Salivary adenoid cystic carcinoma (ACC) is prone to perineural invasion, late recurrence, and distal metastases, with 20-year survival of only 10%. Research defining new targets in ACC has lagged largely due to a dearth of in vitro models. In this thesis, a neurogenic gene signature intrinsic to ACC was characterized revealing genes involved in maintenance, differentiation, and function of non-cancerous neural crest stem cells, including TrkC and SOX10. Analyses of gene expression across tumor types revealed that melanoma, neuroblastoma, glioblastoma, and basal-like breast cancer expressed SOX10 and several SOX10 co-expressed genes, suggesting that tumors derived from cells originating in the neural crest may contain similar populations of stem-like cells. Examination of TrkC signaling revealed that TrkC supported cell migration, invasion, and ACC tumor growth, and that ACC cells produced the TrkC ligand. Optimization of newly-designed culturing techniques allowed for the first time establishment of ACC cell cultures from xenograft and primary ACC specimens. Gene expression analysis revealed that CD133 was co-expressed with SOX10, and remarkably, it marked a subpopulation of ACC cells that preferentially expressed NOTCH1 and SOX10, formed spheroids, and initiated tumors in nude mice. Depletion of NOTCH1 or pharmaceutical inhibition of Notch signaling depleted CD133+ cells, sensitized CD133+ cells to radiation, and suppressed spheroidogenesis and xenograft tumor formation. Optimization of culture techniques provides a framework for future experimentation, and identification of a stem-like subpopulation in ACC suggests that targeting signaling pathways critical for stem cell survival or behavior may provide a new avenue to ACC therapy.

Human papillomaviruses (HPV) are small non-enveloped viruses that infect basal cells. Most HPV infections are mild and develop warts at the site of infection. However, some high-risk serotypes of HPV are able to promote cancer formation. Serotypes 16 and 18 are responsible for the majority of cervical cancer cases [1]. Its early proteins E6 and E7 promote oncogenesis by facilitating the acquisition of 7 hallmark traits necessary for cancer: constant signaling for proliferation, insensitivity to growth suppressors, evasion of apoptosis, limitless replicative potential, angiogenesis, immune evasion, and metastasis [1, 65, 68, 72, 76, 78, 79, 81, 82, 83, 84]. In addition to E6 and E7, specific conditions of an HPV infection seem to increase cancer incidence. Among these conditions are infection at the cervix's transformation zone, HPV genome integration with host chromosomes, inflammation and the presence of estrogen [1, 60, 61, 62, 63, 64, 69, 70, 71]. Estrogen's role in cervical cancer is not well understood. It is possible that it plays a role in the transcription of oncogenes by activating ERα and subsequently activating Sp1 [65]. Specifically, the Sp1 binding site is conserved and necessary for VEGF and hTERT expression [65, 79].

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CANCER BIOLOGY The Role of Toll-Like Receptor 9 in Helicobacter pylori-Mediated Inflammation and Carcinogenesis Matthew Gordon Varga Dissertation under the direction of Dr. Richard M. Peek Jr., MD Helicobacter pylori is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. The H. pylori cag pathogenicity island encodes a type IV secretion system (T4SS), which translocates the pro-inflammatory protein CagA into host cells. Although T4SSs are ubiquitous and facilitate transport of diverse effectors from bacterial cells to the eukaryotic host, only two bacterial species have been shown to translocate DNA into eukaryotic host cells. Furthermore, in DNA-translocating T4SSs, the only proteins transported via the T4SS are proteins associated with the process of DNA transfer. Toll-like receptor 9 is an endosome bound, innate immune receptor that detects hypomethylated CpG DNA motifs. We now demonstrate that the H. pylori cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Genetic deficiency of Tlr9 augments the intensity of IL-17-driven immune responses to H. pylori in vivo, suggesting that engagement of TLR9 by bacterial DNA leads to suppression of inflammation, which may accommodate long-term interactions between pathogenic H. pylori and the gastric epithelium. In support of this hypothesis, analysis of a human population at increased risk for gastric cancer revealed that levels of H. pylori-mediated TLR9 activation were directly related to the severity of cancer risk. These results demonstrate the ever-increasing versatility of T4SS machineries and their involvement in promoting pathogen persistence and modulating disease outcomes.

γKetoaldehydes are generated by free radical peroxidation and cyclooxygenation of phospholipid-esterified arachidonic acid. γKetoaldehydes covalently bind to protein lysine residues and is emerging as a mechanistic link between pathogenic reactive oxygen species and disease progression. However, the questions of whether covalent modification of proteins by γKA are subject to genetic regulation and the identity of γKA-modified proteins remain unclear. This dissertation shows that Nrf2 and Nox2 are key regulators of γKA modification in pulmonary tissue. The identity of these proteins were analyzed by LC-MS following immunoaffinity purification of γKA-modified proteins. Gene ontology analysis revealed that proteins in numerous cellular pathways are susceptible to γKA modification. Although cells tolerate basal levels of modification, exceeding them induces apoptosis. Prominent modification was observed in a murine model of radiation-induced pulmonary fibrosis and in idiopathic pulmonary fibrosis (IPF) patients, two diseases considered to be promoted by gene-regulated oxidant stress. This dissertation found an abundance of γKA modified protein in human IPF compared to control lung tissue, identified collagen 1α1 as one of the highly adducted proteins, and demonstrates that γKA modification impairs MMP1 mediated degradation of collagen. Based on these results the current hypothesis is that γKA modification is a hitherto unrecognized sequelae that contributes to radiation-induced pulmonary injury and IPF.

