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Prostate cancer (CaP) is the most common cancer in men in the UK and its incidence is increasing. The natural history of the disease is very variable, in some men progressing rapidly while in others It will run a more indolent course, in early disease localised to the prostate, radical treatment options offer potential cure however these come with considerable potential morbidity and a significant proportion of patients will relapse despite such interventions. This suggests the presence of microscopic disease beyond the prostate, not clinically detectable using current staging modalities.

The nucleolus is a plurifunctional organelle in which structure and function are intimately linked. Though it is primarily known as the site of ribosomal biogenesis, the nucleolus is also capable of orchestrating the immobilization of a broad range of proteins under specific environmental conditions. This process, known as nucleolar sequestration, contributes to cell viability under stress. Despite the importance of this post-translational regulatory pathway, very little is known about the mechanisms that govern it. Here, we show that heat shock and acidosis, two physiological stimuli associated with nucleolar sequestration, induce the expression of long noncoding RNA (lncRNA) from stimulus-specific loci of the ribosomal intergenic spacer (IGS). These lncRNAs, in turn, immobilize proteins encoding a nucleolar detention sequence (NoDS) within a compartment of the nucleolus termed the detention centre (DC). The DC is a spatially and dynamically distinct region, characterized by an 8-anilino-1-naphthalenesulfonate (ANS)-positive hydrophobic signature. Its formation is accompanied by a redistribution of nucleolar factors and an arrest in ribosomal biogenesis. Silencing of regulatory IGS lncRNA prevents the creation of this structure and allows the nucleolus to retain its tripartite organization and transcriptional activity. Signal termination causes a decrease in IGS transcript levels and a return to the active nucleolar conformation. We propose that the induction of IGS lncRNA, by environmental signals, operates as a molecular switch that regulates the structure and function of the nucleolus.

Introduction: Gastrointestinal stromal tumours (GIST) are the commonest tumour of mesenchymal origin; favour the stomach, and account for a very small percentage of gastrointestinal tract tumours. Methods: In this retrospective audit of GISTs presenting to the Groote Schuur Hospital surgical and oncological multidisciplinary team (MDT) between 2004 – 2019, gastric GISTs were evaluated as regards presentation, gastric anatomical position, histological subtype with risk stratification, management and outcomes. Results: Of 126 GIST tumours presenting to this MDT, 82 originated in the stomach. Complete histopathological records could be obtained for 64. With an average of 59 years (50 male: 32 female), 18 (28%) presented with a herald bleed. Other common presentations included anaemia, epigastric mass and pain. The tumours were predominantly found in the body and fundus (64%), with a spindle cell subtype predominance (41%). The association between cancer cell subtype and gastric position was not significantly different (p=0.728). Cystic degeneration was found on 11 (17%) analyzed and cell necrosis on 12 (18%). These findings were not related to larger tumor size or prognosis. Five required downstaging with Imatinib prior to surgery. Thirty-seven patients underwent a surgical procedure: 24 wedge resections and 12 anatomical resections. Risk stratification was performed with the modified National Institutes of Health (NIH/Fletcher) score. Twenty-eight cases had inaccurate mitotic counts and couldn't be scored, 17 scored high risk, 9 intermediate risk, 9 low risk and 1 very low risk. Ten patients died of metastatic disease, 34 were discharged with no disease progression after 3 years, 1 patient with disease progression currently remains on Imatinib, and 19 were lost to follow up. Conclusion: Gastric GISTs appear to have a predilection for the proximal stomach; it is unsure whether this is purely due the greater surface area. The spindle cell subtype dominated in the proximal gastric GISTs. Cystic degeneration and cell necrosis did not seem to be related to larger tumours or outcomes.

The delivery of immunoreactive macromolecules to tumour cells in solid, heterogeneously perfused tumours is a major problem in the effectiveness of immunotherapy. To help optimize the new experimental treatment method, a published mathematical model of macromolecule transport (Baxter & Jain 1989,1990,199la) was appraised and verified experimentally. Computational and analytical tools were developed to predict the interstitial plasma fluid pressure and velocity distributions in well perfused spherical tumours. Their published analytical solutions of the formulation were found to have some errors and were corrected in this work. To check the validity of the formulation, a series of animal experiments was performed to quantify the total vascular volume, and plasma fluid extravasation rate in SKOV3ipl human ovarian tumour xenografts in nude mice. The results compared well with the theoretically predicted total plasma fluid extravasation rate. Computer codes were also developed to predict the spatial and temporal distributions of intact IgG and its F(ab')₂ and Fab/Fab' fragments in well perfused spherical tumours using the formulation proposed by Baxter & Jain (1989,1990,1991a). The cases of non-binding and binding macromolecules were treated separately. The codes for both the interstitial pressure and macromolecule distributions were written to include a radially variable vessel surface area for transcapillary exchange per unit volume of tumour tissue (SN). The sensitivity of the overall tumour perfusion to variation of (a) the macromolecule m.w., binding affinity, and metabolism, (b) SN, tumour radius, and (c) microvascular permeability were investigated. Comparison of the theoretical predictions with available experimental data leads to the realization of a number of shortcomings in the previously proposed formulations. Finally, a computational method for deriving the effective spherically symmetric spatial distributions for the vascular volume density, and SN from tumour serial sections was developed. This bridges the gap between the actual topology of vascular distributions in tumours and the format of current formulations. / Thesis / Master of Science (MS)

Delta-like ligand 4 (DLL4) is a ligand for the Notch family of receptors. DLL4 is an important regulator of angiogenesis and DLL4 blockade promotes non-productive angiogenesis and delays tumour growth. The aim of this thesis was to investigate the effects of anti-DLL4 therapy in solid tumours in combination with a clinically relevant dose of ionising radiation (5 Gy; IR) and to analyse alterations in the Notch pathway induced by the treatments. Combining both treatments resulted in a greater than additive tumour growth delay in LS174T tumours, compared to either treatment alone. DLL4 blockade dysregulated vasculature and increased necrosis in LS174T and HCT-15 (DLL4-expressing and negative cell lines respectively) tumours within 3 days after treatment, but no changes were observed with IR alone. Additionally, combined IR and anti-DLL4 treatment of FaDu tumours (another DLL4-negative cell line) by our colleagues, also resulted in a supra-additive growth delay. These results show that combining IR with DLL4 blockade is an effective strategy for prolonging tumour growth delay and suggest that the stroma/vasculature provide the main therapeutic target for the anti-DLL4 therapy. Analysis of Notch pathway shows that IR upregulated Jag1 in tumour cells, and may inhibit Notch and downregulate DLL4 in the stroma. These changes may potentially affect tumour vessels and response to anti-DLL4 therapy. In vitro, anti-DLL4 therapy induced proliferation in quiescent contact-inhibited endothelial cells and also appeared to abrogate IR-induced inhibition of migration. These results suggest that DLL4 may be important in maintaining vessel quiescence and that IR may in part decrease migration through Notch signalling. Combining IR and DLL4 blockade to target tumour growth is an effective and well tolerated strategy and warrants further validation and refinement to be translated into clinical practice.

No / Despite promising early data, the natural product dolastatin 10 has not been successful as a single agent in phase II clinical trials. Herein the mechanism of action and efficacy of a synthetic analogue, auristatin PYE, was investigated in 2 human colon adenocarcinoma models, DLD-1 and COLO 205. In vivo efficacy was assessed in subcutaneous xenografts following intravenous administration. Mechanistic studies investigated effects of auristatin PYE on microtubule disruption using immunocytochemistry, whilst cell cycle effects were studied using flow cytometry. Possible effects on tumour functional blood vasculature were assessed in tumour-bearing mice. Auristatin PYE was less potent in vitro than dolastatin 10, but was significantly more effective (p<0.01) in vivo against both tumours. Significant effects on tumour blood vasculature were seen, with optimal shutdown at 6-h post-treatment. Extensive necrosis became more evident over time after treatment. Auristatin PYE caused severe disruption of normal microtubule structure at concentrations and times comparable with the IC50 data, and also instigated a G2/M cell cycle block. Auristatin PYE was more effective in the DLD-1 and COLO 205 models than dolastatin 10, with anti-tumour effects mediated through vascular shutdown. These data suggest that auristatin PYE has good potential as an anti-cancer agent.

