Chimeric antigen receptor (CAR) T cell therapies have been highly effective and clinically approved for treating haematological malignancies, however trials in solid cancers have shown limited efficacy, likely due in part to the increased complexity of the immunosuppressive tumour microenvironment (TME) in solid cancers. CAR T cells are inhibited by immunosuppressive proteins, cytokines or physical barriers deployed by the tumour to evade and avoid destruction by anti-tumour immunity. One such process involves the accumulation of extracellular adenosine (eADO), in the TME which has potent immunosuppressive effects on T cells and other immune cells. eADO has four known G protein coupled receptors, the A1R, A2AR, A2BR and A3R, of which the A2AR is primarily responsible for suppressing T cell function. Our previous studies highlighted a major impediment to pharmacological blockade of the A2AR which was predicted to be hindered by poor solubility and suboptimal in vivo pharmacokinetic profile [1]. This became apparent when comparing the effectiveness with genetic deletion of A2AR in CAR T cells to pharmacological blockade, in which the CAR T cells generated from A2AR-/- mice elicited comparatively greater efficacy in vivo when combined with anti-PD-1 blockade [1, 2]. This thesis therefore investigated multiple gene editing strategies to modulate adenosine receptor signalling, firstly by overexpressing the alternative signalling A1R or A3R in human or mouse CAR T cells. A1R or A3R have been shown to act by the opposing downstream signalling pathway to A2AR, and thus it is hypothesised that A1R or A3R overexpression can reverse suppression and supercharge CAR T cells in the presence of eADO. Interestingly, A1R or A3R overexpression did not confer protection to suppression by eADO in both mouse and human models, but A1R expression instead enhanced effector and terminal differentiation, activation, and baseline cytokine production of CAR T cells. This however translated to higher expression of exhaustion markers, loss of memory associated gene expression and reduced stem-like memory fraction in the CAR T cell product, ultimately leading to reduced persistence in vivo, and limiting the therapeutic efficacy of this approach. Alternatively, a previous publication from our lab briefly examined short-hairpin RNA (shRNA) mediated silencing of A2AR expression [1]. While shRNA-mediated silencing of the A2AR was able to partially reverse suppression by eADO, much like A1R expression, it also led to effector differentiation, activation, and increased baseline cytokine production. Importantly, while shRNA-mediated silencing of the A2AR also resulted in reduced persistence in vivo, it was able to mediate modest anti-tumour efficacy leading to reduced tumour growth and increased mouse survival. Both overexpression and knockdown approaches are limited by sub-optimal persistence in vivo which limited their overall therapeutic efficacies. Yet these results contradicted our prior observations of CAR T cells derived from A2AR-/- mice and from studies in the Lymphocytic choriomeningitis virus (LCMV) setting, whereby A2AR deletion was linked to increased T cell numbers [1, 3]. Therefore, the final gene-editing approach examined in this thesis utilised CRISPR/Cas9 protocols to achieve full deletion of the A2AR in CAR T cells. CRISPR/Cas9 methodologies are currently being used in clinical trials and therefore deleting the A2AR in CAR T cells using this approach is highly novel and clinically translatable. To reasons unknown, CRISPR/Cas9 mediated deletion of A2AR had minimal effects on CAR T cell memory phenotypes and no adverse effects on engraftment or persistence in vivo. Furthermore, CRISPR/Cas9-mediated deletion of A2AR in CAR T cells led to enhanced therapeutic efficacy in both mouse and human models, thus representing a potent approach to targeting the A2AR. In conclusion, future studies comparing full A2AR deletion to A2AR silencing/ pharmacological blockade or A1R overexpression may be of interest to fully elucidate the mechanisms of adenosine receptor signalling on T cell persistence and memory.

Precision oncology refers to the use of sophisticated assays to tailor therapy to an individual patient. The feasibility of implementing such a program in a comprehensive cancer centre in Australia is unknown. The utility of precision oncology also depends on understanding how genomic profiles may evolve over time and differ between tumours in the same patient. A precision oncology program called SEGMENT was designed and implemented in a single centre. The program was popular, recruiting well over the study period. Timely acquisition of samples for sequencing was suboptimal from external pathology providers, and proved increasingly expensive during the study period. Delivery of results in a manner where they could be utilized by the patient was challenging in cases where patients were referred late in their natural history. A custom hybrid capture panel worked reliably. A total of 300 patients were recruited to the study, of which 288 had at least one sample received. Accounting for attrition, 214 patients or 71% went onto the main study. The spectrum of mutations and copy number alterations found in this study was similar to published cohorts. There were few differences between primary and metastatic lesions on average. Paired primary and metastatic samples however displayed discordance for both copy number and mutations. This was the case for actionable alterations in ESR1, ERBB2 and very rarely PIK3CA. Approximately 50% of patients had an actionable alteration. Of these 14% of the overall cohort received a therapy matched to their genomic profile. Five patients received matched therapy off trial and 26 received matched trial therapy. Three were zero responses in the off-trial group, and a response rate of 27% in the matched trial therapy group. To explore genomic heterogeneity in greater resolution, 4 patients with advanced breast cancer underwent rapid autopsies to collect large numbers of metastatic samples. Whole exome sequencing was performed on multiple lesions per patient which allowed inference of the subclonal structure. All patients displayed a monophyletic architecture, with truncal driver alterations giving rise to subclones with differing genomic profiles. One patient with a long disease free interval from primary to metastasis showed the acquisition of a new driver in the metastatic lesion. Driver alterations appeared to shape subsequent evolution.

The appropriate regulation of gene expression programs is essential for normal cellular function. In cancer, mutations derail normal developmental gene expression programs resulting in a malignant epigenetic state. Traditionally, therapies have attempted to kill cancer cells by targeting rapid proliferation or by directly disrupting the mutated protein. More recent therapies, such as BET inhibitors, attempt to directly target the oncogenic epigenetic state. All of these therapeutic approaches are frequently hampered by the emergence of drug resistance. In many cases, resistance is acquired through genetic changes. However, there is increasing evidence that drug resistance can also arise through non-genetic/epigenetic mechanisms. This has major implications for cancer treatment, as the processes that drive genetic and non-genetic resistance are completely different and epigenetic changes, unlike genetic changes, are potentially reversible. Our laboratory previously generated a model of non-genetic resistance to BET inhibitors in acute myeloid leukaemia (AML). Resistance coincided with the acquisition of a less differentiated stem cell like phenotype and was stable upon drug withdrawal. To discover targets that can overcome the resistant epigenetic state, I performed a focused CRISPR-Cas9 screen, which identified the enhancer regulator, LSD1, as key to maintaining the resistance phenotype. Treatment with an LSD1 inhibitor overcame resistance through time-dependent reprogramming of the resistant cells. This epigenetic reprogramming resulted in both differentiation and the formation of new enhancers around critical BET inhibitor target genes. Through functional genomics experiments, I demonstrated that re-sensitization of the resistant cells was driven by the new enhancer formation, rather than differentiation. Mechanistically, the enhancer remodeling is caused by upregulation of Irf8, which together with the pioneer factor, Pu.1, initiates the formation of new enhancers around BET inhibitor target genes, leading to a restored BET inhibitor transcriptional response. Motivated by the poor clinical responses and rapid acquisition of resistance to BET inhibitors in AML, I also sought to develop an assay that could identify new therapeutic targets that may be more effective. Transcription factors (TFs) are ideal targets, however they are difficult to disrupt directly. To circumvent this, I developed an assay that can identify what cofactors (which are often druggable) are required to drive the transcriptional activity of a given TF. This assay works by combining the Gal4 transactivation system with CRISPR-Cas9 screening. Preliminary screens using the transcription factors, VP64 and MYB, were able to identify previously validated cofactors for each TF. VP64 was dependent on MED25 and MYB was dependent on P300. A number of other potential specific cofactors were identified and will be validated in future work. This assay not only provides the potential to identify novel therapeutic targets, but will also provide insight into the poorly characterized interface between TFs and cofactors.

Cancer is the leading cause of disease burden in Australia. Targeted therapy utilises oncogenic mutations in tumours by modulating cancer-promoting proteins and signalling pathways. While these drugs deliver promising initial responses, cancers invariably develop resistance resulting in the recurrence of the cancer. A common theme of resistance mechanisms is the ability of cancer to hijack alternative signalling pathways to evade therapy. The identification and evaluation of these relatively understudied pathways could facilitate the development of novel therapies to overcome resistance development. One such understudied subset of signalling molecules, the WNT-binding RTKs, are at the interface of two protein families frequently dysregulated in cancer – receptor tyrosine kinases (RTKs) and WNTs. RTKs are targets of many drugs used clinically for cancer treatment, often through the inhibition of their kinase activity or their interactions with activating ligands. WNTs are key signalling morphogens in cancer but the complexity of their signalling has made them difficult drug targets. RYK is a WNT-binding RTK pivotal for embryonic development but with unusual biochemical properties that have meant it is understudied in a cancer context. This Thesis sought to uncover a role for RYK in cancer and evaluate its potential as a therapeutic target. This Thesis revealed the overexpression of RYK mRNA in lung squamous cell carcinoma and glioblastoma samples before identifying human tumour cell lines that readily express RYK mRNA and might be reliant on RYK signalling for its tumourigenic nature. Genetic perturbation of RYK through siRNA knockdown and CRISPR/Cas9 inactivation facilitated the discovery of human tumour cells from four different tumour types that were dependent on RYK for their viability. To assess the clinical expression of RYK protein, a novel chicken anti-RYKEC antiserum was generated, validated and utilised to uncover an upregulation of RYK in a subset of human NSCLC tumours. This antiserum also identified RYK expression in tumours from breast cancer patients and found an inverse correlation between RYK expression and the tumour grade of breast cancers. The establishment of a novel bioassay in pre-osteoblast cells dependent on WNT3A/RYK signalling is described. This assay is used to optimise the expression and purification of two RYK/WNT signalling inhibitors and confirm their RYK/WNT-inhibitory activity. One of these inhibitors, a neutralising anti-RYK antibody – RWD1 – then demonstrated an anti-cancer effect by inhibiting the viability of the RYK-dependent human tumour cells in vitro. RWD1 also reduced the growth of A549 NSCLC tumour xenografts, demonstrating the anti-cancer effect of targeting RYK signalling in vivo. RNA-sequencing analysis revealed an upregulation of epithelial to mesenchymal transition (EMT) genes in human tumour cells upon RWD1 treatment, implying RYK signalling supresses EMT. CRISPR/Cas9-mediated inactivation of RYK increased the metastatic potential of A549 tumour xenografts in vivo. This Thesis uncovered a dual role for RYK signalling in a cancer setting. RYK was found to promote the viability and growth of human tumour cells while also suppressing EMT and metastatic growth. The findings of these parallel pro- and anti-tumour functions of RYK signalling, suggest that RYK signalling could be exploited to deliver a therapeutic effect in cancer.

Background: In human brain cancers, glioblastoma stem cells (GSCs) originate from neoplastic transformation of neural stem cells (NSCs) or dedifferentiation of other neural cells. Similar to normal NSCs, GSCs possess stem-cell properties to self-renew and differentiate into multiple neural lineages. However, GSCs are more plastic than normal NSCs, as they can transdifferentiate into other cell types. At the present, we do not fully understand the cellular step-wise conversion from GSCs into other distinct cell lineages, and the molecular mechanism responsible for this event. Drosophila NSCs, called neuroblasts (NBs), also asymmetrically divide to renew themselves, and generate neurons or glia that make up the adult central nervous system. Disrupting either asymmetric cell division or neuronal maintenance allows differentiated cells to dedifferentiate into ectopic NBs, which then continue to proliferate and form tumours. Methods and aims: We utilised several in vivo brain tumour models in Drosophila to study how GSCs function in brain cancers and how they undergo transdifferentiation. In this thesis, I induced loss-of-function of transcription factors Prospero or Nerfin-1 in NB lineages to generate dedifferentiation-driven tumours, and found a class of cells which exhibited glial cell identity. I sought to investigate the behaviours and characteristics of these ectopic glia to elucidate some aspects about GSC transdifferentiation by answering three questions: (1) What are the cell numbers and cell types within the pros- and nerfin-1- tumours. (2) Do ectopic glia arise by transdifferentiation of NBs in the tumours? (3) Which transcription factors and signal transduction pathways drive the formation of ectopic glia, and are ectopic glia required for tumour growth? Results: (1) I found that the expansion of ectopic glia population is correlated with overall tumour growth. Ectopic glia exhibit glial identity and their formation is not dependent on the location of tumours in the central nervous system. (2) By performing live-cell imaging of pros- tumours and molecular marker analysis, I found a subset of NBs switch to glial cell fate. (3) I performed genetic experiments to manipulate the transcription factors dichaete, tailless and glial cells missing (gcm) in pros- tumour and found that they are required for ectopic glial formation and tumour growth. Their target gene reversed polarity (repo) regulates the formation of ectopic glia, which in turn, promote the tumour growth. I showed that Notch promotes tumour growth independently of its effect on ectopic glial formation, as Notch regulates the tumour growth in the absence of ectopic glia. I also showed that FGF signalling pathway promotes tumour growth by regulating ectopic glia formation, as it does not affect the tumour growth in the absence of ectopic glia. Hippo pathway also plays a role in promoting the formation of ectopic glia and tumour growth. Our study of pros- and nerfin-1- tumour models in the context of transdifferentiation may extend our understanding of the biology of NBs and may shed light on GSC behaviours upon their transdifferentiation into different cell types. We can use the underlying mechanisms of these phenotypes to gain a better understanding of the transdifferentiation events at the molecular and cellular levels. As most genes and signalling pathways examined in this study are also found in human brain cancers, this study will enhance the knowledge of how cell fate changes can influence the tumour malignancy.

