PDAC respectively; and are relevant tools to study mechanisms of metastasis and inflammation-driven carcinogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd deadliest cancer in the United States with a projected 12% 5-year survival rate. Acinar cells have been proposed as a potential cell-of-origin for PDAC after undergoing acinar to ductal metaplasia (ADM). In the absence of oncogenic mutations (e.g. Kras), ADM lesions form as an adaptive response and eventually resolve to regenerate the acinar compartment, which we term as adaptive ADM. However, in the presence of oncogenic Kras mutations, the ADM lesions can transform to a pre-invasive state called pancreatic intraepithelial neoplasia (PanIN). Thus, a normally adaptive metaplastic response becomes maladaptive, which we term as oncogenic ADM. The mechanisms that drive PanIN formation in the context of injury and oncogenic mutations are poorly understood, resulting in an absence of targets to combat persistent ADM. This thesis investigates the role of FRA1 (gene name Fosl1) in acinar cell de-differentiation, PanIN transformation, and eventual PDAC tumorigenesis. Through CUT&RUN sequencing of mice undergoing recovery from caerulein-induced acute pancreatitis, we identify FRA1 as the most active transcription factor during KrasG12D mediated acute pancreatitis- mediated injury. We have elucidated a functional role of FRA1 by generating an acinar-specific Fosl1 knockout mouse expressing KrasG12D. Using a gene regulatory network and pseudotime trajectory inferred from single nuclei ATAC-seq and bulk-RNA seq, we hypothesize a regulatory model of the acinar-ADM-PanIN continuum and experimentally validate that Fosl1 knockout mice are delayed in the onset of ADM and PanIN. Furthermore, deletion of Fosl1 in an autochthonous PDAC mouse model revealed that this ADM-initiated delay eventually culminates in a significant survival advantage and a less aggressive tumor phenotype. Through investigation of upstream regulators of FRA1, we identified G-CSF as an ADM-promoting cytokine. Fosl1 depletion prevented the pro-inflammatory effects of G-CSF, indicating that the G-CSF/FRA1 signaling axis can modulate ADM. Using ex vivo acinar cultures, we also showed that G-CSF can induce FRA1 through MEK/ERK signaling. Our findings reveal that FRA1 is a mediator of acinar cell plasticity and contributes to acinar cell de-differentiation and malignant transformation. Although the majority of this thesis focuses on oncogenic ADM, we also include a chapter on the role of Prrx1 in adaptive ADM. Our comprehensive and unbiased approach identified previously the Paired-Related homebox1 (Prrx1) as the most upregulated transcription factor in the intersection of pancreatic ductal development, regeneration, and evolution of PanIN. We have demonstrated previously that Prrx1 can promote a ductal phenotype by binding the Sox9 promotor and inducing its expression during pancreatitis. In this body of work, we present a novel mechanism by which Prrx1 regulates maintenance of adaptive ADM. Using novel mouse models and ex vivo acinar culture systems, we demonstrate that Prrx1 can induce TGFβ signaling and reduce E-Cadherin expression to promote ADM. We do not know if there is any potential epistatic interaction between FRA1 and PRXX1. Overall, we reveal the rippling effects of FRA1 can have during the early stages of pre-neoplasia, and we unveil an alternative function of PRRX1 for stimulating an adaptive response to stress. This thesis presents a new understanding of how acinar cell de-differentiation occurs in the pancreas by revealing novel roles of two transcription factors, FRA1 and PRRX1, and furthers our understanding of tissue regeneration in an injured pancreas.

While single-cell RNA sequencing provides a remarkable window on pathophysiologic tissue biology and heterogeneity, its high gene-dropout rate and low signal-to-noise ratio challenge quantitative analyses and mechanistic understanding. This thesis addresses this issue by developing PISCES, a pipeline for regulatory network-based single-cell analysis of mammalian tissues. PISCES accurately estimates the mechanistic contribution of regulatory and signaling proteins to cell state implementation and maintenance based on the expression of their lineage-specific transcriptional targets, inferring protein activity for a putative set of transcriptional regulators and cell-state markers. Experimental validation assays – including technical analysis via downsampling of high depth data and biological analysis by assessing concordance with CITE-Seq-based measurements – show a significant improvement in the ability to identify rare subpopulations and to elucidate key lineage markers compared to gene expression analysis. The improved ability to identify biologically meaningful cellular subpopulations makes PISCES an ideal tool to deconvolute heterogeneity in a wide variety of biological contexts. A systematic analysis of single-cell gene expression profiles in the Human Protein Atlas (HPA) by PISCES generated tissue-specific clustering and master regulator analyses across 26 human tissues, as well as a publicly available repository of ready-to-use regulatory networks specific to cell-lineages in each tissue. This resource will allow researchers to access the algorithmic advantages of PISCES without requiring prohibitively expensive or technically challenging computational resources. Additionally, PISCES is able to unravel the heterogeneous stromal environment of Pancreatic Ductal Adenocarcinoma, a malignancy defined by a large and complicated stromal compartment. This analysis reveals several novel candidate subpopulations, including a fibroblast subtype that has never been observed in humans, a potential pro-metastatic population of endothelial cells, and a population of immune-suppressing stellate cells. PISCES is also able to deconvolute more continuous forms of heterogeneity, as demonstrated by an analysis of epithelial cells in the developing murine lung. Here, PISCES is able to computationally reconstruct a developmental trajectory between Sox9+ distal cells and Sox2+ proximal cells, which is then leveraged to identify several novel markers of the critical intermediate population. Subsequent analysis suggests that these transition zone cells may share programs similar to those seen in injury repair and identifies a candidate therapeutic target that can drive cells into or out of this transition state. Finally, protein activity measured by PISCES is used to refine faulty experimental labels through differential density analysis. This analysis lead to the development of a machine learning classifier that accurately predicted increased degrees of stemness in experimentally transduced populations. Additionally, the density analysis paradigm has been extended to unsupervised settings, allowing for the detection of stable cellular populations and transitory trajectories.