The apoptotic function of p53 is specifically regulated by iASPP and ASPP2 and their additional modulation of p63, a key regulator of squamous epithelial homeostasis, has been demonstrated in mice. In this study the role of iASPP and ASPP2 in human carcinomas was explored with a particular focus on the development of squamous cell carcinomas. The predominant expression of cytoplasmic ASPP2 is seen in the superficial, terminally differentiated cell layers of normal squamous epithelia and its loss is documented in areas of dysplasia and in squamous cell carcinomas. In addition the absence of nuclear ASPP2 in association with human papillomavirus infection suggests it could be a novel viral target. Furthermore in a novel mouse model of lung tumourigenesis, the somatic deletion of ASPP2 is shown to promote the over expression of markers associated with specific lung progenitor cells and to be associated with the development of non small cell lung carcinomas. Conversely nuclear iASPP expression is seen in p63 expressing, replication competent, basal and parabasal squamous epithelial cells and is increased in areas of dysplasia and in squamous cell carcinomas. Moreover the combination of nuclear iASPP, p63 and loss of ASPP2 expression is seen to be associated with cell survival and characterises the invasive edges of lingual squamous cell carcinomas. The novel interaction of iASPP with ER&alpha; is also demonstrated together with the negative impact of nuclear iASPP on ER positive breast cancer survival, potentially identifying iASPP as a novel player in estrogen signalling and an important factor in breast tumourigenesis. Together the data presented here provide significant corroborative evidence implicating ASPP2 and iASPP in tumourigenesis. Specifically ASPP2 is shown to promote cellular differentiation and inhibit the expansion of proliferative populations whereas nuclear iASPP promotes the survival of potentially neoplastic clones. It is likely that the balance of these proteins is a key factor in determining individual cell fate decisions.

Apoptosis is a physiological form of cell death. It is a highly evolutionarily conserved process that is non-inflammatory or anti-inflammatory in nature. This anti-inflammatory nature of apoptosis is evident by the fact that neutrophils are histologically absent from sites where homeostatic apoptosis rates are high. The rapid phagocytosis of apoptotic cells as a means to prevent the release of noxious inflammatory compounds also accounts for the anti-inflammatory environment of such sites. However, the mechanisms that enable mononuclear phagocytes to migrate to sites where homeostatic apoptosis rates are high, and not granulocytes, the professional phagocytes that accumulate at sites of inflammation, have not been determined yet. Using Burkitt’s lymphoma (BL) as a model of apoptosis, the aim of this thesis was to identify the regulatory mechanisms or factors underlying the non-phlogistic features of sites where homeostatic apoptosis rates are high and in particular, those preventing the recruitment of neutrophils - a major granulocyte subclass to these sites. BL is a highly aggressive B cell lymphoma that is mainly characterised by a high rate of apoptosis. By carrying out a series of in vitro chemotaxis assays and biochemical approaches, it was found in this thesis that BL cells actively inhibit neutrophil migration by releasing factors that were identified to be lactoferrin, a 80 kDa iron-binding glycoprotein with anti-bacterial and anti-inflammatory properties. It was further demonstrated that lactoferrin selectively inhibited migration of granulocytes (both neutrophils and eosinophils) but not mononuclear phagocytes and this effect was irrespective of its iron saturation status and the chemoattractant used. Also, lactoferrin potently inhibited neutrophil migration, as assessed by thioglycollate-induced in vivo model of mouse peritonitis. This anti-inflammatory function of lactoferrin was attributed to its effect on granulocyte signalling pathways that regulate cell adhesion and motility. Finally, it was demonstrated that in cell types of diverse lineages, induction of apoptosis results in de novo synthesis and secretion of lactoferrin. In subsequent proliferation assays determining the in vitro growth of a number of BL cell types, it was demonstrated that lactoferrin is an essential component of BL cells and promotes their proliferation, as its antibody-mediated neutralisation or shRNA-mediated expression knockdown, reduced BL cell growth. Together, the results of this thesis identified lactoferrin as one of the few characterised antiinflammatory components of the apoptosis milieu that negatively regulate granulocyte migration. This effect may provide opportunities for broad therapeutic interventions concerning the use of lactoferrin in chronic inflammatory conditions characterised by aberrant neutrophil influx as well as atopic allergic disorders, such as asthma. Moreover, based on the tumour-supporting role of lactoferrin described in this study, targeting its expression in tumours could lead to tumour regression and thus, be a promising therapeutic molecule in tumour immunotherapy.