Gynaecological cancers (which include cancers of the ovary, cervix, uterus, vagina, endometrium, vulva and fallopian tube) account for 19% of all female cancers, and there are approximately 942,000 new cases diagnosed per year worldwide. Treatment for gynaecological cancer is often multi modal and consists of surgery, radiotherapy and chemotherapy. Current government policy has highlighted the need to focus on improving patient reported outcomes, including the patient experience of all aspects of cancer (including treatment), and the quality of life (QoL) of patients living with and beyond a cancer diagnosis. This thesis focuses on the effects of two different complementary interventions available to patients who were undergoing active treatment for gynaecological cancer at the Royal Derby Hospital between 2010 and 2012. Patient reported outcomes were explored in terms of the patients’ experience of the interventions and their reported levels of quality of life. The first study in this thesis explores the patient experience of an enhanced recovery programme (ERP) which was implemented for gynaecological cancer patients undergoing surgery at the Royal Derby Hospital in 2010. Previous research has found that ERPs (which complement traditional surgery) can decrease length of hospital stay, and they are now being implemented nationwide. However, there is a paucity of research into the patient experience of ERPs, especially in the field of gynaecological cancer. This study utilised a qualitative methodology to explore the experiences of 14 gynaecological cancer patients who took part in the ERP at Derby. Each patient was interviewed using a semi-structured format and the transcripts were analysed using Interpretative Phenomenological Analysis. The analysis highlighted that patients highly value the programme, and four main themes, fundamental to their experience, emerged from the data: taking part in the programme, the role of home, managing expectations and individual experiences outside of the programme. The second part of this thesis explores whether yoga can improve the quality of life (QoL) of patients undergoing treatment for gynaecological cancer when used as a complementary therapy. Previous research has found that participation in yoga can improve QoL in the breast cancer population, although there have been no similar studies conducted with UK gynaecological cancer patients to date. The study presented here utilised a randomised controlled design; 44 patients receiving treatment for gynaecological cancer were randomly allocated into a control group or a 10 week yoga intervention group. Outcomes were measured using the EORTC QLQ C30 questionnaire pre and post trial alongside visual analogue scales that were incorporated into a weekly diary. The results suggest that there was no significant effect of yoga on QoL, although there was encouraging data from one set of tests within the analysis, which suggested that patients on the yoga arm were seeing more improvement in QoL over time compared to the controls. Methodological improvements to clinical trials investigating complementary interventions are discussed in light of the results of this study. The overall findings of these two studies highlight that the utilisation of mixed methods is efficacious when exploring the effects of complementary interventions on the patient reported outcomes of those with gynaecological cancer. The use of qualitative methods to explore the patient experience of the ERP allowed for an in-depth, unique analysis to take place which was specific to the service delivered at The Royal Derby Hospital. The findings and recommendations from this part of the research have been incorporated into the on-going development of the pathway; it has indicated that more use of qualitative methods is needed in health services research to ensure that the patient experience is being fully explored, in line with the current government policy. Similarly, the second part of the research reported here indicates that further research in the area of yoga and gynaecological cancer is warranted. This requires a narrower focus with regards to both cancer type and point of treatment, to ensure that the number of variables is controlled. In addition, appropriate measurement and analysis techniques need to be considered (such as the generalised additive model used in this research) to preserve the richness of the data as this has not been considered (or utilised) in the many previous pieces of research in the area.

Exosomes are extracellular vesicles that carry a variety of RNA and protein cargos, including growth factors, transmembrane proteins, angiogenic factors, proteinases, and microRNA. However, the mechanisms that regulate exosome secretion are still poorly understood. This dissertation work shows that the tumor-overexpressed cytoskeletal protein cortactin promotes exosome secretion. Knockdown or overexpression of cortactin in head and neck squamous cell carcinoma, fibrosarcoma, and breast cancer cells leads to a corresponding decrease or increase in exosome secretion. However, cortactin knockdown does not affect cargo sorting or exosome biogenesis. Rescue experiments carried out by adding purified exosomes back to cortactin-knockdown cells suggest that exosome secretion may account for many functions of cortactin in tumor aggressiveness. Furthermore, live imaging revealed that cortactin controls both trafficking and plasma membrane docking of multivesicular endosomes. Mechanistically, interaction of cortactin with Arp2/3 complex and branched actin is critical for exosome secretion. Also, cortactin, Rab27a, and coronin 1b coordinately control stability of cortical actin multivesicular endosome docking sites along with exosome secretion. Overall, this dissertation identifies molecular and cellular mechanisms important for exosome secretion.

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy