<p>Brain metastases are most common in adults suffering from lung cancer, predicting uniformly poor patient outcome and short survival time. Despite their frequency and severity, very little is known about the tumorigenesis of brain metastases. Previously developed primary brain tumour-initiating cell (BTIC) models were used to determine the presence of a stem-like population in brain metastases from the lung. Use of clinical samples and the NCI-H1915 cell line allowed for the development of useful strategies for study of brain metastasis.</p> <p>The sphere formation capacity and expression of known BTIC markers in brain metastases was suggestive of a self-renewing population. Differentiation studies demonstrated that neither clinical samples nor NCI-H1915 cells had neural lineage potential. Intracranial xenotransplant of clinical samples and NCI-H1915 cells into NOD-SCID mice led to formation of multiple focal masses throughout the ventricles; the tumours were also serially transplantable, further implicating a TIC population. Of known BTIC markers, only CD15 expression levels and patterns were similar enough in clinical samples and NCI-H1915 cells to warrant prospective sorting experiments in the cell line. Use of CD15 failed to identify a CSC or TIC population in NCI-H1915 cells.</p> <p>These findings suggest that a TIC population is present in brain metastases; however, this remains to be identified. It is recommended that due to the limitations of cell surface markers, the study of brain metastasis should use a selective gene expression approach, in order to target genes and pathways essential to metastasis. It was shown that NCI-H1915 cells could be useful for such an approach, studying the effects on proliferation, sphere formation, and tumour formation capacity of brain metastases from the lung. Further study using this model could ultimately lead to the disruption of pathways essential to the metastatic process, transforming a uniformly fatal disease into a more localized and treatable one.</p> / Master of Science (MSc)

<p><strong>Background</strong>: Health-related quality of life (HRQL) studies have inconsistently identified a burden of pain in survivors of brain tumours in childhood, with limited exploration of this morbidity.</p> <p><strong>Objective: </strong>To explore the HRQL, with a focus on pain, in survivors greater than 10 years from diagnosis of a primary brain tumour in childhood or adolescence.</p> <p><strong>Methods:</strong> A cross-sectional study was undertaken using Health Utilities Index (HUI) questionnaires. Location of pain was queried using a homunculus and a colour-analog scale facilitated the reporting of severity. Single-attribute HRQL scores for participants with and without pain were compared. Stability of pain over a decade was established using available HUI2/3 data from the same cohort with imputation for missing variables.</p> <p><strong>Results:</strong> Twelve males and 13 females out of 37 eligible subjects participated in this study. Participants (mean time from diagnosis of 19.7 years) had mean multi-attribute HRQL scores of 0.79 (SD of 0.23) for HUI2 and 0.69 (SD of 0.29) for HUI3. Thirteen (52%) participants reported pain, with ranges in severity and location of the discomfort. Participants with pain had considerably greater burdens of morbidity in sensation and emotion than those without pain. Pain also increased from the initial interview (10 years prior) to the final interview.</p> <p><strong>Conclusion:</strong> As a group, long-term survivors of brain tumours in childhood have diminished overall HRQL. However there is variability between subjects. Pain appeared to be a persistent and significant burden in a subset of individuals, with those experiencing pain reporting greater severity of morbidities in other attributes.</p> / Master of Science (MSc)

A Thesis Submitted to the School of Graduate Studies in the Partial Fulfillment of the Requirements for the Degree Master of Science / Caenorhabditis elegans and one of its close relatives, Caenorhabditis briggsae, are animal models that are commonly used for comparative studies to understand the evolution of developmental mechanisms and gene function. Although the two species appear nearly identical morphologically, comparative genomic analyses have revealed interesting differences between the genomes. Whether such differ- ences contribute to changes in developmental mechanisms and signalling pathways is an active area of research. One of the most well studied phenotypes associated with C. elegans signalling pathways are those that affect the specification of vulval tissue. Within the system of vuval development, mutants that exhibit the Mul- tivulva (Muv) phenotype are important as they show inappropriate divisions of vulva cells, which model tumour formation. Comparing gene function in different species genetic backgrounds can lead to an understanding of how genetic differ- ences contribute to different responses in cancer development. Genetic screens, conducted in our laboratory, yielded several genes whose loss of function results in a Muv phenotype and identified a novel regulator of C. briggsae vulval devel- opment, Cbr-ivp-3. Using the nematode C. briggssae as experimental system, we have characterized the tumour suppressor gene, Cbr-ivp-3, which impacts cell sig- nalling and cell division. I have carried out molecular genetic analyses of ivp-3 in both C. briggsae and C. elegans and have begun to characterize the functional role of Cbr-ivp-3. The findings in this thesis suggest that Cbr-ivp-3 is functioning to negatively regulate EGF/Cbr-lin-3. / Thesis / Master of Science (MSc) / The nematodes Caenorhabditis elegans and Caenorhabditis briggsae, are commonly used for comparative studies to understand the evolution of developmental mechanisms and gene function. Although both species appear morphologically similar, comparative genomic analyses reveal differences between genomes. Comparing gene function in different genetic backgrounds can lead to an understanding of how genetic differences contribute to different responses in cancer development. Genetic screens have yielded several genes whose loss of function results in a Multivulva phenotype, showing inappropriate division of vulva cells, modeling tumor formation. We have carried out molecular genetic analyses of ivp-3, a novel regulator of C. briggsae vulval development, in both species and have found that Cbr-ivp-3 is regulating vulva development by negatively regulating EGF/Cbr-lin-3.

Glioblastoma (GBM) is a very aggressive and invasive tumour that relapses within nine months of diagnosis and remains incurable despite advances in multimodal therapy including surgical resection, chemotherapy and radiation. Poor patient outcome has been correlated to specific markers of brain tumour initiating cells (BTIC) and intratumoural heterogeneity (ITH), which have also been associated with treatment resistance and tumour recurrence. ITH can be explained at the cellular level by the existence of multiple populations of cancer cells, including some which have acquired stemness properties like self-renewal, proliferation, and multilineage differentiation, also known as cancer stem cells (CSCs). In brain tumours, CSCs or BTICs, have been shown to be resistant to both chemotherapy and radiation treatment, allowing them to escape therapy and consequently generate for tumour recurrence. As a result, therapies that focus on targeting the BTIC compartment within the bulk GBM tumour would provide better treatment and prognosis for patients. To profile GBM BTICs we conducted two transcriptomic screens. The first compared GBM BTICs to neural stem cells (NSCs), their healthy counterparts, and for the second we developed a pipeline utilizing a dynamic BTIC patient-derived xenograft (PDX) model of human GBM recurrence allowing for the profiling of GBM BTICs at engraftment, after chemoradiotherapy delivery in a phase we have termed "minimal residual disease" (MRD), and at tumour recurrence. In this study, Dickkopf-1 (DKK1) was identified as a potential therapeutic target for GBM from each transcriptomic screen and was studied using short hairpin knockdowns, blockade with monoclonal antibodies, and subsequent functional stem cell assays. / Thesis / Master of Science (MSc) / Glioblastoma (GBM) is a very aggressive tumour that relapses within nine months of diagnosis and remains incurable despite chemotherapy, radiation, and surgery. Relapse is believed to be caused by the presence of a wide variety of cell types, including cancer stem cells (CSCs), which have been shown to be resistant to both chemotherapy and radiation in GBM. As a result, therapies that focus on targeting the CSCs within the bulk GBM tumour would provide better treatment for patients. In this study, we analyzed this cell population by conducting two screens. The first compared the level at which genes are expressed in GBM CSCs in comparison to how they are expressed in their healthy counterparts, neural stem cells, whereas the second compared the primary patient GBM tumour to its relapsed form in a mouse model of the disease. In this study, the protein Dickkopf-1 (DKK1) was identified and validated as a potential therapeutic target of GBM using well established molecular and stem cell functional assays.

Caenorhabditis elegans and its congener, C. briggsae are excellent animal models for the comparative study of developmental mechanisms and gene function. Gupta lab is using the vulval tissue in these nematodes as a system to investigate conservation and divergence in signal transduction pathways. Genetic screens conducted earlier in our laboratory recovered several mutants that cause multivulva (Muv) phenotype. The Muv genes act as tumor suppressors and negatively regulate the proliferation of vulval precursors. Genetic and molecular work on these genes has revealed that C. briggsae vulva developmental utilizes novel genes representing a new phenotypic class termed ‘Inappropriate Vulva cell Proliferation (IVP)’ (Sharanya et al., 2015). This indicates that the signaling mechanism in C. briggsae specifies vulval cell fates differently from C. elegans. Interestingly, it has been found that Cbr-ivp mutants show higher levels of Cbr-lin-3 (EGF) transcript, indicating that these genes act genetically upstream of Cbr-lin-3, similar to SynMuv family members in C. elegans. Moreover, RNAi knockdown of the Cbr-lin-3 transcript resulted in the suppression of the multivulva phenotype in mutant animals. Similar suppression was also observed when a MAP kinase inhibitor was used in the previous study. In addition, the role of two other novel negative regulators of cell proliferation, Cbr-lin(bh1) and Cbr-lin(bh3) was also investigated. Preliminary findings on these regulators suggested that both Cbr-lin(bh1) and Cbr-lin(bh3) exhibiting a heritable Muv phenotype and are found to be located on Chromosome I and III respectively. Identification of novel genes and further characterization will help us understand the molecular function of genes and their involvement in the regulation of vulval cell differentiation. The findings of my research work will provide a background for future studies to understand the role of novel genes in reproductive system development. Overall, these results provide evidence that although the morphology of vulva is similar in the two nematode species, underlying mechanisms of development appear to have diverged. / Thesis / Master of Science (MSc)

<p dir="ltr">Protein tyrosine phosphorylation is a key post-translational modification that drives numerous cell signaling pathways governing cell proliferation, differentiation, and transcriptional activation. Protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) coordinate the protein tyrosine phosphorylation levels while dysregulated PTKs or PTPs activity results in aberrant protein tyrosine phosphorylation levels and cause multiple human diseases including cancer, diabetes, and autoimmune diseases. Targeting PTKs using small molecule inhibitors or antibodies achieved many successes for various indications. For example, targeting epidermal growth factor receptor for cancer therapy and targeting Janus kinase 2 for autoimmune diseases. Meanwhile, targeting PTPs as therapeutic approaches remains underexplored due to the limited understanding of PTP biology and challenging inhibitor development.</p><p><br></p><p dir="ltr">So far, many substrates and pathological mechanisms of PTPs remain elusive. As an example, although it is well recognized that Phosphatases of Regenerating Liver (PRL) family members PRL1/2/3 are overexpressed in the majority of cancer types and are frequently associated with poor clinical outcomes, the pathological mechanism of PRLs is unclear due to the limited understanding of their substrates. In the presented study, I focused on understanding the oncogenic mechanisms of PRL2 phosphatase and the therapeutic potential of targeting PRL2. I developed the first breast-specific PRL2 deletion mouse model to understand the role of PRL2 in estrogen receptor-positive breast tumorigenesis driven by the aberrant PI3K/AKT signaling, which represents over 30% of the human breast cancer population. I found PRL2 deletion drastically extended the median tumor-free survival of mice harboring PI3K gain-of-function mutant from 377 days to 605 days while such extension was invalidated in the absence of PTEN, a major tumor suppressor and one of the substrates of PRL2.</p><p><br></p><p dir="ltr">PTPs are challenging targets for inhibitor development due to the highly conserved and positively charged active site. Most of the identified PTP inhibitors lack the selectivity as a chemical probe for interrogating the PTP biology and are negatively charged which limits their bioavailability as a therapeutic approach. To overcome these defects, we utilized the proteolysis targeting chimera (PROTACs) technique to generate bifunctional small molecules that recruit the protein of interest to an E3 ligase for protein ubiquitination and proteasome degradation. Compared with the traditional occupancy-based inhibitors, PROTACs have improved efficacy and selectivity due to the catalytic degradation turnover and the necessity of the formation of the target-PROTAC-E3 complex. We developed the first-in-class PTP1B/TC-PTP dual degrader named DU-14 and TC-PTP selective degrader named TP1L for cancer immunotherapy. Protein tyrosine phosphatase 1B (PTP1B) and T-cell protein tyrosine phosphatase (TC-PTP) play non-redundant negative regulatory roles in T-cell activation and tumor antigen presentation. Previous studies have shown that the deletion of these two PTPs elicits potent anti-tumor immunity in vivo. I have shown that DU-14 and TP1L efficiently degrade their corresponding target with outstanding selectivity and elevate the cytokine-mediated phosphorylation of their substrates. As a result, I have shown that DU-14 and TP1L elicit potent anti-tumor immunity using co-culture or in vivo tumor models.</p><p><br></p><p dir="ltr">I also discovered an unexpected but beneficial off-target effect of SHP2 allosteric inhibitors under clinical trial and investigated its therapeutic implications for aberrant RAS-driven cancers. Aberrant activation of RAS-MAPK signaling is common in cancer, and efforts to inhibit pathway components have yielded drugs with promising clinical activities. As a central node essential for receptor tyrosine kinase-mediated RAS activation, SHP2 has emerged as an attractive cancer target. Consequently, many SHP2 allosteric inhibitors are now in clinical testing. I discovered a previously unrecognized off-target effect associated with SHP2 allosteric inhibitors. I showed that off-target autophagy inhibition by SHP2 allosteric inhibitors contributes to their anti-tumor activity. Finally, I exemplified a new therapeutic framework that harnesses both the on- and off-target activities of SHP2 allosteric inhibitors for improved treatment of mutant RAS-driven and drug-resistant malignancies such as pancreatic and colorectal cancers.</p><p><br></p><p dir="ltr">In summary, these studies facilitate the understanding of PTP disease biology and provide examples of successful strategies in developing small molecule PTP inhibitors for cancer therapy.</p>

No / PURPOSE: C-1311 is a member of the novel imidazoacridinone family of anticancer agents. This phase 1 trial was designed to investigate the safety, tolerability and preliminary anti-tumour activity of C-1311. PATIENTS AND METHODS: This was a phase 1, inter-subject dose escalating and pharmacokinetic study of intravenous (IV) C-1311, administered weekly during 3consecutive weeks followed by 1week rest (constituting 1 cycle) in subjects with advanced solid tumours. RESULTS: Twenty-two (22) patients were treated with C-1311, the highest dose given was 640mg/m(2). All subjects experienced one or more treatment-related adverse events (AEs). The most frequently observed treatment-related AEs were neutropaenia and nausea (50% each), followed by vomiting (27%), anaemia (23%), asthenia (23%) and diarrhoea (18%). Most treatment-related AEs were of Common Terminology Criteria for Adverse Events (CTCAE) grades 1-2, except for the blood and lymphatic system disorders, which were primarily of grades 3-4. The recommended dose (RD) of C-1311 administered as once weekly IV infusions for 3weeks every 4weeks is 480mg/m(2), with the dose limiting toxicity (DLT) being grade 4 neutropaenia lasting more than 7days. Treatment at this dose offers a predictable safety profile and excellent tolerability. CONCLUSION: The safety profile and preliminary anti-tumour efficacy of C-1311, observed in this broad-phase dose-finding study, warrants further evaluation of the compound.

Yes / Non-invasive methods to monitor tumour growth are an important goal in cancer drug development. Thermographic imaging systems offer potential in this area, since a change in temperature is known to be induced due to changes within the tumour microenvironment. This study demonstrates that this imaging modality can be applied to a broad range of tumour xenografts and also, for the first time, the methodology’s suitability to assess anti-cancer agent efficacy. Mice bearing subcutaneously implanted H460 lung cancer xenografts were treated with a novel vascular disrupting agent, ICT-2552, and the cytotoxin doxorubicin. The effects on tumour temperature were assessed using thermographic imaging over the first 6 hours post-administration and subsequently a further 7 days. For ICT-2552 a significant initial temperature drop was observed, whilst for both agents a significant temperature drop was seen compared to controls over the longer time period. Thus thermographic imaging can detect functional differences (manifesting as temperature reductions) in the tumour response to these anti-cancer agents compared to controls. Importantly, these effects can be detected in the first few hours following treatment and therefore the tumour is observable non-invasively. As discussed, this technique will have considerable 3Rs benefits in terms of reduction and refinement of animal use. / University of Bradford

Yes / 5,7-Dihydro-3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1, prepared from a dibenzyl ether precursor via Pd-catalysed intramolecular direct arylation, possesses broad-spectrum in vitro cytotoxicity towards various tumour cell lines, and induces vascular shutdown, necrosis and growth delay in tumour xenografts in mice at sub-toxic doses. The biological properties of 1 and related compounds can be attributed to their ability to inhibit microtubule assembly at the micromolar level, by binding reversibly to the same site of the tubulin αβ-heterodimer as colchicine 2 and the allocolchinol, N-acetylcolchinol 4.

Yes / The gadolinium-doped iron oxide nanoparticles (GdIONP) with greater specific power adsorption rate (SAR) than Fe3O4 was developed and its potential application in tumour therapy and particle tracking were demonstrated in transgenic adenocarcinoma of the mouse prostate C1 (TRAMP-C1) tumours. The GdIONPs accumulated in tumour region during the treatment could be clearly tracked and quantified by T2-weighted MR imaging. The therapeutic effects of GdIONP-mediated hyperthermia alone or in combination with radiotherapy (RT) were also evaluated. A significant increase in the tumour growth time was observed following the treatment of thermotherapy (TT) only group (2.5 days), radiation therapy only group (4.5 days), and the combined radio-thermotherapy group (10 days). Immunohistochemical staining revealed a reduced hypoxia region with vascular disruption and extensive tumour necrosis following the combined radio-thermotherapy. These results indicate that GdIONP-mediated hyperthermia can improve the efficacy of RT by its dual functions in high temperature (temperature greater than 45 °C)-mediated thermal ablation and mild-temperature hyperthermia (MTH) (temperature between 39 and 42 °C)-mediated reoxygenation.