Oesophageal adenocarcinoma (OAC) is a lethal disease with a rapidly rising incidence. The standard of care for patients with early stage OAC is surgery combined with either neoadjuvant chemo-radiotherapy or perioperative chemotherapy. However, over 70% of patients present with unresectable or metastatic disease. In this setting, their treatment options are significantly limited, and their overall clinical outcome is exceedingly poor, with a median survival of under 12 months. Therefore, safe and effective therapies for patients with OAC are an urgent and unmet medical need. Mutations in the tumour suppressor gene TP53 are by far the most common genetic alteration in OAC. This phenomenon occurs early in oesophageal carcinogenesis and often results in stabilisation of mutant p53 (mut-p53) protein. The loss of wild-type p53 (wt-p53) tumour suppressor activity together with the acquisition of mut-p53 oncogenic gain-of-function, likely drives tumour progression resulting in poor patient survival. Mut-p53 is thus a potential therapeutic target in OAC. In particular, restoring wild-type tumour suppressor activity to mut-p53 should lead to cancer-specific cell death. APR-246, the prototypical mut-p53 reactivator, has been shown to refold mut-p53 protein into wild-type conformation, restoring wild-type activity leading to transcriptional upregulation of wt-p53 target genes. This drug has proven preclinical efficacy and was recently evaluated in early phase clinical trials. In patients with haematological, prostatic and ovarian malignancies, APR-246 was shown to be well tolerated whilst achieving p53-dependent therapeutic effects. As a result, this drug is currently being further studied in a randomised phase II trial for patients with high grade serous ovarian cancer. Given the prevalence of p53 mutations in OAC (>70%), this thesis firstly investigated the anti-tumour activity of APR-246 in preclinical models of OAC. In cell line and patient derived xenografts of OAC, APR-246 demonstrated significant therapeutic effect. Treatment with APR-246 up-regulated the expression of canonical wt-p53 target genes, inhibited clonogenic survival and induced cell cycle arrest as well as apoptosis in mut-p53 OAC cells. This anti-tumour activity was observed in cancer cells harbouring a wide range of p53 mutants. Interestingly, endogenous levels of mut-p53 protein closely correlated with cellular sensitivity to APR-246. From a translational perspective, APR-246 synergistically enhanced the cytotoxicity of conventional chemotherapies including cisplatin, 5-fluorouracil and epirubicin. Moreover, it also overcame intrinsic chemoresistance to these agents. Importantly, based on genetic knockdown and knockout studies of mut-p53, this thesis uncovered significant p53-independent effects of APR-246. Following in-depth analysis into the p53-independent mechanisms of action of APR-246, this thesis found that APR-246 depleted intracellular glutathione and triggered lipid peroxidative cell death. Extending this, the cellular expression of SLC7A11, a key component of system xC-, which imports cystine for glutathione synthesis, was found to accurately predict and directly modulate tumour response to APR-246. These findings led to the discovery that mut-p53 protein suppresses SLC7A11 expression through entrapping the master anti-oxidant transcription factor NRF2. This results in diminished glutathione synthesis and increased levels of endogenous reactive oxygen species, which predisposes mut-p53 tumours to further oxidative damage. Importantly, genetic and pharmacological inhibition of system xC- was found to preferentially target tumours that accumulate mut-p53 in a synthetic lethal-like manner. Moreover, system xC- inhibitors synergised with APR-246 to induce extensive apoptosis in mut-p53 OAC. Collectively, this thesis has identified a novel therapeutic strategy to target mut-p53 tumours. In parallel, this thesis has established the first isogenic pair of OAC cell lines with differing extents of spontaneous metastatic behaviour. Genome-wide transcriptomic analysis of these cell lines, together with functional in vitro and in vivo assays, has highlighted the importance of E-cadherin, a cell surface adhesion molecule, in regulating metastasis in OAC. These cell lines will enable drug testing in a more clinically relevant system, and also provide a new platform to further understand the pathophysiology underlying metastasis in this disease. Overall, this thesis has shown that mut-p53 is an actionable therapeutic target in OAC. To this end, APR-246 has shown promising anti-tumour activity in preclinical models, and should therefore be further evaluated in patients with advanced oesophageal cancer. Additionally, the finding that mut-p53 tumours are susceptible to oxidative stress, and that this vulnerability can be therapeutically harnessed through inhibition of system xC-, represents an exciting avenue for future drug development. Furthermore, the establishment of novel cell lines that faithfully recapitulate the metastatic process in humans will facilitate the discovery of effective strategies to combat this devastating disease.

