Pancreatic and prostate cancer are both prevalent cancers in the United States with pancreatic being one of the most aggressive of all cancers and prostate cancer being one of the most common, ranking as the number one cancer in men. Treatment of both cancers can be quite challenging as the anatomy of the pancreas and prostate, as well as the development and diagnosis of the disease can greatly limit treatment options. Therefore, it is necessary to develop new cancer treatments to help manage and prevent these cancers. Irreversible electroporation is a new non-thermal focal ablation therapy utilizing short, pulsed electric fields to damage cell membranes leading to cell death. The therapy is minimally invasive, involving the insertion of needle electrodes into the region of interest and lasts less than two minutes. Heat sink effects that thermal therapies experience near large blood vessels do not affect irreversible electroporation. This allows the treatment to be used on tumors near vasculature as well as critical structures without harming these vital regions. While irreversible electroporation is a promising new cancer therapy, further developments are necessary to improve treatment planning models. This work aims to further understand the electric field thresholds necessary to kill different types of cancer cells with a focus on pancreatic and prostate cancer. The work is done using an in vitro tumor (hydrogel) model as this model is better than traditional cell suspension studies, with added benefits over the immediate use of tissue and animal models. / Master of Science

Intracellular hyperthermic therapy may prove to be a unique and novel approach to the management of pancreatic cancer. Utilizing the principle of photothermal destruction, selective killing of cancer cells with minimal injury to normal tissues may be possible. This dissertation investigated the role of antibody targeted metal nanoparticles and the cytotoxic effects of nonionizing radiofrequency fields in pancreatic cancer. Cancer cell death was induced by heat release from intracellular metal nanoparticles after radiofrequency field exposure. Fluorescent and gold nanoparticles were delivered with two antibodies, cetuximab and PAM-4, to pancreatic cancer cells in vitro and mouse xenografts in vivo. Selective delivery of these nanoparticles induced cell death in vitro and decreased tumor burden in vivo after whole animal RF field exposure. This occurred through both apoptosis and necrosis. In addition, activated caspase-3 was increased after antibody treatment and RF field exposure. Furthermore, although there was non-specific uptake by the liver and spleen in vivo, there was no evidence of acute or chronic toxicity in the animals. These results are in agreement with the principle that malignant cells are more thermally sensitive than normal cells or tissues. Selective intracellular delivery of metal nanoparticles coupled with whole body RF field exposure may be a beneficial therapy against micrometastases and unresectable pancreatic cancer in the future. Further studies are planned with more specific antibodies, other nanoparticles, and other cancer targets.

Pancreatic adenocarcinoma (PDAC) putatively arises from the pancreatic duct, thus usage of the normal human pancreatic duct epithelial (HPDE) cell line is an ideal model to examine the successive accumulation of genetic alterations involved in carcinogenesis. KRAS mutations have been reported in 90% of PDACs. Oncogenic KRAS elicits activation of downstream pathways involved in survival, motility, and cell cycle progression. KRASG12V introduction in the HPDE cell line upregulates Lipocalin-2 (LCN2) expression. LCN2 has been identified in numerous carcinomas and is associated with survival, tumorigenicity, and invasion. In this work, LCN2 was found to be commonly expressed in high grade pancreatic duct neoplastic precursor lesions and PDAC illustrating its potential as a biomarker. Moreover, in vitro and in vivo studies demonstrate that high LCN2 expression promotes gemcitabine resistance, MMP-9 activity, angiogenesis, and tumorigenicity. Loss of Smad4 function is found in 55% of PDAC cases. Smad4 is a critical component in the TGF-β signaling which mediates the transcription of genes involved in processes such as cell cycle arrest, apoptosis, and invasion. This work examined the consequences of KRASG12V expression and Smad4 loss in the HPDE model. Cellular invasion was promoted by KRASG12V expression or knocking down Smad4 by 80% in the HPDE model. A TGF-β resistant HPDE cell line, TβR, was shown to lack Smad4 expression due to deletion, promoter methylation, and nonsense mutation. KRASG12V expression in the TβR model (TβR KRAS) promoted neoplastic transformation and tumour formation in immunodeficient mice with complete penetrance. Smad4 expression in the TβR KRAS cell line reinstated TGF-β signaling, delayed tumour formation, and decreased metastatic spread. This study provides evidence that Smad4 acts as a restriction point in the transformation of HPDE cells. Overall, this work examines the contribution of genes involved in transformation, and identifies a potential therapeutic and diagnostic biomarker in PDAC.

It is not known whether pancreatic cancers evolve from a single or multiple cells, or from a particular pancreatic lineage. However, in the Pdx1-Cre; LSL-KrasG12D; LSLTp53R172H mouse model of pancreatic cancer, all pancreatic lineages are susceptible to express mutant KRas and p53. Hence, such mouse model implies a scenario of maximal heterogeneity of cancer cell origins. On this basis, I isolated seven subclones of heterogeneous mouse pancreatic cancer cells from a single tumour; each of them had a distinct morphology and gene expression profile. Notably, they possessed different intrinsic phospho-SMADs downstream of the TGFβ receptor (phospho-SMAD2/3) or the BMP receptor (Phospho-SMAD1/5/8). I discovered that SMAD4, a co-SMAD which is frequently found to be lost in pancreatic caner tissues, upregulated HNF4α via the classical BMP-SMAD1 pathway, when cells were experiencing metabolic stress upon deprivation of serum, or in the presence of excess thymidine. Under serum starvation at a hypoglycemic-like glucose concentration, the HNF4α-expressing sub-clones appeared to be more able to sustain an unstressed morphology than other non-HNF4α-expressing sub-clones. Immunohistochemical staining on pancreatic cancer sections revealed nuclear co-localization of SMAD4 and HNF4α in human (half of the cases) and in mouse samples. As a secondary project conducted during characterization of cells, I also found that three of the subclones more robustly proliferated under anchorage independent conditions, and they relied on the MEK-ERK pathway and the canonical Wnt pathway, to a different degree. Both studies demonstrate for the first time in primary cell culture that pancreatic cancer cells within a tumour could be highly heterogeneous in terms of both morphology and signaling pathways.

Etude à l'aide des outils de transcriptomique de 17 cultures primaires, maintenues à l'état vivant par xénogreffes et cultures cellulaires, et issues de patients ayant présenté un adénocarcinome pancréatique. Par clustering hiérarchique basée sur l'ensemble des gènes du transcriptome tumoral, 5 patients avec dénomination anonyme se sont fortement distingués des autres patients et présentaient de façon similaire des tumeurs peu différenciées sur le plan histologique et une survie péjorative de moins de 8 mois. Dans cette population de « courts survivants », un total de 942 gènes exprimés de façon significativement différentielle a été retrouvé. Parmi ces gènes, 439 gènes sont apparus significativement sous exprimés et 505 gènes significativement surexprimés (fold change ≥2). L'analyse par GO a montré que parmi ces 942 gènes différentiellement exprimés, nous avons retrouvé un enrichissement important chez les courts survivants, des gènes impliqués dans le cycle cellulaire et l'activité mitotique, la réponse cellulaire au stress, le métabolisme cellulaire ainsi que le métabolisme de l'ADN et l'organisation chromosomique. Par ailleurs, nous avons choisi parmi les 17 cultures primaires, les 3 lignées cellulaires les plus sensibles et les 3 les plus résistantes aux drogues selon les résultats des tests de « Chimiogramme ». Plusieurs gènes ont été identifiés comme spécifiquement surexprimés ou sous-exprimés en relation avec une sensibilité ou une résistance particulières des cellules aux drogues utilisées. Nous avons identifié 671 gènes associés à la gemcitabine, 1107 à l'oxaliplatine, 308 au 5-FU et seulement 34 et 46 au docétaxel et au SN38 respectivement. / We developed an efficient strategy in which PDAC samples from 17 consecutive patients were collected by Endoscopic Ultrasound-Guided Fine-Needle Aspiration (EUS-FNA) or surgery and preserved, by an original approach, as breathing tumors by xenografting and as a primary culture of epithelial cells. Transcriptomic analysis was performed from breathing tumors by an Affymetrix approach. We observed a significant heterogeneity in the RNA expression profile of tumors. However, the bioinformatic analysis of this data was able to discriminate between patients with long- and short-term survival corresponding to patients carrying poorly-differentiated PDAC tumors respectively. We identified 942 genes with statically different expression. Among these genes, 439 were under-expressed and 505 genes over-expressed (fold change ≥2) in short survivors. Primary culture of cells allowed us to analyze their relative sensitivity to anticancer drugs in vitro by a "Chemogram", by similarity with the antibiogram for microorganisms, establishing an individual profile of drug sensitivity. As expected, the response was patient-dependent. Interestingly, we also found that the transcriptome analysis predict the sensitivity of cells to the five anticancer drugs the most frequently used to treating patients with PDAC. In conclusion, using this approach, we found that the transcriptomic analysis could predict the sensitivity to anticancer drugs and the clinical outcome of patients with PDAC.

Activating mutations of the kras proto-oncogene are found in more than 90% of human pancreatic ductal adenocarcinoma (PDAC) and can result in increased activity of the NF-κB pathway, leading to constitutive production of proinflammatory cytokines such as TNF-α. Pancreatic cancer progression occurs through a series of pre-invasive lesions, pancreatic intraepithelial neoplasias (PanIN lesions), which progress into invasive carcinoma. The aim of this thesis is to understand the autocrine role of TNF-α produced by premalignant epithelial cells in pancreatic tumour progression. This cytokine has already been shown to be involved in the progression of cancer. The major hypothesis therefore tested was that TNF-α secreted by pre-malignant epithelial cells promotes the early stages of pancreatic carcinogenesis by sustaining an inflamed microenvironment. In the spontaneous kras+/LSL-G12D; pdx1-cre mouse model of pancreatic cancer, concomitant genetic deletion of the TNF-α/IKK2 pathway substantially delayed pancreatic cancer progression and resulted in downregulation of the classical Notch target genes hes1 and hey1. Cell lines from the different PanIN bearing mice were established and used to dissect the cooperation between TNF-α/IKK2 and Notch signalling during PanIN progression. Optimal expression of Notch target genes was induced upon TNF-α stimulation of the canonical NF-κB signalling pathway, in cooperation with basal Notch signals. Mechanistically, TNF-α stimulation resulted in phosphorylation of histone H3 at the hes1 promoter and this signal was lost upon ikk2 genetic deletion. HES1 suppressed the expression of pparg, which encodes for the anti-inflammatory nuclear receptor PPAR-γ. Thus, crosstalk between TNF-α/IKK2 and Notch sustained an intrinsic inflammatory profile of the transformed cells. The treatment of PanIN bearing mice with rosiglitazone, a PPAR-γ agonist, also delayed PanIN progression. A malignant cell-autonomous, low-grade inflammatory process was shown to operate from the very early stages of kras-driven pancreatic carcinogenesis, which may cooperate with the Notch signalling pathway to promote pancreatic cancer progression.

It is imperative that the cancer research community has the means with which to effectively locate, access, manage, analyse and interpret the plethora of data values being generated by novel technologies. This thesis addresses this unmet requirement by using pancreatic cancer and breast cancer as prototype malignancies to develop a generic integrative transcriptomic model. The analytical workflow was initially applied to publicly available pancreatic cancer data from multiple experimental types. The transcriptomic landscape of comparative groups was examined both in isolation and relative to each other. The main observations included (i) a clear separation of profiles based on experimental type, (ii) identification of three subgroups within normal tissue samples resected adjacent to pancreatic cancer, each showing disruptions to biofunctions previously associated with pancreatic cancer (iii) and that cell lines and xenograft models are not representative of changes occurring during pancreatic tumourigenesis. Previous studies examined transcriptomic profiles across 306 biological and experimental samples, including breast cancer. The plethora of clinical and survival data readily available for breast cancer, compared to the paucity of publicly available pancreatic cancer data, allowed for expansion of the pipeline’s infrastructure to include functionalities for cross-platform and survival analysis. Application of this enhanced pipeline to multiple cohorts of triple negative and basal-like breast cancers identified differential risk groups within these breast cancer subtypes. All of the main experimental findings of this thesis are being integrated with the Pancreatic Expression Database and the Breast Cancer Campaign Tissue Bank bioinformatics portal, which enhances the sharing capacity of this information and ensures its exposure to a wider audience.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by an intense desmoplastic stromal response that can comprise 60 to 80% of tumour volume and has been implicated to be a factor in promoting tumour invasiveness and the poor prognosis associated with this cancer type. It is now well established that pancreatic stellate cells, which are vitamin A storing cells found in the periacinar spaces of the stroma in the normal gland, are primarily responsible for this desmoplastic reaction. Studying the interaction between stellate cells and cancer cells could provide for a better understanding of the disease process. During the evolution of PDAC, the stromal proportion increases from 4% in the normal gland to up to 80%. We hypothesised that there is an optimal proportion of stellate cells and cancer cells that modulates tumour behaviour and we attempted to dissect out this probable ‘tipping point’ for stromal composition upon cancer cell behaviour using a well-established in vitro organotypic culture model of pancreatic cancer. The cancer cell-stromal cell interaction led to extra-cellular matrix contraction and stiffening; and an increase in cancer cell number. The stromal stellate cells conferred a pro-survival and pro-invasive effect on cancer cells which was most pronounced at a stellate cell proportion of 0.66-0.83. The expression of key molecules involved in EMT and metastasis such as E-Cadherin and β-catenin showed a reduction and this was found to be most significant again at a stellate cell proportion of 0.66-0.83. Stellate cells altered the genetic profile of cancer cells leading to differential expression of genes involved in key cellular pathways such as cell-cycle and proliferation, cell movement and death, cell-cell signalling, and inflammatory response. qRT-PCR confirmed the differential expression of the top differentially expressed genes and protein validation by immunofluorescence staining using PIGR as a candidate molecule confirmed the experimental findings in human PDAC specimens. This study demonstrates that the progressive accumulation of desmoplastic stromal cells has a tumour progressive (pro-survival, pro-invasive) effect on cancer cells in addition to stiffening (contraction) of the extracellular matrix (maximum effect when the stromal cell proportion is 60-80%). This is mediated through a number of signalling cascades and molecular targets. Dampening this tumour-promoting interaction between cancer and stromal cells by ‘multi-targeting’ agents may allow traditional chemo- and/or radiotherapy to be effective.

This thesis focuses on the use of data mining techniques to investigate the expected survival time of patients with pancreatic cancer. Clinical patient data have been useful in showing overall population trends in patient treatment and outcomes. Models built on patient level data also have the potential to yield insights into the best course of treatment and the long-term outlook for individual patients. Within the medical community, logistic regression has traditionally been chosen for building predictive models in terms of explanatory variables or features. Our research demonstrates that the use of machine learning algorithms for both feature selection and prediction can significantly increase the accuracy of models of patient survival. We have evaluated the use of Artificial Neural Networks, Bayesian Networks, and Support Vector Machines. We have demonstrated (p<0.05) that data mining techniques are capable of improved prognostic predictions of pancreatic cancer patient survival as compared with logistic regression alone.

Pancreatic ductal adenocarcinomas (PDACs) are highly aggressive malignancies, associated with poor clinical prognosis and limited therapeutic options. The KRAS oncogene is mutated in over 90% of PDACs and plays a pivotal role in tumor progression. Global gene expression profiling of PDAC reveals 3-4 major molecular subtypes with distinct phenotypic traits and pharmacological vulnerabilities, including variations in oncogenic KRAS pathway dependencies. PDAC cell lines of the aberrantly differentiated endocrine exocrine (ADEX) subtype are robustly KRAS-dependent for survival. The KRAS gene is located on chromosome 12p11-12p12, a region amplified in 5-10% of primary PDACs. Within this amplicon, we identified co-amplification of KRAS with the STK38L gene in a subset of primary human PDACs and PDAC cell lines. This provided rationale to determine whether PDAC cell lines are dependent on STK38L expression for proliferation and viability. STK38L (also known as NDR2) encodes a nuclear Dbf2-related (NDR) serine/threonine kinase, which shares homology with Hippo pathway LATS1/2 kinases. We show that STK38L expression levels are elevated in a subset of primary PDACs and PDAC cell lines that display ADEX subtype characteristics, including overexpression of mutant KRAS. RNAi-mediated depletion of STK38L in a subset of ADEX subtype cell lines results in decreased cellular proliferation and increased apoptotic cell death. Concomitant with cytostatic and cytotoxic effects, STK38L depletion causes increased expression of the LATS2 kinase and the cell cycle regulator p21. LATS2 depletion partially rescues the cell proliferation and viability effects of STK38L depletion. Lastly, high STK38L mRNA expression is associated with worse patient prognosis compared to low STK38L expression in PDACs. Taken together, our study uncovers STK38L as a candidate, targetable vulnerability in a subset of molecularly defined PDACs. / 2019-11-01T00:00:00Z