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A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2015. / Over the decades, cancer has become a global burden with alarmingly high incidence and mortality rates in both economically developed and developing countries. Characteristically, tumour cells exhibit an over-expression of the 37kDa/67kDa laminin receptor (LRP/LR) in comparison to their normal cell counterparts, with this receptor being implicated in several tumourigenic processes – importantly for the present study, the maintenance of cellular viability and the evasion of apoptosis. This present study aimed to elucidate the role of LRP/LR on the cellular viability of pancreatic cancer (AsPC-1) and neuroblastoma (IMR-32) cells. Flow cytometry revealed that both of these tumourigenic cell lines exhibited LRP/LR on their surface, with further analysis using median fluorescence intensity values showing that IMR-32 cells contain about 70% more cell-surface LRP/LR than AsPC-1 cells. Additionally, Western blotting and densitometry suggested that IMR-32 cells contained about 63% more total LRP/LR than AsPC-1 cells. Western blot analysis also revealed that targeting the mRNA of the 37kDa LRP using a LRP-specific siRNA (siRNA-LAMR1) in AsPC-1 and IMR-32 cells led to significant down-regulation of 90% and 71% in LRP expression, respectively. Consequently, MTT assays showed that LRP knockdown led to reductions of 82% and 65% in the viability of AsPC-1 and IMR-32 cells, respectively. Moreover, use of an alternative LRP-specific siRNA (esiRNA-RPSA) confirmed the specificity and excluded an off-target effect of siRNA-LAMR1 for LRP. BrdU assays revealed that knockdown of LRP reduced the proliferation of AsPC-1 and IMR-32 cells by 76% and 44%, respectively. Confocal microscopy indicated nuclear morphological changes suggestive of apoptosis as the form of cell death occuring in both cell lines after LRP down-regulation. This observation was confirmed using Annexin-V assays, which revealed that AsPC-1 cells underwent 44% more apoptosis than IMR-32 cells post LRP knockdown. Furthermore, caspase-3 activity assays revealed that both cell lines experienced apoptotic induction after siRNA-mediated down-regulation of LRP. Caspase-8 and -9 activity assays suggested that post LRP knockdown, IMR-32 cells undergo apoptosis solely via the extrinsic pathway, whilst AsPC-1 cells use both the intrinsic and extrinsic apoptotic pathways, possibly through a retaliatory feedback loop. Overall, LRP/LR is critical for the maintenance of the tumour cellular viability, making the receptor a promising therapeutic target and proposing the potential use of siRNA technology for treatment of pancreatic cancer and neuroblastoma.

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Cancer is associated with many molecular aberrations that support the malignant phenotype. In that regard, aberrant activation of epidermal growth factor receptor (EGFR), Src, and signal transducer and activator of transcription 3 (Stat3) occur concurrently in pancreatic cancer and are implicated in the disease phenotype. Notwithstanding, increasing evidence indicates that therapies that target only EGFR or Src are rather ineffective in modulating the cancer phenotype. The poor therapeutic outcome of the monotherapies targeting EGFR or Src may in part be due to the increased incidence of signaling cross-talks among aberrant signaling pathways in cancer. Molecular details of the signaling integration between EGFR, Src and Stat3, however, are lacking. Understanding how the aberrant EGFR, Src and Stat3 pathways are integrated in pancreatic cancer would facilitate the design of effective multipletargeted, clinically feasible therapeutic modalities. Our study shows that in pancreatic cancer cell lines, aberrant Src activity promotes abnormal EGFR activation through the phosphorylation of the EGFR motifs, Tyr845, Tyr1068 and Tyr1086. Furthermore, aberrantly-active EGFR and Src together induce constitutive activation of Stat3 in pancreatic cancer cells. Evidence further shows that EGFR, Src and Stat3 physically associated into a heteromeric complex. Significantly, the EGFR, Src and Stat3 heteromeric complex is detectable in the nucleus and functions as a transcriptionallyactive complex to induce the c-Myc gene. Of therapeutic significance, the concurrent inhibition of Stat3 and EGFR or Src promoted greater viability loss and apoptosis of pancreatic cancer cells in vitro, and induced stronger tumor growth inhibition in xenografts of human pancreatic cancer. Altogether, our studies suggest that the iii heteromeric EGFR, Src, and Stat3 complex may serve as an additional novel mechanism of support of the pancreatic cancer phenotype. Furthermore, our studies provide evidence that the concurrent targeting of Stat3 and EGFR or Stat3 and Src could be a more effective therapeutic approach for human pancreatic cancer.

Pancreatic cancer is an abhorrent malignancy with limited diagnostics and response to drug therapy. It is believed that noncoding RNAs (ncRNAs) will further the understanding behind the mechanisms of pancreatic cancer development and progression, providing a novel approach for drug development and biomarker discovery. Therefore, a database of pancreatic cancer ncRNAs was established using bioinformatics and text mining approaches. These ncRNAs were characterized for RNA expression, copy number variation, disease association, single nucleotide polymorphisms, secretome analysis, and identification of protein targets. Exosomal proteins and ncRNA identified through this study provide the basis for noninvasive diagnostic potential. Additionally, a secreted microRNA, MIR3620, emerged from this study as a potential prognostic and diagnostic biomarker for pancreatic cancer. By analyzing MIR3620 and its protein targets, a mechanism of regulation for these genes in contributing to the progression and development of pancreatic cancer was established. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

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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.