Cytotoxic secretory granules (CGs) are unique and specialised lysosomal organelles found specifically in cytotoxic lymphocytes: Natural Killer (NK) cells and CD8+ T cells or Cytotoxic T-Lymphocytes (CTLs). CGs precisely store and deliver perforin and granzymes; molecules that synergistically induce the controlled death of infected and neoplastic cells. The precise molecular mechanisms that govern CG formation remain poorly understood and this work aimed to explore the potential role of the MiT/TFE family of proteins in transcriptionally regulating this process. Three members of the MiT/TFE family (MITF, TFEB and TFE3) are largely recognised as the master regulators of lysosome biogenesis and as the main drivers in the formation of organelles with a lysosomal nature. Under this hypothesis the role of the microphthalmia associated transcription factor (MITF) was studied. CTLs expressing mutant MITF showed a reduced ability to expand and kill target cells in vitro in response to antigen-specific stimulation. However, it was determined that the defect was not intrinsic to the lymphocytes but associated to antigen presenting cells required for CTLs activation. Having ruled out MITF in the formation of CGs, the role of transcription factor EB (TFEB) and transcription factor E3 (TFE3) was explored next. By studying the subcellular trafficking of these two proteins during mouse CTL activation, this work described that TFEB, but not TFE3, translocated from the cytosol into the nucleus. Despite the nuclear TFEB presence, RNA-sequencing of CTLs did not conclusively showed upregulation of canonical TFEB target genes. To gain further understanding on the role of TFEB in CTLs, the lysosomal storage disorder (LSD) Niemann-Pick type C1 (NP-C1) was implemented as a model. This devastating disease, caused by mutations in the lysosomal cholesterol transporter, NPC1 proved to be suitable for this purpose, given that it was shown here that TFEB is retained in the cytosol of CTLs in a mouse model of the disease and this was accompanied by a significant reduction of their cytotoxic function. The implementation of various microscopy techniques led to the discovery that NPC1-defficient CTLs accumulate aberrant autophagic bodies that fuse, or embed, lipid-dense CGs. These observations were corroborated in CTLs derived from a cohort NP-C1 patients. Finally, it was established that reconstituting nuclear TFEB was sufficient to restore autophagic dysregulation, but it was not linked to cytotoxic improvement. Contrastingly, pharmacological depletion of aberrantly accumulated lipids within CGs of NPC1-defficient CTLs, completely restored their cytotoxic function. The work presented here shows that the mechanism behind it is linked to lipid inactivation of the pore forming protein, perforin, and the consequent inability of NPC1-defficient CTLs to kill target cells. Overall, the evidence presented here suggests that TFEB plays a critical role in CTL activation which might be unrelated to its canonical lysosomal biogenesis transcriptional regulation. Moreover, while studying this transcription factor in CTLs in the context of the LSD, NP-C1, a number of phenotypical abnormalities were discovered, including accumulation of autophagic bodies and reduced cytotoxicity. Importantly, the impairment in the autophagic flux in NPC1-defficient CTLs was linked to limited TFEB nuclear translocation while the diminished capacity to kill target cells was associated with lipid accumulation within CGs.

The mammary gland undergoes significant structural changes throughout both development and reproduction, ultimately allowing for breastfeeding of a newborn. The immune context of the mammary gland is known to assist with these processes. In addition, significant evidence now exists supporting a strong correlation between mammary immune infiltration and the development and prognosis of breast cancer. Considering this correlation, this thesis set out to investigate how the immune microenvironment changes during development, reproduction and early stages of breast cancer, and whether this immune environment contributes to the known risk profile associated with developing breast cancer during these times. The post-pregnant (involuting) mammary gland is characterized by widespread apoptosis of milk producing alveoli cells, and remodeling of the ductal tree back to a virgin-like structure. Epidemiological studies have convincingly demonstrated that the period of involution is also associated with a transient increased risk of developing breast cancer that occurs directly following pregnancy. To determine the influence of the immune infiltration on this reproductive time point, immunophenotyping flow cytometry and cytokine bead arrays were performed on involuting and nulliparous mouse mammary glands. The involuting mammary gland displayed significant myeloid driven inflammation, with a modest pro-tumoral effect on mammary tumor growth also observed. These observations were verified in a mouse model of breast cancer development, whereby the immune infiltration, in particular macrophages, was found to be significantly increased prior to and during mammary hyperplasia. Using therapeutic depletion of the macrophages with anti-CSF1R monoclonal antibody treatment, we revealed a significant reduction in mammary tumor growth rate following depletion of macrophages. These findings have demonstrated the importance of mammary infiltrating macrophages during times of increased breast cancer risk, including mammary gland involution and hyperplasia. Ultimately, this work suggests the development and implementation of macrophage-targeted therapeutics could be a useful preventative or neoadjuvant therapy for breast cancer. Once mammary gland involution is complete, epidemiological data suggests that the resting parous mammary gland remains in a state of lifelong decreased breast cancer risk. Little has been done however to model this ‘protection’ afforded by parity in mouse models, for future research into the mechanisms governing parous protection. This thesis showed that the inflammation associated with involution is resolved in the resting parous mouse mammary gland suggesting the transient increased risk period is complete by 10 weeks post weaning. Experiments on tumor growth of breast cancer demonstrated this mouse model accurately mimics parous epidemiological data, with reduced tumor growth observed using a syngeneic ER+ breast cancer cell line. This model will now be able to be used to determine the mechanisms of parous protection, with the long-term aim to develop a preventative medicine for women to take who are known to be in a high breast cancer risk category. Altogether, this work has highlighted the mammary infiltrating macrophages as key early drivers of tumorigenesis and determinants of an increased risk profile. Further, a novel parous mouse model was developed for further studies to determine the mechanisms of parous breast cancer protection. The findings from this thesis provide a strong foundation to investigate macrophage-targeted therapies as a novel preventative/adjuvant therapy or early biomarker of disease for breast cancer.

Breast cancer is the most common cancer in women. Despite advances in treatment options, the spread of breast cancer to distant organs (metastasis) remains the major cause of morbidity and mortality in breast cancer patients. This is attributed primarily to the impairment of function in affected organs. Thus, there remains a vital need for better-targeted treatments that more effectively inhibit the development or progression of metastases. Pim-1 is a serine/threonine survival kinase that has been implicated in the development of metastasis in several haematological and solid cancers. However, little is known about its role in breast cancer. In our laboratory, we previously identified Pim-1 as upregulated in brain metastatic 4T1Br4 syngeneic mouse cells and tumours compared to parental 4T1 cells. This led us to propose that Pim-1 may play a role in mediating breast cancer brain metastasis. Therefore, the overall objective of this project was to examine the expression and functional role of Pim-1 in breast cancer metastasis, with a focus on organ-specific metastasis. We interrogated public databases to show that Pim-1 expression is low to absent in normal breast tissue and increased in breast tumour tissue. Furthermore we show that the murine (4T1Br4) and human (MDA-MB-231Br) brain metastatic breast cancer cell lines and tumours demonstrate the highest expression of Pim-1 mRNA and protein. To investigate the function of Pim-1 in breast cancer metastasis we tested the impact of inhibiting Pim-1, either by gene knock down using short hairpin RNAs or the pharmacological inhibitor SGI-1776, on the ability of 4T1Br4 and MDA-MB-231Br cells to migrate and invade in vitro. 4T1Br4 cells displayed increased migration and invasion propensity after Pim-1 knock down and this was coupled with a decrease in β4 integrin expression. Conversely, MDA-MB-231Br cells showed a decreased ability to migrate and invade after Pim-1 KD, as well as decreased cell surface expression of β1 and β3 integrins. Treatment with SGI-1776 dose-dependently decreased the ability of both 4T1Br4 and MDA-MB-231Br cells to migrate and invade, decreased cell surface expression of β3 integrin in 4T1Br4 cells, and both β1 and β3 integrins in MDA-MB-231Br cells. To examine the effect of Pim-1 inhibition in vivo, we assessed the metastatic spread of Pim-1 knock down MDA-MB-231Br cells in an experimental metastasis assay. After intracardiac injection of Pim-1 knock down cells, we observed a reduction in the number of circulating tumour cells and decreased bone metastasis, indicating a functional role for Pim-1 in breast cancer metastasis to the bone. Data from brain metastasis in this model were inconclusive. In summary, results from this project highlight the importance of Pim-1 in breast cancer metastasis and provide evidence that Pim-1 contributes to the migration and invasion of breast cancer cells both in vitro and in vivo, possibly via regulation of integrin expression, and indicate that Pim-1 is a relevant therapeutic target for the treatment of metastatic breast cancer.

This thesis explores the role of nature in cancer patients’ experiences of health and recovery. Using a 2-Phase mixed-method approach, the investigation aimed to generate new theoretical understanding about cancer patients’ use of nature and how they find nature engagement helpful or not when confronting cancer diagnosis. The project’s translational focus was to produce expert recommendations for nature-based care opportunities in oncology contexts based on patient-reported data. First, a systematic literature review and meta-synthesis was conducted to describe the existing qualitative research evidence base relating to nature experiences and nature-based interventions for cancer populations specifically. The aim was to describe current knowledge about the role of nature in cancer patients’ lives. From eleven eligible publications, seven inter-related core themes were identified as follows: connecting with what is valued; being elsewhere, seeing and feeling differently; exploration, inner and outer excursions; home and safe; symbolism, understanding and communicating differently; benefitting from old and new physical activities; and, enriching aesthetic experiences. Next, an in-depth investigation of cancer patients’ own experiences with nature used primary data to develop a new Grounded Theory describing the underlying and intrapsychic mechanisms of cancer patients’ phenomenal nature experiences. Based on qualitative data collected from semi-structured interviews with 20 cancer patients (9 female), the resulting theory model explores the unique role nature plays when diagnosed with cancer. It constitutes a core category and two inter-related themes, which explain a normalization process in which patients moved towards a state of 'new-normal' (Core Category). Nature functioned in this process as a support structure that repositioned patients and nurtured their inner and outer capacities to respond and connect more effectively (Theme A). Once enabled and comforted, participants could engage survival and reconstructive manoeuvres and explore the consequences of cancer in their present lives and possible futures (Theme B). A dynamic relationship was shown between moving away while, simultaneously, advancing towards the cancer reality in order for patients to incorporate their cancer experiences into a shifting normality. From a place of comfort and safety, patients felt supported to deal differently and more creatively with the threat and demands of cancer diagnosis, treatment, and outlook. The descriptively rich interview data provided further insights into patients’ own recommendations for nature engagement in the oncology context, which were extracted from the transcriptions using deductive content analysis and were consolidated into patient recommendations for nature-based care opportunities. These incorporated using nature for vital sensory stimulation and engagement, using nature for personal space and freedom to enable private and social exploration, using views of nature for distraction and comfort, and accessing nature for physical activity and movement. Three critical factors were determined to avoid adverse experiences: determining appropriate health-care expenditure and resourcing on nature-based interventions, selection of appropriate nature-based design materials, and exercising caution around demanding nature engagement and harsh weather conditions. A questionnaire survey study was conducted following an environmental intervention in an oncology waiting room to assess patient, visitor, and staff responses to design changes, which included the addition of artificial plant materials. Based on 143 returned survey questionnaires consisting of 73 cancer patients, 13 staff, 52 carers, and five ‘other’, it was found that the environmental intervention positively impacted patients, staff, and carers’ perceptions of the oncology waiting room environment. Patients, staff, and carers mostly accepted artificial plants as an alternative design solution to real plants. Comments included positive appraisals and occasional adverse reactions to artificial plants. No significant differences were found between patient, staff, and carer reactions. Insights gleaned from the initial, exploratory phase formed the basis for a second phase investigation comprising an international online Delphi study. The aim was to solicit knowledge from relevant experts drawn from a range of healthcare practitioners, management, designers, and researchers to determine feasible opportunities for, and barriers to, providing helpful nature engagement in oncology settings. Two hundred potential panellists were identified and sent an invitation to participate. Thirty-eight experts were recruited who represented 7 countries: Australia (19), USA (8), UK (3), New Zealand (2), Canada (2), Denmark (3), and Sweden (1). This study followed a structured, iterative feedback process that queried and synthesized expert opinion. Cancer patients’ own recommendations, extracted from phase 1, were used as a starting point for the Delphi panel to brainstorm and develop their own ideas about appropriate nature-based opportunities in oncology settings and the barriers to their provision. In total, 250 separate suggestions for opportunities and 205 suggestions for barriers were collected. Further analysis condensed these into 55 unique items (35 opportunities, 20 barriers). The Delphi panel’s list of recommendations included “Window views from clinical areas onto nature […]” as the highest rated opportunity, and “Building design and site constraints […]” as the highest rated barrier to providing nature-based supports for oncology care. Finally, a synthesis of findings from the overall investigation, which constitutes six publications, is provided to summarize and outline the salient findings and discern the study’s limitations in order to suggest pathways for future research. This synthesis produced a conceptual framework consolidating new theoretical understanding and empirical content from patient and expert-reported data about nature-based care opportunities in the oncology setting. The thesis findings provide evidence for multiple uses of nature as a supportive aid in the cancer care context. Concrete recommendations have resulted to guide the application of nature based concepts in future oncology setting design and may be considered when developing additional supportive care services. The findings may assist healthcare practitioners, designers, researchers, and patients themselves to creatively and practically participate in future oncology care practice and design.

Epidemiological studies demonstrate that women live longer than men following diagnosis of non-small cell lung cancer (NSCLC) even after controlling for prognostic factors. This study examines the one biological trait all patients have, sex, and seeks to understand and generate new knowledge with respect to the impact of sex on survival. Patient and tumour characteristics of women and men are examined from presentation and diagnosis, to staging, with specific focus on the impact of these factors on outcomes in NSCLC treated surgically with curative intent. The study also explores how to include sex differences in medical research more generally. Aim and research question The aim is to examine the impact on survival of the association of sex with the following recognised prognostic patient and tumour characteristics: • age • performance status • smoking history • positron emission tomography maximum standardised uptake value • tumour, node, metastasis (TNM) staging The research question is: “What is the impact of sex on validated and putative prognostic factors in non-small cell lung cancer and how can we translate understandings in sex differences in lung cancer to facilitate a more targeted research and therapeutic approach to improve patient outcomes?” This is answered by sex disaggregated analysis of the prognostic value of host and tumour factors from a detailed clinical dataset, including patient outcomes, and compared to an independent population level validation dataset. Finally, I examine international examples of success in sex differences medical research more broadly, and the policy landscape that is preventing translation of both the findings in lung cancer, and sex differences in health and disease across the health care continuum in Australia. Method A detailed surgical database was developed from patients treated surgically with curative intent from the Peter MacCallum Cancer Centre and St Vincent’s Hospital Melbourne from 2000-2010. An additional cohort of patients was identified and analysed from the Surveillance, Epidemiology and End Results (SEER) database, the US population level database. The SEER database was matched to the Melbourne cohort with respect to surgical treatment with curative intent and date of surgery, to ensure continuity with clinical protocols. Extensive clinical data were collected to allow analyses of the impact of sex differences on prognostic variables in three key areas: 1. Host factors (sex, age, smoking and performance status) 2. The maximum standardised uptake value on Positron Emission Tomography 3. TNM staging The patient outcome was disease specific five-year survival. During this research, a new edition of TNM staging was developed resulting in two different editions of TNM staging being used, with the 7th edition in chapter 3 and 4 and 8th edition in chapter 5. Data were analysed with IBM SPSS Statistics software (SPSS) version 21 (Chapter 4), version 22 (Chapter 3) and versions 25 and R (Chapter 5). Findings There are biological differences between women and men in