The main subject of my thesis is the investigation of mechanisms of glioma tumorigenesis associated with the recently identified mutations in isocitrate dehydrogenase. Gliomas account for 80% of primary brain cancers. They represent a diverse group of tumours, and are graded from I-IV based on histopathological features. Whilst grade I tumours may be curable with surgery alone, grade II and III gliomas inevitably progress to glioblastoma multiforme (GBM), which is highly resistant to current therapies and carries a very poor prognosis. Despite an improved understanding of the pathways and mechanisms involved in the development of glioma and its progression to grade IV disease, current and novel treatments have so far failed to significantly improve outcome. Isocitrate dehydrogenase (IDH) enzymes catalyse the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Somatic mutations in genes encoding IDH1 and IDH2 were first identified in glioma and subsequently in acute myeloid leukemia and other solid tumours. These heterozygous point mutations occur at the arginine residue of the enzymes active site and cause both loss of normal enzyme function and gain-of-function, causing the reduction of α-KG to D-2-hydroxyglutarate (D-2HG), which accumulates. D-2HG may act as an oncometabolite through the inhibition of various α-KG dependent enzymes, stimulating angiogenesis, histone modifications and aberrant DNA methylation. Possibly, IDH1/2 mutations may also cause oncogenic effects through dysregulation of the tricarboxylic acid (TCA) cycle, or by increasing susceptibility to oxidative stress. The exact role of mutant IDH1/2 in tumorigenesis however remains unclear. In the work outlined in this thesis, I have demonstrated that the expression of mutant IDH1/2 in glioma cell lines leads to 2-HG accumultation and a reduction in α-KG production and results in HIF1α accumulation and a reduction in 5hmC production. Furthermore, the brain-specific expression of mutant Idh1 in mice also results in 2-HG accumulation and reduced α-KG production, whilst a reduction in 5hmC levels are also seen. This data appears to support the theory that IDH1/2 mutant activity results in the inhibition of α-KG dependent enzymes, either through the accumulation of 2-HG or due to a reduction in α-KG levels. The brain-specific expression of mutant Idh1 in mice also results in increased cellular proliferation and an increase in the expression of the neural stem cell marker, nestin. However gliomas do not develop, perhaps suggesting that additional mutations are required in conjunction with those occuring in IDH1/2 in order to initiate tumourigenesis. Clinically, IDH1/2 mutations may represent a novel therapeutic target in glioma and may also serve as useful diagnostic, prognostic and predictive biomarkers. However, a better understanding of the pathogenesis of mutant IDH is required, to enable effective IDH1/2 directed therapies to be developed in the future.

The adaptive immune response forms an essential part of the cancer immuno-editing process, whereby nascent malignant cells are detected and destroyed prior to forming tumours. The process is tightly controlled to minimise collateral damage to healthy tissue. One of the mechanisms evolved for this purpose and frequently co-opted by malignant cells is the creation of a microenvironment scarce in essential amino-acids through the use of catabolic enzymes such as Indoleamine 2,3-dioxygenase (IDO) , responsible for the rate-limiting step in tryptophan catabolism. The evolutionary conserved GCN2 and mTORC1 pathways respond to amino-acid starvation by triggering emergency homeostatic response programmes that aim to conserve nutrients by shutting down biosynthetic pathways, slowing cell cycle progression and facilitating autophagy. This research project focuses on elucidating the interaction between IDO activity and these pathways and its implications for the immune-editing process. The role of the mTOR kinase as a regulator of T cell fate following exposure to cognate antigen has recently become apparent. Experiments described herein confirm that in murine and human models of T cell activation exposure to tryptophan starvation results in significant mTORC1 inhibition and a modified phenotype with reduced Tbet expression, altered cytokine secretion profile, greatly impaired proliferative capability and expanded CD4⁺ FoxP3⁺ CD25^high subpopulations. Additional results confirmed that the action of IDO is sufficient to deplete tryptophan from the microenvironment to levels sufficient to depress the mTORC1 axis and trigger GCN2 activity even in tumour cell lines. Lower extracellular tryptophan levels were necessary to perturb these pathways In IDO expressing cell lines, suggesting that compensatory mechanisms allow continued proliferation of malignant cells in the face of conditions that severely impede an anti-cancer immune response. In conclusion, manipulation of the mTORC1 axis via IDO-induced tryptophan depletion is an important tumour immune-escape mechanism that can be a target for cancer immunotherapies.