the disease process in early NSCLC. These differences span elements from patient characteristics to survival outcomes. Irrespective of the parameter examined, male sex was a consistent negative prognostic factor. However, the prognostic value of previously identified tumour and host characteristics was equally valid for men and women. Whilst the finding of poorer survival in men with NSCLC is not new, researchers and clinicians have assumed that this was because women are less likely to smoke; due to ethnicity (there is a distinct variant of NSCLC which is less aggressive and occurs more commonly in women of Asian descent); because women are more likely than men to get adenocarcinoma, with its better survival profile compared with squamous cell cancer; and that women are likely to seek treatment at an earlier stage. The research presented in this thesis demonstrates that these assumptions do not fully explain the observed survival differences. Whilst the observations are correct superficially, the causality is false. Survival differences between men and women persist irrespective of ethnicity, histology and disease stage. These findings have important implications for research design, translation, clinical guidelines and practice. Significance A better understanding of the impact of patient and tumour sex on tumour characteristics has the potential to improve understanding of the biology of lung cancer and may lead to different staging and treatment approaches. Understanding the impact of sex on patient response to treatment may improve patient outcomes for men and women by improving selection of therapies tailored to each sex. In addition, novel targets for therapeutics may be identified. This thesis presents a comprehensive delineation of the differences in survival between men and women with NSCLC. These differences raise important questions with respect to the accuracy and efficacy of TNM staging when applied to clinical decision-making for both sexes and indicate that current therapeutic decision-making driven by stage should be reviewed. There is a significant possibility that the oncological community may be over-treating women and under-treating men. Publications from this study may provide direction for future clinical trials, redefine staging, assist in changing clinical practice by more accurate prediction of tumour behaviour, and support the move to individualised treatment.

All biological systems follow the rules and constraints imposed during their evolution. Current-day gene phenotypes such as gene expression, gene essentiality, gene function and protein localization are linked with the time of evolutionary emergence of genes. In cancer, tumours rely on cellular processes that date back to unicellular ancestors (e.g., cell replication, glycolysis), while dysregulating key pathways linked to the emergence of multicellularity, suggesting that the transition from unicellularity to multicellularity left vulnerabilities in cells that act as guiding principles during cancer development. Therefore, in this thesis I integrate genomics, systems biology and evolutionary biology to investigate fundamental principles of tumourigenesis related to the evolutionary history of genes using gene expression and somatic mutation information across multiple tumour types. First, I coupled the evolutionary age of genes and cellular processes with their expression levels in tumour and normal samples, and found that tumours consistently activate genes from unicellular ancestors while switching off genes related to multicellularity. These consistent patterns were supported by a mutual exclusivity between the activity of genes and transcriptional networks of unicellular and multicellular ancestors, which promoted convergent evolution towards a state of loss of multicellularity. Second, I investigated how somatic mutations disrupted gene regulatory networks. Genes that emerged together with early metazoans were enriched in point mutations and copy- number alterations, indicating that gene innovations that took place at the onset of multicellularity play a fundamental role in cancer development. Importantly, the uncoupling of regulatory networks of unicellular and multicellular ancestors was mostly due to point mutations in gene regulators linking these networks. On the other hand, copy-number aberrations were directly involved in the activation and inactivation of unicellular and multicellular genes, suggesting point mutations and copy-number aberrations play complementary roles in the loss of regulation between unicellular and multicellular transcriptional networks in cancer. Third, I focused on novel transcriptional associations formed during tumourigenesis using gene co-expression module analysis. Significant levels of rewiring between unicellular and multicellular genes were found across tumours. This rewiring was mostly driven by gene amplifications, which promoted the formation of tumour-specific modules composed of novel transcriptional associations between unicellular and multicellular genes, once more linking the genes and regulatory associations evolved at the onset of multicellularity to cancer development. The findings of this work reveal fundamental principles driving cancer development associated with genes and transcriptional networks evolved during the transition from unicellularity to multicellularity. I propose a model whereby activation of programs that date back to unicellular ancestors and the deactivation of multicellular programs is driven by an inherent mutual exclusivity of these genes together with the breakage of regulation between unicellular and multicellular genes by point mutations, whereas the formation of novel transcriptional associations between these genes in tumours is driven by copy-number changes. Finally, I identify potential novel drivers based on their key role in uncoupling unicellular and multicellular transcriptional networks across tumours and suggest novel treatment strategies derived from this evolutionary approach. The results presented in this thesis contribute to our understanding of how past evolutionary events led to vulnerabilities in transcriptional networks that influence cancer development, and highlight the benefits of the integration of evolutionary concepts with genomics and network biology to identify fundamental principles of cancer.