The Receptor Tyrosine Kinase (RTK) Met, overexpressed or mutated in cancer, plays a major role in cancer progression and represents an attractive target for cancer therapy. This study aimed to investigate whether PI3K plays a role in Met oncogenicity. Three cell models were used: (i) NIH3T3 cells expressing WT Met or the constitutively active mutant M1268T Met; (ii) U87MG glioblastoma cells, with endogenous WT Met constitutively activated due to an autocrine loop; (iii) A549 lung cancer cells expressing endogenous WT Met, activated upon binding exogenous HGF. Met dependent Rac1 translocation to the plasma membrane, actin cytoskeleton organisation, cell migration, anchorage independent growth in soft agar and tumour growth were studied in the presence of inhibitors of pan-PI3K / mTOR, various PI3K Class I isoforms, mTOR or Akt, or following siRNA knock-down of PI3K isoforms. We report that PI3K class I (but not class III) regulates Met dependent cell migration. The PI3K class I isoforms required varies among the cell models. Interestingly, the combined inhibition of all p110 Class I isoforms lead to the strongest reduction of Met dependent cell migration. Met dependent phosphorylation of Akt, an effector of PI3K class I, is reduced upon endocytosis inhibition, suggesting that Met signals to PI3K Class I on endosomes. Our results indicate that mTOR is responsible for Met dependent anchorage independent growth and tumour growth in vivo. Surprisingly, PI3K class I (and class III) are not required. Moreover, Rac1 is required for Met dependent mTOR activation, (phosphorylation of mTORC1's effector, p70 S6K) subcellular translocation of mTOR and anchorage independent growth. Finally, our results suggest that this Met-Rac1- mTOR pathway occurs on endosomes. Thus while PI3K class I regulates Met dependent cell migration, mTOR regulates Met driven anchorage independent growth and in vivo tumorigenesis. Thus PI3K Class I / mTOR may be targeted in Met driven cancers.

No description available.

Germ cell tumours (GCTs) are clinically and pathologically heterogeneous neoplasms that arise at gonadal (testicular/ovarian) and extra-gonadal sites. The chemotherapy burden for patients with malignant germ cell tumours (mGCTs) that require treatment results in substantial longterm side-effects, and, furthermore, poor-risk patients have < 50% survival. Consequently, identifying common molecular changes and novel therapeutic targets in mGCTs is of major clinical importance. MicroRNAs are short, non-protein coding RNAs that regulate gene expression. We previously showed that miR-99a-5p/-100-5p and miR-125b-5p are among the most frequently underexpressed microRNAs in mGCTs, regardless of anatomical site, histological type or patient age. The present study investigates the upstream causes and downstream consequences of such under-expression. The mature form of miR-125b-5p is the product of two genomic loci, which form a cluster with either miR-99a-5p (on chromosome 21q) or miR-100-5p (on chromosome 11q). MiR-99a-5p/- 100-5p share identical 'seed' regions (at nucleotide positions 2-7), which determine their mRNA targets. Cross-reactivity experiment revealed that both miR-99a-5p and miR-100-5p probes were highly cross-reactive to each other's target (from 91% to 95%), indicating functional overlap. Linear regression analysis of qRT-PCR data reveals a strong positive correlation between miR-99a-5p/-100-5p and miR-125b-5p levels (R2 =0.989) in mGCTs, strongly suggesting co-regulation. Primary microRNA transcripts (pri-miR-99a/-100 and pri-miR-125b), and other genes that colocalise to these miRNA clusters (e.g. BLID on chromosome 11), were quantified by RT-qPCR in four representative cell lines - TCam2, 1411H, 2102Ep, and GCT44 - which were derived from a range of common histological types of mGCTs. A significant down-regulation (p < 0.0001) of all primary transcripts was observed, suggesting transcriptional repression of the entire cluster regions. Treatment of the cell lines with 5'-azacytidine resulted in significant upregulation of all three miRNAs (p < 0.002), as well as BLID (p < 0.02). The methylation status of potential CpG islands at the region of interest on chromosome 11 and chromosome 21 was therefore investigated by Pyrosequencing. Significant hyper methylation was found in 2102Ep, 1411H and GCT44 cell lines, suggesting that the miR-99a-5p/-100-5p and miR-125b-5p clusters are likely transcriptionally silenced by DNA methylation. To assess the functional relevance of these microRNAs in GCT progression, co-transfection of microRNA mimics (8.3 nM miR-99a-5p/-100-5p + 8.3 nM miR-125b-5p) was performed. A significant decrease in cell growth was seen in 1411H (p < 0.01) and TCam2 (p < 0.03) cells. To identify the mimics' downstream mRNA targets, HumanHT-12 v4 Expression Bead Chip (Illumina) mRNA arrays were used and data analysed using Sylamer. This analysis showed that mimic-treated cells were enriched in downregulated genes involved in pro-proliferative mechanisms. Among those, further functional characterisation focussed in particular on TRIM71, FGFR3, E2F7 and LIN28A. Moreover, restoring miR-99a-5p/-100-5p and miR-125b-5p in TCam2 cells also resulted in G0-G1 accumulation, consistent with a cell cycle effect. These data support a functionally important role for miR99a-5p/-100-5p and miR-125b-5p in GCT progression. They also raise the possibility of a therapeutic replenishment approach for treating these, and potentially other, tumours.

Glioblastoma is a highly malignant tumor which mostly recurs locally around the resected contrast enhancement. However, it is difficult to identify tumor invasiveness pre-surgically, especially in non-enhancing areas. Thus, the aim of this thesis was to utilize multimodal MR technique to identify and characterize the peritumoral progression zone that eventually leads to tumor progression. Patients with newly diagnosed cerebral glioblastoma were included consecutively from our cohort between 2010 and2014. The presurgical MRI sequences included volumetric T1-weighted with contrast, FLAIR, T2-weighted, diffusion-weighted imaging, diffusion tensor and perfusion MR imaging. Postsurgical and follow-up MRI included structural and ADC images. Image deformation, caused by disease nature and surgical procedure, renders routine coregistration methods inadequate for MRIs comparison between different time points. Therefore, a two-staged non-linear semi-automatic coregistration method was developed from the modification of the linear FLIRT and non-linear FNIRT functions in FMRIB’s Software Library (FSL). Utilising the above mentioned coregistration method, a volumetric study was conducted to analyse the extent of resection based on different MR techniques, including T1 weighted with contrast, FLAIR and DTI measures of isotropy (DTI-p) and anisotropy (DTI-q). The results showed that patients can have a better clinical outcome with a larger resection of the abnormal DTI q areas. Further study of the imaging characteristics of abnormal peritumoural DTI-q areas, using MRS and DCS-MRI, showed a higher Choline/NAA ratio (p = 0.035), especially higher Choline (p = 0.022), in these areas when compared to normal DTI-q areas. This was indicative of tumour activity in the peritumoural abnormal DTI-q areas. The peritumoural progression areas were found to have distinct imaging characteristics. In these progression areas, compared to non-progression areas within a 10 mm border around the contrast enhancing lesion, there was higher signal intensity in FLAIR (p = 0.02), and T1C (p < 0.001), and there were lower intensity in ADC (p = 0.029) and DTI-p (p < 0.001). Further applying radiomics features showed that 35 first order features and 77 second order features were significantly different between progression and non-progression areas. By using supervised convolutional neural network, there was an overall accuracy of 92.4% in the training set (n = 37) and 78.5% in the validation set (n=14). In summary, multimodal MR imaging, particularly diffusion tensor imaging, can demonstrate distinct characteristics in areas of potential progression on preoperative MRI, which can be considered potential targets for treatment. Further application of radiomics and machine learning can be potentially useful when identifying the tumor invasive margin before the surgery.

Mutations in the tumour suppressor gene Adenomatous polyposis coli (APC) are found in 80 % of sporadic colorectal cancer (CRC) tumours and are also responsible for the inherited form of CRC, Familial adenomatous polyposis (FAP). In order to identify novel therapeutic targets for the treatment of APC mutated CRC, we have generated an in vitro model of APC mutant CRC using CRISPR-cas9 gene editing. Using the APC wildtype colorectal carcinoma cell line RKO, we targeted the cells with guide RNA (gRNA) targeting exon 2 or exon 15 (encodes 80 % of APC) of the APC gene. We generated isogenic cell lines which differed in the expression of APC, the controls were APC wildtype and the APC mutant (APC Lys736fs) cell lines expressed a truncated ~80 kDa APC protein. We used these cell lines to perform an siRNA screen against 720 kinases and kinase-related genes. We selected seven genes to investigate further, unfortunately none of the potential hits validated. Additionally, we performed an FDA-approved compound screen targeting over 1000 compounds. From this, we identified a group of HMG-CoA reductase (HMGCR) inhibitors known as statins, which selectively cause a greater loss in cell viability in the APC mutated cell lines, compared to the APC wildtype cells. Mechanistically, our data suggests this synthetic lethal relationship is due to a greater decrease in the anti-apoptotic protein survivin. We propose this is due to statins altering the localisation of Rac1, reducing Pak1 activation and reducing the level of Wnt signalling. This results in the reduction of the Wnt target gene survivin. We have successfully identified an FDA-approved family of compounds, which show synthetic lethality with the APC mutation in our in vitro model.

The tumour microenvironment consists of an immunosuppressive niche created by the complex interactions between cancer cells and surrounding stromal cells. A critical component of this environment are myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immature myeloid cells arrested at different stages of differentiation and expanded in response to a variety of tumour factors. MDSCs exert diverse effects in modulating the interactions between immune effector cells and malignant cells. An increased presence of MDSCs is associated with tumour progression, poorer outcomes, and decreased effectiveness of immunotherapeutic strategies. In this project, we sought to quantify and characterise MDSC populations in patients with Acute Myeloid Leukaemia (AML) and delineate the mechanisms underlying their expansion. We have demonstrated that immune suppressive MDSCs are expanded in the peripheral blood and bone marrow of patients with AML. Furthermore, AML cells secrete extra-cellular vesicles (EVs) that skew the tumour microenvironment from antigen-presentation to a tumour tolerogenic environment, through the expansion of MDSCs. We then demonstrated that MDSC expansion is dependent on tumour and EV expression of the oncoproteins MUC1 and c-Myc. Furthermore, we determined that MUC1 signalling promotes c-MYC expression in a microRNA (miRNA) dependent mechanism. This observation lead us to elucidate the critical role of MUC1 in suppressing microRNA-genesis in AML, via the down-regulation of the DICER protein, a key component of miRNA processing machinery. Finally, exploiting this critical pathway, we showed that MDSCs can be targeted by MUC1 inhibition or by the use of a novel hypomethylating agent SGI-110.

Malignancy is associated with a hypercoagulable state manifested clinically by an increased incidence of venous thromboembolism (VTE). Colorectal cancer (CRC) patients who develop VTE have reduced survival. This increased mortality extends beyond the acute VTE event, suggesting that VTE is associated with aggressive tumour biology. Tissue factor (TF) and other clotting factors have been implicated in this process. However, the significance of clotting factors in the tumour microenvironment (TME) remains unknown. The aim of this thesis is to i) determine if a procoagulant TME is a biomarker for poor prognosis and VTE in patients undergoing resectional surgery for CRC and ii) determine the effect of TF, thrombin and FXa on proliferation and migration in vitro in CRC and if their inhibitors have potential as anticancer therapies. In the in vitro studies, epithelial expression of TF had a modest effect on proliferation and migration when quantified using the PrestoBlue proliferation and transwell migration assays. Exogenous TF, FXa and thrombin all increased migration in DLD-1 wild type cells. In addition, exogenous thrombin increased proliferation amongst SW620 wild type cells. This suggests that coagulation factors from the TME, rather than epithelial expression, may influence tumour biology. Moreover, dabigatran, a direct thrombin inhibitor, abrogated the pro-proliferative effects of thrombin, which highlights its potential role as an anticancer therapy. In a multicentre, prospective cohort study of 159 CRC patients undergoing resectional surgery, rates of duplex screen detected deep vein thrombosis (DVT) were correlated to plasma and tumour markers of hypercoagulability. TF is upregulated in the stroma of cancer compared to normal tissue. However, stromal TF expression decreased in more advanced (T4) tumours. This suggests that a procoagulant TME has a role in early tumourigenesis. In total, 5.4%, 7.0% and 9.1% of patients had an asymptomatic DVT pre- operatively, at six weeks post-surgery and after the commencement of adjuvant chemotherapy respectively. The development of a post-operative complication was a risk factor for DVT, whilst locally advanced tumours resulted in a prolonged hypercoagulable state i.e. raised D-dimer at six weeks. This highlights a possible role for pre- and post- operative screening duplex ultrasonography and super-extended VTE prophylaxis in selected patients. In conclusion, this thesis establishes a role for exogenous coagulation factors in promoting tumour biology in CRC. VTE is more common amongst patients undergoing resectional surgery for CRC than previously estimated. The utility of tumour and plasma hypercoagulabilty as biomarkers for survival in CRC will be further analysed when long term follow-up data is available.

Plasma membrane derived microvesicles (MV) are nanoscale particles released from cells both constitutively and in response to stimuli including stress, apoptosis and oncogenic transformation. Due to their mechanism of biogenesis, the majority of MV expose phosphatidylserine (PS) on their surface and as such can be identified by staining with annexin V (AxV). First observed nearly 40 years ago as coagulant ‘dust’ originating from activated platelets, MV were initially studied for their role in thrombosis. In more recent years it has become apparent that MV release is increased in several diseases including cancer; this, in conjunction with their ability to carry cargo such as proteins and nucleic acid species, strongly implicates them in disease pathology. Given their small size it is considered likely that MV are able to travel to distal sites within the body allowing the widespread dissemination of effects otherwise not achievable by their parent cells. In the context of malignancy, the contribution of MV is especially important in that MV have been demonstrated to have roles in oncogenic transformation, promotion of tumour growth and increasing metastatic potential. Although clearly important in pathogenesis, their small size makes qualitative and quantitative analysis extremely difficult. Furthermore, the study of MV has been greatly hampered by a lack of standardised protocols for their isolation and as such the majority of studies have been in vitro. In line with this, the relevance of observed effects to in vivo systems is often questioned; given the high quantities of MV used in in vitro systems, the question of whether these concentrations bear any relevance in vivo remain to be answered. We hypothesise that the high rates of apoptosis observed in many tumours, most notably in the high grade B cell malignancy, Non-Hodgkin’s lymphoma (NHL), provides an environment whereby MV are continually released into the surrounding milieu allowing for an amplification of effects. As apoptosis has been previously implicated in promoting tumourigenesis we propose that this is extended to include MV released from apoptotic tumour cells (aMV). Given the numerous technical challenges involved in MV research, initial studies involved identifying the limitations of the instruments available for MV analysis. Preliminary experiments identified considerable resolution issues with the older style EPICS XL flow cytometer (Beckman Coulter) and so a newer flow cytometer, The Attune™ (Thermo Fisher), capable of higher resolution was utilised for the remainder of the project. Despite this improvement, flow cytometry was demonstrated to be less effective at quantifying MV than nanoparticle tracking analysis (NTA). As the fluorescent capacity of NTA is still in its infancy, it was used in concert with flow cytometry in order to quantify and phenotype MV as accurately as possible. As there is currently no concensus on an optimal method of MV isolation subsequent studies focused on determining a method of MV isolation that was appropriate for our experimental system. To this end, centrifugation, filtration and antibody coated magnetic bead-based methods were all tested and their limitations identified. In terms of bead-based isolation strategies, the generation of AxV, protein S, gla domain and gas 6 fusion proteins was attempted with the intention to conjugate to magnetic beads and provide a novel means to isolate aMV. Unfortunately this aspect of the project was ultimately abandoned due to time constraints and although commerically available antibody coated beads were tested for their ability to isolate MV, later co-culture experiments demonstrated that the beads had off target effects that were deleterious to cells. As a result, centrifugation and filtration methods were next researched and validated extensively. TEM analysis of MV morphology identified damage likely induced by the high-speed centrifugation of a fragile apoptotic cell population. As such, a protocol combining low speed centrifugation and filtration was designed and validated by several methods including TEM and staining with AxV. The surface levels of parent cell markers (CD19 and CD20) and apoptosis associated proteins were compared in aMV and vMV (MV released from viable tumour cells) and results demonstrated that B cell surface markers were off loaded into MV to a greater extent following apoptosis. Additional phenotypic studies extended previous work from the group demonstrating the presence of apoptotic cell associated molecular patterns (ACAMPs) capable of binding a panel of antibodies to LPS. Flow cytometry results confirmed the presence of ACAMPs on aMV and results from co-culture experiments with CD14 positive and negative cells suggested that unlike recognition of LPS, binding via ACAMPs was not CD14 dependent. The protein and nucleic acid content of MV was also studied and interestingly, results demonstrated significantly increased quantities of DNA and RNA in aMV compared to vMV. Furthermore, aMV were also shown to contain the matrix metalloproteinases, MMP2 and MMP12 alluding to a role for aMV in angiogenesis. The final stage of the project was focused on determining the roles of aMV in the tumour microenvironment and effects relating to cell growth, cell cycle and angiogenesis were studied and compared to vMV. Results showed that both aMV conditioned supernatant and aMV concentrated by the centrifugation were able to significantly increase the growth of the parent cell population. Further studies using DAPI staining to determine the cell cycle status of cells co-cultured with aMV demonstrated an increase in DNA synthesis and cell division upon incubation with aMV. An in vitro angiogenesis assay was designed to determine any pro-angiogenic capabilities of aMV given the earlier results demonstrating the presence of MMPs. These results provided some of the most interesting findings of the project and showed that aMV were able to increase the angiogenic potential of human endothelial cells (HUVECs); an effect that was shown to be greatly reduced following storage at either 4 or - 80°C. These results demonstrated that aMV possess factors capable of manipulating the tumour microenvironment to favour disease progression and that previously described pro-tumour functions of MV are increased as a result of apoptosis. These findings have implications both in terms of extending the previously described hallmarks of cancer and also when designing a course of therapy whereby in some instances the generation of large amounts of apoptosis may in fact serve to promote regeneration of the tumour cell population.